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At least 19 recordsLinked to original sources

PubMed1998

Thecal cell-granulosa cell interactions involve a positive feedback loop among keratinocyte growth factor, hepatocyte growth factor, and Kit ligand during ovarian follicular development.

Interactions between mesenchymal-derived thecal cells and epithelial-derived granulosa cells are essential for follicular development in the ovary. These mesenchymal-epithelial cell interactions are in part mediated by keratinocyte growth factor (KGF), hepatocyte growth factor (HGF), and Kit ligand (KL). This study investigates the hypothesis that thecal cell-derived growth factors (e.g. KGF and HGF) regulate granulosa cell function, and granulosa cell-derived growth factors (e.g. KL) regulate thecal cell function. Gonadotropin regulation of this cell-cell interaction is also examined. Sensitive quantitative RT-PCR assays were used to analyze gene expression of KGF, HGF, and KL in the ovary. Thecal cell-derived KGF and HGF stimulated KL expression in bovine granulosa cells. Granulosa cell-derived KL stimulated KGF and HGF expression in bovine thecal cells. These results suggest that thecal and granulosa cells interact in a positive feedback loop mediated by KGF, HGF, and KL. Previous studies have suggested that gonadotropins (i.e. FSH and LH) regulate locally produced growth factor expression in the ovary. Treatment of bovine granulosa cells with FSH and hCG (a LH agonist) directly stimulated KL expression. The LH agonist hCG was also found to stimulate both KGF and HGF expression in thecal cells. The actions of gonadotropins on follicular development may in part be indirectly regulated by KL, KGF, and HGF expression. A novel positive feedback loop was identified between thecal cells and granulosa cells that is mediated by KL, KGF, and HGF. Thecal cell-derived KGF and HGF can stimulate granulosa cell-derived KL expression, and KL, in turn, can stimulate thecal cell-derived KGF and HGF expression. Combined observations support the hypothesis that mesenchymal-epithelial cell interactions between thecal and granulosa cells can play a significant role during ovarian follicular development and mediate gonadotropin actions.

Animals↗

PubMed1990

Estrogen regulation of thecal cell steroidogenesis and differentiation: thecal cell-granulosa cell interactions.

Estrogen regulation of thecal cell steroidogenesis and differentiation was investigated with cells from ovarian antral follicles. Bovine theca interna cells were isolated and cultured in serum-free conditions to evaluate the effects of estradiol on thecal cell production of androstenedione, testosterone, and progesterone. Estradiol increased thecal cell androgen production throughout a 6-day culture period; however, the basal and stimulated levels of androgen production diminished after day 3 of culture. Androstenedione accumulation was approximately 10-fold greater than that of testosterone. In contrast to the stimulatory effects that estradiol had on androgen production, estradiol suppressed progesterone production throughout the 6-day culture period. Comparison of the effects of estradiol and hCG on thecal cells from small (less than 5 mm), medium (5-10 mm), and large (greater than 10 mm) antral follicles demonstrated that estradiol stimulated androgen production to a greater extent than hCG with cells from all of these stages of follicle development. Estradiol stimulation of androstenedione was greater in theca from small follicles than in theca from medium or large follicles. In contrast, suppressive effects of estradiol on progesterone were most apparent on thecal cells from medium and large follicles and less apparent on theca from small follicles. Estradiol stimulated androstenedione production in a dose-dependent fashion, with a minimum effective concentration of 10(-9) M and a maximum effective concentration of 10(-7)-10(-6) M. Concentrations greater than 10(-6) M estradiol resulted in a decline in the stimulatory response and may be important in the preovulatory follicle to suppress thecal cell androgen production and initiate the process of luteinization. Progesterone production was slightly stimulated by 10(-9) M estradiol, whereas higher concentrations (10(-7)-5 x 10(-6) M) resulted in a dose-dependent suppression of progesterone production. Interestingly, combined treatment of thecal cells with estradiol and hCG resulted in a greater than additive stimulation of androstenedione production, and estradiol decreased the ability of hCG to stimulate progesterone production. Observations demonstrate that estradiol can dramatically alter thecal cell production of steroids and support a hypothesis that steroid-mediated interactions between granulosa and thecal cells play an important role in regulating cellular function within follicles. The data provide evidence that a local feedback loop may exist in ovarian follicles, where androgens produced by thecal cells are used as a substrate for granulosa cell aromatization into estrogens, which, in turn, may feed back to stimulate thecal cell production of androgens.(ABSTRACT TRUNCATED AT 400 WORDS)

Androstenedione↗

PubMed2005

Effects of gonadotrophin-releasing hormone agonists on apoptosis of granulosa cells.

Granulosa cells are known to contribute to maturation of oocytes, and most of the growth factors exert their action via granulosa cells. It has been established that granulosa cell death during follicular atresia and luteolysis results from apoptosis. However, the precise mechanistic pathways of granulosa cell apoptosis have not yet been defined. In this study, we determined the proportions of apoptosis in granulosa cells treated with two kinds of gonadotrophin-releasing hormone agonists (GnRHa): buserelin and leuprorelin depot. The incidences of DNA fragmentation of human granulosa cells treated with buserelin and leuprorelin were 54.33% and 39.02%, respectively. The proportions of apoptotic bodies were 6.04% and 4.29%, respectively. There was a significant difference in the proportions of DNA fragmentation between the two kinds of GnRHa-treated granulosa cells. The apoptosis pathway and associated protein expression in granulosa cells treated with GnRHa were also determined. The Bax molecule, a pro-apoptosis protein, was expressed in granulosa cells undergoing apoptosis. In contrast, Bcl-2, an anti-apoptosis protein, could not be detected in the same group of granulosa cells. The distribution of cytochrome c determined via immunostaining showed a diffuse pattern, which most likely indicated that cytochrome c was translocated from mitochondria into the cytoplasm. Western blotting showed the expressions of caspase-9 and caspase-3 in patients' granulosa cells. The GnRHa effects on granulosa cells indicated a higher incidence of DNA fragmentation and apoptotic bodies in the buserelin-treated than in the leuprorelin depot-treated group. The granulosa cells go through the mitochondria-dependent apoptosis pathway; the indicated pro-apoptosis protein Bax was expressed and induced cytochrome c release from mitochondria, which then activated caspase-9 and caspase-3 until cell death occurred.

Adult↗

PubMed1989

Use of low-density and high-density lipoproteins in undifferentiated porcine granulosa cells.

Granulosa cells aspirated from medium-sized porcine ovarian follicles (3-5 mm) in short-term incubation responded to the addition of both low-density lipoprotein (LDL) and high-density lipoprotein (HDL) with increased accumulation of progesterone. HDL was more potent than LDL in enhancing progesterone secretion. When granulosa cells were cultured under serum-free conditions for 72 h, HDL but not LDL exhibited a dose-dependent enhancement of progesterone secretion. Addition of insulin to the cells greatly potentiated the stimulatory effect of LDL on progesterone accumulation, while the response to HDL was only slightly increased. Granulosa cells in culture degraded LDL but not HDL. Addition of insulin enhanced LDL degradation. Exposure of cells in culture to chloroquine, an inhibitor of lysosomal function, completely blocked LDL degradation and LDL-induced stimulation of steroidogenesis. The stimulatory effect of HDL was not affected by chloroquine. We interpret these findings to indicate that granulosa cells derive cholesterol from LDL by means of lysosomal degradation, which is not required for use of cholesterol from HDL. Monensin, a carboxylic ionophore that interrupts recycling of LDL receptors, prevented LDL-enhanced progesterone accumulation but not HDL-induced stimulation. This provides evidence that HDL-induced stimulation of steroidogenesis does not involve LDL receptors. We conclude that HDL present in follicular fluid is capable of providing cholesterol to granulosa cells for steroidogenesis. The stimulatory effect of HDL is not due to the presence of apoprotein E, an HDL component that binds to the LDL receptor. A unique HDL pathway that does not involve LDL receptors and lysosomal degradation may operate in porcine granulosa cells.

Animals↗

PubMed1992

Structure-function relationships during differentiation of normal and oncogene-transformed granulosa cells.

Granulosa cells, which nurse the oocyte, luteinize in response to the preovulatory surge of gonadotropins and become a major producer of progesterone during the normal estrous cycle and pregnancy. This process is characterized by a dramatic change in inter- and intracellular organization and modulation of gene expression that leads to enhanced steroidogenesis. Culturing of human, porcine, and rat cells has permitted detailed studies concerning the microenvironment (hormones, growth factors, and extracellular matrices) that control steroidogenesis in a coordinated fashion both in vivo and in vitro. Reorganization of the cytoskeleton--mainly characterized by down-regulation of the actin network, which is associated with changes in cell contacts and intercellular communication--seems to be a prerequisite for up-regulation of the steroidogenic enzymes. These processes can be investigated now in more detail due to the transfection of rat granulosa cells with specific oncogenes, which leads to their immortalization, concomitantly with the preservation of their capacity for inducible steroidogenesis. The effect of oncogene expression on granulosa cell differentiation suggests that different members of the ras oncogene superfamily may be involved in controlling development and luteinization of both normal and transformed granulosa cells. The identification of the specific forms of the ras protooncogene that may be involved in the differentiation of primary granulosa cells remains a challenging objective.

Animals↗

PubMed2003

Differential expression of GRK isoforms in nonmalignant and malignant human granulosa cells.

Granulosa cell tumors are serious ovarian neoplasms that can occur in women of all ages. While there have been numerous attempts to understand the cause of these malignancies, the pathogenesis of granulosa cell tumors (GCTs) still remains largely unknown. G-protein coupled receptor kinases (GRKs) are important regulators of signal transduction through the process of receptor desensitization and internalization. Receptors that are regulated by GRKs are members of the large family of seven-transmembrane receptors and include the follicle stimulating hormone receptor (FSHR). In granulosa cells, the FSH signaling system is responsible for cell proliferation, differentiation, and steroidogenesis. In the studies presented, we examined GRK mRNA and protein expression in nonmalignant human granulosa cells, in KGN cells, a human GCT cell line, and in a panel of human GCT samples. The KGN tumor cells express significantly less GRK4 alpha/beta protein and higher levels of GRK2 and GRK4 gamma/delta protein as compared to nonmalignant human granulosa cells. In human GCT samples, GRK4 alpha/beta protein was detected in 3 of the 13 tumor samples, whereas gamma/delta proteins expression was detected in all samples. These findings suggest that GRK protein expression is altered in GCTs and may be involved in the pathogenesis of these tumors.

Blotting, Western↗

PubMed1999

Regulation of 11beta-hydroxysteroid dehydrogenase type 1 gene expression by LH and interleukin-1beta in cultured rat granulosa cells.

Granulosa cells from preovulatory follicles show increased expression of 11beta-hydroxysteroid dehydrogenase type 1 (11betaHSD1) at the time of ovulation. As ovulation may be an inflammatory process, this may be a mechanism of local enhancement of the activity of anti-inflammatory glucocorticoids. In this study, we examined direct effects of LH, the proinflammatory cytokine, interleukin-1beta (IL-1beta), and pharmacological activators of protein kinase A (PKA) (forskolin and dibutyryl (db) cAMP) and PKC (LH-releasing hormone and phorbol 12-myristate 13-acetate (PMA)) signalling on the expression of 11betaHSD1 mRNA in vitro. Granulosa cells from immature female rat ovaries were cultured (pretreatment) in serum-free medium 199 containing recombinant human (rh) FSH (1 ng/ml) for 48 h to induce responsiveness to LH. Cell monolayers were then washed and cultured (test treatment) for a further 12 h in the presence of rhLH (0-100 ng/ml), IL-1beta (0-50 ng/ml), or both. Total RNA was extracted from granulosa cell monolayers and taken for quantitative ribonuclease protection analysis of 11betaHSD1 mRNA. The low level of 11betaHSD1 mRNA detectable in unstimulated (control) cultures was increased approximately twofold by the 48-h pretreatment with rhFSH. Subsequent exposure to rhLH (1-100 ng/ml) for a further 12 h dose-dependently increased 11betaHSD1 mRNA expression by an additional two- to threefold. Forskolin (10 microM), db-cAMP (2 mM), LH-releasing hormone (LHRH; 1 microM) and PMA (200 nM) were also stimulatory. IL-1beta (0.05-50 ng/ml) stimulated 11betaHSD1 mRNA expression in a dose-related manner, both in the absence and in the presence of rhLH (3 ng/ml). The interaction between IL-1beta (5 ng/ml) and rhLH (3 ng/ml) was additive. Co-treatment with a 50-fold excess of IL-1 receptor antagonist fully reversed the action of IL-1beta. We conclude that 11betaHSD1 mRNA expression in functionally mature granulosa cells is directly stimulated by gonadotrophins and IL-1beta in vitro, potentially involving post-receptor signalling via PKA- and PKC-mediated pathways. Thus both LH and IL-1beta may serve physiological roles in the upregulation of 11betaHSD1 gene expression by granulosa cells in ovulatory follicles.

11-beta-Hydroxysteroid Dehydrogenases↗

PubMed1982

Estimation of viability of bovine granulosa cells.

Granulosa cells harvested from non-atretic, antral follicles of cow (and pig and sheep) ovaries were incubated over glass cover slips in medium containing 20% (v/v) donor calf serum. Cell attachment to the cover slips was rapid, being in most cases complete within 3 h at 37 degrees C. There was little further change over the next 20 h. The number of bovine granulosa cells which attached to a cover slip was proportional to the volume of cell suspension added to the medium; and the amount of oestradiol secretion by attached cells in a testosterone-enriched medium increased in parallel with their number. Granulosa cells which did not attach within 3 h produced little oestradiol. There was no clear relationship between the number of nigrosin-impermeable cells in suspensions prepared from different follicles and plating efficiency. It is concluded that the 3-h attachment of granulosa cells in culture is a useful measure of the number of viable cells in a suspension and is to be preferred to less direct techniques based on dye exclusion.

Animals↗

PubMed1990

Effect of catecholamines and FSH on progesterone secretion by pig granulosa cells.

Granulosa cells were recovered from small (1-3 mm in diameter) and large (greater than 6 mm in diameter) preovulatory follicles or from follicles of early pregnant pigs (3-5 mm, Day 18). Incubation of these cells (5 x 10(5)) was carried out in a shaking water bath (40 degrees C) for 2 h with or without salbutamol (10(-5) M), isoprenaline (10(-5) M), propranolol (10(-5) M) and FSH (100 ng/ml). Isoprenaline significantly increased progesterone production (P less than 0.05) by granulosa cells of small follicles and large preovulatory follicles but not by granulosa cells of follicles from early pregnant pigs. After blocking the beta-adrenoceptor with propranolol the stimulatory effect of catecholamines was not observed. FSH alone stimulated progesterone production, particularly in granulosa cells of early pregnant pigs (P less than 0.05) but FSH plus catecholamine treatment did not have any effect on progesterone release. These results suggest that catecholamines may play a regulatory role in follicle maturation and this may differ between naturally cyclic and early pregnant animals.

Albuterol↗

PubMed2003

Inhibition of extracellular signal-regulated protein kinase-2 phosphorylation by dihydrotestosterone reduces follicle-stimulating hormone-mediated cyclin D2 messenger ribonucleic acid expression in rat granulosa cells.

Granulosa cell mitogenesis is critical for the development of normal ovarian follicles. FSH and other mitogenic stimuli play a crucial role in this process. We have shown that exposing granulosa cells to 5alpha-dihydrotestosterone (DHT) reduces forskolin-stimulated cyclin D2 mRNA expression, which leads to cell cycle arrest resulting in reduced cell proliferation. The present study investigated the signaling molecules upstream of cyclin D2 in FSH-mediated, cAMP-dependent signaling pathway that may be negatively affected by DHT, leading to inhibition of cell cycle progression. Because ERK is an important molecule in mitogenic signaling, the possible effect of DHT on its phosphorylation was examined. Granulosa cells from 3-d estradiol-primed immature rats were treated with DHT (90 ng/ml) for 24 h and subsequently stimulated with forskolin. DHT treatment reduced forskolin stimulation of ERK phosphorylation. Although DHT exposure did not affect cellular cAMP production in response to forskolin, treating the cells with DHT for 24 h significantly reduced protein kinase A activity. DHT also caused a reduction in ERK-2 phosphorylation in response to FSH similar to that seen with forskolin. Furthermore, blocking ERK phosphorylation as well as DHT treatment resulted in a reduction in FSH-stimulated cyclin D2 mRNA expression. From these results, we conclude that DHT treatment reduces the FSH-mediated ERK phosphorylation in granulosa cells, leading to reduced cyclin D2 mRNA expression that culminates in cell cycle arrest.

Androgens↗

PubMed2001

Characterization of adenylyl cyclases in cultured human granulosa cells.

Granulosa cells play an essential role in follicular development and formation of corpora lutea. Many functions of granulosa-lutein cells are controlled by activation of G protein-coupled receptors and the formation of cyclic AMP (cAMP) by adenylyl cyclase. There are at least nine mammalian adenylyl cyclase isoenzymes, which show different sensitivities towards other signalling systems. The aim of this study was to identify the types of adenylyl cyclase present in human granulosa cells and to investigate its functional regulation by G proteins, calcium and the protein kinase C and A pathways. Granulosa cells were obtained from women undergoing IVF. The cells were maintained in primary culture and they consistently expressed mRNA coding for adenylyl cyclase I, III, VI, VII and IX. The signals for adenylyl cyclase V and VIII were more variable among patients and there was no signal for adenylyl cyclase II. The expression of multiple adenylyl cyclase proteins was confirmed by immunochemistry with subtype-specific antibodies. The formation of cAMP in cultured cells was stimulated many times by hCG (EC(50) value 4.2 iu ml(-1)) and by prostaglandin E(2) (PGE(2); EC(50) = 0.75 micromol l(-1)) in a concentration-dependent manner, thus confirming the presence of receptors coupled positively to G(s). The diterpene forskolin, which stimulates all isoforms of adenylyl cyclase except for adenylyl cyclase IX, increased cAMP formation to higher levels than hCG or PGE(2). The strong stimulation by forskolin indicates that adenylyl cyclase IX is unlikely to be the major source of cyclase activity in these cells. Basal and forskolin- or PGE(2)-stimulated adenylyl cyclase activity was amplified 1.5-2.0 times by phorbol-12,13-dibutyrate, indicating that protein kinase C-sensitive enzymes (for example, adenylyl cyclase types IV, V, VI or VII) may be active in the cells. In contrast, hCG-stimulated activity was inhibited (76 +/- 6%) by phorbol ester. Stimulation of G(i) with the alpha-adrenoceptor agonist clonidine inhibited hCG-induced cyclase activity. This finding indicates that adenylyl cyclase II and IV subtypes, which are stimulated by betagamma subunits released from G(i), are not predominant. Increases in intracellular free calcium concentrations by the ionophore A23187, the calcium-ATPase inhibitor thapsigargin or by fluprostenol, a selective prostanoid FP receptor agonist, which is known to open calcium channels in granulosa cells, or removal of calcium by EGTA, had no significant effects on basal or forskolin-stimulated formation of cAMP. These results indicate that subtypes adenylyl cyclase I, III and VIII, which are activated by calcium, and adenylyl cyclase V and VI, which are inhibited by calcium, are not dominant isoforms in granulosa-lutein cells. The protein kinase A inhibitor H89 had no effects on formation of cAMP; this finding rules out the involvement of adenylyl cyclase V and VI subtypes, which are subjected to negative feedback by protein kinase A. These results indicate that adenylyl cyclase VII is the dominant functional isoenzyme in human granulosa-lutein cells.

Adenylyl Cyclases↗

PubMed1987

[Effects of phorbol ester and forskolin on steroidogenesis and protein phosphorylation in cultured rat granulosa cells].

Granulosa cells obtained from immature estradiol-treated SD rats (10 rats/experiment) were employed in elucidating the control mechanism of steroid secretion. Phorbol 12-myristate 13-acetate (PMA) inhibited estradiol production by cultured rat granulosa cells with IC50 less than 1 nM. PMA, however, stimulated small but significant increases in progesterone production in a dose-dependent manner with ED50 of 14 nM to 3.5-fold above the basal control level. These effects could not be induced by calcium ionophore A23187. Forskolin-stimulated progesterone production was inhibited by the concomitant addition of PMA with IC50 less than 1 nM. The phosphorylation of proteins by [32P] orthophosphate-labelled cells was examined by two-dimensional polyacrylamide gel electrophoresis and autoradiography. Treatment of cells with forskolin altered the intensity of 40 kDa acidic phosphoprotein compared to that of the control. On the other hand, treatment of cells with PMA altered the intensity of 78 and 32 kDa acidic phosphoproteins. These results suggest, therefore, that PMA can modulate steroidogenesis in rat granulosa cells, presumably through activation of Ca2+-activated, phospholipid-dependent protein kinase (protein kinase C).

Animals↗

PubMed1990

Specific nuclear uptake of intracellularly-produced estrogen by rat granulosa cells.

Granulosa cells of the ovarian follicle are unique in that they both synthesize steroid hormones and respond to exogenously-administered steroids. Isolated granulosa cells from ovaries of gonadotropin-primed rats were incubated in the presence of [3H]testosterone, which the cells convert to [3H]estradiol. Nuclear extracts of these cells were analyzed by high-performance liquid chromatography in a system of 40% acetonitrile. When cells were incubated in the presence of [3H]testosterone alone, a significant portion of the radioactivity present in nuclei co-eluted with authentic estradiol. The nuclear binding was considered to be specific, since 50-75% of total binding was suppressed when the incubation medium contained excess unlabeled estrogen. Moreover, when an antibody to estradiol was included in the medium, specific nuclear uptake of [3H]estradiol was not abolished, but rather was increased. Granulosa cells may, therefore, directly utilize endogenously-produced estradiol, a mechanism which may play a role in the regulation of ovarian cells.

Animals↗

PubMed1987

Effects of chondroitin-4-sulphate on ultrastructure of ovarian granulosa cells.

Granulosa cells from medium sized porcine ovarian follicles exhibit inhibition of progesterone secretion after both 3 and 6 days of incubation in the presence of 2 mg chondroitin-4-sulphate (C-4-S)/ml. After 3 days of incubation some relatively small and undifferentiated granulosa cells are present in the above cultures. Six days following initiation of incubation in the presence of 2 mg C-4-S/ml many of the larger granulosa cells taken from medium sized follicles reveal lysis and/or resorption of material (probably lipoprotein) from their originally dense staining bodies as well as other indications of stress and degeneration.

Animals↗

PubMed1981

Hormonal regulation of cytodifferentiation and intercellular communication in cultured granulosa cells.

Granulosa cells from immature hypophysectomized diethylstilbestrol-treated rats displayed pronounced intracellular and intercellular changes after 48 hr of exposure to follicle-stimulating hormone (FSH) in vitro. As determined by light and electron microscopy, most of the FSH-treated cells became highly aggregated and grew in multilayered clusters. Numerous gap junctions were seen between cells, indicating the presence of significant intercellular communication. Microvilli densely covered the surface of the hormone-stimulated cells, which contained enlarged mitochondria with convoluted cristae, characteristic of steroidogenic cells. Luteinizing hormone receptors, identified by autoradiography with 125I-labeled human chorionic gonadotropin, were mainly associated with aggregated cells, whereas single cells were usually free of the labeled hormone. Addition of a gonadotropin-releasing hormone agonist prevented the appearance of luteinizing hormone receptors and markedly impaired cyclic AMP and progesterone production, as well as the morphological changes induced by FSH. The majority of the granulosa cells grown in the absence of either hormone assumed a flattened, and smooth shape and grew primarily in monolayers. The maintenance of cellular aggregation and intercellular communication by FSH, and its inhibition by gonadotropin-releasing hormone, may play an important role in the cytodifferentiation of ovarian granulosa cells.

Animals↗

PubMed1997

Identification of G alpha s messenger ribonucleic acid splice variants in human granulosa cells.

Granulosa cells are essential for follicular development and corpus luteum formation and their functions are regulated by gonadotrophins through G protein-coupled receptors. The dominant second messenger pathway involves the stimulation of cyclic AMP formation by G alpha s-linked receptors. In this paper we have investigated the expression of G alpha s mRNA splice variants in relation to expression of G alpha s protein isoforms in granulosa cells obtained from patients undergoing in vitro fertilization. We have carried out ribonuclease protection assays using cRNA riboprobes which are capable of detecting all G alpha s mRNA isoforms as well as quantifying total amounts of G alpha s mRNA. Granulosa cells express the message for G alpha s-Large and G alpha s-Small and the presence of two distinct protein products was confirmed by immunoblotting using the antibody RM/1. Moreover, the data show that a significant fraction of G alpha s-Large and G alpha s-Small mRNAs contain an extra CAG codon. This should generate proteins with an extra serine residue, resulting in G alpha s variants with the consensus sequence of a protein kinase C phosphorylation site. These results highlight the possible interaction between different signalling pathways in the control of cAMP production and the need to investigate the relationship between G alpha s variants and different adenylyl cyclase isozymes in patients with normal and abnormal ovarian function.

Female↗

PubMed1996

Differential effects of insulin-like growth factor-I and follicle-stimulating hormone on proliferation and differentiation of bovine cumulus cells and granulosa cells.

Effects of gonadotropins and peptide growth factors on 3H-thymidine incorporation, as an indicator of proliferation of granulosa cells and cumulus cells from bovine ovarian follicles during in vitro culture, were determined. Incorporation of 3H-thymidine in cumulus cells during 24-h culture in protein-free medium was about 100-fold greater than in granulosa cells from small follicles. Small but significant (p < 0.05) increases in incorporation of 3H-thymidine in cumulus cells (2.5-fold), but not in granulosa cells, resulted from addition of FSH, LH, or epidermal growth factor to culture media. Much larger, highly significant (p < 0.01) increases in 3H-thymidine incorporation resulted from exposure of both cumulus cells (18-fold) and granulosa cells (6-fold) to insulin-like growth factor-I (IGF-I). Addition of IGF-I together with FSH resulted in additive increases in 3H-thymidine incorporation in granulosa cells over a wide range of each agonist. In contrast, addition of FSH markedly decreased the stimulatory effect of a maximally effective concentration of IGF-I on incorporation in cumulus cells. Addition of estradiol, either alone or with IGF-I, had no consistent effect on 3H-thymidine incorporation in either cumulus cells or granulosa cells. Differentiated functions of cumulus and granulosa cells, including cumulus expansion (an indicator of hyaluronic acid secretion) and progesterone secretion, were investigated under the same culture conditions. FSH was highly effective in stimulation of cumulus cell expansion, with maximal expansion occurring at the lowest concentration tested (0.04 micrograms/ml). LH was effective only at considerably higher concentrations consistent with degree of contamination with FSH. IGF-I was ineffective in inducing cumulus expansion, either alone or together with FSH. FSH and IGF-I by themselves each stimulated progesterone secretion by both cumulus and granulosa cells, with further additive stimulatory effects by the two agents in combination. On a per cell basis, cumulus cells produced considerably more progesterone than granulosa cells under both basal and hormone-stimulated conditions. The results suggest that IGF-I has a dual role in follicle regulation--that it acts additively with FSH as an autocrine regulator of granulosa cell growth and, in the absence of FSH, as a paracrine regulator of cumulus cell proliferation in small antral follicles. The effect of FSH, by inducing cumulus expansion, may terminate further cumulus cell proliferation before ovulation.

Animals↗

PubMed1991

An inducible functional peripheral benzodiazepine receptor in mitochondria of steroidogenic granulosa cells.

Granulosa cell lines, transformed by SV40 T-antigen and Ha-ras oncogene, have recently been established that can produce progesterone at levels comparable to those of highly differentiated cultures of primary granulosa cells (1-4). Here, the hypothesis that these cells contain a mitochondrial benzodiazepine receptor, and that stimulation of the receptor can trigger progesterone production in these cells, was tested. The agonist of the peripheral benzodiazepine receptor, Ro5-4864, produced a 3- to 5-fold stimulation (P less than 0.005) of progesterone production both in differentiated granulosa primary cultures and in the oncogene-transformed cell lines. Ro5-2807 (diazepam, Valium) exerts a similar effect on granulosa cell steroidogenesis while the specific agonist of central benzodiazepine receptor Ro15-4513 was without effect. The effects of Ro5-4864 or Ro5-2807 were not additive to those of gonadotropins and cAMP. Intact isolated mitochondria possessed high-affinity binding sites to [3H]-Ro5-4864 (Kd = 3.03 +/- 0.70 nM), which were enriched by 1 order of magnitude in these organelles compared to total cell homogenate. Bound Ro5-4864 could be competitively displaced with 1 microM unlabeled Ro5-4864 and Ro5-2807, but not with specific ligands of central benzodiazepine receptors Ro15-4513 and Ro15-1788. Prolonged elevation of cAMP in these cells caused a 30% (P less than 0.01) rise in the number of receptors. Mitochondria of NIH 3T3 cells contained only 30-40% (P less than 0.001) of the Ro5-4864 binding sites of mitochondria from steroidogenic cells, whereas yeast mitochondria lacked them completely. The existence of functional peripheral benzodiazepine receptors in mitochondria suggests that they may have a physiological role in the mobilization of cholesterol into mitochondria, and in elevating progesterone production in ovarian cells. The modulation of the interaction between benzodiazepine compounds and the gamma-aminobutyric acid receptor by progesterone metabolites suggests new interrelationships between peripheral and central nervous system receptors sensitive to benzodiazepines.

Animals↗