functional studies of aromatase activity in human granulosa cells from normal and polycystic...

0021-972X/79/4904-0514S02.00/0Journal of Clinical Endocrinology and MetabolismCopyright © 1979 by The Endocrine Society

Vol. 49, No. 4Printed in U.S.A.

Functional Studies of Aromatase Activity in HumanGranulosa Cells from Normal and Poly cystic Ovaries*GREGORY F. ERICKSON, A. J. W. HSUEH, M. E. QUIGLEY, R. W. REBAR, AND S. S.C. YENDepartment of Reproductive Medicine, University of California, San Diego, La Jolla, California 92093

ABSTRACT. To determine the basis of the reduced ovarianestrogen (E) production in polycystic ovary syndrome (PCO),the ability of granulosa cells from normal and polycystic ovariesto aromatize androgens was assessed both in vitro and in vivo.During a 5-h incubation in vitro with graded doses of aromatasesubstrate [androstenedione (A4), 10~'°-10~6 M], a dose-relatedincrease in E production was observed in granulosa cells fromnormal 8- to 15-mm follicles; the minimum and maximum effec-tive doses of A4 were 10~'° and 10~7 M, respectively, and the EDsowas approximately 10~8 M. In contrast, granulosa cells from 4- to6-mm follicles of both normal and polycystic ovaries producednegligible amounts of E when incubated 5 h with graded dosesof A4, suggesting that the aromatase enzyme had not beeninduced in follicles of this size. To test the functional relationshipbetween gonadotropins and E production in PCO and normalgranulosa cells from the aromatase-deficient 4- to 6-mm follicles,

these granulosa cells were cultured in a chemically definedmedium for 48 h with A4 (10~7 M) and FSH (100 ng/ml) or humanLH (100 ng/ml). Cultured granulosa cells from both PCO andnormal subjects responded significantly to FSH by showing 24-and 17-fold increases in E production, respectively. LH had littleor no effect. Similarly, administration of human FSH to patientsin vivo resulted in a rapid and dramatic increase in plasmaestradiol and estrone levels.

These results indicate that granulosa cells from PCO patientshave an inherent ability to respond to the FSH induction ofaromatase activity, but the Graafian follicles never develop tothe size in which the aromatase enzyme is normally expressed.Such data strongly suggest that the absence of aromatase inPCO and the arrested follicle growth may be caused by a reducedlocal concentration of FSH and/or decreased bioactivity. (J ClinEndocrinol Metab 49: 514, 1979)

IN 1961, Short and London (1) examined the steroidcontent in follicular fluid in polycystic ovaries and

found exceedingly high concentrations of androstenedi-one (A4) but no estrone (Ei) or 17/?-estradiol (E2). On thebasis of this finding, they concluded that polycystic ova-ries have a defect in the aromatase mechanism whichprevents the conversion of the Ci9 steroids to Cis estro-gens (1, 2). Confirmatory results using in vitro tissueincubations have been reported by Axelrod and Gold-zieher (3), who found that polycystic ovaries have amarked capacity for A4 production but little if any capac-ity for aromatization. Administration of exogenous FSHto polycystic ovary syndrome (PCO) patients has beenshown to stimulate estrogen (E) excretion and preovu-latory follicle formation (4-10).

Although these findings have provided valuable infor-mation regarding abnormalities in the steroidogenic sys-tems of the polycystic ovary, the cellular site and mech-anism (s) of the abnormal ovarian steroidogenesis haveyet to be established. Studies in rats (11,12) and humans

Received March 19, 1979.Address all correspondence and requests for reprints to: Dr. Gregory

F. Erickson, Department of Reproductive Medicine M-025, Universityof California, San Diego, School of Medicine, La Jolla, California 92093.

* This work was supported by Rockefeller Foundation Grants RF-75029 and RF-76001 and NIH Grant HD-12303.

(13) have shown that FSH plays a crucial role in stimu-lating ovarian E production by acting directly on thegranulosa cell to increase aromatase enzyme activity. Inview of this functional relationship between FSH andaromatase activity together with the reduced FSH to LHratio in PCO patients (14), we have examined the effectof FSH on the aromatase system in granulosa cells fromnormal and polycystic ovaries in an attempt to define, atthe cellular level, the causes of reduced ovarian E pro-duction in PCO.

Materials and Methods

Ovarian follicles for in vitro studies were obtained from fourPCO patients undergoing bilateral wedge resection and fromfour normal women at different stages of the cycle who wereundergoing pelvic surgery for nonendocrine diseases. The invivo experiments with the administration of human pituitaryFSH were performed in three additional well documented PCOpatients by criteria previously described (15). Informed consentwas obtained from all subjects before the study.

Granulosa cell collection

Granulosa cells were collected from individual follicles, aspreviously described (16). In brief, individual Graafian follicleswere dissected intact from control and PCO ovaries, and thediameter of each follicle was measured with a stage micrometer.

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FSH INDUCTION OF AROMATASE ACTIVITY 515

Each follicle was placed in a petri dish containing 5 ml McCoy's5a medium and then cut with an iris knife, and the granulosacells and oocyte were carefully expressed into the medium. Theoocyte from each follicle was isolated and examined with aphase contrast microscope. If the oocyte was spherical andcontained an intact germinal vesicle, the follicle was considerednormal and the granulosa cells were used in the present exper-iments. Follicles with atypical oocytes were considered atreticand the granulosa cells from these follicles were discarded. Inthe PCO experiments, the isolated granulosa cells from a groupof typical follicles (4-6 mm in diameter) were pooled and usedin the in vitro experiments. In the control studies, the normalgranulosa cells were collected from individual follicles (4-15mm in diameter) and studied separately.

The expressed granulosa cells were collected with an orallycontrolled micropipette and transferred to a 15-ml plastic con-ical centrifuge tube (Corning Glass Works, Corning, NY). Thecells were spun at 250 X g for 5 min and the pellet was washedtwice with 5-ml portions of either McCoy's 5a medium (fortissue culture studies) or Hepes-0.1% bovine serum albumin(BSA) buffer (137 mM NaCl, 5 raM KC1, 0.7 mM Na2HPO4) 25mM Hepes, 10 mM glucose, 360 /IM CaCl2, and 1 mg/ml BSA,pH 7.2) for short term incubations. The final cell pellets weredispersed in a known volume of the appropriate buffer andaliquots (50 jul) of the cell suspension were diluted with 50 \i\Trypan blue stain. Samples were taken for counting in a he-mocytometer, after which the volume was adjusted with theappropriate buffer to give a final concentration of 2 X 105 to 2X 10B viable granulosa cells/ml.

Reagents

Hepes, A4, and BSA (fraction V) were obtained from SigmaChemical Co. (St. Louis, MO). McCoy's 5a medium (modified,without serum), penicillin-streptomycin solution (10,000 U pen-icillin base/ml and 10,000 /ig streptomycin base/ml), L-gluta-mine, and Trypan blue stain (0.4%) were obtained from GrandIsland Biological Co. (Santa Clara, CA).

Peptide hormones

Human FSH (NIH-FSH-HS-1; FSH potency equal to 4,990IU/mg and LH potency equal to 187 IU/mg Second Interna-tional Reference Preparation of human menopausal gonadotro-pin by bioassay) and humn LH (LER-960; LH potency equal to4,620 IU/mg and FSH potency equal to 1.07 IU/mg SecondInternational Reference Preparation of human menopausal go-nadotropin by bioassay) were obtained from the NIAMDDPituitary Hormone Distribution Program. Highly purified hCG(CR-119; 11,600 IU/mg) was generously provided by Dr. R. E.Canfield through the Center for Population Research of theNICHHD. Human FSH used for the in vivo studies (NPA lot2; 54 IU/vial) was obtained from the NIAMDD Pituitary Hor-mone Distribution Program.

Short term incubations of granulosa cells

Fifty-microliter aliquots of the final cell suspension werepipetted into 12 x 75-mm glass test tubes containing increasingconcentrations of A4 (10~10-10~6 M) in 300 /il Hepes-0.1% BSA

buffer. The granulosa cells were incubated at 37 C for 3 h in ashaking incubator at 120 oscillations/min. After the incubation,the cells were pelleted by centrifugation (250 X g for 10 min at4 C) and the supernatant was removed and stored at — 20 Cuntil analyzed for steroid content by RIA.

Granulosa cell culture

Granulosa cells from normal and PCO patients were culturedfor 2 days in 30 X 15-mm Falcon tissue culture dishes (FalconPlastics, Los Angeles, CA) in 2 ml McCoy's 5a medium supple-mented with 2 mM L-glutamine, 100 U/ml penicillin, and 100jug streptomycin sulfate. Purified gonadotropins were dilutedwith sterile Hepes-0.1% BSA and added to appropriate culturedishes in 50-ju.l volumes (final concentration, 100 ng/ml). A4 wasdissolved in 100% ethanol and added to appropriate culturedishes in 10-jul volumes. Granulosa cell cultures were main-tained in a humidified 95% air-5% CO2 incubator at 37 C. Afterthe incubation, the media were collected and stored frozen at-20 C until analyzed. The concentration of FSH, LH, Ei, E2,A4, testosterone, and/or progesterone (P) in the samples weremeasured by RIA as previously described (17-20).

Histology

Representative samples of polycystic ovaries were fixed inBouin's solution, dehydrated, embedded in paraplast, sectionedat 10 jam, and stained with hemotoxylin and eosin Y. Histolog-ical examination of representative sections prepared from ova-ries of the PCO patients used in the present study revealed thepresence of a thickened tunica albuginea composed of densecollagenous material and connective tissue elements. No pri-mordial follicles were seen in the fibrous capsule, but a normalcomplement of them was found in the ovarian cortex immedi-ately beneath the thickened capsule. Numerous preantral andantral follicles were seen in the polycystic ovaries, but nocorpora lutea or corpora albacantia were seen. The PCO follicleswere characterized by the presence of multiple medium-sizedGraafian follicles (4-7 mm in diameter) which were packed upagainst the tunica albuginea. These medium-sized follicles con-tained multiple layers of granulosa cells, a normal cumulusoophorous with an oocyte in the germinal vesicle stage, and aprominent theca interna with five to seven layers of thecainterstitial cells which had high cytoplasmic to nuclear ratiosand stained intensely with eosin. Most PCO ovaries containedseveral large cyst-like structures which measured ^1 cm indiameter. The membrana granulosa in these cystic follicles wasreduced to a single layer of flattened squamous epithelial cells,and the basement lamina was hyalinized as is found in atreticfollicles. Granulosa cells from these follicles were not used inthe present study.

Results

Aromatase enzyme activity in normal and PCO granu-losa cells

Normal and PCO granulosa cells were incubated for 5h in vitro with increasing concentrations of aromatasesubstrate (A4). The amounts of E which accumulated in


516 ERICKSON ET AL. JCE&M • 1979Vol49 • No 4

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FIG. 1. Dose-response relationship between aromatase substrate andE production by granulosa cells from normal (control) and PCO ovaries.Granulosa cells (5 X 104 viable cells/tube) were incubated for 5 h at37 C in Hepes-0.1% BSA buffer containing increasing concentrations ofaromatase substrate (A4, 10~IO-10"6 M). After the incubation, the cellswere pelleted by centrifugation, and E in the media was measured byRIA. Data points are the mean ± SE of five separate culture tubes. Noerror bars indicate that the variation was less than the size of thesymbol. The normal follicles were obtained from the following patients:5 and 8 mm from a patient in the midfollicular phase and 15 mm froma patient in the late luteal phase. A, B, C, and D refer to four PCOpatients.

the media are shown in Fig. 1. No E was produced bynormal granulosa cells from the medium-sized (4-6 mm)follicles. In contrast, a A4 dose-related increase in Eproduction was observed in normal granulosa cells fromthe large follicles (8 and 15 mm). The minimum effectivedose of A4 was between 10~9-10~10 M, and the maximumeffective dose was 10~7 M. The ED5o of A4 was 1 x 10~8

M (~3 ng/ml) and 3 X 10~9 M (~1 ng/ml) for cells fromthe 8- and 15-mm follicles, respectively. A small butconsistent decrease in E formation was observed withhigher concentrations of A4 (10~6 M). Maximal E produc-tion per cell was ~ 3-fold greater in granulosa cells ob-tained from 15-mm follicles than that from the 8-mmfollicles.

As shown in Fig. 1, granulosa cells obtained from 4- to6-mm follicles of PCO patients secreted negligibleamounts of E during the 5-h incubation with increasingconcentrations of A4. A small amount of E was producedby PCO granulosa cells obtained from one patient (D),but the production was low.

FSH stimulation of aromatase activity in isolated gran-ulosa cells

The ability of gonadotropins to stimulate E productionby normal and PCO granulosa cells from 4- to 6-mmfollicles during a 5-h incubation with 10"? M A4 was

examined. In these short term experiments, FSH (100ng/ml) either alone or together with LH (100 ng/ml),failed to stimulate E production by the normal and PCOgranulosa cells (data not shown).

To determine if prolonged treatment with gonadotro-pins can stimulate aromatase activity in normal and PCOgranulosa cells from the medium-sized (4-6 mm) follicles,granulosa cells were cultured for 2 days in a chemicallydefined medium containing purified human gonadotro-pins and/or a saturating dose of A4 (10~7 M). AS shown inFig. 2, there was no significant stimulation of E secretionby PCO granulosa cells cultured in the absence of A4;however, in the presence of 10~7 M A4, exogenous FSHcaused a 24-fold increase in E production during the 2-day incubation. Exogenous LH caused a slight stimula-tion in E formation, and this effect of LH could be causedby contaminating FSH in the LH preparation utilized.The granulosa cells from PCO patients secreted insignif-icant amounts of E when cultured in the absence ofaromatase substrate, and under these conditions, theadded gonadotropins failed to stimulate aromatization.Comparable gonadotropin responses were obtained withcultured granulosa cells from ovaries of three additionalPCO patients (data not shown).

Similarly, FSH stimulated estrogen production bygranulosa cells obtained from a normal 6-mm follicle(Fig. 3). In the presence of A4, the normal granulosa cellssecreted negligible amounts of E during the 48-h incu-bation period. The addition of FSH (100 ng/ml) resulted

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Control LH FSH Control LH FSHFIG. 2. Effect of gonadotropins on E production by cultured PCOgranulosa cells isolated from 4- to 6-mm follicles of a patient withclassic Stein-Leventhal syndrome. Granulosa cells (2 X 105 viable cells/dish) were cultured for 2 days in 2 ml culture medium containing oneor a combination of the following hormones: human LH (100 ng/ml),human FSH (100 ng/ml), and 10~7 M A4. E in the media was measuredby RIA. Data points indicate the mean ± SE of four separate culturedishes.



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FIG. 3. Effect of gonadotropins on E production by normal granulosacells isolated from a 6-mm follicle from a control patient at the midlutealphase of the cycle. Granulosa cells (2 x 105 viable cells/dish) werecultured for 2 days in 1 ml McCoy's medium containing 10~7 M A4 andone or a combination of the following hormones: human FSH (100 ng/ml) and hCG (100 ng/ml). After the incubation, the culture media werecollected and E in the media was measured by RIA. Data pointsindicate the mean ± SE of four separate culture dishes.

in a 17-fold increase in E accumulation, whereas hCG(100 ng/ml) had no effect. No further increase in Eproduction was observed when hCG was added togetherwith FSH. Virtually identical results were obtained withgonadotropin-treated granulosa cells in four additionalexperiments using cells from normal 4- to 6-mm follicles(data not shown).

FSH stimulation of E production in PCO patients invivo

Administration of FSH (600 IU) over 3 days to threePCO patients resulted in significant increases in serumE2 within 48 h (P < 0.05) and in serum Ei within 72 h (P< 0.001) after the initiation of FSH administration, reach-ing peak levels of 328 ± 66 and 172 ± 8 pg/ml for E2 andEj, respectively, 24 h after the last FSH injection (Fig.4). Thereafter, serum E gradually declined during theremainder of the study.

In one patient, administration of FSH (300 IU/day) on2 consecutive days resulted in initial increases in E2 andEi of 397 and 173 pg/ml, respectively, followed by aspontaneous secondary increase in E2 to 279 pg/ml andan LH surge 9 days after the initiation of FSH treatment(Fig. 5). This was followed by a gradual increase in serum

P to 20.1 ng/ml, with the onset of menses 14 days afterthe LH surge. Subsequent FSH administration givenduring the luteal phase of the cycle was also associatedwith an increase in serum E2 and Ei, reaching maximumlevels of 352 and 208 pg/ml, respectively, after the lastdosage of FSH.

Discussion

The present experiments demonstrate that granulosacells obtained from medium-sized follicles of women withPCO have little if any aromatase enzyme activity, asevidenced by their inability to convert aromatase sub-strate (A4) to E in vitro (Fig. 1). This finding confirmsand extends earlier in vitro studies which have demon-strated an aromatase deficiency in polycystic ovaries (3,21-23). Granulosa cells from polycystic ovaries, however,are capable of aromatization in vitro when appropriatelystimulated with FSH (Fig. 2). Moreover, treatment ofwomen with PCO with purified FSH results in a markedincrease in circulating E2 levels in vivo (Fig. 5). Thisfinding is consistent with the observation by others thatthe polycystic ovary is highly sensitive to exogenous FSHstimulation (4-10). Since granulosa cells are the onlycells in the ovary known to contain specific FSH recep-

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518 ERICKSON ET AL. JCE&MVol49

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DAYSFIG. 5. Effect of purified FSH on serum gonadotropin and E levels inone patient with PCO. The first administration was apparently followedby ovulation, as documented by an LH surge, elevated P levels, andsubsequent menses. Administration of FSH in the luteal phase resultedin increased circulating E levels but did not produce any apparent effecton luteal function.

tors (24), it seems likely that the observed in vivo Eresponse reflects a FSH action on the granulosa cell.

The absence of demonstrable aromatase activity ingranulosa cells of polycystic ovaries is not, however, aspathological as it seems. First, our study with normalovaries has demonstrated that granulosa cells isolatedfrom normal medium-sized (4-6 mm) follicles also do nothave an active aromatase system, and secondly, as in thePCO cells, FSH specifically induces a marked increase inaromatase activity in the isolated granulosa cells ob-tained from normal medium-sized follicles. This FSH-induced increase in aromatase activity in normal humangranulosa cells in vitro is consistent with the recentreport of Moon et al. (13). On the basis of the presentobservations, we propose that the granulosa cell is thecellular site of aromatase deficiency in the polycysticovary and that this absence of aromatase activity iscausally connected with a defect in quality and/or quan-tity of available FSH in PCO.

Why granulosa cells from medium-sized follicles lackaromatase activity has yet to be determined, but thisabsence could result from inadequate concentrations ofFSH in the follicular fluid. In this regard, it is worthy ofnote that McNatty et al. (25) have reported that theconcentration of FSH in follicular fluid of normal folliclesunder 8 mm is extremely low. In contrast, follicles >8mm in diameter contain significant amounts of FSH and,without exception, these follicles contain high levels of E(25, 26).

In our studies with normal ovaries, we defined thepattern of aromatase activity in the granulosa cells ofindividual follicles. By correlating the granulosa cell aro-

matase activity to the size of the follicle, we obtainedinformation about the development of aromatase activityduring follicular growth. Although the number of folliclesexamined in the present study was small, there was ahigh degree of reproducibility, in that the granulosa cellsfrom all small and medium-sized (>8 mm) follicles con-tained little if any aromatase activity. In contrast, ahighly active aromatase system was evident in granulosacells from all large (>8 mm) follicles, and the enzymeactivity per cell appeared to increase with increasingfollicle size. The active aromatase system in granulosacells from normal large follicles can best be explained bya previous induction of aromatase enzymes in vivo byFSH. The present observation that human granulosacells in large follicles contain aromatase activity confirmsthe original findings of Ryan and coworkers (27, 28).

Previous studies have indicated that the concentra-tions of E2 and Ei in the follicular fluid increase dramat-ically after the developing Graafian follicle reaches about8 mm in diameter (26, 29). Moreover, a marked rise inthe concentration of E2 in the ovarian vein occurs afterthe dominant follicle first achieves a size of 7-8 mm indiameter (26). These data together with our presentobservations strongly suggest that the human granulosacell first acquires a functional aromatase system at orabout the time the developing Graafian follicle reaches8 mm in diameter.

Based on the present observations, the absence ofaromatase activity in ovaries of PCO patients does notappear to be caused by an inherent defect or abnormalityin the ovarian cells but may be entirely accountable byinappropriate gonadotropin stimulation resulting in acessation of follicle growth. When the LH to FSH ratiois elevated, as in PCO (30), there is excessive A4 produc-tion by the theca interstitial cells. Despite the continuedpresence of aromatase substrate, the granulosa cells,because of the FSH-dependent aromatase deficiency, areincapable of forming adequate amounts of E. As a resultof this hormone imbalance, folliculogenesis ceases at themidantral stage and the woman becomes anovulatory.

Acknowledgments

We thank Dr. Howard Judd for kindly providing the ovarian tissuefrom PCO patient D, the NIAMDD and NPA for the purified gonad-otropins, Ms. E. Tucker, Sally Ng, and C. Fabics for excellent technicalassistance, Drs. Bobbi Navickis and Allen Lein for helpful commentsregarding the manuscript, and Ms. Cheri Anderson for typing themanuscript.

References1. Short, R. V., and D. R. London, Defective biosynthesis of ovarian

steroids in the Stein-Leventhal syndrome, Br Med J1: 1724, 1961.2. Short, R. V., Further observations on the defective synthesis of

ovarian steroids in the Stein-Leventhal syndrome, J Endocrinol24: 359, 1962.

3. Axelrod, L. R., and J. W. Goldzieher, Enzymatic inadequacies in



human polycystic ovaries, Arch Biochem Biophys 95: 547, 1961.4. Kettel, W. C, J. T. Bradbury, and F. J. Stoddard, Observations on

the polycystic ovary syndrome, Am J Obstet Gynecol 73: 954,1957.5. Gemzell, C. A., E. Diczfalesy, and G. Tillinger, Clinical effect of

human pituitary follicle stimulating hormone (FSH), J Clin En-docrinol Metab 18: 1333, 1958.

6. Gemzell, C. A., E. Diczfalusy, and K. G. Tillinger, Human pituitaryfollicle stimulating hormone I. Clinical effect of a partially purifiedpreparation, Ciba Found Colloq Endocrinol 13: 191, 1960.

7. Crooke, A. C, W. R. Butt, R. Paulmer, R. Morris, R. L. Edwards,C. W. Taylor, and R. V. Short, Effect of human pituitary follicle-stimulating hormone and chorionic gonadotropin in Stein-Leven-thal syndrome, Br Med J 1 : 1119, 1963.

8. Crooke, A. C, U. D. Sutaria, and P. V. Bertrand, Comparison ofdaily with twice weekly injections of follicle stimulating hormonefor treatment of failure of ovulation, Am J Obstet Gynecol 111:405, 1971.

9. Jewelewicz, R., M. Warren, I. Dyrenfurth, and R. L. van de Wiele,Physiological studies with purified human pituitary FSH (HP-FSH), J Clin Endocrinol Metab 32: 688, 1971.

10. Raj, S. G., M. J. Berger, E. M. Grimes, and M. L. Taymor, The useof gonadotropins for the inductions of ovulation in women withpolycystic ovarian disease, Fertil Steril 28: 1280, 1977.

11. Dorrington, J. H., Y. S. Moon, and D. T. Armstrong, Estradiol-17/?biosynthesis in cultured granulosa cells from hypophysectomizedimmature rats: stimulation by follicle stimulating hormone, Endo-crinology 97: 1328, 1975.

12. Erickson, G. F., and A. J. W. Hsueh, Stimulation of aromataseactivity by follicle stimulating hormone in rat granulosa cells invivo and in vitro, Endocrinology 102: 1275, 1978.

13. Moon, Y. S., B. K. Tsang, C. Simpson, and D. T. Armstrong, 17/?-Estradiol biosynthesis in cultured granulosa cells and theca cells ofhuman ovarian follicles: stimulation by follicle-stimulating hor-mone, J Clin Endocrinol Metab 47: 263, 1978.

14. Yen, S. S. C, P. Vela, and J. Rankin, Inappropriate secretion offollicle-stimulating hormone and luteinizing hormone in polycysticovarian disease, J Clin Endocrinol Metab 30: 435, 1970.

15. Rebar, R., H. L. Judd, S. S. C. Yen, J. Rakoff, G. Vanden Berg, andF. Naftolin, Characterization of the inappropriate gonadotropinsecretion in polycystic ovary syndrome, J Clin Invest 57: 1320,1976.

16. Erickson, G. F., J. R. G. Challis, and K. J. Ryan, A developmentalstudy on the capacity of rabbit granulosa cells to respond to trophichormones and secrete progesterone, Dev Biol 40: 208, 1974.

17. Yen, S. S. C, O. Llerena, B. Little, and O. H. Pearson, Disappear-

ance rates of endogenous luteinizing hormone and chorionic gonad-otropin in man, J Clin Endocrinol Metab 28: 1763, 1968.

18. Yen, S. S. C, L. A. Llerena, O. H. Pearson, and A. S. Littell,Disappearance rates of endogenous follicle-stimulating hormone inserum following surgical hypophysectomy in man, J Clin Endocri-nol Metab 30: 325, 1970.

19. Judd, H. L., and S. S. C. Yen, Serum androstenedione and testos-terone levels during the menstrual cycle, J Clin Endocrinol Metab36: 475, 1973.

20. DeVane, G. W., N. C. Czekala, H. L. Judd, and S. S. C. Yen,Circulating gonadotropins, estrogens and androgens in polycysticovarian disease, Am J Obstet Gynecol 121: 496, 1975.

21. Goldzieher, J. W., and L. R. Axelrod, Clinical and biochemicalfeatures of polycystic ovarian disease, Fertil Steril 14: 631, 1963.

22. Axelrod, L. R., and J. W. Goldzieher, The polycystic ovary. V.Alternate pathways of steroid aromatization in normal pregnancyand polycystic ovaries, J. Clin Endocrinol Metab 25: 1275, 1965.

23. Axelrod, L. R., and J. W. Goldzieher, The polycystic ovary. VIII,Acta Endocrinol (Kbh) 56: 255, 1967.

24. Midgley, Jr., A- R-, A. J. Zeleznik, J. S. Rajaniemi, and L. E.Reichert, Jr., In Moudgal, N. R. (ed.), Gonadotropin and GonadalFunction, New York, Academic Press, 1974, p. 416.

25. McNatty, K. P., W. M. Hunter, A. S. McNeilly, and R. S. Sawers,Changes in the concentration of pituitary and steroid hormones inthe follicular fluid of human Graafian follicles throughout themenstrual cycle, J Endocrinol 64: 655, 1975.

26. McNatty, K. P., D. T. Baird, A. Bolton, P. Chamber, C. S. Corker,and H. McLean, Concentration of oestrogens and androgens inhuman venous plasma and follicular fluid throughout the menstrualcycle, J Endocrinol 71: 77, 1976.

27. Ryan, K. J., and Z. Petro, Steroid biosynthesis by human ovariangranulosa and theca cells, J Clin Endocrinol Metab 26: 46, 1966.

28. Ryan, K. J., Z. Petro, and J. Kaiser, Steroid formation by isolatedand recombined ovarian granulosa and theca cells, J Clin Endo-crinol Metab 28: 355, 1968.

29. Sanyal, M. K., M. J. Berger, I. E. Thompson, M. L. Taymor, andH. W. Home, Jr., Development of Graafian follicles in adult humanovary. I. Correlation of estrogen and progesterone concentration inantral fluid with growth of follicles, J Clin Endocrinol Metab 38:828, 1974.

30. Yen, S. S. C, C. Chaney, and H. L. Judd, Functional aberrations ofthe hypothalamic-pituitary system in polycystic ovary syndrome:a consideration of the pathogenesis, In Serio, M. (ed.), The Endo-crine Function of Human Ovary, Academic Press, London, 1976,pp. 373-385.


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