Riaz Farookhi

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.

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 obt...

B-Type Natriuretic Peptide Receptor Expression and Activity Are Hormonally Regulated in Rat Ovarian Cells1

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 bi...

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 levelsin vivo. Immature mice were injected with either progesterone, testosterone, dihydrotestosterone, 17-β estradiol or 17-α estradiol. Only 17-β estradiol caused a rapid and significant increase in the ovarian E-cadherin mRNA levels. We speculate that this steriod is a key regulator of E-cadherin-mediated epithelial cell interactionsin 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.

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.

Developmental expression and distribution of N- and E-cadherin in the rat ovary.

Abstract The calcium-dependent cell adhesion molecules, cadherins, regulate intercellular junction formation, cell sorting, and the establishment of cell polarity. Their important role in tissue remodeling suggests an involvement in ovarian cellular rearrangements throughout postnatal development. The ovary has a complex topology, and the ovarian follicle undergoes significant cellular rearrangements during its development. Cadherins have been detected previously in whole ovaries and in ovarian cells and cell lines with some immunolocalization in fetal and adult ovaries. This study examines the expression and localization of N- and E-cadherin throughout prepubertal ovarian and follicular development in the rat. We analyzed ovarian cadherin expression in rats from Day 19–20 of gestation to 25 days postpartum, during which follicle formation and folliculogenesis are the dominant ovarian events. Reverse transcriptase polymerase chain reaction detected N- and E-cadherin mRNA expression in the ovaries at all the ages examined. Semiquantification of Western blots of whole ovary extracts confirmed the presence of ovarian N- and E-cadherin protein at all ages with both showing peak expression at 7 days of age. Immunostaining revealed N- and E-cadherin expression in follicular and extrafollicular cell types, but only E-cadherin showed follicle-stage-dependent expression. The changes in cadherin expression, concurrent with ovarian growth and folliculogenesis, suggest a function for cadherins in the morphological and functional development of the prepubertal rat ovary.

Impaired Progesterone Production in Nr5a2+/− Mice Leads to a Reduction in Female Reproductive Function

Abstract NR5A2 is an orphan nuclear receptor involved in cholesterol metabolism and embryogenesis. The high level of expression of NR5A2 in the ovary and its involvement in the regulation of steroidogenic gene expression also suggest a role for this transcription factor in female reproductive function. In vivo evidence for a role for NR5A2 in fertility, however, is still lacking. In order to address this possibility, we used Nr5a2+/− mice to demonstrate that heterozygosity for a null mutation of Nr5a2 leads to a decreased fertility in females. Our results indicate that although Nr5a2+/− mice display normal follicular development, ovulation, and estrogen production, they exhibit altered luteal function. More specifically, we show that the reduced reproductive ability of Nr5a2+/− females arises from a reduction in circulating progesterone concentrations and can be rescued by exogenous progesterone supplementation. This study therefore provides the first in vivo evidence for a role of NR5A2 in reproductive function and steroidogenesis.

Cadherins, steroids and cancer

The cadherins are a family of calcium-dependent cell adhesion molecules which are thought to be key regulators of morphogenesis. This review contains a discussion of the structure, function and regulation of these cell adhesion molecules. In particular, we discuss recent studies that demonstrate the ability of steroids to modulate cadherin levelsin vivo. We speculate that steroids and estrogenic organochlorines exert their diverse morphoregulatory actions on tissues by altering cadherin levels.

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.

LRP5 knockdown: effect on prostate cancer invasion growth and skeletal metastasis in vitro and in vivo.

Prostate cancer (PCa) is a common hormone‐dependent malignancy associated with the development of skeletal metastases. This is due to the increased expression of a number of growth factors, cytokines, and proteases which collectively drive the metastatic cascade in general and increased propensity to develop skeletal metastasis in particular. While a number of signaling pathways have been implicated in PCa progression, the highly complex wnt/β‐catenin pathway is unique due to its ability to regulate gene expression, cell invasion, migration, survival, proliferation, and differentiation to contribute in the initiation and progression of PCa. Members of the wnt family bind to the Frizzle proteins or lipoprotein‐related receptor proteins 5, 6 (LRP5, ‐6) to activate this key pathway. In the current study, we have investigated the role of wnt/β‐catenin pathway in PCa progression, skeletal metastasis, and gene expression using the dominant negative plasmid of LRP5 (DN‐LRP5) and human PCa cells PC‐3. Inactivation of LRP5 resulted in mesenchymal to epithelial shift, lack of translocation of β‐catenin to cell surface, increased tumor cell proliferation, decreased colony formation, migration and invasion in vitro. These effects were attributed to decreased expression of pro‐invasive and pro‐metastatic genes. In in vivo studies, PC‐3‐DN‐LRP5 cells developed significantly smaller tumors and a marked decrease in skeletal lesion area and number as determined by X‐ray, micro (μ) CT and histological analysis. Collectively results from these studies demonstrate the dominant role of this key pathway in PCa growth and skeletal metastasis and its potential as a viable therapeutic target.