Growth factors modulate gonadotropin receptor induction in granulosa cell cultures.

Epidermal growth factor and fibroblast growth factor inhibited follicle-stimulating hormone-dependent induction of luteinizing hormone receptor in cultured ovarian granulosa cells of the rat. In contrast, platelet-derived growth factor potentiated the induction of luteinizing hormone receptor by follicle-stimulating hormone. These results indicate that growth factors, well known for their effects on mitosis and DNA synthesis in cultured mammalian cells, are also able to modulate hormone-dependent differentiation in an endocrine target cell.

A model system for the biochemical study of luteinizing hormone/chorionic gonadotropin receptor synthesis.

A model system for the biochemical study of LH/CG receptor synthesis has been developed. Culture conditions for porcine granulosa cells were adapted that maximized the selective induction of LH/CG receptors by cAMP-inducing stimuli with an elimination of background LH/CG receptor appearance. It was found that the addition of FSH (1.5 micrograms/ml) or cholera toxin (10 ng/ml) 1 day after plating resulted in optimal induction of the LH/CG receptor (20-60 pg [125I]CG bound/micrograms DNA 72 h after addition) with virtually no LH/CG receptor appearance in the absence of added stimuli. Later additions of FSH or cholera toxin required insulin (1.0 microgram/ml) which alone caused background LH/CG receptor appearance in the absence of any additional stimuli. Furthermore, insulin increased the general rate of cellular protein synthesis, whereas FSH or cholera toxin each decreased it. Thus, the use of FSH or cholera toxin, without insulin, may enable one to detect the synthesis of the LH/CG receptor by metabolic labeling techniques where background protein synthesis has been lowered.

[125I]iodo-epidermal growth factor binding and mitotic responsiveness of porcine granulosa cells are modulated by differentiation and follicle-stimulating hormone.

Epidermal growth factor (EGF) stimulates granulosa cell (GC) proliferation of certain species and modulates FSH-induced GC differentiation. The present study was undertaken to characterize the binding properties of the EGF receptor in porcine GCs to determine if the EGF responsiveness of mitotically active porcine GCs was related to their differentiated state and was regulated by reproductive hormones in vitro. Characterization of the EGF receptor-binding properties of porcine GCs revealed that saturation binding was achieved with 10 ng/ml [125I]iodo-EGF after 1 h at 37 C. In all states of differentiation, porcine GCs expressed few (less than 20,000/cell), specific, high affinity EGF receptors with apparent Kd values of 5.5 +/- 0.7 X 10(-10) M (mean +/- SEM; n = 6). Freshly harvested GCs obtained from small follicles were considered slightly differentiated (SDs) and bound, on the average, 2.6-fold more [125I]iodo-EGF than highly differentiated cells (HDs) obtained from large follicles which had further differentiated in vivo. The difference in binding was due to a decrease in receptor number and not to a change in receptor affinity. This relationship observed in freshly harvested cells was maintained in culture for a limited period. At 48 h of culture, the [125I]iodo-EGF-binding capacity of SDs was higher than that of HDs and was inversely related to the state of differentiation, as measured by [125I]iodo-LH/hCG-binding capacity. After 96 h, however, the EGF-binding capacity of HDs increased 3.7-fold from the level of binding at 48 h, while the LH/hCG-binding capacity decreased 10-fold. Conversely, the EGF-binding capacity of SDs decreased 28%, while the LH/hCG-binding capacity remained low. These experiments indicated that the state of GC differentiation was inversely correlated with EGF receptor number and that this relationship was not maintained in culture beyond 48 h. FSH treatment within the first 48 h of culture decreased the EGF-binding capacity of SDs 35% relative to the control value, but estradiol and dihydrotestosterone had no effect. FSH also regulated the mitotic responsiveness to EGF. EGF treatment of cultured SDs stimulated an 84% increase in cell number and a 178% increase in [3H]thymidine incorporation. These effects were suppressed by a high concentration of FSH. Thus, the ability of porcine GCs to bind EGF was changed with differentiation in vivo, while both EGF-binding capacity and mitotic responsiveness were regulated by exposure to FSH in vitro.

The production of transforming growth factor-beta activity by rat granulosa cell cultures.

We have examined whether granulosa cells (GC) secrete transforming growth factor-beta (TGF beta)-like activity using cell cultures prepared from diethylstilbestrol-primed female rats. Our results indicate that a significant level of active as well as latent TGF beta activity is found in defined GC culture medium as assessed by 1) potentiation of FSH-induced differentiation of rat GC, 2) neutralization of its activity by anti-TGF beta immunoglobulin, 3) inhibition of DNA synthesis in mink lung epithelial cells (CCl 64), and 4) activation of latent TGF beta activity by either acid or heat treatment. TGF beta production was more pronounced when the cells were seeded on fibronectin-coated plates. There was no difference in the level of TGF beta secretion by GC preparations derived from either diethylstilbestrol-primed immature or normal immature rats or adult rats. Furthermore, rat GC-conditioned medium contained much more TGF beta activity than medium from normal rat kidney cells (NRK 49-F), human prostatic adenocarcinoma cells (PC-3), or porcine GC. Rat thecal/interstitial cell culture medium contained activity comparable to that of GC medium. We conclude that rat GC preparations secrete a high level of TGF beta activity in vitro. Taken together with previous results, this indicates the possibility that TGF beta may be an autocrine regulator as well as a paracrine one within the ovarian follicle. Moreover, because of the high level of TGF beta activity produced, the rat GC culture system appears to be a useful experimental model for further exploring relationships between TGF beta production and its action.

Developmental coordination of luteinizing hormone/human chorionic gonadotropin (hCG) receptors and acute hCG responsiveness in cultured and freshly harvested porcine granulosa cells.

A fundamental characteristic of ovarian antral follicle development is the progressive differentiation of the granulosa cells, a process marked by an increase in their complement of LH receptors. In this study we investigated the coordinated expression of [125I]iodo-hCG binding sites and hCG-sensitive cAMP production in intact cells from normally differentiating antral follicles of increasing size and in cells maintained in monolayer culture under conditions known to facilitate differentiation. In addition to hCG responsiveness, basal, FSH-stimulated, and cholera toxin-stimulated cAMP production were compared. Granulosa cell [125I]iodo-hCG binding capacity was directly related to follicle size; thus, binding provided a convenient marker of cell maturation, which, in turn, reflected the state of follicle development. Basal and hCG-stimulated cAMP production increased as a function of cellular LH/hCG receptor binding. Whereas basal activity increased linearly and proportionately with LH/hCG receptor binding, hCG-stimulated cAMP production was not linearly proportional. FSH responsiveness in terms of cAMP production declined as a function of LH/hCG receptor binding, exhibiting first order decay. While the patterns of FSH- and hCG-stimulated cAMP production were inversely related throughout much of follicle development, cholera toxin (CTX) responsiveness of cells remained constant until the very late preovulatory stages. Granulosa cells from large follicles (mean diameter, greater than 8 mm), having been exposed to the endogenous LH surge, exhibited high [125I]iodo-hCG binding but severely impaired hCG- and CTX-stimulated cAMP production, suggesting desensitization of adenylyl cyclase. In cultured granulosa cells, increased [125I]iodo-hCG binding induced by insulin and FSH was paralleled by an increased ability to generate cAMP in response to hCG. This coordinated expression of receptor and responsiveness was similar to that observed with progressively higher states of differentiation in freshly harvested cells. CTX-stimulated cAMP production in cells maintained in vitro was elevated and independent of LH receptor levels and, thus, was similar to that exhibited by freshly harvested cells. Granulosa cell cAMP production in response to acute FSH stimulation after chronic FSH treatment during culture was consistently lower than that of freshly harvested cells, a phenomenon that appeared to be related to the chronic dose of FSH used in vitro.(ABSTRACT TRUNCATED AT 400 WORDS)

Platelet-derived growth factor enhances granulosa cell luteinizing hormone receptor induction by follicle-stimulating hormone and serum.

Platelet-derived growth factor potentiated FSH-dependent LH receptor induction in serum-free and serum-containing cultures of rat granulosa cells. Levels of LH receptor binding comparable to pre-ovulatory levels could be achieved in vitro using a combination of FSH, platelet-derived growth factor, and serum. Receptor sites induced under these conditions were capable of mediating an hCG-stimulated increase in progestin secretion. Our results in vitro suggest a possible role for platelet-derived growth factor or related compounds in granulosa cell differentiation in vivo.

The effect of transforming growth factor-beta on follicle-stimulating hormone-induced differentiation of cultured rat granulosa cells.

Growth factors have been shown to modulate differentiation of cultured ovarian granulosa cells. Transforming growth factors (TGFs) constitute a family of polypeptide growth factors capable of reversibly inducing anchorage-independent growth in normal cells. Epidermal growth factor (EGF), which has significant structural homology with TGF alpha, has been shown to modulate differentiation of granulosa cells in vitro. Similarly, TGF beta (TGFB) has been found to have significant structural homology with ovarian follicular fluid inhibin. To examine whether TGFB might affect granulosa cell growth or differentiation, rat granulosa cells were cultured in serum-free medium containing insulin for up to 3 days with varying concentrations of TGFB in the presence or absence of FSH. TGFB caused a dose-dependent increase in FSH-stimulated LH/hCG receptor binding, but had no effect on binding in the absence of FSH; TGFB (10.0 ng/ml) further increased FSH-stimulated LH/hCG receptor binding by 48 +/- 8% (P less than 0.02). Similarly, FSH-stimulated progesterone production was increased by TGFB in a dose-dependent manner; TGFB (1.0-10.0 ng/ml) increased FSH-stimulated progesterone production 2- to 3-fold (P less than 0.02). In contrast, EGF (10.0 ng/ml) decreased FSH-stimulated LH/hCG receptor binding by 93 +/- 1% (P less than 0.02). Neither FSH-stimulated intracellular nor extracellular cAMP accumulations were affected by TGFB treatment. However, EGF (10.0 ng/ml) diminished extracellular and intracellular FSH-stimulated cAMP accumulation at 48 and 72 h of culture. Culture protein and DNA content were not significantly affected by TGFB. These results suggest that TGFB may enhance FSH-stimulated LH receptor induction and steroidogenesis by mechanisms that do not further increase net cellular cAMP accumulation; TGFB and EGF can have opposite effects on gonadotropin-dependent differentiation; and products of the TGFB/inhibin gene family may have a capacity for autocrine or paracrine modulation of granulosa cell differentiation.

Rat granulosa cells express transforming growth factor-beta type 2 messenger ribonucleic acid which is regulatable by follicle-stimulating hormone in vitro.

Freshly harvested granulosa cells (GC) from diethylstilbestrol (DES)-treated rats were examined for the presence of transforming growth factor-beta 1 (TGF-beta 1) and TGF-beta 2 mRNA by Northern analysis. TGF-beta 1 mRNA was not detectable, but hybridization of total RNA with a radiolabeled TGF-beta 2 cDNA probe revealed two mRNA species (5.1 and 3.6 kb) indicative of TGF-beta 2 mRNA. In response to FSH (50 ng/ml), relative TGF-beta 2 mRNA concentrations in cultured GC were 54% of control levels. Furthermore, the conditioned culture medium from FSH-treated GC contained significantly lower (p less than 0.01) TGF-beta-like activity relative to controls. These results demonstrate that rat GC express TGF-beta 2 mRNA which is regulatable by FSH in vitro.

Rat thecal/interstitial cells express transforming growth factor-beta type 1 and 2, but only type 2 is regulated by gonadotropin in vitro.

We previously demonstrated that granulosa cells from diethylstilbestrol-primed immature rats expressed transforming growth factor-beta 2 (TGF beta 2), but not TGF beta 1, mRNA and that its expression was regulated by FSH in vitro. The present studies were designed, therefore, to establish whether thecal/interstitial cells (TIC) from diethylstilbestrol-primed immature rats express more than one subtype of TGF beta mRNA and gene product and whether the levels of expression/production in vitro were regulated by LH/hCG. Reverse transcription-polymerase chain reaction (RT-PCR) analysis of total RNA indicated that TIC expressed both TGF beta 1 and TGF beta 2 mRNA. In response to hCG (200 ng/ml), TIC expressed TGF beta 2 mRNA levels that were 51% of control levels; TGF beta 1 mRNA levels were not altered. In response to cholera toxin (10(-9) M), TIC expression of TGF beta 2 and TGF beta 1 mRNA levels was 10% and 55% of control values, respectively. Western blot analysis established that both TGF beta 1 and TGF beta 2 were secreted in vitro. hCG and cholera toxin reduced secretion of TGF beta bioactivity by 55% and 90%, respectively. In summary, these results indicate: 1) TIC express both TGF beta 1 and TGF beta 2 mRNA; 2) TIC expression of TGF beta 2, but not TGF beta 1, mRNA is regulated by hCG in vitro; 3) TIC secrete both TGF beta 1 and TGF beta 2, and 4) TIC secretion in vitro of TGF beta activity is gonadotropin regulated.

Changes in (125I) labeled human chorionic gonadotropin (hCG) binding to porcine granulosa cells during follicle development and cell culture.

The specific binding of (125I)iodo human chorionic gonadotropin to porcine granulosa cells isolated at two stages of follicle maturation was quantitated immediately after harvest and following 6-7 days of culture. Both porcine LH (pLH, LER 786-3) and hCG inhibited (125I)iodo hCG binding, while pFSH (ler-1132) did not compete for hCG with granulosa cells for 24-48 hours at 37 C did not alter its subsequent ability to bind to fresh cells. The binding of (125I)iodo hCG was a rapid process which was not readily reversible. Scatchard analysis of the equilibrium binding data resulted in linear plots showing the hCG binding capacity of highly differentiated (HD) cells, harvested from large preovulatory follicles, to be significantly greater than that of moderately differentiated (MD) cells isolated from smaller luteal phase follicles, both before (794 vs 93 pmoles/g; P less than 0.001) and after (157 vs 5 pmoles/g; P less than 0.001) culture. A decline in binding capacity occurred in both cell groups during culture and was associated with a significant decrease in progestin production. In contrast, the hCG binding affinity of the granulosa cell was equivalent at both stages of follicle development and remained unchanged after 6 days of culture (mean apparent Kd = 2.9 x 10-10M; n=27. These data indicate that porcine granulosa cells contain a homogeneous class of LH receptors whose number, but not affinity, increases during follicle maturation in vivo. The loss of receptors in vitro suggests the absence of some factors(s), as yet unidentified, critical to the maintenance and development of the receptor population.

Differential effects of epidermal growth factor, somatomedin-C/insulin-like growth factor I, and transforming growth factor-beta on porcine granulosa cell deoxyribonucleic acid synthesis and cell proliferation.

Recent studies have suggested that the mammalian ovary synthesizes epidermal growth factor (EGF), somatomedin-C/insulin-like growth factor I (Sm-C), and transforming growth factor-beta (TGFb) and that these growth factors may in part form a basis for intraovarian regulation of granulosa cell proliferation and differentiation. The studies described herein were initiated to determine to what extent EGF, Sm-C, and TGFb function to regulate DNA synthesis and granulosa cell proliferation during primary monolayer culture. EGF, but neither Sm-C nor TGFb, alone consistently stimulated, in a dose-dependent manner, [3H]thymidine incorporation by porcine granulosa cells under defined conditions (P less than 0.01). Sm-C (10 ng/ml) and TGFb (1 ng/ml) both enhanced EGF-stimulated [3H]thymidine incorporation (56% and 300%, respectively; P less than 0.05). The levels of incorporation obtained with EGF plus TGFb were equal to or greater than those obtained using fetal bovine serum alone. When EGF, Sm-C, and TGFb were combined, [3H]thymidine incorporation was equivalent to that obtained with EGF plus 10% fetal bovine serum, heretofore the most potent stimulatory combination for [3H]thymidine incorporation. Thus, under defined conditions, EGF, Sm-C, and TGFb act synergistically to promote DNA synthesis in primary cultures of porcine granulosa cells. Although DNA synthesis is a requisite step for but is not an accurate measurement of cell proliferation per se, we investigated whether the observed effects of EGF, Sm-C, and TGFb on DNA synthesis were realized in terms of actual cell proliferation. This was accomplished using platelet-poor plasma-derived serum (PPPDS; 0.1-2.5%), which contains reduced levels of endogenous growth factors but not components needed for cell attachment. EGF (P less than 0.05), but neither Sm-C nor TGFb, alone consistently stimulated, in a dose-dependent manner, granulosa cell proliferation, an effect directly related to the PPPDS concentration. Sm-C consistently and significantly (P less than 0.05) enhanced EGF-stimulated cell proliferation in a dose-dependent manner. The facilitative effect of Sm-C was inversely related to the PPPDS concentration, ranging from a 76 +/- 15% increase at 0.1% PPPDS to a 14% increase at 1.0% PPPDS. TGFb exhibited a bifunctional effect on granulosa cell proliferation. At low levels of PPPDS (0.1% and 0.25%) and in the absence of Sm-C, TGFb enhanced EGF-stimulated cell division, an effect which, although small and variable (24 +/- 16%), was consistent.(ABSTRACT TRUNCATED AT 400 WORDS)

Reduction of granulosa cell progesterone secretion in vitro by intraovarian implants of antiandrogen.

Intra-ovarian Silastic implants containing the antiandrogen, Flutamide, or its active metabolite, reduced the level of progesterone secretion in porcine granulosa cell cultures by approximately 50%. These results complement recent observations in vitro suggesting a role for androgen in granulosa cell steroidogenesis during follicular development.

Epidermal growth factor enhances [125I]iodo-follicle-stimulating hormone binding by cultured porcine granulosa cells.

Epidermal growth factor (EGF) has been shown to have diverse effects on granulosa cells (GC). Although a potent mitogen for GC from several species, EGF attenuates many FSH-mediated processes associated with GC differentiation, suggesting that EGF promotes cell proliferation at the expense of cell differentiation. The extent to which EGF effects involve modulation of the FSH receptor level in proliferating GC has not been established. Accordingly, we investigated the effect of EGF on [125I]iodo-FSH binding by porcine GC isolated from small follicles maintained in monolayer cultures. Relative to cells cultured in medium with insulin alone, EGF treatment increased total monolayer [125I]iodo-FSH binding (per culture) 120% (P less than 0.005). This was due to a 40-50% (P less than 0.01) increase in binding per U protein and/or per U cell and a 40-60% (P less than 0.005) increase in both monolayer cell and protein contents. EGF stimulated GC hyperplasia, but not hypertrophy. Optimum EGF doses for increased total monolayer [125I]iodo-FSH binding and binding normalized per U protein or cell were 0.5 and 0.1 ng/ml, respectively. Fibroblast growth factor was 20- to 100-fold less potent than EGF, and thrombin was without effect. Whereas [125I]iodo-FSH binding per U protein or cell was not affected by the serum concentration of the culture medium, the EGF effects on total monolayer binding and cell proliferation were directly related to the serum concentration (P less than 0.005). Thus, EGF-mediated increases in total monolayer [125I]iodo-FSH binding were paralleled by increases in cell number. The equilibrium dissociation constants (Kd) for [125I]iodo-FSH binding to cells cultured with and without EGF were 5.3 and 2.5 X 10(-10) M, respectively. Thus, EGF treatment significantly increased FSH receptor number, but significantly decreased receptor-binding affinity (P less than 0.05). Chronic FSH treatment during monolayer culture decreased total monolayer [125I]iodo-FSH binding and binding per U protein or per cell and attenuated EGF-stimulated cell proliferation, but markedly stimulated cell hypertrophy. Thus, concomitant treatment with EGF and FSH stimulated cell hypertrophy rather than hyperplasia. EGF and FSH each would appear capable of modulating the action of the other with respect to GC function. Our results indicate that EGF-mediated GC proliferation is associated with the expression of FSH-binding sites. This appears to be due to both an increase in FSH receptors among the cell population and an increase in the monolayer cell population.(ABSTRACT TRUNCATED AT 400 WORDS)

Porcine granulosa cells do not express transforming growth factor-beta 2 (TGF-beta 2) messenger ribonucleic acid: molecular basis for their inability to produce TGF-beta activity comparable to that of rat granulosa cells.

In contrast to rat granulosa cells (GC), GC of the pig and cow produce very low levels of transforming growth factor-beta (TGF-beta)-like activity in vitro. Because cultured rat GC predominantly express TGF-beta 2 messenger RNA (mRNA) and secrete high levels of the protein, we hypothesized that TGF-beta 2 mRNA expression by porcine GC might be absent or diminished, thus providing a molecular explanation(s) for their relatively low levels of TGF-beta production. We tested this hypothesis by reverse transcription-polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay analysis. When analyzed by RT-PCR, porcine GC RNA from 1-3 mm follicles did not yield the expected 489 base pair (bp) TGF-beta 2 product but instead generated a smaller than anticipated 240 bp species; porcine testis RNA generated both the 240 and the anticipated 489 bp products. Sequencing these species indicated that the smaller form was not a novel TGF-beta 2 splice variant, and that the 489-bp product was porcine TGF-beta 2. This is the first reported nucleotide sequence for porcine TGF-beta 2; it is 90% and 91% identical to murine and human TGF-beta 2 sequences, respectively. Further RT-PCR analysis of porcine GC RNA resulted in the identification of bp products representing TGF-beta 1 and TGF-beta 3 mRNA. Enzyme-linked immunosorbent assay analysis of porcine GC conditioned medium confirmed the presence of TGF-beta 1 at very low levels. TGF-beta 2 was undetectable. Comparable analysis of GC from the diethylstilbestrol-treated prepubertal rat demonstrated the presence of TGF-beta 1 and TGF-beta 3 mRNA by RT-PCR and very low levels of the corresponding protein products in conditioned culture medium. Collectively, these results suggest that the inability of porcine GC to express TGF-beta 2 mRNA could explain the very low levels of TGF-beta activity secreted by these cells in vitro.

Follicle-stimulating hormone-mediated induction of functional luteinizing hormone/human chorionic gonadotropin receptors during monolayer culture of porcine granulosa cells.

The LH/hCG receptor content of porcine ovarian granulosa cells from 1- to 3-mm follicles can be increases to 4--5 times the preculture level during monolayer culture in serum-containing media supplemented with insulin and FSH. The binding of [125I]iodo-hCG declines during the first 2 days of culture, but then uniformly increases through 6 days, achieving a 14- to 15-fold increase relative to the 2-day level under optimal conditions. Analysis of receptor binding by autoradiography indicates that after 2 days, the number of cells specifically binding [125I]iodo-hCG increases significantly during culture, as does the intensity of binding on receptor-bearing cells. Granulosa cells in monolayer culture exhibit heterogeneous receptor induction, indicating that normalized [125I]iodo-hCG binding data cannot be used to estimate receptor concentration per cell. Receptor affinities in the initial and induced populations are identical. LH/hCG receptors induced in granulosa cells during culture are functional, as demonstrated by specific hCG-stimulated progesterone secretion. 17 beta-Estradiol produces a differential effect in vitro, generally increasing [125I]iodo-hCG binding with respect to FSH-induced levels but consistently depressing the subsequent hCG-stimulated steroidogenic response of cells bearing the induced receptor. The porcine granulosa cell monolayer system thus appears to be a useful model with which to study, in vitro, mechanisms of steroid and gonadotropin regulation of granulosa cell differentiation and overall follicular development.

Human platelet-derived growth factor preparations contain a separate activity which potentiates follicle-stimulating hormone-mediated induction of luteinizing hormone receptor in cultured rat granulosa cells: evidence for transforming growth factor-beta.

The ability of platelet-derived growth factor (PDGF) preparations to potentiate FSH-mediated LH receptor induction in rat granulosa cell cultures was shown to be due to a component distinct from PDGF. Purification of heat-treated platelet lysate by carboxymethyl-Sephadex C-50 and Cibacron blue-Sepharose chromatography, followed by Bio-Gel P-60 chromatography, resulted in the separation of two activities: 1) a growth-promoting activity, P60-PDGF, defined on the basis of increased DNA synthesis in BALB/c-3T3 cells, and 2) a differentiation-promoting activity which enhanced FSH-dependent LH receptor induction in granulosa cells. On the basis of electrophoretic mobility on sodium dodecyl sulfate-polyacrylamide gels, inhibition of tritiated thymidine uptake by epithelial cells, and attenuation of LH/hCG receptor expression in the presence of antitransforming growth factor-beta (anti-TGF beta) immunoglobulin G, the differentiation-promoting component of the preparations appears to be TGF beta. The Bio-Gel fractions that contained TGF beta did not stimulate LH receptor induction of cAMP production in the absence of FSH. PDGF prepared free of TGF beta did not potentiate receptor induction. We conclude, therefore, that the differentiative effects of PDGF previously described in this system are due to TGF beta.

Apoptosis in atretic ovarian follicles is associated with selective decreases in messenger ribonucleic acid transcripts for gonadotropin receptors and cytochrome P450 aromatase.

Although atresia of ovarian follicles is of critical importance during preovulatory follicle selection as well as during normal and premature menopause, the mechanisms underlying atresia remain poorly understood. To study molecular events associated with atresia, we evaluated changes in mRNA levels for cytochrome P450 aromatase, FSH receptor, LH receptor, and a structural protein, beta-actin, during atresia in small (3-mm diameter) and large (6-mm diameter) porcine follicles. In addition, internucleosomal fragmentation of DNA characteristic of apoptosis ("programmed cell death") was assessed in individual healthy and atretic follicles using a sensitive autoradiographic method. Follicles were classified as morphologically healthy or atretic based on the absence or presence of follicular haemorrhagia and the degree of follicular clarity. Morphological signs of atresia in individual follicles were correlated with the occurrence of internucleosomal DNA fragmentation in granulosa cells as well as in thecal cells during advanced stages of atresia. The presence of apoptosis in atretic follicles was also associated with significant decreases in follicular fluid estrogen concentrations compared to those in healthy follicles of the same size. The decline in estrogen synthesis in degenerating follicles was further correlated with decreased levels of a predominant 2.6-kilobase aromatase mRNA. Moreover, substantial declines in both FSH receptor and LH receptor mRNAs were found in atretic follicles, consistent with previous reports of their decreased responsiveness to gonadotropins. The observed decreases in mRNAs for aromatase and gonadotropin receptors could not be attributed to a generalized degradation of cellular RNA during atresia, as evidenced by the presence of intact 18S and 28S ribosomal RNA as well as constitutive expression of beta-actin mRNA in atretic follicles. These data indicate that apoptotic cell death is initiated in both granulosa and thecal cells of porcine follicles during atresia. Associated with internucleosomal DNA fragmentation, decreased transcription of specific ovarian genes or destabilization of their transcripts leads to selective decreases in aromatase and gonadotropin receptor mRNAs. The atresia of ovarian follicles provides an interesting model to further study the molecular events associated with DNA fragmentation and selective mRNA down-regulation during apoptosis.

Prevention of the polycystic ovarian phenotype and characterization of ovulatory capacity in the estrogen receptor-alpha knockout mouse.

Ovarian-derived estradiol plays a critical endocrine role in the regulation of gonadotropin synthesis and secretion from the hypothalamic-pituitary axis. In turn, several para/autocrine effects of estrogen within the ovary are known, including increased ovarian weight, stimulation of granulosa cell growth, augmentation of FSH action, and attenuation of apoptosis. The estrogen receptor-alpha (ERalpha) is present in all three components of the hypothalamic-pituitary-ovarian axis of the mouse. In contrast, estrogen receptor-beta (ERbeta) is easily detectable in ovarian granulosa cells but is low to absent in the pituitary of the adult mouse. This distinct expression pattern for the two ERs suggests the presence of separate roles for each in the regulation of ovarian function. Herein, we definitively show that a lack of ERalpha in the hypothalamic-pituitary axis of the ERalpha-knockout (alphaERKO) mouse results in chronic elevation of serum LH and is the primary cause of the ovarian phenotype of polycystic follicles and anovulation. Prolonged treatment with a GnRH antagonist reduced serum LH levels and prevented the alphaERKO cystic ovarian phenotype. To investigate a direct role for ERalpha within the ovary, immature alphaERKO females were stimulated to ovulate with exogenous gonadotropins. Ovulatory capacity in the immature alphaERKO female was reduced compared with age-matched wild-type (14.5+/-2.9 vs. 40.6+/-2.6 oocytes/animal, respectively); however, oocytes collected from the alphaERKO were able to undergo successful in vitro fertilization. A similar discrepancy in oocyte yield was observed after superovulation of peripubertal (42 days) wild-type and alphaERKO females. In addition, ovaries from immature superovulated alphaERKO females possessed several ovulatory but unruptured follicles. Investigations of the possible reasons for the reduced number of ovulations in the alphaERKO included ribonuclease protection assays to assess the mRNA levels of several markers of follicular maturation and ovulation, including ERbeta, LH-receptor, cyclin-D2, P450-side chain cleavage enzyme, prostaglandin synthase-2, and progesterone receptor. No marked differences in the expression pattern for these mRNAs during the superovulation regimen were observed in the immature alphaERKO ovary compared with that of the wild-type. Serum progesterone levels just before ovulation were slightly lower in the alphaERKO compared with wild-type. These studies indicate that treatment of alphaERKO females with a GnRH antagonist decreased the serum LH levels to within the wild-type range and concurrently prevented development of the characteristic ovarian phenotype of cystic and hemorrhagic follicles. Furthermore, a lack of functional ERalpha within the ovary had no effect on the regulation of several genes required for follicular maturation and ovulation. However, the reduced numbers of ovulations following the administration of exogenous gonadotropins in the alphaERKO suggests an intraovarian role for ERalpha in follicular development and ovulation.

Targeted disruption of the estrogen receptor-alpha gene in female mice: characterization of ovarian responses and phenotype in the adult.

Targeted disruption of the mouse estrogen receptor-alpha gene (estrogen receptor-alpha knockout; ERKO) results in a highly novel ovarian phenotype in the adult. The ERKO mouse model was used to characterize ER alpha-dependent processes in the ovary. Visualization of the ovaries of 10-, 20-, and 50-day-old wild-type (WT) and ERKO mice showed that the ERKO phenotype developed between 20 and 50 days of age. Developmental progression through the primordial, primary, and antral follicle stages appeared normal, but functional maturation of preovulatory follicles was arrested resulting in atresia or in anovulatory follicles, which in many cases formed large, hemorrhagic cysts. Corpora lutea were absent, which also indicates that the normal biochemical and mechanical processes that accomplish ovulation were compromised. Northern and ribonuclease protection analyses indicated that ERKO ovary FSH receptor (FSHR) messenger RNA (mRNA) expression was approximately 4-fold greater than in WT controls. Ovarian LH receptor (LHR) mRNA expression was also higher in the ERKO animals. Cellular localization studies by in situ hybridization analysis of ERKO ovaries showed a high level of LHR mRNA expression in the granulosa and thecal layers of virtually all the antral follicles. Ribonuclease protection analyses showed that ovarian progesterone receptor and androgen receptor mRNA expression were similar in the two groups. These results indicated that ER alpha action was not a prerequisite for LHR mRNA expression by thecal or granulosa cells or for ovarian expression of progesterone receptor mRNA. Ovarian estrogen receptor beta (ER beta) was detected immunohistochemically, was sharply compartmentalized to the granulosa cells, and was expressed approximately equally in the ERKO animals and the WT controls. In contrast, ER alpha staining was present in the thecal cells but not the granulosa cells of the WT animals. The summary findings indicate that in the adult the major cause of the ERKO phenotype is high circulating LH interacting with functional LHR of the theca and granulosa cells. These features result in a failure of the normal maturational events leading to successful ovulation and luteinization and presumably involve both hypothalamic-pituitary and intraovarian mechanisms dependent upon ER alpha action. The presence of ER beta in the granulosa cells did not rescue the phenotype of the ovary.

Estrogen and progesterone production by granulosa cell monolayers derived from in vitro fertilization procedures: lack of evidence for modulation by androgen.

The role(s) of androgens in the steroidogenic regulation of human granulosa cell production of estrogen and progesterone during monolayer culture was studied. These cells were exposed in vivo to human menopausal gonadotropin and hCG gonadotropin with or without clomiphene citrate. Steroid production rates were compared between cells cultured in control medium and those cultured in medium containing a nonaromatizable androgen [dihydrotestosterone (DHT)] or an aromatizable androgen [androstenedione (A'D)]. Some cultures received A'D from 3-12 days; other cultures received DHT alone for 3, 6, or 9 days before the addition of A'D for 3 days. The effect on steroid production during the culture interval before the addition of A'D also was evaluated. Exposure to A'D increased estrogen production over 50-fold compared with that in control cells or those treated with DHT (P less than 0.001). DHT also failed to alter estrogen production when A'D was added to cultures. Furthermore, the delay in introducing A'D to the cultures for up to 9 days did not decrease subsequent estrogen production compared with that in cultures continually exposed to A'D or DHT plus A'D. Progesterone production was substantial for at least 12 days of culture and was unaffected by the presence of androgen. These results do not confirm previous studies using murine or porcine granulosa cells, which suggested that androgen receptor-dependent mechanisms were involved in increasing estrogen and/or progesterone production in vitro. Rather, they indicate that androgen may not be required to maintain aromatase capability per se in human granulosa-luteal cells previously exposed to ovulation-inducing quantities of gonadotropin.