Dichlorodiphenylchloroethylene elevates cytosolic calcium concentrations and oscillations in primary cultures of human granulosa-lutein cells.

1,1-Dichloro-2,2-bis(p-chlorophenyl)ethylene (DDE), a metabolite of DDT (1,1-dichlorodiphenyltrichloroethane), is a persistent hormonally active environmental toxicant that has been found in human serum and follicular fluid. The objective of this study was to determine whether DDE can alter free calcium ion concentrations in the cytosol ([Ca(2+)](cyt)) of human granulosa cells. Changes in [Ca(2+)](cyt) in single cells loaded with Fura-2 were studied using a dynamic digital Ca(2+) imaging system. At a concentration of 100 ng/ml, DDE stimulated small elevations of [Ca(2+)](cyt) accompanied by Ca(2+) oscillations. At 1 microg DDE/ml, there was a biphasic Ca(2+) response with marked elevations of [Ca(2+)](cyt) over time. In Ca(2+)-free medium, cells showed an initial small elevation of [Ca(2+)](cyt), which was magnified after addition of Ca(2+) to the medium. Washing the cells after DDE treatment failed to remove the elevated [Ca(2+)](cyt) and oscillations, both of which were eliminated by addition of EGTA. ATP also induced [Ca(2+)](cyt) elevations and oscillations, and these effects were potentiated when DDE was added. FSH induced transient [Ca(2+)](cyt) elevations, whereas hCG caused a prolonged elevation and marked oscillations in [Ca(2+)](cyt). These results suggest that DDE at concentrations normally found in human tissues induces elevations in [Ca(2+)](cyt) in granulosa-lutein cells. Our data therefore highlight a novel mechanism through which DDE can alter endocrine homeostasis and possibly act as an endocrine toxicant.

Melatonin receptor mRNA expression in human granulosa cells.

We have shown that the melatonin receptor agonist, 2-[125I] iodomelatonin, binds to high-affinity guanine nucleotide-sensitive sites on human granulosa (HG) cell membranes. In order to confirm the presence of melatonin receptors in HG cells, we have now used a reverse transcriptase-polymerase chain reaction (RT-PCR) procedure to examine receptor subtype expression. RT-PCR studies revealed the presence of the mt1 (Mel1alpha) melatonin receptor subtype in ten single or pooled HG cell samples which were obtained from 14 patients. In contrast, expression of MT2 ( Mel1b) mRNA was observed in only two of these HG samples. DNA sequencing of the mt1 PCR product confirmed its identity with the reported human mt1 melatonin receptor. The expression of mt1 and MT2 receptor mRNA in HG cells and the reported presence of melatonin in human follicular fluid indicate a potentially important role for this hormone in regulating human ovarian and reproductive function.

Steroidogenesis in luteinized granulosa cell cultures varies with follicular priming regimen.

During follicular development, a co-ordinated gonadotrophin and endocrine environment is believed to be essential for normal function of the resulting corpus luteum. Whether differences in the gonadotrophins used to promote follicular development can have lasting effects on granulosa cells after they have undergone luteinization and culture, remains to be studied. We measured steroid production under basal and human chorionic gonadotrophin (HCG) stimulation in short and long term cultures of luteinizing granulosa cells obtained from normal ovulatory women undergoing assisted folliculogenesis with either human menopausal gonadotrophin (HMG) or follicle stimulating hormone (FSH). Basal progesterone and oestradiol production by luteinized granulosa cells obtained from follicles stimulated to develop with FSH was significantly greater than that from HMG derived follicles (P < 0.001). In short term cultures, treatment with 10 IU HCG caused a 10-fold increase in progesterone release by cells from FSH stimulated follicles, whereas cells of HMG origin produced only 5-fold more progesterone (P < 0.0001). In cultures that were maintained for 2 weeks, progesterone secretion was reduced, but a similar trend in HCG responsiveness was observed. These experiments demonstrate that the composition of the gonadotrophins used to promote follicular development in vivo leads to differences in granulosa cell steroidogenesis which are evident after luteinization and culture. They additionally support the notion that the environment of follicular development will be reflected in the resulting corpus luteum.

Transforming growth factor alpha localization and role in surface epithelium of normal human ovaries and in ovarian carcinoma cells.

Transforming growth factor alpha (TGF alpha) has been localized by immunohistochemistry in the ovarian surface epithelial (OSE) cells of sections from normal human ovaries and in epithelial cells of surface crypts. An ovarian cancer cell line (HEY) derived from the surface epithelium of a human ovary also exhibited intense staining for the TGF alpha peptide. Using Northern analysis, HEY cells were shown to express a 4.5-kb transcript of TGF alpha, indicating that the TGF alpha peptide was synthesized by these cells and not taken up from the serum in the culture medium and sequestered by the cells. This was confirmed using a radioimmunoassay, which showed that HEY cells in culture secrete TGF alpha peptide, both as a soluble (0.12 +/- 0.02 ng/mg protein) and as a membrane-anchored (0.06 +/- 0.006 ng/mg protein) form. In both normal OSE cells and HEY cells, TGF alpha acted as a growth promoter: TGF alpha significantly stimulated [3H]thymidine incorporation into DNA of both primary cultures of normal OSE cells (2.7-fold) and of HEY cells (2-fold). This study provides the first demonstration of TGF alpha immunostaining in normal surface epithelial cells and in HEY cells, and suggests that TGF alpha, localized in normal and transformed OSE, is an autocrine growth promoter for these cells.

Identification of a transforming growth factor alpha-like molecule in human seminal plasma.

OBJECTIVE: To characterize the putative seminal growth promoting factor serendipitously observed when human seminal plasma was analyzed for bioactive FSH. DESIGN: A pool of human seminal plasma was subjected to sequential Sephadex G-75 (superfine) chromatography and high-performance size exclusion liquid chromatography. The fractions were tested for mitogenic activity using a rat granulosa cell assay and normal rat kidney (NRK) cells. Properties of the factor were established and characterization by gel electrophoresis and neutralization with antibody were accomplished. SETTING: Reproductive Biology Research Laboratory at McMaster University Medical Centre. MAIN OUTCOME MEASURES: Ability of purified fractions of human seminal plasma to augment the uptake of tritiated thymidine into cell DNA. RESULTS: Mitogenic activity of human seminal plasma was augmented in the presence of FSH but not hCG, PRL, E2, T, P, or dihydrotestosterone. The putative growth factor synergized with insulin-like growth factor I (IGF-I), epidermal growth factor (EGF), and transforming growth factor beta (TGF-beta) but not with TGF-alpha. Mitogenic activity was neutralized by a specific TGF-alpha antibody in a dose-dependent manner. The molecular weight of the factor as assessed by gel electrophoresis is 6 kd. CONCLUSIONS: Human seminal plasma contains a mitogen that is similar to TGF-alpha.

Transforming growth factor-alpha: identification in bovine corpus luteum by immunohistochemistry and northern blot analysis.

Transforming growth factor-alpha (TGF-alpha), a product of the thecal cells, has potent mitogenic and steroidogenic influences on cells within the ovarian follicle. Whether TGF-alpha continues to be produced in those follicles that go on to ovulate and form a corpus luteum is currently under investigation. In the present study, TGF-alpha was localized in the bovine corpus luteum by means of immunoperoxidase staining using a monoclonal antibody for TGF-alpha that does not cross-react with epidermal growth factor. In corpora lutea from the mid-luteal phase of the cycle TGF-alpha staining was found predominantly in the large luteal cells. Northern blot analysis using a human TGF-alpha cDNA probe hybridized to the 4.5-4.8 kb TGF-alpha transcript in RNA from the corpus luteum. These studies provide new evidence that TGF-alpha, a potent paracrine regulator within the ovarian follicle, continues to be expressed in the corpus luteum.

Rapid increase in relaxin gene expression in early pregnancy in the pig.

Relaxin mRNA concentrations in porcine corpora lutea were examined during the peri-implantation period and throughout pregnancy using Northern and slot blot analysis. Total RNA was extracted from corpora lutea obtained from pigs of known breeding dates and pregnancy was confirmed by embryo recovery. A 32P-labelled porcine relaxin cDNA probe identified the 1.0 kilobase relaxin transcript. Slot blots were subsequently used to quantify relaxin mRNA concentrations. Relaxin mRNA was detectable in the corpus luteum of the regular cycle and was also present at similar low levels in corpora lutea of days 10, 11 and 12 of pregnancy. In corpora lutea from day 16 of pregnancy onwards 100-fold greater quantities of relaxin mRNA were observed. The intensity remained similar in samples between days 16 and 102 of pregnancy. These studies indicate that elevated relaxin gene expression commences very early in pregnancy and is first detectable in the peri-implantation period.

Immunoreactive adrenocorticotrophin is present in the ovary and in particular the oocyte of several mammalian species.

Proopiomelanocorticotrophin (POMC)-derived peptides have been identified in both male and female reproductive systems. However, there have been few reports of ACTH, the major biologically active POMC product, in the mammalian ovary. We sought evidence for the presence and localization of immunoreactive (ir)-ACTH in ovaries from sheep, humans, cows, pigs, rats and cats using immunohistochemical techniques. Tissue sections were stained with diaminobenzidine following incubation with a primary antibody raised against ACTH1-24. There was positive staining for ACTH in cells scattered throughout the interstitium of ovaries from all species examined. Immunoreactive ACTH was observed in the oocytes of ovaries from humans, cows, pigs, pregnant and non-pregnant sheep, but not from cats or rats. Positive staining of oocytes was associated with all tertiary and secondary follicles, and some primary follicles. There was no apparent difference in the pattern of staining between pregnant and non-pregnant sheep. Staining for ir-ACTH was absent in ovaries from fetal sheep. We conclude that ir-ACTH is present in ovarian tissue, and in particular the oocyte, from several species of mammal. The presence of ir-ACTH within the oocyte is dependent on species and stage of follicular maturation.

Transforming growth factor-alpha in the adult bovine ovary: identification in growing ovarian follicles.

The pituitary gonadotropins and gonadal steroids are required for normal follicular growth and development but neither has been shown to act directly as a granulosa cell mitogen in vitro. A number of polypeptide growth factors, however, are known to have pronounced mitogenic effects on the cells of the follicle. We have localized transforming growth factor-alpha (TGF-alpha), a potent mitogen, in bovine thecal cells via immunoperoxidase staining using a monoclonal antibody for TGF-alpha that does not cross-react with epidermal growth factor. TGF-alpha staining is most intense in the theca of follicles at the discrete physiological stages known to show rapid granulosa cell growth (small follicles of 0.7-2.0 mm diameter). Staining intensity for TGF-alpha declines in large preovulatory follicles, coincident with the known decline in granulosa cell mitosis. These studies provide further evidence for paracrine interactions in the ovary and show that TGF-alpha may play an important role in the regulation of follicular development in the adult bovine ovary.

Bovine thecal cells secrete factor(s) that promote granulosa cell proliferation.

To determine if soluble factors (other than steroids) secreted by bovine thecal cells may be involved in local regulation of follicular development, we examined the effects of thecal cell secretory products on the growth of granulosa cells obtained from the same follicles. DNA synthesis (assessed by the incorporation of 3H-thymidine) by granulosa cells plated on coverslips and cocultured with, but not directly in contact with, thecal cells in organ culture dishes in a serum-free medium was 5-fold greater than controls. The effect of the thecal cell-secreted products on DNA synthesis by granulosa cells was significantly higher than the maximum response produced by epidermal growth factor (EGF). Thecal cell-conditioned medium stimulated 3H-thymidine incorporation into the DNA of granulosa cells and a normal rat kidney cell line in a dose-dependent manner. The increases in 3H-thymidine incorporation into granulosa cell DNA subsequently lead to an increase in cell number. Preliminary characterization studies using ultrafiltration membranes indicated that the mitogenic factor was retained in the greater than 10,000 molecular weight fraction. The activity was stable to heating at 90 degrees C for 5 min and was not extracted in ether. The thecal cell-generated growth factor may act as a paracrine regulator of granulosa cell growth, thus providing the dominant follicle with autonomy over other follicles in the cohort.

Rat thecal/interstitial cells produce a mitogenic activity that promotes the growth of granulosa cells.

To test the hypothesis that the cells outside the basal lamina of the follicle secrete paracrine factors that influence the cells on the inside of the follicle, two ovarian cell populations were obtained from diethylstilbestrol-treated rats. Granulosa cells were obtained by extrusion from the follicles and an ovarian cell preparation, termed thecal/interstitial, was derived from the granulosa cell-depleted ovaries. Light microscopy showed that each cell population in culture had distinctive morphologies. Electrophoretic examination of the radiolabeled proteins secreted by the two ovarian cell preparations revealed that each secreted unique protein components into the culture medium. Rat thecal/interstitial cell-conditioned medium promoted [3H]thymidine incorporation into normal rat kidney cell line (NRK) DNA and into bovine granulosa cell DNA. The growth-promoting activity was stable to heating at 70 degrees C for 5 min whereas native fibroblast growth factor (FGF) lost its activity, showing that the factor was not characteristic of FGF. To further characterize the growth-promoting activity thecal/interstitial cell-conditioned medium was concentrated and the proteins separated by size exclusion high performance liquid chromatography. The growth-promoting activity eluted with an apparent molecular weight between 15,000 and 25,000. The finding that thecal/interstitial cells in culture secrete growth-promoting factors suggests that those cells that are in close proximity to the granulosa cells may secrete protein factors that diffuse into the follicular antrum and influence granulosa cell proliferation.

Aromatase activity in granulosa cells: regulation by growth factors.

This study describes the effects of insulin, insulin-like growth factor 1 (IGF1), and epidermal growth factor (EGF) on the aromatase activity of granulosa cells isolated from immature rat ovaries. None of the growth factors alone influenced the basal level of aromatase activity, but did modulate follicle-stimulating hormone (FSH)-induced aromatase activity. Insulin and IGF1 augmented the action of a sub-optimal concentration of FSH (5 ng/mL) on aromatase activity in a dose-dependent manner. In contrast, EGF (1-10 ng/mL) was effective in inhibiting aromatase activity maximally stimulated by FSH. Since insulin and IGF1 had opposing actions to those of EGF on FSH-induced aromatase activity, we examined the interactions between the growth factors. EGF inhibited the actions of both FSH and insulin on aromatase activity. Both IGF1 and EGF increased the [3H]thymidine incorporation into the DNA of bovine granulosa cells in vitro, IGF1 being a more potent mitogen. Whereas EGF inhibited the actions of IGF1 on aromatase activity, it did not inhibit the effects of IGF1 on the growth of granulosa cells. In summary, growth factors influence both the differentiation and growth of granulosa cells, and may be important regulators of follicular development.

Human granulosa and thecal cells secrete distinct protein profiles.

Granulosa and thecal cells were isolated from follicles of ovaries removed from premenopausal women who underwent salpingo-oophorectomy. The electrophoretic profiles of the [35S]methionine-radiolabeled proteins secreted by the two cell types were quite distinct and showed different major proteins. Untreated granulosa cells secreted a major radiolabeled protein with a molecular weight of 220,000, identified as fibronectin by immunoprecipitation. This protein comprised 21% of the total secreted protein; however, the intensity of labeling was reduced after treatment with 0.5 mM dibutyryladenosine 3':5'-cyclic monophosphate ([Bu]2 cAMP). Fibronectin secretion by control cultures measured by a competitive enzyme-linked immunoadsorbant assay ranged from 15.1 to 16.8 micrograms/mg cellular protein/24 hours and was reduced to 30% after treatment with 0.5 mM (Bu)2 cAMP. In contrast, untreated thecal cells secreted only low levels of fibronectin and a 25,000-dalton protein contributed 20% of the total secreted radiolabeled proteins.

Actions of growth factors in the follicle.

In this paper we have examined the possibility that soluble factors produced by the thecal and granulosa cells may be essential local modulators of follicular development. The observations that insulin could influence both the growth and the differentiation of granulosa cells were important in establishing the concept that peptides could act as amplifiers of the actions of gonadotrophins. Insulin alone did not influence aromatase activity significantly but acted synergistically with FSH to augment aromatase activity in rat granulosa cells. Unlike aromatase activity, insulin alone was able to significantly stimulate 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) activity, the maximum level achieved approaching that obtained with high concentrations of FSH. To determine if insulin could influence other parameters of granulosa cell function in addition to steroidogenesis, we measured a component of extracellular matrix, fibronectin, previously shown to be inhibited by FSH. Treatment with insulin independently inhibited the increase in fibronectin secretion observed in control cultures. Also, insulin alone was able to stimulate quiescent bovine granulosa cells to incorporate [3H]thymidine into DNA under serum-conditions. The concentration of insulin required in these experiments was higher than physiological levels suggesting that other insulin-like growth factors may be involved. Our work and that of others has shown that IGF1 can mimic the actions of insulin and is effective at lower concentrations. A possible source of IGF1 production in the follicle was sought initially by collecting rat granulosa cell conditioned medium, and assessing biological activity and immunoreactivity. Conditioned medium augmented the actions of FSH on aromatase activity and alone stimulated 3 beta-HSD, indicating the presence of insulin-like bioactivity. A positive reaction on immunoblots using specific antiserum confirmed the presence of immunoreactive IGF1. Conditioned medium from thecal cells contained a growth-promoting activity (TcGF) that did not augment FSH-induced aromatase activity. The production of growth factors locally within the follicle may represent the self-amplifying mechanism that enables the dominant follicle to complete its developmental program and ovulate.

Metabolism of androstenedione by Sertoli cell enriched preparations and purified Leydig cells from boar testes in relation to estrogen formation.

The metabolism of [3H]androstenedione in relation to estrogen formation was studied in incubations of Sertoli cell-enriched preparations (30-40% Sertoli cells) and purified Leydig cells (greater than 98%) from testes of mature male pigs. Radioactive metabolites were partitioned by countercurrent distribution (CCD) into unconjugated and 'conjugated' (water-soluble) fractions. Both unconjugated and solvolysed metabolites were separated into neutral and phenolic fractions by CCD with toluene and NaOH. The distribution of radioactivity was examined subsequently for each fraction by partition chromatography on celite columns. Major differences were noted in the products of metabolism from the two cell types. More than half of the radioactivity appeared in the conjugate fraction for Leydig cell incubations, but little or no conjugation occurred in Sertoli cell preparations. Metabolism of androstenedione to other neutral substances was extensive only for Leydig cells, with approx 2% remaining unchanged. No clear evidence of estrogen formation was observed with Sertoli cells; however, both unconjugated and conjugated phenolic for Leydig cell products showed radioactivity corresponding to estrone and estradiol-17 beta on chromatography. About 2-5% of androstenedione was converted to these two estrogens, whereas most of the phenolic material was present as compounds more polar than estradiol.