Hormonal Correlates of Follicular Development in the Human Ovary*

Transcription

1 ! Aust. J. Bii. Sci., 1981, 34, Hrmnal Crrelates f Fllicular Develpment in the Human Ovary* Kenneth P. McNatty Wallaceville Animal Research Centre, Research Divisin, Ministry f Agriculture and Fisheries, Private Bag, Upper Hutt, New Zealand. Abstract This review summarizes recent studies pertaining t fllicular develpment in the human vary. Sme f these studies were cncerned with characterizing the steridgenic capacity f the individual cell types f the fllicle in relatin t whether the fllicle was healthy r atretic. Other studies cvered in this review cncern the cyte and its hrmnal envirnment bth in viv and in vitr, the effects f sterids and gnadtrphins n the steridgenic ptentials f fllicle cells and als sme endcrine and nn-endcrine respnses f thecal and granulsa cells after being separated and then recmbined in vitr. The data are integrated in relatin t the develpment f a fllicle during the fllicular phase f the menstrual cycle. The studies n the islated cellular cmpartments f the fllicle shw that the bisynthetic and mittic r develpmental activities f granulsa cells and thecal tissues, and the behaviur f cytes in vitr, all crrelate with the hrmnal micrenvirnments that they were previusly expsed t in viv rather than thse in peripheral bld. The data suggest that the levels f sterid in fllicular fluid and varian venus bld, cllagen synthesis in the thecal tissue and mittic activity f the granulsa cells are all dependent in part n a functinal interactin between the thecal and the granulsa cells. It is cncluded that the fate f a develping antral fllicle centres arund its ability t generate an estrgen-enriched intrafllicular envirnment while simultaneusly secreting bth andrgens and estrgens int varian-venus bld. Intrductin In the human vary, fllicles begin t frm during the furth mnth f fetal life (Peters et al. 1979). Althugh sme f these newly frmed fllicles start t grw almst immediately, mst remain at rest until sme signal(s) (as yet unknwn) initiate the resumptin f fllicular develpment (see Peters and McNatty 1980 fr review). The resumptin f varian fllicular develpment during fetal, nenatal r adult life begins when the spindle-shaped granulsa cells adjacent t the cyte transfrm int cubidal-shaped granulsa cells and then underg successive mittic divisins cncmitantly with enlargement f the cyte. As the granulsa cells prliferate, the cells immediately utside the basement membrane (the thecal cells) becme cncentrically aligned t the fllicle and fr the remainder f fllicular develpment, the thecal cells develp synchrnusly with ther cmpnents f the fllicular apparatus. Thrughut fllicular develpment, the thecal tissue is vascularized whereas the granulsa cells and cyte are separated frm the bld supply by the basement membrane f the granulsa cells. * The 1980 James Gding Memrial Lecture given at the Annual Meeting f the Australian Sciety fr Reprductive Bilgy, Armidale, N.S.W. in August 1980.

2 250 Kenneth P. McNatty This review summarizes sme recent studies pertaining t fllicular develpment in the human vary. Sme f these studies were cncerned with characterizing the steridgenic capacities f the granulsa and thecal cells in relatin t whether the fllicle was healthy r atretic. Other studies cvered in this review cncern: the cyte and its hrmnal envirnment in vitr and in viv; the effects f sterids and gnadtrphins n the steridgenic ptentials f fllicle cells; and sme endcrine and nn-endcrine effects f the thecal and granulsa cells n ne anther. The data are then integrated in relatin t the develpment f a fllicle during the fllicular phase f the menstrual cycle CD I x 10 a; 5 c UI.2 ::l C Cl '0 ' 0 z...:.:...:.... I L.....: i:.. :!. :e :- 1e : i :.: +'1: :.!::::-: : il -,.;:- i: : F: : 1: : I 1.:!..... i. e.:...:.:.:,.ata.l. 4,! I I I Fllicle diameter (mm) Fig. 1. Number f granulsa cells recvered frm 306 individual human fllicles f knwn diameter which were btained frm the varies (intact r wedge bipsies) f 73 wmen at different stages f the menstrual cycle but wh were underging surgery fr a variety f gynaeclgical disrders. Grwth and Atresia f Ovarian Fllicles Mst human fllicles (99 9 %) which start t grw degenerate at sme stage f their develpment (Byskv 1978). During preantral develpment, the incidence f atresia, as assessed by mrphlgical criteria, increases with fllicular size. Hwever, the ttal number f fllicles which degenerate befre the antrum is frmed is prbably lw (Byskv 1978). In the infant human vary, nly 15 % f all grwing fllicles underg atresia during preantral develpment (Himelstein-Braw et al.1976); hwever, quantitative data are unavailable fr the adult vary. In studies n antral fllicles, McNatty et al. (1979d) have described a series f functinal criteria by which the degree f health r atresia f a fllicle might be assessed: it was prpsed that fllicles with mre than 50 % f their full cmplement f granulsa cells at any fllicular diameter were thse which had sme ptential fr further develpment (healthy fllicles). That is, nly the fllicles with less than 50% f the maximum cell number per fllicle size were thse underging irreversible

3 Hrmnal Crrelates f Fllicular Develpment in the Human Ovary 251 atresia (atretic fllicles). Healthy fllicles were thse which cntained high cncentratins f estradil in fliicular fluid (> 1000 ng/ml) r granulsa celis with the capacity t generate this sterid in respnse t fllicle stimulating hrmne (FSH) r bth, and a germinal-vesicle stage cyte which appears healthy at the level f the dissecting micrscpe (x 100). Atretic fllicles were thse with lw levels f estradil (Ez) in fllicular fluid and granulsa cells incapable f respnding t FSH t generate high levels f intrafllicular E 2 The distributin f granulsa cell ppulatins in human fliicles f different sizes is shwn in Fig. 1. These data indicate that mre than 85 % f the antral fllicles in the human vary are smalier than 10 mm in diameter and, in terms f their granulsa cell ppulatins with respect t fllicle diameter, mst f these small fllicles (i.e. 92 %) culd be cnsidered t be atretic (McNatty et al. 1979d). In cntrast, mre than 50 f the fllicles greater than 10 mm in diameter culd prbably be cnsidered as healthy structures. Cllectively the abve data, based n functinal criteria, suggest that mst human antral fllicles underg atresia during early antral develpment. These findings are cnsistent with the mrphlgical data f Blck (1951). Early Late Prevulatry antral antral 25mm a' II) 200 :I rn r c - 20.s t !E Qj u Early Late Prevulatry antral antral 25mm 100r--I-1 50 c rnu> :10 5 c:.- E X Cl '0 0.5" ci z '.. 0"..... Fig. 2. Change in the weight f thecal tissue, number f granulsa cells, and vlume f antral fluid with respect t the diameter f the healthy antral fllicle. Early Late Prevulatry antral antral 25mm I lr-i-1., II) E :I 1 > 0 5 "0 ':; ;;:: E <C Fllicle diameter (mm) Develpmental Changes in the Thecal Cell, Granulsa Cell and Antral Fluid Cmpartments f the Ovarian Fllicle during Fllicular Develpment These changes are summarized in Fig. 2. Substantial thecal mass accumulates during early antral develpment whereas during the later phases f grwth the rate f accumulatin declines. In cntrast t the theca, the granulsa cells accumulate in

4 252 Kenneth P. McNatty large numbers during bth the early and late antral grwth phases althugh the rate f accumulatin declines when the fllicle reaches the prevulatry phases (see als Delfrge et al. 1972; Bmsel-Helmreich et al. 1979). By the time the fllicle has reached its final phase f maturatin it has accumulated mst f the granulsa cell mass which is ultimately present in a crpus luteum. In cntrast t the fllicular cells, antral fluid cntinues t accumulate until the time f vulatin. Indeed, during the prevulatry grwth phase, fllicular enlargement is due almst entirely t fluid accumulatin r.s 40 1 c:: c:: Q) g 30 "0 25 "e Q) ti 20 '" ::l g 15 Q) > 10.;: <II 6 5 Fig.3 vurtin 4 E2 A Fllicle diameter J, 4/E2 7E2 n = /E2 I - = 50 I 0 01 II _" II,;", "" I L ''--.., " 25 E.s Q) 20 ;:g (5... "0 Q) C. 15 a; > Q) "0 ::l ti 5 Q) '0... Q) a; E <II Ci 1.50[ 1 00,.. <I :c 0.50 ex) '<I" 'c,.9: a; L' '''f Fig.4 n I- 0.0; c=-, 0 n --'..--'-'-...L...L.--.lL-.L, '" n 0.1O n Q; c 0.05t c. 0 0 n c::.;: Q) '" 2 l'oo en 0 80 N 0.60, w 0.40' I,..--.., Fig. 3. Relatinship between the diameter f the largest healthy fllicle and the varian secretins f andrstenedine (114) and estradil (Ez) during the early (EF), mid (MF) and late (LF) fllicular stages and the early (EL), mid (ML) and late (LL) luteal stages f the menstrual cycle. The data fr sterid cncentratins in varian venus bld were determined frm three t seven bservatins at each phase f the cycle. Fig. 4. In vitr prductin f andrstenedine (114), teststerne (T), dihydrteststerne (DHT), estrne (E,) and estradil (Ez) by human granulsa cells cntaining >75, 51-75,26-50 and 0-25 % f their maximum number f granulsa cells per fllicle diameter respectively. Sterid prductin was that amunt prduced per granulsa cell during 48 h culture minus the cellular cncentratin f sterid at the start f culture. The results are expressed as means (histgram) + s.e.m. (vertical bars). Fllicular Develpment and the Ovarian Secretins f Andrstenedine and Oestradil during the Menstrual Cycle Nrmally, healthy antral fllicles greater than 10 mm diameter are nly bserved during the mid- and late-fllicular phases f the menstrual cycle (Fig. 3). Hwever, large healthy fllicles have smetimes been bserved during the late luteal phase

5 Hrmnal Crrelates f Fllicular Develpment in the Human Ovary 253 (Fig. 3; McNatty 1978a). These late-luteal-phase fllicles d nt appear t sustain their develpment int the next fllicular phase since large antral fllicles during the early fllicular phase have been fund t be severely deficient in granulsa cells (McNatty et al. 1975). During the fllicular phase, the vary cntaining an enlarging fllicle simultaneusly increases its utput f andrstenedine (Ll 4 ) and E2 whereas the secretins f these sterids frm the cntralateral vary cntaining small antral fllicles remains lw (McNatty et al. 1976; Seri et al. 1976). As the dminant fllicle enlarges, the utput f Ll4 cntinues t exceed that f E2 althugh the Ll4/E2 rati decreases prgressively (Fig. 3). As a fllicle develps int a prevulatry structure, it emerges as a majr surce f Ll4 (Abraham 1974) as well as the majr surce f E2 (Baird and Fraser 1975). Althugh antral fllicles develp thrughut the luteal phase, the amunt f Ll4 secreted by nn-luteal tissue des nt exceed that generated by varies during the early fllicular phase. Mrever, nn-luteal tissue during the luteal phase secretes nly trace amunts f E2 (Fig. 4). Gnadtrphins and Intravarian Cntrl f Fllicular Develpment: Three Cncepts 1. Fllicular develpment and varian sterid secretin can be stimulated in hypphysectmized wmen, r in wmen with gnadtrphin deficiencies, after the administratin f exgenus gnadtrphin preparatins cntaining FSH and LH activities (see reviews by Vande Wiele et al. 1970; Rss and Lipsett 1978). Antral fllicles have nt been cited in varies f wmen with hypgnadtrphic hypgnadism. Mrever, in these same varies it was bserved that preantral fllicular develpment was severely impaired (Tagatz et al. 1970; Gldenberg et al. 1976). Antral fllicular develpment was als fund t be impaired in wmen wh had taken ral cntraceptives cntaining bth estrgen and prgestins fr at least 2 mnths (i.e. after gnadtrphin secretin has been suppressed fr sme time) (Maque et al. 1972; Starupand Visfeldt 1974). Cllectively, therefre, these data supprt the ntin that 'nce fllicles have started t grw, their cntinued develpment is dependent n a certain level f gnadtrphic supprt' (see als Rss 1974 fr review). 2. Within the vary, the cncentratins f sterid are nt unifrm thrughut the gland (see review by Edwards 1974). Indeed, the hrmnal milieu within each fllicle is different frm that in adjacent fllicles and plasma. Therefre, the cyte and granulsa cells within each fllicle are expsed t a unique endcrine micrenvirnment at anyne time (McNatty 1978b). These findings have led t the suggestin that 'the frmatin f a fluid-filled cavity within the avascular regins f the fllicle prvides the means by which intrafllicuar cells f ne fllicle may be expsed t a different hrmnal micrenvirnment frm that in adjacent fllicles and peripheral bld'. 3. In vitr studies n the islated cellular cmpartments f the fllicle have shwn that the mittic and bisynthetic activities f granulsa cells, the develpmental and bisynthetic activities f thecal tissues and the behaviur f cytes all crrelate with the hrmnal micrenvirnments t which they were previusly expsed in antral fluid rather than thse in peripheral bld (McNatty and Sawers 1975; McNatty et al. 1979d; Kb, McNatty and Ryan, unpublished data). These findings supprt the ntin that 'the hrmnal cmpsitin f a fllicle may determine whether it matures r underges atresia' (Harman et al. 1975; Luvet et al. 1975; McNatty and Sawers 1975; Lipsett and Rss 1976).

6 254 Kenneth P. McNatty Functinal Crrelates f Grwth and Atresia Cause-effect relatinships have been established between varian hrmnes and fllicular develpment in experimental animals. In the hypphysectmized and immature female rat, estrgens have been shwn t enhance fllicular develpment whereas andrgens, such as teststerne (T) have been shwn t antagnize the actins f estrgen and t stimulate fllicular atresia (Rss and Hillier 1979). Althugh such causal relatinships have nt been established fr the human female, high cncentratins f estrgen in antral fluid have been assciated with nn-atretic fllicles whereas lw cncentratins f estrgen have been assciated with fllicles underging degenerative changes (Table 1). Fr example, if an antral fllicle cntains mre than 75 % f its maximal granulsa cell number fr a given fllicular diameter then it invariably cntains an cyte free f degenerative changes [at the level f the dissecting micrscpe (x 100), 'healthy-lking'] tgether with high levels f E2 and Ll4 (Table 1) and detectable levels f FSH (data nt shwn; McNatty et al. 1979d). Table 1. Interrelatinships in the human fllicle between the number f granulsa cells, cyte viability, and the cncentratins f andrstenedine (4)' teststerne (T), dihydrteststerne (DHT) and estradil (Ez) in antral fluid Data (frm McNatty 1980) cllected frm 157 human antral fllicles ranging in diameter frm 1 t 20mm N. N. f granulsa healthy-appearing, Mean hrmne cncn in antral fluid ± s.e.m. (ng/ml) cells (%)A GV-stage cytes/ 4 T DHT Ez N. btained (%)B >95 9/10 (90 0) 1224 ± ± ± ± /18 (72 2) 1453 ± ± ± ± /22 (63 6) 1232 ± ± ± ± /52 (46 2) 1078 ± ± ± ± /55 (29 1) 967 ± ± ± ± 24 A Number f granulsa cells is the rati f the number f granulsa cells recverable frm a fllicle f a given size t the maximum number f recverable cells frm a fllicle f that size. B GV, germinal vesicle. Fllicles with fewer than the maximum number f granulsa cells fr a given fllicle size can be cnsidered t be underging sme degree f atresia: thus the fewer granulsa cells fund, the greater the degree f atresia. In such deppulated fllicles lwer levels f fllicular E2 are fund withut any significant alteratin in the levels f Ll4' T r dihydrteststerne (DHT) (see McNatty et al. 1979d). Mrever, fllicles deficient in granulsa cells are less likely t cntain a 'healthy lking' germinal-vesicle-stage cyte. Meitic Maturatin Ptential f Human Ocytes in vitr The interrelatinships between the ability f the germinal-vesicle-stage human cyte t resume meisis in vitr, the diameter f the cyte and the cncentratins f Ll4 and E2 in the antral fluid frm which the cyte was recvered are shwn in Table 2. Many cumulus-enclsed human cytes were fund t be incapable f resuming meisis in vitr even thugh they had been remved frm all ther fllicular elements. The cumulus-enclsed cytes which did nt resume meisis were the smallest f thse recvered and they came frm fllicles with lw levels f estrgen.

7 Hrmnal Crrelates f Fllicular Develpment in the Human Ovary 255 Thse which resumed meisis and went n t frm plar bdies were amngst the largest f the cytes recvered and they came frm fllicles with high levels f estrgen. Ocytes which lst their germinal vesicles but did nt frm plar bdies were intermediate in size, and were frm estrgen-deficient fllicles. The size f the antral fllicle per se was fund t bear n relatinship t the ability f cytes t Table 2. Relatinship between tbe ability f tbe healtby cyte t resume meisis in "itr, the size f tbe cyte and tbe cncentratins f andrstenedine (8 4 ) and estradil (Ez) in antral fluid A healthy cyte is ne which appears devid f degenerative characteristics at the level f the dissecting micrscpe (x 100) (see McNatty et al. 1979d). Data frm McNatty et af. (1979d). Values sharing a cmmn alphabetical letter are significantly different frm ne anther: "P < 0 01; b,c P < Ocyte status after 48 h culture Healthy GV breakdwn, n plar bdy frmatin A Plar bdy A GV, germinal vesicle. Ocyte diameter (JIm) ± 0 9" ± ± 2 2" Sterid cncn in antral fluid (ng/ml) 84 Ez 825 ± ± ± ± 79 b 87 ± 31 C 1019 ± 31O b,c resume maturatin in vitr. These findings suggest that intrafllicular estrgen may itself be imprtant fr subsequent maturatin f the human cyte, r that fllicular estrgen synthesis reflects sme ther aspect f fllicular maturatin critical t the develpment f the cyte. Table 3. Ability f human cytes t resume meisis in culture medium alne r in culture medium supplemented with 50 % fllicular fluid Fllicular fluid fr each cyte culture was btained frm the same fllicle as the cyte. Ocytes were cultured in 20 J1l f media which cnsisted either f 10 J1l antral fluid plus 10 J1l f medium cntaining Ham's F-lO supplemented with L-glutamine, sdium pyruvate (0 055 mg/ml), penicillin (100 Lu./ml), streptmycin (50 J1g/ml) and 20% newbrn calf serum (referred t as AF + medium) r 20 J1l medium alne. Data frm McNatty et al. 1980c Stage f N. f Ocyte Percentage f cytes at each stage f cyte maturatin cytes treatment maturatin at the end f h at the time culture in vitra f recvery GV GVBD PB Necrtic Unknwn stage Germinal vesicle 18 AF+medium Germinal vesicle 96 Medium alne A GV, germinal vesicle; GVBD, germinal vesicle breakdwn but withut plar bdy frmatin; PB, plar bdy. A nn-steridal substance in human fllicular fluid has been fund t prevent cytes frm resuming meisis in vitr (Channing et al. 1978). Hwever, when human cytes were cultuted in their wn fllicular fluid (50% v/v) supplemented with culture medium (50% v/v), their meitic maturatin ptential still crrelated

8 256 Kenneth P. McNatty with the hrmnal micrenvirnment that they had previusly been expsed t in viv (data nt shwn) and was nt influenced by the presence f fllicular fluid in the culture dish (Table 3). These data suggest that the existence f 'inhibitry substances' in fllicular fluid in sufficient cncentratins t be f physilgical imprtance must remain equivcal at the present time. Fllicular Sterids: Pssible Cellular Origins It seems that the fate f a develping fllicle centres arund its ability t generate an estrgen-enriched intrafllicular envirnment while simultaneusly secreting bth andrgens and estrgens int varian venus bld. A greater understanding f fllicular develpment may therefre be derived frm examining the pssible cellular surces f fllicular andrgen and estrgen and the rle f the gnadtrphins in regulating their secretins. Steridgenesis by Granulsa Cells in vitr Andrgens and estrgens The first 48 h f prductin f andrgens and estrgens by human granulsa cells in a tissue culture medium supplemented with 20 % (vjv) fetal calf serum is shwn in Fig. 4. Irrespective f whether they were recvered frm small «8 mm diameter) r large (>8 mm diameter) fllicles, human granulsa cells were fund t have the bisynthetic capacity t prduce sme A 4, T, DHT, estrne (E 1 ) and E 2 These findings are cnsistent with the earlier results f Ryan and Smith (1965) and Channing (1969). The patterns f secretin f these hrmnes were fund t be related t the health f the fllicle frm which the cells were harvested. When granulsa cells were harvested frm fllicles with mre than 75 % f their maximum cell ppulatin, they preferentially secreted estrgens and smaller amunts f andrgens. Hwever, when the cells were derived frm fllicles with prprtinately smaller ppulatins f granulsa cells per fllicle size, they were fund t be less capable f secreting estrgens but nt andrgens in vitr. When granulsa cells frm healthy fllicles (with >50 % f their maximum granulsa cell ppulatin) between 2 5 and 10 mm in diameter were cultured in the presence f excess A 4, their ability t prduce estrgen was enhanced when they were expsed t additinal amunts f FSH (McNatty et al. 1979c). Hwever, when granulsa cells frm healthy fllicles > 10 mm in diameter were expsed t excess A4 in vitr, their ability t prduce estrgen was nt enhanced by the additin f FSH t the culture medium. Thus in healthy grwing fllicles, it seems that granulsa cells prbably functin as estrgensecreting cells and that the level f armatase (estrgen synthetase) activity in these cells is acutely sensitive t FSH stimulatin during the early antral phases f develpment. In cntrast t the granulsa cells recvered frm healthy fllicles, thse frm atretic fllicles (i.e. with < 50 % f their maximum granulsa cell ppulatin) were generally incapable f metablizing A4 t estrgen. Mrever, even after expsure t additinal FSH their capacity t metablize A4 t estrgen remained lw. Thus as a fllicle degenerates its granulsa cells appear t lse their capacity t secrete estrgen and eventually, the cells lse their capacity t respnd t FSH. The effect f LH n andrgen and estrgen synthesis by granulsa cells is unknwn.

9 Hrmnal Crrelates f Fllicular Develpment in the Human Ovary 257 Prgesterne As a fllicle matures int a prevulatry structure, its granulsa cells transfrm frm an estrgen-secreting tissue int a prgesterne (P)-secreting ne (McNatty and Sawers 1975; McNatty et al. 1979b). The capacity f the granulsa cells t make this transfrmatin appears t depend n their ability t respnd t LH (Table 4) which in turn depends n the apprpriate 'priming' f the cells t FSH and estrgen within the fllicle (McNatty and Sawers 1975; Table 4). High levels fp in the antral fluid f large fllicles befre vulatin are indicative f granulsa cells underging luteinizatin (Fig. 5; Baird et al. 1975). Table 4. Relatinship between the endcrine micrenvirnment f the hmnan antral fllicle and the average daily secretin f prgesterne by granulsa cells expsed LH r LH-free media in "itr Data frm McNatty (1979a) Mean daily secretin rate (range) f Hrmnes in Stage f prgesterne by granulsa cells antral fluid menstrual in vitr (pg/cell) in viva cycleb LHC LH-free media D (30m u./ml) «0 02 m u./ml) -FSH, -E2 F 0 57 ( ) 0 13 ( ) n FSH, -E2 F 0 55 ( ) n 10 +FSH, +E2 MF 0 64( ) 0 12 ( ) n 8 5 +FSH, +E2 LF 5 0 ( ) 0 85 ( ) n 16 5 AE2,estradil-17,B; -FSH, <2 8mu./ml; -E2' <250ng/ml; + FSH, > 2 8 m u./ml; + E2, > 250 ng/ml; n, number f fllicles. B F, any stage f fllicular phase; MF, mid fllicular phase; LF, late fllicular phase. C Culture media cntained 1 7 m u./ml LH, 1 8 m u./ml FSH and 5 ng/ml prlactin. D Culture media cntained < 0 02 m u./ml LH, 2 6 m u./ml FSH and 6 ng/ml prlactin. Cntributin j intrajllicular cells t sterids in antral fluid Granulsa cells are a majr surce f sterid in antral fluid. In the apprpriate tissue culture medium, granulsa cells can generate levels f P, T, E1 and E2 that are similar t thse in antral fluid frm which the cells had been recvered (Table 5; McNatty et al. 1979a). Indeed, f the majr sterids in antral fluid nly Ll4 is present in significantly higher levels than the granulsa cells are capable f prducing in vitr. The cyte-cumulus cell cmplex is als capable f secreting sterids such as T, E1 and E 2, and f mdifying an antral-fluid endcrine envirnment in vitr (McNatty et al. 1980c). Therefre, it is likely that mst (if nt all) f the cell types within the avascular tissues f the fllicle influence the sterid cmpsitin f antral fluid. Steridgenesis by Thecal Tissue in vitr Prductin f andrgens and estrgens by minced human thecal tissue in vitr in the first 48 h is shwn in Fig. 6. In cntrast t the secretry behaviur f granulsa

10 258 Kenneth P. McNatty cells, the majr secretry prduct f the thecal minces was always Ll4 (see als Tsang et al. 1979; Batta et al. 1980) irrespective f whether the tissue was recvered frm healthy r atretic fllicles (Fig. 6). In healthy but nt severely degenerating fllicles, E2 was als a majr secretry prduct. It seems unlikely that the thecal utput f estrgen was due t granulsa-cell cntaminatin, nevertheless cnclusive prf fr E """: ::J E -"C ':; ;:;:: li! -r:: CO r:: r:: '';:::;!!! -r:: Q) (.) r:: (.) :c --I Fig. 5.LH (26) Prgesterne (28) T (9) (6) E.s "0 '5 ;;:... C til.5 c.+::... c (I) c (I) c e (I) 'Iii (I) Cl e a.. :2 IX) 'Ot... Cl.s (I)... <I lillillul....+:: Fig.6 2' lrullill lnruj Q. c.2 "0 C :E e (I) ci EF MF LF VEL Stage f menstrual cycle , I I I I I I Fig. 5. Relatinship between the cncentratins f LH and prgesterne in large antral fllicles (8 mm diameter) and in recently vulated fllicles during the early (EF), mid (MF) and late (LF) fllicular phases and the very early luteal (VEL) phase f the menstrual cycle. The mean cncentratins f LH in plasma are als shwn (x). AIl results are expressed as means + s.e.m. The number f bservatins are in parentheses. The dtted line was the limit f detectin f LH (2 8 m u./ml). The limit f detectin fr prgesterne was 10 ng/m!. Data frm McNatty 1979a. Fig. 6. In vitr prductin f andrstenedine (4)' teststerne (T), dihydrteststerne (DHT), estrne (E1) and estradil (E2 ) by human thecal tissue frm fllicles cntaining > 75, 51-75, and 0 25 % f their maximum number f granulsa ceils per fllicle diameter respectively. Sterid prductin was that amunt prduced per granulsa ceil during 48 h culture minus the ceilular cncentratins at the start f culture. The results are expressed as means (histgrams) + s.e.m. (vertical bars). The number in parenthesis represent the numbers f bservatins. thecal armatase activity must await the islatin f a 'pure line' f thecal cells (McNatty et al. 1979b). On a per unit mass basis, thecal tissue secreted mre sterid than the surrunding strmal tissues under the in vitr cnditins emplyed (McNatty et al. 1979b). The thecal utput f andrgen and estrgen exceeded that f the strma by abut 50- t too-fld. Irrespective f fllicle size, the capacity f a given amunt f thecal tissue t secrete sterid in vitr remained relatively cnstant. Thus,

11 Hrmnal Crrelates f Fllicular Develpment in the Human Ovary 259 during fllicular develpment any increased utput f sterid by thecal tissue may partly be related t the amunt f tissue present. Table 5. Relatinship between sterid prductin by the ttal ppulatin f granulsa cells frm each healthy fllicle in vitr and the levels f sterid in the antral fluid frm which cells were harvested Healthy fllicles were thse cntaining > 50 % f their maximum number f granulsa cells fr the size that the fllicle has reached. a, P<O OI; b, P<O OOI; c, P< Data frm McNatty et al. 1979a Healthy Fllicle Mean sterid cncentratins in the culture medium and antral fluid fllicles A diam. ± I s.e.m. (ng/mi)b (min) P.6.4 T DHT El E2 n In viv <8 36± ± 37" 26±10 29± lob 29± ± 76 8 In vitr <8 29± 8 687±158 a 19± 4 179± 40 b 27± 5 498± 41 8 In viv ;;.8 334± ±IW 41±11 174± ± ±250 4 In vitr ;;.8 269± ±267 e 41± ± ± ±403 4 A In vitr cncentratins are the amunts f sterid prduced by the ttal ppulatin f granulsa cells frm each fllicle int I ml f culture medium cnsisting f 20 % fetal calf serum + 80 % medium Hank's salts + HEPES buffer; in viv cncentratins are mean cncentratins f the sterids in the antral fluid frm which the cells were harvested. B P, prgesterne;.6. 4, andrstenedine; T, teststerne; DHT, dihydrteststerne; E 1, estrne; E2, estradil; n, number f bservatins. Table 6. Amunts f sterid prduced by thecal tissues frm healthy and atretic fllicles in vitr Data frm McNatty et al. 1980b. Median values and 95 % cnfidence limits are given. All numbers sharing a cmmn alphabetical letter are significantly different frm ne anther: a, b,c,d,e,f, P < Abbreviatins P,.6.4, T, E2 as in Table 5 Fllicle N. f Amunt f sterid prduced (ng/15 h) treatment fllicles P.6.4 T E2 Thecal tissue frm healthy fllicles Cntrl 5 1 3" ge (0 8, 1 8) (8 0, 120 1) (0 2, 4 6) (0 7, 3 7) +LH ab cd (10 ng/ml) (I 3, 8 9) (33 8, 220 3) (1 4,6 9) (2 2, 6 2) +LH b d (50 ng/mi) (I 2, 2 2) (11 5, 143 9) (0 5, 2 9) (0 5, 6 1) Thecal tissue frm atretic fllicles Cntrl e (I 3, 2 8) (4 3,29 0) (0 8,2 1) (1 0, 2 2) +LH ef (t ng/ml) (I 3, 8 4) (23 0, 79 7) (1 4, 3 2) (1 2, 3 2) +LH ' (50 ng/ml) (1 1, 10 9) (2 4, 34 0) (1 1,3 5) (0 9, 3 6) Respnsiveness f Thecal Tissue t Pituitary Hrmnes Human thecal tissue is respnsive t LH but nt FSH (Tsang et al. 1979). Hwever, the respnsiveness f thecal tissue t LH in vitr is variable and depends partly n the mass f tissue (i.e. size f the fllicle), the health f the fllicle, and the cncentratin f LH (Tsang et al. 1979; McNatty et al. 1980b). In viv, it prbably als

12 260 Kenneth P. McNatty depends n the nature f the LH stimulus in bld (i.e. a pulsatile stimulus v. cnstant level) (Table 6 and McNatty et al. 1980b). Small incremental increases in LH (frm I t 10 ng/ml) during a 15-h incubatin perid were fund t increase markedly the thecal utput f A4 frm healthy r atretic fllicles r bth. Theca frm healthy fllicles were als stimulated t increase their utput f P and E2 whereas theca frm atretic fllicles prduced mre variable amunts f P and were unable t generate E 2 When theca frm healthy r atretic fllicles were expsed t high cncentratins f LH (50 ng/ml) in vitr, the net utput f mst sterids remained cmparable t thse prduced by the unstimulated cntrls. Table 7. In vitr metablism f andrstenedine by granulsa cells recvered frm healthy fllicles f different sizes Data frm McNatty et al. 1979c Fllicle DHT Oestrgen N. f diameter TP frmed frmed / fllicles (mm) (%)A (%)B (%)C tested A TP(%) is the sum (in percentages) f teststerne (T), dihydrteststerne (DHT) and estrgen [estrne (E,) + estradil (E2 )] frmed after incubatin f granulsa cells with 1 f.lg f andrstenedine f.lg FSH fr 48 h. B DHT(%) is the percentage f DHT in metablite TP. C Percentage f E, + E2 in the metablite TP. The amunt f T (data nt shwn) in themetablitetp is 100% - [DHT(%) + estrgen (%)]. Metablism f Andrstenedine by Granulsa Cells and the Pssible Rle f Andrstenedine Metablites in Atresia Since human thecal tissue is a majr surce f A 4, it has been suggested that the granulsa cells culd utilize this hrmne t frm mre bilgically ptent andrgens r estrgens (see Hendersn 1979 fr review). The ability f human granulsa cells frm different-sized healthy fllicles t metablize A4 t either DHT r estrgen (i.e. E1 and E 2 ) in vitr is shwn in Table 7. The ability f a given ppulatin f granulsa cells t metablize A4 was fund t increase as a functin f fllicle diameter. Granulsa cells frm small fuicles «8 mm diameter) preferentially metablized A4 t DHT irrespective f the amunt f FSH present. Hwever, granulsa cells frm healthy large fllicles (> 12 mm diameter) preferentially metablized A4 t estrgen. The preferential cnversin f A4 t DHT in small fllicles may partly explain why mst small antral fllicles in the human vary underg atresia (see Fig. 7). As demnstrated earlier, substantial thecal-cell mass is frmed befre the fllicle reaches a diameter f 10 mm. During this early phase f grwth the theca is sensitive t changes in the cncentratins f LH and is capable f secreting large amunts f A 4 Cnsequently, excessive r repeated LH stimulatin f the theca, in small fllicles,

13 Hrmnal Crrelates f Fllicular Develpment in the Human Ovary 261 may result in the granulsa cells preferentially metablizing thecal Ll4 t DHT. DHT is knwn t inhibit armatase activity in granulsa cells (Hillier et af. 1980), and t significantly reduce the rate f granulsa cell accumulatin in vitr (McNatty 1980). Because DHT and E2 are detectable in antral fluid f mst fllicles, the ptential exists fr the mutually antagnistic actins f andrgens and estrgen t ccur simultaneusly during antral fllicle develpment. Thus fr small antral fllicles in which the armataseenzyme activity is dependent upn FSH stimulatin, grwth may be dependent n a limited utput f thecal andrgens r the apprpriate balance f LH and FSH in plasma r bth (Brwn 1978; Rss and Lipsett 1978). Small fllicle Elevated Inhibitin f mittic activity Thecal G ranu I sa Oestrgen cells cells D Large fllicle grwth? DHT} Rapid fllicular /' 4.. prevulatry Elevated Oestrgen Thecal cells Granulsa cells Fig. 7. Pssible mechanisms by which LH prmtes atresia f small fllicles and grwth f large fllicles in the human vary. Armatase activity= estrgen synthetase activity. Plasma Prlactin and the Micrenvirnment f Antral Fllicles Prlactin has been shwn t have bth inhibitry and lutetrphic effects n prgesterne synthesis in human and prcine granulsa cells (McNatty et af. 1974; Veldhuis and Hammnd 1980). In the prcine vary these divergent effects can be regulated by estrgen (Veldhuis and Hammnd 1980). In granulsa cells frm the immature, hypphysectmized, estrgen-treated rat, high cncentratins f prlactin have been shwn t suppress basal and gnadtrphin-induced increases in estrgen prductin in vitr (Wang et af. 1980). All the in vitr evidence frm the abve three species suggests that prlactin may, in sme circumstances, have a direct inhibitry effect n the develpment f varian fllicles. Irrespective f whether prlactin exerts an 'antignadal effect' at the level f the vary r via the hypthalamic-pituitary axis (Glass et af. 1975; Bhnet et af. 1976; Jacbs et af. 1976) hyperprlactinaemia can be assciated with a marked reductin in intravarian activity and the extent f this reductin may nt always be apparent frm the levels f FSH and E2 in plasma r frm the numbers f antral fllicles (McNatty 1979b). This latter cnclusin was reached after the levels f prlactin, FSH and E2 were measured in plasma and fllicular fluid f wmen underging hysterectmy. Als in this study the number f granulsa cells were determined in the individual excised antral fllicles. A summary f these data fr varies in the fllicular phase is shwn in Table 8. When the plasma levels f prlactin exceeded 100 ng/ml, 95 % f the fllicles (19 ut f 20) were fund

14 262 Kenneth P. McNatty t cntain undetectable levels f FSH, levels f E2 belw 500 ng/ml, and nne f the fllicles cntained mre than 50 % f their maximal granulsa cell number per fllicle size. In cntrast, when the prlactin levels were belw 100 ng/ml, between 20 and 62 % f the fllicles cntained measurable amunts f FSH, high levels f E2 and > 50 % f their maximum granulsa cell number. Table 8. Relatinship between prlactin levels in plasma, number f human antral fllicles with detectable FSH levels and distributin f fllicles with certain granulsa cell ppulatins during the fllicular phase f the menstrual cycle Plasma N. f N. f N. f fllicles (%) with N. f prlactin fllicles fllicles granulsa cell ppulatins C fllicles D levels at withfsh withe2 >75% 51-75% 26-50% <26% variectmy >1 3 m u./ml?500 ng/ml (ng/ml)a in antral fluid in antral fluid B > < A The lwest and highest prlactin levels recrded were 11 and 260 ng/ml respectively. B Oestradil (E 2) levels? 500 ng/ml indicate active cntributin frm the granulsa cells (McNatty and Baird 1978). C A fllicle with a granulsa cell ppulatin f 100% is ne with its full cmplement f cells fr whatever diameter it has reached. A fllicle with a granulsa cell ppulatin f 0 % is devid f granulsa cells (mdified frm McNatty 1979b). D Fllicles studied ranged in diameter between 4 and 20 mm. Sterid and Nn-steridal Interactins between Granulsa Cells and Thecal Tissues Sterid Interactins The amunts f sterid entering the bldstream frm human fllicles are prbably nt simply the summatin f thse secreted frm each fllicular cmpartment in islatin. As has already been indicated, it is likely that the cells frm each fllicular cmpartment (theca and granulsa) utilize sterid substrates frm ne anther and thereby enhance r lwer the utput f ne sterid ver anther (see Bjersing 1978 and Hendersn 1979 fr reviews). Recent in vitr studies have demnstrated that the utput f P, 4 and E2 frm the recmbined granulsa and thecal tissues frm the same fllicles can smetimes be significantly greater than that generated by the tissues in islatin (Batta et al. 1980; McNatty et al. 1980c). In shrt-term (4 h) incubatins, recmbined granulsa and thecal tissues were capable f prducing greater than additive amunts f E2 but nly when the tissues were recvered frm large fllicles (>8 mm diameter) and expsed t elevated cncentratins f LH and FSH (50 ng/ml f each hrmne; P<0 05). In lng-term incubatins (48 h), granulsa cells plus thecal tissue frm small fllicles prduced significantly greater than additive amunts f 4 in the presence f lw cncentratins f LH and FSH (c:::2 ng/ml f each hrmne; P<O 05) hwever, in the presence f high cncentratins f LH and FSH they prduced significantly greater than additive amunts f E2 (P < 0 05). In cntrast, granulsa cells plus thecal tissue frm large fllicles prduced significantly mre P in the presence f lw cncentratins f LH and FSH (P < 0 05) but significantly mre P, 4 and E2 in the presence f high cncentratins f LH and FSH cmpared with the respective amunts prduced by granulsa cells and thecal

15 Hrmnal Crrelates f Fllicular Develpment in the Human Ovary 263 tissue in islatin (P < 0 05). These findings are cnsistent with the hypthesis that there is increasing cllabratin between granulsa cells and thecal tissue in the prductin f E2 as the fllicle enlarges (McNatty et al. 1979c; Hillier et al. 1980). Mrever, they suggest that peak secretins f E2 frm large fllicles withut cncmitant luteinizatin may be critically dependent n the nature f the gnadtrphin stimulus and the length f time the tissues are expsed t elevated levels f gnadtrphin. In summary they als suggest that peak secretins f all the majr varian sterids (i.e. P, 4 and E 2 ) are the results f synergistic interactins between different varian cell types, namely the thecal cells, the granulsa cells and the strma (McNatty et al. 1980a). Table 9. Numbers f granulsa cells after 6 days f culture after the cells had been cultured alne (G) r in cmbinatin with thecal tissue (G+T) with r withut expsure t elevated cncentratins f LH plus FSH C-culture f granulsa cell with ther minced human tissues such as fetal heart, kidney r skin did nt result in a statistically significant change in granulsa cell number cmpared with that when granulsa cells were cultured alne. Data frm McNatty et al. (1980a) Treatment Fllicle Cell N. f diameter rati A bservatins (mm) G+T < ± 0 45" 43 G < ± 0 22" 43 G+T ±0 53 b 12 G ±0 25 b 12 G + T + LH/FSH < ± 0 0ge 4 G + LH/FSH < ± 0 07e 4 G + T+ LH/FSH G + LH/FSH A Rati f the number f granulsa cells after 6 days f culture t the number f 'live' cells at the start f culture. ", P<O OOl; b, e, P<O Ol (tw-way analysis f variance). Granulsa Cell Accumulatin and Thecal Cllagen Synthesis In additin t the fllicle cell interactins in vitr influencing steridgenesis, granulsa and thecal cells interact t influence the rate f accumulatin f granulsa cells (Table 9) and als the amunt f cllagen frmed in the thecal tissues (Kb, McNatty and Ryan, unpublished data). Apparently, thecal tissue cntains sme substance(s) which can act n the membrana granulsa t enhance cell prliferatin (McNatty et al. 1980a). Thecal tissue is very rich in cllagen: its cncentratins may range frm 30 t 230llg/mg tissue (Kb, McNatty and Ryan, unpublished data). In cntrast the membrana granulsa are entirely free f cllagen. Theca frm healthy fllicles was fund t increase its cllagen cntent by 32 % during 6 days in tissue culture, but if the same tissue was c-cultured with granulsa cells frm the same fllicles, the cllagen cntent in the theca was increased by 93 % (P<0 05). In cntrast, theca frm atretic fllicles either alne r in c-culture with granulsa cells was nt fund t increase its cllagen cntent after 6 days f culture (Kb, McNatty and Ryan, unpublished data).

16 264 Kenneth P. McNatty These findings, althugh preliminary, suggest that prliferating granulsa cells may influence certain structural changes in the thecal cmpartment. Similarly, as the theca accumulates its secretins may enhance granulsa cell accumulatin. Cncluding Remarks On Ovarian Fllicular Steridgenesis It is suggested that steridgenesis during fllicular maturatin can be summarized as fllws: (1) Bth the granulsa and thecal cells have the capacity t synthesize and secrete prgestins, andrgens and estrgens. Therefre, differences in the patterns f steridgenesis between the tw cell types are quantitative rather than qualitative. (2) Thrughut mst f antral fllicle develpment, thecal tissue is respnsive t LH but nt FSH: small increases in LH cncentratins stimulate the prductin f 4 and, t a lesser extent f E 2, frm healthy fllicles but nly 4 frm atretic fllicles; large increases in LH cncentratins (> 50 ngjml) d nt enhance 4 and E2 prductin frm either healthy r atretic fllicles abve the levels generated by unstimulated tissues. (3) Thecal tissue is prbably the majr surce f the 4 in antral fluid. Indirectly, therefre, the theca is likely t exert a majr influence n the intrafllicular levels f T, DHT, E1 and E 2. (4) Cllectively, the granulsa cells, the cyte-cumulus cell cmplex and, t a lesser extent, thecal tissue determine the intrafllicular levels f P, T, DHT, E1 and E 2 In healthy small antral fllicles ( 10 mm diam) granulsa cells are respnsive t FSH and have a capacity t generate high levels f estrgen. In degenerating fllicles granulsa cells eventually lse their capacity t respnd t FSH and althugh they becme incapable f metablizing 4 t E 2, they retain a capacity t metablize 4 t DHT. Prlactin appears t have bth stimulatry and inhibitry effects n steridgenesis by granulsa cells but its rle in the develpment f human fllicles is unknwn. (5) 'FSH-estrgen primed' granulsa cells eventually develp a respnsiveness t LH thereby enabling them t transfrm int luteal cells. (6) Peak secretins fp, 4 and E2 frm human fllicles are prbably the utcme f synergistic interactins between the granulsa and thecal cmpartments. On Integratin f Intravarian and Extravarian Events Assciated with Fllicular Maturatin It is suggested that ur current understanding f fllicular develpment in the human can be summarized as fllws: (1) Fllicular grwth is a cntinuus event. Fllicles leave the nn-grwing pl f small fllicles cntinuusly thrughut childhd and adult life. The initiatin f fllicular grwth is a prcess which is nt interrupted during infancy, pregnancy r any ther perid f anvulatin (Peters et al. 1978). (2) The fllicle which ges n t vulate starts t grw befre the fllicular phase in which it will vulate. At the nset f the fllicular phase, the

17 Hrmnal Crrelates f Fllicular Develpment in the Human Ovary 265 presumptive prevulatry fllicle will already have reached a diameter f abut 4 mm (McNatty et al. 1979d). (3) It is prbably reasnable t assume that the selectin f a fllicle t vulate is made during r befre the early fllicular phase f the menstrual cycle. Presumably, a fllicle is selected because it happens, by chance, t be the mst respnsive f all healthy fllicles t the changing patterns f gnadtrphin secretin at the time when the crpus luteum is regressing. (4) It appears that the successful maturatin f small antral fllicles during the early fllicular phase is dependent n a certain 'threshld level' f FSH (Brwn 1978) with respect t the circulating levels f LH in plasma (Rss et al. 1970). (5) At the beginning f the mid-fllicular phase, the best develped fllicle will have reached a diameter ranging between 8 and 16 mm. At this time the thecal cmpartment f the fllicle is well develped and the granulsa cells have develped sufficient armatase activity t enable them t sustain an estrgen-enriched fllicular micrenvirnment despite an increased utput f thecal andrgen. (6) During the mid- t late-fllicular phase, the prevulatry fllicle will have develped a certain degree f autnmy: its granulsa cells n lnger require stimulatin by FSH in rder t metablize andrgen t estrgen; the capacity f the fllicle t prduce estrgen is limited nly by the amunt f armatizable substrate; and subject t being adequately stimulated by LH (Yen et al. 1974) this fllicle is established as the majr varian surce f Ll4 and E2 (Fig. 3). Thus such a fllicle has the ability t suppress FSH but nt LH secretin (Baird et al. 1975) thereby reducing the pssibility f any ther emerging fllicles being stimulated by FSH t generate r sustain an estrgen-enriched fllicular micrenvirnment. (7) As the late fllicular phase appraches, the rapidly prliferating granulsa cells f the presumptive prevulatry fllicle prbably cntribute markedly t the fllicular utput f estrgen which in turn results in the discharge f prevulatry LH. (8) Finally, the expsure f fllicle cells t prevulatry levels f LH results in a reduced utput f andrgen and estrgen secretin by the theca, luteinizatin f the granulsa cells and the transfrmatin f the fllicle frm an estrgen-secreting tissue int a prgesterne-secreting ne with the cncmitant release f a large healthy cyte. Acknwledgments Many f the studies described herein were made while I was a recipient f a Harkness Fellwship frm the Cmmnwealth Fund f New Yrk at the labratry fr Human Reprductin and Reprductive Bilgy, Harvard Medical Schl, Bstn. I therefre wish t thank the staff f the Cmmnwealth Fund and gratefully acknwledge the cllabrative effrts f my clleagues Drs Anastasia Makris, Dianne Mre Smith, Rapin Osathanndh, Tm Kb and Kenneth Ryan. I als wish t thank Nra S. Sttijn fr typing this manuscript.

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