Hormone Regulation of Elasmobranch Physiology Chris Bedore and Shannon Long

Hormone Regulation of Elasmobranch Physiology

Chris Bedore and Shannon Long

What We’ll Be Covering

• Digestion and Energy Metabolism

• Growth• Stress• Osmoregulation• Physiological Color

Change• Reproduction• Reproduction• Reproduction• Research methods

Things to Remember

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The Players• Secretin—stimulates secretion of bicarbonate-rich

pancreatic juices• Cholescystokinin (CCK)—regulates supply of bile

and pancreatic enzymes• Somatostatin (SS)—suppress production of gastric

acid, inhibit rectal gland secretion, inhibitory regulation of GH from hypothalamus

• Neuropeptide Y (NPY)—promote digestion by increasing blood flow, inhibits gastric acid secretion, pancreatic enzyme release, and gallbladder contraction, inhibit rectal gland secretion

• Bombesin/Gastrin-releasing peptide (GRP)—promote digestion by increasing blood flow, effecting acid/enzyme secretion/gut motility, inhibit rectal gland secretion

• Vasoactive intestinal polypeptide (VIP)—suppress digestion by reducing blood flow, effecting acid/enzyme secretion/gut motility, stimulates salt excretion by vasodilating the rectal gland and increasing cellular cAMP enzyme, used to regulate water and ion balance

• Tachykinins—promote digestion by increasing blood flow, effecting acid/enzyme secretion/gut motility

• Insulin—storage/conversion/uptake of energy substrate, pancreas, regulated by nutrient levels, reduction in circulating amino acid levels, no effect on ketones

• Glucagon—antagonistic to insulin• Thyroid—alter levels of enzymes in amino acid/lipid

metabolism

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The Players

• Growth hormone (GH)—pituitary gland, regulated by GHRH, promotes somatic and skeletal growth

• Growth hormone-releasing hormone (GHRH)—regulates GH, stimulatory from hypothalmus

• Insulin-like growth factors (IGF-I)—promotes growth of vertebral column

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The Players

• Chromaffin tissue—masses of neurosecretary cells on kidney, cells secrete epinephrine and norepinephrine, response to acute stress

• Catecholamines—epinephrine and norepinephrine, promote mobilization, of energy reserves, increase blood pressure, blood flow to gut reduced, increase oxygen uptake in gills

• Hypthalamo-Pituitary-Interrenal axis (HPI axis)—mid-axis lengthwise down body, helps to regulate ions

• Corticosteroids—interrenal body, regulated by ACTH, promote nutrient movement through body, inhibit growth and energy storage, aid in retention of sodium

• Adrenocorticotropic hormone (ACTH)—regulates corticosteroids, stimulated by CRF, induces hyperglycemia

• Corticotrophin-releasing factor (CRF)—hypothalamic compound, stimulates ACTH, regulate interrenal production of 1α-OHB

• 1α-Hydroxycorticosterone (1α-OHB)—corticosteroid produced only in elasmobranchs, stimulate retention of sodium and chloride

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The Players• Renin-angiotensin system (RAS)—used to regulate water

and ion balance, series of biochemical steps1. convert hepatic glycoprotein angiotensinogen to

ANG I2. cleavage to ANG I by ACE makes ANG II

• Angiotensin I (ANG I)—inactive form• Renin—enzyme used to convert to ANG I, secreted by

juxtanglomerular cells of kidney• Angiotensin converting enzyme (ACE)—promotes

cleavage in ANG I to make ANG II• Angiotensin II (ANG II)—biologically active, receptors in

interrenal gland/gill/rectal gland/intestine, modulate HPI axis, stimulates 1α-OHB secretion, promotes sodium retention, influence electrolyte balance by reducing GFR and UFR, inhibits salt release from rectal gland, increase drinking rate

• C-type natriuretic peptide (CNP)—used to regulate water and ion balance, stimulates production of VIP and rise in salt secretion, expressed in heart and brain, responds to increased cardiac pressure, directly affects rectal gland epithelial cells, binds to NPR-B to increase cGMP and PKC, dilates rectal gland to cause increase in salt release

• Natriuretic peptide type-B (NPR-B)—in rectal gland epithelium

• Cyclic granosine monophosphate (cGMP)• Protein kinase C (PKC)• Arginine vasotocin (AVT)—regulates

osmoregulation, reduces diuresis

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The Players

• Melanocyte—type of chromatophore, contains melanosome which has brown-black melanin

• α-Melanocyte-stimulating hormone (α-MSH)—regulates physiological color change, produced in neurointermediate lobe of pituitary, when removed- sharks lighten in color, when expressed- sharks darken, controlled by neural signals to hypothalamus

• Melatonin—induces skin pallor• Prolactin (PRL)—in par distalis of pituitary, in

freshwater ray, regulates physiological color change

What Goes On Inside

diverse breeding strategies in elasmos

therefore, probably diverse regulatory mechanisms

this area is largely unknown and mostly hypotheticalinformation is from hormone concentration at aspecific time in mating season and deduced

from other vert spp.

well studied in only a few species (ex: D. sabina, S. tiburo)

Reproductive Endocrinology- Overview

Brain-Pituitary-Gonad (BPG) axis: primary endocrine regulation, initiated by env. stimuli

Reproductive Endocrinology- Overview

gonadal steroids:regulate gametogenesis, modulate reproductivebehavior, modulate development and function of2⁰ sex characteristics

influence production of GnRH and GTH (neg. feedback)steroid binding sites in hypothalamus

potential to alter production of relaxin, calcitonin,thyroid

most cycle throughout mating season

Reproductive Endocrinology- Overview

Reproductive Endocrinology- Anatomy

Reproductive Endocrinology- Anatomy

Gonadotropin-releasing hormone:

many forms (7?), therefore many functionsdfGnRH, sGnRH, cIIGnRH, mGnRH

GnRH and GnRH-BPs present in systemic circulationdirect action on gonads?

different forms present in different parts of the brain

GnRH

Hypothalamus/Forebrain: regulate GTH

Pituitary/Forebrain: regulate pituitary/gonads, conveyenv. info to initiate BPG

GnRH

Terminal nerve: regulate repro. processes?!

GnRH

Midbrain/Hindbrain: regulate sensory sensitivity duringreproduction (ie-e-reception)? clasper movement

GnRH

gonadotropin hormone

pituitary gland

partially regulate steroidogenesis, gametogenesis(systemic GnRH)

response to GTH may depend on env. stimuli (H2O temp,photoperiod) and reproductive stage

2 types found so far in elasmossimilar structure to FSH, LH in tetrapods?*future research*

GTH

3 major gonadal steroids:

17β-estradiol (estrogen)- E2

Progesterone- P4

Testosterone- T

Female Steroids-Gonadal

Female Steroids-Gonadal

E2

Female Steroids-Gonadal

2 peaks1. pre-ovuation/follicle development stimulate vitellogenesis regulate development of oviducal gland

2. late gestation (Squalus acanthias) regulate vitellogenesis regulate secretion of histotroph (Dasyatis sabina)

P4

Female Steroids-Gonadal

suppress vitellogenesis (opposes E2)

viviparous: peaks close to ovulation↓ late gestation-permits next follicles to develop

(S. acanthias)

oviparous: regulate timing of oviposition (↑=oviposit)

T and DHT (androgens)

Female Steroids-Gonadal

↑ during follicle development

precursor to E2 synthesis

modulate copulatory behavior

sperm storage (T)

regulate oviposition?

*future research- distribution of receptors*

Female Steroids-Other

Relaxin (Rlx)

Female Steroids-Other

found in ovary of several species

implant and removal experiments-↑ cervical area- prep for parturition

probably aids pupping and oviposition

maintains uterus during gestation (↓ contractions)

Sphyrna tiburo- participates in ova release?

Thyroid (T3 and T4)

Female Steroids-Other

interacts with BPG axis, role unknown

↑ during ovulation and gestation (D. sabina)associated with > metabolic costs at this time?S. tiburo highest levels- placental formation

Thyroid (T3 and T4)

Female Steroids-Other

embryo- passed to young through yolk (McComb et al. 2005)regulate rate of development> concentration found in yolk from populations with:

larger birth sizefaster rate of developmentgreater size at maturityhigher maternal investment

Calcitonin (CT)

Female Steroids-Other

produced in ultimobranchial glandmuscles between pharynx and pericardial cavity

↑ in response to E2 binding on gland

maternal gill: ir-cellsregulate Ca2+ homeostasis during gestation?mechanism/role unclear

Calcitonin (CT)

Female Steroids-Other

S. tiburo: peaks during yolk dependent stageasstd w/digestion of yolk? ir-cells in duodenum &pancreas of early embyros

Calcitonin (CT)

Female Steroids-Other

D. sabina: peaks during histotroph productionno ir-cells in embryo, therefore not involved inembryo nutrition

Male Steroids-Gonadalmostly produced in Sertoli cells (in testes)

Leydig cells: supplements gonad steroids for regulatingstages of spermatogenesis (epididymis and ductus deferens)

trends differ among spp, but all regulate aspects of repro.

Male Steroids-Gonadal4 major gonadal steroids:

Testosterone (T)

Dihydrotestosterone (DHT)

Progesterone (P4)

17β-estradiol (E2)

Male Steroids-Gonadal

T and DHT (androgens)

Male Steroids-Gonadal

↑ middle to late spermatogenesis= peak GSI (high # spermatocytes in testes)

influence development of spermatogonia and repro. ducts

routes of hormone transfer between testes and urogenitalsystem:

systemic circulation, binding sites in spermatozoa,enzymes in semen

T and DHT (androgens)

Male Steroids-Gonadal

claspers/cartilage: calcification due to androgens?

indirect evidence, but no direct evidencecoincides with androgen peakenlarged S. tiburo cephalofoil during malepubertal development

but- implant/removal experiments showed norelationship

mediated through GH and IGF-1 (after E2 peak)?*future studies*

T and DHT (androgens)

Male Steroids-Gonadal

E2

Male Steroids-Gonadal

unsure of role in males- some spp show cycling patterns (D.sabina), irregular variations in others (S. tiburo)

D. sabina:receptors in epididymis and seminal vesicles

maintain repro. tract function?

peak early-middle spermatogenesisregulate early spermatogenesis?

*future studies*

P4

Male Steroids-Gonadal

cycle mirrors androgens (S. tiburo)substrate for androgen synthesis?

but- pubertal S. tiburo & D. .sabina:peak precedes androgen peak

regulate spermiogenesis and/or spermiation?

Relaxin (Rlx)

Male Steroids-Other

produced by gonads

other verts: regulate male fertility

S. tiburo:↑ during late spermatogenesis and copulatory period

[relaxinsemen]=1000x [relaxinblood]

facilitate insemination through contractability ofrepro tract of postmated female?

Male Steroids-Other

seasonal cycling

peak spring and fall

aid in ↑ metabolic needs during migration?

*future studies*

Thyroid (T3 and T4)

Gonadal Steroid & Development

*effect of steroid hormones on development needs moreresearch!*

E2, P4, T transferred from female to yolkE2, T probably utilized during development

BPG axis activated during maturationsteroidogenesis

stage specific increases of hormones (S. tiburo), butroles unclear

Hormones and Behavior

androgens peak in males during copulatory period in somespecies (ex: D. sabina)

may influence certain behaviors- aggressionex: protracted mating period/aggression D. sabina

may also affect sensory sensitivitiesex: electroreception in D. sabina

Dasyatis sabina

Tricas et al., 2000spermatogenesis:

production and development of spermatozoa

spermiogenesis: last stage; produces mature spermatozoa

spermiation: release of spermatozoa from Sertoli cells

spermatogonium → spermatocytes → spermatids →spermatozoa

Dasyatis sabina

Tricas et al., 2000

male steroid hormones:

4 phases:1. androgen suppression

2. PAI, peak E2, P4

3. androgen decrease

4. SAI

Dasyatis sabina

phase 1:

androgen suppressionbetween mating seasons

Dasyatis sabina

phase 2:

primary androgen increase

E2, P4 increases

maximum spermatocyte development (max GSI)

Dasyatis sabina

phase 3:

androgen decrease following maximum testisgrowth and spermatocyte development

E2 and P4 also decrease

Dasyatis sabina

phase 4:

secondary androgen increase

peak sperm maturation

copulatory period

Dasyatis sabina

Female hormones

androgens

small, brief peaks winter, spring

aggression during matingperiod?

Dasyatis sabina

Female hormones

E2:

1st peak- maturation of oocytes (March); synthesis and uptake of vitellogenin

2nd peak- late development, parturitiontransition yolk to histotroph? *future studies*

Dasyatis sabina

Female hormones

P4:

1st peak: near ovulation

2nd peak: parturition

Dasyatis sabina

androgens, behavior, and protracted mating period:

courtship behaviors- males follow females nose to ventchase, bite fins

females- enhance mate choice by fleeing

Immunocytochemistry, Immunohistochemistry(ICC, IHC):

ir-staining of desired hormone

ex: GnRH neurons inhypothalamus or terminal nerve

Reproductive Endocrinology- Methods

ICC, IHC:-collect tissue samples

Reproductive Endocrinology- Methods

ICC, IHC:

- section tissue

Reproductive Endocrinology- Methods

ICC, IHC:

- apply primary antibody, apply secondary antibodywith enzyme, provide substrate, then counter-stain

Reproductive Endocrinology- Methods

Reproductive Endocrinology- Methods

P. marinus GnRH neurons (top-female, bottom-male) before (left column) and after(right column) electro-stimulation

Control

Male

Female

Electro-Stimulated

Radioimmunoassay (RIA)

Reproductive Endocrinology- Methods

Radioimmunoassay (RIA)

uses radiolabeled (ex: 3H) antibody for competitive binding to hormone

calculate % bound radiolabel to determine hormoneconcentration in a sample

Reproductive Endocrinology- Methods

hormone implants

ex: Sisneros and Tricas, 2000

surgically implant hormone tablet/capsule into body

wait for it to take effect (usually a # of days)

check for changes (RIA, behavior, electrophysiology)

Reproductive Endocrinology- Methods

hormone implants

Reproductive Endocrinology- Methods

organ removal (ie pituitary, hypothalamus)

similar to hormone implant, but remove organ

wait for it to take effect

check for changes (RIA, behavioral, electrophysiology)

Reproductive Endocrinology- Methods

Conclusion/Future Research

hormone regulatory mechanisms seem to be similarto other vertebrates

many vertebrate hormones first appeared in elasmobranchs

MUCH research is needed to determine roles of manyhormones. There is less information on elasmo hormones than any other vertebrate group, despiteevolutionary role. We need to catch up!

Literature CitedChung-Davidson, Y.-W., M.B. Bryan, J. Teeter, C.N. Bedore, and W. Li. 2007. Neuroendocrine

and behavioral responses to weak electric fields in adult sea lampreys(Petromyzon marinus). Hormones and Behavior. (In Review)

Gelsleichter, J. 2004. Hormonal Regulation of Elasmobranch Physiology. Pp. 287-324 in Biologyof Sharks and Their Relatives (J.C. Carrier, J.A. Musick, and M.R. Heithaus, eds). CRC Press, Boca Raton.

Kajiura, S.M., J.P. Tyminski, J.B. Forni, and A.P. Summers. 2005. The sexually dimorphic cephalofoil of bonnethead sharks, Sphyrna tiburo. Biol. Bulletin 209: 1-5

Sisneros, J.A. and T.C. Tricas. 2000. Androgen-induced changes in the response dynamics ofampullary electrosensory primary afferent neurons. J. Neurosci. 20(22): 8586-8595.

Tricas, T.C., K.P. Maruska, and L.E.L. Rasmussen. Annual cycles of steroid hormoneproduction, gonad development, and reproductive behavior in the Atlantic stingray.Gen. Comp. Endocrin. 118: 209-225.

Trivett, M.K., T.I. Walker, D.L. Macmillan, J.G. Clement, T.J. Martin, and J.A. Danks. 2002. Parathyroid hormone-related protein (PTHrP) production sites in elasmobranchs.J. Anat. 201: 41-52.