A functional bone morphogenetic protein system in the ovary.
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Bone morphogenetic proteins (BMPs) comprise a large group of polypeptides in the transforming growth factor beta superfamily with essential physiological functions in morphogenesis and organogenesis in both vertebrates and invertebrates. At present, the role of BMPs in the reproductive system of any species is poorly understood. Here, we have established the existence of a functional BMP system in the ovary, replete with ligand, receptor, and novel cellular functions. In situ hybridization histochemistry identified strong mRNA labeling for BMP-4 and -7 in the theca cells and BMP receptor types IA, IB, and II in the granulosa cells and oocytes of most follicles in ovaries of normal cycling rats. To explore the paracrine function of this BMP system, we examined the effects of recombinant BMP-4 and -7 on FSH (follicle-stimulating hormone)-induced rat granulosa cytodifferentiation in serum-free medium. Both BMP-4 and -7 regulated FSH action in positive and negative ways. Specifically, physiological concentrations of the BMPs enhanced and attenuated the stimulatory action of FSH on estradiol and progesterone production, respectively. These effects were dose- and time-dependent. Furthermore, the BMPs increased granulosa cell sensitivity to FSH. Thus, BMPs have now been identified as molecules that differentially regulate FSH-dependent estradiol and progesterone production in a way that reflects steroidogenesis during the normal estrous cycle. As such, it can be hypothesized that BMPs might be the long-sought "luteinization inhibitor" in Graafian follicles during their growth and development. (+info)
Human granulosa cells express integrin alpha2 and collagen type IV: possible involvement of collagen type IV in granulosa cell luteinization.
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Previously, it has been shown that integrin alpha6beta1 expressed on human granulosa cells regulates luteinization in co-operation with its ligand, laminin. In this study, integrin alpha2 was immunohistochemically demonstrated to be expressed on granulosa and large luteal cells. It was also detected on luteinizing theca interna cells after ovulation. Immunoreactive collagen type IV, which is one of the ligands for integrin alpha2beta1, was detected around granulosa cells in the pre-ovulatory follicles and its expression was rapidly increased during ovulation. By flow cytometry, collagen type IV was detected on the cell surface of luteinizing granulosa cells isolated from pre-ovulatory follicles, confirming the physiological interaction between granulosa cells and collagen type IV. Collagen type IV in follicular fluid was positively related with progesterone concentration. In 4-day cultures of granulosa cells, collagen type IV in the media was significantly increased by human chorionic gonadotrophin (HCG). The progesterone production was significantly attenuated when granulosa cells were cultured on collagen type IV-coated dishes, suggesting that collagen type IV suppresses granulosa cell luteinization. These findings show that collagen type IV, a ligand for integrin alpha2beta1, is rapidly produced around luteinizing granulosa cells during ovulation, probably under the control of luteinizing hormone (LH) and suggest that collagen type IV is a new parameter and/or regulator of granulosa cell luteinization in the periovulatory phases. (+info)
Stimulation of early embryonic development in cattle by coculture with surfactant.
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PURPOSE: Our purpose was to determine the efficacy of Surfacten, a bovine pulmonary surfactant, on the maturation of in vitro bovine ova. METHODS: We used Surfacten as a supplement to the coculture media both at the onset of coculture and after cleavage in bovine ova had been determined. The controls received no Surfacten. RESULTS: The maturation rate in bovine embryos to the blastocyst stage statistically improved (P < 0.05) in the series in which Surfacten was added to the media at the onset of coculture, compared with the controls and the series in which Surfacten was added after cleavage had been determined. CONCLUSIONS: Surfacten, a commercially available surfactant which is a naturally occurring phospholipid that dramatically increases in the cervical mucus and the ampullaris of the oviduct at or near the time of ovulation, improves the maturation of bovine embryos in vitro by making the coculture medium approach the conditions found in the oviducts. (+info)
Evidence for an inverse relationship between apoptosis and inducible nitric oxide synthase expression in rat granulosa cells: a possible role of nitric oxide in ovarian follicle atresia.
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Ovarian follicle atresia is thought to be induced through apoptosis of granulosa cells. This study was designed to investigate the possible involvement of nitric oxide (NO) in granulosa cell apoptosis. In immature rat ovaries obtained 48 h after pregnant mare serum gonadotropin administration, immunohistochemistry and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL), a method to detect apoptotic cells, revealed that inducible NO synthase (iNOS) was predominantly localized in granulosa cells in most healthy immature follicles with TUNEL-negative granulosa cells. In contrast, all atretic follicles with TUNEL-positive granulosa cells were iNOS-negative whatever the developmental stage of the follicle. In cultured granulosa cells, the addition of S-nitroso-N-acetyl-DL-penicillamine (SNAP), an NO generator, directly inhibited spontaneously occurring apoptosis. These results suggest that NO produced by iNOS in granulosa cells of immature follicles may prevent ovarian follicle atresia by inhibiting granulosa cell apoptosis in an autocrine/paracrine manner. (+info)
Inhibition of salmon calcitonin on secretion of progesterone and GnRH-stimulated pituitary luteinizing hormone.
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The effects of salmon calcitonin (sCT) on the production of progesterone and secretion of luteinizing hormone (LH) were examined in female rats. Diestrous rats were intravenously injected with saline, sCT, human chorionic gonadotropin (hCG), or hCG plus sCT. Ovariectomized (Ovx) rats were injected with saline or sCT. In the in vitro experiments, granulosa cells and anterior pituitary glands (APs) were incubated with the tested drugs. Plasma LH levels of Ovx rats were reduced by sCT injection. Administration of sCT decreased the basal and hCG-stimulated progesterone release in vivo and in vitro. 8-Bromo-cAMP dose dependently increased progesterone production but did not alter the inhibitory effect of sCT. H-89 did not potentiate the inhibitory effect of sCT. Higher doses of 25-hydroxycholesterol and pregnenolone stimulated progesterone production and diminished the inhibitory effects of sCT. sCT did not decrease basal release of LH by APs, but pretreatment of sCT decreased gonadotropin-releasing hormone (GnRH)-stimulated LH secretion. These results suggested that sCT inhibits progesterone production in rats by preventing the stimulatory effect of GnRH on LH release in rat APs and acting directly on ovarian granulosa cells to decrease the activities of post-cAMP pathway and steroidogenic enzymes. (+info)
Evidence for ovarian granulosa stem cells: telomerase activity and localization of the telomerase ribonucleic acid component in bovine ovarian follicles.
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We have previously postulated that granulosa cells of developing follicles arise from a population of stem cells. Stem cells and cancer cells can divide indefinitely partly because they express telomerase. Telomerase is a ribonucleoprotein enzyme that repairs the ends of telomeres that otherwise shorten progressively upon each successive cell division. In this study we carried out cell cycle analyses and examined telomerase expression to examine our hypothesis. Preantral (60-100 microm) and small (1 mm) follicles, as well as granulosa cells from medium-sized (3 mm) and large (6-8 mm) follicles, were isolated. Cell cycle analyses and expression of Ki-67, a cell cycle-related protein, were undertaken on follicles of each size (n = 3) by flow cytometry; 12% to 16% of granulosa cells in all follicles were in the S phase, and less than 2% were in the G(2)/M phase. Telomerase activity (n = 3) was highest in the small preantral follicles, declining at the 1-mm stage and even further at the 3-mm stage. In situ hybridization histochemistry was carried out on bovine ovaries, and telomerase RNA was detected in the granulosa cells of growing follicles but not primordial follicles. Two major patterns of staining were observed in the membrana granulosa of antral follicles: staining in the middle and antral layers, and staining in the middle and basal layers. No staining was detected in oocytes. Our results strongly support our hypothesis that granulosa cells arise from a population of stem cells. (+info)
Comparison of protein synthesis patterns in mouse cumulus cells and mural granulosa cells: effects of follicle-stimulating hormone and insulin on granulosa cell differentiation in vitro.
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Successful development of mammalian oocytes requires correct interactions between developing oocytes and associated granulosa cells. Development of oocyte-granulosa cell complexes from preantral follicles in vitro does not produce oocytes competent to develop to blastocysts at the same frequency as for oocytes that develop in vivo. Addition of either FSH or insulin to cultures of oocyte-granulosa cell complexes does not improve the frequency of blastocyst development, and the combination of both insulin and FSH is deleterious. Here, high-resolution 2-dimensional PAGE (2D-PAGE) and computerized gel image analysis were used to compare patterns of protein synthesis in cumulus cells and mural granulosa cells of small antral follicles, and then to assess effects of FSH and insulin on the differentiation of oocyte-associated granulosa cells (OAGCs) in vitro. Culture of OAGCs without FSH or insulin resulted in failure to synthesize many proteins at rates characteristic of cumulus cells. Either hormone used alone caused many cumulus cell proteins that were decreased in control cultures to be synthesized at nearly normal cumulus cell rates, and also caused the synthesis of other proteins to be increased or decreased. The two hormones added together produced the greatest change in protein synthetic pattern, including overexpression or underexpression of many proteins not affected by either hormone alone. Addition of these hormones to culture media thus appeared insufficient to elicit a normal cumulus cell phenotype in OAGCs and could lead to complex changes in protein synthesis that may be deleterious to oocyte development. The high-resolution 2D-PAGE approach described here should be a valuable tool in studies on oocyte and granulosa cell development in vitro, since phenotype can be evaluated globally through the display of over 1000 newly synthesized proteins rather than relying upon the expression of just a few genes. (+info)
Leptin directly stimulates aromatase activity in human luteinized granulosa cells.
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Leptin, the obese (ob) gene product, is secreted by adipocytes and regulates appetite through interaction with hypothalamic leptin receptors. Leptin may also have a stimulatory effect on reproductive function. Furthermore, leptin receptor mRNA is expressed in the ovary, suggesting a direct effect on its function. The present study examines the direct role of leptin on the oestrogen-producing activity in human luteinized granulosa cells. The cells were obtained from in-vitro fertilization pre-ovulatory follicles, precultured for 24 h in the presence of 5% charcoal-treated serum, and incubated for 48-96 h in a serum-free medium containing recombinant human leptin, follicle stimulating hormone (FSH), and/or insulin-like growth factor-I (IGF-I). A single addition of leptin (0. 5-10 ng/ml) stimulated aromatase activity with the incubation time of up to 96 h. The addition of leptin (1 ng/ml) further augmented the stimulation by a single addition of FSH (100 ng/ml) or IGF-I (100 ng/ml), or a combination of both. A single addition of leptin (1 ng/ml) or a combination of leptin (1 ng/ml), FSH (100 ng/ml), and IGF-I (100 ng/ml) gave rise to an increase in each parameter of oestrogen-producing activity measured, i.e. P450arom mRNA level, P450arom protein level, aromatase specific activity, and the oestradiol concentration in the culture supernatant. However, the production of progesterone did not change. These results indicate that leptin stimulates oestrogen production by increasing P450arom mRNA and P450arom protein expression and, consequently, aromatase activity by its direct action on the human luteinized granulosa cells. (+info)