Embryonic poly(A)-binding protein (EPAB) is required for granulosa cell EGF signaling and cumulus expansion in female mice

Embryonic poly(A)-binding protein (EPAB) is requiredfor granulosa cell EGF signaling and cumulusexpansion in female mice

Cai-Rong Yang, Ph.D, Katie M. Lowther, Ph.D, Maria D. Lalioti, Ph.D,Emre Seli, M.D

Department of Obstetrics, Gynecology and Reproductive Sciences, Yale School of Medicine, 310 CedarSt, New Haven, CT 06510.

Embryonic poly (A)–binding protein (EPAB) is the predominant poly(A)-binding protein in Xeno-pus, mouse, and human oocytes and early embryos prior to zygotic genome activation (ZGA). EPABis required for translational activation of maternally-stored mRNAs in the oocyte and Epab-/- femalemice are infertile due to impaired oocyte maturation, cumulus expansion, and ovulation. The aimof this study was to characterize the mechanism of follicular somatic cell dysfunction in Epab-/- mice.Using a co-culture system of oocytectomized cumulus oophorus complexes (OOXs) with denudedoocytes, we found that when WT OOXs were co-cultured withEpab-/- oocytes, or when Epab-/- OOXswere co-cultured with WT oocytes, cumulus expansion failed to occur in response to epidermalgrowth factor (EGF). This finding suggests that oocytes and cumulus cells (CCs) from Epab-/- micefail to send and receive the necessary signals required for cumulus expansion. The abnormalitiesin Epab-/- CCs are not due to lower expression of the oocyte-derived factors growth differentiationfactor 9 (GDF9) or bone morphogenic protein 15 (BMP15) since Epab-/- oocytes express theseproteins at comparable levels to WT. Epab-/- granulosa cells (GCs) exhibit decreased levels ofp-MEK1/2, p-ERK1/2, and p-p90RSK in response to LH and EGF treatment, as well as decreasedphosphorylation of the EGF receptor (p-EGFR). In conclusion, EPAB, which is oocyte-specific, isrequired for the ability of CCs and GCs to become responsive to LH and EGF signaling. These resultsemphasize the importance of oocyte-somatic communication for GC and CC function.

In the mammalian ovary, oocytes reside within folliclesand are arrested at prophase of the first meiotic division

(1). The growth of immature early antral follicles to thepreovulatory stage is promoted by the pituitary gonado-tropin follicle stimulating hormone (FSH) (2). At the pre-ovulatory stage, a surge of luteinizing hormone (LH) trig-gers the resumption of meiosis, cumulus expansion, andovulation (3). During this process, cumulus cells (CCs)and granulosa cells (GCs) are reprogrammed to expressspecific genes required for their terminal differentiation;and CCs produce hyaluronic acid, which expands thespace between the cells, embedding them in a mucinousmatrix (4, 5). These events are coordinated so that a de-velopmentally competent egg is ovulated into the oviductto await fertilization (6).

Bidirectional communication between the oocyte andsomatic compartment is essential for normal folliculogen-esis. CCs and GCs provide nutrients and regulatory signalsto the oocyte required for metabolism, oocyte growth, andmeiotic and developmental competence (7, 8). In turn, theoocyte controls GC differentiation and function (9, 10).One important way in which the oocyte contributes to thesomatic cells is by secreting soluble growth factors, such asGDF9 and BMP15. GDF9 and BMP15 belong to the trans-forming growth factor-beta (TGF-�) superfamily (11, 12),are expressed exclusively in the oocyte (13, 14), and arecritical local regulators of ovarian function (15–17). Theseoocyte-secreted factors lead to activation of SMAD2/3and MAPK signaling in CCs, which in turn regulate CCgene expression and key cumulus functions (18). GDF9

ISSN Print 0013-7227 ISSN Online 1945-7170Printed in USACopyright © 2015 by the Endocrine SocietyReceived February 9, 2015. Accepted October 16, 2015.

Abbreviations:

O R I G I N A L R E S E A R C H

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and BMP15 also enhance hyaluronic acid synthesis inCCs, an essential step in ovulation (19, 20). Therefore, theappropriate cooperation between oocyte-derived growthfactors and somatic cells is required for folliculogenesis,cumulus expansion, and ovulation.

In addition, accumulation of EGF-like growth factorsand transactivation of EGF receptor (EGFR) signaling arecritical events for LH-induced cumulus expansion andovulation (21–23). In support of this, the disruption of theEGF signaling network in mice leads to impairment ofLH-induced ovulation in vivo (24). EGF-like growth fac-tors amphiregulin (AREG), epiregulin (EREG), and beta-cellulin (BTC) are rapidly induced by LH, or its analoghuman chorionic gonadotropin (hCG) in mural GCs, andfunction in an autocrine and paracrine manner to transmitLH signals via EGF receptors in GCs and CCs (22, 25–27).Cumulus expansion and oocyte meiotic resumption ofCOCs can be induced upon LH, EGF or AREG treatmentin vitro, which act in a manner similar to that observed byLH/hCG in ovarian follicles in vivo (28–30). The mostprominent downstream target of EGFR signaling is theMAPK cascade, which is activated by EGF-like factors,and subsequently elicits distinct biological effects, such ascumulus expansion and ovulation (23). Collectively, thesefindings support the critical role for the EGF network inintegrating the function of GCs/CCs/oocytes within thepreovulatory follicle.

A complex network of translational activation and re-pression of dormant maternal mRNAs is required for nu-clear and cytoplasmic maturation of the oocyte (31–33).Since fully-grown oocytes are transcriptionally silent,oocyte maturation and early developmental processes relyexclusively on maternal mRNAs that are synthesized andstored in advance. Translational activation of stored ma-ternal mRNAs occurs primarily by cytoplasmic polyade-nylation (31, 34), which drives the oocyte’s reentry intomeiosis and controls the gene expression during oocytematuration, fertilization and early embryo development,until zygotic gene activation (ZGA) (34–37). This processrequires CPEB1 (cytoplasmic polyadenylation elementbinding protein 1), which becomes phosphorylated uponstimulation of oocyte maturation, and leads to the elon-gation of the poly(A) tail of bound transcript by activatinga poly(A) polymerase complex (38). It has been demon-strated that several dormant mRNAs in the oocyte haveshort poly(A) tails, which elongate in response to an ex-ogenous cue (eg, hormonal stimulation or fertilization),and become translated (39–41). The process of transla-tional activation during oocyte maturation is complex(42) and additional pathways independent of cytoplasmicpolyadenylation, such as those involving DAZL (deletedin azoospermia-like) (43–45), have also been identified.

Embryonic poly (A)-binding protein (EPAB) is the pre-dominant poly(A) binding protein in Xenopus, mouse,and human germ cells and early embryos until zygoticgenome activation (ZGA), when it is replaced by the so-matic cytoplasmic poly(A) binding protein, PABPC1 (42,46, 47). EPAB is associated with both polyadenylation-dependent (CPEB1-SYMPK-CPSF) (48) and -independent(DAZL-Pumilio) (44) complexes that mediate translationactivation in the oocyte. In Xenopus oocytes, EPAB pre-vents deadenylation of mRNAs (49), promotes cytoplas-mic polyadenylation (48), enhances translation initiation(50), and is required for maturation (51). Epab-deficientfemale mice are infertile due to impaired translational ac-tivation of maternal mRNAs and oocyte maturation (52).In addition, Epab-deficient mice exhibit defective cumulusexpansion and ovulation in vivo, suggesting that the LH-mediated preovulatory changes fail to occur in the somaticcells of the follicle (52).

The aim of the current study was to characterize the roleof EPAB in regulating granulosa/cumulus cell function.We hypothesized that EPAB is involved in promoting anoocyte-specific signal that is communicated to the sur-rounding somatic cells and is required for cumulus expan-sion. We used an established coculture system of denudedoocytes and oocytectomized cumulus oophorus com-plexes (OOX) to assess the interactions between theoocyte and the somatic cells in Epab-/- mice. We found thatcumulus expansion failed to occur both in the WT-OOX/KO-oocyte and the KO-OOX/WT-oocyte coculture sys-tem, suggesting that not only do Epab-/- oocytes fail tosend necessary signals that promote cumulus expansion,but that the Epab-/- CCs fail to become responsive tooocyte-derived signals. EPAB acts through a pathway thatis independent of the oocyte-derived GDF9/BMP15 sys-tem, as the protein expression of these factors are similarin WT and Epab-/- oocytes. Importantly, the expression ofdownstream targets are significantly reduced in responseto LH, EGF, and AREG in the GCs of Epab-/- mice, whichcould in part be due to lower phosphorylation levels of theEGF receptor. Overall, these findings demonstrate thatoocyte-specific EPAB is required for the competency of thesomatic compartment.

Materials and Methods

MiceMice were bred and maintained according to the Yale Uni-

versity animal research requirements. All animal protocols wereapproved by the Institutional Animal Care and Use Committee(protocol 2011–11 027) prior to the initiation of the studies. TheEpab-/- knockout mice were generated as previously described(52).

2 EPAB is required for EGF signaling Endocrinology


Isolation and culture of cumulus oophoruscomplexes (COCs), oocytectomized COCs (OOXs),denuded oocytes (DOs), and evaluation of in vitrocumulus expansion

Denuded oocytes and COCs were collected from the ovariesof 12 week old WT (Epab�/�) and Epab-/- mice 44–48 hoursafter intra-peritoneal (IP) injection of 5 IU pregnant mare serumgonadotropin (PMSG; Sigma-Aldrich, St. Louis, MO) Ovarieswere punctured with a 261⁄2-gauge needle and COCs were re-leased from the follicles in M2 medium (Sigma-Aldrich). De-nuded oocytes were isolated from COCs by repeat pipetting witha pipette. Oocytectomized complexes (OOXs) were producedusing microsurgical technique in order to remove oocytes fromthe COCs, as previously described (53, 54).

For coculture experiments, 10 WT OOXs or Epab-/- OOXswere cultured with 10 WT or Epab-/- oocytes in a 20 �l drop ofbasic medium [�-MEM supplemented with 75 �g/ml penicillinG, 50 �g/ml streptomycin sulfate, 0.23 mM pyruvate, and 3mg/ml BSA (Sigma-Aldrich)] with or without 10 ng/ml EGF(SRP3196, Sigma-Aldrich), covered by liquid paraffin oil(Sigma). WT COCs were used as a positive control. Complexeswere incubated for up to 16 hours in a humidified atmosphere at37°C with 5% CO2, and evaluated for cumulus expansion.

Cumulus expansion was assessed using a previously de-scribed scoring (scores of 0–4) system (55). Score of 0–1 indi-cates no expansion or minimum expansion; score of 2 indicatesthat cells in the outer two layers began to expand; score of 3indicates expansion of all layers of the cumulus except coronaradiate cells; and score of 4 indicates expansion of the wholecumulus including corona radiate cells. After scoring, CCs wereremoved from COCs and OOXs and the expression of cumulusexpansion-related transcripts was determined by real-time PCR.

Isolation and culture of GCsTo test the response of GCs to LH, EGF, and AREG stimu-

lation in vitro, GCs were isolated from ovaries of 12 week oldWT and Epab-/- mice 48 hours after IP injection of 5 IU PMSG(Sigma-Aldrich). Ovaries were punctured using a 261⁄2-gaugeneedle allowing the release of COCs and GCs from follicles. TheCOCs were collected and CCs were isolated via mechanical ma-nipulation in HEPES-buffered medium containing 1 mg/ml hy-aluronidase (Sigma-Aldrich). The remaining GCs were filteredthrough a 0.4-�m strainer and centrifuged for 5 minutes at1500�g. For in vitro stimulation experiments, GCs were resus-pended in Dulbecco’s modified Eagle’s medium (DMEM) me-dium (Invitrogen) supplemented with 5% fetal bovine serum(FBS, Invitrogen) and 1% antimycotic - antibiotics (Gibco). Theywere cultured in a humidified atmosphere at 37°C with 5% CO2

to allow the cells to adhere to the bottom of the dishes. At 70%confluence, they were serum-starved for 12 hours stimulatedwith either 1 �g/ml LH (39341–83–8, EMD Chemicals, Inc, SanDiego, CA), 10 ng/ml EGF (SRP3196, Sigma-Aldrich Co., St.Louis, MO), or 100 ng/ml AREG (989-AR-100/CF, R&D Sys-tem, Inc, Minneapolis, MN).

Western BlotGCs were washed with PBS and incubated in lysis buffer [20

mM Tris-HCL (pH 8.0), 5 mM MgCl2, 10 mM EGTA (pH 8.0),1% Triton X-100, 1 mM Na3VO4, 50 mM NaF, complete pro-tease inhibitor cocktail (Roche Diagnostics) per 10 mL of buffer]

for 15 minutes on ice with shaking. Lysates were cleared bycentrifugation at 12 000�g for 30 minutes at 4°C. Samples wereprepared with 2� Sodium dodecyl sulfate (SDS) sample buffer,separated by 10% SDS-PAGE (Bio-Rad Laboratories), andtransferred to polyvinylidene fluoride membrane (PVDF, Bio-Rad Laboratories) at 85 V for 2 hours. The membrane wasblocked with 5% BSA in TBS-T for 1 hour at room temperatureand incubated with primary antibody diluted in 5% BSA inTBS-T at 4°C overnight. Then membrane was washed threetimes in TBS-T and incubated with horseradish peroxidase-con-jugated secondary antibody (1:5000; Chemicon) diluted inTBS-T for 1 hour at room temperature. After washing three timesin TBS-T, the protein signals were detected using SuperSignalECL (Pierce) and exposed to film (Kodak). Primary antibodiesare listed in Supplemental Table 1. Image J (National Institutesof Health) software (56) was used to measure the intensity ofprotein bands on the film. Levels of GDF9 and BMP15 werenormalized to GAPDH. Phosphorylated proteins were normal-ized to total protein and �-Actin served as a loading control.

Real-Time PCRTotal RNA from GCs, OOXs or CCs of COCs was extracted

using the RNAqueous-Micro kit (Ambion) and reverse tran-scribed using RETROscript (Ambion). The quantitative RT-PCR was carried out on an iCycler (Bio-Rad Laboratories) andassayed in triplicate. Each 10 �l reaction contained 5 �l of SYBRGreen supermix (Bio-Rad Laboratories), 3 �l of H2O, 0.5 �l ofeach primer, and 1 �l of cDNA. The 2-��Ct (cycle threshold)method was used to calculate relative expression levels after nor-malization to �-Actin or Rpl19 levels. Results are reported as afold change in gene expression. The linear dynamic range andPCR efficiency of each primer set was determined using standardcurve and one single melting curve analysis was used to excludenonspecific amplifications. The primers used for Real-Time PCRare given in Supplemental Table 2.

Statistical analysisEach result represents at least three independent experiments.

Values were analyzed either by Student’s t test, One-wayANOVA, or Two-way ANOVA, as described in each figure leg-end. All statistical analyses were performed using Graph PadPrism software and significance was assessed at P � .05.

Results

Epab-/- oocytes fail to promote expansion of WTCCs and Epab-/- CCs fail to expand in response tostimuli from WT oocytes

To determine whether the defect in cumulus expansionin Epab-/- mice is due to factors inherent to the oocyte, wecultured three different groups: 1) WT-OOX/WT-oocytegroup (WT OOXs cocultured with WT denuded fullygrown oocytes), 2) WT-OOX/KO-oocyte group (WTOOXs cocultured with Epab-/- denuded oocytes), and 3)KO-OOX/WT-oocyte group (KO OOXs cocultured withWT denuded fully grown oocytes). After 16 hours of co-culture in the absence of EGF (control), the OOXs adhered

doi: 10.1210/en.2015-1135 press.endocrine.org/journal/endo 3


to the tissue culture plate and assumed a fibroblastic ap-pearance and cumulus expansion was not observed in anyof the three groups (Figure 1A). When cocultured in thepresence of EGF, over 50% of the OOXs from the WT-OOX/WT-oocyte group maintained a spherical appear-ance and expanded into a three-dimensional, gelatinoussphere, which comprised CCs reaching to almost full ex-pansion (level 3) (55)(Figure 1A and 1B). These results aresimilar to COCs with intact oocytes cultured in EGF-con-taining media (data not shown). However, the degree ofOOX expansion in the WT-OOX/KO-oocyte group andtheKO-OOX/WT-oocytegroupwas significantly reduced(Figure 1A and 1B). Only 3.33% of WT-OOXs culturedwith KO oocytes reached a score of 3, while 56.67% and40% of WT-OOXs remained at a score of 0/1 and a scoreof 2, respectively. Furthermore, 78.33% and 21.67% ofKO-OOXs cultured with WT oocytes displayed a score of0/1 and a score of 2, respectively. level 3 expansion was notobserved in the KO-OOX/WT-oocyte group.

We also tested whether the expression of genes knownto regulate cumulus expansion is altered in Epab-/- mice.After 16 hours of the coculture, OOXs were collected forqRT-PCR to assess the levels of Ptgs2, Btc and Tnfaip6.Compared to control group (no EGF treatment), CC ex-pression of transcripts encoding PTGS2 (Figure 2A), BTC(Figure 2B), and TNFAIP6 (Figure 2C), showed signifi-cant increase in the WT OOX/WT-oocyte group in re-

sponse to EGF treatment, but there was no change in theWT-OOX/KO-oocyte or KO-OOX/WT-oocyte groups(Figure 2A-C). These observations demonstrate that theoocytes of Epab-/- mice fail to send the necessary signals toWT CCs that promote cumulus expansion. In addition,CCs from Epab-/- mice are unable to respond to oocyte-derived signals from WT oocytes.

To confirm that the CCs from Epab-/- ovaries are prop-erly differentiated, mRNA levels for CC markers (Amh,Slc38a3, and Ar) (57) was determined by qRT-PCR (Sup-plemental Figure 1). The expression of these CC tran-scripts in Epab-/- CCs was comparable to WT.

EPAB-deficiency has no effect on the expression ofGDF9 and BMP15

Since Epab-/- oocytes fail to communicate with the sur-rounding CCs, it is possible that the expression of oocyte-derived factors is altered in Epab-/- oocytes. GDF9 andBMP15 are two key mediators of oocyte-cumulus cell in-teractions (58, 59), and decreased expression could ex-plain the abnormalities observed in Epab-/- ovaries. Toexamine this, 300GVoocyteswere collected fromWTandEpab-/- mice at 12 weeks of age. GCs were collected as anegative control. We found that the expression of GDF9and BMP15 were not altered in Epab-/- oocytes (Figure 3Aand B). As expected, the GCs did not display any immu-noreactive bands. Since follicle growth and cumulus ex-

pansion requires timely expressionof oocyte factors GDF9 and BMP15,it is possible that EPAB’s effect isdownstream of these factors, or thatEPAB is required for the activation ofan independent signaling pathwaythat regulates folliculogenesis.

GCs of Epab-/- mice exhibit animpaired response to LH, EGF,and AREG stimulation

Next, we tested whether failed cu-mulus expansion could also be due toa defect in downstream signalingevents in GCs in response to LH. TheLH surge causes dramatic functionaland structural changes in COCs thatlead to mucification with a hyaluro-nan-rich matrix. During this process,LH induces the rapid expression ofthe EGF-like factors amphiregulin(AREG), epiregulin (EREG) and be-tacellulin (BTC) that act on the EGFreceptor (EGFR) expressed by GCsand CCs (6–8). MEK1/2, ERK1/2,

Figure 1. Epab-/- oocytes fail to promote cumulus expansion and Epab-/- CCs fail to undergoexpansion in the presence of WT oocytes. Oocytes and OOXs were obtained from COCs of 12-week-old WT or Epab-/- mice and cultured in combinations, as indicated, for 16 hours in thepresence (�) or absence (-) of 10 ng/mL EGF. (A) Representative images of OOX/oocytecoculture combinations in the presence or absence of 10 ng/ml EGF. WT OOXs cultured with WToocytes showed cumulus expansion in the presence of EGF. Conversely, WT OOXs cultured withKO oocytes or KO OOXs cultured with WT oocytes did not undergo expansion despite EGFtreatment. Scale bar � 10 �m. (B) Cumulus expansion of OOXs was scored after 16 hours ofcoculture with WT or Epab-/- denuded oocyte in the presence of EGF The grade of cumulusexpansion was assessed as previously described (55). A score of 0/1 indicates no detectableresponse (0) or the minimum observable response (1). A score of 4 indicates the maximumdegree of expansion in which the cumulus oophorus and corona radiate have undergoneexpansion. A score of 2 or 3 indicate intermediate levels of expansion between 0/1 and 4. Dataare presented as mean � SEM from three independent experiments. Bars with different lettersare significantly different (P � .05). Significance was determined by one-way ANOVA.



and p90RSK are downstream targets of the EGF networkand regulate the expression of genes involved in hyaluro-nan synthesis and accumulation (60).

To determine whether the LH signaling pathway is af-fected in GCs of Epab-/- mice, GCs were collected from theovaries of PMSG-primed WT and Epab-/- mice at 12 weeksof age, cultured to preconfluence, serum-starved for 12hours, and treated with LH (1 �g/ml) for 5 minutes and 10minutes. The expression of downstream mediators wasthen examined by Western blot analysis. An immunopo-sitive signal for total and phosphorylated MEK1/2,ERK1/2, and p90RSK, was observed in GCs from WT andEpab-/- mice (Figure 4A). In WT GCs, LH strongly acti-vated ERK signaling such that phosphorylation ofMEK1/2 and p90RSK (Figure 4B, D) was significantlyincreased following 10 minutes of LH treatment, andphosphorylation of ERK1/2 (Figure 4C) was significantlyincreased following 5 and 10 minutes of LH treatment.However, the phosphorylation of MEK1/2, ERK1/2, andp90RSK did not increase significantly in Epab-/- GCs inresponse to LH. While there was a small increase inpMEK1/2 and pERK1/2 in Epab-/- GCs, it was not signif-icantly different from the control. Furthermore, whencompared to WT, the levels of pMEK1/2, pERK1/2, andp-p90RSK were significantly reduced in the GCs of Epab-/-

mice after 10 minutes of LH treatment (Figure 4B-D).Since the ERK cascade is an important downstream

mediator of EGF pathways, we tested whether MEK/ERK/p90RSK activation in response to EGF stimulation is alsoaffected by EPAB deficiency. GCs were collected as statedabove and stimulated with 10 ng/ml EGF for 5 minutesand 10 minutes (Figure 5A). EGF-stimulated phosphory-lation of MEK1/2 (Figure 5B), ERK1/2 (Figure 5C) andp90RSK (Fig. D) was significantly increased in WT GCsbut not in Epab-/- GCs following 10 minutes of treatment.

These results demonstrate that the GCs of Epab-/- mice areincapable of activating the ERK cascade in response to LHand EGF.

The response to Amphiregulin (AREG) was also eval-uated since it is a member of the epidermal growth factor(EGF)-like growth factor family and a likely mediator ofLH action in vitro (22, 26). Cultured GCs were treatedwith AREG (100ng/ml) for 5 minutes and 10 minutes.Western blot analyses showed that AREG strongly phos-phorylated MEK1/2, ERK1/2, and p90RSK in GCs of WTmice (Supplemental Figure 2). However, phosphorylationof MEK1/2, ERK1/2, and p90RSK were significantly re-duced in GCs of Epab-/- compared to WT within 5 or 10minutes of AREG stimulation (Supplemental Figure 2).

If the differentiation or maturation of GCs from Epab-/-

mice was affected, it may explain the impaired signalingresponse to LH and EGF. The expression of GC markers(Lhcgr, Cyp11a1, and Cd34) was evaluated by real-timePCR and was similar between WT and Epab-/- GCs. How-ever, Cyp19a1 was significantly lower in Epab-/- GCs(Supplemental Figure 1). Although Epab-/- GCs expressmarkers of GC differentiation, lower expression ofCyp19a1 suggests that some aspect of their maturationhas been affected.

GCs of Epab-/- mice exhibit impaired EGFRphosphorylation in response to EGF

LH induced oocyte maturation and ovulation requirestransactivation of EGFR in the somatic cells (61), and it ispossible that impaired MEK/ERK/p90RSK activation ob-served in Epab-/- GCs is due to abnormalities in EGF re-ceptor activation. We examined the expression of totalEGFR and p-EGFR in the GCs of PMSG-primed WT andEpab-/- mice after 5 minutes and 10 minutes EGF stimu-lation (10 ng/ml). As expected, EGFR phosphorylation

Table 1. Antibody

Antibody Isotype Dilution Catalog NO. Company

(p)-ERK1/2 Rabbit monoclonal 1.430 555 556 # 4370p Cell signaling Technologyp-MEK 1/2 Rabbit monoclonal 0.736 111 111 # 9154p Cell signaling Technologyp-p90RSK Rabbit monoclonal 0.736 111 111 # 9335p Cell signaling TechnologyERK1/2 Rabbit monoclonal 0.736 111 111 # 4695 Cell signaling TechnologyMEK1/2 Rabbit monoclonal 0.736 111 111 # 8727 Cell signaling TechnologyT-90RSK Rabbit monoclonal 0.736 111 111 # 9355 Cell signaling Technology�-Actin Rabbit monoclonal 3.513 888 889 # 5125s Cell signaling TechnologyGAPDH Rabbit monoclonal 3.513 888 889 # 3683s Cell signaling TechnologyEGFR Rabbit monoclonal 0.736 111 111 # sc-03 Santa Cruz Companyp-EGFR Rabbit monoclonal 0.736 111 111 # 2234s Cell signaling TechnologyLHR Rabbit monoclonal 0.736 111 111 # PA5–21 271 Thermo Fisher ScientificGDF9 Goat monoclonal 0.736 111 111 # sc-12 244 Santa Cruz CompanyBMP15 Rabbit monoclonal 0.736 111 111 # sc-28 911 Santa Cruz CompanyCREB Rabbit monoclonal 0.736 111 111 # 9197 Cell Signaling Technologyp-CREB Goat monoclonal 0.736 111 111 # sc-7978 Santa Cruz Company



significantly increased in the GCs of WT mice after EGFstimulation (Figure 6). However, this increase in p-EGFRin response to EGF treatment was not observed in the GCsof Epab-/- mice (Figure 6). Therefore, impaired EGFR

phosphorylation observed in Epab-/- GCs may be a con-tributing factor leading to failure of MEK/ERK/p90RSKactivation in response to LH, EGF, and AREG.

While EGF mediates many of the responses of LH, LH-induced activation of ERK signaling can also occur in theabsence of EGF receptor activation (25, 62). Thus, LH actsthrough additional pathways downstream of cAMP to ac-tivate ERK. To address whether Epab-deficiency affectsERK activation independently of EGF signaling, we ex-amined p-CREB levels in WT and Epab-/- GCs followingLH treatment. We did not observe CREB activation in WTor Epab-/- GCs after 5 or 10 minutes of LH treatment(Figure 7). Since p-CREB activation appears to requirelonger LH treatment, it is likely that impaired ERK acti-vation observed in Epab-/- GCs in response to 5 and 10minutes of LH treatment is due to impaired EGFRphosphorylation.

Discussion

In our previous study, we demonstrated that EPAB-defi-cient mice are infertile due to impaired oocyte maturation,cumulus expansion and ovulation. In this study, we in-vestigated the cause of defective cumulus expansion andovulation in EPAB-deficient mice. We first demonstratedthat Epab-/- oocytes fail to promote cumulus expansion inWT CCs. However, this is not due to decreased expressionof oocyte-derived factors GDF9 and BMP15. Addition-ally, we found that the CCs from Epab-/- mice fail to un-dergo expansion even in the presence of WT oocytes. Fi-nally, we demonstrated that MEK/ERK/p90RSKactivation in response to LH and EGF signaling is im-paired in GCs as a result of Epab deficiency.

Since EPAB is oocyte-specific, we hypothesized that thedysfunction in GCs from Epab-/- mice is due to factorsinherent to the oocyte. We utilized a coculture system ofOOXs with denuded oocytes under EGF stimulation. Ourresults show that WT-OOXs expand in WT-oocyte-con-ditioned media in response to EGF treatment, consistentwith previous reports (53), and is similar to that observedwith intact WT COC complexes. However, when WT-OOX/KO-oocyte group were treated with EGF, cumulusexpansion did not occur and the expression of mediatorsof cumulus expansion (Ptgs2, Btc, and Tnfaip6) did notchange. Since EPAB is required for translational activationuponstimulationofoocytematuration (34), it is likely thatthe failure of promoting cumulus expansion of Epab-/-

oocytes is due to impaired translation of maternaly-de-rived mRNAs in the oocyte. Consequently, Epab-/- oocytesfail to express optimal levels of specific proteins that arerequired by the somatic cells in order to achieve cumulus

Figure 2. The expression of transcripts encoding proteins thatmediate cumulus expansion do not increase in response to EGF in CCsfrom WT OOX/KO oocyte and KO OOX/WT. Oocytes and OOX’s wereobtained from COCs of 12-week-old WT or Epab-/- mice 44–48 hoursafter PMSG injection. The expression of Ptgs2 (A), Btc (B) and Tnfaip6(C) in CCs after 16 hours of coculture in the presence or absence of10 ng/mL EGF was assessed using qRT-PCR. Expression of target geneswas normalized to �-actin levels and results are shown as the fold-change in gene expression between EGF stimulation (�) and no EGFstimulation (-). Data are presented as mean � SEM from threeindependent experiments. (*) indicates significance between transcriptlevels with or without EGF treatment (P � .05). Significance wasdetermined by t test.



expansion. Additionally, the oocyte’s production of cu-mulus enabling factors is developmentally regulated suchthat oocytes incapable of spontaneous germinal vesiclebreakdown (GVBD) are unable to promote cumulus ex-pansion (55). Epab-/- oocytes fail to undergo GVBD andmature to metaphase II, and therefore may not be at theright stage to produce the appropriate factors required forcumulus expansion.

Evidence emerging from earlier studies demonstratesthat oocyte specific factors including GDF9 and BMP15are indispensable for regulating somatic cell functions.

Gdf9-null mice are infertile due to an arrest of follicledevelopment at the primary follicle stage (63). GDF9 pro-motes GC proliferation and differentiation leading to theformation of the two-layer preantral follicle and subse-quent recruitment of the thecal layer (58). GDF9 also pro-motes cumulus expansion by stimulating Ptgs2 and Has2mRNA expression and suppressing urokinase plasmino-gen activator (uPA) (17, 64). Thus, in addition to playinga key role during early folliculogenesis, GDF9 functions asan oocyte-secreted paracrine factor that regulates key en-zymes involved in cumulus expansion. Unlike Gdf9-null

mice, Bmp15-null mice are fertilebut display impaired terminal cumu-lus-oocyte maturation and de-creased ovulation rates. BMP15 hasbeen reported to regulate cumulusexpansion via a mechanism requir-ing EGFR signaling (16, 65). In ad-dition, oocyte-derived BMP15and/or GDF9 suppress LH signalingin CCs by preventing Lhcgr mRNAexpression (17, 64, 66, 67), therebyleading to heterogeneity in LH recep-tor expression between cumulus andmural GCs in preovulatory follicles.In our study, WT and Epab-/- oocytes

Figure 3. The expression of GDF9 and BMP15 is not altered in the oocytes of Epab-/- mice. A,GDF9 and BMP15 expression was detected by Western blot analysis using 300 GV oocytes fromWT or Epab-/- mice. Granulosa cells (GC) from WT mice were used as a negative control. (B)Band intensities were analyzed using densitometry and normalized to GAPDH. Data arerepresented as the mean � SEM from three independent experiments. There was no significantdifference in GDF9 or BMP15 expression between WT and Epab-/- oocytes. Arrow designates WTGC bar.

Figure 4. The response to LH stimulation is impaired in GCs of Epab-/- mice. A–D, Western blot analysis was performed to compare LH-inducedphosphorylation of MEK1/2, ERK1/2 and p90RSK in GCs from WT and Epab-/- mice. GCs were collected from WT and Epab-/- mice at 12 weeks ofage 44–48 hours after 5 IU PMSG injection, cultured, serum-starved, and treated with LH (1 �g/ml) for 5 minutes and 10 minutes. RepresentativeWestern blots of total and phosphorylated proteins are shown in (A). The intensity of Western blot bands was analyzed using densitometry. Theratio of p-MEK1/2 (B), p-ERK1/2 (C), and p-p90RSK (D) to total protein were normalized to untreated WT control. Data are presented as mean �SEM from 3 separate experiments. (*) above bars indicates significant difference between groups (*P � .05, **P � .01, and ***P � .001).Significance was determined by two-way ANOVA followed by Tukey’s Multiple Comparisons Test.



did not show a difference in GDF9 or BMP15 expression,suggesting that the failure of cumulus expansion of EPAB-deficient mice is not due to the absence of these oocytefactors. Therefore, EPAB is either downstream of thesefactors, or EPAB could be required for the activation of anindependent signaling pathway that regulates folliculo-genesis. Another possibility is that EPAB-deficient oocytesproduce the necessary factors, but are unable to transportthem to the somatic compartment. Perhaps EPAB is im-portant for maintaining communication between theoocyte and somatic cells, either by regulating the expres-sion of components required for junction formation or theassembly of transzonal processes.

Interestingly, Epab-/- OOXs fail to expand in responseto EGF even when cocultured with WT oocytes. This sug-gests that impaired cumulus expansion is also due to adefect in Epab-/- CCs. If the CCs from Epab-/- mice werenot properly differentiated, they would be incapable ofsupporting cumulus expansion. This is an important con-sideration since the oocyte drives the CC lineage (30, 57,68) and also because EPAB is required in the oocyte at thepreantral stage of folliculogenesis prior to CC differenti-ation (69). The expression of cumulus markers (Amh,Slc38a3, Ar) (Supplemental Figure 1) suggests that Epab-/-

CCs are phenotypically normal; however, other tran-

scripts may be abnormally expressed. Another possibilityis that in addition to GCs, CCs from Epab-/- mice fail toactivate MEK/ERK signaling, thereby leading to impairedcumulus expansion.

Failure of cumulus expansion in Epab-deficient mice invivo could also be due to impaired signaling in GCs inresponse to LH. The LH receptor (LHR) is a G protein-coupled receptor (GPCR) expressed on theca and muralGCs. Since CCs and oocytes are insensitive to direct LHstimulation due to lack of LH receptors (70, 71), they relyon signals initiated by the GCs. Specifically, LH stimulatesmeiotic maturation, cumulus expansion, and ovulation byinducing expression of EGF-like factors in GCs (26). EGF-like factors, in turn, activate the EGFR signaling pathwayin CCs to induce COC expansion and oocyte maturation(60). In vitro, soluble EGF (an AREG and EREG analog)stimulates mRNA expression of Ptgs2, Tnfaip6 and Has2in primary GC cultures (72). These genes are necessary forsynthesis and stabilization of the extracellular matrix ofCCs and are required for cumulus expansion (73–75).EGF also acts as a potent stimulator of cumulus expansionin intact cumulus-oocyte complexes in mice (24, 76).However, EGF cannot induce cumulus expansion in theabsence of the oocyte, which suggests that oocyte secreted

Figure 5. The response to EGF stimulation is impaired in GCs of Epab-/- mice. A–D, Western blot was performed to compare EGF-inducedphosphorylation of MEK1/2, ERK1/2 and p90RSK in GCs from WT and Epab-/- mice. GCs were collected from WT and Epab-/- mice at 12 weeks ofage 44–48 hours after 5 IU PMSG injection, cultured, serum-starved, and treated with EGF (10 ng/ml) for 5 minutes and 10 minutes.Representative Western blots of total and phosphorylated proteins are shown in (A). The intensity of Western blot bands was analyzed usingdensitometry. The ratio of p-MEK1/2 (B), p-ERK1/2 (C), and p-p90RSK (D) to total protein was normalized to untreated WT control. Data arepresented as mean � SEM from 3 separate experiments. (*) above bars indicates significant difference between groups (*P � .05, **P � .01, and***P � .001). Significance was determined by two-way ANOVA followed by Tukey’s Multiple Comparisons Test.



factors in vitro are necessary for the CCs to undergo ex-pansion in response to EGF (53).

Disruption of the EGFR signaling in mice compromisescumulus expansion and ovulation, indicating that activa-tion of this pathway is essential for LH-induced ovulation(28, 62). Mice in which ERK1 and ERK2 have been dis-rupted in GCs exhibit normal follicle growth, but in re-sponse to LH, the COCs fail to expand, oocytes fail tore-enter meiosis, and follicles fail to either ovulate or lu-teinize (77). The phenotypes of mice with genetic disrup-tion of EGFR or ERK1/ERK2 in GCs show significantsimilarities to the phenotype of Epab-/- mice, which exhibitfemale infertility due to impaired cumulus expansion andovulation, in addition to an inability to achieve oocytematuration (52). Importantly, phosphorylation ofERK1/2 in Epab-/- GCs was completely abolished in re-sponse to EGF, while the effect was less prominent (al-though significant) in response to LH (Figures 4 and 5).This finding, combined with the observation that EGFRphosphorylation is suppressed in Epab-/- GCs (Figure 6),while LHR expression is unchanged (Supplemental Figure1), suggests that the main defect in Epab-/- somatic cellsinvolves impaired EGF signaling and ERK activation.

LH and EGF-like factors work together in a coordi-nated fashion to induce oocyte maturation and cumulus

expansion and both result in MAPK (MEK/ERK) activa-tion. LHR is coupled primarily to Gs protein and activa-tion of adenylyl cyclase. Thus, the primary signal emanat-ing from LHR is the accumulation of cAMP. In GCs,ERK1/2 phosphorylation in response to LH, forskolin,and 8-Br-cAMP is blocked by H89, a potent and selectiveinhibitor of PKA (78), suggesting that stimulation ofERK1/2 phosphorylation in GCs by LH is mediated bycAMP-dependent PKA activation. The cAMP/PKA path-way leads to production of EGF-like factors that will sub-sequently bind and activate the EGFR (79) and result inERK1/2 phosphorylation (25). Importantly, LH-inducedERK1/2 phosphorylation is only partially dependent onEGFR, suggesting that MEK/ERK pathway is activatedupstream (or in parallel) as well as downstream of theEGFR (25, 62). Thus, additional pathways downstream ofcAMP contribute to LH-mediated activation of MAPK. Inour study, we did not observe activation of CREB in re-sponse to 5 minutes and 10 minutes LH treatment in WTor Epab-/- GCs. Thus, rapid ERK activation in response to5–10 minutes of LH treatment appears to occur indepen-dently of the cAMP pathway and is the result of EGFRsignaling. This finding supports the conclusion that im-paired phosphorylation of MEK1/2, ERK1/2, and

Figure 6. The activation of EGFR is impaired in GCs of Epab-/- mice.(A-C) Western blot analysis was performed to compare EGF-induced

phosphorylation of EGFR in GCs from WT and Epab-/- mice. GCs werecollected from WT and Epab-/- mice at 12 weeks of age after 44–48hours of 5 IU PMSG injection, cultured, serum starved, and treatedwith EGF (10 ng/ml) for 5 minutes and 10 minutes. RepresentativeWestern blots are shown in (A). The intensity of Western blot bandscorresponding to total EGFR (B) and p-EGFR (C) were analyzed usingdensitometry. The ratio of p-EGFR to total EGFR was normalized tountreated WT. Data are presented as mean � SEM from threeseparate experiments. (*) above bars indicates significant differencebetween groups (*P � .05; ***P � .001; ***P � .0001). Significancewas determined by two-way ANOVA followed by Tukey’s MultipleComparisons Test.

Figure 7. CREB activation does not increase in response to short-termLH treatment in GCs from WT or Epab-/- mice Western bot analysiswas performed to compare LH-induced phosphorylation of CREB inGCs from WT and Epab-/- mice. GCs were collected from WT andEpab-/- mice at 12 weeks of age 44–48 hours after 5 IU PMSGinjection, cultured, serum-starved, and treated with LH (1 �g/ml) for 5minutes and 10 minutes. Representative Western blots of total andphosphorylated CREB are shown in (A). The intensity of Western blotbands was analyzed using densitometry (B). The ratio of p-CREB tototal CREB was normalized to untreated WT control. Data arepresented as mean � SEM from 3 separate experiments.Phosphorylation of CREB was not significantly different in WT orEpab-/- GCs after 5 or 10 minutes of LH treatment.



p90RSK observed in Epab-/- GCs is most likely due toinsufficient EGFR activation.

The inability of Epab-/- GCs to properly respond to LHand EGF could be due to abnormal GC maturation duringfolliculogenesis. While the expression of the LH receptor,Cyp11a1 and Cd34 were similar to WT, the expression ofCyp19a1 was significantly lower in Epab-/- GCs. Thisfinding suggests that some aspect of GC maturation ordifferentiation is affected by EPAB deficiency. Cyp19a1expression is regulated by FSH (80), which is necessary toinduce growth and maturation of ovarian follicles to thepreovulatory stage. FSH induces a complex pattern ofgene expression in GCs that is mediated by several differ-ent signaling cascades including ERK, MAPK, and PI3K(81). FSH also leads to the production of EGF-like growthfactors in CCs and results in meiotic resumption (82).Therefore, it is possible that FSH signaling is also impairedin GCs from Epab-/- mice and will be examined in futurestudies.

Overall, EPAB-deficient mice display impaired cumu-lus expansion, ovulation and follicle development (52). Inthe current study, we demonstrate the basic molecularmechanisms of follicular somatic cell dysfunction inEpab-/- mice. Since EPAB is expressed exclusively in theoocyte, the underlying cause is likely to be failed commu-nication of oocyte specific factors that ultimately affectsthe differentiation and function of the somatic cells. EPABpromotes CC responsiveness to EGF as well as the acti-vation of the ERK cascade in GCs in response to LH andEGF. The findings reported here open a new perspectiveon oocyte-somatic cell communication and cooperation.Studies are in progress in order to further understand howoocyte-specific EPAB regulates cumulus/granulosa celldifferentiation and function.

Acknowledgments

We thank the Lalor Foundation for providing Katie Lowtherwith a postdoctoral fellowship.

Address all correspondence and requests for reprints to: EmreSeli, M.D., Department of Obstetrics, Gynecology, and Repro-ductive Sciences, Yale University School of Medicine, 310 CedarStreet, LSOG 304B, New Haven, CT 06 520–8063., Phone203–785-7873, Fax: 203–785-7134, E-mail:[email protected].

Disclosure Summary: The authors have nothing to disclose.This work was supported by Award R01HD059909 to E.S.

from the National Institute of Health (NIH). K.M.L was sup-ported by a fellowship from Lalor foundation. M.D.L was partlysupported by grant KL2-RR024138 from the National Center

for Research Resources (NCRR) and the National Center forAdvancing Translational Science (NCATS).

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Embryonic poly(A)-binding protein (EPAB) is required for granulosa cell EGF signaling and cumulus expansion in female mice