High expression of insulin-like growth factor binding protein-2 messenger RNA in epithelial ovarian cancers produces elevated preoperative serum levels.

The molecular etiology of epithelial ovarian cancer remains unclear. Using microarray expression analysis, we recently reported that expression of the insulin-like growth factor binding protein-2 (IGFBP-2) gene is elevated in advanced epithelial ovarian cancers. The aim of this study was to further delineate the role of IGFBP-2 in the pathoetiology of epithelial ovarian cancer and determine if elevated ovarian cancer IGFBP-2 gene expression is reflected in serum. Relative IGFBP-2 expression was measured using quantitative real-time polymerase chain reaction in 113 epithelial ovarian cancers and 6 normal ovarian surface epithelial samples. Preoperative serum IGFBP-2 levels were measured by radioimmunoassay in 84 women (42 ovarian cancers, 26 benign gynecological conditions, and 10 healthy female controls). Ovarian cancers demonstrated 38-fold higher mean IGFBP-2 expression than normal ovarian epithelium (P < 0.01). Serum IGFBP-2 levels were elevated in women with early- and advanced-stage ovarian cancer compared to controls and patients with benign gynecological conditions (P = 0.05 and P < 0.01, respectively). Epithelial ovarian cancers express high levels of IGFBP-2 relative to normal ovarian epithelium, and this is associated with elevated serum IGFBP-2 levels compared to both normal controls and patients with benign gynecological disease. Our findings provide further support that the insulin-like growth factor pathway plays a significant role in epithelial ovarian cancer pathogenesis. Further, IGFBP-2 may represent an additional serum biomarker with utility in detection and monitoring of epithelial ovarian cancer.

2-Methoxyestradiol, an endogenous estradiol metabolite, differentially inhibits granulosa and endothelial cell mitosis: a potential follicular antiangiogenic regulator.

2-Methoxyestradiol (2-ME) is an estradiol metabolite with antiangiogenic and antitumor activity. It is formed by granulosa cell (GC) catechol-O-methyltransferase activity and is present in the normal follicle at high concentrations. In this unique microenvironment, it may regulate selected cell types via autocrine and/or paracrine action. To assess the possibility that 2-ME or estradiol might exert differential mitotic and/or apoptotic effects on endothelial cells and GCs, we compared their actions on primary cultures of hormone- and/or growth factor-stimulated porcine GCs (pGCs) as well as two types of endothelial cells, primary cultures of porcine endothelial cells (pECs), and a spontaneously transformed rabbit endothelial vascular cell (REVC) line. The 2-ME, but not estradiol, dose dependently suppressed tritiated thymidine ((3)H-T) incorporation into epidermal growth factor (EGF)-stimulated REVCs and EGF/insulin (INS)-stimulated pECs. In contrast, 2-ME did not attenuate incorporation in FSH/INS-stimulated pGCs. It reduced incorporation by approximately 50% in EGF/INS-stimulated pGCs, indicating that responsiveness to 2-ME in normal cells can be modulated by hormone and growth factor treatment. Estradiol was not antimitotic to pGCs. As indicated by 4',6-diamido-2-phenylindole hydrochloride nuclear staining, estradiol was nonapoptotic in either cell type, and 2-ME significantly increased apoptosis of REVCs, but not of pGCs. In a cell migration assay, REVC movement was attenuated by 2-ME, but not by estradiol. In summary, the results show that antimitotic as well as proapoptotic responses to 2-ME vary with cell type and, in the case of pGC antimitotic activity, with the regulatory microenvironment. Thus, they provide a rationale for autocrine and/or paracrine action of 2-ME at its site of production in vivo, and they strongly support the concept of 2-ME as a candidate ovarian angiogenesis inhibitor.

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.

Restricted expression of WT1 messenger ribonucleic acid in immature ovarian follicles: uniformity in mammalian and avian species and maintenance during reproductive senescence.

WT1 is a zinc finger protein with transcriptional repressor activity on several growth factor and growth factor receptor genes. In the ovary, a potential role for WT1 in the suppression of the development of immature follicles has been demonstrated. Here, gel retardation assays further showed that recombinant WT1 protein interacted with consensus DNA sequences in the inhibin-alpha gene promoter. We investigated the pattern of WT1 expression in a wide variety of species and also over the reproductive life span in rats. In chicken ovaries, Northern blot analysis revealed the presence of WT1 transcript in small healthy white follicles (1-5 mm in diameter) and its absence in small yellow (6-12 mm in diameter) or larger follicles (F1-F5). In pig and monkey ovaries, WT1 expression was limited to granulosa cells of preantral follicles, as shown by in situ hybridization analysis. In rats, Northern blot analyses demonstrated the presence of WT1 transcript in the ovaries of young (3-mo-old) and middle-aged (9-mo-old) rats on the proestrous day, with a decrease in old (12-mo-old) rats in persistent estrus. In situ hybridization analysis further suggested that the decrease in WT1 expression in aging ovaries was associated with fewer immature follicles. Thus, WT1 expression is restricted to immature follicles in diverse avian and mammalian species and over the reproductive life span in rats. These data demonstrated that WT1 is a marker for immature follicles and suggested a potential role of this transcriptional repressor in the slow growth of early follicles.

Estrogen receptor gene disruption: molecular characterization and experimental and clinical phenotypes.

The estrogen receptor (ER) is thought to play a crucial role in the regulation of many life processes, including development, reproduction and normal physiology. Because there have been no known mutations of the estrogen receptor in normal tissue of humans and animals, its presence and tissue distribution is thought to be essential for survival. Using the techniques of homologous recombination, we have disrupted the ER gene and have produced a line of transgenic mice possessing the altered ER gene (ERKO). The mouse ER gene was disrupted by inserting a 1.8 kb PGK-Neomycin sequence into exon 2, approximately 280 bp downstream of the transcription start codon. The correct targeting of the disruption was demonstrated by Southern blot analysis and PCR. Western blot analysis of uterine preparations from ERKO females showed no detectable ER protein. Heterozygotes had one half the level of ER protein compared to wild-type animals. Estrogen insensitivity was confirmed using estrogen agonists, estradiol, hydroxy tamoxifen, diethylstilbestrol treatment for 3 days which resulted in a 3-4-fold increase in uterine wet weight and vaginal cornification in wild-type females, while ERKO mice were totally unresponsive. These data were further supported by the failure of estrogen or EGF treatment to induce DNA synthesis in uterine tissue of similarly treated mice. Lactoferrin, an estrogen-responsive gene in the uterus, was also assayed by Northern blot. Wild-type mice treated with a single estradiol injection showed a 350-fold induction in lactoferrin mRNA. while ERKO females showed no detectable response. Both male and female animals survive to adulthood with normal gross external phenotypes. As expected, females are infertile and demonstrate hypoplastic uteri and hyperemic ovaries with no apparent corpora lutea. Males are also infertile, with atrophy of the testes and seminiferous tubule dysmorphogenesis. Although the reproductive capabilities have been altered with a dramatic effect on the gonads, prenatal development of the reproductive tracts of both sexes appear to be independent of an ER-mediated response. Analysis of the mammary glands of the ERKO females at 4 months of age showed a primitive ductal rudiment rather than the fully developed ductal tree seen in wild-type siblings. Also absent were the terminal end buds seen during normal ductal morphogenesis. Both sexes show a decrease in skeletal bone density, supporting a direct role for ER action in bone. A single patient is described who is homozygous for a point mutation in the human ER gene at codon 157. The mutation produces a truncation of the ER protein and results in estrogen insensitivity syndrome. Most significant of the clinical findings are effects on skeletal bone density and retarded bone age. Findings from the patient and mice suggest that the absence of functional ER is not lethal. Mutation in the ER gene is present in the human population. Further characterization of the mice and identification of additional patients will be required to more fully understand the consequences of ER gene mutations.

Transforming growth factor beta modulates gonadotropin receptor expression in porcine and rat granulosa cells differently.

Almost without exception, the studies to date describing the effects of transforming growth factor beta (TGF beta) upon various ovarian cell types have utilized the subtype TGF beta 1. Since TGF beta 1 and TGF beta 2 have been demonstrated to influence major developmental processes differentially during hematopoiesis and embryogenesis, we investigated in two species (rat and pig) whether they might also differentially modulate principal regulatory processes of ovarian cellular differentiation, specifically FSH and/or LH receptor (FSHR, LHR) expression. TGF beta 1 plus FSH significantly stimulated LHR binding levels by cultured granulosa cells (GC) from prepubertal diethylstilbestrol-treated rats. TGF beta 2 produced the same effect. In porcine GC from 1-3-mm antral follicles, TGF beta 1 and TGF beta 2 again acted similarly, but the direction of the response was opposite to that in the rat GC system. This difference could not be ascribed to the fact that the GC utilized represented different stages of follicular development in vivo because TGF beta 1 also potentiated FSH-dependent LHR induction in GC from antral follicles of cycling rats at all stages of the estrous cycle. The major effect of TGF beta 1 on FSHR expression in the rat system was to increase binding by attenuating the down-regulatory action of cholera toxin (CTX) or FSH. In the porcine system, TGF beta reduced FSHR binding at FSH or CTX concentrations that enhanced expression, and it did not attenuate the down-regulatory effect of FSH or CTX at higher doses. In summary, TGF beta up- or down-regulated LHR and FSHR binding coordinately within species.(ABSTRACT TRUNCATED AT 250 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.

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.

Effects of diethylstilbestrol on rat granulosa cell and thecal/interstitial cell transforming growth factor-beta 2 mRNA expression in vivo: analysis by reverse transcription-polymerase chain reaction.

Recent studies indicate that reproductive hormones may stimulate target cell growth via the actions of growth factors. In this study, reverse transcription-polymerase chain reaction analysis was used to assess the effects of in vivo diethylstilbestrol (DES) treatment on granulosa cell (GC) and thecal-interstitial cell (TIC) transforming growth factor (TGF)-beta 2 mRNA expression. In response to DES, GC expression of TGF-beta 2 mRNA was stimulated approximately 6.8-fold. By contrast, DES did not significantly alter TIC expression of TGF-beta 2 mRNA. Data were normalized relative to glyceraldehyde-3-phosphate dehydrogenase mRNA levels, which were unaffected by DES. These results extend our earlier findings, which demonstrated that GC and TIC expression of TGF-beta 2 mRNA is regulated by gonadotropins in vitro, to include steroidal regulation in vivo of intraovarian growth factor expression. Collectively, the present study and previous results support the following concepts: 1) intraovarian TGF-beta 2 mRNA expression is regulated by both gonadotropins and steroids, and 2) GC and TIC expression of TGF-beta 2 mRNA are differentially regulated.

Human granulosa-luteal and cumulus cells express transforming growth factors-beta type 1 and type 2 mRNA.

Human granulosa-luteal cells and cumulus cells obtained from women undergoing in vitro fertilization and embryo transfer (IVF-ET) were examined for the presence of TGF-beta 1 and TGF-beta 2 mRNA by reverse transcription-polymerase chain reaction (RT-PCR) analysis. RT-PCR analysis revealed that both follicle cell types express mRNA for both TGF-beta subtypes. Verification of RT-PCR products was done by restriction enzyme digestion analysis. These results suggest a role(s) for TGF-beta 1 and TGF-beta 2 in the development of human granulosa-luteal cells and the oocyte-cumulus cell complex.

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.

Acute and subacute effects of naturally occurring estrogens on luteinizing hormone secretion in the ovariectomized rat: Part 2.

Acute intravenous administration of the phytoestrogen genistein (G) blocks the gonadotropin-releasing hormone-(GnRH)-induced rise of luteinizing hormone (LH) in ovariectomized rats. The present experiments were performed to determine whether subacute administration of G or the mycoestrogens zearalenone and zearalenol would affect GnRH-induced or progesterone-induced LH secretion in ovariectomized rats. Charles River CD rats were ovariectomized and used 2 to 5 weeks later. Blood samples were obtained either via decapitation or via intraatrial cannulae three days after compounds were injected subcutaneously in sesame oil or corn oil vehicle. LH was measured by RIA. Pretreatment with estradiol benzoate suppressed LH levels at 1200 h, while G had no effect. Challenge with progesterone (8 mg/kg BW, sc) evoked LH release at 1600 h in rats pretreated with estradiol benzoate, but LH levels did not change in rats pretreated with G, zearalenone, or zearalenol. While GnRH-induced LH secretion was preserved in rats pretreated with estradiol, no LH response was detected in rats pretreated with the higher dose of G (8 mg/kg BW) or either dose of zearalenol (0.8 mg/kg BW or 8 mg/kg BW). We conclude that in the ovariectomized rat 1) subacute administration of G, zearalenone, or zearalenol do not inhibit tonic LH secretion, 2) G, zearalenone, and zearalenol do not provide "estrogenic priming" for progesterone-induced LH secretion; however, 3) G and zearalenol do block GnRH-induced LH secretion. The seemingly selective neuroendocrine effects of these naturally-occurring dietary estrogens emphasize that actions of each putative estrogen must be characterized for each "estrogenic" endpoint.

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.

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.

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.

Synergistic effect of insulin and follicle-stimulating hormone on biochemical and morphological differentiation of porcine granulosa cells in vitro.

Insulin and follicle-stimulating hormone (FSH) have been shown to facilitate granulosa cell differentiation in vitro. To gain insight into this process, we evaluated the effects of these hormones, alone and in combination, upon the biochemical parameters of luteinizing hormone/human chorionic gonadotropin (LH/hCG) receptor induction and progesterone secretion concomitantly with morphometric analysis of granulosa cell ultrastructure and LH/hCG receptor distribution by quantitative autoradiography under light microscopy. Granulosa cells isolated from small antral follicles (controls) cultured in the absence of exogenous hormones exhibited few microvilli and gap junctions; the mitochondria, endoplasmic reticulum, and Golgi complex were all poorly developed. Progesterone secretion was negligible and the cells bound little [125I]iodo-hCG. Insulin treatment increased gap junction formation, and the extent of smooth and rough endoplasmic reticulum and Golgi complex development (all p less than 0.05) but did not affect mitochondrial ultrastructure or volume. Insulin treatment modestly but significantly increased [125I]iodo-hCG binding and progesterone secretion relative to controls (p less than 0.001). FSH treatment had a similar effect to insulin on cell ultrastructure and additionally enhanced development of the mitochondria and smooth endoplasmic reticulum as well as formation of the microvilli (p less than 0.05). FSH significantly increased [125I]iodo-hCG binding and progesterone secretion relative to insulin-treated samples (p less than 0.001). Combined treatment with insulin and FSH markedly increased gap junction and microvilli formation and enhanced the development of the smooth endoplasmic reticulum and the Golgi complex relative to treatment with either hormone alone (p less than 0.05). Additionally, the combined treatment produced larger mitochondria with tubular christae. Consistent with the morphological development, the combined treatment of insulin and FSH significantly increased progesterone secretion and [125I]iodo-hCG binding (p less than 0.001). Autoradiographic analysis showed that aggregated cells in general exhibited higher LH/hCG receptor density than nonaggregated cells, and a significantly higher overall receptor density compared to nonaggregated cells or to cells treated either with insulin or FSH alone. Our results indicate that insulin and FSH facilitate morphological differentiation of the granulosa cell in a synergistic manner, stimulating gap junctions and microvilli formation and enhancing development of the mitochondria, endoplasmic reticulum, and Golgi complex.(ABSTRACT TRUNCATED AT 400 WORDS)

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)

[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.