Salt Inducible Kinases are Negative Regulators of Follicle Stimulating Hormone in

Ovarian Granulosa Cells

BY

MARAH ARMOUTI B.S., Benedictine University, 2012

THESIS

Submitted as partial fulfillment of the requirements for the degree of Doctor of Philosophy in Physiology and Biophysics

in the Graduate College of the University of Illinois at Chicago, 2020

Chicago, IL

Defense Committee: Carlos Stocco, Advisor Chong Wee Liew, Chair Henar Cuervo Grajal Mark Brodie Joanna Burdette, Department of Pharmaceutical Sciences

ii

ACKNOWLEDGEMENTS

In the name of God, the most Merciful. I am grateful for His countless blessings and the

privilege I have been given to pursue a higher degree.

I would like to thank all those with whom I have worked with in the Department of

Physiology and Biophysics over the past five years, particularly my advisor, Dr. Carlos Stocco,

who has provided me with great mentorship and direction. I am thankful for the opportunity to

explore ideas in the lab, make mistakes, and to have been made to feel like a colleague whose

suggestions and ideas are valued. I would also like to thank Dr. Nikki Winston, who was a

refreshing presence in the lab. I enjoyed exchanging ideas, talking about life, and many different

topics such as how to overcome challenges and dead-ends in research. To Dr. Meena Rao,

who encouraged and nurtured my interest in teaching. Finally, I would like to thank my thesis

committee—Dr. Chong Wee Liew, Dr. Henar Cuervo Grajal, Dr. Mark Brodie, and Dr. Joanna

Burdette—for their kindness and assistance. In my tendency to get nervous and frigid during

presentations, they would encourage me to show the excitement I feel about my results.

I am grateful for my parents, family, and friends along the way who supported me

throughout my graduate school journey. I would always tell my dad, Dr. Husein Armouti, that it is

his fault I ended up in a Ph.D. program, to which he would chuckle and say: “if it was easy,

everyone would do it”. Thank you, baba, now I see why you were eager to have me in the

“Ph.D. club”. To my mom, Majida, who gave me unwavering support, tough love, and

encouragement. She would always listen to me talking about my days. Whenever I felt

overwhelmed, she would do little things to uplift me or help me in any way she could. Mama,

your selfless love got me through this. To my siblings: Jena, Lena, and Abood, you three keep

me grounded. Thank you for helping keep my sense of normalcy throughout this journey. Jena

was someone I could turn to when I needed a laugh. She’s a fiery soul who always helps me

see a different perspective and challenges my thinking. Lena was the best support; she would

listen to me every night after coming back from lab and successfully calmed down many panic

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ACKNOWLEDGMENTS (continued)

attacks. We explored Chicago together and she took me to nice places to get my mind off of

things. She’s intuitive and gives amazing advice. Although I often consider myself like a second

mom to my brother Abood, I always end up learning more from him. He’s a creative and

intelligent man who puts himself out there and has the kindest heart. To my friend Taliha, I am

so thankful for your presence down the hall, and for our shared experience of being visibly

Muslim women in science. Your fearlessness and confidence helped me in more ways than I

have expressed. I am so excited for your bright future.

I would also like to thank my uncle and aunt, Dr. Said Al-Hallaj and Dr. Catherine

O’Connor, for their support throughout this journey. I am thankful to have you as family. To the

Abusalem family, thank you for being a constant and reliable safe place in my life.

I feel that teachers are often not given the credit they deserve. My undergraduate

Physiology professor, Dr. Jayashree Sarathy, made Physiology so much fun to learn about. My

undergraduate Physiology Lab professor, Dr. John Mickus, challenged me to truly understand

the subject, and not merely memorize facts about the body. Thank you both for instilling in me

the love of Physiology and the desire to pursue it further.

And to my future husband, Waleed. I thank God every day that He put you in my life.

Thank you for supporting me every single day and believing in me. Your calming energy and

encouragement are exactly what I needed in the final stretch of this journey. I cannot wait to see

what the rest of our lives brings.

MHA

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TABLE OF CONTENTS

CHAPTER PAGE

I. INTRODUCTION: OVARIAN PHYSIOLOGY AND HUMAN FERTILITY……………………….1 1. Infertility……………………………………………………………………………………….1 2. The Ovary…………………………………………………………………………………….3

1. The Follicle……………………………….……………………………………........…….3 a) Follicle Formation…………………………………………………………………….3 b) General Structure…………………………………………………………………….3

2. The Oocyte………………………………………………………………………………..4 3. Granulosa Cells…………………………………………………………………………..5 4. Theca Cells………………………………………………………………………………..5 3. Folliculogenesis……………………………………………………………………………...8 a) Primordial follicle recruitment and activation……………………………………...8 b) Primary, secondary, and pre-antral follicles……………………………………….8 c) Antral follicles…………………………………………………………………………9 d) Dominant follicle……………………………………………………………………...9 e) Ovulation………………………………………………………………………………9 f) Corpus luteum……………………………………………………………………….10 g) Follicular atresia…………………………………………………………………….10 h) Timing………………………………………………………………………………..11 1. Steroidogenesis and the Two Cell Theory..………………………………………….11 4. Endocrine and Paracrine Signaling in Granulosa Cells………………………………..13 1. Follicle Stimulating Hormone…………………………………………………………..13 2. Insulin-like Growth Factors…………………………………………………………….14 3. Salt Inducible Kinases………………………………………………………………….16 5. Statement of Hypothesis and Aims………………………………………………………19

II. MATERIALS AND METHODS…………………………………………………………………….21 1. Human granulosa cell processing and culture………………………………………….21 2. Rodent granulosa cell isolation and culture……………………………………………..22 3. Messenger RNA (mRNA) quantification…………………………………………………22 4. Treatments and inhibitors…………………………………………………………………25 5. Western blotting…………………………………………………………………………….25 6. Animal handling…………………………………………………………………………….26 7. Genotyping………………………………………………………………………………….26 8. Fluorescent immunocytochemistry……………………………………………………….30 9. Immunohistochemistry…………………………………………………………………….30 10. Luciferase assay……………………………………………………………………………30 11. 17b-Estradiol measurement in rat granulosa cell culture media………………………31 12. Ovulation assay…………………………………………………………………………….31 13. Statistical analyses…………………………………………………………………………31

III. CONDITIONAL KNOCKDOWN OF IGF1R IN THE GRANULOSA CELLS IMPAIRS STEROIDOGENESIS AND AKT ACTIVATION……………………………………………………...32

A. Introduction……………………………………………………………………………………...32 B. Results…………………………………………………………………………………………..33

1. Expression of Cre-recombinase under both Cyp19a1 and Ers2 promoters leads to undetectable levels of IGF1R in GCs…………………..…………………………….33

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TABLE OF CONTENTS (continued)

CHAPTER PAGE

2. The IGF1R is necessary for FSH-induced steroidogenesis and differentiation of GCs in vivo…………………………………………………………..………………….33

3. The IGF1R does not affect FSHR expression in vivo………………………………….34 4. A lack of IGF1R impairs FSH-induced AKT phosphorylation in vivo…………………34

C. Discussion……………………………………………………………………………………….41 IV. SALT INDUCIBLE KINASES OPPOSE FSH ACTIONS IN CULTURED GRANULOSA CELLS……………………………………………………………………………………………………42

A. Introduction……………………………………………………………………………………...42 B. Results…………………………………………………………………………………………..42

1. Human and rodent granulosa cells express SIK1, SIK2, and SIK3…………………..42 2. SIKs inhibition in rodent granulosa cells enhances FSH actions……………………..43 3. Steroidogenesis in primary human granulosa cells is inhibited by SIKs activity…….43 4. SIKs inhibition recovers aromatase production in IVF patients with

different etiologies………………………………………………………………………….44 C. Discussion……………………………………………………………………………………….51

V. SALT INDUCIBLE KINASE 2 ATTENUATES FSH ACTIONS………………………………...52 A. Introduction………………………………………………………………………………………52 B. Results……………………………………………………………………………………………52 1. SIKs inhibition potentiates FSH-induced steroidogenesis in vivo………………..…...52 2. Knockdown of SIK2 enhances FSH actions in vitro………………………………..…..53 3. Granulosa cells of GC-specific SIK2 knockdown mice have increased steroidogenesis………………………………………………………………..54

4. Effect of SIKs inhibition or SIK2 knockdown on ovulation……………………………..54 C. Discussion………………………………………………………………………………………62 VI. UNDERSTANDING THE MECHANISM OF SIKS ACTIONS………………………………..63

A. Introduction…………………………………………………………………………………...…63 B. Results…………………………………………………………………………………………..63

1. SIKs actions are downstream of cAMP signaling………………………………….…...63 2. FSH does not induce SIKs expression…………………………………………………..64 3. SIKs involvement in the IGF1 receptor pathway………………………………………..65 4. Role of GSK3b on the interaction between FSH and SIKs in GCs…………………...66 5. Effect of SIKs inhibition on PKA downstream targets………………………………….66

C. Discussion……………………………………………………………………………………….76 VII. GENERAL CONCLUSIONS AND FUTURE DIRECTIONS…………………………………78 VIII. APPENDICES……………………………...…………………………….………………………84

A. Appendix A………………………………………………………………………………………84 B. Appendix B………………………………………………………………………………………85 C. Appendix C……………………………………………………………………………………...86 D. Appendix D……………………………………………………………………………………...88

IX. CITED LITERATURE…………………………………………………………………………….89 X. VITA..………………………………………………………………………………………………97

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LIST OF TABLES

TABLE PAGE TABLE I: QUANTITATIVE PCR PRIMERS……………………………………..……………………24 TABLE II: TREATMENTS, ACTIVATORS, AND INHIBITORS…………………………..………...27 TABLE III: WESTERN BLOT ANTIBODIES…………………………………………………..……..28 TABLE IV: GENOTYPING PCR PRIMERS…………………………………………………………..29

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LIST OF FIGURES

FIGURE PAGE

1. Summary of reproductive trends…………………………………………………….…………2

2. Changes in oocyte numbers during fetal and postnatal life…………………….…………...6

3. Follicle development and structure………………………………………………….…………7

4. Steroidogenesis in the ovary…………………………………………….……………………12

5. SIK structure and placement in the cAMP pathway………………………………………...18

6. Knockdown of IGF1R expression in GCs………………………….………………………...35

7. Fertility effects of IGF1R knockdown in GCs………………………………….…………….36

8. Fertility effects of IGF1R knockdown in GCs……………………………….……………….37

9. Relative expression of the main differentiation markers in GCs of control and IGF1Rgcko mice……………………………………………….…………..38

10. Relative expression of the FSH receptor in GCs of control and IGF1Rgcko mice…………………………………………………..………..………39

11. Lack of IGF1R in GCs leads to diminished AKT activation…………….…………………40

12. Expression of SIKs in rat and human GCs………………………….……………………….46

13. Expression of SIKs in the rat ovary…………………………………..………………………47

14. SIKs inhibition enhances FSH actions in primary rat GCs…………….…………………..48

15. SIKs inhibition with HG enhances FSH actions in primary human GCs…………….……49

16. SIKs inhibition rescues FSH actions in human GCs from patients with different etiologies of infertility………………………………………….……...50

17. Effect of SIKs inhibition in mouse GCs……………………………………………….……...56

18. SIKs inhibition enhances FSH actions in vivo………………………………………….……57

19. SIK2, not SIK3, pharmacological inhibition augments aromatase expression………………………………………………………………….………58

20. SIK2 knockdown mimics the pharmacological inhibition of SIKs activity…………………59 21. SIK2 knockdown in GCs augments steroidogenesis in vivo……………………………….60

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LIST OF FIGURES (continued)

FIGURE PAGE

22. SIKs inhibition or SIK2 knockdown does not increase ovulation………………………….61

23. SIKs actions are downstream of the FSH receptor…………………………………………68 24. SIKs activity is downstream of PKA…………………………………………………………..69

25. FSH does not induce SIKs expression……………………………………………………….70

26. SIKs and the IGF1R pathway…………………………………………………………………71

27. SIKs and the IGF1R pathway………..………………………………………………………..72

28. GSK3b inhibition does not potentiate FSH actions…………………………..……………..73

29. SIKs inhibition does not increase AKT phosphorylation…………………………………...74

30. SIKs inhibition does not increase CREB phosphorylation…………………………………75

31. Summary of SIKs actions and placement in granulosa cells………………………………83

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LIST OF ABBREVIATIONS

17b-HSD 17-beta Hydroxysteroid Dehydrogenase 3b-HSD 3-beta Hydroxysteroid Dehydrogenase 8CPT ‘8CPT-2Me-cAMP, an Epac activator AC Adenylate Cyclase AEW NVP-AEW451, an IGF1R inhibitor AKT v-akt murine thymoma viral oncogene homolog/protein kinase B (PKB) AMH Anti-Müllerian Hormone AMPK Adenosine Monophosphate activated protein Kinase ANOVA Analysis of Variance ART Assisted Reproductive Technologies BMP15 Bone Morphogenetic Protein 15 BSA Bovine Serum Albumin cAMP Cyclic Adenosine Monophosphate CHIR CHIR-99021, a GSK3 inhibitor cDNA Complimentary Deoxyribonucleic Acid CL Corpus Luteum COC Cumulus-Oocyte Complex CoC Compound C CRE CREB-Response Element Cre Causes Recombination CREB cAMP Response Element Binding Protein CRTC CREB-Regulated Transcription Coactivator (also TORC) CYP11A1 Cholesterol Side Chain Cleavage Enzyme (also P450scc) CYP19A1 Aromatase

x

LIST OF ABBREVIATIONS (continued) dbcAMP Dibutyryl Cyclic Adenosine Monophosphate, a cAMP analog DMEM/F12 Dulbecco’s Modified Eagle Medium/Nutrient Mixture F-12 DMSO Dimethyl Sulfoxide DNA Deoxyribonucleic Acid ECM Extracellular Matrix Epac Exchange Protein Activated by cAMP ERK Extracellular Regulated Kinases ERS2 Estrogen Receptor beta F/F Floxed/Floxed FOXO Forkhead family transcription factors FSH Follicle-Stimulating Hormone FSHR Follicle-Stimulating Hormone Receptor FSK Forskolin, an adenylate cyclase activator GC Granulosa Cell GCKO Granulosa Cell Knockout GDF9 Growth Differentiation Factor 9 GnRH Gonadotropin-Releasing Hormone GPCR G-protein Coupled Receptor GSK3b Glycogen Synthase Kinase-3 beta H&E Hematoxylin and Eosin hCG Human Chorionic Gonadotropin HDAC Histone Deacetylase HG HG-9-91-01, a SIKs inhibitor IGF Insulin-like Growth Factor

xi

LIST OF ABBREVIATIONS (continued)

IGF1R Insulin-like Growth Factor 1 Receptor IHC Immunohistochemistry IRS Insulin Receptor Substrate i.p. Intraperitoneally IVF in vitro Fertilization KO Knockout LH Luteinizing Hormone LHR Luteinizing Hormone Receptor LKB1 Liver Kinase B1 MAPK Mitogen-activated Protein Kinase mRNA Messenger Ribonucleic Acid MRT MRT67307, a SIKs inhibitor P450scc Cholesterol Side Chain Cleavage Enzyme (also CYP11A1) PBS Phosphate Buffered Saline PCR Polymerase Chain Reaction PI3K Phosphatidylinositol-3 Kinase PKA Protein Kinase A PMSG Pregnant Mare’s Serum Gonadotropin qPCR Quantitative real-time Polymerase Chain Reaction RIPA Radioimmunoprecipitation Assay RNA Ribonucleic Acid RPL19 Ribosomal Protein L19 SEM Standard Error of the Mean SIK Salt-Inducible Kinase

xii

LIST OF ABBREVIATIONS (continued) StAR Steroidogenic Acute Regulatory Protein TC Theca Cell TORC Transducer of Regulated CREB (also CRTC) WT Wildtype YKL YKL-05-099, a SIKs inhibitor

xiii

SUMMARY

Infertility affects 12% of couples in the United States. About 40% of the seven million

women suffering from fertility issues have ovulatory dysfunctions. The ovary serves as a site of

gamete formation, the oocyte, and steroid hormone production. Oocytes are housed at the

center of the follicle before they are released from the ovary at ovulation, which is the

culmination of a long process of follicle growth and maturation called folliculogenesis. Within

each follicle, the oocyte interacts with surrounding granulosa cells (GCs) that act as nurse cells

and produce the steroid hormone estradiol. GC differentiation into highly steroidogenic cells is

critical for proper follicle development. Towards the end of the folliculogenesis process, GCs

differentiate into two populations: the less differentiated cumulus cells directly surrounding the

oocyte, and the more differentiated mural cells at the follicular periphery. Pituitary-secreted

follicle-stimulating hormone (FSH) induces mural GC differentiation and is the main factor

controlling estradiol synthesis. FSH actions require paracrine factors such as insulin-like growth

factors (IGFs). Here, I study the signaling crosstalk between the FSH receptor (FSHR) and IGF1

receptor (IGF1R).

First, I showed that the IGF1R is required for FSH actions in vivo since previous studies

focused on in vitro models. I confirmed findings that the FSHR and IGF1R pathways converge

on AKT activation and that the crosstalk between the two pathways is required for female

fertility. Female mice lacking IGF1R expression in GCs have impaired folliculogenesis, do not

ovulate, and are infertile. Their GCs lack the expression of key differentiation genes while

expressing increased markers of apoptosis. Finally, AKT phosphorylation is impaired,

confirming that FSH and IGF1 converge on AKT activation in vivo.

Next, I examined the interaction between FSHR and IGF1R signaling. The FSH receptor

activates cAMP signaling leading to PKA-dependent activation of CREB. As described above,

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SUMMARY (continued)

FSH activates AKT in an IGF1R dependent manner. Thus, I evaluated the role of factors

downstream of AKT and CREB. This analysis suggested that salt-inducible kinases (SIKs) may

play a role in the control of GC function. SIKs are known attenuators of cAMP actions in

osteocytes and macrophages but have not yet been studied in ovarian GCs. SIKs are also

inhibited by factors downstream of AKT signaling. In light of this, we investigated SIKs actions in

ovarian GCs and whether they control the FSHR and IGF1R pathways.

Firstly, I characterized the expression of SIK1, SIK2, and SIK3 and observed they are

highly expressed in rodent and human GCs. Moreover, I found that inhibition of SIK activity

attenuates FSH actions in vitro in primary human and rat GCs. This effect of SIK was also

observed in vivo. Thus, wild-type mice injected with a SIK inhibitors and FSH had a significantly

higher increase in estradiol production when compared to mice injected with FSH alone. Next, I

determined the role of each SIK gene by knocking down SIK1, SIK2, or SIK3 separately using

shRNA and observed that only SIK2 knockdown increases GC steroidogenesis. Based on these

results, we developed a mouse model of GC-specific SIK2 knockdown. These mice have

increased steroidogenesis in response to FSH when compared to control mice. However, the

number of oocytes ovulated was comparable to wildtype mice, suggesting the presence of other

controlling factors that link steroidogenesis and ovulation.

The last chapter of this thesis aimed to investigate the placement of SIKs activity within

the FSHR/IGF1R pathway. SIKs inhibition potentiated estradiol synthesis in the presence of

cAMP or a PKA activator demonstrating that SIKs actions occur downstream of cAMP and PKA.

We also determined that FSH treatment does not induce SIKs expression and that SIK does not

affect AKT phosphorylation. Finally, I showed that SIK inhibition does not alter FSH activation of

CREB. I concluded that SIKs target the FSH pathway by affecting factors located between

cAMP/PKA and CREB and propose that SIKs control the activity of CREB cofactors.

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SUMMARY (continued)

Taken together, the results show that SIKs, particularly SIK2, are critical modulators of

ovarian function. My findings demonstrate for the first time that SIKs blunt the response of GCs

to FSH and cAMP. Because FSH actions are crucial during folliculogenesis, my findings place

SIKs as crucial players in the regulation of female fertility.

1

I. INTRODUCTION: OVARIAN PHYSIOLOGY AND HUMAN FERTILITY 1- Infertility

Infertility is defined as the inability to become pregnant after twelve months of unprotected

intercourse, or six months for women aged 35 or older. In the United States, 15.5% of

reproductive-age women are infertile (1). Among the 12% of couples struggling with infertility, the

problems are equally distributed between the woman, the man, or a combination of both in

addition to unexplained causes (2). The emotional and mental burden that infertility poses are

also important problems. For instance, women that unsuccessfully undergo infertility treatments

are more likely to experience psychiatric disorders and to abuse intoxicants (3).

In women, a key factor contributing to infertility is the natural decline in the reproductive

capacity that starts around the age of 32 and rapidly decreases after the age of 35 (Figure 1A).

Additionally, older women are more likely to experience pregnancy loss (4). A consequence of

this decline is an increase in infertility as women are waiting longer to have children. Between

1970 and 2012, the number of women having their first child at 35-39 years old increased six-fold,

while first births to women aged 40-44 years rose four-fold in the same time (5) (Figure 1B).

As a result of the increasing prevalence of infertility, more women are resorting to assisted

reproductive technologies (ART). The primary type of ART is in vitro fertilization (IVF), in which

ovulation is stimulated and monitored to maximize the production of mature oocytes, which are

extracted, fertilized in the laboratory, and transferred back into the uterus. In 2017 alone, there

were 284,385 ART cycles performed in the United States that resulted in 68,908 live births,

yielding 78,052 live-born infants (6). The number of babies born with the help of IVF clinics

doubled over the past two decades, and today, about 1.7% of all infants born in the US every

year are conceived through ART (7).

The most common cause of infertility in women is the failure to ovulate, which accounts

for 40% of all potential causes (7). Therefore, a better understanding of ovarian function and its

control is vital to better understand infertility and to design new and more effective treatments.

2

Figure 1: Summary of reproductive trends

(A) Natural conception: schematic demonstrating trends in pregnancy and miscarriage rates according to age (8).

(B) First birth rates by selected age of mother: United States, 1970-2012 (5).

A

B

3

2- The Ovary The ovary is the gonad of the female reproductive tract, which stores gametes (also

known as oocytes) and plays a central role in the regulation of the female reproductive cycle. The

follicle is the functional unit of the ovary and is the structure that houses the oocyte and the

somatic cells: granulosa cells (GCs) and theca cells (TCs). These cells produce the sex steroids

estradiol and progesterone require to coordinate the functionality of the female reproductive tract

and its preparation for conception. Each follicle contains one oocyte, GCs, and TCs.

2.1 The Follicle

2.1.a: Follicle Formation

During embryonic development, primordial germ cells originate at the proximal region of

the epiblast and migrate to the genital ridge by six weeks of gestation. At the genital ridge,

primordial germ cells, now called oogonia, are critical for the development of the ovary and

undergo mitosis, increasing their numbers to peak at 6-7 million oogonia by 20 weeks of

development (9). At birth, all oogonia have either entered meiosis to become primary oocytes or

undergone atresia. Those forming primary oocytes acquire a single surrounding layer of somatic

GCs and form a new structure called the primordial follicle. The combination of a reduced rate of

mitosis and an increased rate of oogonia/follicular atresia causes the primordial follicle count to

fall to 1-2 million at birth. By puberty, the follicular pool is reduced to around 300,000, of which

only 400-500 are ovulated during a woman’s reproductive life span, while the rest undergo

atresia. The follicular pool is exhausted by menopause (Figure 2).

2.1.b: General Structure

The follicle goes through progressing stages of growth from the primordial phase, leading

to ovulation. The oocyte is at the center of the primordial follicle, with a single layer of flattened

GCs surrounding it. When the follicle progresses to the primary stage, the GCs change

morphology to become cuboidal shaped. At the secondary stage, the GCs have proliferated and

formed several layers within a basement membrane. At this stage the follicle also acquires a TC

4

layer outside the basement membrane (Figure 3). The theca layer forms when GCs secrete kit

ligand that binds to its receptor expressed on TCs, causing the organization of this layer (9). After

this, a fluid-filled cavity begins to form within the GC layer called the antrum, forming the antral

follicle. When an antral follicle is selected to progress to the pre-ovulatory stage, a rapid increase

in the volume of the antrum divides GCs into two populations (see 2.3 Granulosa cells).

2.2 The Oocyte

The oocyte is the female gamete housed in the follicle. In primordial follicles, the oocyte is

arrested at prophase I. It remains so until a few hours before ovulation, when the follicle is

exposed to the LH surge, causing the oocyte to complete meiosis I, extrude the first polar body,

and to enter meiosis II. Now, the “secondary oocyte” is arrested at metaphase II due to its

production of high amounts of MAPK pathway proteins that maintain its arrest (10). Secondary

oocytes complete meiosis II only after fertilization.

Although the primary oocyte is arrested at prophase I during the various follicular stages,

it still goes through several phases of maturation. In the primordial follicle, the primary oocyte

begins transcription and translation to produce proteins necessary for oocyte survival and

completion of meiosis (9). Towards the antral stage, the oocyte secretes extracellular matrix

glycoproteins to form the zona pellucida, a protective layer surrounding the oocyte that contains

the binding site for sperm during fertilization (11). By the pre-ovulatory follicular stage, the primary

oocyte accumulates enough cell cycle proteins, making it competent to complete meiosis. Thus,

the oocyte is not a passive occupant of the follicle, but rather it plays an important role in follicular

function through secreting various paracrine factors that regulate follicle growth and development

(12,13).

5

2.3 Granulosa Cells

Granulosa cells are located within the basement membrane of the follicle and are

connected to each other and the oocyte via gap junctions. GCs nurture the oocyte, sustain its

maturation, and deliver the hormones required to synchronize uterus receptivity with the release

of mature eggs. GCs are the only cells that closely interact with the oocyte from the moment the

follicle forms until the release of the oocyte at ovulation (14,15). For example, GCs produce

cAMP that maintains the oocyte in a state of meiotic arrest. Also, the oocyte secretes growth

differentiation factor 9 (GDF-9) and bone morphogenetic protein 15 (BMP-15) that influence GC

function (16). Throughout this long relationship, both the oocyte and the GCs go through

significant functional and morphological changes.

By the pre-ovulatory stage, GCs have differentiated into two populations: the more

differentiated mural cells at the periphery of the follicle and the less differentiated cumulus cells

directly surrounding the oocyte. Pituitary-secreted follicle-stimulating hormone (FSH) causes GC

proliferation and differentiation into mural cells, which are steroidogenic cells that mainly produce

estradiol. The oocyte secretes factors that prevent the surrounding cumulus cells from

differentiating into mural cells. Cumulus cells are released with the oocyte during ovulation and

facilitate its uptake by the fallopian tubes. After ovulation, the remaining mural cells in the follicle

undergo a process of terminal differentiation, called luteinization, and form the corpus luteum

(CL). The CL produces progesterone for the remainder of the ovarian cycle and sustains early

pregnancy.

2.4 Theca Cells

Theca cells (TCs) are located outside the basement membrane of the follicle and play an

important role in maintaining follicular structure. Importantly, TCs produce androstenedione,

which is the precursor of estradiol. The proper interaction between the oocyte, GCs, and TCs is

critical for the maintenance of the follicular structure and the proper growth and maturation of the

follicle during a process called folliculogenesis (17).

6

Figure 2: Changes in oocyte numbers during fetal and postnatal life. The total number of oocytes is a reflection of the balance between active proliferation and oocyte/follicle atresia (9).

7

Figure 3: Follicle development and structure During follicular development, the primordial follicle initially contains one layer of flattened GCs surrounding the oocyte. When follicular growth is activated, the single GCs layer of the primary follicle changes morphology to become cuboidal cells. These GCs proliferate and form multiple layers in the secondary follicle. A fluid-filled cavity begins to form within the GCs, called the antrum. Once a single dominant follicle is selected within the cohort, it becomes the pre-ovulatory follicle. Here, the antrum grows, and the GCs differentiate into the mural cells lining the follicular wall, and the cumulus GCs that immediately surround the oocyte. (GCs = granulosa cells).

PrimordialFollicleFlattened

layer of GCs

PrimaryFollicle

Single layer of cuboidal GCs Secondary/preantral

FollicleMultiple layers of GCs;

TCs present

Early Antral Follicle

Preovulatory FollicleAntrum formed; Cumulus and mural

GCs present

Initiation/Recruitment

SelectionCumulus GCs

Antrum

Mural GCs

8

3- Folliculogenesis

Ovulation involves the release of the oocyte from the follicle. It is the culmination of a

lengthy process of growth and maturation, called folliculogenesis, that starts with the activation of

a group of primordial follicles and ends with a selection of one dominant pre-ovulatory follicle. The

different stages of folliculogenesis are described below:

a) Primordial follicle recruitment and activation: Primordial follicles are quiescent and can remain

so from birth until menopause. They are formed when one primary oocyte acquires one

surrounding layer of flattened GCs within a basal lamina. Primordial follicles exit quiescence

and start the growth process in groups. Once it is activated, the layer of flat GCs become

cuboidal. At this point, the follicle is considered a primary follicle. Interestingly, the factors

causing primordial follicle activation are unknown, but it is believed that intraovarian factors

play a large role (12,18).

b) Primary, secondary, and pre-antral follicles: Once primary follicles form, GCs begin to

proliferate. Follicles with two or more layers of GCs around the oocyte are known as

secondary follicles. A key feature of secondary follicles is the formation of a thecal cell layer

outside the basement membrane. Once a TC layer is acquired, the pre-antral follicle moves

towards the inner ovarian cortex, closer to the vasculature of the medulla, and secretes

angiogenic factors, which stimulates the vascularization of the follicles (9). Notably, growth

until the pre-antral stage is gonadotropin-independent, since the follicle is still avascular.

Rather, the oocyte guides this process, which was demonstrated in experiments where

oocytes from mouse secondary follicles grafted into primordial follicles increased the growth

rate of the recipient follicles (19).

Growing pre-antral follicles and early antral follicles secrete anti-Müllerian hormone

(AMH). AMH plays an important role in maintaining the follicular pool as it suppresses the

recruitment of primordial follicles (20). AMH secreted by the GCs is detected in the follicular

fluid and serum and can be used in the clinic as an indicator of the ovarian reserve. AMH

9

gene expression levels decline towards the pre-ovulatory stage, indicating that pre-

antral/early antral follicles are the main source of AMH secretion (20).

c) Antral follicles: As pre-antral follicles continue to grow, they accumulate a fluid forming a

cavity called the antrum. GCs are responsible for the formation of the antrum in response to

FSH; they transport ions into the follicular space, creating an osmotic gradient and allowing

the transcellular movement of water through aquaporins 7,8, and 9 (21). The antrum serves

as a site for nutrient exchange and waste removal (22), facilitates the expulsion of the oocyte

at ovulation, and divides the GCs into the mural and cumulus populations.

The antral follicle is dependent on pituitary gonadotropins for its viability, steroidogenesis,

and rapid growth. Luteinizing hormone (LH), induces thecal cells to produce androstenedione

and testosterone, and FSH induces aromatase expression by mural GCs (see 3.1

steroidogenesis), which produce estradiol. Mural GCs also produce inhibin B, which together

with estradiol negatively feeds back on pituitary FSH secretion. This step is important to

detect the follicle that is most responsive to FSH.

d) Dominant follicle: During the follicular phase, a cohort of antral follicles rapidly grows under

the control of the gonadotropins leading to a progressive increase in estradiol production. The

consequent decrease in FSH levels due to estradiol negative feedback causes most of the

cohort to undergo follicular atresia until one follicle, which can survive in the presence of

decreasing FSH, is left. This dominant follicle is characterized by having the highest FSH

receptor (FSHR) expression. FSH also induces LH receptor (LHR) expression in the mural

cells, allowing them to respond to the upcoming LH surge that triggers ovulation (9).

e) Ovulation: As the dominant follicle secretes high levels of estradiol, it exerts a positive

feedback effect on pituitary gonadotropins stimulating mainly the secretion of LH. Towards the

middle of the cycle, LH reaches a peak level, called the LH surge, that affects the follicle in

several ways (23). First, it causes the expulsion of the oocyte with the surrounding cumulus

cells within 32-36 hours in humans. This is accompanied by the release of inflammatory

10

cytokines as the follicular and ovarian walls are ruptured. Second, it causes the terminal

differentiation, or luteinization, of the remaining mural and thecal cells and formation of the

corpus luteum (CL). The CL is highly steroidogenic and produces large amounts of

progesterone that prepares the female reproductive tract for implantation and gestation (10).

The LH surge also suppresses aromatase expression; therefore, estradiol production

decreases, and progesterone becomes the dominant hormone in circulation during the luteal

phase. Finally, the LH surge causes the oocyte to complete meiosis I, enter meiosis II, and

arrest at metaphase II. Ovulation marks the mid-point of the ovarian cycle.

f) Corpus luteum: After ovulation, leftover debris from the ovarian rupture is removed by

macrophages. The remaining mural and theca cells enlarge and become filled with lipids.

They occupy the follicular space, have limited proliferation, and form the corpus luteum. The

CL is formed during the second part of the ovarian cycle, the luteal phase, and in humans is

viable for about 14 days if no pregnancy occurs (10). It produces high levels of progesterone,

which causes the uterus to secrete nutrients in preparation for implantation and to support

blastocyst viability. If pregnancy does not occur, then progesterone levels decline and the CL

regresses into a scar-like structure called the corpus albicans. At this point, menses ensues,

and another ovarian cycle begins. If pregnancy does occur, then the implanting embryo

secretes human chorionic gonadotropin (hCG) that maintains the CL during the first trimester.

After that, the placenta takes over progesterone production.

g) Follicular atresia: As mentioned previously, most follicles in the ovary undergo atresia at

different stages of folliculogenesis. Atresia occurs spontaneously due to the absence of key

trophic factors at critical times during folliculogenesis, or in response to environmental factors

(24,25). During this process, the GCs and oocytes undergo apoptosis, and the theca cells

persist in the ovarian stroma. The fas-fas ligand system is an important mediator of follicular

atresia; fas-deficient mice have an increased number of secondary follicles, decreased

numbers of large antral follicles, and a defective GC and oocyte cell death response to fas

11

ligand (26). Other pro-apoptotic signaling proteins are implicated in follicular atresia, such as

bax and caspases 2,3,9,11, and 12 (27,28). Caspase 3 function is necessary to maintain

appropriate GC apoptosis; mice lacking its activity display aberrant follicular atresia. The rate

of primordial follicle activation and appropriate follicular atresia both determine a woman’s

ovarian reserve and consequently her reproductive life span.

h) Timing: Follicular growth from the primordial to the pre-ovulatory stage takes approximately

one year to complete (9). The majority of this time is spent in the gonadotropin-independent

stage, prior to antrum formation. Circulating gonadotropins only guide the last 50 days of the

maturation process.

3.1 Steroidogenesis and the Two-Cell, Two-Gonadotropin Theory

Theca cells and mural GCs are needed for estradiol production (Figure 4A). TCs and

mural GCs express the genes needed to produce progesterone, such as Steroidogenic Acute

Regulatory Protein (StAR, which transports cholesterol into the mitochondria), cholesterol side-

chain cleavage enzyme (P450scc or CYP11A1, which catalyzes the first step of steroidogenesis),

and 3b-Hydroxysteroid Dehydrogenase (3b-HSD, which catalyzes the conversion of the precursor

pregnenolone to progesterone) (10). However, only TCs produce androgens by expressing

cytochrome P450 17A1 (CYP17A1), whereas GCs express CYP19A1 (aromatase) and can

convert androgens into estrogens. In fact, aromatase is a marker of GC differentiation and is

highly expressed in mural GCs and at low levels in cumulus GCs. Aromatase is critical for female

fertility; aromatase knockout mice are infertile because their follicles arrest at the antral stage of

development and do not reach ovulation (29). In the two-cell, two-gonadotropin system, LH

stimulates TCs to produce androgens that diffuse to the GC layer and are converted to estrone or

estradiol by FSH-induced aromatase. Aromatase converts testosterone to estradiol and

androstenedione to estrone. GCs express 17b-Hydroxysteroid Dehydrogenase (17b-HSD), which

converts estrone into the more potent estradiol (Figure 4B).

12

Figure 4: Steroidogenesis in the ovary

(A) The two-cell, two-gonadotropin theory: TCs produce LH-induced androgens that diffuse to GCs and are converted to estradiol by FSH-induced aromatase. Ch=cholesterol. P=progesterone.

(B) Key enzymes and products in the ovarian steroidogenic pathway

Antral Follicle

IGFs

Ch P

Ch P Androgens

EstradiolCYP17A1

StARP450scc3b-HSD

Aromatase

FSH

StARP450scc3b -HSD

Granulosa Cells

Theca Cells

Androgens

Estradiol

Basement Membrane

LH

A

Cholesterol

Pregnenolone

Estrone

Testosterone

Androstenedione

Progesterone

Estradiol

CYP11A1/P450scc

3b-HSD

CYP17A1

17b-HSD 17b-HSD

CYP19A1/aromatase

CYP19A1/aromatase

B

13

4- Endocrine and Paracrine Signaling in Granulosa Cells

4.1 Follicle Stimulating Hormone Signaling

The effects of FSH actions in the ovary have been previously described. FSH is a

heterodimeric glycoprotein consisting of an a and b subunit. It is released from the anterior

pituitary gland in response to pulsatile gonadotropin-releasing hormone (GnRH) secretions from

the hypothalamus. FSH shares the same a subunit as pituitary luteinizing hormone (LH) and

thyroid-stimulating hormone (TSH), but they each have a unique b subunit (30). The glycosylation

of these hormones is important for subunit assembly, stabilization, secretion, circulatory half-life,

and biological activity (30).

FSHR is a G-protein coupled receptor (GPCR) consisting of an N-terminal extracellular

domain that specifically binds the b subunit of FSH, seven transmembrane segments connected

by three extracellular loops and three intracellular loops, and a C-terminal intracellular domain.

Expression of FSHR is necessary for female fertility; mice lacking FSHR have small ovaries, thin

uteri, and are infertile because their follicles do not develop past the pre-antral stage (31). Also,

these mice have enlarged pituitary glands with significantly more FSH-positive cells due to a lack

of FSH responsiveness. Binding of FSH to the FSHR stimulates adenylyl cyclase (AC) activity

(32), which activates the second messenger cyclic AMP (cAMP), and consequently protein kinase

A (PKA). A canonical target of FSH-induced PKA activation in GCs is the phosphorylation of

cAMP response element-binding protein (CREB). The phosphorylated CREB, a transcription

factor, binds CREB response elements (CRE) at the promoter regions of several GC-

differentiation genes and upregulates their transcription (33).

It has been shown that FSH stimulation of PKA triggers the activation of different signaling

cascades downstream of PKA, other than CREB. For example, PKA rapidly phosphorylates

histone H3, which results in the activation of several FSH-induced GC differentiation genes such

as inhibin-a (34). Another pathway involved in FSH-stimulated GC differentiation is the

14

phosphatidylinositol-3 kinase (PI3K) pathway, which leads to AKT activation. The PI3K-AKT

pathway is necessary for the induction of aromatase and other steroidogenic genes (35,36). PKA

has been shown to enhance the activity of other signaling cascades such as the extracellular

regulated kinases (ERKs) and p38 mitogen-activated protein kinases (MAPKs) (34). The

integration of these pathways to regulate GC differentiation is still not fully understood (37).

FSH-induced aromatase transcription requires CREB binding to the ovary-specific PII-

promoter region. It has been shown that two other transcription factors are required for aromatase

production: liver receptor homolog 1 (LRH1) and steroidogenic factor 1 (SF1). All three

transcription factors are expressed in GCs and are necessary for their differentiation (38,39). In

addition, FSH induction of CREB activity is modulated and is not an all or nothing response. FSH

has been reported to activate calcium signaling, which through Ca2+-calmodulin activity activates

the phosphatase calcineurin. Calcineurin could indirectly modulate CREB activity by

dephosphorylating/activating the CREB co-activator CREB-regulated transcription coactivator,

formerly named transducer of regulated CREB (CRTC/TORC). Calcineurin and CRTC have been

shown to increase the stimulation of StAR, CYP11A1, 3b-HSD, and aromatase in response to

FSH and TGFb1 signaling (40,41).

4.2 Insulin-like Growth Factors

Although FSH is the endocrine driver of follicle development, several other paracrine

factors are also involved. Of those, the insulin-like growth factors (IGFs) have a critical role in

folliculogenesis. IGF1 and IGF2 are polypeptide hormones that have several important functions.

The main source of IGF1 in circulation is the liver, and IGF1 secretion is potentiated by pituitary

growth hormone (GH) (42). In target tissues, IGF1 binds to and activates the IGF1 receptor

(IGF1R) to promote growth, cell proliferation, and survival (43). IGF2 is also secreted by the liver

and functions as the main growth factor in fetal development, although it is also expressed in

15

different adult tissues, including the liver (44). The systemic activity of liver-released IGF2 is not

well known (44).

Of the two known IGFs, IGF1 is the predominant IGF in the ovaries of rodents, while IGF2

is the predominant factor in humans. IGFs are released by GCs of antral follicles (45) and are

abundant in preovulatory follicles (45,46). Both IGF1 and IGF2 bind the IGF1-receptor (IGF1R)

(47), a receptor tyrosine kinase consisting of two ligand-binding a subunits and two intracellular b

subunits. Once activated, tyrosine residues in the catalytic loops of the b subunits become

phosphorylated, which enhances the tyrosine kinase activity of the b subunits themselves (48).

The activated intracellular subunits serve as signal transducers and docking sites for additional

scaffold proteins such as insulin receptor substrate (IRS) and activate pathways such as the

mitogen-activated protein kinases (MAPK) and PI3K/AKT pathway. IGF1R is expressed in the

GCs of primary to pre-ovulatory follicles (49).

Our laboratory demonstrated that IGFs augment FSH activity in GCs. Thus, in mouse or

human GCs treated with FSH plus IGFs, aromatase expression is significantly higher than in cells

treated with FSH alone (35,50). IGFs not only augment FSH actions but are required for FSH-

induced steroidogenesis. Thus, IGF1R inhibition blocks FSH stimulation of mouse and human

GCs (35). Mice with a conditional deletion of the IGF1R in GCs have impaired folliculogenesis:

their follicles do not develop past the pre-antral stage (49). Consequently, these mice do not

ovulate and are infertile.

The in vitro and in vivo effects of IGFs have been demonstrated in the clinic, where high

follicular IGF1 levels are correlated with better embryo quality and higher implantation rates (51).

In fact, IGF1 levels in the follicular fluid of poor responding patients (to FSH) are significantly

lower (52). A study correlating IGF1 levels in the follicle with the number of FSH ampoules

administered showed that patients with higher follicular IGF1 received fewer doses of FSH (53).

16

In light of this, the crosstalk between the FSH and IGFs signaling pathways was

investigated, and the two pathways were shown to converge on the phosphorylation and

activation of AKT (35,36). However, the downstream interactions linking AKT activation to GC-

differentiation gene pathways are not understood.

4.3 Salt Inducible Kinases

FSH targets GCs and enhances estrogen synthesis, a process that is essential for normal

ovarian function. To better understand this process, we examined the current literature in search

of factors that might negatively regulate FSH signaling and therefore estrogen synthesis. As

mentioned above, FSH activates receptor-associated Gα proteins, which stimulate adenylate

cyclase activity and the production of cyclic AMP (cAMP) (54). Analysis of the literature for factors

that might regulate FSH signaling revealed that salt-inducible kinases (SIKs) are strong

candidates. SIKs are serine/threonine kinases whose major biological role is to control gene

expression in response to extracellular cues that increase intracellular levels of cAMP (55,56).

The physiological significance of SIKs in cAMP signaling is exemplified by their regulation of

several cAMP-centered systems. For instance, in macrophages, SIKs oppose cAMP signaling

stimulated by prostaglandin E2 (57,58). In osteocytes, inhibition of SIKs mimics the effects of

parathyroid hormone, which is known to activate cAMP signaling (59). In hepatocytes,

glucagon/cAMP induction of gluconeogenic genes is accompanied by inactivation of SIK activity

(60). In melanocytes, SIK inhibition strongly induces melanin synthesis, which is known to be

controlled by cAMP (61). Finally, in adrenal cells, SIK activity inhibition is sufficient to increase the

expression of steroidogenic genes including StAR and CYP11A1 (62-67). These findings suggest

that SIKs are active under basal conditions and cellular response to cAMP signaling improves

when their activity is inhibited.

The founding member of the SIK family, SIK1, is induced in the adrenal glands of rats fed

a high salt diet (68), an effect giving rise to their name. Currently, there are three subfamily

members: SIK1, SIK2, and SIK3. They are encoded by separate genes, SIK2 and SIK3 are linked

17

to chromosome 11 in humans and chromosome 9 in mice (69). SIK1 is highly expressed in the

adrenal glands, SIK2 and SIK3 are ubiquitously expressed although SIK2 is highest in adipose

tissue and SIK3 is highest in the brain (70). The SIK family members share three common

domains: an N-terminal kinase domain, a sucrose non-fermenting-1 homology (SNF) domain, and

a C-terminal domain containing multiple sites for PKA phosphorylation (Figure 5A). The SNF

domain is a hallmark of all AMPK family members (71). Additionally, all AMPK members,

including SIKs, are phosphorylated and activated by the master regulator liver kinase B1 (LKB1)

at the activation loop of the N-terminal kinase domain (72) (Figure 5B)

SIKs have two notable substrates: class IIa histone deacetylases (HDACs) and the

aforementioned CREB-regulated transcription co-activators (CRTC). SIKs phosphorylate these

substrates and sequester them in the cytoplasm by increasing their association with 14-3-3

chaperone proteins. When de-phosphorylated, these substrates enter the nucleus and regulate

gene expression. Class IIa HDACs inhibit myocyte enhancer factor-2 (MEF2) gene expression

and notably activate the forkhead family transcription factors (FOXO) (73). CRTCs enhance

cAMP actions by acting as CREB co-activators (40,41)

SIKs activity is modulated by several different signaling pathways, including the

cAMP/PKA and PI3K/AKT pathways. SIK1 and SIK3 contain two PKA phosphorylation sites,

while SIK2 contains four PKA phosphorylation sites (74). When phosphorylated, these residues

serve as docking sites for the inhibitory 14-3-3 proteins. Additionally, the PI3K/AKT pathway

inhibits SIK activity through inhibiting glycogen synthase kinase-3b (GSK-3b), a SIK activator

(75,76). SIK has also been shown to regulate its own activity through an autophosphorylation

mechanism (76).

18

Figure 5: SIK structure and placement in the cAMP pathway (A) Representation of human SIK1, SIK2, and SIK3 proteins and their conserved

domains, proposed upstream kinases and identified phosphorylation sites (55).

(B) Canonical SIKs actions in the cAMP pathway

A

GaS

cAMP

AC

ATP

PKA

SIKs

CRTC HDAC

-----------------------------------------------------------

p p

CREB

P CRTC

MEF2

HDAC

B

19

Although it has been well established that SIKs substrates are CRTCs and class IIa

HDACs, it has been shown that SIKs act through other signaling cascades and targets additional

substrates. For example, increased SIK2 activity is implicated in ovarian cancer metastasis

through the PI3K pathway (77), and SIK2 has been shown to phosphorylate insulin receptor

substrate-1 at S794 in adipose tissue (78). SIK activity has been studied in the male reproductive

system, where SIK1 impedes steroidogenic StAR expression in testicular cells (64). However,

neither SIK expression nor its activity has been investigated in the ovaries, where both cAMP and

PI3K signaling are crucial in regulating GC functions.

5- Statement of Hypothesis and Aims

The reported capacity of SIKs to regulate cAMP signaling led to the hypothesis that SIKs

influence the response of GCs to FSH. We speculate that SIKs are negative regulators of ovarian

function, specifically we postulate that SIKs hinder the response of GCs to FSH and IGFs.

Determining whether SIKs play a role in the ovaries could be a potential gateway to

understanding the mechanisms of FSH action and its control of follicle development. This

knowledge could be useful in the clinic to optimize the response of poor responding patients or

older patients to FSH.

Aspects of follicle development in the ovary are still not properly understood. The IGF1R is

required for FSH-induced GC differentiation and steroidogenesis, a process that is crucial for

proper follicle development. Important work in the lab has shown that the two pathways converge

on the activation of AKT in cultured GCs. The downstream integration of cAMP and AKT signaling

remains to be elucidated. We postulate that salt-inducible kinases are negative regulators of FSH

actions and that these factors are controlled by pathways downstream of the IGF1R. The central

hypothesis of this thesis is that SIKs attenuate FSH actions in the ovary by inhibiting

differentiation and steroidogenesis in GCs and that they integrate the FSHR and IGF1R pathways

downstream of AKT. To test this hypothesis the following aims were pursued:

20

Specific Aim 1: Demonstrate that the IGF1R is required for follicular response to FSH in

vivo. We hypothesized that fertility and AKT activation would be impaired in the absence of

IGF1R activity in mice. To test this, we developed a GC-specific IGF1R knockout mouse model

and observed the effect on different aspects of fertility and AKT signaling (Chapter III).

Specific Aim 2: Determine if SIKs attenuate FSH actions in granulosa cells. We

hypothesized that SIKs inhibition enhances GC response to FSH. To test this, we inhibited SIKs

activity in cultured GCs and mice, then determined the effect on FSH-induced steroidogenesis.

We also developed a GC-specific SIK2 knockdown mouse model and explored the effect on

steroidogenesis and some aspects of fertility (Chapter IV and V).

Specific Aim 3: Investigate the mechanism of SIKs actions and their placement within the

FSHR and IGF1R pathways. Our hypothesis is that regulation of SIKs activity is a key step

involved in the crosstalk between the FSHR/cAMP and IGF1R signaling pathways. To test this,

we investigated where along the FSHR pathway does SIKs inhibition enhance GC differentiation

gene expression. We also explored the effect of FSH and IGF1R on SIKs expression and

whether SIKs inhibition overcomes the lack of IGF1R activity. Finally, we evaluated whether SIKs

target CREB or AKT phosphorylation (Chapter VI).

21

II. MATERIALS AND METHODS 1. Human granulosa cell processing and culture

Primary cumulus granulosa cells were collected from women undergoing IVF treatment at

the University of Illinois at Chicago fertility center and the Fertility Centers of Illinois with the

approval of the Institute Review Board. Patients underwent controlled ovarian hyperstimulation,

then were injected with human chorionic gonadotropin (hCG) to trigger ovulation. After 35 hours,

follicles were aspirated to retrieve the cumulus-oocyte complexes (COCs). The COCs were

separated from the follicular aspirate and the cumulus cells mechanically separated from the

oocytes. The cumulus cells from one patient were pooled together, treated with 80 U/mL

hyaluronidase (Sigma), gently pipetted to break up clusters, and incubated at 37°C for 5 minutes.

Then cells were centrifuged at 1000xg for 2 minutes and the pellet resuspended in red blood cell

lysis buffer to remove erythrocytes and incubated at room temperature for 5 minutes, then

centrifuged at 1000xg for 2 minutes. The pellet was resuspended in phenol red-free, serum-free

DMEM/F12 media, and an aliquot was used for cell counting using a hemocytometer. Cells were

seeded at a minimum density of 1.6x104 cells/cm2 on pre-coated tissue culture plates and/or

dishes and incubated at 37°C with 5% CO2.

The media used was phenol red-free and serum-free DMEM/F12 (Sigma-Aldrich D2906)

supplemented with 0.2% w/v bovine serum albumin (BSA, Sigma A4503), sodium bicarbonate

(14mM), 2x Antibiotic Antimycotic Solution (Corning 30-004-Cl), 1x GlutaMAX (Gibco 35050-061),

and 1.6 ug/mL Amphotericin B (Gibco 15290-018) at 7.2 pH, passed through a 0.22 µM vacuum

filter. 1x insulin, transferrin, and selenium (ITS, Sigma), 30nM estradiol, 50ng/mL IGF2, 5ng/mL

bFGF2, and 1x extracellular matrix (BD MatrigelTM) were added to the media before seeding.

Cells were incubated for at least 48 hours before treatments were initiated. Each experiment

represents results from a single patient since cells from different patients were cultured

separately.

22

2. Rodent granulosa cell isolation and culture

Immature Sprague Dawley rats (Charles River Laboratories) aged 24 days old were

subcutaneously injected with 1.5 mg estradiol suspended in sesame oil (Sigma) per day for three

days to promote granulosa cell proliferation and to inhibit differentiation. Immature 21-23 days old

C57BL/6 mice were subcutaneously injected with 0.5mg estradiol per day for three days. On the

fourth day, rats or mice were euthanized using isoflurane followed by cervical dislocation before

dissection of the ovaries. The ovaries were cleaned of surrounding tissues and incubated in

EGTA (6.8 mM) for ten minutes at 37°C, followed by a ten-minute incubation in sucrose (0.5 M)

also at 37°C. This pre-treatment disperses the granulosa cells and yields more viable and better-

quality cells (79). Afterward, the ovaries were placed in phenol-free, serum-free DMEM/F12

media, and follicles punctured using 25g needles to release the granulosa cells. The cells were

resuspended in culture media and seeded on pre-coated tissue culture plates and/or dishes and

incubated at 37°C in a 5% CO2 atmosphere.

3. Messenger RNA (mRNA) quantification Gene expression was measured by the quantification of mRNA levels using polymerase

chain reaction (PCR). For this purpose, total RNA was isolated using TRIzol reagent (Invitrogen)

according to the manufacturer’s protocol. 1µg of RNA was reverse-transcribed using anchored

oligo-dT primers (IDT) and Moloney Murine Leukemia Virus reverse transcriptase (Invitrogen) at

37°C for 2 hours. The resulting cDNA was diluted to 10 ng/µL in H2O, and 50ng used in a qPCR

reaction. The number of mRNA copies of the target gene was quantified using a standard curve

containing serial dilutions of the purified PCR product ranging from 6x106 to 9.6x103 copies per

tube. An internal control gene, ribosomal protein L19 (RPL19), was also quantified in each

sample and the results for each target gene are reported as the ratio between the copies per tube

of the gene of interest and RPL19. Intron-spanning primers were used to amplify the target gene

(Table I).

23

The qPCR reaction was carried out using the Bio-Rad MyiQTM Cycler Real-Time PCR

machine under the following conditions: pre-incubation at 95°C for 2 minutes, followed by 40

cycles of denaturation at 95°C for 5 seconds, annealing at 60°C for 10 seconds, and extension at

72°C for 40 seconds. To ensure that unintended products were not being amplified, the melting

curve for each amplification reaction was measured on all determinations.

24

TABLE I - QUANTITATIVE PCR PRIMERS

Species Gene Forward Reverse Human RPL 19 GCT GTG GCA AGA AGA AGG TCT GG TGT TTT TCC GGC ATC GAG CCC Human CYP19A1 GCT GGA CAC CTC TAA CAC GCT CAG GTC ACC ACG TTT CTC TGC T Human STARD1 GGC TCA GGA AGG ACG AAG AAC C ATC ACA GCC TGT TGC CTC AGC Human CYP11A1 GTG ATG ACC TGT TCC GCT TTG C AAG GTT GAG CAT GGG GAC GC Human SIK1 GAG TCA CCA AAA CGC AGG TTG C ATG TGA TGG TCG TGA CAG TAC TCC Human SIK2 TTC ACC GAA CAT GAG GCT GC TGC GTG TGG ACT GAA ATG CC Human SIK3 TGG GGA AAA TGA GGA ATG TGG GG AAG GGC AAT TTG GCA CAA CGC Human AMH GCT GCC TTG CCC TCT CTA C GAA CCT CAG CGA GGG TGT T Human IGF2 AGT CCG AGA GGG ACG TGT TGG ACT GCT TCC AGG TGT

Rat RPL 19 TGC CTT CAG TTT GTG GAT GTG C CCT GGA TGC GAA GGA TGA GG Rat CYP19A1 CAC CCA GCC TGT CCA AAT GC CTC CAG ATT CGG CAG CAA GC Rat STARD1 TGG CTG GCG AAC TCT ATC TGG GGG AGA TGC CTG AGC AAA GC Rat CYP11A1 TGA ACT TGG TCC CCA CAT CAC G GCC AAA ACA CCA CGC ACT TCC Rat SIK1 TGT CTT ACC TCC TGT CAG CTT CC CCT CGC GTT TTT CCT TAG CTG C Rat SIK2 TTG CTG AAC AAA CAG TTG CC TCA AGC AGA CAG CCA TTC AC Rat SIK3 AAA CTC CCG CTA TCC AGC TAC G ACA TGG CAA AAG TCC CTG GC Rat FSHR GCT TTT GCA AAC TTG AAG CGG C GAC CCT GAG GAT GTT GTA CCC C

Mouse RPL 19 CAA TGA GAC CAA TGA AAT CG GCA GTA CCC TTC CTC TTC C Mouse CYP19A1 CAA GTC CTT GAC GGA TCG TT GAC ACA TCA TGC TGG ACA CC Mouse STARD1 GCG AAC TCT ATC TGG GTC TGC G TTT TGG GGA GAT GCC GGA GC Mouse CYP11A1 GAT GTT CCA CAC CAG TGT CCC AGG GTA CTG GCT GAA GTC TCG C Mouse SIK1 GCA CAG CCG TCT TAC CTC CC GGG AGT TCG GAC GGA GGA CT Mouse SIK2 AGC TAT GAC CCA CTG GCC CT CCC AGC TTC TCT CTG CAG CC Mouse SIK3 CCC TAC GGA CAC CAG CCA AC AGG CAT CGT CGC TGT TCT GG Mouse IGF1R GGA CAT TGG AGG AGA AGC CAG CAC TCG TTG TTC TCG Mouse LHR TGT AAC ACA GGC ATC CGG ACC ACT CCA GCG AGA TTA GCG TCG Mouse FSHR GTG CAT TCA ACG GAA CCC AGC CGC CTC CAG TTT GCA AAG GC Mouse Inhibin a CCC AAC CTT ATT ACT CAA CAC TGT

GC GGG TGG AGC AGG ATA TGG ATC C

Mouse Inhibin Ba AGC TTC ATG TGG GTA AAG TGG GG GAC AGG TCA CTG CCT TCC TTG G Mouse Inhibin Bb CTG AAC CAG TAC CGC ATG CG ACA CTC CTC CAC GAT CAT GTT GG

25

4. Treatments and inhibitors Rat, mouse, and human granulosa cells were cultured for at least 48 hours before

treatment. The different treatments are listed in Table II. Additionally, the in vivo mouse and rat

injections used to stimulate ovulation, stimulate GC proliferation, and inhibit SIKs activity are

listed in the same table.

5. Western Blotting Cumulus cells were harvested in ice-cold RIPA buffer supplemented with 1x Protease

Inhibitor Cocktail Set I (Calbiochem), 5mM NaF, 2nM Na3VO4, and 1nM phenylmethylsulfonyl

fluoride (PMSF). Total protein concentration was quantified using Pierce BCA Protein Assay Kit

(Thermo Scientific) using a BSA standard. Approximately 10 µg-30 µg total protein was separated

on 12% bis-Tris-PAGE gels in 50mM MOPS, 50mM Tris, 1mM EDTA, 5mM sodium bisulfite, and

0.1% sodium dodecyl sulfate buffer.

After separation, the protein was transferred to either a methanol-activated polyvinylidene

fluoride (PVDF) or nitrocellulose membrane in transfer buffer (25mM Tris, 0.19M glycine, 20%

methanol). Membranes were then blocked against unspecific binding using either 5% nonfat dry

milk (BD Difco) in TBS-T (2mM Tris, 15mM NaCl, 0.1% Tween-20, pH 7.6) or WestVision Block

and Diluent buffer (Vector Laboratories). The primary antibodies (Table III) were diluted in the

blocking buffer and incubated on the membrane overnight at 4°C. After washing three times for

10 minutes each in TBS-T, membranes were incubated with the appropriate horseradish

peroxidase (HRP)- conjugated secondary antibody (according to the species of the primary

antibody) for two hours in blocking buffer at room temperature. The membranes were washed

three times for 10 minutes each with TBS-T and visualized using the Bio-Rad ChemiDoc imaging

system. The visualizing substrate used was either the SuperSignal West Pico or Femto

Chemiluminescent Substrate (Thermo Scientific), depending on the strength of the signal. The

26

bands were quantified using Image Lab software (Bio-Rad Laboratories) and intensities adjusted

relative to loading controls (Table III).

6. Animal handling Animals were treated following the NIH Guidelines for Care and Use of Laboratory

Animals, and all protocols were approved by the University of Illinois at Chicago Animal Care

Committee. Mice of the following strains were used: IGF1R F/F, IGF1R KO, Cyp19-Cre (provided

by Dr. Joanne Richards), Ers2-Cre (provided by Dr. Jay Ko), and SIK2 F/F (provided by Dr.

Hiroshi Takemori).

7. Genotyping The genotype of mice was determined using PCR on total DNA isolated from tails of less

than 16-day-old mice. A total of 1.5 µg DNA was used per reaction. The PCR buffer consists of

1x Taq Buffer (GenScript), 1x Cresol red dye (as a loading buffer), 0.2mM dNTPs, and 0.1 U/µL

Taq polymerase (GenScript). Genotyping primers are listed in table IV. The PCR reaction

consisted of the following steps: 98°C for 2 minutes, followed by 35 cycles of 10 seconds at 95°C,

30 seconds at 57°C and one minute at 72°C, and a final incubation at 72°C for 2 minutes.

The PCR products were separated by electrophoresis in 3% agarose gel and visualized

with GelRed Nucleic Acid Gel Stain (Biotium) or GreenGlo Safe DNA Dye (Deville Scientific)

under UV light.

27

TABLE II - TREATMENTS, ACTIVATORS, AND INHIBITORS

Treatment Company Function Concentration

Mouse and rat injections Pregnant mare’s serum gonadotropin (PMSG)

Sigma Stimulates folliculogenesis, mimics FSH

4 IU/injection

Human chorionic gonadotropin (hCG)

Sigma Triggers ovulation, mimics LH

5 IU/injection

Estradiol Sigma Stimulates GC proliferation and inhibits endogenous FSH secretion

1.5 mg/injection (rats) 0.5 mg/injection (mice)

YKL-05-099 MedChem Express

SIKs inhibitor 0.2 mg/injection (10 mg/kg)

Culture treatments Recombinant FSH (human, mouse GCs)

Serono Stimulates GC differentiation

50 ng/mL

Ovine FSH (rat GCs) NIH Stimulates GC differentiation

100 ng/mL

Forskolin (FSK) TOCRIS Adenylyl cyclase activator

2 μM

Dibutyryl cyclic adenosine monophosphate (dbcAMP)

Sigma cAMP analog 0.5 mM

NVP-AEW451 (AEW) TOCRIS Inhibits IGF1R 0.5 μM HG-9-91-01 (HG) TOCRIS Pan SIK inhibitor 0.3, 1, 3 μM MRT 67307 (MRT) TOCRIS Pan SIK inhibitor 0.3, 2, 4, 6 μM Compound C (CoC) TOCRIS SIK2 inhibitor 0.1, 0.3, 3 μM rac Pterosin b TOCRIS SIK3 inhibitor 100, 300 μM CHIR-99021 (CHIR) TOCRIS GSK3-b inhibitor 0.5, 1 μM ‘8CPT-2Me-cAMP (8CPT) TOCRIS Epac activator 10 μM

28

TABLE III - WESTERN BLOT ANTIBODIES

Antibody Company Dilution Used

b-actin (control) Proteintech 1:1000 Aromatase (CYP19A1) Abcam 1:1000 FSHR Epitomics 1:1000 SIK1 LS Bio 1:1000 SIK2 (Western Blot) Cell Signaling 1:1000 SIK2 (IHC) LS Bio 1:1000 SIK3 LS Bio 1:500 AKT Cell Signaling 1:1000 phospho-AKT (Ser473) Cell Signaling 1:1000 IGF1- Receptor b Cell Signaling 1:1000 CREB Cell Signaling 1:1000 Phospho-CREB (Ser133) Cell Signaling 1:1000

29

TABLE IV - GENOTYPING PCR PRIMERS

Gene Forward Reverse Expected PCR

Product Size IGF1R F/WT

CGG TGG AGA CTT TAA CTA CA

TTA GAG AAA GGA GGT TCT GG

WT: 215 bp Floxed: 315 bp

IGF1R KO

CAT GGA ACA GTA ATG TGT GG

TTA GAG AAA GGA GGT TCT GG

250 bp

SIK2 F/WT

TAT TGT GCT TAA TGC CTA CC

CAG TGT CCT TTG TCA TTG AT

WT: 391 bp Floxed: 487 bp

CYP19-Cre

GGA ATG CAC GTC ACT CTA CCC

GGT TTT GGT GCA CAG TCA GC

500 bp

ERS2-Cre

CTT AGT TAC TCC GGC AGC TTG AAC

AGG GGA AGT AAG GCT TGA TGG TGA CAG GTG CTG TTG GAT GGT CTT C

Ers2Cre: 401bp WT: 181 bp

30

8. Fluorescent Immunocytochemistry Rat granulosa cells were plated on ECM pre-coated 8 chamber tissue culture microscope

slides and cultured as described above. After treatment, slides were fixed using 4% formaldehyde

in PBS for 10 minutes, rinsed with PBS twice, and permeabilized using 0.1% Triton X-100 in PBS

for 10 minutes. Slides were then washed 3 times with PBS and blocked with 1% BSA and 0.1%

tween-20 in PBS for 10 minutes. Slides were incubated for 1 hour at 37°C with specific primary

antibodies diluted in 1% BSA in PBS. Slides were then washed 3 times with PBS and twice with

1% BSA in PBS. Afterward, slides were incubated in a fluorochrome-coupled secondary antibody

diluted in 1% BSA in PBS for 1 hour at room temperature in the dark. Slides were then washed

three times with PBS and coverslips mounted on the slides using DAPI-containing antifade

mounting medium (Vectashield H-1500). Slide images were taken on a Zeiss LSM 880 confocal

microscope.

9. Immunohistochemistry Rat ovaries were embedded in paraffin to prepare 5 μm sections, which were stained

using primary antibodies diluted in PBS. Antibodies were detected using Vectastain Elite (Vector

Laboratories) and counterstained with Gill’s hematoxylin. Histological studies of ovarian sections

were performed after hematoxylin and eosin staining.

10. Luciferase assay The CYP19ov-Luc reporter was generated by cloning the +1 to -320 region of the human

CYP19A1 ovarian promoter followed by the firefly luciferase cDNA. Lentivirus containing this

construct was subcloned into the pTY-CMV lentivirus transfer plasmid and the virus produced in

human embryonic kidney HEK 293FT cells. Empty plasmids were used as controls. Cells were

infected with lentivirus and treated after overnight incubation. After 48 hours, cells were lysed

using 1x Passive Buffer (Promega), then frozen at -80°C for 10 minutes (to achieve better lysis).

The cells were then thawed, scraped, and 50-70 μL of the lysate transferred to a reading plate.

31

An equal amount of luciferin substrate (Promega) was added and luciferase activity was

measured and quantified.

11. 17b-Estradiol measurement in rat granulosa cell culture media Granulosa cells from immature rats were isolated and cultured as previously described

above. Testosterone (50 nM) was added to the cell culture media 4 hours before harvesting the

cells. Media was collected from each well, and 17b-estradiol levels were measured in undiluted

media using an estradiol enzyme-linked immunosorbent assay (ELISA) kit according to the

manufacturer’s protocol (DRG Instruments).

12. Ovulation assay Immature female mice 21-25 days old were injected intraperitoneally (i.p.) with 4 IU

pregnant mare’s serum gonadotropin (PMSG, Sigma-Aldrich) to mimic FSH actions. After 48

hours, they were injected i.p. with 5 IU hCG to stimulate ovulation. After 17 hours, the mice were

sacrificed, and their oviducts were dissected. The cumulus-oocyte complexes were extracted

from the oviducts and the number of oocytes counted.

To test the effect of SIK inhibition on ovulation, the SIK inhibitor, YKL-05-099 (10 mg/kg)

was injected i.p. two hours before PMSG injection and the injection repeated with the PMSG

injection. Each injection consisted of 0.2 mg inhibitor diluted in 2uL dimethyl sulfoxide (DMSO)

and 100 µL PBS. The pre-injection was done to maximize the effect of the inhibitor, which is

stable for about two hours in vivo (80). The general well-being of the mice was monitored

throughout the ovulation assay in response to the SIKs inhibitor.

13. Statistical analyses All experiments were performed at least three times and data presented as mean values ±

standard error of the mean (SEM). Statistical comparisons of mean values between groups were

done with t-tests and multiple comparisons performed using one-way analysis of variance

(ANOVA) using GraphPad Prism. Groups were found to be significantly different if the P-value

was <0.05.

32

III. CONDITIONAL KNOCKDOWN OF IGF1R IN THE GRANULOSA CELLS IMPAIRS STEROIDOGENESIS AND AKT ACTIVATION

A. INTRODUCTION

IGFs levels in the follicular fluid have a significant impact on the response of patients to

exogenous FSH stimulation. Extensive work in our lab determined that in GCs across different

species (mouse, rat, human) IGF-1 augments FSH stimulation of GC-differentiation genes such

as aromatase, StAR, 3b-HSD, and P450scc. This was shown at the aromatase promoter level,

mRNA transcription, protein expression levels, as well as estradiol levels (35). Importantly, IGFs

stimulation alone does not cause a change in the expression of these genes, but rather it

enhances the effect of FSH. We also demonstrated that in vitro, IGFs enhance the response of

the GCs to FSH without increasing the expression of the FSH receptor (35). In fact, IGFs

enhance aromatase stimulation by compounds that mimic FSH signaling such as forskolin (an

adenylyl cyclase activator) and dbcAMP, an analog of cAMP (35).

Strikingly, our laboratory demonstrated that IGFs not only augment FSH actions in GCs

but also that the activity of the IGF1R is needed for FSH stimulation of GC differentiation and

steroidogenesis. Thus, in the presence of an IGF1R activity inhibitor, FSH fails to stimulate the

expression of aromatase, P450scc, StAR, and LHR (35). IGF1R inhibition has no effect on FSHR

expression. Similar effects were seen when IGF1R expression was knocked down. Additionally, it

was shown that endogenous IGF secretion by the follicle is sufficient to augment FSH actions

since sequestration of IGFs using IGF binding protein 2 (IGFBP2) blocked aromatase and

P450scc production.

Taken together, these in vitro findings prove a crucial role of the IGF system on the

stimulation of GCs function by FSH. Whether this role of IGFs is conserved in vivo is not known. It

has been shown that mice that are null for the igf1 gene are infertile. IGF1 knockout mice,

however, exhibit significant growth deficiency and depending on the genetic background only

10% to 60% survive to adulthood (81). This evidence suggests that infertility in IGF1-knockout

33

mice could be caused by multiple factors that contribute to a decrease in the well-being of the

animals. On the other hand, IGF1R knockout mice die at birth (81); therefore, the role of the

IGF1R in ovarian function and female fertility remains to be determined. Consequently, we

utilized a cre-lox system to develop a GC-specific IGF1R knockout mouse (IGF1Rgcko). For this

purpose, we used Cyp19a1 and Ers2 promoters as cre-recombinase drivers. We hypothesized

that these mice have impaired steroidogenesis and defective AKT activation in their GCs.

B. RESULTS

1. Expression of Cre-recombinase under both Cyp19a1 and Ers2 promoters leads to

undetectable levels of IGF1R in GCs

Mice expressing either driver alone have partial IGF1R knockdown (49). However, the

simultaneous use of both promoters led to a complete knockdown of the IGF1R in GCs (Figure

6). Complete knockout of IGF1R in GCs causes the mice to have impaired folliculogenesis,

leading to anovulation and infertility (Figure 7). The follicles express increased markers of

apoptosis and a decrease in proliferation markers compared to control follicles (Figure 8A). They

also have significantly reduced estradiol production (Figure 8B) (49).

2. The IGF1R is necessary for FSH-induced steroidogenesis and differentiation of GCs in

vivo

Next, I explored the role of the IGF1R on the expression of key steroidogenic genes and

differentiation markers in the GCs of mice stimulated with PMSG for 48 hours. The induction of

steroidogenic genes by PMSG treatment was significantly compromised in the GCs of IGF1Rgcko

mice when compared with control animals (Figure 9). Thus, the expression of StAR, Cyp11a1,

and Cyp19a1 in the GCs of IGF1Rgcko mice was lower by seven-fold, 2.5-fold, and nine-fold,

respectively, when compared with control GCs. The expression of markers of GC differentiation

including luteinizing hormone receptor (LHR), inhibin-a, inhibin-Ba, and inhibin-Bb were also

significantly lower in GCs of IGF1Rgcko mice when compared with controls.

34

3. The IGF1R does not affect FSHR expression in vivo

Since a lack of IGF1R impairs the ability of GCs to respond to FSH, I measured the

expression of the FSH receptor to determine if the IGF1R knockdown impairs FSH-induced

steroidogenesis and differentiation through decreasing the availability of the receptor. Control and

IGF1Rgcko mice were injected with PMSG, then 48 hours later the mRNA and protein expression

of FSHR were measured. In contrast with steroidogenic and differentiation gene expression, the

knockdown of the IGF1R in the GCs had no effect on FSH receptor expression. Thus, the GCs of

control and IGF1Rgcko mice expressed comparable levels of FSH receptor protein and mRNA,

indicating that the IGF1R acts downstream of the FSHR in vivo (Figure 10).

4. A lack of IGF1R impairs FSH-induced AKT phosphorylation in vivo

The previous in vitro findings demonstrated that FSH and IGF1R signaling converged at the

activation of AKT (35,36,50). Therefore, I next examined whether GCs of IGF1Rgcko mice have

defective expression or activation of AKT. The protein levels for total AKT and phospho-S473-

AKT were measured in the GCs of control and IGF1Rgcko mice treated with PMSG for 1 hour. AKT

phosphorylation was extremely low in the GCs of IGF1Rgcko mice compared with controls;

however, a noticeable decrease in total AKT was also observed (Figure 11). Analysis of these

data revealed a significant reduction in the ratio of phosphorylated to total AKT in the GCs of

IGF1Rgcko mice when compared to controls, suggesting that the expression of the IGF1R in the

GCs is crucial for the activation of AKT by FSH in vivo.

35

Figure 6: Knockdown of IGF1R expression in GCs (A) IGF1R mRNA expression levels in GCs from PMSG-treated animals expressed

relative to mouse ribosomal L19 (the average of at least six samples per genotype is shown). Bars represent mean ± SEM (*p < 0.05).

(B) Immunohistochemistry for the IGF1R protein in ovaries of 21-25-day old controls and

IGF1Rgcko PMSG-stimulated mice (n=3 for each genotype; a representative image is shown).

A

B

36

Figure 7: Fertility effects of IGF1R knockdown in GCs (A) Representative hematoxylin and eosin staining of ovaries of control and IGF1Rgcko

mice treated with PMSG for 48 hours.

(B) The number of pups per litter was determined in control mice and experimental animals over 6 months. Four or more females were used for each genotype. Columns represent the average ± SEM of the number of pups per litter (*p < 0.05; **p < 0.01 vs. controls). IGF1Rgcko females produced no pups.

A

B

37

Figure 8: Fertility effects of IGF1R knockdown in GCs (A) Estradiol levels in control and experimental mice treated with PMSG for 48 hours.

Bars represent the mean ± SEM of measurements from at least five animals per genotype. Different letters denote differences between genotypes (a-b, b-c p < 0.05; a-c p < 0.01).

(B) Coimmunostaining for markers of proliferation (Ki67) and apoptosis (cleaved caspase 3) were performed in control and IGF1Rgcko ovaries from 23-day-old PMSG treated mice. Cleaved caspase 3 staining is depicted in pink, Ki67 staining is depicted in brown, and counterstaining by hematoxylin is depicted in light blue. Arrowheads indicate secondary follicles, black arrows indicate early antral follicles, and yellow arrows indicate follicles with structural distortion. High magnification (x20) sections are indicated (n=3 for each genotype; a representative picture is shown).

Estradiol A B

38

Figure 9: Relative expression of the main differentiation markers in GCs of control and IGF1Rgcko mice. Total RNA was extracted from GCs isolated after PMSG treatment in control and IGF1Rgcko females. The expression of genes important for GC differentiation was measured by quantitative PCR. Three or more animals were included for each genotype. Columns represent the mean SEM (***p < 0.01 vs. control).

39

Figure 10: Relative expression of the FSH receptor in GCs of control and IGF1Rgcko mice. FSHR mRNA (left) and protein levels (right). Blots are representative of three different animals. BACT, b-actin.

Control IGF1Rgcko

40

Figure 11: Lack of IGF1R in GCs leads to diminished AKT activation. Western blots for phospho-S473 and total AKT in control and IGF1Rgcko mice treated with PMSG for one hour. *p < 0.001 vs. control, n=3.

41

C. DISCUSSION

These findings demonstrate in vivo that GCs require the IGF1R to undergo differentiation

upon FSH stimulation. Thus, FSH stimulation of steroidogenesis and differentiation was

completely blocked in the absence of IGF1R expression in GCs. This in vivo evidence supports

the presence of parallel and cooperative pathways between FSHR and IGF1R in the stimulation

of folliculogenesis and fertility.

GC-specific IGF1R knockout mice have FSHR expression levels that are comparable to

those found in control animals. This finding suggests that the IGF1R is not involved in the

regulation of FSHR in vivo and that the lack of IGF1R impacts mechanisms downstream of the

FSH receptor. This confirms our previous in vitro studies demonstrating that FSHR expression is

not affected when IGF1R activity is inhibited (35). Since the most prominent FSH-induced

steroidogenic and differentiation genes depend on cAMP signaling and the activation of the cAMP

response element-binding protein (CREB), we postulate that the crosstalk of the two signaling

pathways occurs downstream of cAMP, but the exact mechanisms remain to be determined.

In this chapter, I have demonstrated an impairment of FSH-induced AKT phosphorylation

in the GCs of IGF1Rgcko mice. Because AKT is essential for the differentiation of GCs, the failure

of FSH to stimulate AKT phosphorylation in vivo is likely the most relevant defect that prevents

the induction of GC differentiation in IGF1Rgcko mice.

Since AKT phosphorylation is impaired in GCs of IGF1Rgcko mice, it is reasonable to

conclude that multiple signaling components are involved in the crosstalk between the two

pathways. It has been shown that FSH alone targets AKT phosphorylation through the activation

of the PI3K pathway (82), but it is also known that AKT activation alone is not sufficient to cause

GC differentiation without the presence of FSH (83). Therefore, more studies are needed to

understand the integration of the two signaling pathways, especially linking AKT and CREB

activation.

42

IV. SALT INDUCIBLE KINASES OPPOSE FSH ACTIONS IN CULTURED GRANULOSA CELLS

A. INTRODUCTION

FSH activates receptor-associated Ga proteins, which stimulate adenylyl cyclase activity

and the production of cAMP (54). As previously described, analysis of the literature for potential

factors that might regulate FSH signaling revealed a subfamily of AMP-activated protein kinases,

salt-inducible kinases (SIKs) as plausible candidates. Since the major biological role of SIKs is to

control gene expression in response to cues that increase intracellular levels of cAMP (62-67), we

hypothesized that SIKs play a role in influencing GC response to FSH stimulation. In this chapter,

I describe the expression of all the members of the SIK family in rodent and human GCs and

reveal that SIKs repress FSH actions in GCs by attenuating steroidogenesis.

B. RESULTS 1. Human and rodent granulosa cells express SIK1, SIK2, and SIK3

Since the expression of SIKs in the ovary has not been previously investigated, we quantified

the mRNA levels of the three SIK genes in rat and human GCs. The mRNA for all SIKs was

detected in the GCs of both species, although the relative expression of Sik1 and Sik2 mRNAs

was lower than the expression of Sik3 (Figure 12A). Western blot analysis confirmed the

expression of all SIK proteins in rat GCs (Figure 12B).

As we observed at the mRNA level in rat GCs, immunohistochemical studies of rat ovaries

showed a robust signal for SIK2 and SIK3 proteins while the SIK1 protein signal was substantially

lower (Figure 13). These studies also showed that GCs express both SIK2 and SIK3, while the

interstitial tissue and the theca cells express mostly SIK3 (Figure 13). The expression of SIKs in

rat GCs was further visualized using immunofluorescence, which showed a strong signal for SIK2

and SIK3, while the SIK1 signal was significantly lower and almost undetectable (Figure 12C).

43

2. SIKs inhibition in rodent granulosa cells enhances FSH actions

To test whether SIKs activity regulates GC function, we ablated SIK activity in rat GCs using

HG-9-91-01 (HG), a SIK inhibitor whose potency and specificity have been well-characterized

(84). SIKs inhibition potentiated FSH-stimulation of aromatase in a concentration-dependent

manner (Figure 14A); however, a stronger potentiation was observed with the lower

concentrations (0.3 and 1 µM) than with the higher concentration (3 µM) of the inhibitor used. In

the absence of FSH, treatment with 1 or 3 µM of the SIKs inhibitor increased mRNA levels.

Comparable findings were seen using a second SIKs inhibitor, MRT (Figure 14B).

To determine whether the effect of SIK inhibition on Cyp19a1 mRNA levels is mediated by an

increase in the expression of the Cyp19a1 gene, rat GCs were infected with a reporter controlled

by the Cyp19a1 proximal promoter. Luciferase activity was detectable but low in the absence of

FSH, while FSH stimulated reporter activity by 20-fold (Figure 14C). Co-treatment with the SIKs

inhibitor enhanced the stimulatory effect of FSH on aromatase promoter activity in a

concentration-dependent manner. No activity was observed in cells infected with an empty

reporter. Treatment with the SIK inhibitor alone increased Cyp19a1 promoter activity.

Aromatase drives the production of estradiol, a steroid hormone playing a central role in the

regulation of all aspects of female reproductive activity. Consequently, we examined the effect of

SIKs inhibition on estradiol production by GCs. Inhibition of SIKs activity using either HG or MRT

potentiated the stimulation of estradiol production by FSH in a dose-dependent manner (Figure

14D).

3. Steroidogenesis in primary human granulosa cells is inhibited by SIKs activity

Next, we examined the effect of SIKs inhibition in cultured primary human GCs. As in rats,

SIKs inhibition in human GCs potentiated FSH stimulation of CYP19A1 mRNA expression (Figure

15A) and CYP19A1 promoter activation (Figure 15B). We also examined the impact of SIKs on

the expression of insulin-like growth factor 2 (IGF2), which is expressed exclusively in human

44

GCs and strongly stimulated by FSH (50). The addition of SIKs inhibitors to the media enhanced

the expected stimulatory effect of FSH on IGF2 mRNA expression although the difference was

not statistically significant when compared to cells treated with FSH alone (Figure 15C).

Interestingly, SIK inhibition by HG in the absence of FSH increased IGF2 mRNA levels

significantly when compared to controls (Figure 15C).

SIKs inhibition also potentiated the stimulatory effect of FSH on StAR and CYP11A1 mRNA

levels (Figure 15 D and E), whose expression is known to increase after treatment of human GCs

with FSH (35,36). Moreover, as observed for CYP19A1, treatment with HG alone was enough to

increase StAR and CYP11A1 mRNA levels.

To further examine the role of SIK in human GCs, a second inhibitor and a larger cohort of

patients (n = 16) were used to study the effect of SIK activity on CYP19A1 mRNA levels. In this

larger experiment, the combination of FSH plus MRT significantly increased CYP19A1 mRNA

levels by 3-fold when compared to cells treated with FSH only (P<0.0001) (Figure 15F). In

contrast to HG, treatment with MRT alone did not affect CYP19A1 mRNA levels.

4. SIKs inhibition recovers aromatase production in IVF patients with different etiologies

Next, we examined the effect of SIKs inhibition on CYP19A1 protein expression in the GCs of

patients with normal (tubal, malefactors, endometriosis) or abnormal (polycystic ovarian

syndrome (PCOS), anovulation) ovarian function. In patients with normal ovarian function, FSH

strongly increased CYP19A1 protein levels, an effect that was potentiated by the inhibition of SIK

activity (Figure 16A). In the absence of FSH, SIK inhibition stimulated CYP19A1 protein

expression in three of the four patients with normal ovarian function.

In contrast, FSH was unable to stimulate CYP19A1 in two of the three patients with PCOS,

while SIK inhibition rescued FSH induction of CYP19A1 in these two patients (Figure 16B). In the

PCOS patient that responded to FSH alone, the presence of a SIK inhibitor potentiated FSH

45

actions. In the patient with anovulation, FSH stimulated CYP19A1 mRNA levels marginally, while

HG alone or in the presence of FSH stimulated CYP19A1 strongly (Figure 16B).

46

Figure 12: Expression of SIKs in rat and human GCs

(A) SIK1, SIK2, and SIK3 mRNA levels in rat and human GCs expressed as relative expression to ribosomal L19 protein (Rpl19). Bars represent mean ± SEM, N ³ 10.

(B) Representative blots of SIK1, SIK2, and SIK3 protein levels in rat GCs treated with vehicle (C) or FSH (50 ng/mL) for 48 hours, N=3. BACT: b-actin.

(C) Representative immunofluorescent imaging of SIK1, SIK2, and SIK3 (green) in cultured primary rat GCs (N=3). Nuclei were stained with DAPI (blue).

Rat

SIK1 SIK2 SIK30.0000.0010.0020.0030.0040.200.250.300.350.40

Rel

ativ

e toRpl19

Human

SIK1 SIK2 SIK30.00000.00010.00020.00030.00040.0005

0.050.060.070.080.090.10

Rel

ativ

e toRpl19

A

B C

47

Figure 13: Expression of SIKs in the rat ovary Representative stains of SIK1, SIK2, and SIK3 (brown) in rat ovaries. Slides were counterstained with hematoxylin (blue), N = 5.

48

Figure 14: SIKs inhibition enhances FSH actions in primary rat GCs Rat GCs were treated with vehicle, FSH, FSH plus (A) HG-9-91-01 (HG) or (B) MRT67307 (MRT), or SIKs inhibitors alone. CYP19A1 mRNA levels were quantified 48 hours later and expressed relative to Rpl19. a-bp<0.005, b-cp<0.0001, b-dp<0.0001, b-

ep<0.0001. (C) Lentivirus was used to deliver an empty luciferase reporter or a reporter carrying the

ovarian CYP19A1 promoter. Cells were treated with vehicle, FSH, FSH+HG, or HG alone 48 hours after the addition of the virus. Luciferase activity was quantified 48 hours after the initiation of treatments. a-bp<0.05, b-cp<0.005, a-cp<0.0001.

(D) Rat GCs were treated with vehicle, FSH, FSH plus HG or MRT, or SIKs inhibitors alone. Estradiol concentration in the media was determined by ELISA 48 hours after the initiation of treatments. a-bp<0.0001, b-cp<0.0001, b-dp<0.0005.

Different letters represent significant differences between groups, one-way ANOVA followed by Tukey, p<0.05, N ³ 5.

C 0 0.3 1 3 0.3 1 30.000

0.005

0.010

0.015

Rel

ativ

e to

Rpl19

FSH

Aromatase

ab

c

d

e

b

ee

HGuM

C 0 0.3 2 4 6 0.3 2 40.000

0.005

0.010

0.015

0.020

0.025

Rel

ativ

e to

Rpl19

FSH

b

c

d

a

c

a

dd

Aromatase

MRTuM

C F FHG HG C F FHG HG0

500

1000

1500

2000

Empty-Luc CYP19-Luc

a

b

c

a

Luci

fera

se A

ctiv

ity

LUC CYP19ov

A

C D Luciferase Activity

B

a

b

c c

b d

49

Figure 15: SIKs inhibition with HG enhances FSH actions in primary human GCs (A) Aromatase mRNA and (B) promoter activity in primary human GCs after treatment with vehicle, FSH, FSH+HG, or HG alone for 48 hours. HG was used at 1 µM concentration. (C) IGF2, (D) StAR, and (E) CYP11A1 relative mRNA expression in primary human GCs after treatment with vehicle, FSH, FSH+HG, or HG for 48 hours. Mean ± SEM, N ³ 7. (F) Aromatase expression in primary human GCs after treatment with FSH, FSH+MRT, or MRT alone. Mean ± SEM, N = 16. Different letters represent significant differences. One-way ANOVA followed by Tukey. a-bp<0.05, b-cp<0.005, a-cp<0.0001.

50

Figure 16: SIKs inhibition rescues FSH actions in human GCs from patients with different etiologies of infertility (A) Primary GCs were obtained from patients with normal ovarian function or (B) patients diagnosed with PCOS or anovulation. Cells were treated with vehicle, FSH, FSH+HG, or HG alone for 48 hours. CYP19A1 and b-actin (BACT) protein levels were measured 48 hours after the initiation of treatment by Western Blot. The intensity of the CYP19A1 and BACT bands was quantified and the data expressed as a ratio between CYP19A1 and BACT. Different letters represent significant differences. One-way ANOVA followed by Tukey test. *p < 0.05. N ³ 3. a-bp<0.008,b-cp<0.05, a-cp<0.005.

A

B

51

C. DISCUSSION

These findings reveal a novel role for SIKs in the control of GC differentiation in humans

and rodents. I describe the expression of all SIK genes in the ovary and GCs, leading the way to

understand the role of each individual gene in regulating fertility. It is clear that in human and

rodent GCs, SIKs oppose cAMP actions through attenuating GC production of aromatase,

estradiol, and other CREB-dependent steroidogenic genes.

My results are clinically significant in at least two possible ways. First, the inhibition of

SIKs activity could improve the response of poor responders or older patients to controlled

ovarian stimulation. Although SIKs inhibition would not compensate for the scarcity of follicles

found in these patients, they may benefit from the exploitation of pathways aimed to maximize the

growth and response of the follicles that remain viable in their ovaries. In support of this idea, our

results show that SIK inhibition rescues FSH sensitivity in primary human GCs extracted from

PCOS and anovulatory patients. If SIKs inhibition improves FSH responsiveness of these

patients, then it could contribute to alleviating the financial burden of IVF by reducing the amount

of FSH needed. Second, pharmacological inhibition of SIKs activity is being explored as a novel

therapy in several types of cancers, including breast cancer (85). Therefore, the use of SIKs

inhibitors must be monitored closely in patients with estrogen receptor-positive tumors as the

treatment also increases estradiol production.

52

V. SALT INDUCIBLE KINASE 2 ATTENUATES FSH ACTIONS A. INTRODUCTION

Since SIKs activity limits the ability of both rodent and human granulosa cells to respond

to FSH in vitro, our next question was to understand whether SIKs modulate GC differentiation

and steroidogenesis in vivo, and if this has an impact on fertility. To answer these questions, we

injected wildtype mice with SIKs inhibitors and determined the response of GCs to FSH.

Additionally, we utilized genetic models to understand the role of the individual SIK genes, and

accordingly developed a mouse model to further characterize the effect of SIK inhibition. Here,

we show how SIK2 inhibition augments GC response to FSH, partially modulating

folliculogenesis.

B. RESULTS 1. SIKs inhibition potentiates FSH-induced steroidogenesis in vivo

To study the role of SIKs in ovarian function using genetic models, I first examined the effect

of SIKs inhibition on FSH actions in GCs isolated from wild-type mice in vitro. The results of these

experiments mirrored those in rat and human GCs. Thus, as shown in Figure 17, inhibition of SIK

activity potentiated the stimulation of CYP19A1, StAR, and CYP11A1 mRNA levels by FSH in a

dose-dependent manner.

As mouse GCs responded similarly to rat and human cells, we examined the effect of SIKs

inhibition in vivo. Since the inhibitors used above have a short half-life in vivo, it was necessary to

test SIK inhibitors suitable for in vivo studies. We examined the effect of YKL-05-099 (YKL) that

achieves free IC50 serum concentrations for SIKs inhibition for more than 16 h, reduces the

phosphorylation of known SIKs substrates in vivo, and is more tolerable and soluble than other

SIKs inhibitors (80). In vitro experiments demonstrated that YKL enhances the stimulatory effect

of FSH on CYP19A1 expression in a concentration-dependent manner (Figure 18A).

53

Next, we injected immature 23-day-old female mice with vehicle or YKL before the

administration of PMSG, a hormone with FSH activity. The mRNA levels for CYP19A1, StAR, and

CYP11A1 were high in the GCs of animals treated with PMSG. Treatment with YKL enhanced the

stimulatory effect of PMSG on CYP19A1, StAR, and CYP11A1 significantly (Figure 18B). To

determine whether this stimulatory effect occurs at the level of the FSH receptor (FSHR) or IGF1

receptor (IGF1R), I measured the mRNA expression levels of both genes and determined that

neither receptor is impacted in vivo by SIKs inhibition (Figure 18C).

2. Knockdown of SIK2 enhances FSH actions in vitro

To gain insight into the role of individual SIK genes, rat GCs were treated with Compound C

(CoC), which has been previously shown to prevent SIK2-mediated suppression of a cAMP

reporter without suppressing SIK1 and SIK3 activity (86). Figure 18A shows that CoC strongly

potentiated the stimulatory effect of FSH on aromatase. However, CoC alone failed to upregulate

aromatase mRNA levels. We also treated both human and rat GCs with rac Pterosin b, a

compound shown to inhibit SIK3 activity (87,88). The inhibition of SIK3 alone did not significantly

potentiate the FSH stimulation of aromatase (Figure 19B).

These pharmacological findings suggest that SIK2 but not SIK3 plays a crucial role in

regulating FSH actions in GCs. To confirm these results, we utilized small interference RNAs to

selectively knockdown SIK1, SIK2, or SIK3. Rat GCs were infected with lentivirus carrying small

hairpin (sh) RNA specific for each form. FSH or vehicle was added to the media 48 hours after

virus infection. Then, cells were incubated for 48 hours before the determination of gene

expression. Each Sik shRNA significantly knocked down its respective SIK gene when compared

to cells infected with a control shRNA (Figure 20A). Each Sik shRNA targeted specifically the

intended gene without affecting the others (Figure 20A).

The knockdown of Sik1 or Sik3 had no effects on CYP19A1, StAR, or CYP11A1 mRNA

expression in the presence or absence of FSH (Figure 20B). In contrast, Sik2 knockdown

54

potentiated the stimulatory effect of FSH on the three genes, suggesting a leading role of SIK2 in

the regulation of the response of GCs to FSH.

To further confirm the role of SIK2 on aromatase expression, GCs were infected with

increasing amounts of anti-Sik2 shRNA (shSIK2) and cultured in the presence of FSH for 48

hours. We observed an increase in aromatase mRNA levels proportional and concomitant with a

decrease in Sik2 mRNA levels (Figure 20C).

3. Granulosa cells of GC-specific SIK2 knockdown mice have increased steroidogenesis

Since shRNA experiments showed that SIK2 plays a significant role in regulating GC

differentiation, I developed a GC-specific SIK2 knockout mouse utilizing the cre-lox system to

determine the effects of a lack of SIK2 expression in GCs. As with the GC-specific IGF1R

knockout mice (Chapter III), I used both the aromatase (CYP19A1) promoter and estrogen

receptor beta (ER-β) promoter to drive the expression of cre-recombinase. Figure 21A shows that

animals carrying both CYP19A1-Cre and ER-β-Cre promoters and the SIK2 floxed allele have a

significant knockdown of Sik2 in GCs without impacting the expression of Sik1 or Sik3. Using

either CYP19A1-Cre or ER-β-Cre alone also caused a significant knockdown of Sik2 compared to

controls.

To determine whether SIK2 knockdown in GCs affects steroidogenesis and differentiation,

control and CYP19A1-Cre + ER-β-Cre SIK2 F/F (SIK2gcko) mice were injected with PMSG to

mimic FSH actions. SIK2gcko mice had significantly increased expression of aromatase, StAR, and

p450scc expression compared to controls (Figure 21B), indicating that the knockdown of SIK2

improves GC responsiveness to FSH.

4. Effect of SIKs inhibition or SIK2 knockdown on ovulation

Our findings demonstrate that inhibition of SIKs activity or the knockdown of SIK2 in vivo

potentiates the stimulatory effect of FSH. Therefore, we next tested whether inhibition of SIKs

activity also leads to an increase in the number of oocytes released at ovulation. For this purpose,

55

wild type mice were injected with PMSG or PMSG plus YKL. After 48 hours, mice were injected

with human chorionic gonadotropin (hCG) to stimulate ovulation. After 17 hours, mice were

sacrificed, their oviducts collected, and the number of oocytes ovulated was counted. Despite that

SIKs inhibition potentiates the effect of FSH, there was no significant increase in ovulation in the

YKL-treated mice when compared to mice treated with FSH alone (Figure 22A).

Next, we examined ovulation in GC-specific SIK2 knockdown mice to determine if the

increase in FSH-induced steroidogenesis observed in the GCs of these mice leads to more

oocytes ovulated. For this experiment, we use mice carrying floxed SIK2 alleles in the presence

of CYP19-CRE, ER2-CRE, or both CYP19/ER2-Cre. As shown in figure 22B, the number of

oocytes ovulated by animals carrying either CYP19-CRE, ER2-CRE, or CYP19/ER2-Cre was

comparable to the numbers of oocytes ovulated by control animals.

56

Figure 17: Effect of SIKs inhibition in mouse GCs Primary mouse GCs were treated with vehicle, FSH, or FSH plus increasing concentrations of HG. CYP19A1, StAR, and CYP11A1 mRNA levels were quantified 48 hours later and expressed as relative to RPL19. Different letters represent significant differences. Mean ± SEM, N=5, one-way ANOVA followed by Tukey, a-b, b-cp < 0.05, a-cp < 0.01.

57

Figure 18: SIKs inhibition enhances FSH actions in vivo (A) Primary rat GCs were treated with vehicle, FSH, FSH plus YKL, or YKL alone.

CYP19A1 mRNA levels were quantified 48 h later and expressed as relative to Rpl19. Different letters represent significant differences (mean ± SEM, N = 5, one-way ANOVA followed by Tukey, p < 0.05) Immature 23-day-old female mice were injected i.p. with YKL or vehicle (PBS). Two hours later, animals were treated with PMSG or PMSG plus YKL. The expression of (B) CYP19A1, StAR, and CYP11A1 and (C) FSHR and IGF1R was quantified 48 hours later. * p < 0.05, t-test.

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Figure 19: SIK2, not SIK3, pharmacological inhibition augments aromatase expression (A) Rat GCs were treated with Compound C (CoC, a SIK2 inhibitor) for one hour before

treatment with FSH. Aromatase expression was determined 48 hours after the addition of FSH (mean ± SEM, N = 3, one-way ANOVA followed by Tukey,*p< 0.05, ****p<0.0001).

(B) Rat (left) and human (right) GCs were treated with rac Pterosin b, a SIK3 inhibitor, for

one hour before treatment with FSH. Aromatase expression was determined 48 hours after the addition of FSH (mean ± SEM, N = 3, one-way ANOVA followed by Tukey, *p < 0.05, **p< 0.01).

A

Aromatase

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Figure 20: SIK2 knockdown mimics the pharmacological inhibition of SIKs activity (A) SIK isoforms expression in rat GCs exposed to scrambled oligos (shSCR) or anti-

SIK1 (shSIK1), SIK2 (shSIK2), or SIK3 (shSIK3) shRNAs. * p<0.05, ** p<0.01, t-test, N = 3.

(B) CYP19A1, StAR, and CYP11A1 relative mRNA expression levels after exposure to shSCR, shSIK1, shSIK2, or shSIK3. Cells were treated with vehicle (white bars) or FSH (black bars). *** p<0.001, t-test, N = 3.

(C) Inverse correlation between Sik2 knockdown with CYP19A1 expression in rat GCs. Cells were exposed to increasing concentrations of shSIK2 for 48 hours and then treated with FSH for an additional 48 hours, N = 3. One-way ANOVA followed by Tukey, a-b, b-cp < 0.05, a-cp < 0.01.

s h L U C s h S IK 1 s h S IK 2 s h S IK 30 .0 0

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FSH Control

60

Figure 21: SIK2 knockdown in GCs augments steroidogenesis in vivo (A) mRNA expression of SIK1, SIK2, and SIK3 was quantified in GCs of immature 21-23-

day-old mice carrying two SIK2 floxed alleles and CYP19A1-Cre (19-Cre), ER-β-Cre (β-Cre), or both (19+β-Cre). Different letters represent significant differences. One-way ANOVA followed by Tukey, a-bp<0.05.

(B) Immature 21-23-day-old control and CYP19A1-Cre + ER-β-Cre SIK2 F/F (SIK2

GCKO) mice were injected with 7.5 IU PMSG. mRNA expression of aromatase, StAR, and CYP11A1 was quantified 48 hours later. *p <0.05, **p <0.01, t-test.

WT 19-Cre β-Cre 19+β-Cre0.00

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Figure 22: SIKs inhibition or SIK2 knockdown does not increase ovulation

(A) Immature 21-23-day-old control mice were injected i.p. with vehicle or YKL-05-099 (YKL, a SIKs inhibitor). Two hours later, animals were treated i.p. with PMSG or YKL+PMSG. After 48 hours, animals were injected i.p. with hCG (to trigger ovulation), and 17 hours later the oocytes ovulated were counted. t-test was used, no significant difference was found.

(B) Ovulation was stimulated in CYP19A1-Cre (19-Cre), ER-β-Cre (β-Cre), or both (19+β-Cre) as described and oocytes counted. One-way ANOVA followed by Tukey. No significant differences found.

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62

C. DISCUSSION

The inhibition of SIKs activity in vivo through YKL injections confirmed in vitro experiments

showing that the response of GCs to FSH increases with lower SIKs activity. Steroidogenesis

also increased in the GCs of wildtype mice, indicating that acute systemic treatment with SIKs

inhibitors increases the ability of GCs to respond to FSH, an important step in folliculogenesis.

The impact of decreased SIK2 expression on fertility was evident through achieving similar

potentiation of steroidogenic genes in the knockdown mice, especially because the expression of

neither SIK1 nor SIK3 was impacted in SIK2gcko mice. Finally, since the levels of FSHR and

IGF1R were unchanged by acute SIKs inhibition, the effect of SIKs on GC differentiation appears

to take place downstream of these receptors by mechanisms that still need to be elucidated (see

chapter VI).

Interestingly, although SIK2 knockdown mice have an increased response to exogenous

gonadotropins, the number of oocytes ovulate by these mice is comparable to those of control

mice. I propose that SIK2 is one of several mechanisms involved in the control of ovulation. Thus,

the lack of SIK2 may not be enough to augment follicle recruitment by endogenous gonadotropins

since other pathways may maintain ovulation within physiological parameters. Several different

possibilities need to be studied. For instance, since FSH and LH are needed for proper ovulation,

it is possible that SIK2 knockdown does not potentiate LH actions. Another possibility is that SIK1

or SIK3 may also participate in the regulation of ovulation and that their activity compensates for

decreased SIK2 expression. Finally, it would be interesting to gauge if SIK2 knockdown increases

folliculogenesis; it may be that decreased SIK2 expression increases the number of recruited

mature follicles, but that their release at ovulation is defective.

In conclusion, in this chapter, I demonstrate that SIK2 is the main gene regulating the

response of GCs to FSH. These findings were confirmed in vivo using a pharmacological inhibitor

of SIKs activity and by the conditional knockdown of SIK2 in GCs. However, the contribution of

SIK2 to ovulation and female fertility remains to be examined.

63

VI. UNDERSTANDING THE MECHANISM OF SIKs ACTIONS A. INTRODUCTION

Our findings thus far demonstrate that SIK2 activity attenuates FSH actions both in vitro

and in vivo. This chapter seeks to increase our understanding of the molecular mechanisms

controlled by SIKs in GCs. First, I investigated at which level on the FSH receptor signaling

pathway SIKs act. The FSH receptor primarily activates Ga protein, which in turn stimulates

adenylate cyclase activity and the production of cyclic AMP (cAMP). We postulate that SIKs may

affect either the expression of the FSH receptor or the production and effects of cAMP.

Additionally, I initially proposed SIK as the link between the signaling pathways of the FSH

receptor and the IGF1 receptor. Therefore, this chapter also attempts to determine if SIKs activity

affects IGF1 receptor signaling.

B. RESULTS 1. SIKs actions are downstream of cAMP signaling

Since SIKs attenuate the effect of FSH in GCs, I next examined whether SIKs decrease GC-

response to FSH by decreasing FSH receptor expression, I either inhibited SIKs activity using

HG-9-91-01 or reduced SIKs expression through shRNA knockdown. Neither approach affected

FSH receptor expression (Figure 23A), indicating that SIKs actions occur downstream of the FSH

receptor. Since FSH activates adenylyl cyclase (AC), I next treated the cells with forskolin, a

specific AC activator. Forskolin by itself induced aromatase and StAR expression. As with FSH,

the stimulatory effect of forskolin was also significantly potentiated by the inhibition of SIKs

activity (Figure 23B).

Adenylyl cyclase activation leads to an increase in intracellular cAMP concentration. Because

SIKs inhibition enhances the effect of forskolin, I next examined whether SIKs inhibition also

augments the stimulatory effect of cAMP on aromatase. For this purpose, I treated the GCs with

dibutyryl-cAMP (dbcAMP), a cell-permeable analog of cAMP, which alone induced aromatase

64

and StAR expression (Figure 24A). Again, as with FSH and forskolin, SIKs inhibition potentiated

dbcAMP-induced aromatase and StAR (Figure 24A).

Finally, cAMP effects are mediated mainly by two effectors containing cAMP-binding

domains: Protein Kinase A (PKA) and the exchange protein directly activated by cAMP (Epac)

(89). Therefore, I next examined whether SIKs inhibition modifies the effects of the

pharmacological activation of Epac. To specifically activate Epac, I treated GCs with 8-(4-

Chlorophenylthio) adenosine 3’5’-cAMP (8-CPT-cAMP), a compound that activates Epac but not

PKA. Epac did not induce aromatase or StAR expression (Figure 24B), whereas SIKs inhibition

using HG-9-91-01 alone induced these genes. The combination of HG and 8CPT treatment did

not induce aromatase or StAR more than HG treatment alone did (Figure 24B). Taken together,

these results suggest that SIKs actions occur downstream of cAMP and PKA, and that Epac is

not involved in the stimulation of aromatase in GCs.

2. FSH does not induce SIKs expression

Since knocking down SIK2 increases the expression of steroidogenic genes, my next

experiment was to determine whether FSH treatment regulates the expression of SIKs in GCs. I

hypothesized that one mechanism by which FSH may induce aromatase expression is by the

inhibition of SIKs expression. To test this, I treated cultured rat GCs with FSH and measured

aromatase, StAR, SIK1, SIK2, and SIK3 expression at 1, 3, 6, 12, 24, and 48 hours after the

initiation of the treatments. Predictably, FSH induced aromatase and StAR expression

significantly after 48 hours of treatment (Figure 25). However, SIK2 expression remains

consistent throughout the experiments and it did not decrease at the peak of aromatase

expression (Figure 25). Interestingly, FSH induces a transient increase of SIK1 expression after

one hour of treatment, a similar phenomenon that occurs in adrenal glands after stimulation with

ACTH (90). A transient increase of SIK3 expression occurs only after 3 hours of FSH treatment

65

(Figure 25). These results suggest that FSH has no effects on the expression of SIKs in GCs.

However, it remains to be determined whether FSH regulates the activity of SIKs.

3. SIKs involvement in the IGF1 receptor pathway

I hypothesized that SIKs are the link between the FSH and IGF1 pathways. Moreover, since

SIKs inhibition and IGF1 receptor activation enhance the effect of FSH in GCs, I postulated that

IGF1 receptor activation inhibits SIKs. Therefore, my next experiments were designed to

determine whether SIKs are downstream of IGF1R. I postulated that if the IGF1 receptor inhibits

SIKs activity, blocking SIKs activity in the presence of an IGF1 receptor inhibitor would rescue

FSH actions. For this purpose, I treated cultured primary human GCs with AEW, an IGF1R

inhibitor, in the presence or absence of HG, the SIKs inhibitor.

As we have previously demonstrated (35,36), AEW completely inhibited FSH induction of

aromatase but did not affect StAR or P450scc expression (Figure 26A). In cells lacking IGF1

receptor activity, SIKs inhibition did not recover aromatase (Figure 26A). Moreover, AEW

inhibited HG-induced aromatase expression suggesting that IGF1 receptor activity is downstream

of SIKs in GCs. SIKs inhibition significantly increased StAR expression after AEW treatment, but

not P450scc expression (Figure 26A).

To test whether AEW-inhibition of IGF1 receptor activity affected SIKs expression in these

cells, I measured SIK1, SIK2, and SIK3 mRNA levels after 48 hours of treatment with AEW. The

results showed no significant difference between control and AEW treated cells for all three

genes (Figure 26B).

Contrary to the in vitro data showing that AEW inhibition of IGF1 receptor activity causes no

change in SIKs expression, GCs of IGF1Rgcko mice have significantly higher expression of all

SIKs compared to control mice (Figure 27A). Because of this high SIKs expression, I postulated

that the significantly reduced steroidogenesis observed in the GCs of IGF1Rgcko mice (49) can be

rescued by inhibiting SIKs activity. For this purpose, I injected IGF1Rgcko mice with the SIKs

66

inhibitor YKL-05-099 and PMSG and then measured aromatase, StAR, and P450scc mRNA

levels. As expected, treatment with PMSG stimulated aromatase expression strongly in control

mice but failed to do so in IGF1Rgcko mice (Figure 27B). Strikingly, SIKs inhibition did not rescue

PMSG actions as there was no significant increase in aromatase expression levels in IGF1Rgcko

mice treated with YKL+PMSG versus PMSG alone (Figure 27B).

4. Role of GSK3b on the interaction between FSH and SIKs in GCs

Earlier work showed that FSH and IGF1 signaling converge on AKT activation. As previously

described, AKT is a known inhibitor of GSK3, which is a SIKs activator (75,76,91). Therefore, I

hypothesized that inhibiting GSK3 would also potentiate FSH actions. I co-treated primary human

GCs with FSH and CHIR 99021, a GSK3 inhibitor. CHIR did not potentiate the stimulation of

aromatase, StAR, or P450scc by FSH (Figure 28). The results suggest that GSK3b does not

participate in FSH induction of gene expression in GCs.

5. Effect of SIKs inhibition on PKA downstream targets

Activation of PKA has been shown to activate AKT and CREB in GCs (35,92). I next sought to

determine if SIKs impact the activation of these proteins. Primary rat GCs were pre-treated with

HG for one hour and then stimulated with FSH for one hour. Whole-cell lysates were used for

total AKT and phospho-S473-AKT determination through Western blotting. As expected, FSH

increased AKT phosphorylation significantly (Figure 29). The inhibition of SIKs activity with HG

did not modify the effect of FSH on AKT. SIKs inhibition with HG had no effects on AKT

phosphorylation (Figure 29). The results suggest that SIKs activity is not involved in the regulation

of AKT activation in GCs.

CREB is phosphorylated by PKA at S133, which facilitates the recruitment of co-activators

causing an increase in CREB’s transcriptional activity (93). To understand the mechanism by

which SIKs inhibition increases CREB-dependent gene transcription, I tested whether SIKs

inhibition affects CREB phosphorylation. Primary rat GCs were pre-treated with HG or vehicle for

one hour and then stimulated with FSH or vehicle for one hour. Whole-cell lysates were used for

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total CREB and phospho-S133-CREB determination through Western blotting. As expected, FSH

alone increases CREB phosphorylation at Serine 133 (Figure 30). This stimulatory effect of FSH

was not affected by the simultaneous inhibition of SIKs activity. Accordingly, SIKs inhibition alone

did not increase CREB phosphorylation (Figure 30).

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Figure 23: SIKs actions are downstream of the FSH receptor

(A) SIKs were inhibited (left) or their expression was knocked down (right) in cultured rat GCs. After 48 hours, the FSH receptor expression was measured. No significant differences were found. One-way ANOVA followed by Tukey. N=3.

(B) Cultured rat GCs were pre-treated with vehicle or HG for one hour, then treated with FSH or forskolin (FSK), an adenylate cyclase activator. After 48 hours, aromatase and StAR mRNA expression levels were determined. One-way ANOVA followed by Tukey. *p < 0.05, **p < 0.01. N=3.

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** **

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Figure 24: SIKs activity is downstream of PKA

(A) Cultured rat GCs were pre-treated with vehicle or HG for one hour, then treated with FSH or dbcAMP. After 48 hours, aromatase and StAR mRNA expression levels were determined. One-way ANOVA followed by Tukey. *p < 0.05. N=3.

(B) Cultured rat GCs were pre-treated with vehicle or HG for one hour, then treated

with FSH or 10µM 8-CPT-cAMP (8CPT), an Epac activator. After 48 hours, aromatase and StAR mRNA expression levels were measured. One-way ANOVA followed by Tukey. **p < 0.01, ***p < 0.001. N=3.

Aromatase

C 0 1 0 1 10.000

0.005

0.010

0.015

0.0200.15

0.20

0.25

0.30

FSH dbcAMP 0.5mM

HGuM

Rel

ativ

e to

Rpl19

*StAR

C 0 1 0 1 105

10152025

150

200

250

FSH dbcAMP 0.5mM

HGuM

Rel

ativ

e to

Rpl19

*

C 0 1 0 1 10.0000

0.0005

0.0010

0.00150.020

0.025

0.030

0.035

0.040

Rel

ativ

e to

Rpl19

Aromatase

***

FSH 8CPT

HGuM

C 0 1 0 1 10

1

2

3

15

20

25

Rel

ativ

e to

Rpl19

StAR

**

FSH 8CPT

HGuM

A

B

70

Figure 25: FSH does not induce SIKs expression Cultured rat GCs were treated with FSH at different times, then the mRNA expression levels of aromatase, StAR, SIK1, SIK2, and SIK3 were measured 1, 3, 6, 12, 24, and 48 hours later. One-way ANOVA followed by Tukey. *p < 0.05, N=3.

Timecourse

C 1 3 6 12 24 480.00

0.02

0.04

0.06

0.08

0.10

20

40

60AromataseSIK1SIK2SIK3StAR

Hours

Rel

ativ

e to

Rpl19

*

*

*

*

71

Figure 26: SIKs and the IGF1R pathway

(A) Cultured human GCs were pre-treated with HG or AEW (an IGF1R inhibitor) for one hour. Then, cells were treated with FSH for 48 hours and mRNA expression levels of aromatase, StAR, and P450scc were determined. One-way ANOVA followed by Tukey; different letters represent significant differences. a-b p<0.05, b-cp<0.05, c-dp<0.001. N=4.

(B) Cultured human GCs were treated with vehicle or AEW for 48 hours, then expression levels of all SIK genes were measured. No significant differences were found. t-test. N=4.

0 0 1 0 1 1 10

5

10

15

Aromatase

Rel

ativ

e to

Rpl19

FSH

AEW

+ + + +

+ + +

HG uM

a

b

c

a a a

b

0 0 1 0 1 1 10

1

2

3

4

5

StAR

Rel

ativ

e to

Rpl19

FSH

AEW

+ + + +

+ + +

HG uM

ab

c

b

d

aa

0 0 1 0 1 1 10

50

100

150

Rel

ativ

e to

Rpl19

p450scc

FSH

AEW

+ + + +

+ + +

HG uM

a

bb b

bb

b

C AEW0.0000

0.0001

0.0002

0.0003

0.0004

SIK1

Rel

ativ

e to

Rpl19

C AEW0.000

0.002

0.004

0.006

0.008

SIK2R

elat

ive

to Rpl19

C AEW0

10

20

30

SIK3

Rel

ativ

e to

Rpl19

A

B

72

Figure 27: SIKs and the IGF1R pathway (A) SIK1, SIK2, and SIK3 mRNA expression levels were measured in GCs of wildtype

mice and GC-specific IGF1R knockout mice (IGF1RGCKO). t-test. **p < 0.005, ***p < 0.001. N=3.

(B) Wildtype and IGF1Rgcko mice were injected i.p. with vehicle or YKL, a SIKs inhibitor, for two hours. Then, mice were injected i.p. with PMSG or YKL+PMSG. After 48 hours, the expression levels of aromatase, StAR, and P450scc were measured. t-test. **p < 0.005

SIK1

WT IGF1RGCKO0.000

0.001

0.002

0.003

0.004

Rel

ativ

e to

Rpl19

***

SIK2

WT IGF1RGCKO0.000

0.001

0.002

0.003

0.004

0.005**

Rel

ativ

e to

Rpl19

SIK3

WT IGF1RGCKO

0.000

0.002

0.004

0.006

0.008

0.010

**

Rel

ativ

e to

Rpl19

A

B

PMSG PMSG YKL+PMSG0.0

0.1

0.2

0.3

Aromatase

Rel

ativ

e to

Rpl19

WT IGF1R GCKO

**

PMSG PMSG YKL+PMSG0.00

0.02

0.04

0.06

StarR

elat

ive

to Rpl19

WT IGF1R GCKO

**

PMSG PMSG YKL+PMSG0.0

0.5

1.0

1.5

2.0

2.5

p450scc

Rel

ativ

e to

Rpl19

WT IGF1R GCKO

73

Figure 28: GSK3b inhibition does not potentiate FSH actions Cultured human GCs were pre-treated with CHIR 99021 (CHIR), a GSK3 inhibitor, at different doses for one hour then FSH for 48 hours. mRNA expression of aromatase, StAR, and P450scc were then determined. One-way ANOVA followed by Tukey, **p < 0.005. N=4.

C 0 0.5 1 0.5 10

1

2

3

4

Aromatase

Rel

ativ

e to

Rpl19

FSH

CHIR uM

**

C 0 0.5 1 0.5 10.0

0.1

0.2

0.3

0.4

StAR

Rel

ativ

e to

Rpl19

FSH

CHIR uM

C 0 0.5 1 0.5 10.00

0.01

0.02

0.03

p450scc

Rel

ativ

e to

Rpl19

FSH

CHIR uM

74

Figure 29: SIKs inhibition does not increase AKT phosphorylation Cultured rat GCs were pre-treated with HG for one hour and then stimulated with FSH for one hour. Whole-cell lysates were used for total AKT and phospho-S473-AKT determination by Western blotting. A representative blot is shown. One-way ANOVA followed by Tukey. N=3.

C F F+HG HG

pAKT

AKT

C F F+HG HG0.0

0.2

0.4

0.6

0.8

pAKT/AKT

75

Figure 30: SIKs inhibition does not increase CREB phosphorylation Cultured rat GCs were pre-treated with HG for one hour and then stimulated with FSH for one hour. Whole-cell lysates were used for total CREB and phospho-S133-CREB determination by Western blotting. A representative blot is shown. One-way ANOVA followed by Tukey. N=3.

C F F+HG HG

pCREB

CREB

C F F+HG HG0.0

0.5

1.0

1.5

2.0

2.5

pCREB/CREB

76

C. DISCUSSION Despite clear evidence demonstrating that inhibiting SIKs activity augments FSH actions,

understanding the mechanisms and the signaling pathways involved have been more elusive.

Our findings suggest that SIKs act downstream of the FSHR and cAMP, as evidenced by the

ability of HG to potentiate aromatase and StAR expression induced by various activators of this

pathway. I have shown that from the two exclusive cAMP targets, PKA and Epac, SIKs activity

appears to target proteins downstream of PKA. Epac alone does not induce aromatase or StAR

expression, and it does not affect HG’s tendency to stimulate these genes on its own. Thus, our

findings demonstrate that SIKs actions are exclusively downstream of PKA. How SIKs and PKA

interact remains to be studied in the GCs.

I hypothesized that FSH regulates its activity by decreasing SIKs expression. However,

the results show that FSH does not significantly affect SIKs expression up to 48 hours after

treatment, which is the peak of FSH-induced aromatase and StAR expression. It could be that

FSH regulates SIKs activity instead of its expression, which is supported by the presence of PKA

phosphorylation sites on all SIK proteins.

The answer to whether SIKs actions are also downstream of the IGF1R remains obscure.

SIKs inhibition did not recover aromatase expression after pharmacological inhibition of this

receptor in vitro; thus, aromatase levels remained extremely low in cells treated with AEW or

AEW+HG. Since AEW inhibited HG-induced aromatase expression, I postulated that IGF1R

activity might be downstream of SIK. However, SIKs inhibition did not increase AKT

phosphorylation, indicating that SIKs are not involved in AKT regulation. Thus, I suggest that SIK

is downstream of AKT in the IGF1R signaling pathway.

Although GSK3 is a known activator of SIK and is known to be inhibited by AKT, the

findings demonstrate that GSK3 is not relevant in the regulation of FSH-induced steroidogenesis

in GCs. We expected that GSK3 inhibition would have prevented SIKs activation, which

theoretically should have the same effect as SIKs inhibition. However, GSK3 inhibition has no

77

effects on the FSH-induction of aromatase. Further studies are needed to understand the function

of GSK3 in GCs since this protein is rapidly phosphorylated by FSH. Our findings demonstrate

that GSK3 is not involved in SIKs activation at least in ovarian GCs.

However, a puzzling finding suggesting that SIKs are a potential link between FSHR and

IGF1R is that GCs of IGF1Rgcko mice have higher expression levels of all three SIK genes, a

phenomenon that was not observed in vitro after 48 hours of pharmacological IGF1R inhibition. A

potential explanation of this discrepancy is that 48 hours may not be enough to impact SIKs

expression, while IGF1Rgcko mice never expressed the IGF1R in GCs which may have facilitated

the upregulation of SIKs expression. Although SIKs expression is higher in IGF1Rgcko mice,

inhibition of its activity in vivo did not significantly increase or recover steroidogenesis, indicating

that there are other factors downstream of the IGF1R that regulate the response of GCs to FSH

stimulation, and that SIKs inhibition simply is not enough to overcome the lack of IGF1R activity.

Finally, the mechanism by which SIKs inhibits CREB-dependent genes still needs to be

understood. I have shown that inhibiting SIK does not impact CREB phosphorylation/activation.

However, SIKs inhibition potentiates CREB-dependent genes such as aromatase and StAR.

Therefore, it stands to reason that SIK activity impacts CREB coactivators recruitment to the

promoter of these genes. Previous reports have demonstrated that SIKs are strong inhibitors of

CREB-regulated transcription coactivators (CRTC) (40,41,57,63,64,86) blocking several CREB-

dependent systems, such as steroidogenesis in the adrenal glands in response to ACTH (94). I

propose that in GCs, SIKs inhibition increases the recruitment of CRTC to the promoter of

aromatase and StAR. Further experiments are needed to determine the involvement of CRTC in

the regulation of FSH action in GCs.

78

VII. GENERAL CONCLUSIONS AND FUTURE DIRECTIONS

Infertility is an issue that affects about 12% of reproductive-aged couples. About one-third

of infertility cases are caused by fertility problems in women. Key factors adding to this growing

problem are both the natural decline in reproductive capacity for women after the age of 32, and

the tendency for women to wait longer until they have their first child. This is evidenced by the

fact that the average age of first-time mothers has increased six-fold between 1970 and 2012,

and first births to women above age 40 are at an all-time high. As a result, more women are

resorting to assisted reproductive technologies.

The most common cause of infertility in women is the failure to ovulate, so understanding

the process of normal ovarian function and control is vital to better design effective treatments.

The most common procedure used in assisted reproductive technologies is in vitro fertilization

(IVF). During IVF, ovulation is stimulated and monitored to maximize the number of mature

oocytes in one cycle. For this purpose, patients undergoing IVF receive exogenous FSH to

stimulate follicle growth and oocyte maturation. After that, the oocytes are extracted, fertilized in

the laboratory, and transferred back into the uterus. Although FSH is the main driver of follicle

growth, it relies on intra-ovarian factors whose secretion enhances follicular response to FSH.

The main factor we have studied in the lab are the IGFs, and we have shown that FSH requires

IGFs activity in the follicles to stimulate their growth. Patients with lower IGFs in their follicular

fluid have a decreased response to FSH and require higher doses to retrieve the same number of

oocytes. Currently, more than a third of patients respond poorly to FSH stimulation. That is

typically due to advanced maternal age, a low reserve of follicles, ovarian dysfunction, or

unexplained causes. The current solution for this problem is to administer higher doses of FSH;

however, this does not always cause an increased response, and patients are at a higher risk of

ovarian hyperstimulation syndrome (OHSS). Additionally, this approach increases significantly the

cost of the treatments due to the high cost of producing recombinant FSH.

79

Extensive work in the lab has determined that GCs require IGF1R activity to respond to

FSH stimulation, which was measured through the induction of steroidogenic genes, mainly

aromatase. Here, we developed a mouse model with undetectable levels of IGF1R in the GCs.

GC-specific IGF1R knockout mice (IGF1Rgcko) express diminished steroidogenic and

differentiation markers such as aromatase, StAR, LH receptor, and the inhibin genes.

Consequently, these mice do not produce estradiol, have severely impaired folliculogenesis, do

not ovulate, and are infertile. Additionally, their follicles express increased apoptosis markers and

decreased proliferation markers.

To understand the mechanism of action of the IGF1 receptor, and why it is needed for

GCs to respond to FSH, we measured the expression levels of the FSH receptor in IGF1Rgcko

mice and determined that IGF1R does not increase or alter FSHR expression. Therefore, we

explored various intracellular pathways that are affected by the lack of IGF1R activity. We

determined that AKT phosphorylation and activation was compromised in IGF1Rgcko mice

compared to controls and that the activities of both FSHR and IGF1R converge on the activation

of AKT in vivo. This supports the previous in vitro work done in the lab.

Since the most prominent impairment in IGF1Rgcko mice occurs in genes downstream of

the cAMP pathway, we concluded that the crosstalk of the two pathways occurs downstream of

cAMP. Also, since IGF1R and FSHR converge on AKT activation, we postulated that there is a

link between AKT and cAMP (mainly CREB). After performing a literature search, we zoned in on

a likely candidate that is downstream of AKT and is a known inhibitor of CREB activity: the salt

inducible kinases. SIKs function as negative regulators of cAMP signaling in several systems (see

introduction) but their function was not explored in ovarian granulosa cells. Here, we found that all

SIK genes, SIK1, SIK2, and SIK3 are expressed in GCs of mice, rats, and humans and also

function as negative regulators. I showed this in several experiments where GCs were stimulated

with FSH and simultaneously SIKs activity was inhibited. I showed that SIKs inhibit FSH-induced

cAMP signaling, which consequently attenuates steroidogenic gene expression. Since aromatase

80

is the main product of FSH and the marker of its stimulation, I explored the role of SIKs inhibition

at several levels: promoter activation, mRNA expression, protein production, and estradiol

production. SIKs inhibition increased aromatase promoter activation, consequently causing

increased aromatase protein levels and estradiol. My results confirm that SIKs are negative

regulators of cAMP signaling, a phenomenon that has been observed in several systems

including adrenocorticotropic cells responding to ACTH stimulation. SIKs act as signaling

modulators of cAMP actions, including ovarian GCs (Figure 31).

The most exciting part of these results is that primary granulosa cells obtained from IVF

patients also responded to SIKs inhibition by producing more aromatase. Cells from patients with

no ovarian issues that responded “normally” to FSH by producing aromatase expressed even

higher levels in response to SIKs inhibition. Remarkably, cells from poor responding patients with

no aromatase expression after FSH stimulation were able to recover their aromatase protein

levels after treatment with SIKs inhibitors. This was a step forward in understanding the

underlying issues behind poor responding patients.

The effects of SIKs inhibition were also observed in vivo; mice injected with the SIK

inhibitor YKL-05-099 expressed increased levels of steroidogenic genes in response to FSH

stimulation. Additionally, YKL injections had no adverse effects on the injected mice, since they

appeared to behave normally and were not distressed. This supports the possibility of using SIK

inhibitors as acute treatments to enhance steroidogenesis.

To better understand the role of all SIK genes, and to better understand the reproductive-

specific effects of SIK inhibition, we utilized genetic tools to knockdown each SIK gene separately

and determined the effect on FSH stimulation. From these experiments, we observed that

inhibition of SIK2 specifically increased FSH-induced steroidogenic gene expression, so we

proceeded to develop a mouse model with GC-specific SIK2 knockdown. SIK2gcko mice had a

baseline increased expression of steroidogenic genes in response to FSH stimulation than

wildtype mice, but their ovulation count was not significantly increased. Even SIKs inhibition using

81

YKL did not increase ovulation in wildtype mice. This was a puzzling finding since the potentiation

of FSH responsiveness was very robust. However, my analysis is that ovulation is a complex

process that depends on the actions of pituitary LH in addition to FSH. It could be that SIKs

activity in the thecal cells also impacts total ovarian response to ovulation stimulation. Since SIKs

are also expressed in the stromal cells, there needs to be more research done to determine the

role of all SIK genes in each ovarian cell type. It could be that SIKs inhibition enhances

folliculogenesis and increases the number of follicles activated, but that internal control

mechanisms limit the number of oocytes released so as not to exceed the uterine capacity of the

mother (95). To better understand the role of SIK3, our lab recently obtained SIK3 floxed mice to

develop a GC-specific knockout model. Perhaps there is a complex interaction between all SIK

genes where they compensate for one another in the granulosa, theca, and stromal cells.

SIKs do not regulate FSHR or IGF1R expression in vivo since mice injected with YKL and

FSH did not have altered expression of either receptor compared to mice injected with FSH

alone. Thus, it is reasonable to conclude that SIKs act downstream of the cAMP pathway

because inhibition of their activity enhanced FSH activity. This was further confirmed by SIKs

inhibition having a similar effect on the actions of activators downstream of the FSHR that also

induced steroidogenic gene expression, and by showing that SIKs attenuate PKA activity by

inhibiting CREB-dependent genes (Figure 31). These genes (aromatase and StAR) were not

induced by the second cAMP target, Epac, indicating that SIKs are involved with PKA activity. All

SIK genes contain multiple inhibitory PKA phosphorylation sites. Since I observed that FSH

activity does not induce or reduce SIKs expression, I propose that FSH decreases the activity of

SIKs via a PKA-mediated phosphorylation process. It would be interesting to see the effect on

CREB-dependent genes after FSH stimulation of GCs overexpressing SIKs containing a mutated

PKA phosphorylation site.

Although I have shown that inhibiting SIKs activity does not overcome IGF1R inhibition,

throwing into question the hypothesis that SIKs activity is downstream of the IGF1R, I speculate

82

that I have not observed an effect because the IGF1R inhibition was for a short period of time.

Although acute IGF1R inhibition in vitro using AEW did not alter SIKs expression, GCs of

IGF1Rgcko mice have increased SIKs expression. This leads me to hypothesize that FSHR

modulates SIKs activity, while IGF1R modulates SIKs expression (Figure 31). This needs

extensive studying, and it would be interesting to develop a double knockout mouse model that

lacks both IGF1R and SIKs expression to understand whether SIKs are at all involved with the

IGF1R pathway. Acute SIKs inhibition using YKL was not enough to overcome the inability of

IGF1Rgcko mice to ovulate, leading me to think that either SIKs are not involved in the pathway, or

that their acute inhibition was not enough to overcome the absence of the IGF1R.

Noteworthy, in all my in vitro SIKs inhibition experiments, the SIKs inhibitor HG-9-91-01

alone caused an increase in steroidogenic gene expression, sometimes to levels comparable to

those found with FSH stimulation alone. This further supports my hypothesis that SIKs activity is

normally in the “on” state and it is inhibited by the cAMP pathway; so, inhibiting its activity alone

has significant effects on the CREB dependent genes. The substrates of SIKs still need to be

studied in the GCs. The two main targets are the CREB co-activator CRTC, or the class IIa

histone deacetylases (HDACs). I have shown that SIKs do not target CREB, as observed when

CREB phosphorylation was not significantly changed between cells treated with FSH or FSH+HG

(Figure 31. I have also shown that SIKs do not target AKT phosphorylation, indicating that SIKs

are not involved in AKT regulation.

In my concluding thoughts, I have come to realize that my research contributed to

understanding the underlying problems leading to infertility. The practical implications of my

research could aid in fine-tuning the IVF experience for patients. Perhaps, we can relieve the

financial burden of gonadotropin administration by treating patients with SIK inhibitors, which

could help decrease the dosage of FSH needed to stimulate follicle growth or maximize the

response to FSH in poor responder patients consequently leading to an increase in pregnancy

rates.

83

Figure 31: Summary of SIKs actions in granulosa cells and open questions that remain to be addressed in future research

IGFsFSH

cAMP

PKA

pCREB -- CRE

SIKs

GC diff. genes

-------------------------I

I

Activity?

----------------------------I

Express

ion?AKT

-------I?

CRTC

----------------I?

Granulosa Cell

84

VIII. APPENDICES Appendix A

Office of Animal Care and Institutional Biosafety Committee (OACIB) (M/C 672) Office of the Vice Chancellor for Research 206 Administrative Office Building 1737 West Polk Street Chicago, Illinois 60612

Phone (312) 996-1972 • Fax (312) 996-9088

4/4/2020 Carlos Stocco Physiology & Biophysics M/C 901 Dear Dr. Stocco: The protocol indicated below was reviewed in accordance with the Animal Care Policies and Procedures of the University of Illinois at Chicago and renewed on 4/4/2020. Title of Application: Salt-Inducible Kinase Regulation of Ovarian Granulosa Cells (Form G) ACC NO: 19-045 Original Protocol Approval: 4/8/2019 (3 year approval with annual continuation required). Current Approval Period: 4/4/2020 to 4/4/2021 Funding: Portions of this protocol are supported by the funding sources indicated in the table below. Number of funding sources: 1

Funding Agency Funding Title Portion of Funding Matched NIH Salt-Inducible Kinase Regulation of Ovarian Granulosa

Cells (Institutional # 00458980) Form G protocol linked to UIC 17-216 and University of Colorado-HSC- 00161

Funding Number Current Status UIC PAF NO. Performance Site Funding PI RO1 HD097202 (A1 version years 1-5)

Funded UIC and Other Univ of Colorado-HSC

Carlos Stocco

This institution has Animal Welfare Assurance Number A3460.01 on file with the Office of Laboratory Animal Welfare, NIH. This letter may only be provided as proof of IACUC approval for those specific funding sources listed above in which all portions of the grant are matched to this ACC protocol. Thank you for complying with the Animal Care Policies and Procedures of the UIC. Sincerely,

Amy Lasek, PhD Chair, Animal Care Committee AL/kg cc: BRL, ACC File, Raj Kumar

85

Appendix B

Phone (312) 996-1972 • Fax (312) 996-9088 • www.research.uic.edu

Office of Animal Care and Institutional Biosafety Committees (MC 672) Office of the Vice Chancellor for Research 206 Administrative Office Building 1737 West Polk Street Chicago, Illinois 60612-7227

May 3, 2018 Carlos Stocco Physiology & Biophysics M/C 901 Dear Dr. Stocco: The protocol indicated below has been reviewed in accordance with the Institutional Biosafety Committee Policies of the University of Illinois at Chicago on 4/12/2018. The protocol was not initiated until final clarifications were reviewed and approved on 5/3/2018. Protocol expires 3 years from the date of review (4/12/2021). This protocol replaces protocol 15-041 which has been terminated. Title of Application: Regulation of Gene Expression in Ovarian Cells IBC Number: 18-023 Highest Biosafety Level: 2 You may forward this letter of acceptable IBC verification of your research protocol to the funding agency considering this proposal. Please be advised that investigators must report significant changes in their research protocol to the IBC office via a letter addressed to the IBC chair prior to initiation of the change. If a protocol changes in such a manner as to require IBC approval, the change may not be initiated without IBC approval being granted. Thank you for complying with the UIC’s Policies and Procedures. Sincerely,

Randal C. Jaffe, Ph.D. Chair, Institutional Biosafety Committee RCJ/mbb Cc: IBC file

86

Appendix C

87

Appendix C (continued)

88

Appendix D Data in Figure 6, Figure 7, Figure 8, Figure 9, Figure 10, and Figure 11 were previously published (49) in Endocrinology by ENDOCRINE SOCIETY and reproduced with permission following their self-archiving policy. I collected material, conducted experiments, performed analyses, and edited the manuscript. Dr. Sarah Baumgarten collected material, conducted experiments and analyses, and edited the manuscript. Dr. CheMyong Ko provided materials for experiments. Dr. Carlos Stocco oversaw the project and wrote and edited the manuscript. Data in Figure 12, Figure 13, Figure 14, Figure 15, Figure 16, Figure 17, Figure 18, Figure 19, and Figure 20 were previously published (96) in Endocrinology by ENDOCRINE SOCIETY and reproduced with permission following their self-archiving policy. I collected the material, conducted the experiments, performed the analyses, and edited the manuscript. Dr. Nicola Winston provided material for experiments and helped edit the manuscript. Dr. Osamu Hatano and Dr. Hiroshi Takemori provided material and conducted experiments. Dr. Elie Hobeika, Dr. Jennifer Hirshfeld-Cytron, and Dr. Juergen Liebermann provided material for experiments. Dr. Carlos Stocco oversaw the project and wrote and edited the manuscript.

89

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X. VITA Marah Armouti Ph.D. Candidate Email: marmou3@uic.edu Department of Physiology and Biophysics University of Illinois at Chicago, College of Medicine Education 2015 - present Graduate Education in Medical Sciences Program, Ph.D. Student, Department of Physiology and Biophysics, University of Illinois at Chicago College of Medicine 2008 - 2012 B.S., Biology, Benedictine University, magna cum laude, Scholars Program Teaching Experience 2017 - 2019 Instructor, Endocrinology Lectures, Physiology Course for the Summer

Pre-matriculation Program (SPP) at University of Illinois at Chicago College of Medicine Urban Health Program

2018 Teaching Assistant, GEMS 500 Physiology, University of Illinois at Chicago

College of Medicine 2009 Teaching Assistant, Calculus Lab, Benedictine University College of

Science 2009 Teaching Assistant, Chemistry Lab, Benedictine University College of

Science Leadership Experience 2019 Trainee Organizing Committee, Illinois Symposium on Reproductive

Science (ISRS) 2017 – 2019 Student Representative, Graduate Student Council, University of Illinois at

Chicago 2018 Outreach Committee, Department of Physiology and Biophysics, University

of Illinois at Chicago

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Research Experience 2015 – present Ph.D. Thesis Research University of Illinois at Chicago, Carlos Stocco, Ph.D. Salt Inducible Kinases Negatively Regulate Follicle Stimulating Hormone

Actions in Ovarian Granulosa Cells 2012 – 2013 Research Assistant Biomedical Acoustics Research Company at Rush University Medical

Center Awards 2019 Honorable Mention in the Graduate, MD/Ph.D., and combined degree

students category, College of Medicine Research Forum, University of Illinois at Chicago

2018 Kate Barany Award, Department of Physiology and Biophysics, University

of Illinois at Chicago 2018 CCTS Pre-Doctoral Education for Clinical and Translational Scientists

(PECTS) Fellowship, Center for Clinical and Translational Science, University of Illinois at Chicago

Publications Armouti M, Winston N, Hatano O, Hobeika E, Hirshfeld-Cytron J, Liebermann J, Takemori H, Stocco C. Salt-inducible Kinases Are Critical Determinants of Female Fertility. Endocrinology. 2020;161(7). Hobeika E, Armouti M, Fierro MA, Winston N, Scoccia H, Zamah AM, Stocco C. Regulation of Insulin-Like Growth Factor 2 by Oocyte-Secreted Factors in Primary Human Granulosa Cells. J Clin Endocrinol Metab. 2020;105(1). Hobeika E, Armouti M, Kala H, Stocco C. Ovarian Hormones. In: Litwack G, ed. Hormonal Signaling in Biology and Medicine: Comprehensive Modern Endocrinology: Elsevier Inc.; 2019. Hobeika E, Armouti M, Kala H, Fierro MA, Winston NJ, Scoccia B, Zamah AM, Stocco C. Oocyte-Secreted Factors Synergize With FSH to Promote Aromatase Expression in Primary Human Cumulus Cells. J Clin Endocrinol Metab. 2019;104(5):1667-1676. Stocco C, Baumgarten SC, Armouti M, Fierro MA, Winston NJ, Scoccia B, Zamah AM. Genome-wide interactions between FSH and insulin-like growth factors in the regulation of human granulosa cell differentiation. Hum Reprod. 2017;32(4):905-914. Baumgarten SC, Armouti M, Ko C, Stocco C. IGF1R Expression in Ovarian Granulosa Cells Is Essential for Steroidogenesis, Follicle Survival, and Fertility in Female Mice. Endocrinology. 2017;158(7):2309-2318.

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Convissar S, Armouti M, Fierro MA, Winston NJ, Scoccia H, Zamah AM, Stocco C. Regulation of AMH by oocyte-specific growth factors in human primary cumulus cells. Reproduction. 2017;154(6):745-753. Poster Presentations Armouti M, Fierro M, Winston NJ, Scoccia H, Zamah AM, Stocco C. Salt Inducible Kinase is a Negative Regulator of Granulosa Cell Differentiation in Humans and Rodents. 2017 GEMS Symposium, University of Illinois at Chicago. Chicago, IL Armouti M, Hobeika E, Winston NJ, Stocco C. Salt Inducible Kinase is a Negtaive Regulator of Granulosa Cell Differentiation in Humans and Rodents. Endocrine Society ENDO 2018. Chicago, IL Armouti M, Kala H, Hobeika E, Winston NJ, Hirshfeld-Cytron J, Stocco C. Salt Inducible Kinase is a Negative Regulator of Granulosa Cell Differentiation in Humans and Rodents. 2018 UIC Student Reseach Forum, University of Illinois at Chicago. Chicago, IL Armouti M, Hirshfeld-Cytron J, Alvarez J, Winston NJ, Stocco C. The Inhibition of Salt Inducible Kinase Potentiates Follicle Stimulating Hormone Stimulation of Granulosa Cell Differentiation. 2018 Reproductive Science and Medicine Summit, Northwestern University. Chicago, IL. Armouti M, Hirshfeld-Cytron J, Alvarez J, Winston NJ, Stocco C. Salt Inducible Kinase is a Negative Regulator of FSH in Granulosa Cells. 2018 GEMS Symposium, University of Illinois at Chicago. Chicago, IL Armouti M, Hobeika E, Hirshfeld-Cytron J, Alvarez J, Winston NJ, Stocco C. Salt Inducible Kinase is a Negative Regulator of FSH in Granulosa Cells. 2018 College of Medicine Research Forum, University of Illinois at Chicago. Chicago, IL Armouti M, Hobeika E, Hirshfeld-Cytron J, Alvarez J, Winston NJ, Stocco C. Salt Inducible Kinase is a Negative Regulator of FSH in Granulosa Cells. 2019 Midwest Reproductive Symposium International, The Drake Hotel. Chicago, IL. Armouti M, Winston NJ, Hirshfeld-Cytron J, Alvarez J, Stocco C. Salt Inducible Kinases (SIKs) Are Negative Regulators of Follicle Stimulating Hormone (FSH) in Ovarian Granulosa Cells. 2019 GEMS Symposium, University of Illinois at Chicago. Chicago, IL. Armouti M, Winston NJ, Hobeika E, Hirshfeld-Cytron J, Liebermann J, Stocco C. Salt Inducible Kinases (SIKs) Are Negative Regulators of Follicle Stimulating Hormone (FSH) in Ovarian Granulosa Cells. 2019 College of Medicine Research Forum, University of Illinois at Chicago. Chicago, IL. Honorable Mention Award. Oral Presentations The Interactions of FSH and IGF Signaling Pathways in Ovarian Granulosa Cells. 2017 Departmental Seminar. Department of Physiology and Biophysics, University of Illinois at Chicago. Chicago, IL.

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Salt Inducible Kinase is Negative Regulator of Granulosa Cell Differentation in Humans and Rodents. 2018 Natural Sciences Research Program Guest Speaker, Benedictine University. Lisle, IL. Salt Inducible Kinase is a Negative Regulator of FSH in Ovarian Granulosa Cells. 2018 Departmental Seminar. Department of Physiology and Biophysics, University of Illinois at Chicago. Chicago, IL. Salt Inducible Kinase is a Negative Regulator of FSH in Ovarian Granulosa Cells. Endocrine Society ENDO 2019. New Orleans, LA. Salt Inducible Kinases are Negative Regulators of FSH in Ovarian Granulosa Cells. 2019 Illinois Symposium on Reproductive Science, Northwestern University. Chicago, IL. Salt Inducible Kinases Regulate Ovarian Granulosa Cell Function and Female Fertility. 2020 Midthesis Departmental Seminar. Department of Physiology and Biophysics, University of Illinois at Chicago. Chicago, IL.