Develop. Growth and Differ., 29 (l), 1-12

8/14/2019 Develop. Growth and Differ., 29 (l), 1-12

1/12

Develop . Growth and Differ., 29 ( l ) , 1-12 (1987)

Review17a , 20~-Dihydroxy-4-pregnen-3-oneA TeleostMaturation-Inducing Hormone(17a,20~-dihydroxy-4-pregnen-3-one/maturation-inducingormone/oocyte maturation/ovarian folliclelteleosts)YOSHITAKA NAGAHAMALaboratory of Reproductive Biology, National Institute forBasic Biology, O kaza ki 444, Japan1. Introduction

In most vertebrates, full-grown postvitellogenic oocytes in the ovary are still in prophaseI of meiosis an d canno t be fertil ized. Fo r th e oocytes to be fertil ized they must und ergo finaloocyte ma turation . This process consists of t he breakdown of th e germin al vesicle (GVBD),chrom osom e co nd en satio n, assembly of the first meiotic spindle, and extrusion of the firstpolar body. Th ree major med iators of oocyte maturation have been described in teleostsand amphibians: gonadotropin, maturat ion-inducing hormone, and maturat ion-promotingfactor (1-6). These mediators function sequentially at the levels of the follicle layer, theoocyte surface and th e oocyte cytoplasm (Fig. 1). Am ong these three m edia tors , gonadot ro-pins have been purified in many vertebra te species. In contrast, although m aturation-inducing horm one and m aturat ion-prom oting factor have been known to exist for more than15 years, only a limited progress has been made for their purification and characterization.

O ve r the past several years, a series of studies in our labor atory using a salmonid fish, the

Gonadotropin

Maturation-inducing hormoneI Receptor I

I / .ct

,

Maturationpromoting factor

IOocyte maturation /

Fig. 1 . Hormonal control of oocyte maturation inamphibians and teleosts. Three major mediators,gonadotropin, maturation-inducing hormone andmatu ration-prom oting factor are involved.

1


2/12

2 Y. AGAHAMA

amago salmon, Oncorhynchus rhodurus, as a main experimental organism has providedimportant new information about the chemical nature of maturation-inducing hormone andits synthetic control by gona dotro pin. A s a result of these studies, we have identified thematuration-inducing hormone of amago salmon, for the first time in any vertebrate, as17a , 2Op-dihydroxy-4-pregnen-3-one17a ,20P-diOHprog) (7). It is the purpose of thisarticle to review some of ou r studies o n th e identification of maturation-inducing hormonean d the mechanism by which gonado tropin regulates ovarian synthesis of 1 7 a, 20P-diO Hprog .2.

The primary hormone involved in triggering oocyte maturation in salmonids, as inothe r verteb rates, is gonadotropin (8) . Th e num ber of gonadotropins present in the teleostpituitary gland has been debated and is presently unresolved. A glycoprotein-rich gonad ot-ropin with a molecular weight of 25,000-40,000 has bee n purified in several teleosts (9 , 10).This type of gonado tropin, termed ma turat ionalgonadotropin, has been reported tostimulate almost all gonad al activities. W e purified a gonado tropin from a glycoproteinfraction of aceto ne dried pituitaries of the chum salmon, Oncorhynchus ketu,using affinitychromatography o n C on-A S epharose, ion exchange column on D EA E-Seph acel , and gelfiltration in Sephacryl S-200 (11) . The molecular weight of this gonad otropin was estimatedto be 38,000-40,000by gel filtration and that of the two subunits to be ab out 15,000 an d20,000. This gonadotropin induces oocyte maturation and stimulates ovarian and testicularsteroidogenesis in amago salmon. A second gonadotropin, carbohydrate-poor (10) o rcarbohydrate-rich (12) gon ado tropin , has also been purified from t he pituitaries of severalteleosts including chum salmon. It is unkno wn, how ever, whethe r these second gonado tro-pins possess a function different from the maturational go nadotropin. In this review, th eterm gonadotropin refers to the glycoprotein-rich maturational gonadotropin.

Primary Hormone Triggering Oocyte Maturation

3 . Identification of Maturation-Znducing Ho rm on eAlthough it was known for som e time that both gonadotropin and steroid hormones can

induce oocyte maturation and ov ulation in verteb rate oocytes in vivo and in vitro, it was notuntil th e late 1960s that in anu ran am phibians, gonadotropins were found t o stimulate thefollicular tissues to s ecre te a ho rmo ne, maturation-inducing horm one , which in tu rn acts onthe oocytes to initiate maturation. These findings have facilitated investigations of thehormo nal regulation of final oocyte m aturation in a variety of ver teb rate s (13-16). Wh ile thespecific steroid produced by amphibian ovarian follicle cells to induce oocyte maturation inresponse to gon adotropin has not yet be en identified, a variety of evidence strongly suggeststhat the maturation-inducing hormone of amphibians is progesterone or a similar steroid.This hypothesis has been reinforced by the recent results of measurements of blood o r ovarianconcentrations of progesterone during oocyte maturation (17-19).Follicle-enclosed full-grown postvitellogenic oocytes of some teleosts undergo GVBD invitro when they are incubated with gonadotropin. How ever, denud ed oocytes are incapableof responding to gonado tropin (20). Cy an ok eto ne, a specific inhibitor of 3P-hydroxy-n5-steroid dehydrog enase, completely abolished the m aturational effects of partially purifiedchinook salmon gonadotropin (SG-G1 00) and pregnenolone, but not of steroids such asprogesterone or its metabolites (21). Th us, the action of gonadotropin in inducing oocyte


3/12

M A TU RA TI O N - I N D U CI N G H O R M O N E 3

229 269

maturat ion app ears to be depend ent on the synthesis of a second steroidal m ediator of meioticma turation . In agreem ent with this, we have shown that defolliculated oocytes of amago

I

Fig. 2. High-perform ance liquid chroma togra-phy and maturation-inducing (MI) activity of the50% methanol phase. (see NAGAHAMAndADACHI7] or details).

Lt J . - Y II 11 .

5 10 15 20Fraction number (3mVfr)

Sample

5 0 100 150 200 250 300 ( m l e )Fig. 3.pregnen-3-one standard.Mass spectra of fraction 10 (Fig. 2) of th e 50% methanol phase and 17a , 20g-dihydroxy-4-


4/12

4 Y.NAGAHAMAsalmon undergo GVBD when incubated in media in which folliculated oocytes had beenpreviously incubated with partially purified chum salmon gonadotropin (SGA, Syndel Lab.)indicating media of folliculated oocytes contain a factor(s) which induces meiotic maturation.We then attempted to purify and chemically characterize the natural maturation-inducinghormone of amago salmon from these incubation media (7). In this study, maturation-inducing activity of residues at various steps of purification was assessed by a homologous inv i m assay. Ether extracts of the media from the incubates showed high maturation-inducingactivity. Yolk and oil droplets were first removed from the ether extract by partition withequal volumes of 50% methanol and n-hexane. Maturation-inducing activity was found onlyin the 50% methanol phase. Reversed-phase high performance liquid chromatography(HPLC) was then employed to fractionate the 50% methanol phase. Of the 20 fractionsprepared, only one fraction exhibited maturation-inducing activity (Fig. 2). This fractionhad a retention time that coincided exactly with authentic 17a, 2OP-diOHprog standard anddiffering appreciably from the retention time of the other steroid standards including 17a, -20a-dihydroxy-4-pregnen-3-one.The purity and final characterization of the residues of thisfraction were further confirmed by a comparison with authentic 17a,20P-diOHprog using thinlayer chromatography and mass spectroscopy. The mass spectrum of the fraction wasidentical to that of standard 17a, 20P-diOHprog, the molecular ion peak being at m/e 332 andbase peak at m/e 287 (Fig. 3).

17a, 20P-DiOHprog was first identified in the plasma of mature adult sockeye salmon(22). In collaboration with Dr. A. Kambegawa (Teikyo University), a specific radioimmu-noassy for 17a, 20P-diOHprog was developed in our laboratory and has been applied tomeasure blood concentrations of this steroid during the sexual maturation of female amagosalmon (23). 17a, 20P-DiOHprog levels were less than 0.5 ng/ml in vitellogenic females(June-September) and in those with full-grown immature oocytes collected in early October.A dramatic increase (50-70 ng/ml) occurred in mature and ovulated females collected in midOctober. This increase in plasma 17a,20P-diOHprog levels correlated well with a dramaticrise in plasma gonadotropin levels. The peak of plasma 17a, 20P-diOHprog levels was

PrecursorFH3

.- OHPo1 7 0 hydroxyprogesterone

Fig. 4. Maturation-inducing hormone of amago sal-mo n. 17a ,20,9-Dihydroxy-4-pregnen-3-ones synthe-sized from its precursor, 17a-hydroxyprogesterone, bythe action of the enzyme 208-hydroxysteroid dehyd-rogenase.

1 7a,20p-dihydroxy-4-pregnen-d-one


5/12

MATURATION-INDUCING HORMONE 5

observed 2-4 days prior to ovulation, coincident with the occurrence of GVBD of oocytes.The relative effectiveness of a range of pregnene derivatives in inducing GVBD wasinvestigated in vitro using amago salmon oocytes. Of the steroids tested, 17a,20P-diOHprog was found to be the most effective inducer of GVBD (24). Taken together, theseresults indicate that 17a,20P-diOHprog is the major naturally occurring maturation-inducinghormone in amago salmon (Fig. 4). Further investigations from our laboratory and otherssuggest that 17a, 20P-diOHprog functions as the maturation-inducing hormone common toseveral species of salmonids (25-31). It is also possible that 17a, 20P-diOHprog acts as animportant steroidal mediator of oocyte maturation in some nonsalmonid teleosts (20, 24,32-39).4. Mechanisms of Gonadotropin Regulation of Follicular 17a, 20P-DiOHprog BiosynthesisA. Two-cell type model

The identification of the maturation-inducing hormone in amago salmon has facilitatedresearch on gonadotropin control of ovarian synthesis of this steroid. Significantly elevatedlevels of 17a, 20P-diOHprog were found in media in which folliculated oocytes of amagosalmon were induced to mature in vitro with gonadotropin (23,40), suggesting that the majorsource of ovarian 17a, 20P-diOHprog biosynthesis is follicular tissue.

Ovarian follicles of teleosts, like those of other vertebrates, are composed of two majorlayers, an outer thecal layer and an inner granulosa layer, which are separated by a substantialbasal lamina (41). The thecal layer consists of fibroblasts, collagen fibers and capillaries, andin some species, large glandular cells known as special thecal cells, whereas the granulosalayer consists of a single layer of uniform cells. Special thecal cells possess the ultrastructuralcharacteristics of a steroid-producing cell, such as abundant smooth endoplasmic reticulum,mitochondria with tubular cristae, and lipid droplets. A restricted occurrence of varioussteroidogenic enzymes has been histochemically demonstrated in these cells. In contrast,granulosa cells contain organelles suggestive of protein synthesis, i. e., rough endoplasmicreticulum, Golgi apparatus, and mitochondria with lamellae cristae, although histochemical

GonadotropinI GTH-receptor I

CholesterolPregnenolone

17a -Hydroxyprogesterone

0 17a-Hydroxyprogesterone3G

POP-HSD

0

h

Fig. 5. Two-cell type model for theproduction of 17a , 20b-dihydroxy-4-pregnen-3-one in the ovarian follicle ofsalmonids. (see the text fo r details).


6/12

6 Y . NAGAHAMAevidence suggests the presence of steroidogenic enzymes in these cells of several teleosts.

Development of a simple dissection technique to separate the ovarian follicles ofsalmonids into two layers, the thecal and granulosa layers, has made it possible to elucidatethe relative contributions of each layer and gonadotropin in the overall process of 17a, 20p-diOHprog production in two species of salmonids, amago salmon and rainbow trout, Salmogairdneri (8, 42, 43). Intact follicles and co-culture preparations produced large amounts of17a, 20P-diOHprog in response to salmon gonadotropin. Neither isolated thecal norgranulosa layers alone were capable of producing substantial amounts of 17a, 20P-diOHprogin response to gonadotropin, although in some cases thecal layer preparations producedsignificant amounts of 17a, 20P-diOHprog in response to gonadotropin (SGA or SG-G100).These results indicated that both thecal and granulosa layers are involved in the gonadotro-pin-induced follicular production of 17a, 20P-diOHprog. Measurement of 17a-hydroxyprogesterone concentrations of media from the same experiment revealed thatisolated thecal layers produced large quantities of 17a-hydroxyprogesterone, ut granulosalayers did not respond to gonadotropin. In contrast, levels of 17a-hydroxyprogesterone inmedia from intact follicles and co-culture incubations, peaked at 12 hr and rapidly decreasedconcomitant with a rapid rise in 17a,2OP-diOHprog levels. The presence of 20p-hydroxysteroid dehydrogenase (20P-HSD), the key enzyme involved in the conversion of17a-hydroxyprogesterone to 17a, 20P-diOHprog, has been demonstrated in the granulosalayers, since this layer produced 17a, 20P-diOHprog when incubated with exogenous17a-hydroxyprogesterone. When all these in vifrodata are combined, a two-cell type modelhas been proposed, for the first time in any vertebrate, for the follicular production ofmaturation-inducing hormone, which is summarized in Fig. 5 . In this model, the thecal layerproduces 17a-hydroxyprogesterone that traverses the basal lamina and is converted to17a, -20P-diOHprog by the granulosa layer where gonadotropin acts to enhance the activityof20p-HSD. However, the extent to which thecal layers contribute to the production of17a, 20P-diOHprog is still not clear, since in some cases thecal layer preparations producedthis steroid in response to gonadotropin. Although our granulosa layer preparations arecompletely free of attached thecal layers, it is extremely difficult to obtain pure thecal layerpreparations from postvitellogenic follicles.

In previous in vitro studies, we have presented data indicating a similar interaction ofthecal and granulosa layers for the production of estradiol-17P by ovarian follicles ofsalmonids during vitellogenesis (44, 45). In this model, the thecal layer contributes toestradiol-17P production by synthesizing two aromatizable androgens (androstenedione andtestosterone) which are aromatized in the granulosa layer to estradiol-17P. Thus, theproduction of two biologically important steroids, estradiol-17P and 17a, 20/?-diOHprog, insalmonid reproduction depends on the interaction of the thecal and granulosa cell compart-ments. It is of particular interest that in both cases the thecal layer secretes precursorsteroids, whereas the granulosa layer is the principal site of the conversion of the precursors tothe physiologically active steroids. Similar two-cell type models for follicular estradiol-17Pbiosynthesis have been demonstrated in several species of mammals (46).

Studies of in v i m estradiol-17P and 17a, 20p-diOHprog production by amago salmonfollicles at different stages of development showed that vitellogenic follicles predominantlysecrete estradiol-17p in response to gonadotropin stimulation (47), whereas the capability of


7/12


the intact follicles to respond to gonadotropin by synthesizing and secreting 17a, 20P-diOHprog was acquired immediately prior to th e natural m aturatio n period of oocytes (23).These in vitro data is consistent with the observed shift from estradiol-17P to 17a,20P-diOHprog in plasma immediately prior to or during oocyte ma turation . In the light of thenewly proposed two-cell type model, we have attempted to explain mechanisms involved inthis steroidogenic shift. O ur previous in vitro studies clearly showed tha t a sha rp decrease inthe activity of the aromatizing enzyme in granulosa layers occurred after the completion ofvitellogenesis (48). In view of th e enh anc ed ability of the thecal layer to produce testoster-one in postvitellogenic follicles (47), the d ecre ased follicular secretion can b e explained by thedecreased arom ata se activity in th e granulosa layer. This is in contrast to the situationreported in mammalian ovaries where the decreased secretion of estradiol-17P immediatelyafter ovulation has been explained by a diminished supply of aromatizable androgens d ue todecreased activity of ovarian 1 70-hydroxylase an d C17-C20 lyase (49). In amago salmon,gonadotropin caused a slight, but significant stimulation of 20P-HSD activity (enh ance dconversion of exog enous 170-hyd roxyproge sterone to 17 a, 20P-diO Hprog ) in granulosa cellscollected from mid-vitellogenic follicles. How ever, th e thecal layer only begins secreting17a-hydroxyprogesterone immediately prior to the maturation of oocytes in vivo (Kanamor i ,unpub lished). Resu lts from these in vitro studies can be in terprete d to m ean that in additionto 20P-HSD activation in the granulosa cells by gona do trop in, th e availability of 1 7a -hydroxyprogesterone may also play a m ajo r role in th e enhancing action of gonadotropin onfollicular biosyn thesis of 17a , 20P-diOHprog.B . Gon adotropin receptors

In th e two-cell type model fo r the production of 17a, 20P-diOHprog described above,gonadotropin has at least two sites of action, the thecal an d granulosa layers. This suggeststh e existence of gonad otropin receptors in both cell layers. W e have investigated gonad otro-pin binding to both thecal and granulosa layers of am ago salmon. Purified chum salmongonadotropin (11) was iodinated with the aid of Iodogen (1, 3, 4, 6-tetrachloro-3a,6a-dipheny l glycoluril). Cr ud e me m bra ne preparations derived from 23,000 g pellet ofhomogenates of thecal or granulosa layer preparations were obtained from postvitellogenicamag o salmon follicles. T he binding studies with 121- chum salmon gonadotropin d emons-trated its specific binding to both thecal and granulosa layers. T he inability of other peptidehormones of chum salmon such as growth hormone, prolact in and a-MSH to competeeffectively with iodinated chum salmon gonadotropin for binding sites further de mo nstrate sthe high specificity of this receptor. Scatchard analysis of this binding suggests that thecaland granulosa layers possess a single class of binding sites with similar characteristics(Kanam ori, unpub lished). T he physiological significance of the existence of these bindingsites in thecal and granulosa layers is unknown and is the subject of further investigation.

C . Adenylate cyclasekyclic A M P systemThe earliest effect of gonadotropin on amago salmon thecal layers is receptor-mediatedactivation of adenylate cyclase and formation of cyclic AMP (Kanamori, unpublished).Go nad otrop in action to enhance 17a-hydroxyprogesterone production by the thecal layer wasmimicked by forsk olin, an aden ylate cyclase activator, cyclic A M P analogs an d phosphodies-terase inhibitors such as 3-isobutyl-1-methyl-xanthine (IB M X) an d theophy lline. Bothgonadotropin and forskolin caused a rapid accumulation of cyclic AMP in the thecal layer


8/12

8 Y . NAGAHAMA

with maximal levels at 30-60 min (Ka nam ori, unpublished). Th ese findings are consistentwith the view tha t cyclic A M P is the second m essenger for gon ado tropin action in th e thecallayer. O u r preliminary studies furth er suggest the majo r site of action is at the steroidogenicstep between cholesterol and pregnenolone.

Th e granulosa cells of salmonids provide a unique system in which to study go nad otrop inregulation of a steroidogenic enzy me , since precursor steroids cann ot be prod uced by thesecells. This system allows th e indirect quantification of 20P-HSD activity by the mea sure-ment of 17 a, 20P -diOH prog when these cells are incubated with exog enous 17 a-hydroxyprogesterone. T he functional role of the adeny late cyclasekyclic A M P system in thegonadotropin-induced activity of 20P-HSD in the ama go salmon granulosa cells has beeninvestigated (50). When granulosa cells were incubated with forskolin in the absence of17a-hydroxyprogesterone, 17 a, 20P-diOHp rog levels were very low. In contras t, in thepresence of 17a-hydroxyprogesterone,he production of 1 7a , 20P-diOHp rog was strikinglystimulated by forskolin. Similarly, dibutyryl cyclic A M P and phosphod iesterase inh ibitorsstrikingly enhan ced the production of 1 7a, 20P-diOHp rog w hen 17a-hydroxyprogesteronewas added to the incubation media, whereas dibutyryl cyclic GMP had no such action.Fur therm ore, both go nadotrop in and forskolin caused a rapid accumulat ion of cyclic AM P inthe granulosa layer with maximal levels at 30-60 min (Ka nam ori, unpublished). Ta ke ntogether, these findings suggest that in the amago salmon granulosa cells, the adenylatecyclase/cyclic A M P system acts as an intracellular mediator in the activation of 20P-HSD bygonadot ropin.D . Protein and R N A synthesis

A n extremely pe rtinen t area concerns th e genetic contro l of follicular biosynthesis of thematuration-inducing horm one . A t the present t ime there exist n o relevant da ta on this topic.We have examined the effects of inhibitors of protein synthesis (cycloheximide an d purom y-cin) and RNA synthesis (actinomycin D, cordycepin and a-aman it in) on the product ion of17 a, 20P-diOHprog and i ts precursor, 17a-hydroxyprogesterone by gonad otropin (SG A) inthe amago salmon follicles (51). Both RNA synthesis and protein synthesis inhibitorsblocked gonadotropin-induced 17 a, 20P-diOHprog production by intact follicles. Th eseresults co rrelate well with earlier findings in rainbow trou t which show ed tha t go nad otrop in-induced GVBD in vitro was inhibited by both transcriptional and trans lational inhibitors (37).Th us, the observed inhibition of GV B D can apparently b e accounted for by the suppressionof foll icular 17 a, 20P-d iOH prog production by these inhibi tors. In contrast , gonad otropin-induced 17a-hydroxyprogesterone production by intact follicles was not abolished by acti-nomycin D, but was abolished by cycloheximide. Nev ertheless, in rainbow trou t actinomy-cin D was repo rted to be effective in blocking gonadotropin-induced G V B D (37). Th us , i t islikely tha t in salmonids the significance of 17 a-hydro xypro gesterone is its position as theimmediate precursor o f 1 7a , 20P -diO Hp rog biosynthesis rather tha n it serving as a matura-tion-inducing hormone. Fur thermore , these in vitro data suggest that postvitellogenicamago salmon o varian follicles already contain th e R N A s necessary for t he biosynthesis ofprecursors such as 17a-hydroxyprogesterone.Effects of protein and RNA synthesis inhibitors on gonadotropin-induced 20P-HSDactivation were also determ ined (51). In this experim ent, isolated granulosa cells wereincubated with various concentrations of actinomycin D , cordycepin, a -am anitin, cyclohex-


9/12

MATURATION-INDUCING H ORMONE 9imide or puromycin in the presence of bo th SGA and 17a-hydroxyprogesterone, nd 17a,20P-diOHprog levels in the medium were determ ined . Go nad otrop in markedly enhanced17a, 20P-diOHprog production by granulosa cells incubated with 17a-hydroxyprogesterone.The enhancing effect of gonadotropin was completely blocked by all of the five inhibitorsused. Furth er time course studies revealed that RN A synthesis inhibitors added - 1 , O , 3, and6 hr after th e addition of 17a-hy droxyprog esterone an d go na do trop in, totally abolishedgonado tropin-induced 20/3-HSD, but not 9 hr after ho rm on e addition. With cycloheximidetotal inhibition was observed when added in the period of 1 hr before to 9 hr after th e start ofthe incubation. Th ese time course results suggest tha t de n o w synthesis of 20P-HSD in v i min response t o gonadotropin occurs, and consists of gene transcriptional events within the first6 hr of gonadotropin exposure and translational events 6-9 hr after gonadotropin exposure.More recent studies from our laboratory have also shown that both RNA synthesis andprotein synthesis inhibitors totally preven ted dibutyryl cyclic AM P-induced 20P-HSD activa-tion in granulosa cells (N ag aha m a, unpublished). Th us, the se results suggest that gonad ot-ropin causes the de n o w synthesis of 20P-HSD in the ama go salmon granulosa layer througha mechanism depe nde nt on R N A synthesis. Figure 6 summ arizes our working hypothesis for20P-HSD activation by amag o salmon granulosa cells. T he first ste p of the stimulatory

Precursor( 17a-Hydroxyprogesterone)

I I GTH 1. . . I- IYReceptorc Cell membraneAdenylate cyclase

4-pregnen-3-oneI

CAMPeProtein k inasecPhosphorylation-..

mRNA-.~ R N Ak-...

I

-.m=i

CAMPeProtein k inasechosphorylation-..

mRNAINAk-...

I Nucleus

Granulosa cellSecretion

Fig. 6 .cell via the receptor-adenylate cyclase-cyclic AMP mechanism.gonadotropin.Diagram of 20B-hydroxysteroid dehydrogenase activation of the amago salmon granulosaER, endoplasmic reticulum; GTH,


10/12

10 Y. AGAHAMAeffects of gonadotropin on granulosa cel ls appears to be receptor mediated act ivat ion ofadenylate cyclase and formation of cyclic A M P . It is generally believed tha t th e elevatedlevels of cyclic AMP induced by gonadotropin activate specific protein kinases which addphosp hate group s to key regulatory proteins. Since R N A synthesis and protein synthesisinhibitors completely block b oth the gon ado tropin - and cyclic AM P-induced 20P-HSDenhancement , we suggest that gonadotropin promotes de novo synthesis of 20P-HSD by amechanism involving RNA synthesis. Th us , th e induction of 20P-HSD activity by gonad ot-ropin in am ag o salmon granulosa cells is a good example of differentiated functions expressedby the target cells in response to peptide hormone stimulation.5 . Conclusion

Pituitary g on ad otr op in is of primary impo rtance in regulating th e steroidogenic functionsof the gonads. T h e gon ada l steroid s, in turn , exert significant influences, both directly andindirectly, in controlling germ cell growth and maturation. 170,20P-DiOHprog was iden-tified as the ovarian m ediato r of gonadotropin-induced oocyte ma turation in amago salm on.Th e interaction of two en doc rine follicular layers, the thecal an d granulosa layers, is necessaryfo r 17a, 20P-diOHp rog secretion. Th e ma jor action of go nad otrop in for the production ofthis steroid is to enhance the activity of 20P-HSD in th e granulosa cells. This action ofgonadotropin is mediated by the aden ylate cyclasekyclic A M P system. Ho we ver, the natureand the mechanism of action of the intracellular messengers generated by cyclic AMP aretotally obsc ure. Evidence presented in this review suggests that gonadotropin p rom otes denovo synthesis of 20P-HSD by a mechanism involving R N A synthesis. U p to this point , weknow nothing about the molecular aspects of the gene expression process, but i t seems thatthe amago salmon granulosa cell system will provide an excellent model for the studies onmolecular mechanisms underlying gonadotropin-induced changes in cell function and also thegenetic control of cell differentiation.

A t presen t, nothing is known abo ut the mo de of action of 170, 20P-diOHprog to induceoocyte m aturat ion. 170, 20P -D iOH pro g has been foun d to be ineffective in inducing oocytematuration when microinjected into full-grown immature oocytes of goldfish, but waseffective when applied externally (Kishimoto and Nagah am a, unpublished). Th ese da tasuggest that the site of action of steroidal inducers is at the oocyte surface. Certainly, theoocyte surface receptor of 170, 20P-diOHprog merits further study. Whi le our modeldescribed in this article relates specifically to salmonids, it should provide a basis for ourunderstanding at the molecular action of gonadotropin in other vertebrates.

I thank D r. T. F. Hourigan for reading the m anuscript. Th e studies from ou r laborato ry described in this articlehave been aided in part by Grant-in-Aid for special Project Research (Project No. 58119008 and 61 134041) andScientific Research from the Ministry of Education, Science and Culture. Japan.

R E F E R E N C E SI . WASSERMAN,. J . and L. D . SMITH, 1978. In The Vertebrate O vary (ed. R. E.JoNEs), pp. 443-468.2 . Int. Rev. Cytol. , 57, 185-282.3. SCHUETZ, . W., 1979. J . Steroid Biochem.. 11, 695-699.

Plenum Press, New York.M A S U I , . n d H . J . C L A R K E ,979.


11/12


4. KANATANI,. and Y. NAGAHAMA,980.5. MALLER,. L. and E. G. KREBS, 980.6. NAGAHAMA,., 1987.NORRIS nd R. E. JONES),Plenum Press, New York (in press).7. ~ , and S. ADACHI,985.8.-G. YOUNG, . UEDA,H. KAGAWAnd S. ADACHI,985.IWAMOTOnd S. SOWER),p. 8-19.9. BURZAWA-GERARD,., 1982. Biomed. Res., 1, 273-291.Curr. Top. Cell Reg., 16, 271-311.In Hormones and Reproduction in Fishes, Amphibians and Reptiles (eds. D. 0.Develop. Biol., 109, 428-435. In Salmonid Reproduction (eds. R . N .Washington Sea Grant Program, University of Washington, Seattle.Can. J. Fish. Aquat. Sci., 39 , 80-91.

Academic Press, New York.10. IDLER, . R. and T. B. NG, 1983. In Fish Physiology (eds. W. S. HOAR,D. J. RANDALLnd E. M .11.12.

DONALDSON),ol. IXA, pp. 187-221.UEDA,H. , Y. NAGAHAMAnd H. TAKAHASHI,984.KAWAUCHI,., K. SUZUKI, . NAGAHAMA,. ADACHInd N. NAITO, 986.Gland-Structure, Function and Regulation (eds. F. YOSHIMURAnd A. GORBMAN),p. 383-390.Sci. Pub.13. MASUI,Y., 1967. J. Exp. Zool., 166, 365-376.

14. SCHUETZ,. W., 1967.16. SMITH, . D. , R. E. ECKERnd S. SUBTELNY,968.17. FORTUNE,. E.,P. E. CONCANNONnd W. HANSEL, 975.18. MCCREERY,. R. and P. LICHT,1983.19. LIN,Y.-W. P. and A. W. SCHUETZ,985.20. GOETZ, . W., 1983. In Fish Physiology (eds. W. S. HOAR, . J. RANDALLnd E. M. DONALDSON),ol.

IXB, pp. 117-170.21. YOUNG, ., H. KAGAWAnd Y. NAGAHAMA,982. J. Exp. Zool., 224, 265-275.22. IDLER ,D. ., U. H. M. FAGERLANDnd A. P. RONALD, 960. Biochem. Biophys. Res. Commun., 2,133-137.23. YOUNG, ., L. W. CRIM,H. KAGAWA,. KAMBEGAWAnd Y. NAGAHAMA,983. Gen. Comp. Endocrinol.,24. NAGAHAMA,., K. HIROSE,G. YOUNG,. ADACHI, . SUZUKInd B. TAMAOKI,983. Gen. Comp. Endocri-

nol., 51, 15-23.25. CAMPBELL,. M., A. FOSTIER, B. JALABERTnd B. TRUSCOIT, 980. J. Endocrinol., 85, 371-378.26. FITZPATRICK,. S . , G. VANDERKRAAKnd C. B. SCHRECK,986.27. FOSTIER, ., B. BRETON,. JALABERTnd 0 .MARCUZZI,981.28. SCOTT,A. P., E . L. SHELDRICKnd A. P. E. FLINT, 982.29. TRUSCOIT, ., D. R. IDLER,Y. P. So and J. M . WALSH, 986.30. UEDA,H., 0.HIROI, A. HARA,K. YAMAUCHInd Y. NAGAHAMA,984. Gen. Comp. Endocrinol., 53,31. WRIGHT, . S. and S. M. V. HUNT, 982.32. SCOTT, . P. , D. S. MACKENZIEnd N . F. STACEY,984.33. KACAWA,., G. YOUNGnd Y. NAGAHAMA,983.34. STACEY,. E., R. E. PETER,A. F. COOK,. TRUSCOTT,. M. WALSH nd D. R. IDLER, 983. Can. J. Zool.,35. KOBAYASHI,., K. AIDA nd I. HANYU,985.36. LEVAVI-ZERMONSKY,. and Z. YARON,986.37. JALABERT,., 1976.38. IWAMATSU,., 1980. J. Exp. Zool., 211, 231-239.39.40.41. NAGAHAMA,., 1983.42. ~ , 1984. Guma Symp. Endocrinol., 21, 167-182.

Gunma Symp. Endocrinol., 21, 21-35.In Pars Distalis of the Pituitary

Elsevier

Proc. SOC.Exp. Biol. Med., 124, 1307-1310.15. ~ , 1967. J . Exp. ZOO^., 166, 347-354.

Develop. Biol., 17, 627-643.Biol. Reprod., 13, 561-567.

Biol. Reprod., 29, 863-871.Gen. Comp. Endocrinol., 58, 421-435.

Academic Press, New York.

51, 96-105.

Gen. Comp. Endocrinol., 62, 437-451.C. R . Acad. Paris Ser. 111, 293, 817-820.

Gen. Comp. Endocrinol., 46, 444-451.Gen. Comp. Endocrinol., 62,99-110.

203-21 1.Gen. Comp. Endocrinol., 47, 475-482.

Gen. Comp. Endocrind., 56 , 349-359.Bull. Jpn. SOC.Sci. Fish., 49, 1783-1787.

61, 2646-2652.Bull. Jpn. SOC. Sci. Fish., 51, 1085-1091.

Gen. Comp. Endocrinol., 62, 89-98.J. Fish. Res. Board Can., 33, 974-988.

GREELEY,. S. JR., D. R . CALDER, . H. TAYLOR,. HOLS nd R. A. WALLACE,986.rinol., 62, 281-289.YOUNG, ., H. UEDA nd Y. NAGAHAMA,983.IXA, pp. 223-275. Academic Press, New York.

Gen. Comp. Endoc-Gen. Comp. Endocrinol., 52 , 329-355.

In Fish Physiology (eds. W. S. HOAR,D. J. RANDALLnd E. M. DONALDSON),ol.


12/12

12 Y . NAGAHAM A43. YOUNG, . , S. ADACHInd Y . NAGAHAMA,986.44. KAGAWA ,H . , G .YOUNG,. ADACHInd Y. NAGAHAMA,982.45. KAGAWA, . , G. YOUNGnd Y. NAGAHAMA,985.SOW ER) ,p. 20-25.46. DORRINGTON,. H . and D . T. ARMSTRONG,979.47. KAGAWA, . , G . YOUNGnd Y . NAGAHAM A,983.48. YOUNG, . , H . KAGAWAnd Y. NAGAHAMA,983.49 . SUZUKI,. and B . TAM AOKI ,980. Endocrinology, 107, 2115-2116.5 0 . NAGAHAM A,. , H . KAGAWA, . ADACHInd G. YOUNG,985.51 . ___ G . YOUNGnd S . ADACHI ,985.

Develop. Biol., 118, 1-8.Gen . Comp. Endocrinol., 47, 440-448.

In Salmonid Reproduction (eds. R . N. IWAMOTOnd S .Washington Sea Grant Program, University of Washington, Seattle.Recent Prog. Horm. Res., 35, 301-342.

Biol. R eprod., 29, 301-309.Biol. Reprod., 29, 310-315.

J . Exp. Zool., 236, 371-375.Develop. Growth and Differ., 27, 1213-1221.

(Received November 4 , 1986)

Develop. Growth and Differ., 29 (l), 1-12

Documents