Regulation of MMP2 and MMP9 metalloproteinases by FSH and growth factors in bovine granulosa cells

Matrix metalloproteinases (MMP) are key enzymes involved in tissue remodeling. Within the ovary, they are believed to play a major role in ovulation, and have been linked to follicle atresia. To gain insight into the regulation of MMPs, we measured the effect of hormones and growth factors on MMP2 and MMP9 mRNA levels in non-luteinizing granulosa cells in serum-free culture. FSH and IGF1 both stimulated estradiol secretion and inhibited MMP2 and MMP9 mRNA abundance. In contrast, EGF and FGF2 both inhibited estradiol secretion but had no effect on MMP expression. At physiological doses, none of these hormones altered the proportion of dead cells. Although we cannot link MMP expression with apoptosis, the specific down regulation by the gonadotropic hormones FSH and IGF1 in vitro suggests that excess MMP2 and MMP9 expression is neither required nor desired for follicle development.     

排卵前期卵泡颗粒细胞端粒酶的表达及其影响因素

用端粒酶重复扩增酶联免疫吸附分析法（telomeric repeat amplification protocol-enzyme linked immunoadsordent assay,TRAP-ELISA）观察体外培养的大鼠排卵前期卵巢颗粒细胞中端粒酶活性的表达及其影响因素,并用放射免疫分析法（radioimmunoassay,RIA）同步测定培养液中雌二醇（estradiol,E2）、孕西阿（progesterone,P0）含量的变化及MTT（四甲基偶氮唑盐）法测定颗粒细胞增殖指数,分析颗粒细胞中端粒酶活性的表达以及端粒酶活性表达的影响因素。本实验中大鼠排卵前期卵巢颗粒细胞中有端粒酶活性表达,且在人绒毛膜促性腺激素（human chorionic gonadotropin,HCG）、卵泡刺激素（follicle-stimu1ating hormone,FSH）、二丁酰环磷腺苷（dbcAMP）及维拉帕米（verapamil）作用下活性明显升高,而在反义c-myb作用下活性明显降低。RIA测定培养液中雌激素及孕激素含量发现,在verapamil及FSH作用下E2与P0分泌量明显升高,在dbcAMP及HCG作用下分泌量无明显改变,而在反义c-myb作用下分泌量明显降低,在不同作用因素下的端粒酶活性与它相对应的E2及P0分泌量无相关性。MTT法测定显示,反义hTERT能明显抑制颗粒细胞的增殖。由此可以证实,排卵前期卵巢的颗粒细胞中表达有端粒酶活性,其活性受FSH、HCG、verapamil、dbcAMP及癌基因的影响,并且端粒酶活性与颗粒细胞增殖功能相关。     

Production and validation of a polyclonal serum against bovine FSH receptor

In ovarian granulosa cells, follicle-stimulating hormone (FSH) regulates the proliferation and differentiation events required for follicular growth and oocyte maturation. FSH actions are mediated exclusively through the FSH receptor (FSHR). In cattle, the FSHR gene expression pattern during folliculogenesis and the implications of this receptor in reproductive disorders have been extensively studied. However, the limited availability of specific antibodies against bovine FSHR has restricted FSHR protein analysis. In the present study, we developed an anti-FSHR polyclonal serum by using a 14-kDa peptide conjugated to maltose binding protein. The antiserum obtained was characterized by western blot of protein extracts from bovine follicles, BGC-1 cells and primary cultures of granulosa cells stimulated with testosterone. Also, the blocking effect of serum on estradiol secretion and cell viability after gonadotropin stimulus was characterized in a functional in vitro assay. A 76-kDa protein, consistent with the predicted molecular size of full-length FSHR, was detected in ovarian tissue. Besides, two immunoreactive bands of 60-kDa and 30-kDa (only in cultured cells) were detected. These bands would be related to some of the isoforms of the receptor. Therefore, immunohistochemical assays allowed detecting FSHR in the cytoplasm of granulosa cells and an increase in its expression as follicles progressed from primordial to large preantral follicles. These results suggest that the anti-FSHR serum here developed has good reactivity and specificity against the native FSHR. Therefore, this antiserum may serve as a valuable tool for future studies of the biological function of FSHR in physiological conditions as well as of the molecular mechanism and functional involvement of FSHR in reproductive disorders.     

Regulated expression of sterol carrier protein 2 in the ovary: a key role for cyclic AMP.

Sterol carrier protein 2 (SCP2) is believed to play an important role in the intracellular movement of cholesterol in steroidogenic cells. We examined the distribution of SCP2 gene expression in the rat ovary and the role of gonadotropins and cyclic AMP in the regulation of SCP2 mRNA levels. In situ hybridization revealed that the most steroidogenically active ovarian compartments (e.g., corpora lutea and theca cells) contain significant amounts of SCP2 mRNA whereas granulosa cells have modest levels. Gonadotropins, which promote follicular growth and luteinization, increased the ovarian content of SCP2 mRNA as assessed by Northern blotting along with increases in cytochrome P450scc mRNA. Using steroidogenic transformed rat granulosa cells (Grs-21), a cyclic AMP analogue (8-Br-cAMP) was found to increase SCP2 mRNA and protein levels within 24 h of treatment. P450scc mRNA was also induced whereas actin mRNA levels were not affected. The 8-Br-cAMP stimulation of SCP2 mRNA accumulation was completely inhibited by actinomycin D and cycloheximide. The cyclic AMP analogue also increased SCP2 mRNA levels in a non-steroid hormone producing transformed rat granulosa cell line Gs-8. We conclude that SCP2 gene expression in the ovary is correlated with the state of differentiation of granulosa cells. Gonadotropic hormones which stimulate luteinization of the cells increase SCP2 gene expression. These actions of gonadotropins appear to be mediated at least in part by cyclic AMP through a mechanism requiring ongoing RNA and protein synthesis. However, SCP2 gene expression is not obligatorily coupled to steroidogenic activity, as cyclic AMP analogues can increase SCP2 mRNA in a line of transformed ovarian granulosa cells incapable of synthesizing hormones.     

Hormonal regulation of ovarian cellular proliferation

The steroid hormone estradiol, and the glycoprotein hormones follicle-stimulating hormone (FSH) and luteinizing hormone (LH), are known to be essential for the growth and differentiation of follicles in the ovary. The present study was conducted to determine quantitatively the effects of estradiol, FSH and LH on proliferation of different ovarian cell types (granulosa and theca cells). The immature female hypophysectomized rate sequentially primed with estradiol, FSH and LH was used as the experimental model. Proliferation was assessed by examining changes in total DNA, incorporation of 3H-thymidine into DNA and labeling index in specific cell types. Estradiol and FSH each acted on follicles at different stages of development to stimulate proliferative activity of both granulosa and theca cells. Continued administration of either hormone caused a decrease in the proliferative activity of both cell types. These observations have been interpreted to indicate that estradiol and FSH can each alter the length of the specific phases of the cell cycle. A luteinizing dose of LH caused a cessation of proliferation in luteinizing granulosa cells while stimulating a limited proliferation of theca cells. Absence of the appropriate hormonal stimulus caused both granulosa and theca cells to stop proliferating and the follicles to undergo atresia. These results indicate that, depending upon the state of differentiation of granulosa and theca cells, estradiol, FSH and LH can stimulate or inhibit the ability of these cells to proliferate.     

Estrous cycle-regulated expression of CYP1B1 mRNA in the rat ovary

CYP1B1, a member of the cytochrome p450 superfamily, is expressed constitutively in the steroidogenic tissues of mammals and is inducible by peptide hormones, cAMP and aromatic hydrocarbon receptor (AHR) ligands. The mechanism of induction of this cytochrome p450 is similar to that for CYP1A1, i.e. through the aromatic hydrocarbon receptor (AHR) signaling pathway. We have recently reported that CYP1B1, but not CYP1A1, is expressed in rat granulosa cells (GC) in the absence of any external stimulus. The induction of CYP1B1 mRNA in rat GC by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in vitro was followed by an increase in AHR and estrogen receptor (ER-beta) RNA levels. Estrous cycle-dependent expression of AHR, AHR-nuclear translocator (ARNT) and ER-mRNAs in the rat ovary was reported. We suggest that CYP1B1 may play a major role in the regulation of rat ovarian function/cycle but until now this has been unexplored experimentally. The present study was therefore aimed at examining the expression of CYP1A1, CYP1B1 and ER-mRNA in rat ovarian tissues throughout the estrous cycle to establish any correlation in the expressions of these mRNAs in rat ovary. Total RNA was extracted from the ovary and liver of cycling adult rats and the mRNAs were analyzed using relative RT-PCR with gene-specific primers for the target mRNA and for RPL 19 or S16 primers as an internal control. The results indicated that in the ovary, CYP1B1 mRNA increased significantly on the evening of proestrus and dramatically decreased on the morning of estrus, while ER-mRNA remained unaltered throughout the estrous cycle. CYP1A1 mRNA in the ovary and both CYP1A1 and CYP1B1 mRNAs in the liver were undetectable. That the sudden decrease of ovarian CYP1B1 mRNA on the morning of estrus was not an effect of the LH surge was verified in vitro using our short-term GC culture model. GC prepared from rats super-stimulated with equine chorionic gonadotropin (eCG) were cultured for 6 h with or without LH and TCDD. It was observed that both CYP1A1 and CYP1B1 mRNAs were induced by TCDD with no apparent effect of LH. It is suggested that the high level of CYP1B1 mRNA expression on the evening of proestrus in rat ovary might be involved in metabolism of estrogens to catecholestrogen (a known effect of CYP1B1), and that expression is unaffected in GC by LH.     

Brca1 Mutations Enhance Mouse Reproductive Functions by Increasing Responsiveness to Male-Derived Scent

We compared the gene expression profiles of ovarian granulosa cells harboring either mutant or wild type Brca1 to follow up on our earlier observation that absence of a functional Brca1 in these important regulators of menstrual/estrous cycle progression leads to prolongation of the pre-ovulatory phase of the estrous cycle and to increased basal levels of circulating estradiol. Here we show that ovarian granulosa cells from mice carrying a conditional Brca1 gene knockout express substantially higher levels of olfactory receptor mRNA than granulosa cells from wild type littermates. This led us to hypothesize that reproductive functions in mutant female mice might be more sensitive to male-derived scent than in wild type female mice. Indeed, it is well established that isolation from males leads to complete cessation of mouse estrous cycle activity while exposure to olfactory receptor ligands present in male urine leads to resumption of such activity. We found that Brca1 ^-/- female mice rendered anovulatory by unisexual isolation resumed ovulatory activity more rapidly than their wild type littermates when exposed to bedding from cages where males had been housed. The prime mediator of this increased responsiveness appears to be the ovary and not olfactory neurons. This conclusion is supported by the fact that wild type mice in which endogenous ovaries had been replaced by Brca1-deficient ovarian transplants responded to male-derived scent more robustly than mutant mice in which ovaries had been replaced by wild type ovarian transplants. Our findings not only have important implications for our understanding of the influence of olfactory signals on reproductive functions, but also provide insights into mechanisms whereby genetic risk factors for breast and extra uterine Müllerian carcinomas may influence menstrual activity in human, which is itself an independent risk factor for these cancers.     

Regulation of gonadal androgen secretion

The various mechanisms regulating testicular and ovarian androgen secretion are reviewed. Testicular androgen secretion is controlled by luteinizing hormone (LH) and follicle stimulating hormone (FSH), which influence the Leydig cell response to the LH. The contribution of prolactin, growth hormone and thyroid hormones to the Leydig cell function is discussed. The ovarian androgen secretion is regulated in a very similar fashion as the Leydig cell of testis. Prolactin, however, has an inhibitory effect on androgen secretion in the ovary. The intratesticular action of androgens is linked to spermatogenesis. Sertoli cells, by producing the androgen-binding protein, contribute to the intratubular androgen concentration. Inhibin production of the Sertoli cell is stimulated by androgens. In the ovary, androgens produced by the theca interna are used as precursors for the aromatization of estradiol, which stimulates together with FSH the mitosis of granulosa cells. The feedback control of androgen secretion is complicated, as the direct feedback mechanisms are joined by indirect feedback regulations like the peptide inhibin, which can be stimulated by androgens. Intragonadal mechanisms regulating androgen production are the cybernins for testicles and ovaries. In the testicle, estrogens from the Sertoli cells regulate the Leydig cell testosterone biosynthesis. In the ovary, nonaromatizable androgens are potent inhibitors of the aromatization activity in the granulosa cell. A peptide with a FSH receptor binding inhibiting activity is found in male and female gonads. Finally, LH-RH-like peptides have been found in the testicle, which are capable of inhibiting steroidogenesis. These gonadocrinins are similarly produced in granulosa cells of the ovary.     

Levels of alpha-inhibin in aging female mice.

alpha-inhibin was immunocytochemically localized in granulosa cells of different stages of developing follicles, freshly formed corpora lutea, and scattered interstitial cells (pigmented or ceroid cells) in ovaries of 6-, 14-, and 23-25-mo-old C57BL/6NNia mice. Developing follicles exhibited the greatest amount of staining. Quantitation of the stain using an image analysis system indicated the staining intensity within ovarian follicles of 14-mo-old mice was greater than that in 23-25-mo-old mice. The levels of plasma alpha-inhibin and estradiol (E2) decreased with age. The number of follicles present in ovaries of middle-aged mice was comparable to those of 6-mo-old mice, yet plasma levels of FSH were significantly higher than those of 6-mo-old mice. This may be due to an age-related loss in the sensitivity of the hypothalamus and/or pituitary of middle-aged mice to ovarian hormones. In contrast, ovaries of 23-25-mo-old mice contained few antral follicles and consequently produced little alpha-inhibin. There appeared to be little negative feedback regulation of FSH secretion in 23-25-mo-old mice as a result of age-related ovarian impairments. This study supports an earlier hypothesis from our laboratory [Biol Reprod 1985; 32:989-997] that the primary defect(s) limiting age-related reproductive performance in mice appears to reside within the hypothalamo-hypophyseal axis, whereas secondary defects arise from the ovary.     

Initiation of the expression of peroxisome proliferator - activated receptor gamma (PPAR gamma) in the rat ovary and the role of FSH

PPARgamma is highly expressed in granulosa cells by 23 days post-partum (pp) and is down-regulated in response to the LH surge. We tested the hypothesis that high levels of FSH during the neonatal period trigger the expression of PPARgamma. To determine when PPARgamma expression is initiated, ovaries were collected from neonatal rats. Messenger RNA for PPARgamma was undetectable on day 1, low from days 5-14, and increased by day 19 pp (p < 0.05). PPARgamma was detected in select granulosa cells in primary/early secondary follicles. Messenger RNA for the FSH receptor was detected as early as day 1 and remained steady throughout day 19 pp. The FSH receptor was detected by immunoblot analysis in ovaries collected 1, 2, and 5-9 days pp. In a subsequent experiment, neonatal rats were treated with acyline (GnRH antagonist) which significantly reduced FSH (p < 0.05) but not levels of mRNA for PPARgamma. The role of FSH in the induction of PPARgamma expression was further assessed in ovarian tissue from FORKO mice. Both mRNA and protein for PPARgamma were identified in ovarian tissue from FORKO mice. In summary, the FSH/FSH receptor system is present in granulosa cells prior to the onset of expression of PPARgamma. Reducing FSH during the neonatal period, or the ability to respond to FSH, did not decrease expression of mRNA for PPARgamma. These data indicate that FSH is not a primary factor initiating the expression of PPARgamma and that other agents play a role in activating its expression in the ovary.     

Gonadotropin regulation of RIP140 messenger ribonucleic acid expression in the rat ovary

Female mice null for receptor-interacting protein 140 (RIP140) are infertile because of the failure of follicle rupture. The present study examined gonadotropin regulation of RIP140 expression in immature rat ovary. Treatment with PMSG increased ovarian RIP140 mRNA and protein levels. In contrast, hCG treatment rapidly inhibited RIP140 mRNA and protein levels within 1-3 h. RIP140 mRNA was detected in theca cells of growing follicles in untreated ovary and in granulosa cells in PMSG-treated ovary. Interestingly, hCG treatment reduced RIP140 mRNA levels in granulosa cells of preovulatory follicles, but not of growing follicles. Neither treatment of immature rats with diethylstilbestrol in vivo nor of immature granulosa cells with FSH in vitro affected RIP140 mRNA levels. Treatment of immature granulosa cells with 17beta-estradiol in vitro, however, stimulated RIP140 mRNA levels. In cultured preovulatory granulosa cells, RIP140 mRNA levels were stimulated at 1 h and then declined to below control levels by 3 h after LH treatment. Treatment with MDL-12,330A, an inhibitor of adenylate cyclase, or chelerythrine chloride, an inhibitor of protein kinase C (PKC), inhibited LH-stimulated RIP140 gene expression. Furthermore, forskolin or TPA treatment for 1 h mimicked the stimulatory action of LH, indicating the involvement of both adenylate cyclase and PKC pathways. These results demonstrate the stimulation by PMSG and inhibition by hCG of RIP140 expression in granulosa cells of preovulatory follicles in the rat ovary.     

Autoradiographic localization of aromatic hydrocarbon receptor (AHR) in rhesus monkey ovary

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is the most toxic congener of a large class of manmade pollutants that persist in the environment. TCDD exerts its toxic effects, in part, by binding to its receptor known as the aromatic hydrocarbon receptor (AHR). TCDD is estrogen modulatory and in some systems its receptor associates directly with estrogen receptors via co-activator molecules. TCDD inhibits steroid synthesis in human ovarian granulosa cells and AHR is found in these cells. We have previously shown that AHR is found in whole rhesus monkey ovary, but have yet to establish its location. In the present study, we set out to show that radiolabeled TCDD binds to monkey ovarian follicles and that this binding is receptor mediated. Ovaries from Macaca mulatta were sectioned on a cryostat at 10 micro m; and sections were incubated with either control vehicle, (3)H-TCDD, or (3)H-TCDD plus alpha-naphthoflavone (ANF), a known receptor-blocking agent. Here, we show for the first time specific binding of TCDD to the granulosa cells of antral follicles and other regions of the rhesus monkey ovary. Our data indicate a 60-fold increase in binding with (3)H-TCDD over that of control, and that this binding is reduced to the levels seen in controls with the addition of the competitive antagonist ANF. These findings support the hypothesis that TCDD directly affects primate ovarian function via the AHR.     

Spatiotemporal expression of epidermal growth factor receptor messenger RNA and protein in the hamster ovary: follicle stage-specific differential modulation by follicle-stimulating hormone,luteinizing hormone,estradiol, and progesterone

Spatiotemporal expression, endocrine regulation, and activation of epidermal growth factor receptor (EGFR) in the hamster ovary were evaluated by immunofluorescence and in situ hybridization localization. Whereas granulosa cells (GC) of primordial through large preantral (stage 6, 7-8 layers GC) follicles had low immunoreactivity, granulosa cells of antral follicles, theca, and interstitial cells had intense EGFR immunoreactivity. EGFR expression in GC of primordial and small preantral follicles increased progressively from estrous through proestrous, but a significant increase occurred in mural GC of antral follicles following the gonadotropin surge. Interstitial cells around small preantral follicles had strong immunofluorescence, and the intensity increased significantly in fully differentiated thecal cells. Distinct EGFR protein was localized in the nucleus of the oocytes and granulosa cells. FSH significantly stimulated EGFR expression in the GC, especially the mural GC, theca, and interstitial cells in hypophysectomized hamster. Estrogen stimulated EGFR expression in preantral GC as well as in interstitial cells. Progesterone and hCG effect was limited to theca and interstitial cells. EGFR expression correlated well with EGFR activation following endogenous or exogenous gonadotropin exposure. Receptor mRNA expression closely followed the protein expression, with increased mRNA expression in mural GC of antral follicles. These results suggest that low levels of EGF signal as a consequence of low levels of receptors in preantral GC may be critical for cell proliferation, but higher receptor density may evoke increased signal intensity due to activation of other intracellular signal pathways, which activate cellular processes related to granulosa, theca, and interstitial cell differentiation. The spatiotemporal cell type and follicle stage-specific expression of receptor mRNA and protein and EGFR activation is critically regulated by gonadotropins and ovarian steroids, primarily estradiol.