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Study Objective: Recent findings have shown mechanical fragmentation of ovarian cortex and ovarian drilling could promote follicle growth in patients with premature ovarian insufficiency and polycystic ovarian syndrome, respectively. A common element shared by these treatments is the mechanical disturbance of ovarian extracellular matrix tissues. We thus hypothesized a simplified whole-ovary laparoscopic incision (WOLI) procedure may provide the intrinsic stimuli needed to activate resting follicles in patients with an extremely poor ovarian response (EPOR) who had negligible chance of becoming pregnant with their own oocytes via modern in vitro fertilization practice., Design: Retrospective pilot study., Setting: The study was conducted in a research medical center in Taiwan., Patients: Women who had multiple canceled ovarian stimulation cycles due to the lack of follicle growth were recruited. A total of 6 patients with EPOR received the WOLI procedure, which covers the whole surface of ovaries, in 2015 to 2017., Interventions: After receiving an outpatient WOLI procedure, ovarian response and follicle growth were monitored for 90 days with or without gonadotropin stimulation. Embryo quality and clinical outcomes were analyzed., Measurements and Main Results: After the WOLI treatment, 5 of 6 patients had significant increases in serum estradiol level and improved follicle growth (p = .001). Multiple oocytes were retrieved from each of these patients, and it led to thawed embryo transfer (ET) cycles in 4 patients (p = .010). On average, the duration from the WOLI procedure to the first ovum pickup was 24 days (11 to 58 days). After ET, 2 patients became pregnant and delivered healthy babies. Two other patients received ET, and 1 led to a chemical pregnancy. One patient had cryopreserved embryos with pending transfer., Conclusion: The standardizable WOLI procedure restored hormonal responses in a majority of patients with EPOR. Further validation of this novel and yet simple laparoscopic procedure, which requires only 1 laparoscopic surgery, may provide a practical option to reactivate the aging ovarian environment in patients with EPOR and premature ovarian insufficiency., (Copyright © 2022 AAGL. Published by Elsevier Inc. All rights reserved.)

Mammalian oocytes remain dormant in the diplotene stage of prophase I until the resumption of meiosis characterized by germinal vesicle breakdown (GVBD) following the preovulatory gonadotropin stimulation. Based on genome-wide analysis of peri-ovulatory DNA microarray to identify paracrine hormone-receptor pairs, we found increases in ovarian transcripts for endothelin-1 and endothelin receptor type A (EDNRA) in response to the preovulatory luteinizing hormone (LH)/human chorionic gonadotropin (hCG) stimulation. Immunohistochemical analyses demonstrated localization of EDNRA in granulosa and cumulus cells. In cultured preovulatory follicles, treatment with endothelin-1 promoted oocyte GVBD. The stimulatory effect of endothelin-1 was blocked by cotreatment with antagonists for the type A, but not related type B, receptor. The stimulatory effect of hCG on GVBD was partially blocked by the same antagonist. The endothelin-1 promotion of GVBD was found to be mediated by the MAPK/ERK pathway but not by the inhibitory G protein. Studies using cumulus-oocyte complexes and denuded oocytes demonstrated that the endothelin-1 actions are mediated by cumulus cells. Furthermore, intrabursal administration with endothelin-1 induced oocyte GVBD in preovulatory follicles. Our findings demonstrate a paracrine role of endothelin-1 in the induction of the resumption of meiosis and provide further understanding on the molecular mechanisms underlying the nuclear maturation of oocytes induced by the preovulatory LH surge.

Optimal maturation of oocytes and successful development of preimplantation embryos is essential for reproduction. We performed DNA microarray analyses of ovarian transcripts and identified glial cell line-derived neurotrophic factor (GDNF) secreted by cumulus, granulosa, and theca cells as an ovarian factor stimulated by the preovulatory LH/hCG surge. Treatment of cumulus-oocyte complexes with GDNF enhanced first polar body extrusion with increase in cyclin B1 synthesis and the GDNF actions are likely mediated by its receptor GDNF family receptor-alpha1 (GFRA1) and a co-receptor ret proto-oncogene (Ret), both expressed in oocytes. However, treatment with GDNF did not affect germinal vesicle breakdown and cytoplasmic maturation of oocytes. During the preimplantation stages, GDNF was expressed in pregnant oviducts and uteri, whereas GFRA1 and Ret were expressed in embryos throughout early development with an increase after the early blastocyst stage. In blastocysts, both GDNF and GFRA1 were exclusively localized in trophectoderm cells, whereas Ret was detected in both cell lineages. Treatment with GDNF promoted the development of two-cell-stage embryos into blastocysts showing increased cell proliferation and decreased apoptosis mainly in trophectoderm cells. Our findings suggest potential paracrine roles of GDNF in the promotion of completion of meiosis I and the development of early embryos.

Hormonal factors secreted by embryos and reproductive tracts are important for successful development of preimplantation embryos. We found expression of brain-derived neurotrophic factor (BDNF) and neurotrophin-4/5 (NT-4/5) transcripts at its highest levels in the blastocyst stages. The transcripts for their receptor, TrkB, were detectable throughout the early embryonic stages with an increase after the early blastocyst stage. Both BDNF and TrkB are expressed in trophectoderm cells, whereas ligand-binding studies indicated specific binding of BDNF to trophectoderm cells. Furthermore, BDNF and NT-4/5 were produced in pregnant oviducts and uteri. Treatment with BDNF promoted the development of two-cell-stage embryos into blastocysts showing increased proliferation and decreased apoptosis. The effects of BDNF were blocked by the TrkB ectodomain or a Trk receptor inhibitor, K252a. Studies using specific inhibitors demonstrated the roles of the PI3K, but not the ERK, pathway in mediating BDNF actions. Under high-density embryo cultures, treatment with the TrkB ectodomain or K252a alone also inhibited embryonic development and survival, suggesting potential autocrine actions of BDNF produced by the embryo. In vivo experiments further demonstrated that K252a treatment suppressed early embryo development by inhibiting blastocyst cell numbers, and increasing blastocyst apoptosis. Our findings suggested that BDNF signaling plays important paracrine roles during blastocyst development by promoting the development of preimplantation embryos.

The UCN homologues SCP and SRP bind specifically to the CRFR2 receptor, whereas UCN binds to both CRFR1 and CRFR2. We have previously demonstrated that all three peptides are cardioprotective, and both the Akt and MAPK p42/44 pathways are essential for this effect. Here we tested the hypertrophic effects of these peptides. We examined the effects of the peptides on cell area, protein synthesis, and induction of the natriuretic peptides ANP and BNP. All three peptides were able to increase all the markers of hypertrophy examined, with SCP being the most potent of the three, followed by UCN and SRP last. In addition, we provide a mechanism of action for the three peptides and show that Akt phosphorylation is important for their hypertrophic action, whereas MAPK p42/44 is not involved in this effect.

Cryptorchidism is the most frequent congenital anomaly of the urogenital tract in the male. Although in Western countries 1-2% of males at the age of 3 months are diagnosed with this condition, its aetiology is still unknown. Animal models suggest a possible genetic basis for this disorder. Recently, the INSL3 (Leydig insulin-like peptide) gene and its cognate receptor, LGR8, were found to be important in testicular descent by regulating gubernacular development. Male mice null for either INSL3 or LGR8 genes exhibited bilateral cryptorchidism. Because earlier studies indicated that mutation of the INSL3 gene is not associated with the development of human cryptorchidism, this study analysed whether mutations in the LGR8 gene could be associated with this disorder. Sequencing of 18 exons of the LGR8 gene in 23 cryptorchid Finnish patients and a group of 33 control subjects allowed the identification of three nucleotide changes in exons 12 and 17, showing single base substitutions from A to G at positions 957, 993, and 1810 of LGR8. Among the three changes, only the 1810 A to G substitution is associated with an amino acid change from isoleucine to valine (Ile604Val) located in the fifth transmembrane domain of this seven-transmembrane receptor. This change was more frequent in a control group of normal fertile adult males and infant boys than in the group of cryptorchid males. The change is not associated with altered receptor signalling, thus suggesting the presence of a polymorphism unrelated to the cryptorchid phenotype. These data indicate that mutations involving the human LGR8 gene do not represent a frequent cause of cryptorchidism in the Finnish population.

Objective: The entire human genome will be sequenced in September 2000. Facing the exponential increase of data in the GenBank at the National Center for Biotechnology Information, reproductive biologists are being bombarded with massive amounts of information on diverse genes. It is becoming increasingly difficult for individual investigators to sort out the diverse genetic and physiologic information on the localization and function of different genes in the ovary. To alleviate the present situation, we have taken advantage of the accessibility of the Internet and initiated a project that serves the entire ovarian research community., Results and Discussion: The Ovarian Kaleidoscope database provides information regarding biologic function, expression pattern, and regulation of genes that are expressed in the ovary. In addition, it serves as a gateway to other online information resources relevant to ovarian research by offering results from original papers and data about nucleotide and amino acid sequences, and human and murine mutation phenotypes. All references are linked by hypertext to PubMed and additional links to sequence databases are also included. This information is accessible online and searchable not only by gene name but also by criteria such as the cellular and ovarian function of the gene product, the expression of genes in different ovarian cell types, or their association with specific ovarian phenotypes.

The second messenger cAMP has been implicated in the regulation of mammalian and amphibian oocyte maturation. Although a decrease in intraoocyte levels of cAMP precedes germinal vesicle breakdown (GVBD), the gonadotropin induction of ovulation and oocyte maturation is associated with major increases of cAMP in ovarian follicles. In the mammalian system, isolated oocytes undergo spontaneous maturation in vitro but this process is blocked by treatment with a phosphodiesterase (PDE) inhibitor, IBMX, which increases intraoocyte cAMP levels. In contrast, the same inhibitor, when added to cultured follicles for a brief time, increases follicle cAMP levels, followed by the induction of GVBD. To resolve the paradoxical actions of this PDE inhibitor on the maturation of isolated and follicle-enclosed oocytes, we hypothesized that meiotic maturation requires opposing fluctuations of cAMP levels in the somatic granulosa and germ cells. Such opposing fluctuations may result from selective expression and regulation of PDEs in the somatic and germ cell compartments of the follicle. To test this hypothesis, PDE activity was manipulated in different follicular cells using type-specific inhibitors. The impact of the ensuing changes in cAMP levels in the two compartments was monitored by the induction of GVBD. In isolated oocytes, spontaneous GVBD was blocked by two inhibitors of type 3 PDE (cGMP-inhibited: CGI-PDE), milrinone and cilostamide. In contrast, treatment with an inhibitor for type 4 PDE (cAMP-specific), rolipram, was ineffective. These findings suggest that the oocyte expresses type 3 but not type 4 PDE and that increases in intraoocyte cAMP suppress GVBD. This hypothesis was confirmed by in situ hybridization studies with PDE3 and PDE4 probes. PDE3B mRNA was concentrated in oocytes while PDE4D was mainly expressed in granulosa cells. In cultured follicles, LH treatment induced oocyte maturation but the gonadotropin action was blocked by inhibitors of type 3 but not the type 4 PDE inhibitors. Furthermore, treatment with the type 4, but not the type 3, PDE inhibitor mimics the action of LH and induces oocyte maturation, presumably by increasing cAMP levels in granulosa cells. Our findings indicate that PDE subtypes 4 and 3 are located in follicle somatic and germ cells, respectively. Preferential inhibition of PDE 3 in the oocyte may lead to a delay in oocyte maturation without affecting the cAMP-induced ovulatory process in the somatic cells. Conversely, selective suppression of granulosa cell cAMP-PDE may enhance the gonadotropin induction of ovulation and oocyte maturation. Thus, in addition to the well-recognized differential expression and regulation of adenylate cyclase in the somatic and germ cell compartments of the follicle, we suggest that selective regulation and expression of PDEs may be involved in the regulation of cAMP levels and control of oocyte maturation in the preovulatory mammalian follicle.

Inhibin suppresses the pituitary secretion of FSH but not LH. The two forms of inhibin are composed of a common alpha subunit linked to either a beta A or a beta B subunit. The mouse inhibin alpha gene was isolated and shown to have two exons spanning a 1.7 Kb intron. The proximal 5' flanking region has neither TATA and CAAT boxes nor GC-rich area. Using the 5' flanking region of mouse inhibin alpha gene linked to luciferase gene, transfection of rat granulosa cells indicated that the first 165 bp of the promoter region is required for basal expression. The mouse inhibin alpha genomic clone should be useful for analysis of hormonal control of inhibin alpha transcription and the generation of mice with targeted deletion of this gene.

The potential role of transforming growth factor-beta (TGF-beta) as an intragonadal regulator in the testis was investigated by studying the effect of TGF-beta on testosterone (T) production by neonatal rat testis cells in primary cultures. After 3 days of preincubation in serum-free medium, testis cells were treated with hormones for 3 additional days. Human chorionic gonadotropin (hCG) treatment (0.3-30 ng/ml) of testis cells elicited a dose-dependent increase of T levels with maximum values greater than 9-fold over baseline. Although TGF-beta alone did not affect T levels, a dose-dependent inhibition of hCG-stimulated T production was observed when cells were cotreated with TGF-beta. Maximal inhibition was greater than 85%, and the IC50 value was 5 ng/ml (2 x 10(-10) M; n = 5 experiments). This inhibitory effect was evident 48 h after the initiation of treatment and could be reversed 1 day after the cessation of TGF-beta exposure of cells. TGF-beta also reduced forskolin and (Bu)2cAMP-induced T production (greater than 85% decrease), indicating that TGF-beta can inhibit steroidogenesis distal to the formation of cAMP. The conversion of exogenously added androgen precursors (progesterone (P) and 17 alpha-hydroxyprogesterone) to T by hCG-stimulated cells was suppressed by the addition of TGF-beta. In contrast, endogenous P accumulation did not change in cultures treated with TGF-beta. Because TGF-beta-like activity has been found in the testis, the observed inhibitory effect of TGF-beta suggests a potential intratesticular regulatory role of this growth factor.

RU-486 is a recently synthesized steroid with anti-progesterone and anti-glucocorticoid properties. Its direct anti-progesterone action on the uterus is believed to be the basis for its ability to induce menstruation and early abortion. RU-486 likely antagonizes progesterone action on the uterus progesterone receptor. We have studied the binding of RU-486 to rat ovary steroid receptors in order to learn whether this interesting synthetic compound binds to ovary steroid receptors and thus learn if this antagonist can be used to better define the mechanism(s) of steroid actions on the ovary. Ovaries from estrogen stimulated hypophysectomized immature female rats were homogenized in buffer. The 100,000 x g supernatant was incubated with tritiated steroid agonists (R5020, dexamethasone, or testosterone) alone or with unlabeled steroids including RU-486. Receptor bound, and free steroid, were separated by Sephadex G-200 columns. The relative abilities of the various tested steroids to bind to the rat ovary progesterone receptor were: RU-486 greater than or equal to R5020 greater than progesterone. RU-486 bound to the ovary glucocorticoid receptor with an affinity equal to that of dexamethasone. The affinity of RU-486 for the rat ovary androgen receptor was only about 9% that of testosterone. Thus, RU-486 binds with very high affinity to the rat ovary progesterone and glucocorticoid receptors. This steroid receptor antagonist offers a new tool by which the mechanism(s) of action of these steroids on the ovary can be tested.

Progestins have recently been shown to augment gonadotropin-stimulated progesterone and 20 alpha-hydroxypregn-4-en-3-one (20 alpha-OH-P) biosynthesis in cultured rat granulosa cells. The mechanism by which progestins autoregulate ovarian progestin biosynthesis was investigated by studying the modulation of pregnenolone biosynthesis as well as the activities of the enzymes 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) and 20 alpha-hydroxysteroid dehydrogenase (20 alpha-HSD). Granulosa cells obtained from immature hypophysectomized, estrogen-treated rats were cultured with FSH and/or progestins. Pregnenolone production was measured in the presence of cyanoketone (10(-6) M) to inhibit 3 beta-HSD activity. Enzymatic activities of 3 beta-HSD and 20 alpha-HSD were determined in cell homogenates by direct enzyme assays. FSH stimulated pregnenolone production, while treatment with progesterone or R5020 alone was ineffective. Concomitant treatment with the progestins further enhanced FSH-stimulated pregnenolone production in a dose-dependent manner with minimal effective doses of 10(-8) and 10(-7) M for R5020 and progesterone, respectively. In FSH-primed cells, LH increased pregnenolone accumulation, and concomitant treatment with R5020 also enhanced the LH action. Furthermore, the gonadotropins stimulated the activity of 3 beta-HSD, and this effect was further enhanced by concomitant treatment with either R5020 or progesterone in a dose-dependent manner. In addition, the 20 alpha-HSD activities were enhanced by progestins in cells treated with FSH but not with LH. Thus, both natural and synthetic progestins stimulate the gonadotropin-induced progesterone production in cultured granulosa cells via enhancing the 3 beta-HSD enzyme as well as pregnenolone biosynthesis.

The direct effects of keoxifene (LY 156758), a benzothiphene-derived antiestrogen, on granulosa cell differentiation were examined in vitro using primary cultures of rat granulosa cells. The effect of keoxifene on FSH-stimulated aromatase activity was biphasic in pattern, enhancing at lower concentrations (up to 10(-8) M), and inhibiting at higher concentrations (greater than 10(-6) M). Keoxifene inhibited FSH-stimulated progestin production in a dose-dependent fashion with an IC50 of 5 X 10(-8) M. FSH-induced LH receptors were also inhibited by keoxifene with an IC50 of 5 X 10(-7) M. These findings suggest that keoxifene has direct pharmacologic actions at the ovarian level. In contrast to the earlier reported estrogenic actions of triphenylethylene antiestrogens at the ovarian level, the benzothiphene-derived antiestrogens may be clinically more useful in the management of estrogen-dependent malignancies.

The effects of glucocorticoids on the steroidogenesis of ovarian granulosa cells were investigated. Cortisol and dexamethasone inhibited the increase in aromatase activity induced by FSH in cultured rat granulosa cells. In the same cultures progesterone production was stimulated to a maximum of 167% of the control level. This differential effect of glucocorticoids on estrogen and progesterone production by the granulosa cells indicates that glucocorticoids exert specific inhibition of the induction of aromatase by FSH and do not cause a general suppression of granulosa cell activity. In contrast to their inhibition of the FSH induction of aromatase enzymes, glucocorticoids did not interfere with the activity of pre-existing aromatase enzymes. In granulosa cells containing full aromatase activity, treatment with cortisol and dexamethasone did not inhibit aromatization of androstenedione to estrogens whereas two known aromatase inhibitors (dihydrotestosterone and 4-androstene-3, 6, 17-trione) were effective. These results indicate that the glucocorticoids exert a selective inhibition of the FSH-induction of aromatase activity in rat granulosa cells by a mechanism other than directly interfering with the aromatization reaction.

Gamma-aminobutyric acid (GABA), GABA synthesizing enzyme and GABA binding sites were measured in rat ovaries. The concentration of GABA in the ovary (0.56 microgram/mg protein) was less than that in the brain (1.2-3.4 microgram/mg protein), but was six-fold higher than any other non-neuronal tissue examined. Glutamate decarboxylase, the GABA synthesizing enzyme was also found in high concentrations in whole ovarian homogenate but not in enriched ovarian granulosa cells, testis, anterior pituitary or muscles. Furthermore, high affinity (Kd = 15-21 nM), specific GABA binding sites were identified in the ovaries by specific [3H]muscimol binding and the majority of GABA binding sites were associated with the granulosa cells. These data suggest a possible role of GABA in the regulation of ovarian functions.

Ovarian steroids and growth factors are intragonadal modulators which augment a key endpoint of follicle-stimulating hormone (FSH) action in granulosa cells: the induction of aromatase activity. Studies of these paracrine hormones that enhance FSH-stimulated estrogen biosynthesis by cultured rat granulosa cells, have led to the development of a sensitive and specific in vitro bioassay for FSH. This newly developed granulosa cell aromatase bioassay (GAB) allows for the measurement of bioactive FSH levels in serum and urine of humans and animals with various physiological and pathological conditions. These studies have demonstrated that the GAB assay is useful in detecting possible changes in the molecular forms of FSH. The adaptation of this method for urine samples allows for the measurement of bio-FSH levels in situations where venipuncture is not practical or in species for which specific radioimmunoassays are not available.

Rat ovaries produce a novel ovarian trypsin-like protease that is regulated during follicular development. The protease extracted from the ovaries of immature gonadotropin-treated female rats was unstable to denaturation, but was recoverable after non-denaturing electrophoresis. The activity was inhibited by synthetic serine protease inhibitors but not by aprotinin or soybean trypsin inhibitor, thus distinguishing the enzyme from pancreatic trypsin. Treatment with pregnant mare's serum gonadotropin (PMSG) significantly increased the levels of enzyme in the ovarian granulosa cells (Control, 0.0027 units/10(6) cells; PMSG-treated, 0.0062 units/10(6) cells) which was also secreted by these cells. The novel enzyme described here may be important for matrix remodelling during follicular growth.

The ability of gonadotropins from six mammalian species to stimulate estrogen and progesterone production was investigated in granulosa cells of hypophysectomized estrogen-primed immature female rats. Granulosa cells were cultured for 2 days in the presence of delta 4-androstenedione (10(-7) M) with or without various gonadotropin preparations. Treatment with follitropin (follicle-stimulating hormone, FSH) from human, rat, ovine, porcine, equine, and bovine origins resulted in dose-dependent increases in steroidogenesis from negligible amounts to maximal levels of approximately 4-8 and 12-30 ng/10(5) cells for estrogen and progesterone, respectively. The ED50 values of the FSH preparations for stimulation of steroidogenesis were: human: 1-4 ng/ml; ovine: 2.5-30 ng/ml; rat: 1.6-4.0 ng/ml; porcine: 7.5-20 ng/ml; equine 2.5-6 ng/ml; and bovine greater than 100 ng/ml. Lutropin (luteinizing hormone, LH) from rat, ovine, bovine, and porcine origins, human chorionic gonadotropin (hCG), the alpha-subunit of human FSH and the beta-subunit of human LH were ineffective in stimulating steroidogenesis, indicating the specificity of the assay system for FSH. In a high concentration (600 ng/ml), the beta-subunit of human FSH-stimulated steroidogenesis to a small extent. Furthermore, pregnant mare serum gonadotropin and equine LH also caused a dose-dependent stimulation of estrogen and progesterone production, the half-maximal response values (ED50) being 1.8-4 and 7.5-10 ng/ml, respectively. This is consistent with previous in vivo and in vitro findings, showing the potent FSH activities of these hormones. Thus, the cultured rat granulosa cell system provides a sensitive assay for measuring FSH activities of gonadotropins from various mammalian species.

The influence of androgens on the FSH modulation of progestin biosynthetic enzymes was studied in vitro. Granulosa cells obtained from immature, hypophysectomized, estrogen-treated rats were cultured for 3 days in a serum-free medium containing FSH (20 ng/ml) with or without increasing concentrations (10(-9)-10(-6)M) or 17 beta-hydroxy-5 alpha-androstan-3-one (dihydrotestosterone; DHT), 5 alpha-androstane-3 alpha,17 beta-diol (3 alpha-diol), or the synthetic androgen 17 beta-hydroxy-17-methyl-4,9,11-estratrien-3-one (methyltrienolone; R1881). FSH treatment increased progesterone and 20 alpha-hydroxy-4-pregnen-3-one (20 alpha-OH-P) production by 10.2- and 11-fold, respectively. Concurrent androgen treatment augmented FSH-stimulated progesterone and 20 alpha-OH-P production in a dose-related manner (R1881 greater than 3 alpha-diol greater than DHT). In the presence of an inhibitor of 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD), the FSH-stimulated pregnenolone (3 beta-hydroxy-5-pregnen-20-one) production (a 20-fold increase) was further enhanced by co-treatment with R1881, 3 alpha-diol or DHT. Furthermore, FSH treatment increased 4.4-fold the activity of 3 beta-HSD, which converts pregnenolone to progesterone. This stimulatory action of FSH was further augmented by concurrent androgen treatment. In contrast, androgen treatment did not affect FSH-stimulated activity of a progesterone breakdown enzyme, 20 alpha-hydroxysteroid dehydrogenase (20 alpha-HSD). These results demonstrate that the augmenting effect of androgens upon FSH-stimulated progesterone biosynthesis is not due to changes in the conversion of progesterone to 20 alpha-OH-P, but involves an enhancing action upon 3 beta-HSD/delta 5,delta 4-isomerase complexes and additional enzymes prior to pregnenolone biosynthesis.

The effects of cell plating density on granulosa cell sensitivity to follicle-stimulating hormone (FSH) were investigated, using a serum-free culture of cells obtained from immature, estrogen-treated rats. The cells were incubated at densities of 0.25 to 5 X 10(5) cells/dish with increasing concentrations of FSH for 2 days, and medium estrogen and progestin accumulation were measured by radioimmunoassay. Per-cell estrogen and progestin production rose with increasing FSH concentration and cell density up to 2 X 10(5) cells/dish. At a higher density (5 X 10(5)/dish), per-cell estrogen production fell; progestin production remained constant, although the major progestin produced was no longer progesterone, but rather its metabolite, 20 alpha-hydroxy-progesterone. The effects of changing cell density could not be accounted for by medium steroids or cytotoxic substances. It is concluded that in vitro plating density can markedly affect granulosa cell sensitivity to FSH. In vivo, changing intrafollicular cell densities may thus affect the ability of the whole cell complement to respond to gonadotropin.