reproductive endocrinology of female elasmobranchs: lessons from the little skate (raja erinacea)...

JOURNAL OF EXPERIMENTAL ZOOLOGY 284:557–574 (1999)

© 1999 WILEY-LISS, INC.

Reproductive Endocrinology of FemaleElasmobranchs: Lessons From the Little Skate (Rajaerinacea) and Spiny Dogfish (Squalus acanthias)

THOMAS J. KOOB,1,3* AND IAN P. CALLARD2,3

1Skeletal Biology, Shriners Hospital for Children, Tampa, Florida 336122Department of Biology, Boston University, Boston, Massachusetts 021153Mount Desert Island Biological Laboratory, Salsbury Cove, Maine 04672

ABSTRACT Conventional classification of reproductive modes in female elasmobranchs failsto account for the diversity in ovarian dynamics that operate during oviparous and viviparouscycles. Delineating this diversity is crucial for understanding the endocrine regulation of the mani-fold physiological mechanisms utilized to retain and protect eggs and developing embryos, to fuelembryogenesis, and to manage the intrauterine milieu. Oocyte development and follicular ste-roidogenesis overlap with egg retention and pregnancy in some species, whereas in others thefollicular phase of the cycle is temporally separated from the gravid period. A luteal phase pre-dominates the post-ovulatory period in viviparous species. In oviparous species, the luteal phaseoverlaps with the follicular cycle. This heterogeneity in ovulatory cycles suggests that the endo-crine system evolved a transmutable system for regulating steroidogenesis and the control of thereproductive events. The reproductive biology and endocrinology of the oviparous little skate andlecithotrophic viviparous spiny dogfish are reviewed in order to derive a working hypothesis thatexplains the complex nature of endocrine patterns observed in species utilizing disparate repro-ductive modes. An understanding of the adaptations in ovarian dynamics to particular ovulatorycycles is key to developing theories about the evolution of reproductive strategies in female elas-mobranchs. J. Exp. Zool. 284:557–574, 1999. © 1999 Wiley-Liss, Inc.

Diverse reproductive modes operate among con-temporary elasmobranch species, all of which pro-duce relatively large offspring over protractedreproductive cycles (see Wourms, ’77, ’81; Wourmset al., ’88; Koob and Callard, ’91; Hamlett and Koob,’99). An oviparous mode in which large, well-pro-tected eggs are produced intermittently over a sus-tained egg laying period is utilized by nearly halfof all elasmobranchs, including all skates and nu-merous shark species. Other shark species utilizeone or a combination of several viviparous repro-ductive modes, including lecithotrophic, placental,uterotrophic, or oophagous viviparity (Wourms etal., ’88). All rays reproduce by lecithotrophy coupledwith highly developed uterotrophic viviparity inwhich a histotroph is provided in the uterine lu-men. Thus, the diversity in reproductive modes de-rives from the manifold physiological means ofprovisioning embryos with the nutrients requiredfor development and the site in which the embryosdevelop. The initial energetic investment accruesin the developing follicles in nearly all species asthe accumulation of yolk during follicular cycles. In

Grant sponsor: National Science Foundation.*Correspondence to: Thomas J. Koob, Skeletal Biology, Shriners

Hospital for Children, 12502 North Pine Drive, Tampa, FL 33612.E-mail: [email protected]

matrotrophic species, supplemental nutrientsderive from extra-ovarian sites, are supplied viamorphogenetic adaptations of the uterus, andare subsequently sequestered by the embryothrough commensurate morphological adapta-tions in the embryo itself or derivatives of theyolk sac.

Ovaries in most elasmobranch species, notableexceptions occurring in only a few viviparoussharks and rays, accumulate substantial amountsof yolk in the developing follicles, resulting in rela-tively large ova at ovulation. In oviparous forms,like Raja erinacea, the yolk in the oviposited eggcontains essentially all of the organic materialsnecessary for development to hatching. A similarcondition obtains in lecithotrophic viviparous spe-cies like Squalus acanthias in which embryonicdevelopment relies exclusively on yolk provisions

558 T.J. KOOB AND I.P. CALLARD

but occurs entirely within the uterine lumen. Al-though the uterus does not supply nutrients, itdoes regulate the intrauterine milieu (Kormanik,’93). Matrotrophic viviparous species supplementthe nutrients supplied in the fertilized ova via in-trauterine mechanisms during the later gesta-tional phases, resulting in offspring considerablylarger than what would be attained if the yolkwere the sole nutrient supply. Yolk fuels initialdevelopment; then after the yolk is exhausted,supplementary means of nutrient provisioning areactivated in the uterus. In addition to nutritionalrequirements, the uterus in matrotrophic speciesmust also regulate intra-uterine conditions by pro-viding oxygen, removing wastes, and regulatingthe ionic and osmotic milieu. In some viviparoussharks, exemplified by Sphyrna tiburo, placentalstructures derived from the yolk sac and uterineendometrium develop around the time the yolk isconsumed. Nutrients are subsequently madeavailable from the maternal circulation for use bythe developing embryo via placental exchange. Theexact materials and mechanisms for placental ex-change, however, remain enigmatic. Viviparity inrays is characterized by production of histotrophby the specialized uterine trophonemata follow-ing yolk depletion, secretion of histotroph into theuterine lumen, and direct utilization of histotrophby the developing embryos. The Atlantic stingray,Dasyatis sabina, typifies this reproductive mode.

The physiological and morphological mecha-nisms central to each of these reproductive modesare likely regulated by endocrine factors, with re-spect to both morphological differentiation and in-duction of requisite metabolic pathways. Basedon available evidence, the ovary, as in other ver-tebrates, plays a pivotal regulatory role duringreproductive events in female elasmobranchs. Fol-licular compartments differentiate and subse-quently activate specific steroidogenic pathwaysduring ovulatory cycles and pregnancy (Callardet al., ’93). In the few species that have been ad-equately examined, biosynthesis and secretion ofeach steroid is temporally restricted to certain pe-riods of the reproductive cycle, suggesting steroid-specific regulatory actions associated with eventsparticular to that period.

The reproductive biology and endocrinology oftwo elasmobranch species utilizing distinct repro-ductive modes, the oviparous little skate andlecithotrophic viviparous spiny dogfish, are de-scribed here. Observations on these species areintegrated into a review of the current knowledgeof the endocrinology and physiology of elasmo-

branch reproduction. Our principal goal is to de-rive a working hypothesis about the nature of en-docrine control of reproduction in extant speciesof female elasmobranchs.

Before discussing the endocrine aspects, we willbriefly review reproductive and ovulatory cycles.This survey is necessary because the conventionalclassification of reproductive modes fails to ac-count for distinctions in ovarian dynamics thatare entirely relevant when considering endocrinecontrol. An account of what is known about theendocrinology of reproduction in female elasmo-branchs, focusing on ovarian derived steroids, willfollow. Experimental studies on the source andfunction of circulating steroids in Raja erinaceaand Squalus acanthias will be described next. Inthe final section, these discrete phenomena willbe integrated to develop hypotheses that we hopewill prompt further investigations into the physi-ology and evolution of elasmobranch reproduction.

REPRODUCTIVE CYCLESAnnual reproductive cycles among elasmobranchs

can be grouped into three types made up of distinctspecies assemblages (Fig. 1). Continuous breedersare reproductively active throughout the year. Manyviviparous sharks are continuous breeders: preg-nancy lasts nearly a full year, with mating and thesubsequent pregnancy ensuing soon after partu-rition. In general, mating, pregnancy, and parturi-tion are coupled with environmental factors; eachevent occurs consistently at certain times of theyear, which results in synchronization of the repro-ductive cycle within a population.

Seasonal breeders are reproductively active foronly a portion of the annual cycle. In seasonallybreeding oviparous species like Raja eglanteria,egg laying occurs during a limited six-month pe-riod of the year (see Rasmussen et al., ’99). Thisgroup also includes most species of rays, includ-ing Dasyatis sabina, which are all viviparous, andsome small viviparous shark species, for example,the bonnethead shark, Sphyrna tiburo (Manire etal., ’95). Pregnancy in seasonally viviparous formspersists for several months; the remainder of theyear is spent non-pregnant. As with continuousbreeders, mating, pregnancy, and birth correlatewith environmental factors and recur at approxi-mately the same time each year with the entirepopulation synchronized.

Viviparous species that undergo punctuatedcycles are pregnant for approximately a full year,but an intervening year or two is spent non-preg-nant. This pattern occurs in both sharks and rays.

REPRODUCTIVE ENDOCRINOLOGY OF FEMALE ELASMOBRANCHS 559

Prionace glauca (Pratt, ’79) and Carcharhinusplumbeus (Springer, ’60; Wass, ’73) produce offspringevery two years. Pregnancy lasts one year and dur-ing the intervening year females are non-pregnant.The reproductive cycle in Torpedo marmorata com-prises a one-year pregnancy separated by two non-pregnant years (Mellinger, ’74). Oocyte developmentis arrested during pregnancy, then requires nearlytwo years to attain ovulatory size. With few excep-tions (e.g., Gollum attenuatus, Yano, ’93), cycles inthese species are likewise synchronized within thepopulation and associated with specific times ofyear. Punctuated cycles may also occur in someoviparous forms like Raja erinacea and Scyliorhinuscanicula. However, the reproductive term of indi-viduals within northern populations has not beenunequivocally determined.

OVULATORY CYCLESDespite the wide array of reproductive modes

and diverse reproductive cycles, ovulatory cycles

fall naturally into a limited set of distinct pat-terns. However, ovulatory cycles do not correlatedirectly with reproductive cycles or modes. Fol-licle development is either incessant throughoutthe reproductive cycle or restricted to a portion ofeach cycle (Fig. 2). Within the group of continu-ous breeders, the timing of follicle developmentdiffers. In some species, follicle development con-tinues unabated during pregnancy. This patternof continuous follicle development throughout theadult life of the individual occurs in many vivipa-rous sharks. In other species, like Squalus acan-thias, follicle development is restricted during theinitial half of pregnancy but then proceeds apacethrough the remainder of gestation. In both groups,a significant proportion of follicle development forthe subsequent pregnancy occurs concomitant withgestation, thereby allowing ovulation to occur soonafter parturition. In species with seasonal and punc-tuated cycles, the bulk of follicle development istemporally separated from pregnancy and occurs

Fig. 1. Illustration of the generalized patterns of annualreproductive cycles in female elasmobranchs with represen-

tative species listed for each type. Note that these cycles donot segregate species by reproductive mode.


well after parturition, primarily in the monthsimmediately preceding the subsequent pregnancy.

Oviparous species typify another group, inter-mittent ovulators, in which follicular developmentcontinues throughout the protracted ovulatory pe-riod (Fig. 3). In captivity, most oviparous specieslay two egg capsules every few days for severalmonths. The ovaries in egg laying females con-tain follicles of various size classes. A large re-serve of uniformly small, pre-vitellogenic folliclesis present throughout the cycle. Developing fol-licles are present only in egg laying females. Thesize distribution of developing follicles in ovariesof egg laying Raja erinacea is typical for ovipa-rous species (Koob et al., ’86). One pair of eggs,either in separate ovaries or together in one ovary,is near the ovulatory size at 20 mm. Pairs of fol-licles to be ovulated in subsequent egg layingevents are smaller by 2 mm increments.

Following ovulation, the eggs are encapsulatedin the shell gland. Capsule formation requires ap-proximately 12 hr. The capsules are then held inthe uterus for several days before oviposition. Fol-lowing oviposition, several days pass before thenext ovulation and encapsulation. During thistime, each pair of developing follicles grows by 2mm with one pair reaching ovulatory size. Theexact duration of each segment of the ovulatorycycle varies among individuals (see Callard and

Koob, ’93). Several oviparous shark species retaineggs in utero before ovipositing the lot. Recurrentovulatory cycles are superimposed on egg reten-tion in these species.

A few viviparous shark species similarly ovu-late eggs intermittently, generally over a relativelybrief period at the beginning of pregnancy. En-capsulated embryos in pregnant Ginglymostomacirratum vary substantially in developmentalstage, indicating that ovulatory cycles must beseparated by several days and therefore resemblethose in oviparous species (Castro, ’97). In oopha-gous species, eggs ovulated intermittently duringthe initial stages of pregnancy serve as a nutri-tional source for the developing embryos in utero(see Gilmore, ’93). Thus, an ovulatory cycle is su-perimposed on the initial stages of pregnancy inthese species.

In the preponderance of viviparous species, fol-licle development and ovulation is a coordinatedprocess leading to ovulation of an entire group ofidentically sized oocytes. A limited number of pre-vitellogenic follicles, the number being species-spe-cific and unrelated to reproductive mode, isselected by some unidentified signaling pathwayfor development to ovulation. These follicles growand oocytes accumulate yolk at the same rate, andthen are ovulated as a group or consecutively overa brief period. In Squalus acanthias, the eggs are

Fig. 2. Follicular cycles in viviparous species. In continu-ous breeders, follicle development is either restricted to thelater gestational phase (upper panel) or continues throughout

pregnancy (lower panel). Follicle development in seasonal andpunctuated breeders occurs exclusively between pregnancies,generally during the months immediately preceding ovulation.


ovulated as a group, and are transported to thenidamental gland through the oviduct, where theyare encapsulated in a single egg capsule. In otherspecies, typified by Sphyrna tiburo and Dasyatissabina, eggs are ovulated and encapsulated indi-vidually, in a conveyor belt fashion. Ovulation ofall the eggs in one litter takes place rapidly sinceall the embryos in one litter are essentially iden-tical with respect to developmental stage.

Another important endocrine tissue functioningduring ovulatory cycles in elasmobranch ovaries isthe corpus luteum. Corpora lutea differentiate fromfollicles after ovulation in all species examined todate; however, the functional life of the corpus lu-teum differs among species using distinct reproduc-tive cycles. We know less about the cyclical dynamicsof luteal morphogenesis and function since corporalutea are not as easily distinguished as developingfollicles and their physiological significance can only

be determined by measurement of functional ac-tivities, the latter having been accomplished inonly two species, Raja erinacea and Squalusacanthias. Corpora lutea are the source of ste-roids and possibly peptide hormones in some spe-cies. They have been implicated in the regulationof a variety of physiological mechanisms, includ-ing vitellogenesis, egg retention, pregnancy, ovi-position, and parturition. More will be said aboutcorpora lutea in the sections to follow.

Follicular cycles can be temporally separatedfrom pregnancy, superimposed on pregnancy, orconcomitant with continual egg production. Thesewidely varying follicular cycles raise questionsabout the role of the developing follicles and cor-pora lutea in regulating reproductive events. Noparadigm is common among the various reproduc-tive modes or even within a single group, withthe possible exception of oviparous species. Sincethe follicles and corpora lutea are responsible forthe circulating reproductive steroids, as describedbelow, both the control of steroidogenesis and thefunctional role of the steroids themselves mustdiffer while sharing some functional attributesamong the various reproductive modes.

ENDOCRINE CYCLESCirculating steroids have been measured through-

out female reproductive cycles in only a few elas-mobranch species, but these include oviparous andviviparous species, both continuous and seasonalbreeders (Figs. 4 and 5). Elevated estradiol titersare associated with the follicular phase in all spe-cies examined to date, regardless of when follicledevelopment takes place during the cycle. In Rajaerinacea, increasing estradiol titers correlate withfollicle recrudescence in preparation for the pro-tracted egg laying season. Moreover, circulatingestradiol levels rise during the follicular phase ofeach ovulatory event, then decline as ovulationapproaches. Thus, elevated estradiol titers are as-sociated with growing follicles in this as well asother oviparous species (Scyliorhinus canicula,Sumpter and Dodd, ’79; Raja eglanteria, Ras-mussen et al., ’99; Hemiscyllium ocellatum,Heupel et al., ’99)

In viviparous species, elevated estradiol titers aresimilarly correlated with follicle development, eventhough the timing with respect to the follicularphase and gestation differs in these species. Folli-cular development overlaps with pregnancy in con-tinuous breeders whereas it occurs during thenon-pregnant phase in seasonal and punctuatedbreeders. Estradiol levels are chronically elevated

Fig. 3. Reproductive cycles in oviparous species. (A) Fol-licle development during ovarian recrudescence in prepara-tion for ovulatory cycles. (B) Ovulatory cycles during the egglaying period.


during the latter half of pregnancy in the leci-thotrophic spiny dogfish, Squalus acanthias, whenthe preponderance of follicle development for thesubsequent pregnancy occurs. In the seasonallybreeding, placental bonnethead shark, Sphyrnatiburo, estradiol titers rise as follicle developmentproceeds prior to ovulation, peaks in the pre-ovula-tory period, then declines in the peri-ovulatory pe-riod (Manire et al., ’95). Circulating estradiolremains at relatively low levels during pregnancy,parturition, and the post-partum period until fol-licle development begins for the subsequent preg-nancy. A similar rise in estradiol is concomitant withthe final stages of follicular development in the pre-ovulatory phase in the seasonally breeding Atlan-tic stingray, Dasyatis sabina (Snelson et al., ’97).Circulating estradiol rises again during the secondhalf of pregnancy and thereafter remains chroni-cally elevated until just before parturition. This ges-tational rise in estradiol, however, is not coupledwith follicle development.

Measurement of plasma estradiol at specifictimes during the reproductive cycle in other fe-male elasmobranchs support the conclusion thatestradiol levels rise coincident with follicle devel-opment during sexual maturation, ovarian recru-

Fig. 4. The source and circulating titers of estradiol (E),testosterone (T), and progesterone (P) during the ovulatorycycle in Raja erinacea. The principal source of each steroid is

indicated by the solid bold line in the upper panel: additionalsources are indicated with the dashed lines.

descence, and ovulatory cycles. Elevated estradioltiters are correlated with advancing sexual ma-turity in the lemon shark, Negaprion brevirostris,tiger shark, Galeocerdo cuvier, and hammerheadsharks (Rasmussen and Gruber, ’90, ’93). High lev-els of circulating estradiol were found in repro-ductively active females of several viviparousspecies, including lemon sharks with ripe ovarianfollicles and during mating, hammerhead sharkswith pre-ovulatory follicles, and blacknose sharkswith large ovarian follicles (Rasmussen andGruber, ’90, ’93). In comparison to other stages,highest estradiol titers were found in plasma fromTorpedo marmorata approaching the final stagesof follicle maturation leading to ovulation andpregnancy (Fasano et al., ’92).

Current evidence indicates that testosterone isan important ovarian derived steroid during re-productive cycles in female elasmobranchs (Sump-ter and Dodd, ’79; Koob et al., ’86; Tsang andCallard, ’88, ’92; Rasmussen and Gruber, ’90;Rasmussen and Murru, ’92; Manire et al., ’95;Rasmussen et al., ’99). Circulating testosteronetiters are elevated at specific times during repro-ductive cycles and in some cases quantitativelysurpass those of estradiol. Testosterone titers are


similarly temporally elevated and track closelywith fluctuations in circulating estradiol duringovulatory cycles. Elevations in testosterone coin-cide with follicle development and are generallyelevated in the pre-ovulatory period.

In contrast to estradiol and testosterone, thetiming for elevated progesterone titers differsamong oviparous and viviparous species. Proges-terone is elevated for only a brief 1–2 day periodduring the pre-ovulatory period in the little skate,Raja erinacea (Koob et al., ’86). Progesterone re-mains at relatively low levels during ovulation,egg encapsulation, egg retention, and oviposition.In the clearnose skate, Raja eglanteria, the brief,circumscribed rise in progesterone titers occursafter ovulation (Rasmussen et al., ’99). It is notclear whether this post-ovulatory elevation is as-sociated with the next ovulatory event, since thisspecies lays eggs every four days.

In viviparous species progesterone remains el-evated during the initial stage of pregnancy.Progesterone titers are high during the first halfof pregnancy in Squalus acanthias, decline there-after to low levels for the second half of pregnancywhen estradiol levels are high (Fig. 5). In Sphyrnatiburo and Dasyatis sabina, progesterone levels

Fig. 5. Patterns of circulating estradiol (E) and progesterone (P) titers during the repro-ductive cycles of three viviparous species.

rise in the peri-ovulatory period and remain el-evated during the first part of pregnancy, then de-cline and remain at relatively low levels for theremainder of pregnancy. The conclusion thatprogesterone is associated with the peri-ovulatoryperiod and pregnancy in viviparous species is sup-ported by isolated measurements on several spe-cies (Rasmussen and Gruber, ’90, ’93).

Little attention has been directed to the poten-tial dynamics in circulating titers of other steroidsduring reproductive cycles in female elasmo-branchs. However, recent evidence has raised thepossibility that steroids other than estradiol, test-osterone, and progesterone, particularly androgens,are intimately involved in reproductive cycles. Thedata derive primarily from measurement of thesesteroids in the peripheral circulation. Manire et al.(’95) reported on the circulating titers of dihydro-testosterone in the viviparous female bonnetheadshark. Dihydrotestosterone levels were relativelyelevated during the pre-ovulatory phase and in theperi-ovulatory period, then declined to a low levelaround the time of implantation, followed by aslight rise near parturition. More recent work hasfound significant levels of 11-ketotestosterone, 11-ketoandrostenedione, and dihydroprogesterone in


the peripheral circulation of female bonnetheadsharks (Manire et al., ’99). Elevated levels of 11-ketoandrostenedione were present from matingthrough the first part of gestation, falling slightlyat parturition. An apparent rise in 11-keto-testosterone titers occurred around the time ofimplantation. Dihydrotestosterone also circulatesin female stingrays, Dasyatis sabina, during theirreproductive cycle (Snelson et al., ’97) and Rajaeglanteria during the egg-laying season (Ras-mussen et al., ’99).

SOURCES OF CIRCULATING ESTRADIOL,TESTOSTERONE, AND PROGESTERONEEarly work on steroids in elasmobranch ovaries

identified estradiol, estrone, estriol, and progest-erone in extracts of ovaries from both viviparous(Squalus acanthias, Torpedo marmorata) andoviparous (Scyliorhinus canicula) sharks (Wotizet al., ’58, ’60; Chiefffi and Lupo di Prisco, ’63;Simpson et al., ’64). That the ovarian tissuesthemselves synthesized these steroids was sub-stantiated by histochemical methods that identi-fied steroidogenic enzymes. In addition, in vitromethods showed that ovarian tissues were capableof de novo steroid synthesis (Raja erinacea,Squalus acanthias, Callard and Leathem, ’65,Tsang and Callard, ’82; Scyliorhinus stellaris, Tor-pedo marmorata, Lupo di Prisco et al., ’65; Scylior-hinus canicula, Dodd et al., ’83). Only recentlyhave the ovarian compartments responsible forbiosynthesis and secretion of the predominant cir-culating steroids been analyzed with contempo-rary methods and this in only two species, Rajaerinacea and Squalus acanthias.

The ubiquitous correlation between elevated es-tradiol levels and follicle development in all speciesexamined to date, both oviparous and viviparous,clearly indicates that estradiol is a product of thefollicular compartments. Rising estradiol titers cor-relate with ovarian recrudescence and follicle de-velopment in the little skate, and are elevated onlyduring the follicular phase of each ovulatory cycle(Koob et al., ’86). In Squalus acanthias, elevatedlevels of estradiol titers correlate with follicle de-velopment that occurs during the latter half ofpregnancy (Tsang and Callard, ’88). In season-ally breeding viviparous species, estradiol levelsrise during the follicular phase leading to ovula-tion, and thereafter decline and remain low dur-ing the initial stages of pregnancy (Sphyrnatiburo, Manire et al., ’95; Dasyatis sabina, Snelsonet al., ’97).

In in vitro experiments, de novo steroid biosyn-

thesis by follicles cultured for brief periods hasestablished the source of estradiol and testoster-one in Squalus acanthias and Raja erinacea.These experiments allow analysis of steroidogenicpotential of the various size classes of follicles aswell as the distinct follicular compartments thatconstitute these follicles. In the viviparous spinydogfish, estradiol synthesis by granulosa cells cor-related directly with circulating titers (Tsang andCallard, ’92). Small follicles from ovaries of earlystage A pregnancy produced relatively smallamounts of estradiol in vitro. Granulosa cells fromthe middle term of pregnancy, stages B and C,synthesized 15- to 40-fold more estradiol. Nearthe end of pregnancy, the rate of estradiol syn-thesis had declined, but remained 15-fold higherthan that of follicles from early pregnancy. Thesedata indicate that the follicles undergoing devel-opment for the subsequent ovulatory cycle andpregnancy are the principal source of the elevatedestradiol titers during the second half of the ex-isting pregnancy.

A different question about which follicularcompartment is responsible for estradiol syn-thesis arises when considering follicular cyclesin oviparous species. A graded series of folliclesizes is continually present in egg laying fe-males. Measurement of in vitro estradiol syn-thesis by various size classes of ovarian folliclesfrom Raja erinacea determined that the me-dium-sized follicles (11–19 mm diameter) syn-thesized estradiol at a 5-fold greater rate thanthat of either small (<10 mm) or large follicles(20–25 mm) including the largest, immediatelypre-ovulatory follicles (Fileti and Callard, ’90;Callard et al., ’93). Experiments with isolated fol-licular compartments established that estradiolis produced primarily by the granulosa cells. Incontrast to follicles, corpora luteal tissue did notsynthesize estradiol in vitro.

Testosterone titers similarly correlate with fol-licle development in the little skate and spiny dog-fish, implicating follicles as the principal source.Granulosa cells from follicles of pregnant spinydogfish begin synthesis of testosterone duringstage B, then increase production throughoutpregnancy to reach highest rates in stage D nearparturition (Tsang and Callard, ’92). All develop-ing follicles in Raja erinacea produced significantamounts of testosterone in vitro (Fileti andCallard, ’90; Callard et al., ’93). In contrast to es-tradiol synthesis, however, rates of testosteronesynthesis by the largest follicles (20–25 mm di-ameter) was greater than that of the smaller size


classes. Testosterone is the predominant productof the theca. However, in vitro experimentsshowed that luteal tissue can also produce test-osterone, suggesting that both the pool of devel-oping follicles and corpora lutea together areresponsible for the dynamics of circulating titersof testosterone.

Progesterone titers in viviparous species corre-late with the functional life of the corpora lutea.Progesterone titers begin to rise in the peri-ovu-latory period and remain elevated during the ini-tial stages of pregnancy (Tsang and Callard, ’87a;Manire et al., ’95; Snelson et al., ’97). Luteal tis-sue from spiny dogfish ovaries produces progest-erone in vitro, whereas follicular granulosa cellsdo not (Tsang and Callard, ’87b; Tsang andCallard, ’92). Based on in vitro measurements ofsteroidogenesis, the principal source of progester-one in the little skate is luteal tissue (Fileti andCallard, ’88; Callard et al., ’93). Unstimulated de-veloping follicles fail to produce progesterone invitro. It would appear then that the principal sourceof progesterone in both oviparous and viviparousspecies is luteal tissue. Since the circumscribed el-evations in progesterone titers differ with respectto the ovulatory cycle, the role of the corpus luteumis likely to be a pivotal one in the evolution of re-productive modes (Callard et al., ’92).

Although it is clear that the source of progester-one in oviparous species is luteal tissue, the timingof the brief elevation in circulating progesteronewith respect to the ovulatory cycle is puzzling. Theprogesterone peak occurs well after oviposition andprecedes the next ovulation by one to two days. Thecorpora lutea formed from one pair of ovulated fol-licles must be responsible in large part for the pro-duction of progesterone after egg retention andoviposition of their oocytes. It appears that theprogesterone peak originating from the corporalutea of one ovulatory event is coupled with thefollicular phase of the subsequent ovulation. Thispattern argues for different regulatory pathwaysfor progesterone biosynthesis between oviparousand viviparous forms. In addition, progesteronemay have distinct functional roles that are spe-cific to each reproductive mode.

ENDOCRINE REGULATIONTwo fundamental distinctions are central to un-

derstanding the role of endocrine control duringreproductive cycles in female elasmobranchs.First, the source of the organic elements neces-sary for embryonic development to term differsin oviparous and viviparous species, likewise dif-

fering substantially even among viviparous spe-cies. Second, the site of development coupled withthe requisite means of packaging and deliveringnutrients differs depending upon the reproductivemode. In oviparous species and lecithotrophic vi-viparous species, the yolk is the sole source of nu-trients, thus biosynthesis of yolk macromoleculesand oocyte accretion of yolk during follicle devel-opment are the principal targets for endocrineregulation. These same mechanisms operate inmost matrotrophic viviparous species, but accrualof significant amounts of nutrients also takes placein utero. The uterus as well as extra-uterine sitesare potential targets for endocrine modulation ofnutrient metabolism. Complex morphological ad-aptations are associated with both physiologicalmechanisms. Based on the mammalian and avianparadigms, parsimony dictates that we assumeendocrine factors are instrumental in regulatingthese morphological and physiological mecha-nisms. However, sufficient data to support thisassumption are available for only a limited num-ber of species and processes. Thus, it must be em-phasized that the following generalizations andresulting hypotheses that enlist endocrine factorsare based on too few observations and must beviewed with guarded enthusiasm.

Nutritional demandsThe organic nutrients for the initial stages of

embryonic development accumulate in the devel-oping oocytes in the form of yolk during the folli-cular phase of ovulatory cycles. Yolk acquisitionoccurs in nearly all elasmobranch species, al-though the absolute amount of yolk accumulatedin the oocyte differs. This difference, at least interms of the relationship between the amount ofyolk in the ovulated oocyte compared to the sizeof the neonate, correlates with reproductive mode.Thus, the endocrine regulation of follicle develop-ment and yolk acquisition is a basic physiologicalmechanism that is likely shared by all membersof the taxon. The various reproductive modes mustoperate within this constraint. We focus here onregulation of yolk acquisition since the control offollicle development and steroidogenesis by fac-tors such as pituitary hormones, cytokines, andautocrines is well beyond the scope of the presentdiscussion (see Tsang and Callard, ’87a, ’88, ’92;Fileti and Callard, ’88, ’90, ’91; Sherwood andLovejoy, ’93; Wright and Demski, ’93).

Although compositional analyses of elasmo-branch yolk are rudimentary, it is clear that he-patic vitellogenin and its metabolites, lipovitellin


and phosvitin, are major yolk components and keyplayers in oocyte development (see Perez and Cal-lard, ’92, ’93). In other non-mammalian vertebratesthat utilize vitellogenin as the principal source ofyolk nutrients, this complex lipophosphogly-coprotein is synthesized in the liver, transportedto the ovary through the peripheral circulation,and taken up by the oocyte by receptor mediatedendocytosis (see Wallace, ’85). This basic verte-brate pattern is likely to operate in elasmo-branchs as well. Vitellogenin has been identifiedin the blood of Scyliorhinus canicula, Raja erina-cea, and Squalus acanthias; and elevations in cir-culating levels occur during the follicular phaseof ovulatory cycles when oocytes are accumulat-ing yolk (Craik, ’78a,b; Ho et al., ’80; Perez andCallard, ’92, ’93). Elevated plasma vitellogeninlevels also correspond with the high circulatingtiters of estradiol and testosterone which accom-pany follicle development, suggesting that one orboth of these steroids regulates vitellogenesis inthe liver (Craik, ’78c, ’79; Ho et al., ’80; Perezand Callard, ’92).

Experimental studies support the conclusionderived from the correlative observations de-scribed above that estradiol regulates hepaticvitellogenin synthesis. Craik (’78d) showed thatchronic administration of estradiol increasedthe amount of circulating vitellogenin in Scyli-orhinus canicula. Estradiol administration toSqualus acanthias females in late pregnancysimilarly increased circulating vitellogenin lev-els (Ho et al., ’80). More recently, estradiol hasbeen shown to induce hepatic vitellogenin syn-thesis in both female and male Raja erinacea(Perez and Callard, ’93). Moreover, an estrogenreceptor has been unequivocally identified inthe little skate liver (Paolucci and Callard,unpublished). These observations indicate a cas-cade involving the differentiation of the steroid-ogenic pathway for estradiol synthesis, andsubsequent elevations in circulating estradioltiters, which in turn induce and maintain vi-tellogenesis in the liver during the follicularphase of ovulatory cycles (Fig. 6).

Progesterone is also involved in the regulationof vitellogenin production. There is a clear inverserelationship between circulating progesterone ti-ters and follicle/oocyte development in viviparousspecies. In the spiny dogfish, follicle developmentis attenuated during the first half of pregnancywhen progesterone values are chronically elevated.The bulk of follicle growth occurs only afterprogesterone levels have declined to baseline val-

ues (Tsang and Callard, ’87). Moreover, estradioltreatment fails to upregulate vitellogenin synthe-sis in this species when administered during thefirst half of pregnancy while progesterone valuesare elevated (Ho et al., ’80). In viviparous sea-sonal breeders, like Sphyrna tiburo (Manire etal., ’95) and Dasyatis sabina (Snelson et al., ’97),progesterone levels are elevated during the ini-tial phase of pregnancy. Follicle growth and oo-cyte maturation are suspended during this time,only to resume following parturition, again sug-gesting that progesterone may inhibit vitellogeninsynthesis. Recent studies from this lab haveshown that progesterone administration blocks theupregulation of vitellogenin synthesis by estradioltreatment (Perez and Callard, ’93). In addition, aspecific progesterone receptor has recently beenidentified in the liver of the little skate (Paolucciand Callard, ’98).

A model that is consistent with the dynamicsof estradiol and progesterone synthesis describedearlier and the patterns of follicle growth and oo-cyte maturation posits that estradiol induces andmaintains hepatic vitellogenin synthesis, therebyeffectively mediating oocyte maturation, whileprogesterone inhibits hepatic vitellogenin synthe-sis and consequently follicle growth (Fig. 6). Howdoes this model fit with the various reproductivemodes? In viviparous species for which data areavailable, the model accounts for the apparentparadox in which the follicular phase impingeson pregnancy. In the spiny dogfish, follicle devel-opment and oocyte growth occur almost entirelyduring the second half of pregnancy. Overlap ofthe follicular phase with a significant proportionof pregnancy in this species is tolerated for tworeasons. First, progesterone titers that are el-evated during the first half of pregnancy declineto chronically low levels during the second half,thereby allowing increasing estradiol titers toupregulate vitellogenin production for follicle de-velopment. Second, the developing embryos inutero rely only on yolk and do not obtain supple-mentary nutrients from extra-ovarian sites at anytime during pregnancy.

Another means of avoiding conflicts between thenutrient requirements of developing follicular oo-cytes and those of developing embryos in utero isutilized by other viviparous species. Oocytegrowth is temporally separated from pregnancy.In seasonal breeders like Sphyrna tiburo andDasyatis sabina, oocyte maturation and elevatedestradiol titers occur between pregnancies. Al-though very little is known about endocrine cycles


in species utilizing punctuated cycles, it is clearthat follicle development and oocyte growth donot overlap with pregnancy.

The situation in oviparous species is not so clear.Acquisition of yolk by oocytes in developing fol-licles appears to be a continuous process duringthe egg-laying period. However, estradiol levelsfluctuate during each ovulatory cycle and a briefbut substantial elevation in progesterone occursin the peri-ovulatory period (Koob et al., ’86;Rasmussen et al., ’99). In Raja erinacea, the peakof circulating progesterone occurs one to two daysbefore ovulation. Given the experimental resultsdescribed above, it seems probable that hepaticsynthesis of vitellogenin is inhibited by progest-erone during this time. No evidence is availableto explain why it would be necessary to inhibithepatic vitellogenin synthesis for a brief periodprior to ovulation in the little skate or other ovipa-rous species.

Taken together, these observations raise ques-tions about the function of progesterone in bothoviparous and viviparous species. Does progest-erone function solely to regulate vitellogenin syn-thesis? In those viviparous species that have beenexamined in detail, progesterone is elevated inthe peri-ovulatory period and for only a portion

of pregnancy, with highest levels during the ini-tial stages. This is the period when embryonicdevelopment is fueled by the yolk contained inthe ovulated egg. It also is a time when follicledevelopment and oocyte growth have ceased. Byinhibiting follicle development, progesterone pre-vents the initiation of the subsequent ovulatorycycle. It seems less clear why progesterone medi-ated inhibition of vitellogenin synthesis would beimportant in oviparous species.

Unfortunately, little is known about the sourceof nutrients in matrotrophic species, even the na-ture of the nutrients remains uncharacterized. Itis generally assumed that in placental species nu-trients cross the uterine-placental unit via vas-cular exchange. Whether the putative nutrientsoriginate on demand by endocrine induction inspecialized tissues like the liver is not known. Itis just as likely that the nutrients are simply pas-sively supplied from the normal circulating poolof nutrients. In species utilizing uterotrophicmechanisms, the uterus itself produces the histo-troph. De novo biosynthesis of nutritional elementsby the specialized uterine tissues nonetheless re-quires that the metabolic precursors be suppliedto the cells via the circulation. Again the sourceof these precursors could be recruited from spe-

Fig. 6. Regulation of vitellogenin biosynthesis in Squalusacanthias by estradiol (E) and progesterone (P). Steroid ac-tions on the liver are mediated by estrogen (ER) and proges-

terone (PR) receptors. Plus marks indicate induction ofvitellogenin biosynthesis; minus marks indicate inhibition.


cialized sources under endocrine control or fromthe general circulating nutrient pool.

Based on the three viviparous species in whichcirculating steroids have been examined in detail,estradiol could be involved in regulating the sup-ply of nutrients during pregnancy to both thegrowing ovarian oocytes and the developing em-bryos in utero. Estradiol rises in late pregnancyin all three species (Squalus acanthias, Tsang andCallard, ’87, ’88; Sphyrna tiburo, Manire et al.,’95; Dasyatis sabina, Snelson et al., ’97). In thespiny dogfish, estradiol appears to be involved ininducing hepatic vitellogenin synthesis during thelatter half of pregnancy for the development ofoocytes intended for the subsequent pregnancy. Asmentioned above, there is no conflict with the nu-tritional demands of the developing fetuses sincethey rely exclusively on yolk reserves. Thus, es-tradiol does mediate nutrient acquisition duringpregnancy, but not for the fetuses in utero, ratherfor the subsequent generation.

In the placental bonnethead shark, estradiollevels rise around the time of implantation andremain elevated until parturition. Since folliclesdo not develop in this species until well after par-turition, estradiol could function late in pregnancyto facilitate nutritional acquisition through pla-cental exchange. Exactly where estradiol wouldhave this effect is not clear. However, since onlysmall metabolites cross the egg envelope/pla-cental barrier in this species (King et al., ’98),estradiol potentially regulates liver metabolismand/or maternal placental metabolism in orderto make these metabolites plentiful and avail-able. A marked and chronic elevation in estra-diol titers in late pregnancy in the Atlanticstingray correlates with the development andmetabolic activity of the uterine trophonemataproducing histotroph, which are the principalsource of nutrients for the remainder of preg-nancy. It seems reasonable to speculate that es-tradiol during the latter half of pregnancy in thisspecies regulates trophonemata activity, either byregulating circulating nutrient availability vialiver metabolism or by inducing the productionof uterine histotroph.

Site of embryonic development andmeans of packaging and delivering nutrients

The reproductive tract has adopted distinctfunctions for attaining the numerous and diver-gent reproductive modes in female elasmobranchs.Common to all species is the role as a conduit for

transporting the future progeny from the ovaryto the external environment. Along the passage,eggs are fertilized, encapsulated, retained, and de-livered. For oviparous species, encapsulation isthe dominant process. For viviparous species, en-capsulation, while still maintained, takes on asubordinate role to intrauterine mechanismsadapted for embryonic development to term, in-cluding regulation of the intrauterine milieu andprovision of nutrients. We have in the past pre-sented justification based on correlative and ex-perimental studies for postulating functional rolesfor estradiol and progesterone in regulating cer-tain of these reproductive tract activities (Kooband Callard, ’91; Callard and Koob, ’93; Callardet al., ’93). For detailed analyses, the reader isdirected to these reviews. For the purposes of thisdiscussion, a brief description of the conclusionsreached in these studies will be necessary to placethe present discussion on endocrine regulation inproper context.

Nearly all elasmobranchs encapsulate eggs inmaterials synthesized, secreted, and assembledby the shell, nidamental, or oviducal gland (seeHamlett et al., ’98). The shell glands in all ovipa-rous species, as well as some lecithotrophic vi-viparous species like the whale shark and nurseshark, produce substantial egg capsules that, be-cause of the rapid and repeated production of cap-sules, require considerable morphological andmetabolic investments. In matrotrophic vivipa-rous species, nidamental glands manufacture athin egg envelope that persists either through-out gestation, as in placental sharks, or functionsonly during the initial stages of pregnancy, as inrays. The shell gland is almost certainly underendocrine control, first to induce and maintainsynthesis of the structural proteins and enzymes,and, second, to cause secretion and assembly ofthe capsule precursors around the descending oo-cytes. Based on correlative and experimentalstudies, the former is regulated, at least in largepart, by estradiol during the follicular phase ofeach cycle. Nidamental gland growth is coincidentwith follicle development and elevated estradioltiters in the little skate (Koob et al., ’86) and es-tradiol treatment causes shell gland enlargementand capsule protein synthesis in Scyliorhinuscanicula (Dodd and Goddard, ’61). In addition,since elevated estradiol titers are characteristicof the pre-ovulatory phase when nidamental glandgrowth is required in preparation for capsule for-mation, as described above for Raja eglanteria,Squalus acanthias, Sphyrna tiburo, and Dasyatis


sabina, it seems reasonable to conclude that es-tradiol is critically involved in nidamental glandfunction in these species as well. However, thisconclusion is a broad and nebulous one, sincethere are numerous aspects to regulating ovidu-cal gland function, including differentiation of thecomplex array of cells required for capsule bio-synthesis (Hamlett et al., ’98); induction of geneexpression for the many structural proteins andenzymes that must be coordinately produced inthe proper relative amounts (Koob and Cox, ’93);and morphogenesis of the extensive tubules, baffleplates, spinnerets, and lamellae that assemble thecapsule precursors (Knight et al., ’96).

What factor induces egg capsule secretion andassembly is entirely unknown and unexplored, butit must be endocrine related since egg capsule for-mation does not require passage of the eggthrough the gland. The synchronous formation ofcapsule pairs in oviparous species begins prior toovulation and is approximately half completedwhen the ova enter the capsule. Moreover, ovi-posited capsules that lack ova are occasionally pro-duced by oviparous sharks and skates. Thesecapsules, while smaller, are otherwise structur-ally complete, including the egg jelly that occu-pies the capsule lumen.

In addition to egg capsule production, the shellgland is thought to be the principal site for spermstorage (Pratt, ’93). Extended storage of sperm isadvantageous in that it de-couples mating from ovu-latory events. This is particularly important inoviparous species that ovulate, encapsulate, and ovi-posit eggs over several months. The same is truefor populations of viviparous species, like Sphyrnatiburo, in which mating is temporally separatedfrom ovulation and pregnancy. Whether endocrinefactors are involved in sperm storage and activa-tion is uncertain. However, it has been suggestedthat one role for the ubiquitous but enigmatic el-evations in testosterone levels and those of relatedandrogens during the follicular phase of ovulatorycycles is direct regulation of the quiescence and ac-tivation of stored spermatozoa (Manire et al., ’95).

The uterine region of the reproductive tract im-mediately caudal to the shell gland in the littleskate and spiny dogfish is morphologically andfunctionally specialized. In Raja erinacea, imme-diately before ovulation and encapsulation, theuterine wall is predominated by loose, hydratedconnective tissue with very little muscle (Kooband Hamlett, ’98). Compliance of this region isnecessary in order to accommodate passage of themechanically friable and chemically untanned

capsule (Koob et al., ’81). The uterus here is alsospecialized for biosynthesis and secretion of me-tabolites and for facilitating vascular exchange.The morphology of this region changes during theovulatory cycle and preliminary observations sug-gest that progesterone influences the alteration.This region of the spiny dogfish uterus must simi-larly function for capsule tanning. It also func-tions as a valve to close off the major uterinecompartment in which the embryos develop toterm (see Koob and Callard, ’91). Both activitiesmust be under endocrine regulation; however,little evidence is available to support speculation.We have shown that estradiol administration tospiny dogfish increases the compliance of the tis-sue (Koob et al., ’83). It should also be empha-sized that comparable morphologically specializedand endocrine regulated regions of the upperuterus probably function in other elasmobranchs.Unfortunately, this region of the reproductivetract has received scant observation since Wida-kowich (’07) first drew attention to these struc-tures nearly 100 years ago.

The principal function of the uterus is to housethe oocytes and developing fetuses prior to deliv-ery. In oviparous species, egg retention allowscompletion of the capsule sclerotization process.Eggs are held in utero for only a brief period beforeoviposition, e.g., 2–7 days in Raja erinacea (Callardand Koob, ’93). The activity of the uterus duringthis time is critical for the development of the physi-cochemical properties of the capsule, since the tan-ning process is extinguished if the capsule isoviposited early or if untanned capsule material isremoved and incubated in sea water. The uterusmust also propel the capsule along the tract, even-tually moving it to the urogenital sinus for oviposi-tion. Based on the increased relative proportion ofsmooth muscle over ciliated epithelium, activity ofthe muscularis must be responsible for movementin the distal region of the uterus (Koob and Hamlett,’98). Since the capsule moves into this region, isretained for several days, and is then moved out,uterine muscular activity must be under humoralcontrol. We have speculated based on circulatingsteroid titers that progesterone is involved inregulating egg retention in the little skate; more-over, experimentally manipulated chronic eleva-tions of progesterone caused early oviposition inthis species (Callard and Koob, ’93). We have alsoidentified estradiol receptors in the skate uterusindicating that estradiol regulates uterine functionduring ovulatory cycles (Reese and Callard, ‘87).

In viviparous species, the uterus must attain asize sufficient to accommodate the large volume


of ovulated eggs. Uterine morphogenesis occurs dur-ing sexual maturation and ovulatory cycles coinci-dent with follicle development, suggesting thatovarian derived factors mediate morphogenetic dif-ferentiation. During pregnancy, the size and physi-cal properties of the uterus are altered in order toaccommodate the increasing volume of the concep-tus. In a few viviparous species, particularly pla-cental species, the uterus undergoes furtherstructural adaptations in the formation of uterinecompartments that separate individual embryos.Although the initial morphogenesis correlates withelevated estradiol and testosterone titers, subse-quent structural modifications during pregnancy areless well correlated with endocrine factors. Embryo-related structural specializations in the uterus suchas uterine folds and placenta could also be causedby factors originating in the embryo itself.

One uterine tissue that has received experimen-tal study is the myometrium in the spiny dogfish(Sorbera and Callard, ’95). Regulation of uterinecontractions could be important for two reasons:during early pregnancy, the capsule containing thedeveloping embryos is easily broken by mechanicalstresses; later in pregnancy after the embryos havebroken out of the capsule, the uterus periodicallyflushes the lumen with sea water. Endogenousspontaneous myometrial activity in early preg-nancy when circulating progesterone levels areelevated is relatively low, suggesting progest-erone may modulate contractions. The fre-quency of spontaneous uterine contractions bothin vivo and in vitro were inhibited in late preg-nancy by administration of homologous, ova-rian-derived, dogfish relaxin. This effect waspotentiated by estradiol. The proposed modelexplaining these observations includes proges-terone inhibition of uterine contractions in earlypregnancy. Attenuated uterine contractionswould protect the encapsulated embryos earlyin pregnancy. Estrogen-facilitated relaxin mod-ulation of the frequency of contractions in latepregnancy would allow myometrial generateduterine flushing but at the same prevent earlyparturition (Sorbera and Callard, ’95).

The uterus in viviparous species not only retainsand harbors the developing fetuses, but also differ-entiates pathways for regulating the intrauterinemilieu. Vascularization of the uterine endometriumis presumed to mediate oxygen transfer, ionic andosmotic homeostasis, and waste removal. In matro-trophic species, morphological and biochemicalmechanisms for providing nutrients operate throughmuch of gestation, but function principally after the

yolk reserves have been depleted. Placentae oper-ate in matrotrophic sharks to mediate nutrienttransfer. Trophonemata are the immediate sourceof nutrients in rays. Given the abundant observa-tions on these structural specializations in a vari-ety of viviparous species, it is surprising thatnothing is known about the endocrine control of thespecific mechanisms involved. It is not knownwhether ovarian derived endocrine factors regulateuterine vascularization, morphogenesis and activ-ity of placentae, or development and metabolism oftrophonemata. Only correlative studies on circulat-ing steroid titers and reproductive tract dynamicscan provide data for speculation. Delineating endo-crine control mechanisms will necessarily rely on abetter understanding of the physiology and biochem-istry of these inadequately investigated tissues andprocesses.

In all three viviparous species in which circu-lating steroids have been accurately tracked withreproductive events, progesterone titers are el-evated during the initial phase of pregnancy(Tsang and Callard, ’88; Manire et al., ’95; Snelsonet al., ’97). As indicated above, elevated progest-erone may function to inhibit hepatic vitellogeninproduction and thereby follicle and oocyte develop-ment. Progesterone may also inhibit either directlyor indirectly ovarian estradiol and testosterone syn-thesis and be responsible for the lowered levels ofthese steroids while progesterone is elevated. Asprogesterone rises in the bonnethead shark and At-lantic stingray, estradiol levels decline. Althoughprogesterone levels are elevated, estradiol and tes-tosterone levels are low. Progesterone may alsofunction to regulate uterine metabolic activity aswell; however, no evidence exists to suggest whichactivity might be affected by progesterone, espe-cially since the physiological function of the uterusduring this time remains an open question.

Estradiol titers rise during late pregnancy inSqualus acanthias, Sphyrna tiburo, and Dasyatissabina. This elevation is particularly significantin the spiny dogfish and Atlantic stingray, sug-gesting estradiol might function to regulate eventsoccurring in utero at this time. As noted above,estradiol in the late pregnant spiny dogfish in-duces hepatic vitellogenin production in order tofuel oocyte maturation in the developing follicles.In the Atlantic stingray, elevated estradiol circu-lates in the absence of developing follicles in latepregnancy, suggesting that pre-vitellogenic fol-licles produce estradiol, which has a collateral rolerelated to gestation. Concurrent with the elevatedestradiol titers is the shift in embryonic nutrient


supply from the yolk reserves to the histotroph pro-duced by the trophonemata. Perhaps estradiol regu-lates trophonemata activity either by modulatingnutrient metabolism in the liver or by controllingthe production of histotroph. In the bonnetheadshark, the small elevation in estradiol levels corre-lates with the formation of the placenta and theresulting shift from yolk to placental exchange asthe principal source of nutrients. As in the Atlanticstingray, estradiol could control availability of cir-culating nutrients for placental transfer.

Taken together, these observations suggest thatone of the major roles of estradiol in elasmobranchreproductive cycles is to regulate nutritional sup-ply for the developing offspring. The initial invest-ment in the oocyte is yolk derived from estradiolmediated hepatic vitellogenin production. In ovipa-rous species such as Raja erinacea, yolk is thesole source of nutrients, and estradiol levels re-main low during egg retention in utero, except incases in which growth of the subsequent set offollicles impinges on egg retention, e.g., in Rajaeglanteria. In the lecithotrophic viviparous spe-cies that are also continuous breeders, the spinydogfish being the best example, estradiol-inducedvitellogenesis takes place in the latter half of preg-nancy. Follicle development in the bonnetheadshark and Atlantic stingray does not take placeduring pregnancy. Control of nutrient apportion-ing by estradiol has shifted from yolk productionfor oocytes to induction of nutrient supply directlyto the developing embryos in utero.

Finally, oviposition and parturition require al-terations in the properties of the uterine cervixjoining the uterus with the urogenital sinus. Thecervix maintains the isolation of the uterine con-tents from the urogenital sinus and external en-virons. In the little skate, the cervix is inextensibleduring egg retention, but becomes compliant atoviposition. Based on experimental studies, ova-rian derived relaxin may be involved in regulat-ing cervical compliance in Raja erinacea. In vivotreatment with the structurally related analogue,insulin, caused a significant increase in the ex-tensibility of the cervix, and estradiol potentiatedthe insulin effect (Callard and Koob, ’93). In thespiny dogfish, insulin had similar effects on ren-dering the cervix compliant and, in addition,caused early delivery (Koob et al., ’85). Thus, regu-lation of cervical compliance is critically impor-tant in maintaining pregnancy in this viviparousshark. It seems likely that similar structures op-erate in all viviparous species to control not onlyingress of environmental elements but also the

proper timing of parturition. We predict that thecervix is a structure common to all elasmobranchsthat functions to maintain the integrity of theuterine contents and that endocrine factors arepivotal for regulating its mechanical propertiesand therefore the proper timing of oviposition andparturition.

SUMMARYThe follicular compartments of the elasmo-

branch ovary synthesize and secrete the predomi-nant circulating steroids, estradiol, testosterone,and progesterone, in temporally circumscribedpatterns during reproductive cycles. Follicles pro-duce estradiol and testosterone during oocyte de-velopment; corpora lutea produce progesterone.Although circulating estradiol and testosteroneare elevated, progesterone levels remain low.When progesterone levels increase, estradiol andtestosterone titers decline. Estradiol and testoster-one are associated with follicle development;progesterone is associated with egg retention andpregnancy.

Circulating estradiol titers are elevated duringthe phase of reproductive cycles when nutrientsare being supplied to the future progeny, eitherin the form of yolk to the developing oocytes, oras yet uncharacterized nutrients through placen-tae or trophonemata to developing embryos inutero. Empirical evidence has established that es-tradiol induces hepatic vitellogenin synthesis andthereby yolk accumulation in the developing oo-cyte during the follicular phase of ovulatory cycles,regardless of when during the cycle follicle devel-opment occurs. Less evidence is available to as-cribe a direct role to estradiol in regulatingnutrient availability to the developing embryos inutero in matrotrophic species. However, elevatedestradiol titers during pregnancy in these speciesoccur in the absence of oocyte development, sug-gesting that estradiol may influence embryo nu-trition after the yolk is depleted.

The reproductive tract must be prepared beforeovulation to process and house eggs and embryos.Differentiation and morphogenesis of the reproduc-tive tract occurs during the follicular phase of thecycle in both oviparous and viviparous species.Estradiol is elevated during this time and is un-doubtedly involved in regulating many of these re-productive tract functions. Estradiol plays a role inregulating nidamental gland morphogenesis andcapsule precursor biosynthesis; however, the exactmechanisms involved are not clear. Estradiol maycontrol the metabolic activities and physicochemi-


cal properties of the isthmus connecting the nida-mental gland with the uterine portion of the repro-ductive tract. Estradiol could be involved in inducingpre-ovulatory morphogenesis of the uterine special-izations like vascularization, connective tissue andmuscle properties, and endometrial cell activity.However, at present, we have little direct evidenceto assign any specific function for estradiol in re-productive tract morphogenesis.

Progesterone titers are elevated around thetime of ovulation in both oviparous and vivipa-rous species, continuous and seasonal breeders.In oviparous species, transitory elevations in cir-culating progesterone precede ovulation; levelsremain low before encapsulation, egg retention,and oviposition. In viviparous species, circulat-ing progesterone remains elevated through theinitial stages of pregnancy. Progesterone inhibitshepatic vitellogenin production and consequentlyoocyte development, suggesting that it delays thesubsequent ovulatory cycle during egg retentionin utero. It appears that progesterone is requiredto maintain the initial phase of pregnancy. Howit does so remains an open question.

Figure 7 summarizes these conclusions and pre-sents a model for endocrine regulation in femaleelasmobranchs. This is a working model that wehope will focus attention on the critical need formore investigations on reproductive mechanismsat all biological levels. Physiological and biochemi-cal studies on essentially every aspect of repro-duction are needed before definitive experimentaland molecular approaches to endocrine regulationcan be devised. More information on the mecha-

nisms utilized to supply nutrients to the develop-ing oocyte, but more importantly, on the nature,source, and means of providing nutrients to thedeveloping embryos in utero are clearly needed.Feedback mechanisms that operate through theendocrine system and involve signals from the re-productive tract and embryos must be examined.Endocrine-mediated environmental influences onreproductive cycles are clearly evident but littleexplored. We have only just begun to lay thegroundwork for understanding the reproductiveendocrinology of the diverse reproductive modesoperating in contemporary elasmobranchs.

ACKNOWLEDGMENTSWe thank Charlie Manire and Jim Gelsleichter

for the invitation to participate in the symposiumand contribute to this volume. The empirical workon the little skate and spiny dogfish reviewed herewas supported in large part by the National Sci-ence Foundation. Manuscript preparation was ac-complished under the auspices of the Shriners ofNorth America.

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reproductive endocrinology of female elasmobranchs: lessons from the little skate (raja erinacea)...

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