Medically Assisted Conception: An Agenda for Research (1989)

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E= ME R~CN ~ Em: away Jo M. 13ann~ Icon The grow and ~velc~: of ache ovarian follicle entails a final process of call r—licati~ arm differ~iatic~n churl Whim the Infer of marl arms lining the follicle may increase by several logs arm daughter alas eve With a Sniffers ~~ of horn rancors arx] st~id~enic enzymes (retrim in Richards, 1980~. Purim this process ma~raticmaleven~cs=~e several folli~1ar~-~a granola am come - mast be mused. Purim the r - motive life of news these ells are cI—rly strait deco the pity signals of the r~pr~ive Cycle. E~ver, few follicles are able ~ row to we signals and Mach an arable state. :En minion, a ax~sic3erable body of data SLIMS that initial stages of folli~,lar grouch begm before the Cyclic hy~mic~pi - Entity f~c~k Manic are establish art that the initiation of folli=~1ar grew entire to scare extent Hegira periods in Thick pyclic~1 gana~r~n secretion is in ran not (Ri~, 1980~. _ ~ ~ = ~ ~ - =_ ~ ~ _~ ~ Awe pysiologica1 facts r~i~ marian Physiologists to idols illtramarian control systems ran term authorize or paracrine Anion, lc~ before ~ Am; were ~velc~ and polarized. The definition of we ~i~ has earl as me of the mint tics ~ Ovarian arm In the last Clyde. In rat years, refine in vitro systems arx] Swear appeal; to identify arx] characterize sum ~i~ have ally an unpr~ rate of data activation. It is near generally agreed that paracrine ~i~ are critical to follicle development an] ~]ecci=~, but c3efiniti~re proof of this hypes reman elusive. To care to grips win this amount of information it has been rosary to limit the ~ of this Alien. Tic ~ to air con r~r~ area - paracrine and autocrine Mongol of samtic cells of Me Ovarian follicle by ~tr~ arm gnash factors - have bee ~hasiz~, arm air own data have been use1 as exiles for nary fits pro. D;-~=i~ of Ante mz~turatia~ wit be left to irxlivi~nals at this ~ ;ium ~ o have been more directly involved in defining these mechanisms. For a survey of paracrine mechanisms in the corpus luteum, the reader is referred to another recent review (KhanrDawcod and Dawcod, 1986~. In reviewing paracrine nechariscs in folli=~1ar cell function several classes of regulators have been identified. These are distinguished ~ part by then' biochemical nature and in part by the strategies amply n Air elucidation. 2'1 -

, Ovarian ~i~ It was `~= in Me 1940's that Cambrian sten~ids wan strong Midair for ovarian regulatory prirx:~les (P~arz, 1940; Simian et al, 1941~. this his Is canfin~ arm refine by a rower of critical In viva e~peri~nts perform in he 1970's (Golderiberg et al, 1972a,b arm 1973; Fret et al 1975; Peso et al, 1978~. -these studies, ungirt varietal-= c~inatic~s of ~ins, art, ~tr~ and slid antacid immature a~/or ~ys~ized rats ~~~ a rib for follia~ar <3evelc~ and~cr - In action; In general, a~r~ were found to be inhibitory or attic. he effects of tr~ an gears cull r—licatial In viva Are particularly impressive and these c~;ervatiaY; 1~ to ~ e~ro1utia~ of Me -tatted rat new which has Jaw provided biological malaria for Mantles sties of granule at function In villa. In addition, ~tr~ ~ found ~ interact synergistically with Mains in pm folli=~lar cell diff~ntiatic~n, with Ill r~r irruption Selkirk as a convenient and widely shied end point (See Richards, 1980~. Are or 1~= sin~tan~sly, the obligatory Aeration of {H~t at bilks In p~idi~ serrate for FSH~t grid outran synthesis was defined (Pelvic far Dorrir~ 1977; Byan 1979~. C~11ecti~rely these c~vatians pelvic the frank for Current i~ir~ ~ the induration of steroid bills and action, In the Ovarian follicle. ^m rent c~vatic=; have peril a mx~ pore detailed picture of the action of steroids an follicle ~11 faction. EN action, In particular, has been investigated ~ Hirable Am. Data on estrogen rectors ark their r~ulaticn In Cat ovarian ells ~ particularly adamant arm has led ~ a p~;ive ~ for these birdie sites as an Nor cent regulatory 1~'= for had interaction (Richards, 1975~. Aver, a recent ientia~s effort to - 1 ineate ~tn~ rectors ~ Mar Ovarian Ails fail to disclose sum sty why i~r~ai~1 tennis; 1Hi~d-Petito et al, 1988~. These studies;, alar' th the failure to ~~ the m~t~enic action of ~tr~ In yivo In several species other than the Cat (Kim arm ~1d, 1987) may inclicate significant differer~es ~ the nature arx] ir~ta~ of Ate effects an He Ovary of variants As. He stogie; perform ~ by laboratory and else by J*~a~es Veldt; are typical of Variants Al By several groups whim have pir~poir~ 03 action of ~tr~ cm the client s~id~ic pathway ~ Ovarian ells. ~;esh~ies, Nurtured porcine gran~106acelis, rev~leda ted irAibitozy action of ~tradiol (E2) (Veldts and Eland, 1980) which smears to be Aviated at the 3~roid d~ydr~ step (Veldinlis, et al 1986,a) and whit is followed by a brisk stipulatory acticu1 Of ~m ~ E~in bits (Veldht,;~ and ~d, 1980; Veldt et al, 1981). Ibe latter effects are ti _ t, inhibited by anti~tz~, andinvol~rebi~sofs~idog~icenzyD~, ~ole~ero1 ~ ~~ _

aid ~~a:Lr~ cleavage ~x~r~*s ~ particular Oreld~s arx] me, 1980, Trellis et al 1981, 1982a,b; Staff et al 1983; 1985). In amidic, a Cal Grease In spate foxy to ~ side ~ camp sip ~ generated by agrarian In patters of lip~r~ein utilization (pelvis et al, 1982b, 19847. EffectE; of ~ In this stun are parti=~1ary ~ ~ the pow of Strains (Veldts et al 1981; 1982a;b). :Cn the Maine system this intmactia, BERM to Mar p~inantly, if net allusively at a Ices distal to Me ger~ral:ian of cAMP: it has been infusible far us to ~ an effect of ~ art bum or F5H~b chap levels (Spicer ark Hand, 1988,b) and E' significantly is the effects of chap anal~; (Veldtmis et al, 1981~. In no - t z~s, Air sties ~ the porcine grams of system agree well with data ~~— stipulatory effects of E2 an terms in rat graraIl~ cello: shim were ~ at the sane tine in atoner laboratories (Fitful et al, 1984~. In a~itic:'n, ~= resets camp the sties of Richards arx] of who have Straw i~rtarTt stipulatory i~ractic~ns of g0ad~r~ins ark ~ at Me level of Ibid proteins ark of f~tic~al plated kinase activity (Rata~h arx] Richards, 1985~. Awe latter results suggest a plausible ir~ra~lular manic By whim the ~i~ic-actic~ns of might be ;. Wile the initial germ print leadir~ to these Strom effects has net been det~i~, the Ire distal effects are ran w~l ~ ~;iderable de - I. Are went shies; ~ Char laboratory have fc=~ an the cat~=l~trc~, an irrtriguir~ cleric of chucks which mav fur~tion to ~1 - ~^ =~ ,~ em; =+ ~ a, _~ ~_: - ~ - an: ~~ ~¢ _~ ,~ "Lucas: ashy ~1 u~ ~ ~ Al ~ al ~ Woo bale AL ~~ ~uc~ens. Me activity of ~tr~2~y1~-~ (E-2 ~ the enzyne responsible for Overtire E to cat ~ olestrogens, is as high or higher in ~ e pa ~ ulatory porcine follicle than in other extrahepatic tissues; importantly, the activity of E-2-M dramatically enhanced An the walls of large well-differentia~c~ ovarian follicles, in the mPmbrana granulnFa in particular, with levels 10-100 fold lower measured in immature follicles and corpora lutea (Hammond et al, 1986~. A physiologist function for the brief surge of catecholestroqan biosynthesis in the preavula~cory follicle has been suited By air sties of the actions of ca~lestr~ens con Turk porcine granule Owls. These studies have shun Bat the cat~olestr~s, in the presence of FUJI, have pi Ale to that of E2 in stipulating pro~ne ~etion; hover, their ~ of actia, ~ ite di~tir~b - the two claims- of steroids are signific~y a`3di~cive at dimly effective ~tra~cicms and the actions of ca~lestn~ens are rut inhibited By anti estr~Fns, Bile those of E2 are Copier am ~xl, 1987~. Further the cat~lestr~Fns have minatory effects cm AMP levels arx] ~rgic rectors which Writ be mimic ~ the parents est~ (spider ark HI, 1988a,b). Incus the stage is set for an; - tidal autocrine/paracrine Cysts in the pr~vulatory follicle by chick lamlly so catec~ol~ mild function, all with ~ to; - lify the stipulatory acticns of ~r~ins al a lock level. Al~h space does not permit a Sails review, both alarm arx] Heir also have cI—rly Titrated ream and actions can ovarian (reprised ~ HA et al, 1984~. - 213

Ih sunny, Aids of several claim- can be r~l as established paraded armor a~crire radiators of cwarian furls by He criteria Aim can be fitly applied in Die field. }A the bigness and antis of these ~ are w~t~ In ~iderable ~tail. In general, me ~`kvl of Base Em—can be in~ra~ced into a Went and plausible ~t of Vary E~siolc~y dung me rq~tive ~e. War. · ~ __ ~ ~ a · _ _ ~nc AL 1 - ~~ Ron to be resolved Knin the bib al Rices of ~ and ~tr~ and their relative i~rtar~e ~ vivo. on cre hand, ~ skids serve as a Ned for ~ levy of ~ii~ whim to be aliens Ernie many of me ovarian Eyes He secticms. on 03 our hand, the anus of p~ister~t Ir~i~ point At st ~ on, difficulties in ~ trapolati*~ fink ~ in vitro to ~ e complex milieu of the ovary ~ Viva. The Role of Ovarian Grcw~h Factors As d;-rn-Cc~ above, gFxiYk~bro pins and gonadal studies have unambiguous stipulatory effects on follicular growth and granule== cull replication in viva. However, at least in cur hands, these hormones lack significant mitogenic actions when added to cultured granulate calls in viler (Hammond and English, 1987). In contrast, peptide growth factor,; have been keen to exert pi ~tcgenic effects an ventured granule cells for at least a ~de. ~us, Air In beret, and that of of, in the Ovarian Growth factor was stimulated in part By the speculation fat such factors might provide the His by whip pib';tary and g~1 hinges regulated Ovarian ce11 r—1ication. In the years which have folded, immortal actions of Growth factors =~ ~adiffermiciatia~ of Ovarian cells have ^~ beeline augment. In the E~r~s whip follow, He eviderxx snip palatine effaces of Oral growth factor fairies—the insul~like growth factors (IGFs), the epid~mal growth factor (~-~/tra=;formir~ growth factor -a pa family, the TO - eta fairy, and the fibrc~last growth factom (add;) are retrim. For each of He flies He minim criteria of Utica n folli=~lar cells and Em ~ the ovarian follicle have beg Am. Her, it has ret yet been pcessible to page Cat ardor of Pose p~ptide; are obligatory nE,diatom of follicalar grca~h. Perfuse of Air awn card it and ~ airs- of the amount of evidence available, the IGFs are considered ~ greatest detail. Ir~;uli~like ~ Fencers. The IGFs (exultation factors, satire were originally disxx~w~ned and named And on their insulin-like effects and on their presumed role as the circulating mediators of growth hormone action a~ cartilage Levied ill I)ll~ay, 1984~. E~v~, it he also He clear that IGF~ are synthesized widely in peril tic and recent interest In the field has Realized their role as lom1 mediators of tissue gram and differ~ii~tion {U~d et al., 1986~. King the organs for which an auto or paracrine Fannie of the IGFs has been Cat—, the cat ~ their effects In the ovary may be the Nat ca~r~bensi~re and persuasive. - 214 -

Sina3 air original A~ripticn of the effects of rat ISF-II (MSA) on the level of orn~thi~ ~1~ ~ porcine grad ells ~elc~hu;~ Hi, 1979), are Man 50 pipers have been public ~nL~ the action of the I~ ~ the ad. filly, an Chic review Is ignoble In the Averment Apace. Briefly stated, it ad: Cat these pepti~= have a Amatory action art every edging whim has On did. these Cremate readily into the general areas of gr~-related actions arx] effects on differentiated function. the first area include stipulatory effects of the I0Fs ox ornithir~ Bylaws (Veldbu;-= ark Ed, 1979), cell remeet Are (Savian et al., 1981; Ed et al., 1983), thymidine in~rporati~ into 1~ (Baranao arm }I, 1984; }A arm English, 1987) , glad a~cidatim (fir and Subarea, 1988), and stipulation of olysa~ride Venetian (~3a~hi et al., 1986c). In Haitian ~ these Greed F~, ir~;ul~ arm the IGEs have been fang to errand cliff~iated furx~ic~n of both theta As (Barbieri et al., 1986; Bra arx] ~field, 1988) arm Gould ~Is. With gram Ails, me IGFs or insulin seen to be essential for optimal effects of AH on IH receptor ir~tic:~n (Mash) et al., 198Sc), ger~rati~ of cyclic Ad (Ada~hi et al., 198Eib) and for FIJI antic on sternid~enesis (I~ranao arx] Hand, 1984, 198fib; ~3adhi et al., 198Sb). Se; specific points in the sten~ici~ genie pathway am Chad by Ids including i~alizatioa' arm pressing of ex~xls ^)l~terl:~1 (Veldht];~ et at., 1987), the sit clangs ~ (Veldhuis et al., 1986b) , arm ar~atase (Ada~hi et al., 1985a; I~voren et al., 1985; Veldts et al., 1985~. Ad con the magnitude of the effects ;, it ~1d Bar that the clifferentiative influence; of the IGFs on folli~1ar cells are at let as important as then' m~tc~enic actions. In Batik, the relative inportar~e of the gr~ch-relat~ and c~iffer~iative effects of ~ I0Fs ~ an the level of Greta cliff~tiati~ am the influx of Aver hordes and grad factors ~ et al., 1988b): ~ the presence of AH and In the milieu of the pr~vulatory follicle, IGFs prance sterai~;is; in the presence of other growth factom m~togenes~s p~irmtes. For Be actions of the IGFs to be mediated in an autccrine or p~crine fashion, it was redry for them to be present anchor secreted ~ the marian follicle. this Chic has ~n a particular inb~b of air I^ - ratory, and Cal' s ~ ies have Rae Gently been confir ~ d and extended Or ot~ r groups. Fegardin3 ovarian IGF levels in viva, our studies have indicated that I) IGFs and IGF binding prcte ~ were -~-~ y reasuc able ~ porcine folli~,lar fluid OKam~on] et al., 1982; 1983), 2) IGF-II-like ma1~les and IGF-~-like in~Lncactivity were both present piamnond et al., 1985, Mcrdschein et al 1988c), 3) the ococantrations of the IGFs were sufficient to tunic the actions of Be Patti - ; in folli=~lar Be so (~d et al., 1983,1985), 4) me oonc~rati~ of T~ in folli~1ar fluid imposed Purim folli~1ar Gnat (~d et al., 1985, disdain et al 1988c), ark 5) the concentration of IGF-] in follim3lar flog was Creased for grins (~xl et al., 1988a), ark grad homage (Bryan et al., 1988) . Related dad n ovarian I=-I levels in the rat ark Weir stimulation by growth horde have bed develc~ through organ ex~ctic~ by Da~roren ark ~ueh - 215 -

(1986). In Portia,, high ticks of I~-II have }on famd in }Oman follic-,lar fluid, harm et al., 1986). Elided Urns he station of IGFs By Ovarian ~1 Is and its Hannibal r~ulati~ has ban plaids ore directly ~ cultured grar~lc~ cells of porcine and Ban species. Our data with porcine cells s—lest hat IGF secretion ~ Staid urger senm~free a~i~cia~ for at lost 10 days In culture (A et al., 1985), and that inactive IGF-I paired Over IG]?-II~s~ein et al, 1988c). lee secretion of im~nx~reactive IG]?-I Cold be Shard by AH, estradiol, Is, growth Laurie (Lou and ~d, 1987a,b), and ,hI_~- ~~ ~ ~d, 1988). ~ i, ~fo~ growth factor~eta airs to inhibit IGF secretion in vitro except at very lcx~~ntratic=; p~mand~d, 1988c;M0sdm~net al, 1988a). Our data with folliallar fluid, rightly collected by analysis of ovarian Ned levels, Ant At I~-I and II am both synthesized In the porcine ovary ~= et al 1988; And et al unpublished). Ihis situation may differ Frau that ~ the hen ~ assays for Be aseptic and their A; At that the dminant Ovarian IGF Is I0F-II (R~ and Li, 1987; Va~tila~nen and Miller, 1987). In fist, hybridization data fmn Ovarian extracts (gnat et al., 1987a; Arty et al., 1987) sagest that IGF-I is Be dominant, if AL the exclusive, Ovarian IGF in the rat. Be diversity of expression of th—;e peptide; ~ to= Ovary ~ ~C:i~X is intriguing and scx~hat Al. Be evil reviewed above Air s;~1tanea~s section arx! action of IGFs ~ the follicle sorely sits a rule for IS In folli~1ar grouch ark desrelc~nt. Hover, it zag; ~aller~ir~ to prove that they are c~ligatory n~;ators In thee pros. St~ies with ~clonal antibodies to the I=s have putrid an ~;ticxlal approach. In air porcine granule ~11 culture system (HINDU arrt HA 1987b), go hogan, E2 arid Perkins interact syr~istically to stimulate IGF-I and pz~te~ secretion. Rent studies have An that the effects of these hoDnes on steraid~enes" can be ~tantially diminish by a n~l~al antibody to IGF-I fin et al, 1988b). obese data constitute a direct tration of an autocrine action of IGF in vitro. Related studies, ir~icati~ an inhibitory effect of the ISF-T antibody can the sten~i~enic actions of folly fluid] have also An the illporta~e of IGFs ~ the stipulatory milieu of Be preavulatory follicle fin et al, 1988b). these data are consistent with a role for IGF in folli~1ar devel~nt in vivo ark cystic the mat direct evic3erx~e available for a critical Hale for l IGFs as 1~l amplifiers of the effect; of pih~ita~ ark gonac~al hormones. TO air kr~l~ge, sum ions= here rot been USA for the cipher Arm factors. H-~ and ELF- PeptideE;. As reviewEx1 by ethers (greets ark Z~i, 1986; Cohen, 1987) , the primary stroke of ED ~ as well as Pat of its rotor, hoe been Leon for sag the. Bile many ells ~ Be bog ~ to this peptide, the nature ark sage of the As ligate for these actions Is unclean Stable levels of ~pr~;orn~were fag in ~illary gland, kinky, Awry gland, As, small i~;tine, - 216 -

pit~;tary, lured ppleen, b~' arxt Ovary ~ order of d~asir~ ~a~ ~1 et al., 1985). ~ Critics to pepsin similar t;o salivary ~F, relay peptic have been ~zed tory the use of radior~xptor assays for Em. Ire must windy shxli.3d of these is 3= alma. This factor has been fat Gainly In trar~fo~ ~11 my;, but Patti—: him alar to be similar ham been fat ~ platelets (Assoian et al., 1984), pituitary (s~ar et al., 1986), and Emery epithelium (Val~rerius et al., 1987) . As filed ~~ - J. I~F alla Is to be ~ Fr-~—like provide ~ the ovary. The Masonic actions of Em c' varian ~Is, first ~at"1 by r~n.cz et al. (1977a) In bovine grarnOosa ~Is, have mew been In granul=;a cells of c*her species arx] In a variety of =~ ~itions, (~ncz arm Bial~ki, 1979, F~i~t et al., 1986; Hat and Erotic, 1987~. ~ brash to me cot' stipulatory actions of =t an ~1l ~plicatico, me actions of ELF on differ~ia~ function are Ire complex and generally inhibitory. Severn1 gram have demonstrated a r - Dative effect cn Men genetic by grammar calls (~ et al., 1981; Knot arx] C~tt, 1983a) arx] ~ FSH-ir*~ ~ rectors ~s~= and Sc~a~rg, 1981; it a=] Ott, 1983b). As arIticipat~, Arable results were adhie~red with '~-alpha (~3a~hi am] Rennin, 1986) whim birds to tile sane rotor. me multiple inhibitory effects of Em an FSH action ~ me possibility of a gimbal antacid of F5H effects. ~ever, Be- can erhance FI;H birxti~ to pig granulosa ~11s (My et al., 1987), arm me data or1 ELF effects and ~F/DiH interactions An me priests bi~thetic pathway have bed quite var;~hie. Ibe latter results are at least partially at for by cx~znita~ stipulation of peters metabolism to 20—ydr~p~ter~e and sti~:tatic~n of averts preen bills at me sidereal cleavage reaction arm the 3 - Rid d~y~na~ step (Jones et al., 1982~. me data rubric above Dede EGF-like peptic; attractive mr~idat~ as intraovarian regulatory ~1 es. Ihis poesibili~r has been further sup~r~ by red shies In several laboratories indicating Ad- or -alpha like activity In Me Ovary. Using a radiator Ably which realizes both Em- and 1~ alpha, we detected ~ysiologi~1ly significant levels of -like activity in porcine folli~llar fluid (Hd;u et al., 1987~. me ~wrtra~cio'= In folli~lar fluid wed ~canbially higher than those In porcine plum and higher in fluid f`~ "nail man fmn pr~vulatory follicles. Are Ray, these studies have bed ED r other groups do have In that TGF aplha is secret" by Rod ~ cells (Skinner et al 1988) and localized to ~ Owls 18s~_i~1ly (~1~ et al, 1988~. Collectively, these data DElke it Try ~ ly lilmly that EX~-like pumpkin-= will be assigned an autocrine or paracrine function ~ ovarian physiology. We postulate that their dot inant effects will be exerted in cnF11 follicles where they should act to promote ~11 replication and restrain differentiation. However, an understanding of the true significance of these pqpkiAFc will ~ more detailed studies of their nature and regulation. l~nsformir~ Growth Factors beta was initially defined as a peptide whim, in (~xljUrXItiOll off EXJF or ~~ a, allied reversible transformation of normal rat kidney Ills to anchorage irk growth (Spore et al., 1986). S~ral analyses have In a s~cia1 duke of seye - 217

l~y been l - ~a, irhibins, activins arm Mullerian irhibitir~ tang, 1987). Toyota Is new In to be p= t in, or s~ by, a rower of reseal as ~1 as nonplastic cell types and is print in al in platelets (Assoian et al., 1984). Ite action of this factor is bi~tial, faci~i~cati~ grog in ~ ~11 go; ark ,~llb~ cities, but inhibiting growth ~ our ci~ (lies et al., 1984; Alerts et al., 1985). As purified preparation; of Taffeta beg available, its effects con cat granary Ills Ire ever; Ire or 1~ si~ltar~sly by several laboratories (A`3ashi and Pesni~k, 1986; get et al., 1986; Yips et al., 1986; Susan ark go, 1987). In the adulate, these studies a sti~la~ry action of 'yucca An AH acetic, reflect ~ an Age in espy secretion (Mash) ark Resrlick, 1986; Yir~ et al., 1986), IT }or activity (Kit, et al., 1986; rag and Sag, 1987), are] pr~t~e bi~yr~is (Dysan and Sag, 1987). Ire recently, a stipulatory infraction of Qua ~ FSH con INA Rests by rat granule cells has also been Strafed (Dorri~ et al, 1988~. While the stipulatory actions of Thea can rat gram owls seem clan', the work of ~t et al. (1987) has mien that tom effects dam critically an the ~ntration of the grub factor and of FSH. At high FI;H ~xr*rati~ a d~3ent irhibitoryacti~of TGr~etz~was Scam. In a~rporuine grayly cell culture system, ~ whim z~licatic~, thymidine incorporation, IGF secretion, arm Eli _ t pr~t~e suction Here storied, an inhibitory effect of 1~ was fat on all the parroters ermine Rein and }go, 1988; Rein et al., 1988a). Me effects of '~-beta in this system were particularly caviar= when the particle was Cabined with stipulatory ~tratic~ns of or ~ (offs ~ to thymidil~e ir~rE=n~tion. ark IGF-I satiny or with FSH (with rears to pr~e #accretion). Our ruts are In aghast with the grub inhibitory effects of ~ - em on bovine Granule Delis (Skinned et al., 1987) as well as with the inhibitory effects of TGF—eta an grub ark differ~iatic~n of abler retrial arxi nonplastic ~11 types; (grief ~ go; et al., 1984) ir~c1~i~ Article Ells (cotta and Baird, 1986~. In go, TGF - eta has bun fat to have paint hat variable effects on bath replication art diff~tdilt~ fur~ticn of Adrian grar~losa ells. It ~r; that the action of this peptize will depend critically cm the sties ard,/or the preserve or absence of other speculators of grate call faction. This ~ reflective, An the Ace herd, of the ~1 trim biE~eia1 nature of T~a's action ~ n3~s ~11 types; art, cm the Aver, of the duplex Iranian of - ~ltip1e facts Aids are irrvolv" in cFrarian cellular dial. six i~rtar~ of these actions of TG[ - eta i'; ~or" by rent data fray several "arm; ir~icatir~ probable s~reti~ of ]~ - pica ~ the ovary. skier et al. (1987) have Rated the gig of in am reactive Area by Future bovine are Nat theca Ems. In Ratio, the MA for Strata has bun fa=1 in the chary by I~rynd}c and Rhee (1987) are ~rnar~z et al. (1987b). Alto the details of the secretion of Rota by ovarian Ells r'= to be worm At, this Gram factor can probably be r~r~ as an important autocrirx3/paracrine influence in the Ovary A the buts of present dam. 218

Fibr~last Ah Factor. Me ~ of Im was ~ ~ the Botanic activity of pi - Italy Tracts ark partially purified pituitary handle pr~ratic~ns. Since these original st~ies He peptize has been purified to hey fmll a ~ of hiss, including We ovary, and its Binary 6tr~re analyzed (retrim by Bird et al., 1986; Wick et al., 1986b). Be OF peptic= have patent gr~r~ti~ activities an many cams of Hal origin, are the my - enic action of me Few on Lial cells, Implying angi~enic activity, has attract particular a~ic~n. this action may be of Rare ~ me develc~ of ~ ovarian follicle as sell as the corms 1~. shortly after purified preparatic~ns of F0F ~ available, the Halide fang to be m~tog~ic for granulosa cells f~ Ovine (~ar~icz et al., 1977a), rabbit, porcine, An, are guide pig follicles (~ar~icz and Biale~ki, 1979). Ells free corpora lutes also retained sensitivity to OF but not ~ (~ar~icz et al., 1977b). In addition, [~t shorts the Garth of luteal filial calls ((~ar~icz et al., 1986a). Effects of E~ ~ diff~tiated fur~tion and s~ic~;is by ovarian cells have net been stied as Thematically, but the available data Hats that the net effects are generally inhibitory (Savior and ~ar~icz, 1980, Baird et al., 1986, Hi et al, 1988). me fist clef' Cation of [ - In the Calvary was by ~ar~icz et al. (1985) who purified an attic factor Mom bovine corms luteum that had binning, biological, arm Hex homology to pituitary For. Ore meetly, Neufeld et al. (1987) have also An that bovine granulosa Ells prime a basic E~ similar to that fm~pit~uit~ ~ Tare el~tz~hore~cic arm reactor bit activity. Related data have been pulpy Birded al. (1986) whc) extracted an [~-likepeptide fin rr~luteiniz~ rat ovary. the r~aticmship of He well characterized liar species to atoner peroxides which EYK~= me or Ore of the pry of Em rum; a subject of active Elation. A hepari~birdi~ Al mit~angiogenic factor, d~ated tar Claris et al. (1984), aE~ar~y has scare similarities to ~ ~ edict Tuition (M&kr~s, E~1 Fornication). In Action, other facto=; rem grant cell~iti~l Odium love ar~iogenic activity (Ken;, 1986~. With the i~E~t ~ specific receptor and Whys for Me EGFs, a Ire dint Parish of Me factors to ] - ~d be fo~irg. In spry, ovarian growth factors have been intently investigated an reagent year';. Be data available irxlicat~ that ~rera1 Ha; of gram factors are Rested, active, arm probably Important ~ the ovarian follicle. Eta Hurt the IGFs ~ mart complete, arm our level of ~i.rq of me role these phi - ; rivals Cat achieve for steroids. With Rae to the IGFs, the Rim picture Hats an Ovarian an~lificati~ An interfaced with pituitary arm goal horns at several levels whim ~c! erharx~e the ~velc~nt of follicles sell for ovulation. coca Nordic the secretion arm action of our Growth factors Is I^~c at, but it Is actuating rapidly. A ~~ Hub picture of the p~siolc~i~1 i~ortarx~e of He peptides ~ ld be Fusible shortly. - 2Ig

Our ~ Prides , _ _, , _ be Ovarian Follicle men I first reprised this an for a sy~6ium ~ 1980 (and, 1981), thinking In be field was dominated by a lumber of factors, identified in folli~llar fluid or ovarian e~ccracts and fined In tenrd; of In vitro bits whim were assured to reflect paracrine relati~hip6 In me ovary cx~yte ma~ratic~n ir~ibitortt, ~luteinizati~ inhibitors, ttl~Tteinization ~imdatort', ~cz - in birding ir~ibitors, arm Gr~H-like suntan (~c Adonis and S—al, 1982). At the Tim, these factors berm ~E;iderable city ham a, the as~ion that they ~ unique ovarian Riptides which, when EA~rifi", w ~ d allow safe and specific a ~ roac ~ ; to f ~ iffier regulation. Particularly frum this historian perspective, progress ~ the definition of these factors has been disappointing. Although the phenomenclogi~1 and conceptual framework of these observations has persisted (ace reviews by Tbnetta and deZeriga, Tsafriri and Pomerantz, and Khan-Dawcod and Dawc ad, in Franchlmcnt, 1986) none of the peptizes themselves has been purified sufficiently to permit structural analys ~ or definition of mechanism of action. In the He of sew coca, it seems Educible that sum effects may be redated for other, mare rightly defined paracrine regulator=, acting indivisibly or in concert. In particular, we found that most of the stipulatory activity, for granule ~1l steruidc~enes" in folli~llar fluid Frau preavulatory follicles, previa~sly termed "~xteinization stimulator" (l~witz-Rigby am Rightly, 1983), could be neutralize By a mor~oclona1 arrtib~ t:o IGF-I. Apices of severe of the other grew factors described ate overlap the inhibitory effects ascribed to variants follit-,lar factors on somatic cells of the follicles, and the Offs and EGF also have well described effects on oocyte maturation (Dekel and 5heriz~y, 1985, Feng et al, 1988~. Of the follicular factors, ~ data is most abundant concerning a GnRH-like pepkide identified and partially purified by Behrman and oolleagues (Aten et al, 1986), and a broad spectrum inhibitor of steroidbgenes ~ named Follicular Regulatory Fepkide by deZeriga and colleagues (reviewed ~ Tboetta and diZerega, 1986). Both of these factors appear to represent discrete entities; however, their biochemical nature and similarity or diccim;larity to other proteins previously identified in the gonad or elsewhere remains to be determined. The cu~bersoca bioassays for apparently unique ovarian peekides may ~ Retinue to hamper purification efforts. However, it would be unfortunate if such efforts were totally suspended, since they set l offer the best change to isolate peptides unique to the gonad and useful for regulation of gonad function. As proved to be the CAMP for inhibin, these difficult purifications should be technically forcible when cl~-ccical bi^~=C^y prooedure6 are reinforced with modern fractionation and mal ~ alar techniques. A., ~ ore recent years, l~66 at Me clefiniticm of peptide radiators In the gcmad has pro with pros and techniques quite different than ewhi~urx3erlay these initial efforts. The tacit a~bic~n of most Parr studies Is Mat peptide radiators id~ifiec] arm purified f~ other organs are pro and inpor~cant in the Ovary. Ire utilizatic~n of i~i~1 and ~1~1 ar probes for sum previously identified pepti - = has maw Foible a~nJatic~n of information cn goal E~i~X at a lance previously infusible. Variants of this strategy led to identification of many of the ovarian growth factors described previously. Aversely, surveys - 220 -

of other ti~PC for go gene pc~t6, e.g. irhib=, indicated the F~i~ for a Max broker physiological hale than initially ~ (punier et al, 1988). Finally, sup techniques have ~ possible a Dare rapid Edit of the relaticm of gimbal Stir to relate gene fPmi 1 ies of considerable interest to ovarian physiologists. As again exemplified By Me ir~ibirVactiv~n field, ~ of Eve woo urban at Me initiaticn of Hose sh~ies (Ying, 1988~. Other global peptic identified by these strategies include transferrin, fibm~tin, ar~iatensm lI, Stile Y. Fiord patio, vasc~ressm, growth lxn~3 relying hormone and Ear Amps factor. It Is inedible to Graze the sta~of-~art Zaire eat of Muse regulatory Scam Am. Sam of He topics have been ~ ire our remet review that may be helpful ~ Bring areas ~ apply develop hue —.~ et al., 1984; Hs~, 1986; Rosetta and —7:^riga, 1986; ~afr~i are F~antz, 1986; Smith and Fiery, 1988, Syrups, 1988~. Sugary of Current Efforts and Fire Ares It will be clear fzan the forgoing rev)"' that the aut~rine/paracrine influerx~; which impinge can ovarian sells are axY;i~rably more n ~ s and ~ t certainly as important as the classic endocrine feedback loops. On the other hands the endocrine, autocrine' and paracrine relationships involved in ovarian development have reached a level of complexity that currently defies comprehensive analysis, and the true situation is urdkLb*eCly substantially more complex. While continuing the descriptive phase, concerned principally Pith identification and characterization of new regulatory mechanisms in the ovary, we also need to integrate this information into pysiologir~1 coo structs, to establish criteria for judging which of the putative regulatory systems are most important, and to begin to develop new investigative strategies which may ultimately lead to phar~oologi~1 control of such Units . by general Dent the minis criteria for Avis paracrine/aut~ine regulatory systems is loam solution err] local action of putative regulatory principles. these criteria have Ben met for the principles considered In this redries with the e ~ tion of Are of the more recently discovered peptizes mentioned in the previous paragraph. However, considerably more detailed information about the mechanisms involved In the synthesis and action of effect or molecules ~~ needed for most of the paracrine systems. Of many topics deserving of mention, two are particularly intriguing to me: 1) the ?n~1tiplicity of mRNHk species an4/or large pepkide Arm; for several ovarian factors ~ Desist with biotic arx3/or pot - ranslatia~al r~ati~ whim Alla be Dime Pacific art hormone rearm; 2) the biochemical Manic whim govern the interplay between z - licati~ arm differentiation of ovarian cells represents a critical topic for further ~ y. A =ivatia, of pret ~ s may be Are fruitful a Breach ~ this area. Next, it seems essential to understand the manner in which the secretion and action of these local regulators is inberfared with the hormonal signals of the reproductive cycle and coordinate] with developmental changes in the several clarion compartments. These issues have been best addressed with regard to steroids and the IGFs. Increasingly refined Nell culture · ~ ~( ~ _

ties will ~i~ to trite ~ Air ~i~ of Me s~retic~n arx] action of paracrine facts and the ~l of 0#;e pro far Me cl;~.==ic Ovarian regulator pituitary Horton and genial steroids. VItima+^ly, it ~ By to indurate the information fray these ~ vim= sties, In whim pros has been not ~ rapid, with the situation in viva Analy~;~s of foUiadar Of; tragic of Me adrenal said _ e an! peptide regulator; arming folli~,lar grow has }me a clonic strategy. Ever, this ~rutegy has I~ to an Chasm ~ the role of such factors an the te spinal s ~ es of folli=,lar ~ velc ~ nt and different lotion. To n-C~== the likely importance of such influences in folli~logenesis and the initiation of follict3lar grcw~h, other techniques will be required, the most chin ~ being ~ ~~rchise~che=~ca~ and yeas for relevant peptizes and hybridization histochemiE try for their TERNS. It is acknowledged that such studies are intrinsically ~P=rripkive and correlative and unable to clearly delineate cable and effect. Ninety=, a good deal Ore of this information will be retire to pin Beeper a Brat pysiologio=1 picture of the role ~ immortal of Be factors. Ultimately it will be Wary to develc~ niques to bitt or amplify the effects of sum factor. In the firm instant, such strategies are needy to clergy determine which of ache marry putative radiators are Rental to ovarian function. fly, these pilot studies may pomt Me way to a fore systematic application of such Knit; to regulate fertility. One approach is illustrate by Air use of D=rxx:~a1 antibodies to _ . . . . . . . . ~ . . . IGF In veto and ex viva (ate folli~1ar fluid). With are widesDread availability, ar~i~iesto the ligar~sar~Vor their r~rs ~dprOvide useful tools for Eharma~logi=1 ablation of these Systems In vitro, and, urger scale Is, in merino. Ultimat-1y, i~i~a~ techniques may be splat by Briar Studs sum as birdie of functional RNAs by mici~e pro; am sit~di~ ~= of ovarian pepti - ; or their Captors;; sum techniques are in relatively widespread practice in the stay of grouch factor effects in transfon~ call lines. ~ever, even with He availability of appropriate blacking agents, Somatic ~nipulati~ of these pa~crire relati~ipe In viva son rue. Several of the limitation; a, these strategies are intrinsic to the very nature of the pezacrine/auto~ine His arm may be untruly difficult to averoalle. A Ordinal feature of these relationships is their restriction In tin art Apace to ally their impact to be specific. At present, we lack the topological capability to deliver either the regulators or blc~kir~ agues in a Drably Pacific fashion. In Bib n, all of the ovarian paracrine regulator disc~rer{~1 to date exist ~ or intact Am, many if not tact body tissues. 1~ ifici~ In their Ovarian tactics cS-rive fran Heir interaction with pituitary henries or ~K3al Bids whim Other embark their s~ticm or modify their effects. As Hi - - ax, Me Stick of Ark sordid ark petite regulators an ovarian apes am quite variable, off the effects Countered critically ~c ~ the Exits preserve of cir~latir~ hare, other lo- radiators anchor the level of differentiation of the ovarian cells Big. -these complex relationships; c- - n well designed for fine ~1 of the Whelming ovarian follicle, ho they may prove fzustratir~ to ovarian physiologists who seek to manipulate than. 222

~,,3 I a~wledge the helpful its of leer. J.S. disc - In arm devat" service of Mrs. icy Muir, i. Saga Clan, is. saga ~ aced - . ME Of Em. 223 -

Lily Ci~ 3ashi, E.Y. arm C.E. Pssni~k. 1986. ~is~c ~aticnE;;of Conforming grow factors In we r~atic~n of gram cell differ~ia~ci=~. B~rir~logy ll9:I879.- Ada~hi, E.Y., C.E. Res;nic~k, C.S. Croft, J.V. May, ~a~icz. 1988. R - sic fibs grow factor as a repair of Ovarian grands call diff~bian: a r~1 r~nit~enic role. Em. ~11. Endocrinology. 55:7. 3a~hi, E.Y., C.E. P`esni~k, A.M.H. Brcxlie, M.E. Spa, arxt J.J. Van Eye. 1985a. Sc~na~i~C~;a~ ~t~biaticm of follicl~sti~latir~ hor~i~ ar~atase activity of ablaze rat grmn~lc~ Owls. Er~rinalogy 117:2313. H3a~hi, E.Y., C.E. Schick, M.E. Spa, and J.J. Van talk. 198Sb. Sanatcrr - uric sy~izes Pith folliclff~;n',Qatir~ hormone In the acquisition of pr~estan bic~;ynthetic opacity< by c~1t~1 rat granule cat Is. Er~rinalc~ 116: 2135 . Adashi, E.Y., C.E. Resnick, M.E. Svrhf~, and J.J. Van Wyk.1985c. sGcedszKatin-c enhances induction of luteinizing hormone receptors by follicle-st;~lating hormone in cultured rat granule== cells. Endocrinology 116:2369. Aten, R.F., A.T. Williams, H.R. Behrman. 1986. Ovarian gonadotropin-rel^~=ing hormcne-like prose ~ (s): dklrx~stration and characterization. Endocrinology 118:1148. Adashi, E.Y., C.E. Resnick, M.E. Svaboda and J.J. Van Wyk. 1986b.SomatccE3~rn-C as an amplifier of follicle-stimu~ating hormone action: E~!iu~oed accumulation of adenosine 3', 5'-moncphosphate. Endocrinology 118:149. Adashi, E.Y., C.E. Resnick, M.E. Svaboda, J.J. Van Wyk, V.C. Hascall, and M. Yanagishita. 1986c. Inlk~x~xdent and ~ gistic actions of somatrmed;~-C in the stimulation of prcteoglycan biosynthesis by ventured rat granLl06a m-1 Is. Endocrinology Il8:456. Assoian, R.K., A.B. Rcberbs, L.M. WaXefield, M.A. Anzano, and M.B. Sporn.1984. T=ns forming grow factors In nc~lastic t;~s and weir rule In Trolling All grow. En: J. Fc~mi~, B. Ozarme, arm C. Stiles (Ends.) fir owls 3/~ Factors arm 1~ansformatic~n, An. 59 64. Cold S~?rir~ Har3x~r I~borator~e;, Cold Sprig Hirer, Now York. }gird, A., F. Escort, P. Muzzle, N. Am, N. 1 - , P. Allen, S.-~. Yips, W.B. rg, arm R. Guill ~in. 1986. - ~ ~ liar characterization of fibs blast growth factor: Distribution and biological activities in various ti=C~-C. Rec. Prog. Harm. Res. 42:143. - 224

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