Granulosa cell fusion allows heterologous receptor stimulation of adenylate cyclase and progesterone accumulation.

The hallmark of the preovulatory follicles in the rat ovary appears to be the presence of receptors for both LH and FSH on follicular granulosa cells. We have tested the possibility that both gonadotropin receptors could share a common adenylate cyclase system utilising cell fusion techniques. Adenylate cyclase (and concomitant stimulation of progesterone synthesis) was inactivated in granulosa cells possessing both FSH and LH receptors by incubation of the cells with N-ethylmaleimide. This treatment did not affect hormone binding to the cells. Subsequent fusion, using polyethylene glycol, of the cyclase-inactivated cells with granulosa cells possessing FSH receptors only and an active cyclase restored LH stimulation of cAMP and progesterone production. These findings support the hypothesis that the LH and FSH receptors on granulosa cells of preovulatory follicles share a common adenylate cyclase system.

Contact-dependent cell interactions determine hormone responsiveness and desensitization in rat granulosa cells.

The maintenance of associations between granulosa cells (GCs) is necessary for FSH-stimulated induction of LH receptors. In cultures in which these associations have been disrupted, FSH fails to induce LH receptors. As FSH exerts its action in GCs via cAMP, we have examined if the aggregation state of GCs plays a role in modulating FSH-stimulated cAMP production. GCs were obtained from the ovaries of diethylstilbestrol-primed immature rats. Cells were prepared as aggregate or dispersed populations by isolating GCs in either the presence or absence of Ca2+. Nonviable cells were removed by a brief exposure to trypsin. We have shown previously that trypsin treatment in the absence of Ca2+ removes a class of cell adhesion molecules, termed cadherins, from the plasma membranes of GCs. Hence, the dispersed GCs are incapable of reaggregating. Dispersed or aggregate GC preparations were incubated with different doses of human FSH (0-1 microg) for 0-60 min in the presence of isobutylmethylxanthine, a phosphodiesterase inhibitor. Incubations were terminated, and the cAMP accumulated was measured using a specific RIA. As desensitization to hormonal stimuli is a characteristic property of many G protein-coupled response systems, cAMP production of cell aggregates and dispersed cells in response to a repeated stimulation with FSH was assessed. Our results indicate that aggregate GCs have a significantly attenuated cAMP response to all doses of FSH compared with dispersed GC preparations. Changing cell densities did not alter the nature of these responses, indicating that nonspecific cell interactions were not responsible for this difference. The number of FSH receptors and their affinity were unaltered in the two cell preparations. Cholera toxin- and forskolin-stimulated cAMP production were similar in the two preparations, demonstrating that the changes in responsiveness did not arise from alterations in G protein activation or adenylate cyclase activity. Only the aggregate GCs could be desensitized. The dispersed cells displayed undiminished cAMP responsiveness to a second FSH stimulation. Finally, culture of the GC preparations with cholera toxin induced LH receptors in GC aggregates only. LH receptor induction in dispersed cell cultures required the addition of estradiol. These results indicate that contact-dependent cell interactions can modulate GC cAMP production in response to FSH. cAMP responses, however, were not the sole determinant of cell differentiation, as assessed by LH receptor induction. We speculate that cell-cell interactions within the follicular epithelium are important determinants for cell differentiation leading to follicle selection for ovulation or atresia.

B-type natriuretic peptide receptor expression and activity are hormonally regulated in rat ovarian cells.

Natriuretic peptides form a family of structurally related peptides known to regulate salt and water homeostasis and to cause vasodilation. Synthesis of atrial (ANP), brain (BNP), and C-type (CNP) natriuretic peptides occurs mainly in the heart and brain and has been identified recently in the female reproductive tract. The expression of ANP and CNP as well as their cognate guanylyl cyclase receptors (NPR-A and NPR-B, respectively) have been detected in the rat ovary. We have shown previously that the expression of the natriuretic peptides and their receptors in the rat ovary appears to be modulated by the estrous cycle. In the present study we have evaluated the expression of the natriuretic peptide system (peptide and receptor) in ovarian cells (granulosa and thecal-interstitial cells) obtained from immature female rats treated with either diethylstilbestrol (DES), an estrogen analog, or equine CG (eCG), a gonadotropin that possesses both LH and FSH activity. Using a whole cell RRA, we found that CNP binding was increased by 2-fold in granulosa cells taken from animals treated with either DES or eCG. Semiquantitative RT-PCR revealed that granulosa cells from DES- or eCG-treated animals have increased levels of NPR-B messenger RNA (mRNA) transcripts, which was in good agreement with the increased binding. The activity of the receptors was assessed by ligand-dependent stimulation of cGMP release. CNP, but not ANP, stimulated the release of cGMP from granulosa cells obtained from DES-treated, but not from eCG-treated, animals. The relative levels of CNP mRNA in granulosa cells were unaltered by either DES or eCG treatment. In contrast, CNP mRNA levels were increased more than 2-fold, but only in theca-interstitial from the eCG-treated animals. Our results indicate that CNP and NPR-B are expressed in the ovary, and their expression is responsive to hormonal treatments. Furthermore, expression of these components of the natriuretic peptide system appears to be compartmentalized, with CNP being derived from the extrafollicular compartment and acting, through NPR-B, on the granulosa cells.

Cellular heterogeneity in the membrana granulosa of developing rat follicles: assessment by flow cytometry and lectin binding.

The hormone-mediated maturation of ovarian follicles is apparently accompanied by position-specific differentiation of cells of the membrana granulosa. We have assessed the extent of this cellular heterogeneity by flow cytometry using a variety of fluorescein isothiocyanate-labeled lectins as probes. Follicular development was stimulated in immature rats by treatment with either diethylstilbestrol (DES) or equine CG (eCG). Lectin binding to monodispersed rat granulosa cells was then analyzed by flow cytometry. Our results demonstrate that there are two distinct populations of small (4-7 microM) and large (9-12 microM) granulosa cells in follicles from DES- and eCG-treated animals. Both populations appear to be mitotically active and show specific lectin-binding characteristics. Six lectins (canavalia ensiforms, triticum vulgaris, maclura pomifera, erythrina cristagalli, jacalin, and vicia villosa) bind equally to both small and large granulosa cells from the DES- and eCG-treated rats. In contrast, no binding to either cell population was detected with six other lectins (dolichos biflorus, griffonia simplicifolia-II, lycopersicon esculentum, datura stramonium, solanum tuberosum, and ulex europaeus). Furthermore, four galactose-binding lectins (bauhinia purpurea, glysine maximus, griffonia simplicifolia-I, and arachis hypogaea) were found to identify specific subsets of granulosa cells. Three of these lectins (bauhinia purpurea, glysine maximus, and griffonia simplicifolia-I) bind to only small granulosa cells from either DES- or eCG- treated immature rats. The fourth lectin (arachis hypogaea) identifies subpopulations of both small and large granulosa cells. Application of the four galactose-specific lectins to fixed sections of frozen ovaries demonstrated binding to the perioocyte and cumulus granulosa cells. We conclude that cellular heterogeneity exists within the follicular epithelium at various stages-specific lectin-binding sites.

Estrogens potentiate the stimulatory effects of follicle-stimulating hormone on N-cadherin messenger ribonucleic acid levels in cultured mouse Sertoli cells.

Gonadal steroids and FSH are key regulators of Sertoli cell function. N-Cadherin (N-cad) is a calcium-dependent cell adhesion molecule that mediates Sertoli cell-germ cell interactions. We recently demonstrated that steroids, in particular estradiol, are potent regulators of testicular N-cad messenger RNA (mRNA) levels in vivo. In view of the cooperative effects of steroids and FSH on Sertoli cell-germ cell interactions, we examined the combined effects of these hormones on N-cad mRNA levels in cultured mouse Sertoli cells. FSH was capable of increasing N-cad mRNA levels 2-fold in these cells. The effects of FSH on N-cad mRNA levels in cultured Sertoli cells were mimicked by cAMP-inducing agents. Treatment of the Sertoli cell cultures with FSH and estradiol stimulated N-cad mRNA levels 3-fold, whereas steroids alone had no effect on N-cad mRNA levels. These studies demonstrate that FSH and estradiol in combination are required to achieve maximal N-cad mRNA levels in cultured Sertoli cells. The results obtained from these studies substantiate the hypothesis that estrogens play a pivotal role in regulating spermatogenesis.

Uterine oxytocin gene expression. I. Induction during pseudopregnancy and the estrous cycle.

We have recently demonstrated that the gene encoding the hypothalamic peptide oxytocin (OT) is highly expressed in the rat endometrial epithelium during the last 4 days of pregnancy. Here, we show that uterine OT gene expression is also induced during the proestrous phase of the estrous cycle and after induction of pseudopregnancy. In mature female rats, OT mRNA levels increased more than 10-fold between diestrus and proestrus and remained elevated at estrus. The levels attained at estrus corresponded to about 1/20th of the levels present at term. In immature rats rendered pseudopregnant by treatment with pregnant mare serum and hCG, uterine OT mRNA levels rose steadily and reached a maximum on day 14 of pseudopregnancy, corresponding to about 1/8th of the levels observed on day 21 of normal pregnancy. Oil-induced decidualization of the left uterine horn prolonged pseudopregnancy and maintained OT mRNA levels in both uterine horns until day 19 of pseudopregnancy. These changes were tissue specific, as hypothalamic OT mRNA levels remained essentially unaffected. The present findings demonstrate that either spontaneous or induced changes in endogenous steroid levels are capable of eliciting important changes in uterine, but not hypothalamic, OT gene expression.

Uterine oxytocin gene expression. II. Induction by exogenous steroid administration.

As we have recently shown, the gene encoding the hypothalamic nonapeptide oxytocin (OT) is expressed in the rat endometrial epithelium during late pregnancy and the estrous phase of the estrous cycle. To investigate the role of ovarian steroids in the regulation of uterine OT gene expression, Silastic capsules containing estradiol or progesterone were implanted into immature ovariectomized rats. Exposure to estradiol alone for 2 days caused a significant rise in OT mRNA. Administration of progesterone alone was without effect. However, a strong synergism was observed when the two hormones were applied together; progesterone potentiated the effect of estradiol by a factor of 7. In animals treated with steroids for 4 days, the removal of either the estradiol or progesterone capsule after day 2 led to a decrease in the total amount of OT mRNA accumulation, implying that the continued action of both steroids was required to achieve maximal OT mRNA levels. Immunocytochemical analysis demonstrated that the main site of steroid-induced uterine OT gene expression is the endometrial epithelium, the same site where endogenously induced OT gene expression occurs at the end of pregnancy. The OT mRNA levels achieved after 4 days of treatment with both steroids were comparable to those achieved at estrus or during pseudopregnancy, but corresponded to less than 20% of the levels present in the uterus on day 21 of pregnancy. These data suggest that in the uterus, the synergistic action of ovarian steroids represents an important, but probably not exclusive, regulator of OT gene expression.

Estradiol regulates E-cadherin mRNA levels in the surface epithelium of the mouse ovary.

E-cadherin is a calcium-dependent cell adhesion molecule which is present in the surface epithelium of the mouse ovary. This cell adhesion molecule has been implicated as a suppressor of tumorigenesis. The regulators of E-cadherin mRNA levels in the ovary have not been identified. We have examined the ability of steroids to influence ovarian E-cadherin mRNA levels in vivo. Immature mice were injected with either progesterone, testosterone, dihydrotestosterone, 17-beta estradiol or 17-alpha estradiol. Only 17-beta estradiol caused a rapid and significant increase in the ovarian E-cadherin mRNA levels. We speculate that this steroid is a key regulator of E-cadherin-mediated epithelial cell interactions in vivo. We also discuss the possibility that the carcinogenic effects of estrogens on the ovary may be related to their ability to regulate E-cadherin levels in this tissue.

Plasma patterns of LH, FSH and prolactin in rats with a polycystic ovarian condition induced by oestradiol valerate.

The patterns of plasma LH, FSH and prolactin concentrations were investigated in rats with a polycystic ovary condition (PCO). The condition was induced by treatment with oestradiol valerate 9 weeks before blood sampling. Serial blood samples were taken at 10-min intervals for 4 h from ten rats with PCO. All samples were assayed for LH, those from five animals for FSH and those from the remaining five animals for prolactin. In addition, five control animals with normal oestrous cycles were sampled during oestrus and the samples assayed for LH. Mean concentrations of LH, FSH and prolactin in rats with PCO were 140 ng/l, 76 micrograms/l and 7.6 micrograms/l respectively. All three hormones exhibited an episodic pattern. The mean peak amplitudes of LH, FSH and prolactin were 120 ng/l, 25 micrograms/l and 3.5 micrograms/l respectively. All three hormones exhibited a similar mean frequency of four or five episodes per 4 h. The LH and FSH patterns were closely synchronized; nearly all FSH peaks coincided with LH peaks. The prolactin pattern did not, however, correlate with that of the gonadotrophins. Despite the persistent oestrous condition of the animals with PCO, it was clear that their pattern of LH did not resemble that of cyclic animals in normal oestrus; in the normally cyclic animals in oestrus the pulse period was nearly twice as long and the pulse amplitude was more than sixfold greater than those in animals with PCO. We conclude that the unique episodic patterns of gonadotrophins are more important than mean blood concentrations of these hormones in establishing and maintaining the polycystic ovary syndrome.

Luteinizing hormone receptor induction in dispersed granulosa cells requires estrogen.

Studies on granulosa cell responses in vitro have routinely utilized cell preparations in which intercellular gap-junctions are maintained. The present study was conducted to determine if disruption of these junctions, prior to culture, would affect subsequent follicle-stimulating hormone (FSH)-stimulated luteinizing hormone (LH) receptor induction on these cells. Granulosa cells were expressed from ovaries of diethylstilbestrol (DES)-primed immature rats after a short incubation of the excised ovaries in culture medium alone or medium containing 6.8 mM EGTA. The latter procedure disrupts gap-junctions between granulosa cells thus providing a predominantly mono-dispersed cell suspension. The two cell preparations were cultured, separately, for 72 h in sterile polypropylene tubes in media containing either FSH or FSH plus various steroids (estradiol, testosterone, or 5 alpha-dihydrotestosterone (DHT)). LH receptor content of cells was determined at 72 h of culture. Both the cell yield and the proportion of viable cells obtained were enhanced by the EGTA pretreatment. LH receptors were induced in all FSH-containing cultures of non-dispersed granulosa cells. In the dispersed cell cultures, FSH alone failed to induce LH receptors. The inclusion of either estradiol or testosterone but not DHT with FSH, however, restored LH receptor induction to levels comparable to non-dispersed cultures. LH receptors were not induced in cultures of either cell preparation with steroids alone. Aromatase activity, however, was stimulated in both cell preparations by FSH alone. These results suggest that cell-cell communication may be necessary for LH receptor induction in granulosa cells and that estradiol (or an aromatizable androgen) can promote intercellular interactions if this communication has been disrupted.

Hormonal regulation of N-cadherin mRNA levels in rat granulosa cells.

We have examined the ability of hormones to modulate the steady-state levels of N-cadherin mRNA transcripts in aggregated and dispersed rat granulosa cell populations. Estradiol and follicle-stimulating hormone (FSH) had no effect on the levels of N-cadherin mRNA transcripts in aggregated granulosa cells. In contrast, these two hormones stimulated N-cadherin mRNA levels in dispersed granulosa cells. This is the first report that estradiol and FSH are capable of regulating N-cadherin mRNA levels. The results also suggest that the N-cadherin mRNA levels in dispersed and aggregated granulosa cells are regulated by different mechanisms.

Effects of aromatizable and nonaromatizable androgen treatments on luteinizing hormone receptors and ovulation induction in immature rats.

The effects of androgen pretreatment on follicle-stimulating hormone (FSH)-stimulated luteinizing hormone (LH) receptor induction in ovarian granulosa cells was examined. Immature female rats were treated with various doses (0.1-5 mg/rat) of testosterone (T), 5 alpha-dihydrotestosterone (DHT), 5 alpha-androstane-3 alpha,17 beta-diol (3 alpha-diol), or 5 alpha-androstane-3 beta,17 beta-diol (3 beta-diol). Subsequent follicular development was stimulated by treatment with ovine FSH. LH receptor induction in granulosa cells and ovulatory responses to 10 IU human chorionic gonadotropin (hCG) were examined. Since LH receptor induction requires the synergistic action of both FSH and estradiol, the effects of the androgen pretreatment on FSH-stimulated estradiol production were also examined. Dihydrotestosterone treatment at doses greater than 1 mg inhibited LH receptor induction by approximately 70%, which resulted in absent ovulatory responses. Treatment with 1 mg or more of T or 3 alpha-diol had no effect on LH receptor induction, yet the hCG-stimulated ovulation rate was reduced to 40% of that seen in vehicle-treated controls. 3 beta-Diol, at a dose of 1 mg/rat, did not affect LH receptor induction but did reduce hCG-stimulated ovulation responses. No significant effects of androgen treatment on ovarian or uterine weight or FSH-stimulated estradiol production were observed. These results suggest that androgens can act at multiple sites to inhibit ovarian follicular development and function. In addition these studies demonstrate that, although LH receptor induction is necessary, it may not be a sufficient condition to ensure ovulation of ovarian follicles.

Estrogen-binding parameters of cytoplasmic and nuclear receptors in an established rat endometrial cell line and tumor.

We have consistently found receptors for estradiol in both the cytosol and nuclear extracts of a rat endometrial cell line and in transplantable tumors derived from this cell line. The equilibrium dissociation constants (Kd) and the rate constants for the receptor-estradiol interaction in these cells and tumors did not differ significantly from those of the cytosol receptor in the rat uterus. A mean Kd of 3 x 10(-10) M with a rate of association (Ka) of 3 x 10(5) M-1sec-1 and a rate of dissociation (Kd) of 1.5 x 10(-5) sec-1 were obtained for nuclear and cytosol receptors for both tumors and cells. For uterine cytosol, a Kd of 8 x 10(-10) M, ka = 2.8 x 10(5) M-1sec-1 and kd = 1 x 10(-5) sec-1 were obtained. Although no differences were seen in equilibrium and kinetic parameters for estradiol-17beta binding between the nuclear and cytosol receptors of tumors and cells, an apparent difference in the relative affinities of nuclear and cytosol receptors for estrone was detected. This suggests that the binding site in nuclear receptors may have been modified. Implications of this observation with regard to receptor translocation and the mechanism of action of sex hormones are being considered.

PIP: Estrogen-binding parameters of cytoplasmic and nuclear receptors in an established rat endometrial cell line and tumor were investigated. The equilibrium dissociation constants (Kd) and the Rate constants for the receptor-estradiol interaction in these cells and tumors were similar to those of the cytosol receptor in the rat uterus. A mean Kd of 3 X 10 less than -10 greater than M with a rate of association (ka) of 3 X 10 less than 5 greater than M second and a rate of dissociation (kd) of 1.5 X 10 less than -5 greater than/second were obtained for nuclear and cytosol receptors for both tumors and cells. For uterine cytosol a Kd of 8 x 10 less than -10 greater than M, ka of 2.8 X 10 less than 5 greater than /M/ second and kd of 1 X 10 less than -5 greater than/second were obtained. No differences were seen in equilibrium and kinetic parameters for estradiol-17beta binding between the nuclear and cytosol receptors of tumors and cells but an apparent difference in the relative affinities ofnuclear and cytosol receptors for estrone was detected. These results suggest that the binding site in nuclear receptors may have been modified. Implications of this observation are being considered with regard to receptor translocation and the mechamism of action of sex hormones.

A granulosa cell differentiation-stage-specific cytotoxic activity in bovine and rat sera.

Heat-inactivated serum is cytotoxic to granulosa cells from preantral follicles but not to cells from preovulatory follicles. A dominant feature of the granulosa cells of preovulatory follicles is the presence of luteinizing hormone (LH) receptors on the surface of the cells. In the present study, we have examined the relationship between the process of LH receptor induction and the acquisition of serum tolerance in granulosa cells in vitro. Granulosa cells from the ovaries of immature rats primed with diethylstilbestrol (DES) were cultured in a 1:1 mixture of Ham's F-12 and Dulbecco's modified Eagle's medium containing 30 ng of ovine follicle-stimulating hormone (oFSH; NIH-15). At either 0, 24, or 48 h of culture, heat-inactivated fetal bovine serum (FBS) was added (10% by volume) to separate groups of culture tubes. All cells were cultured for a total of 72 h, at which time the cultures were assessed for LH receptor (specific 125I-human chorionic gonadotropin [hCG] binding) and DNA content. LH receptors were induced in all FSH-containing serum-free cultures by 48 h. Receptors were not induced, however, when serum was added after either 0 or 24 h of culture. Furthermore, serum addition at these times resulted in a cell loss (assessed by DNA) of 40-60%. Serum addition at 48 h to FSH-containing cultures resulted in an inability to detect LH receptors at 72 h and with no significant effect on the culture DNA content. Addition of a protein extract of FBS at the initiation of cell culture prevented FSH-stimulated LH receptor induction and was cytotoxic. A lipid extract of FSH did not interfere with receptor induction and was not cytotoxic.(ABSTRACT TRUNCATED AT 250 WORDS)

E- and N-cadherin expression and distribution during luteinization in the rat ovary.

Cadherins, a family of Ca(2+)-dependent cell adhesion molecules, play an important role in ovarian tissue remodelling processes. The aim of this study was to examine the expression pattern of E- and N-cadherin in rat preovulatory follicles, luteinizing follicles and corpora lutea. Immature female rats were treated with equine chorionic gonadotrophin (eCG) to promote preovulatory follicle development. At 48 h after eCG treatment, the rats were injected with an ovulatory dose of hCG. Ovaries were analysed by western blot analysis and immunofluorescence for E- and N-cadherin expression at 48 h after eCG injection, and at 24 and 72 h after hCG injection. Ovaries of cyclic adult rats were examined to assess whether the changes in the expression pattern of cadherin were in agreement with those of the gonadotrophin-treated rats. Finally, expression of E-cadherin in luteinizing granulosa cells in vitro was assessed by RT-PCR and western blot analysis. Immunofluorescence results indicate that E-cadherin is expressed in the theca-interstial cells surrounding preovulatory follicles. N-cadherin expression is prominent in the membrana granulosa of these follicles. The initiation of luteinization with hCG leads to a decreased expression of N-cadherin in the membrana granulosa, whereas expression of E-cadherin starts within the luteinizing follicle. Both cadherins are prominently expressed in the fully formed corpus luteum at 72 h after hCG treatment. Immunofluorescence results revealed that the patterns of E- and N-cadherin expression in the gonadotrophin-treated rats were similar to those of the cyclic adult rats. Western blot analysis reflected similar changes for N-cadherin in the ovaries of both the cyclic adults and gonadotrophin-treated rats; however, they were different in E-cadherin expression. The expression of E-cadherin mRNA and protein was induced in vitro in luteinized granulosa cells. These results support the hypothesis that modulation of cadherin expression is an integral component of remodelling processes, including corpus luteum formation, in the ovary. The results also indicate that expression of E- and N-cadherin in granulosa-lutein cells appear to be under hormonal control.

Estradiol stimulates cadherin expression in rat granulosa cells.

We have investigated the ability of hormones to modulate cadherin expression by differentiating cells. Immunocytochemical and immunoblot techniques were employed to analyze the effects of estradiol and follicle-stimulating hormone on cadherin expression in rat granulosa cells. Estradiol was shown to stimulate the expression of cadherin by these cells. This is the first report of a hormone regulating the levels of cadherin in differentiating cells.

Pattern of human chorionic gonadotropin binding in the polycystic ovary.

The histologic evolution of polycystic ovaries in the estradiol valerate-treated rat coincides with the development of a unique plasma pattern of luteinizing hormone. To assess the role of luteinizing hormone in polycystic ovaries, it is necessary to evaluate the luteinizing hormone sensitivity of the specific tissues in the polycystic ovary. Therefore, we examined the pattern of luteinizing hormone binding sites in polycystic ovaries. Rats at 4 or 8 weeks after estradiol valerate treatment each received an intrajugular injection of iodine 125-labeled human chorionic gonadotropin. Some rats also received a 1000-fold excess of unlabeled human chorionic gonadotropin in the same injection. Ovaries were prepared for autoradiography. Dense accumulations of grains occurred over the theca of normal and atretic secondary follicles in all ovaries and over clusters of secondary interstitial cells. The iodine label was variable over the typically hypertrophied theca of precystic follicles. The theca of definitive cysts showed little or no label. These results indicate that cyst formation coincides with the loss of luteinizing hormone/human chorionic gonadotropin binding to the affected follicles.