8/14/2019 Int. J. 0.,. BioI. 38: 217-229 (1994)

1/13

In t. J. 0 .,. B io I. 3 8: 2 17 -22 9 (1 99 4) 21 7

ReviewEndocrine regulation of gametogenesis in fish

YOSH ITAKA NAGAHAMA*Laborat or y o f Reproduc ti ve B io lo gy , Na ti onal I ns ti tu te f or Ba si c B io lo gy , Okazak i, J apan

ABSTRACT The pituitary.gonadal axis plays an important role in regulating gametogenesis invertebrates. In m ost cases, gonadotropins act through the biosynthesis of gonadal steroid horm oneswhich in turn mediate various stages of gametogenesis. A series of studies in our laboratory usingseveral species of teleost fishes as experimental animals has provided new information about theendocrine regulation of gam etogenesis. including oocyte grow th, oocyte m aturation. sperm atogenesisand sperm maturation. This article briefly reviews our findings on the identification of steroidalmediators involved in each process of gametogenesis, and the sites and mechanisms of action of them ediators. These observations collectively dem onstrate the appropriateness of using teleost fishes asvalid models for examining hormonal influences on gametogenesis. Such models could also haveapplications and validity for vertebrates in general.K EY WORDS: gam flogfllfSis, ~(m ad(JlroJ)il/, steroid horm ones, fiJh

IntroductionIt is w ell e stab lishe d th at in ve rte bra tes, g ona do trop ins are the

prim ary h orm on e to re gula te g am eto ge ne sis. H ow ever I i t a ppea rsthat gonadotropins do not act directly. but work through thegonadal biosynthesis 01 steroid horm ones which in turn m ediatevarious stages of gam etogenesis. Over the last several years, aseries of studies in our laboratory using several fish species asexperimental animals has provided new information about theh orm on al re gu la tio n o f g am eto ge ne sis . T his a rtic le b rie fly re vie wsour results on the horm onal regulation 01 oocyte grow th, oocytem atu ra tio n, s pe rm ato ge ne sis, a nd sp erm matu ra tio n in fish (F ig ,1 ).Gonadotropin

R ece nt stu dies h ave ind icate d th at fish pituitaries, like th ose 0 1o th er ve rte bra te s, se cre te tw o k in ds o f g on ad otro pin s. T wo d istin ctcarbohydrate gonadotropins, designated as G TH -I and G TH -li,ha ve b ee n isola te d a nd cha ra cte rize d fro m p itu itaries o f spa wnin gc hum s almon, Oncorhynchus keta(Suzuki et a l ., 1988a ,b ; Kawauch ie t al., 1989). Each of these gonadotropins consists 01 a. and ~subunits, the B subunits having am ino acid sequence identities of31% . GTH-I and GTH-Ii have also been purified in coho salm on,Onco rh yn ch us k is utc h, (Swanson et al., 1991) and they arechem icaliy sim ilar to the chum salm on gonadotropins. Both GTH -I a n d G TH -II a re ste ro id og en ic (S uz uk i et a l ., 1 98 8c), a nd d iffe r o nlyin their relative potencies. Plasm a levels of G TH -I show an exten-de d in crea se du ring vite llo ge ne sis/sp erm ato ge nesis. an d a d ecli-ne at the time of spawning. On the other hand, plasma levels ofGTH-II are constantly low throughout vite liogenesis/s pe rm ato ge ne sis a nd in cre as e d rama tic ally a tth e tim e o f s p aw nin g

in th e la te a utu mn, T aken log ethe r, i t i s su gg este d th at in a dd itio nto the e stab lish ed ro le 0 1 G TH -Ii du rin g fin al g on ad al m atu ra tio n,G TH -I m ay p la ya ro le d urin g vite lio ge ne sis a nd sp erm ato ge ne sis.In this review, the term gonadotropin is taken to refer to theg lyc op ro te in . ric h m atu ra tio na l g on ad otro pin o r G TH -II.Hormona l re gu la tio n o f o ocyte g rowth (v ite llo gene sis )

Oocytes of nonm am malian vertebrates grow while arrested inth e first m eio tic p ro ph ase . Th e princip al e ve nts re spo nsib le fo r theenorm ous grow th 01 f ish oocytes occur predom inantly during thephase of development termed vitellogenesis, and involve thesequestration and packaging of a hepatically derived plasmap re cu rs or. v ite llo ge nin . in to y olk p ro te in , E stro ge n, e stra dio l-1 7~ inmost cases, produced by the ovary under the influence ofg on ad otr op in s, is in tro du ce d in to th e v as cu la r s ys tem and s timu la te sth e h ep atic s yn th esis a nd se cre tio n o lv ite llo ge nin (F ig . 2 ). In je ctio nw ith p itu ita ry extra cts or va rio us prep aration s o f go nad otro pin sin duces estra diol-1 7B synthe sis a nd su bseq uen t vite llo gen esis.P lasm a levels ot estradio l-17~ and vite liogenesis are positivelycorre lated and increase in parallel. Exogenous estrogen caninduce vitellogenin synthesis in the liver and its appearance in theplasma.

V ite llogenin is selectively taken up from the blood stream byde ve lo pin g o ocytes (W alla ce , 1 98 5). V ite llo ge nin recep to rs w ere

. ibbrn ' ;a l ;mu IHrd jll t h i J /mf"'": I lu.20B- IW. 17( I, :?OlHl ihydrux~+prq::cn-3-one; l ic e. h uman c lu lr io ni c g on ad c) (r op in ; G \ "Bn , h n'.. kc lnw ll o f g e rm in aln '.~ ic 1e ; \IIH . m all lr~ lIi oll -in d1 U:i n~ h nn no l1 c'; \ IP F, m atu ra ti on p ro mo ti ngEu : lo r ; : !Of i-S , [ lu.2011 , ~1 -t rih )c I roxy. '1 -pr t'g ('n .3 -ol \ t' .

-A ddress for reprints: Laboratory of Reproductive Biology. N ational Institute for Basic Biology, O k a z a k i 4 44 , J ap an . F A X : 81-564-55-1556.0214-6282/94/503.00e UHC Pr~"f'rinL~din Spain

8/14/2019 Int. J. 0.,. BioI. 38: 217-229 (1994)

2/13

21X Y. Nuguhu/I1u

Cholesterolt tPregnenoloneH(Progesterone)~ 1 \",

o#C"

CH ,

oc&d'OH17a,20p-Dihydroxy-4-pregnen..3.one(Oocyte maturation,Sperm matu ra tion )

-7a..Hydroxyprogesterone -----.., ,Androstenedionep C"

oC66Testosterone/"'-..

oays HO~1 1-K etotestosteron e E strad iol-171 3(Sperm atogenesis) (O ocyte grow 1h)

Fig. 1. Steroidogenic m ediators of oocyte growth. oocyte maturation.spermatogenesis and sperm maturation in teleosts.

characterized in isolated oocyte membranes of coho salmon(Stifanl e l a l., 1990) , t ila pia , O reoc hr om is n ilo tic us (Chan et al..1991) and rainbow tro ut, O nco rh ynch us m ykiss (L an cas te r an dTyler, 1991; Le M enn and N unez R odriguez, 1991). S aturation andScatchard analyses reveal only a single class of binding site. Intilapia, the num ber and affinity of these bindings greatly increasef r o m the previtellogenic to the vitellogenic stage, and rem ainunchanged until the preovulatory stage at w hich tim e the affinity ofthe bindings is also highest.Several hormones. s u c h as gonadotropins, thyroxine,triiodothyronine. i n s u l i n , and growth hormone, have been impl ica tedin regulating oocyte grow1h, although the role of any ot thesehormones has ye t to be determ ined. GTH-I, but not GTH-II,stimulates in v itr o upt ake o f v ite ll og en in i n t o ova rian f olli cle s o fi nt ac t v ite llo geni c r ain bow tr ou t, a s we ll a s i n v it ro i nco rpo ra tion o fvitellogenin by partially denuded follicles (Tyler, 1991). Usingfo llic le -fr ee o oc yte s o f ra in bow tr ou t, w e h av e s hown th at in su linand thyrox ine s ign if icant ly inc rease v it el logen in up take (Sh iba ta etal., 1993). In contrast, neither chum salmon GTH-I nor GTH-II iseffective. M ature oocy1es incorporate only 1/15 of vitellogenin.In su lin a ls o h as n o e ttec t o n th e m atu re oo cy1 es . It is h yp othe siz edfrom these findings that the previously observed stimulation ofvite llo ge nin u pta ke in to in tac t fo llic le s o r p artia lly de nu ded o ocy te sof rainbow trout is most likely mediated by the actions ofgonadotropins on t he ova ri an fo ll ic le s.Es trad io l-1713 p roduc tion

The te leos t o va ry consi sts o f oogonia , o ocyte s, and sur roundingfo llic le c ells , s up po rtin g tis su e o r s tr oma, a nd v as cu la r a nd n erv etissue. W ith the grow1h of the oocytes, follicle cells m ultiply and

form a continuous follicular layer (granulosa cell layer).Simultaneously, t h e s u rr o un d in g s t r o m a l connective t is s u e e le m e n t sa l s o b e c o m e o r g a n iz e d to f o rm t h e d i s t in c t o u t e r la y e r of thef ollic ula re nvei ope ( th e the ca l l ayer ). The re fo re , v it el lo genic oocytes ,like those of other vertebrates, are composed of two major celll a y e r s , a n o u te r t h e c a l c e l l l a ye r a n d a n in n e r g r a n u l o s a c e ll l a ye rwhich are separated from each other by a distinct basementm em brane. The thecal layer contains fibroblasts, collagen fibers,and capillaries, and, in some species, special thecal cells(N agaham a, 1983). A sim ple dissection technique to separate theovarian follicles of salm onids into two cell layers, the thecal andgranulosa layers, has m ade it possibie to elucidate the role of eachla ye r a nd g on ad otro pin s in th e o ve ra ll p ro ce ss o f s te ro id p ro du ct io n(Kagawa e t a l., 1 98 2). O ur in v ilr o stud ies in dic ate th at th e the ca la n d g ra n u lo s a c e ll la y e r s c oo p e ra te in th e p ro du c t io n of stero idalm ediators of oocyte grow 1h and m aturation (see below ).

A two -c ell ty pe mod el in th e p ro du cf io n o f fo llic ula r e st ra dio l-1 7Bhas been proposed. In this m odel, the thecal cell layer, under thei n f lu e n c e o f g o n a d o t r o p i n , s e c r e t e s t h e a n d r o g e n s u b s t r a t e(pro ba bly te sto ste ro ne) w hich d iffu se s into th e g ra nulo sa c ell la ye rw here the arom atase is localized exclusively (K agaw a el al., 1 9 8 5 ;Nagaham a, 1987b; Adachi el al., 1 99 0). R es tric te d d istrib ution o tt e s t o s te r o n e a n d e s t r a d i o l - 1 7 B w a s c o n f i rm e d immuno-histochem ically in vitellogenic ovarian tollicles of rainbow trout(Schulz, 1986). W e have found that the thecal cell layer fromama go sa lm on (O n co rh yn chus r hodu ru s) and the granulosa celllayer from rainbow trout could produce the sam e effect as has beenreported using a com bination ot thecal and granulosa cell layersf r o m th e s a m e s p e c ie s . T h e r e c i p r o c a l u s e o f a m a g o s a lm o ng ran ulo sa a nd ra inb ow tro ut th eca l c ell lay ers is also e ffe ctiv e. T hisfinding im plies that there m ay be little species specificity ot each ofth es e c ell la ye rs amo ng s alm on id s (N ag ah am a, 1 987 b). H ow ev er,the tw o-cell type m odel described above does not seem to be validf or t he Fundulus heleroc l itus(k i ll i tish) and medaka , Oryz ias l al ipes.In these species, in w hich special thecal cells are not evident in thethecal layer, the follicle cells (granulosa cells) are the m ajor site ofsteroid synthesis and the production of estradiol-17B does notrequire the involvem ent of tw o cell types (O nitake and Iw am atsu,1986; Pet ri no et a l. , 1 989) .

Gon ad ot ro pin a cts o n th ec al c ell la ye rs to s tim ula te te sto st ero nep rod uc tio n th ro ug h a re ce pto r-m ed ia ted a den yl a te c yclas e-c AMPs y s t e m . M o r e r e c e n t s t u d i e s h a v e d e m o n s t r a t e d t h a t o t h e ri n t r a c e l lu la r s i g n al l in g m o le c u l e s i n c lu di n g c a lc iu m , c a lm o d u li n .p ro 1 e i n k in as e C , a n d a r a c h id on ic a c id a r e i n v o lv e d i n g o na d ot r o pi n -in du ce d te stos tero ne pro du ction by inta ct pre ov ula to ry fo llic le s ofgoldfish, Ca ra ss iu s aura lu s (Van Der Kraak, 1990, 1991; Van DerKraak and Chang, 1990). It is interesting to note the presence ofgonadotropin receptors in the granulosa cell ot amago salmonv it ello ge nic fo llic le s. it w as fo un d th at in th is s pe cie s g on ad otr op in sa nd s ev era l a g e n t s k n o w n t o in c r e a s e in t r a c e l l u l a r c A M P le v e lfailed to enhance aromatase activity in granulosa layers at alldevelopmental stages. Nevertheless, aromatase activity ingranulosa cell layers m arkedly increased during vitellogenesis.Thus, the mechanism ot the activation of the amago salmong r a n u l o s a c e l l a r o m at a s e s y s t e m i s u n k n o w n a t p r e s e n t .

W e a ls o in ve s t ig a te d t h e m e c h an is m o f a ro m at a s e a ct i v a t io n inearly vitellogenic follicles of medaka, a species in which thes e q u e n c e o f e v e n t s s u c h a s v i t e l l o g e n e s is , o o c y t e g r o W th . m e io t icm aturation, and ovulation can be tim ed accurately (Sakai ef al.,1987). Using this model, we have demonstrated a progressivein cre as e in a roma ta se a ctiv it y, a ss es se d in dir ec tly b y t he c on ve rs io n

8/14/2019 Int. J. 0.,. BioI. 38: 217-229 (1994)

3/13

o f e xo ge no us te sto ste ro ne to e stra dio l~ 17 B, in v ite llo ge nic fo llic le sisolated betw een 28 and 20 h before spaw ning (S akai et al " 1988),In this species, aromatase activ ity is markedly enhanced bypreg nan t m are se ru m go na dotrop in (PMSG) a fte r 18 h of incub atio no f v ite llo ge nic to llicle s iso la te d a t 3 2 h b eto re sp aw nin g, T his a ctio nof PMSG is m im icked by forskolin and dbcAMP which are knownto raise the cellular level ot cA MP , This enhancing effect of PM SG ,forskolin and dbcA MP on arom atization is com pletely inhib ited byactinomycin 0 and cyclohexim ide, These results suggest thatPMSG ind uce s a ro matase activ ity in m ed aka vite llo ge nic fo llic le svia an a de nyla te cyclase~ cAMP syste m, F urthe rm ore th is actio n ofP MS G is dependent upon both transcriptional and translationalp ro ce ss es (N agah ama e t a l., 1991).Oocyte ma tu ra tio n

A p erio d o f oo cyte g ro wth is fo llo wed b y a process ca lle d o ocytem atura tion w hich o ccu rs p rior to o vu latio n a nd is a p re req uisite lo rsuccessful fertilization. Oocyte maturation is defined as there in itia tio n a nd c om ple tio n o f th e first m eio tic d iv isio n, s ub se qu en tprog re ssio n to m etap ha se II. In so me sp ecie s such as g oldfish , thefirst vis ib le event of oocyte maturation is the m igration of thege rm ina l v esi cl e (GV) toward the anim al pole and is often used asan indicator of the onset of oocyte m aturation. The m echanism s ofth is process involve changes in cytoskeleta l networks such asm icro tu bu le d istrib utio n (L essm an et al " 19 88; Jian g et al.,u np ub lis he d). F ollo win g th e m ig ra tio n o f t he G V, s ev era l p ro ce ss esoccur in the nucleus and cytoplasm of the oocyte, including thebreakdown of the germ inal vesicle (GVBD) (which indicates theend of prophase I), condensation of the chrom osom es, form ationof a spindle, and extrusion of the first polar body (w hich m arks thecom pletion of meiosis I) (M asui and C larke, 1979; Goetz, 1983;N agaham a, 1987c). S tudies from our laboratory and others haveindicated that in te leosts, as in am phibians. oocyte m aturation isregulated by three major mediators, namely, gonadotropin,m atu ra tio n~ in du cin g h orm on e (M IH ), a nd m atu ra tio n~ promo tin gfactor (M PF) (N agaham a, 1987c; R edding and P atino, 1993) (F ig.3) .P rima ry media to r of ooc yte ma tu ra tio n - gonadotropin

In v it ro in cu ba tio n s tu die s h av e d emons tra te d t ha t g on ad ot ro pini nd uc es me io ti c ma tu ra tio n in th e fo llic le -e nc lo se d p os tv ite llo ge nicfish oocytes, but no t in defollicu lated oocytes (Goetz, 1983;N agah ama , 1 98 7c ). C ya no ke to ne , a s pe cif ic in hi bit or o f 3B~h yd ro xy ~" ,5 ~s te ro id d eh yd ro ge na se , c omp le te ly a bo lis he d th e ma tu ra tio na le ffe cts o f g on ad otro pin (Y ou ng et al " 1982; D eM an no and G oetz,1 98 7). T ak en to ge th er, th es e fin di ng s s ug ge ste d t ha t g on ad otro pi nstrig ge r m atu ra tio n b y stim ula tin g th e fo llicle ce lls to sy nth es ize a ",4 ~steroid tha t a cts directly on th e oo cyte t o c au se ma tu ra tio n.Seconda ry media to r o f o o cy te matu ra tio n - Ma tu ra tio n-inducinghormone (MIH)

Num erous studies have been conducted to test the effects ofvariou s ste roids o n the in du ctio n of oo cyte m atu ra tio n i n v it ro (seerev iews, Jalaber t, 1976; Iw am atsu , 19 78 ; Fo stier et al., 1983;Goe tz , 1 98 3; N ag ah ama e t a l. , 1983 ; G ree le y e t a l. , 1986 ; Cana ri oand Scott, 1988; Trant and Thomas, 1988). Am ong the C18, C19,and C 21 steroids tested so far, the C21 steroids have alw ays beenrep orte d to h ave m ore p ote nt m atura tlon ~in du cing activ ity tha n the

H orm ones and gam etogencsis injish 21 9other two groups of steroids. The M IH of am ago salmon waspurifie d a nd cha ra cte riz ed from media in whic h immatu re b ut fu ll-g rown fol licu la ted oocytes had been incubated w it h part ia lly puri fi eds almon gonadotro pin (Nagahama and Ada ch i, 1 985). Among 20fra ctio ns s epara te d b y re ve rs ed pha se h ig h perfo rmance liq uidchrom atography, m aturation-inducing activity w as found only inone fraction w hich had a retention tim e coinciding exactly w ith1 7a ,2 0B~d ih yd ro xy~4~p re gn en~3~one ( 17 , 20B~DP) . T he p urity a ndfin al ch ara cte riza tio n o f th is a ctiv e fra ctio n w ere fu rth er c on firm edb y t hin la ye r ch roma to gra ph y a nd mass sp ectrome try w ith a uth en tic17a,20B~DP. Plasm a levels of 17a,20B~DP were found to be highonly in m ature and ovulated am ago salm on fem ales and were verylow or nondetectable (less than 30 pg/m l) in females during therema in in g p erio d o fth eir life (Y ou ng et a l .. 19 83 a).ltw as a lso fo un dtha t a mon g the steroid s te sted , 1 7 ,2 0B ~D P w as th e m ost effe ctiveinducer of GVBD in amago salmon postvite llogenic oocytes(Nagahama e t a l., 1983). 17,20B~ DP was thus identified as thenaturally occurring MIH in am ago salm on (Nagaham a, 1987a).F urth erstu die s from o ur la bo ra to ry a nd o th ers su gg est th at 17a,20B~DP functions as the M IH com mon to several other te leost fishesin clud in g m ost sp ecie s o f s alm on id s (Jalab ert, 19 76 ; G oe tz, 1 98 3;Yamauchi e t a t.. 1984; Canario and Scott, 1988).

Mo re re ce nt ly , 17 a,2 0B ,21 ~ trihy drox y~ 4~p re gn en ~3 ~on e (20 B~S ) has been identified as a naturally occurring M IH of the A tlanticcroaker, a m arine perciform fish (Trant et al.. 1986; Trant andT homa s, 1 98 9a ). in th is sp ec ie s, 20B~S was found to be one of them ost potent steroids to induce G VB D i n v it ro (T ra nt a nd T homa s,1 988 ). E xpo su re of m atu rin g ova ria n follicles to H CG in v it ro greatlyi nc reased t he ir p roduc ti on o f 2 G B . S , w ith a con com itan t in crea se int he r ate o f o oc yte ma tu ra tio n in v it ro . Furthermore, in v it ro productionand plasma levels of 17a,20B~DP during the period of oocytematuration were very Jow in this species (Trant and Thomas,1 989 b). Th ese re su lts stron gly su pp ort th e h yp othe sis th at 20B~S

~tuitaryGonadotropin

Testosterone 0 0 :: :~ O~ ~ T hecal cell ... >::>~-

8/14/2019 Int. J. 0.,. BioI. 38: 217-229 (1994)

4/13

220 Y. Nagalw lI1ai s t h e n a t u r a l , m a jo r M IH in A tla n t ic croaker. The invo lvementof20B-S in the induct ionof oocyte m a tu ra tio n is a ls o suggested in ac l o s e l y r ela te d sp ec ie s. sp otted se atr ou t, C yno sc ion nebo lu su s(T hom as and Trant. 1989). H ow ever, 208-S does not appear to bei n v o l v e d in the induct ionof oocyte m a tu ra tio n in s alm o n id s , s in cet h e r e is no evidence f o r t h e p r e s e n c e o f la r g e a m ou n t s o f t h iss t e r o id in t h e b lo od o f f e m a le s alm o n id s u nd er g oi n g m a tu r a t io n orovu latio n (S co tt and C anario, 1 987).17a ,20B -DP p roduc ti onUsin g in cu ba tio n te ch niq ue s o f is ola te d s almon id fo llic ula r

p r e p a r a t i o n s s im ila r to t h o s e used f o r t h e s tu d ie s o n f o ll ic u l a re str ad io l- 17 8 p ro du cti on , a two- ce ll t yp e mode l has been proposed,f o r t h e f i r s t t im e in a n y v e r t e b ra t e , f o r th e f o ll i c u la r p ro du c ti o n ofMIH. In this model, the thecal cell layer produces 17a-hydroxyprogesterone that traverses the basal lam ina and isc on ve rte d to 17a,208-DP by the granulosa cell layer wheregonadotr op in acts t o enhance t he activ it y o f 208 -h yd ro xy st er oiddehydrogenase (208-HSD), the key enzym e involved in theconve rs ion o f 17a-hyd roxyp roges te roneto 17a,208 -DP (Young etal.. 1986 ; Nagahama, 1987a ,b ).The f irs t s tep o t th e s timu la tin g effect o f gonadot rop in in thecalcell layers i s receptor -media ted activa t ion of adenylate cyclase an dfo rmatio n o f c AMP, th e majo r s ite o f a ctio n p ro ba bly o cc urrin g a tth e s te ro id o g en ic s te p between cho le st er ol and p r e g n e n o l o n ein v o lv in g c h o le s te r o l s id e - c h a in c l e a v a g e e n z y m e s y s t e m s( Kanamor i and Nagahama, 1988a ,b ).The action of gonadotropin on 208-H SD enhancem ent ing ra nu lo sa c ells is m im ic ke d b y forsk olin , b y dbcAMP, but notdbcGMP , and by two phosphodie st era se inhib ito rs (Nagahama etal.. 1 98 5a : K anamo ri a nd N aga hama , 1 988 b). F urth erm ore ,gonadot rop in and forsko lin caused a rapid accumu la tio n o f cAMPw it h maximum leve ls a t 30- 60 m in . These fin dings a re consis tentw ith th e v iew th at c AMP is th e s ec on d mes se ng er ofgonadotropinaction in granulosa c e l l s .

In v it r o e x p e r i m e n t s utilizingboth p r o te in s y n th e s is inhibitors( cy cl oh ex imid e and pur omyci n) an d RNA syn th esis inh ib ito rs(actinomycin D, co rdycep in and a -amani tin ) have revealed tha t thegonadot rop in and cAMP induct ion o f 208-HSD ac ti vi ty in g ranulosac ell la ye rs is d ep en de nt o n th e a ctiv atio n o t n ew RNA and p ro te ins y n th es is . O u r t im e c ou rse s tu die s fu rth er s ug g e s t t h a t d e n o v osynthesis of 208-H SD in vitro in re spo ns e to g on ad otro pin an ddbcAMPoccurs, and consists ofgene transcriptional v e n ts w i t h inthe first 6 h ot exposure to gonadotropin and dbcAMP andt ransc ri pt ional even ts 6 -9 h affer t he exposure to gonadot rop in andcAMP (Nagahama e t a l. , 1985b ). Taken toge ther , th ese re su ltssuggest th at gonadotr op in causes the d e n ov o s yn thes is o f 208 -H SD in th e am a go sa lm on g ra nu lo sa c ell thro ugh a m ec ha nismdependent on RNA syn thes is .S te ro id og en ic s hift o ccu rrin g in fo llic le cells immediatelyp rio r to o oc yte m atu ra tio nT he ca pac ity o f in ta ct fo llic les to p ro du ce e strad io l-1 78 inr esponse to gonado trop in s t i m u l a t i o n increases during oocytegrowth, but r ap id ly dec reases in assoc ia tio n w it h the ability o f t heoocyte to m atu re in re sp ons e to go na do tro pin (K ag aw a et at "1983). Tes to ste rone p roduct io n by t he thecal la ye r p repa ra tio nso bta in ed e ac h mon th d urin g oocyte g rowth a nd matu ra tio n h asrevealed that the capaci ty of the theca llayer to produce testosterone

in re sp on se to g on ad otro pin gra du ally in cre as es d uring th e c ou rseof oocyte g ro wth an d p ea ks du rin g th e p os tv itello ge nic p erio d: th iscapacity of thecal cell layers is m aintained by the period of oocytem aturation and ovulation (K anam ori et at.. 1 98 8). A roma ta seactivity ingranulosa c e l l l a y e rs i nc reas es during v it el logenes is anddecreases rapidly in association w ith the ability of the oocyte tom atu re in re sp on se to g on ad otro pin (Y ou ng etat., 1983b : Kanamo rie t a /., 1988). This decrease in arom atase activity appears to becoincident w ith the decreased ability of intact follicles to produceestradio l-17B in response to gonadotropin . S ince tes tos teroneproduction in thecal cell layers do not decline during this tim e, there du ce d p ro du ctio n o f e stra dio l-1 78 b y p os tv ite llo ge nic fo llic le s isdue. i n part , t o dec reased aromatase act iv it y in granulo sa c ellayers.

Immediately prio r to o oc yte mat ur ati on , in ta ct o va ri an f olli cle s ofs alm on id fish a cq uire a n in cre as ed a bility to p ro du ce 1 7a ,2 08 -D Pin response to gonadotropin. T hecal cell layers do not develop theability to produce 17C1-hydroxyprogesterone in response tog on ad otr op in u ntil immed ia te ly p rio r to oocyte maturat ion . A lthoughgranulosa ce ll l aye rs f irst acquire th e abil ity to convert exogenous17C1-hydroxyprogesterone to 17a,208-DP in response togonadotropin about 2 months prior to oocyte m aturation, anincrease inendogenous 2 0 B -H S D a c t iv i t y i n granulosa c el ls o ccur sduring oocyte m aturation. Thus, a decrease in C17-20 lyase andior 178-hydroxysteroid dehydrogenase activity in thecal cells andan increase in 208-HSD in granulosa cells appear to be the m ajorf ac to rs re sp on sib le f orth e ra pid in cre as e in 1 7a ,2 08 -DP p ro du ct io nb y in ta ct fo llic le s d urin g oocyte maturation.

S in ce th e s te ro id og en ic s hiff fro m e stra dio l-1 78 to 17 a,2 08 -D Poccurs in association w i t h increasing concentrations ofg on adot ropi ns . it is impor ta nt t o in ve st ig at e t he mo le cu la r eventsby which the shift is regulated by gonadotropins. The first steptoward elucidation of this problem is th e id entific atio n a ndcharacterization o f c D N A c lo n es e n c o din g s t e r o i d o ge n ic enzymesresponsible for estradiol-178 and 17a,208-DP biosynthesis. W ehave recently isolated and cloned full-length cDNA insertscom plem entary to the m RN As encoding rainbow trout cholesterolside-chain cleavage cytochrom e (P -450scc) (T akahashi et al..1 99 3), 3 8-h yd ro xy ste ro id d eh yd ro ge na se (3 8-H SD ) (S ak ai e t a l. ,1993), 17a-hydroxylaseiC17-20 lyase cytochrom e (P-450c17)(Sakai et at., 1992), and arom atase cytochrom e (P-450arom )(Tanaka et al.. 1992b). Th e cDNA inserts were confirmed toencode each steroidogenic enzyme by introducing it inton on ste ro id og en ic COS-1 monk ey k id ne y tumo r c ells . U nto rtu na te ly ,unlike m ost otthe ovarian steroidogenic enzym es, 208-H SD cD NAh a s n o t b e e n c lo ne d in a ny a nim al s pe cie s. T he refo re . a s a firststep, we isolated a cDNA encoding pig testis 208-HSD, sincepuritied preparations of 208-H SD is available only in pig (N akajine t a l., 1988). U sing synthetic oligonucleotides deduced from thepartially determ ined am ino acid sequences, we have, for the firstt ime, isolated and cloned cDNA encoding 208-HSD from a pigte stis c DNA lib ra ry (T an aka e t a l., 1992a). The cDNA contains anopen r eadin g f rame p redic te d t o e nc od e 2 8 9 a m in o a c i d residues.S urprisingly it has 85% am ino acid hom ology w ith hum an carbonylreductase. We are now using this cDNA clone to isolate cDNAsencod ing r ain bow trout 2 0 B - H S D from a r a in bo w trout ovariancDNA lib ra ry .

The mRNA levels of P.450scc, 38.HSD, and P-450c17 areb are ly d ete cte d in th e fo llicle s d urin g th e m id -v ite llo ge nic stage,and abundant in follicles during the postvitellogenic stage andoocyte ma tu ra tio n (T ak ah as hi e t a l., 1 99 3; S ak ai et al., 1992,

8/14/2019 Int. J. 0.,. BioI. 38: 217-229 (1994)

5/13

1993). The P-450arom RNA transcripts are present in the ovaryd urin g a ctive vite llo gen esis, bu t a re ab sen t in th e ovary in th e s ta geof oocyte maturation or in the ovary c ont ai ni ng post ov ul at or yf ol li cl es ( Tanaka e t a l. , 1992b). These results are consistent w iththe ra pid de cre ase in a ro ma ta se a ctiv ity in th e g ra nulo sa ce ll la yersduring the postovulatory period (Young et a l.. 1983b). It is thusc on clu de d th at th e a bility o f th e g ra nu lo sa ce lls to p ro du ce e stra dio l-17B is regulated by the amount of the RNA transcripts present(Tanaka etal., 1992b ). Ou r p re lim ina ry r es ul ts i nd ic at et ha tf or sk o li n-induced 17a,20B-DP production is accom panied by a dram aticdecrease in P -450arom m RNA levels by granulosa cells isolatedtro m p ostvitello ge nic follic le s. A 2- to 3 -fo ld incre ase in P -4 50sccand 3B-HSD m RNAs and a slight decrease in P-450c17 m RNA area ls o o bs erve d d urin g th e fo rs ko lin -in du ce d 1 7a ,2 0B -D P p ro du ctio n(T ana ka a nd N ag ah am a, un pu blish ed). O ur prelim in ary N orth ernhybrid ization analysis using the pig 20B-H SD cD NA as a probe hasrevealed that 20B-HSD m RNA transcripts are low in granulosacells prior to oocyte m aturation, but m arkedly increased duringn atu ra l a nd g on ad otro pin -in du ce d o ocy te m atu ra tio n.

S ite of M IH a ctionPrevious in v it r o studies in fish h ave sh ow n tha t the m ech anism

o f a ctio n invo lve d in th e ste ro id -stim ulation of oo cyte m atu ra tio nhas specia l characteristics not typical of the classic steroidm ech an ism o f action (D ettla ff an d S ko blin a, 1 969 ; Jalab ert, 1 976 ;DeMan no and Goetz, 1987). We also found that 17a,20B-DP isin effective in in du cin g o ocyte m atu ra tio n w he n m icroin je cte d in tofully grow n im mature oocytes of goldfish, but w as effective w hena pplied exte rn ally. T ake n to ge the r, the se in v itr o r es ul ts s ugges tthat the site of M IH action in inducing meiotic maturation in fishoocytes is at or near the oocyte surface.

More direct evidence for the existence of M IH receptors inoocyte plasm a m em branes has been obtained by binding studiesusin g la be lled M IH . W e h ave id entified an d ch ara cterize d spe cificbinding of ['H )17a,20B-DP to plasm a m em branes prepared fromd efo llicu la te d o ocyte s o f ra in bo w tro ut (Y osh iku ni e t a l., 1993).Scatchard analysis reveals two different binding sites: a highaffin ity binding site with a K d at 18 nM and a 6max of 0.2 pmoleslmg protein and a low affin ity binding site with a Kd 01 0 .5 ~M anda 6max of 1 pmolefmg protein. Maneckjee et al. (1 98 9) a lsore po rte d 1 7a,20 B-D P bind in g a ctiv ity in the zo na ra dia ta -oo cytemembran e c omple x of ra inbow trout and brook trout, Salvelinusfontinalis. Recently, we also described a specific 17a,20B-DPreceptor for 17a,20B-DP in the oocyte cortices of the Japaneseflounder, Para li ch thy s o li baceus (Yoshikuni e t a /., unpublished).This 17a,20B-D P binding increases and reaches an equilibriumwithin 1 h. Scatchard analysis reveals a single binding site w ith aKd of 63 nM and a 6max of 25 fmoleslcortex. The 17a,20B-DPre ce pto r is p re se nt in oo cyte cortice s from b oth po stvite llo gen icoocytes (500-600 ~m in diameter) and ovulated eggs, but not inth ose from e ith er m id-vite llo gen ic o ocyte s (42 5-5 00 11 m) or e arly-vite llogenic oocytes 425 ~m ).

The binding 01 20B -S to plasm a m em branes from the ovaries ofspotted seatrout was also characterized (Patino and Thom as,1 99 0a). S ca tcha rd an alyse s su gg est a sin gle cla ss of h igh affin ity(K d, 1 O~ 'o M), lo w.ca pa city (1 0~13-1 0~12 molfg ovary) binding sitesfor 20-S. A specilic b inding site for 20B-S exists in plasmam em branes from spotted seatrout ovaries (Palino and Thom as,1 99 0b). T he co nce ntra tio ns o f 2 0B -S re ce pto rs in ova ria n p lasm am em branes are three tim es higher in seat rout undergoing oocyte

H orm ones and gam etogencsis in fish 22 1matu ra tio n th an in vitelJo gen ic fem ale s. T his in cre ase in re cep to rco nce ntratio ns in fish collecte d d urin g th eir n atural spa wn in g cycleis sim ilar to that previously observed in seatrout undergoing finalm atu ra tion fo llo win g LH RH in je ctio ns (T ho ma s an d P atin o, 1 991 ).T he se o bserva tion s su gge st th at cha nge s in the co nce ntra tio n ofMIH re ce pto rs in th e o va rie s a re o f p h ys io lo gica l im po rta nc e d urin gnatural oocyte m aturation. Sim ilarly. a specific progesterone (aproposed MIH of amphibian oocytes) receptor has beendemonstrated on the surface of Xenopus oocytes (Sadler andM aller, 19 82). Ta ken to gethe r, th ese o bse rva tio ns e mp ha size th atth e a ct io n o f m atu ra tio n- in du ci ng steroid on the oocyte is rathern ove l in the ligh t of co nve ntio nal co nce pt fh at ste ro id s a ct th ro ug hcytoso lic or n uclea r rece ptors. C lea rly, fu rth er w ork is n ece ssary tofu lly cha ra cte rize ste ro id re ce pto rs o n o ocyte m em bra ne s.Acquirement of oocytes to m ature in response to hor-monal s timu la tion

It has been reported that intra lo llicu lar oocytes 01 severalte leosts develop the ability to undergo GV6D in response toh ormona l s timu la ti on immedi at el y p ri or to th e fi na l o oc yte ma tu ra tio nsta ge . Fo r e xa mp le , m eda ka fo llicles acq uire th e a bility to u nd erg om aturation in response to gonadotropin 28 h before the expectedtim e of spaw ning (S akai e t a l., 1 98 7). In o oc yte s at kisu (Sillagojaponica, Kobayashi et al.. 1 98 8), d ra go ne t (Repomucenusbeniteguri, Zh u et al.. 1989) and Atlantic croaker (Patino andThomas, 1990b,c), there is a stage in which they can undergooocy te ma tu ra ti on i n v itr o in re sp on se to g on ad otro pin stim ula tio nbut not to M IH. However, these oocytes becom e sensitive to M IHif e xp ose d to g ona dotro pin i n v itro . These results suggest thatgonadotropin is responsib le for the induction of m aturationalcompetence of o oc yte s. F urth erm ore , H CG -in du ce d m atu ra tio na lcom petence was inhib ited by cyclohexim ide and actinom ycin 0(P atin o a nd T ho ma s, 1 990 c). T ho ma s an d P atin o (19 91) e xam in edthe relationship betw een 20B -S receptor concentrations and thedevelopment of maturational competence using an ovarianincu ba tion system . T rea tm en t o f sp otted sea t ro ut o va rian fo lliclesw it h gonadot rop in causes a tw o-fold increase in 20B-S receptorco nce ntratio ns a nd, con com ita ntly, fu ll-g ro wn o ocyte s a cqu ire th ea bility to m atu re in re spo nse to 20 B-S .ln co ntra st, 2 0B -S tre atm en tdoes not induce an increase in receptor concentrations or thed ev elo pm en t o f o oc yte m atu ra tio na l comp ete nc e. S im ila rly, i n v it rotreatm ent of Japanese flounder follic les w ith HCG also causes athree-fo ld increase in 17a,20B-DP r ec ep to r c oncent ra ti ons.M ore ove r, th is HCG -in du ce d e le va tio n in re ce pto r c on ce ntra tio nsis accom panied by the appearance of m aturational com petence inth e fo lli cle -e nc lo se d o oc yte s (Yo sh ik un i etal., u np ub lis he d). T ak entogether, these results suggest that a gonadotropin-inducedincrease in M IH receptor concentrations is responsib le for thedevelopm ent of oocyte m aturational com petence in seatrout andthe Japanese flounder, and is not mediated by increases in thep ro duction 0 1 th e M IH .Tertia ry med ia to r o f o oc yte maturation - M aturation-prom oting factor (M PF)

In a p re vio us s tu dy , D ettla ff et al. ( 1977) repor ted MPF-lik eact iv i ty inthe sturgeon, Acipenser stellatus. oocytes . However . t hi sac tiv ity was no t d ete cfe d in the pre sen ce 0 1 c yclo he xim ide , aprote in synthesis inhib itor. so that pretranslat ional factors upstreamo f t rue MPF act iv ity we re no t exc luded . To de te rm ine whethe r MPF

----

8/14/2019 Int. J. 0.,. BioI. 38: 217-229 (1994)

6/13

222 Y . Nagahama

~U;la'y

Gonadotropin

.. '17,,-Hydroxyprogesterone0 0 ~ f ~Maturation-inducing hormo~: o~~ Theca l ce ll17".2011-Dlhydroxy- 0 ~~~ (4.pregnen-3-one ~~ G ra nu lo sa ce ll. " !1.~Maturar;on-pmmor;ng K\~.. . ~ ;l . . factor \~'\j.. K ( cd c2 k ln a se ..

CYClin B.I

~~~ e D~R t ~ .. Germinalesiclee ce p o r .J .~~ Oo cy le m alm al lo n o~

~ ~~~Fig. 3. Hormonal regulation of oocyte maturation in teleosts.a c t iv i t y is p r e s e n t in t h e m a t u r e f is h o o c y t e , w e e x t r a c t e d M P Fa c ti v it y f r o m h u m a n c h o r io n ic g o n a d o tr o p in ( H C G ) - in je c t e d m a tu rego ld fish oo cytes and n atu rally sp aw ned carp o ocy tes (N agah am aand Yamashita, 1988). These mature oocytes were crushed byultracentrifugation at 100,O OO xg for 1 h in a buffer containingphosphatase inhibitors and E GT A . A fter centrifugation, the fivem a jo r la y e r s o b t a in ed , f r o m c e n t r ip e t a l t o c e n t r i f u ga l p o le , w e r e :s u pe r n a t a n t , g r e e n la y e r , y o l k , c h o r io n , a n d c o rt i c a l a lv e o l i . T h eg r e e n la y e r w a s e n r i c h e d w i t h c e l lu la r o r g a n e l le s s u c h a sm it o c h o n d r ia a n d e n d o p la s m ic r e t ic u l u m . M P F a c t iv i t y o f e a c hl a y e r w a s d e t e r m in e d u s in g th e Xenopus o o c y te m i c r o in je c tio na s s a y . M P F a c t iv i t y w a s lo ca l i z e d in t h e s u p e r n a t a n t a n d t h e g r e e nla y e r ; m ic r o in je c t io n o f t h e g r e e n la y e r c a u s e d c e l l l y s is . M P Fa c t iv i t y w a s a ls o e x t r a c t e d f r o m o o c y t e s m a t u r e d in v it r o by1 7a,20 ~-D P, b ut no t fro m immature oo cytes o r activ ated o ocy tes.M P F a c t iv i t y e x t r a c t e d f r o m f is h w a s a ls o e f f e c t iv e w h e n i n je c t e din t o im m a tu r e f is h o o c y t e s u n d e r c o n d i t io n s o f in h i b i t e d p ro t e ins yn th es is . T h e in je c te d o oc yt e s m a tu re d m u ch f a s t e r t h an o oc yt e sinduced to m ature by incubation w ith 17a,20B -D P: G VBD50 w asaround 2.5 h in MPF-induced maturation, but about 6 h wheninduced by 17a,20B-D P. GV BD usually occurred at the center oft he MPF-injec ted oocytes, because the movement o f t h e G V t o t h ea n im a l p o le d id n o t t a k e p la c e. G V m ig ra t io n a lw a y s o cc u r r e d i n t h eo o cy te s m a tu re d n a t u ra lly , i n v iv o b y H C G , o r in v it ro b y 1 7 (, ( ,2 0 B -D P . T h u s , p o s t - t r a n s l a t io n a l M P F a c t iv i t y is p r e s e n t in m a t u r eg old fis h an d c arp o oc yte s.MPF from m ature oocytes at goldfish induced GVBD w h e nin je c t e d in to X e n o p u s o oc y te s a nd v i c e versa. I t w as a ls o s h o w np r e v io u s ly t h a t g o ld f is h M P F in d u c e d m a t u r a t io n o f im m atu r eoocytes of the starfish, As te ri na pec ti ni fe ra (Ki sh imo to , 1 98 8) .These f in d i n g s a r e c o n sis t e n t w it h t h e n o t io n t h a t M P F is s im i la ramong vert ebra te s and invert ebra te s (Kishimoto, 1988).MPF pur ific atio n a nd c ha ra cte riz atio n

I n s p i t e o f e f f o r t s in s e v e r a l l a b o r a t o r ie s , p r o g r e s s in MPFpur if ic ati on h ad b ee n v er y s low unt il v e ry r ec en tly . T he b re ak th ro ughin pu rification w as achieved b y L oh ka et al. ( 19 88 ), who pur if ie d

XenopusMPF app rox ima te ly 3 ,000- fo ld ; t hi s MPF f ract ion cont ai nedtwo predominant p r o t e i n s , w it h r e l a t iv e m o l e c u la r m a s s o f 3 4 a n4 5 k D a , r e s p e c tiv e l y . T h e former r o te in h as b ee n id en ti f i e d a s t hXenopus h o m o lo g o f a f is s io n y e a s t p r o t e i n e n c o d e d b y a g e n e( cd c2+) , wh ic h i s re qu ir ed f or th e G2 /M t ra ns it io n in m it ot ic c ell c yc l(Gautier e t a l. , 1988) . The p roduct o f t he c dc 2+ g en e i s a 3 4 k Ds e r in e / t h re on in e p ro te in k in a s e, d e s ig n a te d p 34 cd c 2 orcdc2 kinasA noth er compo nen t o f t he Xenopus MPF, the 45 kD a protein, habeen identified as a Xenopus B- type cyc li n (Gau ti er e t a l., 1 99 0)Similar M PF has also been purified in starfish (Labbe et al1989a,b).

W e re ce ntly p urifie d a nd c ha ra cte riz ed MPF (Y am as hita e t a l1992a) and histone H1 kinase (Y am ashita et al., 19 92 b) fro m1O O , O O O x g supernatant ofcrushed, natu,al ly ovulated, unferti lizee g g s o f c a r p u s in g f o u r c h r o m a t o g r a p h ic s t e p s i n c lu d in g QSepharose Fast-Flow , P13'""-affinity Sepharose, M ono S, andS up e r o se 1 2. M P F a c tiv i t y o f v ar io us f r a c tio ns d u r in g p u r i f i c at io nw a s d e t e rm in e d b y t h e Xenopus o o c y t e m i c ro in je c tio n a s s a y .A nalyses by SD S-polyacrylam ide gel (PAG E) show ed that themost a c t iv e f r a c t io n a f t e r M o n o S c o n t a in e d f o u r p r o t e in s , w it hmolecular masses of 33, 34, 46, and 48 kDa. W hen the activefractions after M ono S w ere applied to Superose 12, M PF eluted asa single peak w ith an apparent m olecular w eight of about 100 kD aSD S-PA GE of the active fraction after Superose 12 revealed thesame f o u r p r o t e in s a s t h o s e o b t a in e d a f t e r M on o S , i n d i c a t in g t h a tthese proteins form com plex(es) of about 100 kD a in their nativef o rm . T h e f i n a l p r e p a r a t io n w as p u r i f i e d o v e r 1 , O D D - f o ld w i t h ar e c ov e ry o f a b o u t 1%.

It is suggested from the comparison of the components op urified carp M PF w ith tho se of Xenopus MPF that am ong the foupro teins fou nd in c arp M PF , th e34 -kD a pro tein, an d th e 4 6- and 48kD a p r o t e i n s c o r r e s p o n d t o c d c 2 k in a s e a n d c y c l i n B o f X e n o p u sMPF, respec tively . To f ull y cha ra cte riz e t he se fou r p ro te in s, weisolated cD NA clones encoding fish hom ologs of M PF- relatedp ro te in s, in clu din g c dc 2 k in as e (K ajiu ra et a l. , 1993), c dk2 k in as e(Hiraiet a l. , 1992b), cyel in A (Katsu et a l ., unpublished) , and cyc linB (H ira i e t al., 1992a) f rom cDNA li br ar ie s o f gold fis h oocyte s.Monoclonalantibodies against the C- termina l sequence of goldfishcdc2 k inase (Ka jiu ra e t a l., 1 99 3), a u niq ue s tre tc h o f 1 6 amin oacid s (EGVPSTA IRE ISLLKE , t ermed t he PSTA IR sequence ) t hais p e r f e c t l y c o n se r v e d in c d c 2 k in a s e a n d i t s h o m o lo gs f r o m y e a s tto m an (Y am as hita et al., 1 99 1), th e C -te rm in al s eq ue nce ogo ld fi sh cdk2 k inase (H irai et a l ., 1992b), an d E.co li -produced fu llle ngth gold fis h c ye lin A (Ka tsu et a l. , unpub lis hed) and B (Hir ai eal., 1992a ) we re r ais ed .

T h e s e m o n o c lo n a l a n t ib o d i e s w e r e u s e d t o c h a r a c t e r i z e t h ep u r i f i e d ca rp MPFwh ich contains 33 -, 34 -, 46 -and 48-kDaproteins .Both the 33- and 34-kDa proteins are recognized by the anti-PSTA IR antib ody , in dic atin g t ha t t he y a re the c yc lin -dependentk inases . The an ti -cdc2 C- te rm ina l an ti body reac ts w ith the 34 -kDa ,b ut n ot th e 3 3-kDa p ro te in . T he la tte r is re co gn iz ed w ith th e a nti-cdk2 C- te rm ina l an ti body (H irai et a l ., 1992b ). The 46- and 48- kDapro te ins a re recogn ized by the an ti -f ul l l eng th cycl in B an ti body, bu tnot b y an anti- cy clin A antib ody (Yamashit a et a l. , 1992a ; Ka tsu etal., unpub li shed) . I t i s conc luded f rom these f ind ings tha t ca rp MPFisa complex of cdc2 k inase (34 -kDa) and cycl in B (46 -and 48-kDa).MPF activation

I t has been r e p o r t e d t h a t in im m atu r e o o c y t e s of Xenopus an dstar fish, cdc2 k inase forms a c omple x w ith c yc lin B as p re -MPF. In

8/14/2019 Int. J. 0.,. BioI. 38: 217-229 (1994)

7/13

c on tra st, immatu re g old fish oocytes c on ta in th e 3 5.k Da in ac tiv ecdc2 kinase but no cyclin B, and mature oocytes contain both the35-kDa inactive and the 34-kDa active cdc2 kinases and cyclin B(Hirai e t a /., 1 992a; K atsu e t a l., 1993). The appearance of the 34-kD a a ctiv e c dc 2 k in as e c oin cid es w ith th e a pp ea ra nc e o f c yc lin 8just betore GVBD. Anti-cyclin B immunobiots of the p13'""precip itates and anti-cdc2 kinase im munoblots of anti-cyclin Bimmunoprecip itates show that the binding of cdc2 kinase andcyclin B coincides w ith the appearance of cyclin B and the 34-kD aactive cdc2 kinase. The cyclin B which appears during oocytem aturation is labeled w ith 35S -m ethionine (H ira i et a/., 1992a),demonstrating de novo synthesis d uring oocyte m atu ration . O n th eath er h and , an ti-cyclin B immun oprecip itates from m ature oocyteextracts som etim es contain the 35.kD a inactive cdc2 kinase (H iraie t a l., 1992a), and the 35-kDa cdc2 kinase, as well as the 34-kDaform , can bind to cyclin B in a cell-free system (Y am ashita e t a /.,unpublished). Therefore, it is m ost likely that the 35-kD a inactivecdc2 kinase binds to de novo synthesized cyclin B at first, then israpid ly converted into the 34-kD a active form (Fig. 4).

The preceding results indicate that the appearance ot cyclin Bis requ ired and is sufficien t for in du cin g oocyte m aturation ing old fish . T o con firm th at th e ap pea ran ce of cyclin B is suffic ie nt forind ucing oo cyte m atu ra tio n, E . coli-p ro duce d fult le ng th g old fishcyclin B p ro te in w as in jec ted in to immatu re o oc yte s. E ven u nd erco nd itio ns o f in hibite d p ro te in synthe sis, injecte d cyclin B ind uce soocyte m atu ration w ith in 1 h r after in jection in a dose-d ep end entm anner. Introduction of goldfish cyclin B into im mature goldfishoocy te ext ract s a l s o in du ce d cdc 2 k in as e a cti va ti on , c on cu rr en tw ith the shift in apparent m olecular w eight of cdc2 kinase from 35-to 34-kDa, as found in oocytes matured w ith 17a,20B-DP.Phosphoam ino acid analysis shows that threonine (Thr)phosphorylation of the 34-kDa cdc2 kinase and serinep hosp ho ryla tio n of cyclin B are a ssocia te d w ith the a ctiva tion . Th esam e phosphorylation is found in oocytes m atured by 17a,20B-D P. C yclin B -ind uce d cd c2 kin ase activa tion is n ot o bse rve d w he nthreonine phosphorylation of cdc2 kinase and serinep ho sp ho ry la tio n o fc yclin B a re in hib ite d b y p ro te in kin ase in hib ito rs,a lthough the binding of the 35-kD a cdc2 kinase to cyclin B occurseven in the presence of the inhib itors. In contrast, cdc2 kinase isa ctiv ate d b y m uta nt cy clin s th at u nd erg o n o s erin e p ho sp ho ryla tio nd urin g th e activa tion . T he site o tth reo nin e p hosp ho ryla tio n o n cd c2kin ase w as m ap ped to re sid ue Th r16 1. T he se find in gs ind icate th atthe Thrl61 phosphorylation of cdc2 kinase, but not serinephosphorylation of cyclin B, is required for cdc2 kinase (MPF)a ctiv atio n in g old fish o ocy te s.

Recently we have isolated a cDNA clone encoding a goldfishhom olog of p 40M0 15 , t he c ata ly tic su bu nit o f a p ro te in kin ase w hic hh as b een sh ow n to a ctiva te cd c2 kina se th ro ugh p hosp ho ryla tionofThrl6t, from a goldfish oocyte cD NA library (O noe e t a l.. 1 993) .Northern and W estern blot analyses reveal that both p40M015mRNA and protein are already present in gO ldfish immatureoocytes and do not appear to exhib it any measurable changesduring horm onally induced m aturation (O noe et a /.. 1 993 ; O noe ,unpublished). Thus, it is suggested that p40M015 may itself bere gu la te d b y su bu nit a sso cia tio n a nd /o r p ro te in p ho sp ho ryla tio ns .T he su bstra te p re fe re nce o f p 40M0 15 a lso n ee ds to b e d ete rm in ed .

In Xenopus oo cytes, de pho sph oryla tio n of th re on in e (T hrI4 )an d tyro sine (T yr15 ) is re quire d fo r cdc2 kina se activa tio n (M alter,1991). However, it is doubtful fhat tyrosine (Tyr15)d ep ho sp ho ry la tio n o f c dc 2 kin as e is a p re re qu is ite fo r its a ctiva tio ndu rin g goldfish oocyte m atu ration , s in c e t h e a c t iv a t i o n i s n o t

H orm on es a nd ga meto gen esis in fish 22 317 a.2OI!-Dlhydroxy-4--pregnen-3-oneIe de 2 k in a se ~sma rTWImbrane~ ~ I A e ce P I~ 15 161I ~ e de 2 k ln n e

i

,,~I ~/~+ ~ L-ID Cyelln B,CyeUnBmANA

Cy el ln B s yn th es isS er in a k in a se( cd e2 k in a se ) T hr eo ni ne k in as e(p40.("')

! !

Fig. 4. M echanism s of M PF activation during 17f1.20B-DP-inducedmeiotic maturation in goldfish oocytes.

in hib ite d b y v an ad ate , a p ro te in p ho sp ha tas e in hib ito r common lyu se d fo r in hib itin g c dc 25 a ctiv ity th at d ep ho sp ho ry la tes ty ro sin e(Ty rt 5 ) o fc dc2 k in as e, t he re by i nh ib it in g i ts a ct iv at io n. Fu rt he rmo re ,anti-phosphotyrosine antibodies do not cross-react w ith the 35-kD a cdc2 kinase, as w ell as the 34-kD a form , w hich binds to cyclinB . A lso, an antibody w hich recognized the d ephospho rylated formof Tyr15 cross-react w ith both the 34- and 35-kD a cdc2 kinase.Therefore, the 35-kD a cdc2 kinase found in goidfish immatureoocytes m ay be already de phosphorylated on Thr14 and Tyr15and the threonine phosphorylation (T hrI61) m ay only be requiredfor the activation. Taken together, it is strongly suggested that17a,2 0B -D P induces oocytes to synthesize cyc/in B , w h ich in turnactivates preexisting 35-kD a cdc2 kinase through its threonine(T hr 16 1 ) p h os ph ory la tio n, p ro du ci ng t he 3 4- kDa a ctiv e c dc 2 k in as e.T he se m ec han ism s o f MP F ac tiv atio n in fish ap pa ren tly d iffer fromt h o s e in Xenopus a n d s t a r f i s h , in w h ic h c y c l in B is p r e s e n t inimm ature oocytes and forms a complex with cdc2 kinase (pre-MPF).S tru ctu re o f tele os t te stis

T he testis of teleosts, as that ot other vertebrates, is com po sedo f in te rs tit ia l a nd l ob ula r (t ub ula r) c omp ar tme nts . T he in te rs titi umbetween l obul es c on si st s o f i n te rs ti ti al c el ls ( Leyd ig c e ll s) , t ib rob la st s,a nd b lo o d a nd ly m p h vessels. V a r io u s e n z y m es in vo lv ed in s te ro idhorm one synthesis have been dem onstrated by histochem istry inthe L eydig cells of a num ber of teleosts. T hese cells possessu lt r a s t r u c t u ra l f e a t u re s c om m o n ly fo u n d in s te ro id -p ro du c in g c e l l ss u c h a s a g r a n u la r e n d o p la s m ic r e t i c u lu m a n d m it o c h o n d r ia w it htu bu la r c ri sta e (Na ga hama, 1 98 3) . T he l ob ula r ( tu bu la r) c omp on en tof the teieo st testis contains tw o cell types: germ cells and distinctsomatic cell s l in ingthe periphery of the lobule, Ser to l icel ls . I t i s st il luncertainwhetherS e rto li c e l l s c a n p ro d u c e s te ro id s . U l t r as tr u c tu ra le v i d e n c e g e n e r a lly d oe s n ot in dic ate stero id s yn th etic c ap ac ity to rthese cel is . However, cer tain species do seem t o have s ome o f t heappropriate histochem ical and ultrastructural features forsteroidogenesis ( Nagahama, 1983 ).

8/14/2019 Int. J. 0.,. BioI. 38: 217-229 (1994)

8/13

224 Y. N agalwma

Testis

v1tary

Gonadotropin~

Basement membrane

S er toll c ellActl vln B

Spermatogonium ,Spermatogenesis

Fig. 5. Hormonal regulation of spermatogenesis in the eel testis.

S te roid p rod uc tio n d ur in g sp ermato gen esisI n a n u m b e r o f t e l e o s t species, a s in o t h e r t e le o s t species,plasm a levels of testosterone and 11-ketotestosterone are high

during the later stages of sperm atogenesis and rapidly declineaher the onset of sperm iation (Fostier e f a l., 1 98 3; U ed a ef a l..1983a), In vifro Incubation studies on 11-ketotestosteronep ro du ct io n b y te st ic ula r f ra gmen ts a t d if fe re nt s ta ge s o f d e ve lo pmen thave revealed that the capacity of the fragments to produce thissteroid in response to gonadotropin is high during the later stagesof spermatogenesis and rapidly deciines after the onset ofspe rm ia tio n (Saka i ef ai" 1989). T he se resu lts su gg est th atandrogens are invo lved in sp erma to ge ne sis in ama go sa lm on .Hormona l r eg ula tio n o f s pe rma to gene sis

Sperm atogenesis in teleost fish takes place within testicularc ys ts f orme d b y Ser1 0li c ells . W it hin e ac h c ys t g erm c ell d ev elo pmen to cc ur s s yn ch ro no us ly (G rie r, 1 98 1; B illa rd e f a l. . 1982 ; Nagahama,1983; C allard. 1991 ).In the cultivated m ale Japanese eel. Anguillajaponica. the only germ cells present in the testis are the type A ande arly ty pe B sp erm atog on ia w hic h a re p rim itive s pe rm ato go nia th athave n o t b e g u n t o p r o l i f e r a t e . T h u s , s tu d i e s u s i n g t h e s e a n im a lsmay provide considerable advantages over the use of highlyc o m p l e x spermatogenic s y s t e m s in h i g h e r v e r t e b r a t e s . A s i n g le

in jection of H CG to these m ales induced sperm atogenesis w ithin1 8d ay s (M iu ra e f a l. , 1991a). W ithin 1 day aherthe H CG injection.L ey dig c ells a nd Ser 10 ii c ells a re mar ke dly a ct iv ate d. S ub se qu en tly(3 da ys a fte r H CG in je ctio n). s pe rm ato go nia b egin pro lifera tio n.and type B spermatogonia appear. Following approxim ately 10m i t o t ic d iv is io ns . spe rma togonia b eg in me io si s. Spermatocytesw ith s yna pto nema l c om ple x first a pp ea r in tes te s 12 d ays a fter th einjection. Spermatids and spermatozoa are observed aher 18days.

A serum -free. chem ically defined organ culture system for eeltestes has been developed, and used to investigate the effect ofHCG and various s t e r o id h o rm o n e s o n t h e i n d u c t io n o fs p e r m a t o g e n e s i s i n v it ro . C u l t u r e s o f t e s t ic u l a r f r a g m e n t s in m e d i acontaining H CG induced the entire process of sperm atogenesisf r o m p r e m i t o t i c s p e rm a t o g o n ia t o s p e rm a t o z o a w i t h in 2 4 d a y s(Miura e f al_ . 1991c). This HCG -induced sperm atogenesis wasaccom panied by a m arked activation of Ser10li cells and Leydigcel ls , o ccur ri ng p rio rt o t he beg in ni ng o f spe rma togoni al p ro li fe ra tio n.L ey dig c ell a ctiv atio n w as c ha ra cte riz ed b y u ltra str uc tu ra l fe atu re sof active steroid production. Fur1herm ore. the addition of HCG toc u l t u r e m ed ia c o n t a in in g t e s t ic u la r f r a g m en t s i n d u c e d a m ar k e d ,r a p i d i n c r e a s e i n 1 1 - k e t o t e s t o s t e r o n e p r o d u c t io n . T h e s e r e s u l t sin dic ate t ha t g on ad otro pin trig ge rs s pe rmato ge ne sis in th e J ap an es ee el. a nd fu rth er su gg est th at th is e ffe ct of g on ado tro pin is m ed ia te dth ro ug h th e a ctio ns of te stic ula r s om atic c ells, p rob ab ly th ro ugh thep ro du ctio n o f 1 1-k eto te sto ste ro ne b y L ey dig c ells . T his s ug ge stio nw a s c o n f i rm ed b y o r g a n c u l t u r e e x p e r im en t s d e m on s t r a t i n g t h a t1 1 -k e t o t e s t o s t e r o n e c a n in d u c e a l l s ta g e s o f s p e rm a t o g e n e s i si n c l u d in g s p e r m ato g o n i a p r o l i f e r a t io n , m eio t i c d i v is io n an dspermiogenesis in v if ro wit hin 2 1 d ay s (M iu ra e f a l. , 1 9 9 1 d ) ( F i g . 5 ) .1 1 -k e t o te s t o s t e r o ne a ls o c a u se s a m a r k e d c y t o l o g ic a l a c t iv a t io n ofS er10li cells, but not Leydig cells. suggesting that the action of 11-k e t o t e s t o s t e r o n e o n s p e r m ato g e n e s is is m ed ia t e d t h r o u g h theaction of Ser10li cells. This is the first anim al system in which theentire process of sperm atogenesis has been induced by horm onalmanipulation i n v it ro .Ac ti vi n B : a pot en tia l medi ator o f gonadotrop in -inducedspermatogenesisA s d esc rib ed ab ove, m orp ho log ica l ch an ges in g erm ce lls.L ey dig c ells . a nd Ser1 0li c ells a re firs t v is ib le o ne d ay a he r HCGtre atm ent. W e ha ve u se d su btrac tive hyb ridiza tio n to id en tifyg e n e s t h a t a re e x p r e s s e d d if f e re n ti a ll y i n e e l t e s te s in t h e f i r s t 24 hafter HCG treatm ent in vivo, w hich ultim ately inducessp erm ato ge nesis. O ne up -reg ula te d cDNA w as iso la te d froms ub tra ctiv e cDNA lib ra rie s d eriv ed from mRNA e xtra cte d fromc on tro l te ste s a nd te ste s o ne d ay a fte r a s in gle in je ctio n o f HCG .F rom it s deduced am ino acid sequence. t his c lo ne was iden tifie das cod ing fo r th e activ in B subun it. The nuc leot id e sequence o f ee la ctiv in B cDNA is 3 _3 k b. A meth io nin e c od on a t n uc le otid e 1 57 2in itiates a lo ng o pe n re ad ing frame spe cifyin g a p ro te in of 3 95

a m in o a c id s . U s in g N o r th er n b lo t a n a ly s is a nd i n s it u h y b r i d i z a t i o nt e c h n iq u e s , w e e x a m in e d s e q u e n t i a l c h a n g e s in t r a n s c r ip t s o ft e s t i c u la r a c t i v in B d u r in g H C G -in d u c e d s p e r m a t o g e n e s is . N ot r a ns cr ip ts f o r a c t i v in B w e r e f o u n d in t e s t e s p rio r t o H C G in je c t io n .In con tra st. 3 .3 kb mRNA tr ansc rip ts fo r a ct iv in B were p rom inen tin t e s t e s o ne d ay a ft e r t h e in je c t io n .The t ransc ri pt concen trat ion began to decrease three days a fterth e in je ctio n. fo llowe d b y a fu r1 he r s ha rp d ro p b y n in e d ay s. T heHCG -d ep en de nt a ctiv in B mRNA e xp re ss io n in the testes was

8/14/2019 Int. J. 0.,. BioI. 38: 217-229 (1994)

9/13

co nfirm ed b y i n s it u h yb rid iz atio n u sin g a d igo xig en in -Ia bele d RNAp rob e; the s ign al w as re stricte d to S erfo li c ells in te ste s tre ate d w ithHCG for one fo three days.

T ake n to geth er. the se tin din gs s ug ges t th e fo llo wing se qu enc eef the hormonal induction of spermatogenesis in the eel.Gonadotropin stimulates the Leydig cells to produce 11-ketotestosterone. which, in turn, activates the Sertoli cells toproduce activin B . A ctivin B then acts on sperm atogonia to inducem itos is le ad ing to the fo rm atio n o f s pe rm ato cyte s.S teroid production during the final testicular m aturation

17a,20B-DP has been reported to be high in the plasma orserum of several species of salm on ids during sperm iation (Scottand Baynes, 1982; Ueda etal., 1 983 a,b, 1 98 4). F urth erm ore, th ecapacity of testicular fragm ents to produce 17a,20B-DP is lowduring spermatogenesis and increases sharply at the time ofs pe rm ia tio n (U ed a e t a i, 1 98 3b ; S ak ai et al " 1 98 9) . T hu s, a d is tin ctshift in the steroidogenic pathw ay from androgens to 17a,20B -D Pappears to occur in the testis of amago salmon around the onseto f s perm ia tio n (S aka i e t a l., u np ub lis hed ). T he se re su lts su gg estthat 17a,20B-DP plays a role in the process of final testicular orsperm m aturation in som e teleosts.

U sing three different testicular preparations (intact testicularfragments, sperm-free testicular fragments and spermpreparations), we e xamin ed t h e r o l e s of testicular som atic andgerm cell elem ents in the gonadotropin-induced testicular productionof 1 7a ,2 0B -D P (U ed a e t a l., 1984). I ntac t tes ti cu la r f ragmen fsproduced a large amount of 17a,20B-DP in response to gonadotropinor 17a-hydroxyprogesterone. G onadotropin did not stim ulate1 7a,2 0B -D P p ro du ctio n b y e ith er s pe rm -fre e te stic ula r fra gm en tsor sperm preparations, but markedly stimulated 17a-hyd ro xypr oges te rone p roduct io n b y spe rm - fr ee tes tic ula r f ra gmenf s.N either sperm -free testicular fragm ents nor sperm preparationsalone were capable of producing 17a,20B-DP in response togonadotropin. 17a-H ydroxyprogesterone m arkedly stim ulated17a,20B -D P production by sperm preparations, buf not by sperm -Iree te stic ula r p re pa ra tion s. F ina lly , 1 7a ,2 0B -D P w as ide ntified a sthe o nly m ajo r m eta bo lite w he n s pe rm p re pa ra tio ns w ere inc ub ate dwi th ["C]17a-hydroxyprogesterone.

T hese results suggest that the site of 17a,20B -D P production issperm , but its production depends on the provision of a precursorste ro id (p ro ba bly 17 a-hy dro xy pro ge ste ro ne ) w hic h is p ro du ce d b ytesticular som atic cells under the influence of gonadotropin. T heresults also suggest that the distribution of 20B -H SD in the testis ofsp erm ia tin g ra in bo w trou t is lim ite d to o nly to s perm . M ore re cen tly ,Asahina e t a l. (1990) reported that isolated sperm of fhe commoncarp contain 20a-HSD. In contrast, Loir (1988) reported that insp erm ia tin g ra inb ow tro ut th e va rio us te sticu la r c ell p re pa ra tio nsmostly devoid of sperm secrete 17a,20B-DP regardless ofgonadot rop in s timulat ion .Spermiation

A lth ou gh fis h s pe rm ato zo a in th e te stis h ave a lre ady c om ple te dtw o m eio tic d iv is io ns , the y m ay n ot b e fe rtile . F or e xamp le, s alm on idsperm atozoa in the testis are immotile, and acquire their m otilityduring passage through the sperm duct (M orisaw a and M orisaw a,1986) . Fu rt he rmo re , spe rm ia tio n, wh ich is p re requ is it e f or succe ss fu lfe rtiliz atio n, g en era lly o cc urs in te le os ts immed ia te ly b efo re o rdu ri ng spawn ing pe riod .

Hormones Gnd gam l'logenesis in fish 225

vitary

GOnadcroPin

Somatic cells " I17 a .Hydroxyprogesterone------Sperm~tozoon 20p-HSD

17a,2~.Dihydroxy.4-pregnen.3-one

In cre ase in s pe rm duct pHtIncre ase in intraspe rm cAMPtSperm motility

-j"E~Q)c-rnFig. 6. Hormonal regulation of acquirement of sperm motility insalmonids.

T he results described above suggest the possible involvem entof 17a,20B-DP in the process, of spermiation. This is stronglysupported by our studies dem onstrating that a single injection ofg on ad otr op in t o n on -s pe rm ia tin g amago s almon in du ce d p re co cio usspermiation 1-2 months prior to the normal spermiation period,concom itant-w ith a m arked increase in plasm a levels of 17c..t,20B -D P. S im ilarly, tw o successive injections of 17C l,20B -D P causedprecocious spermiation, but the response to 17a,20B-DP was ofle ss er m ag nitu de th an to gonadotropin. Neither testosterone norll-k eto te sto ste ro ne w as e ffe ctive (U eda e t a l. . 1985 ) . Conside redtogefher, these results provide evidence to suggest that 17a,20B -DP is a testicular steroidal m ediator of gonadotropin-inducedspermiation in salmon ids. A sim ilar suggestion has also beenm ad e for g old fis h (K ob ay ash i e t a l., 1 98 6).Spe rm motility

In s alm on id fish es su ch a s m asu sa lm on, Onc orh yn ch us m as ou ,sperm in the testis and sperm duct are not motile. If sperm ductsperm are diluted w ith fresh w ater, then for the first tim e, they gainm otility. If testis sperm are diluted w ith fresh w ater, how ever, theyw ill not becom e m otile. T hus, tw o separate processes are involvedin th e ind uc tion o f s pe rm motility in s alm on id s. O ne is th e a cq uis itio nof m otile ability w hen shifting from the testis to sperm duct, and theother is the initiation of m otility w hen diluted w ith fresh w ater. E arly

8/14/2019 Int. J. 0.,. BioI. 38: 217-229 (1994)

10/13

226 Y. Nagahamain the breeding sea son, h ow ever, sperm from the sperm du ct d o no tgain motility even when they are d ilu ted w ith fresh water. Thischaracteristic, namely the period during which sperm becom emotile, makes the masu salmon a good system for studying theendocrinological m echa nism s invo lved in the acquisitio n of spe rmmotility.

T he effects of tw o successive daily intram uscular injections o ft h r e e s te ro id hormone s on the a cquisition of spe rm m otility in m asusalm on d uring the ea rly breeding season w ere e xamin ed (M iu ra etal., 1991 b). Injections of 17a,20B-DP significantly raised thepercentage of m otile sperm and the duration of sperm motility. Incontrast, the other two steroids showed no significant effect onsperm mot il ity . S imilar s t imula tory ef fec ts of 17a ,20B-DP on spe rmmotility were observed in the Japanese eel (M iura ef al., 1991 e).These resul ts sugges tt ha t 1 7a,2 0 B -D P is in vo lv ed i n the acqu is it ionof sperm m otility in masu salmon and eel.

W ethe n exam ined the effects of 17a,20B-DP on the deve lo pmentof sperm motility in vitro. In this case, sperm duct immotilesperm atozoa did not develop m otility when they w ere incubated inartificial sem inal plasma at pH 7.4 containing various doses of17a,20B-DP, suggesting that 17a,20B-DP has no direct action onth e development of sperm motility. The next experiment wasdesigned to determ ine the effects of horm one injections on the pHof sem ina l plasma in the sperm duct, since we found that the pHsem inal plasma of m ale m asu salm on during the early breedingseason, when spermatozoa in the sperm duct are still imm otile ,was significantly lower (pH 7.4-7.5) than that of the sperm duct ofmales during the active breeding season (pH 8.5). Both SGA and1 7a,2 0B -DP s ig nif ica ntly increased the pH of the sperm duct from7.4 to 8.0. In contrast, these injections did not increase the pH ofth e te stis . N eith er 1 1-k eto te sto ste ro ne n or te sto ste ro ne in je ctio nswere effective in raising the pH of the testis or sperm duct.C ollectively, these results suggest that th e actio n of 17a,20B -D Pon the acquisition of spe rm m otility is m ed iated through a n incre asein pH of the sperm duct.In th e next experiment. the effect of pH on the acquisition ofsperm m otility was investigated. W hen imm otile sperm atozoac olle cte d fro m th e te stis w ere in cu ba te d in a rtific ia l s em in al p la sm aof va rying pH , these sperm atozoa acquired the capacity for m otilityin artificial sem inal plasm a of pH higher than 7.8. Furtherm ore,sperm atozoa incubated in the pH range 8.0-9.0, which is the sam epH range af w hich the acquisition of sperm m otility occurred, hads ig nific an tly e le va te d le ve ls o f c AMP. Immotile te stis s pe rm ato zo aalso acquired m otility when incubated w ith dbcAM P, but not withd bcGMP (M iura e f a l., unpubl ished).

T aken together, the findings described above led us to concludethe follow ing sequen ce for the acq uisition of sp erm m otility in m asus alm on ; g on ad otro pin s tim ula te s th e te stis to in du ce th e p ro du ctio nof 17a,20B-DP, which acts to increase sperm duct pH , this in turnincre ases cAMP in sperm allow ing the acquisition o f sperm m otility(F ig . 6 ).Conclusion

T his a rtic le p re se nts a c urre nt p ic tu re o f t he h orm on al re gu la tio no f g am eto ge ne sis in te le os ts . S te ro id al m ed ia to rs o f o o cy te g ro wth ,o ocyte m aturation, sperm atogene sis, an d sperm m aturation haveb ee n id en tifie d in s ev era l te le os t s pe cie s, p artic ula rly in s alm on id s.In both females and males, the steroidogenic shifts that occurimmediately prior to oocyte and sperm maturation seem to bep rerequisite step s for grow ing germ c ells to e nte r th e fin al sta ge of

maturation. These sw itches require a com plex and integratednetw ork of gene regulation involving cell-specificity, horm onalre gu la tio n, a nd d ev elo pmen ta l p att er nin g. Ou r c urr en t e ffo rt s c en te ron the cloning and sequencing of the genes encoding severalsteroidogenic enzymes responsible for estradiol-17B, 11ketotestoste rone and 17a,20B-DP biosynthesis. W e w ill theninvestigate how gonadotropin and other factors act on gonadalsom atic cells to turn on and turn off the expression of these spe cificgenes at specific tim es during various stages of g am etogen esis.O th er n on -s te ro id al m ed ia to rs w hic h re gu la te fis h g am eto ge ne sishave also been determ ined. These include MPF (a mediator o1 7a ,2 01 3-D P a ctio n o n o oc yte m atu ra tio n) a nd a ctiv in B (a m ed ia to rof 11-ketotestosterone action on sperm atogenesis). These findingson teleosts m ay be one of the best docum ented exam ples of th ero le of steroid hormones in the regulation of gametogenesis invertebrates, and w ill certainly provide the basis for a study at them olecular levels o f horm onal reg ulation of germ ce ll de velopm entand maturat ion .

AcknowledgmentsI am in de bte d to m y c olle ag ue s w ho have c ol la bo ra te d w ith me ovarious aspects of the w ork described herein. F inancial support from theM in ist ry o f E d u ca ti on , S cie nc e, a nd C ul tu re o f J a pa n ( 02 10 20 10 ). t he N ait oF ou nd at io n. a nd th e J ap an He alt h S cie nc es F ou nd at io n is also apprecia ted .

ReferencesA DA CH I, S ., K AJIU RA , H ., K AG AW A, H . and N AG AH AM A, Y . (1 990 ). Ide ntificatio n

o f a ro ma tiz ab le a nd ro ge ns s ec re te d b y Is ola te d th ec al c ell la ye rs fro m v ite llo ge nico varia n fo llicles o f a teleost, am ag o sa lm on (O nc orh yn ch us rh od uru s). B io me dRes. 11 : 359-363 .

ASAHINA, K., BARRY, y,p, AIDA, K.. FUSETANI, N. and HANYU, r. (1990).Biosynthesis of 17a,20a-dihydroxy-4-pregnen-3-one from 17a-hydroxyprogesterone by sperm atozoa of the comm on carp. Cypr in us c ar pi o. JE xp . Z oo 12 55 : 2 44 -2 49 .

BillARD, R ., FO ST IE R, A ., W ElL , G . and B RE TO N, B . (19 82). T he e ndo crine controlo f s pe rm ato ge ne sis in te le os t fis h. C an . J. Fish. A quat. S ci. 39 : 6 5 -7 9.

G AL LA RD , G .v . (1 99 1). S pe rm ato ge ne sis . In Ve rt eb ra te Endo cr in o lo g y: F undament al sa nd B iome dic al I mp fic at io ns , Y ol. 4A (E ds. P .K .T. P an g and M .P . S chre ibm an).Academ ic Press, N ew York, pp. 303-341.

C AN AR IO , A .Y .M . an d S CO TT , A .P . (1 988 ). S tru cture-a ctivity rela tionsh ip s o f C 21s te ro id s in a n i n v it ro o o cy te m atu ra tio n b io as sa y in ra in bo w tro ut, Salmogairdneri.G en . C om p o E nd oc ri no !. 71 : 338-348 .

C HA N, S .L ., T AN , C .H ., P AN GM , K .M , a nd L AM , T .J . (1 99 1). Y ite llo ge nin p urific atio nan d de velo pm en t o f assay for vitelloge nin recep to r in oocyte m em branes of thetilapia (Oreochromis n i lo t icus, Linnaeus 1766). J. E xp . Z oo !. 2 57 : 9 6- 1 09.

DEMAN NO, D.A. and GOETZ F.W . (1987). The effect of forskolin, cAMP, andcyanoketone on steroid-induced meiotic maturation of ye llow perch (Percht!avescens) oocytes in v itr o. G en . C ompo E nd oc rin ol. 66: 2 33 -2 43 .

DETTLAFF, T.A. and SKOBLlNA, M.N . (1969). The role of germ inal vesicle in theprocess of oocyte ma tu ra ti on i nAnu ra a nd Ac ip en s er id a e. Ann. Embryol. Morphog.S up pl .1 : 1 33 -1 51 .

D ETTLAFF, T.A., FElG ENG AUER, P.E. and GH ULlTZKAYA, E.V. (1977). Effect ocyclo hexim ide on disintegra tion of th e germ inal vesicle m em bra ne an d ch ang eso f th e c ort ic al la ye r i n t he o oc yte s of Xenopus laevisand Acipenser stel/atusduringt he ir m a tu ra tio n u nd er th e in flu en ce o f a cti ve c yt op la sm . Ontogenesis 8:478-486.

F OS TIE R, A ., JAlABERT, B., BILLAR D, A., BRETON , B. a nd ZO HAR , Y. (1983). Thego nad al steroidog ene sis. In Fish Physiology, Vol. IXA (Eds. W .S. Hoar, D .J.R an da ll and E .M . D ona ldso n). A ca de mic P ress, N ew Y ork, pp. 2 77-3 72 .

GAUTIER, L., M INSHULL, J., lOHKA, M., G lOTZER, M., HUNT, T. and MALLER,J.L. (199 0). C yclin is a co mpon en t of m aturatio n-pro moting factor from Xenopus.GeI/60:487-494.

GAUTIER, L., NORBURY, S., LOHKA, M.. NURSE, P. and MALLER, J. (1988).P urified m aturatio n-p rom otin g fa cto r co ntains the prod uct of a Xenopus homologof the fission ye ast cell cycle control gen e cdc+ . G el /5 4: 4 33 -4 39 .

8/14/2019 Int. J. 0.,. BioI. 38: 217-229 (1994)

11/13

GOE TZ , F .W . (1 98 3). H orm on al co ntr ol 0 1 o ocy te f in al m atu ra tio n an d o vu la tio n infis he s. In Fi sh Phy si o logy , V ol. IX A ( Eds. W .S. H oar, D .J. Randall and E.M .D on ald so n). A cad em ic P res s, N ew Y ork , p p. 1 17 .1 70 .

GRE elE Y, M .S ., C ALDER, D .A ., T AY lOR, M .H ., HOlS , H .H . an d WALLACE, A .A .( 19 86 ). O oc yte m atu ra ti on in th e m umm ic ho g (Funau lu s he /e rOC/i tu s ): e ff ec ls o fs te ro id s o n g er min al v esicle b re ak do wn o f in tac t fo llic le s in v itr o. G en . C am p.Endocrinol. 62 : 281-289 .

GR IER, H .J . ( 19 81 ). C el lu la r o rg an iz at io n o f t he te sti s a nd s pe rm ato ge ne sis in f is he s.Am . Zool . 2 1: 34 5 -3 57 .H IR AI, T ., Y AMASHIT A, M ., Y OS HIK UN I, M ., lO U, Y .-H . and N AGAHAMA, Y .( 1992a ). CycJ in B i n f is h OOCy1es : ts cDNA and am ino a ci d s equ en ce s, a pp ea ra nc e

during ma turat ion , and induct ion 01p34cdc2act iva ti on. M o l. R ep ro d. D ev . 3 3: 1 31 -140.

H IRAI ,Y ., YAMASHITA, M. ,YOSHIKUNI, M. , TOKUMOTO, T ., KAJ IURA, H ., SAKAI,N . a nd NAGAHAMA , Y . ( 19 92 b) . I so la tio n a nd c ha ra ct er iz ati on o f g ol df is h C dk 2,a co gn ate v aria nt o f t he ce il cy cle re gu la to r c dc 2. D e v. B io i. 152 : 113-120 .

IWAMATSU , T . ( 19 78 ). S tu di es o n o oc yt e m atu ra ti on o ll he m ed ak a, Oryzi a s l a ti pes .V .On t he s tr uc tu re o f s te ro id s t ha t in du ce d m at ur ati on in v it ro . J . E xp . Z oo l. 2 04 :401-408.

JALABERT, B. (1976). In v i t rooocyte maturat ion and ovul at ion in ra inbowtroul (Salmogairdneri), no rt he rn p ik e (Esox l uci us ) , a nd gol df is h ( Ca ra ss iu s a ur at us ). J . F is h.R es. B oa rd C an. 33:974-988.

K AGAWA, H ., Y OUNG , G . and N AGAHAMA, Y . (1 983). R elationship betw eens ea so na l p la sm a e stra dio l-1 7B a nd tes to ster on e lev els a nd i n v it ro p roduc ti on byovarian fo ll icles01amago salmon (Oncorhynchus r hodur us ). B io i. Repr od . 29 :301.309.

KAGAWA , H ., YOUNG , G ., ADACH I, S . a nd NAGAHAMA , Y . ( 1982 ). E str ad io l.1 7Bproduct ioninamago salmon (Oncorhynchus rhodurus)ovar ian fo ll ic les : ro le of Ihet hecal and g ranulosa ce ll s. Gen. CompoEndocr ino l . 47: 440-448.K AG AWA, H .. Y OUNG , G ., A DA CH I, S . a nd N AG AH AMA, Y . (1 98 5). E stro gens yn th es is in th e te le os t o va ri an f ol li cl e: t he t wo -c el l ty pe mode l i n s almoni ds . I nSalmon id Reproduc tion (E ds . R .N . Iw am oto a nd S . S ow er). W as hin gto n S eaGran t P rog ram , Un ive rs it y o f Washing ton , Seat tl e, pp . 20 -25 .KAJIUAA . H ., YAMASHITA , M . ,KATSU, Y .and NAGAHAMA, Y . ( 1993) . J so la ti onandcharacterization ofgoldf ishcdc2, a cataly ticcomponent of maturation-promotingfactor. Dev. Grow t h D i ff er . 35: 647-654.KANAMORI, A. and NAGAHAMA, Y. (1988a) . Developmenta lchanges inthe proper tiesofgonadotrop in receptors in the ovarian fO ll ic lesof amago salmon (Oncorhynchusrhodurus) tochumsalmongonadot rop induring oogenes is. Gen.Comp. Endocr ino l .72: 25-38.KANAMORI,A.and NAGAHAMA,Y.(1988b). Involvement of3 ',5 '-cyc l ic adenOSinemonophos ph at e i n t he c on lr ol o f f ol li cu la rs te ro id ogenes is o f amago s almon(Oncorhynchus rhodurus) . Gen. Compo Endocr ino l. 72:39-53.KANAMORI, A ., ADACHI , S . and NAGAHAMA, Y . ( 1988) . Deve lopmen ta l changes ins te ro id og en ic re sp on se s o f o var ia n fo llic le s o f am ag o sa lmon (Oncorhynchusrhodurus)to chumsalmon gonadot rop in dur ing oogenes is. Gen. CompoEndocr inol.72: 13-24.KATSU , Y ., YAMASH ITA, M ., KA JIURA, H . a nd NAGAHAMA , Y . ( 1 993) . B ehav io r of

th e c om po ne nts o fm a tu ra ti on -p romo ti ng f ac to r, c dc 2 k in as e a nd c yc lin B, du ri ngoocyte m aturation of goldfish. Dev . B io I. 160: 9 9.1 0 7.

KAW AUCHI, H., SUZUKJ, K.,ITOH, H., SW ANSO N, P., NAITO , N., NAGAHAMA, Y.,OZAKI, M ., NAKAI, Y. and ITOH, S. (1989). The d ua Ji ly o f t el eo st g on ad ot ro pi ns .F is h P hy sio l. B io ch em . 7 : 2 9 .3 8 .

K IS HIM OT O, T . (1 98 8). R eg ula tio n o f m eta ph ase by a ma tu ra ti on .p romot in g f ac to r.Dev. Grow t h D i ff er . 30: 105-115.K OB A Y A SA HI, M ., AID A, K . a nd H AN YU , J. (19 86). E ffects of H CG on m ilt am ount an d

p la sm a le ve ls o f s te ro id h orm on es in m ale g old fish . Bull. J pn . S oc . S ci. F is h. 52 :755.

KOBAYASAHI, M ., AIDA, K., FURUKAWA, K., LAW, Y.K., MORIWAKI, T. andH AN YU , I. (1 98 8). D ev elo pm en t o f s en sitivity to m aturation-induC ing steroids inth e o oc yt es o f t he d ail y s pawn in g te le os t, t he K is u Siflagojaponica . Gen. CompoEndocrinol. 72 : 264-271 .

LABBE, J-C., CAPONY, J-P., CAPUT, D., CAVADORE, J-C., DEAANCOUAT, J.,KAGHAD, M., lEUAS, l.M., PICARD, M. and DOREE, M. (1989a). MPF fromstarfish oocytes at first m eiotic m etaphase is a heterodimer containing onem ole cu le o f c dc 2 a nd o ne m olec ule of cy clin B . EM BO J. 8 : 3 053 - 3058.

LABBE, J.C., PICARD, A., PEAUCElllER, G., CAVADORE, J.C., NURSE, P. andD O R E E , M . (1989b). P urifica tion of M PF f r o m s ta rfis h: Id en tilica tio n a s th e H 1h is to n e k in as e p34cdc2 a nd a p oss ib le m ec ha nis m fo r its p erio dic a ctiv atio n. Cell57 : 253-263 .

Hormones and gametogenesis ill fish 22 7

LANCASTER , P .M . a nd TYLER , C .R . (1 99 1). Is ola tio n o f a m em bra ne b ou nd p ro te inw ith a n a ffin ity fo r v ite llo ge nin fro m ra in bo w tro ut (Oncorhynchus mykiss) follicles.In P ro ce ed in gs o f th e 4 th In te rn atio na t S ym po siu m o n R ep ro du ctiv e P hy Sio lo gyo f F is h (Eds. A .P. Scon, J.P. Sumpter, D.E. Kime and M .S. Rolfe). FishSymp 91,S he ffie ld , p . 1 28 .

lE M ENN, F. and NUNEZ RODRIGUEZ, J. (1991). Receptors m ediate endocytosisof VT G i n f is h f olli cl e. In Proceedings of the 4th International Sym posium onR ep ro du ctive P hy sio lo gyo t F ish (Eds. A.P. Scott, J.P. Sumpter, D .E. Kime andM .S . Rolle). F ishSymp 91, Sheffield, pp. 300.302.

lESSMAN, CA, HABIBI, H.A. and MACRAE, T.H. (1988). Effect ot m icrotubulereac tiv e d ru gs o n s te ro id a nd c en trifu ga tio n-in du Ce d g erm in al ve sic le m ig ra tio nd ur in g g ol dfi sh o oc yte m eio si s. Bioi. C e/l 64 : 2 93 -2 99 .

lOHKA, M .J., HAYES, M .K and MAllER, J.l. (1988). Pu ri fi ca ti on o f ma tu ra ti on -p romo tin g f ac lo r, a n in tr ac ell ula r r eg ula to r o f e a rly m ito tic events. Proc . Natl . Acad .S ci . U SA 8 5:3 00 9- 30 13 .

lOIR, M . (1988j. Trout Sertoli and Leydig ce lls: isola tion, separation, and culture .Gamete Res . 20 :437-458 .M AllER, J.l. (1991). M ilotic control. Curr o O pin . C ell B io I. 3 :2 69 -2 75 .MANECK JEE, A ., WE IS BART , M . a nd ID LER , D .A . ( 19 89 ). T he p re se nc e of 170..208-

dihydroxy-4.pregnen-3-one receptor activity in the ovary of the brook trout,Sa lv e li nus t on t ina li s, d urin g te rm in al s ta ge s o f o oc yte m atu ra tio n. Fish Physio/.Biochem. 6 : 1 9 -3 8 .

M ASU I, T . and CLARKE, H .J . ( 19 7 9) . Oo cy te ma tu ra ti on . I nt . R e v. C y to l. 5 7: 18 5- 282.M IU RA , T., YA MAU CH I, K., N AG AH AM A, Y . and T AK AH AS HI, H . ( 1991 a). Jn ductionof sperm atogen esis in m ale Ja panese eel, Angui ll a j aponi ca , b y a s in gle in je ct io n

0 1 human cho ri on ic g o nado tr op in . Zool. Sci. 8 : 6 3 -7 3 .MIURA, T ., YAMAUCHJ ,K .. TAKAHASAHI , H . a ndNAGAHAMA, Y .( 1991b ). TheroJe

o f h orm on es in th e a cq uis itio n o f sp erm m otility in sa lm on id fish . J . E xp . Z oo l. 261:359-363.

MIURA, T., YAM AUCHI, K., TAKAHASHI, H. and NAG AHAM A, Y. (1991C). Humanc ho rio nic g on ad otro pin in du ce s a ll s ta ge s of spermatogenesis i n v i tro in th e m aleJapanese eel (Anguilla japanica). D ev. B ioi. 146: 2 58- 262 .

M IU RA , Y ., Y AM AU CH I, K ., TA KA HA SH I. H . a nd N AG AH AM A, Y . ( 19 91d) . Ho rmona linduction in v ir ro of all stages of spermatogenesis in the male Japanese eel(Anguillajaponica). Proc. Natl. Acad. $ci. USA 8 8: 5 77 4- 57 78 .

M IU RA . Y ., Y AMAUCHI , K ., T AKAHASHI , H . a nd NAGAHAMA, Y . ( 19 91 e) .ln vo lv em en tof s tero id hormone s i n g o nado tr op in .i nd uc ed t es ti cu la r maturation i nma l e J apane seeel (A ngu illa ja pon ica ). B io me d. R es. 1 2:2 41-2 48 .

M OA IS AW A, S. and M OR ISA WA , M . ( 1986). A cquisition of potentia! for sperm m otilityi n r a inbow trout a nd c hu m sa lm on . J. E xp . B io i. 1 26 : 8 9- 96 .

NAGAHAMA, Y. (1983). The functional morphology of teleost gonads. In FishPhysiology, Vol. IXA (Eds. W .$. Hoar, D.J. Randall and E.M . Donaldson).Academic Press, New York, pp. 223-275.

NAGAHAMA, Y . ( 1 987 a) . 1 7 a. 20B -D i hyd ro xy- 4- p re g nen -3 -o n e: a te leost ma tu rat ion -i nd uc in g h ormone . Dev . G ro wth D if fe r. 2 9: 1 .1 2.

N AGAHAMA, Y . ( 19 87 b) . G on ad ot ro pin a ctio n o n g am et og en es is a nd s te ro id og en es isin t ele os t g on ad s. Zool . S c i. 4 : 209. 222 .

NAGAHAMA , Y . ( 19 87 c) . E nd oc rin e c on tr ol 0 1 o oc y1 e maturation. In Hormones an dReproduction in Fishes, Amphibians, and Reptiles (Eds. 0.0. Norris and A.E.Jones ). P lenum Press, Ne w York, pp . 1 71 -202.

NAGAHAM A, Y. and ADACHI, S. (1985). Identification o f a ma tu ra ti on -i nd uc in gste ro id in a te le os t, th e a ma go s alm on (Onc orhy n ch us r ho d urus ). D ev. B io f. 1 09 :428-435.

NAGAHAMA, Y. and YAM ASHITA, M. (1988). M echanism of synthesis and action of1 70 ., 20 f3 -d ih yd ro xy -4 -p re gn en -3 -o ne , a t ele os t m atu ra tio n. in du cin g s ub sta nc e.Fish P hy si ol . B io ch em . 7 : 1 93- 200 .

NAGAHAM A, Y., HIROSE, K., YOUNG, G., ADACHI, S., SUZUKI, K. and TAM AOKI,B. (1983). R elative in vitro e ffe ctiv en es s o f 17a,20B-dihydroxy-4-pregnen-3-oneand other pre gnene derivatives on germ inal ve sicle bre akdo wn in oocytes of ayu(P lecog lossus a lt ive li s) , ama go s alm on ( On co rh yn ch us r ho du ru sJ , r ai nb ow tr ou t( Sa /m o g ai rd ne ri ) and go ld f ish (Carassius a ur at us ). G en . C om p o End oc ri no l. 5 1:15-23.

NAGAHAMA, Y., KAGAWA, H. and YOUNG, G . (1985 a). S tim ulatio n of 170.,2013-dih ydro xy.4 .p reg nen -3.on e p ro du ction in th e granulosa ce lls of am ago salm on,Oncorhynchus rhodurus, b y c yc li c n uc le ot id es . J. Exp. Z ool. 236: 371-375.

NAGAHAMA, Y., MATSUHISA, A., JWAMATSU, T., SAKAI, N. and FUKAOA, S.(1991). A m~chanism for the action 01 pregnant mare serum gonadotropin ona ro ma ta se a ctivity in th e o va ria n 1 0U ic ie o f t he m ed ak a, Oryzias latipes. J . E xp .Zoo!. 2 59 : 5 3-5 8.

8/14/2019 Int. J. 0.,. BioI. 38: 217-229 (1994)

12/13

22R Y. NagalwmaNAGAHAMA, y" YOUNG. G. and ADACHI, S. (1985b). E ffect o f actin om ycin D an dcycloheximide on gonado trop in - induced 17a,2013-dihydroxyA-pregnen-3-one

production by intact fo llicles and granulosa cells of the amago salmon,Oncorhynchus rhodurus. Dav. G rowth Differ. 2 7: 2 13 -2 21 .

N AKAJ IN , S ., O HNO, S . a nd S HIN ODA, M . ( 19 88 ). 2 0B -H yd ro xy stero id d eh yd ro ge na seo f n eon at al p ig t es ti s: p urific atio n a nd s om e p ro pe rtie s. J . B io chem. 1 04 :5 6 5- 569.ONITAKE, K. and IWAMATSU, T. (1986). Immunocytochemical demonstration ofsteroid horm ones in the granulosa cells of the m edaka, O ryzias latip es. J. E xp .Zool.239:97-103.

ONOE, S., YAMASHITA, M., KAJIURA. H., KATSU, Y., JIANOUAQ, J. andNAG AHAM A, Y. (1993). A fish hom olog of the cdc2-related prote in p40M 015: itscD NA clo nin g and expre ssion in oo cyte s. B iome d. R es . 1 4:4 41 -4 44 .

PATINO, R. and THOMAS, P. (1990a). Characterization of membrane receptora ct iv it y fo r 1 7( 1,2 0f 3, 21 -t rih yd ro xy -4 -p re gn en -3 -o ne in o va rie s o f s po tt ed s ea tr ou t( Cy no sc io nn eb ulo su s) . G en . C om p. E nd oc rin ol . 78:204-217.

PATINO , R. and TH OM AS, P. (1990b). Induction of m aturation of Atlantic croakeroocytes by 17c l, 20f3 ,2Hr ihyd roxy -4 -pregnen -3 -one i n v itr o: co nsid eratio n ofsome b io log ica l and expe r imen ta l va ri ab les . J . E xp . Z oo !. 255: 97-109.

P AT IN O, R . a n d T HOMA S, P . ( 19 90 c). E ffe cts o f g on ad otro pin o n o va ria n in tra fo llic ula rprocesses during the development of oocyte maturational competence in ate leo st, the A tlan tic croa ker: eviden ce for tw o d istinct stag es of g ona dotro picc on tr ol o f f in al o oc yt e m at ur ati on . Bioi . Reprod . 43: 818-827.

PETRINO, T.R., GREELEY, M.S.Jr., SELMAN, K., LlNM, Y.W .P. and WALLACE,A .A. ( 1989 ). S t eroi dogenes is in Fundulus heteroclitus. II. P ro du ctio n o f 1 7( 1-h yd ro xy -2 0f3 -d ih yd ro pro ge ste ro ne , te sto ste ro ne , a nd 1 71 3-e stra dio l b y v ario usc om po ne nts o f th e o va ria n fo llic le . G en . C am p. E nd oc rin ol. 7 6: 2 30 -2 40

R EO OIN G,J.M . and P ATIN O, R . (199 3). R epro ductive p hysiolo gy. In The Physio logyo f F is he s (Ed. D.H. Evans). CRG Press, Boca Raton, pp. 503-534.

SADLER, S .E . a nd MAL LER, J .L . (1982). I de n ti fi ca ti on o f a s te ro id r ec ep to r o n t hes urfa ce o f Xenopusoocytes b y pho to aHi ni ty l ab e ll in g . J. B io i. C h em . 2 57 :3 55 -3 61 .

S AK AI, N ., IW AM A T SU , T ., Y AM AU CH I, K . a nd N AG AH AM A, Y . ( 19 87). D eve lop mento f th e stero id oge nic cap acity of m eda ka (Or yz ia s l at ip es ) o va ria n f oll ic le s d ur in gv ite llo ge ne sis a nd o oc yt e m a tu ra tio n. G en . C am p. E nd oc rin ol . 66 : 333-342 .

SA KA I, N ., IW AM AT SU , T ., Y AM AU CH I, K ., SU ZU KI, N . a nd N AG AH AM A, Y. (1988 ).In fluen ce of fo llicula r d evelopm ent on stero id prod uction in fh e m edaka (Oryziaslatipes) ovarian follicle in response to exogenous substrates. Gen. Camp.Endocrino!. 7 1: 5 16 -5 23

SAKAI, N ., T ANAKA, M ., A DACH I, S .. M IL LER , W .L . a nd NAGAHAMA, Y . (1992).Ra in bow f ro u f c yt oc h rome P-450c17 (17a-hydroxy lase/17,20- lyase): cDNA clon ing.e nz ym atic p ro pe rtie s a nd te mp ora l p atte rn o f o va ria n P -4 50 C1 7 m R NA e xp re ss io nd u ri ng o ogene s is . F EB S L et!. 301: 6-64.

SAKA I, N., TANA KA, M ., TAKA HASHI, M., A DACH I, S. and NAG AHAM A. Y. (1993 ).Iso latio n a nd e xpression of ra inb ow fro ut ( On co rh yn ch us m yk is s) o va ria n c DNAe nc od in g 3 B- hy dr ox ys fe ro id d eh yd ro ge na se /ll 4- 5- is om er as e. F is h P hy sio l.Biochem. 11 : 273 -279 .

S AK AI, N ., U ED A, H" SUZUKI, N. and NAG AH AM A, Y. (1989). Stero id production byam ag o s al mo n (Onc or hy nc hu s r hodur us )t es te s a t d i ff ere nt d ev el opme nt al s ta ge s.G en. C om po Endocrinol. 75:231-240.

SCHULZ , R . ( 1986) . Imm un oh is to lo gic allo ca li za fio n o f 1 7B -e str ad io l a nd t es to st er on ein the ovary of the rainbow trout ( Sa lm o g ai rd ne ri Richardson) during thep re ov ula to ry p er io d. C ell Tissue R es. 245: 6 29 -6 33 .

SC OTT, A.P. and BAYN ES, S.M . (1982). Plasm a levels of sex steroids in relation toovulation and sperm iation in ra inbow trout. In P ro ce ed in gs o f th e I nt er na ti on alS ym po siu m o n R ep ro du ctiv e P hy sio lo gy o f F is h. Cent re f or Ag ri cu lt ur al P u bl is h in ga nd D ocum entation , W ag ening en, p p. 10 3-10 6.

S CO TT , A .P . and G AN AR IO , A .V .M . (1 987 ). S ta tus o f oocyte m atu ration -in ducingstero ids in teleosts. In Proceedings of the Third International Sym posium onR epro ductive P hysiolo gy of Fish (Eds. D.R. Id ler, L .W . C rim and J.M . W alsh).M em oria l U niversity P ress, S I. John 's, N ew foun dla nd, pp . 224-234.

S HIB AT A, N ., Y OS HIK UN I, M . a nd N AG AH AM A, Y . (1 99 3). V ite llo ge nin in co rp ora tio nin to o oc yte s o f ra in bo w tro ut, Onco rhynchu s my ki ss , in v i tro : e ff ec t o f ho rmoneso n d en ud ed o oc yte s. Dev . G roW1h D if fe r. 35: 1 1 5- 12 1 .

STIFANI, S., LE MENN. F., RODRIGUEZ, J,N. and SCHNEIDER, W. (1990).R eg ula tio n o f o og en es is : th e p is cin e re ce pto r o f v ite llo ge nin . B io ch im B io ph ys .Acta 1045:271 -279.

SUZUKI, K., KAWAUCHI, H . and NAGAHAMA, Y. (1988a). Isolation andc ha ra cte riz atio n o f t wo d is tin ct g on ad otro pin s fro m c hu m s alm on p itu ita ry g la nd s.G en . C om po E nd oc rin ol. 71 : 292-301 .

SUZUKI, K., KAWAUCHI, H. and NAGAHAMA, Y. (1988b). Isolation andc ha ra cte riz atio n o f s ub un its o f tw o d is tin ct s alm on g on ad otro pin s. G en. C om poEndocrinol. 7 1: 3 02 -3 06

SUZUKI, K" N AG AH AM A, Y . a nd K AW AU CH I, H . (1 98 8c ). S te ro id og en ic a ctiv itie s otw o d is tin ct s alm on g on ad ot ro pi ns . Gen . C omp o E nd oc rin ol. 71 : 452 -458 .SWANSON, P ., SU ZU KI, K ., K AW AU CH I, H . a nd D IC KH OFF, WW. (1991 ). I so la ti on

and characterization of two coho salm on gonadotropins, G TH I and G TH II. Bioi.Reprod. 4 4: 2 9- 38 .

TAKAHASHI, M., TANAKA, M ., SAKAI, N" ADACHI, S., MILLER, W.l. andNAGAHAMA, Y . (1 99 3). R ain bo w tro ut o va ria n c ho le ste ro l s id e-c ha in c le av ag ecytochrome P450 (P450scc): cDNA cloning and mRNA expression duringoogenesis. F EB S L eft. 319:45-48.

T ANAKA, M ., O HNO, S" A DA CH I, S ., N AKAJ IN , S" SH IN OD A, N . and N AG AH AM A,Y . (1 99 2a ). P ig te stic ula r 2 0B -h yd ro xy ste ro id d eh yd ro ge na se e xh ib its c arb on ylreductase-like structure and activity: cDNA cloning of pig testicular 2013-h yd ro x ys te ro id d e hy d ro g ena se . J . B io i. C he m. 2 67 : 1 34 51 -1 34 55

TA NA KA , M ., T EL EC KY , T.M ., F UK AD A, S ., A DA CH I, S ., C HEN , S . and N AG AH AM A,Y. (1992b) , C lo nin g a nd s eq ue nc e a na ly sis o f t he c DN A e nc od in g P -4 50 a ro ma ta se(P 45 0a rom) from a ra in bow tro ut (Oncorhynchus mykiss ) o