Sodium-dependent regulation of steroidogenesis in rat granulosa cells: possible involvement of a Na+/H+ antiport

The possible role of Na+/H+ antiport in the gonadotropic regulation of steroidogenesis was examined in rat granulosa cells incubated for up to 6 h in a chemically defined medium in the absence or presence of Na+ (128 mM), gonadotropin (FSH or LH; 0-500 ng/ml), dibutyryl cyclic AMP [Bu)2cAMP; 2 mM) and amiloride (0-1 mM). Replacement of Na+ (Na+0) in the incubation medium with choline chloride resulted in a marked decrease in basal and LH-, FSH- and (Bu)2cAMP-stimulated progesterone and 20 alpha-hydroxypregn-4-en-3-one (20 alpha-OH-P) synthesis in vitro. The Na+/H+ exchange inhibitor, amiloride significantly suppressed basal and hormone-stimulated progestin production dose-dependently in the presence of Na+0. However, it was without effect in Na+-deficient medium. The effect of the inhibitor on progestin production appeared to be directed at specific step(s) involved in the synthesis of pregnenolone, as concentrations of amiloride which inhibited progesterone production failed to influence the metabolism of exogenous pregnenolone to progestins. Cell viability and the incorporation of [3H]leucine into acid-precipitable material were not affected by amiloride. Our findings support the contention that extracellular sodium is important for steroidogenesis in rat granulosa cells. The inhibition by amilordie indicates an involvement of the Na+/H+ exchange in the regulation of this granulosa cell function.     

Microtubules and the calcium-dependent regulation of rat granulosa cell steroidogenesis

The possible relationship between calcium and microtubules in the regulation of granulosa cell steroidogenesis was assessed by using agents known to alter microtubule-tubulin equilibrium together with the ionophore A23187, an antibiotic that facilitates the movement of calcium across plasma membranes. Using immunofluorescence and morphometric analysis, we determined alterations in microtubule organization and overall cell shape, respectively, in response to ionophore-stimulated production of progesterone and 20 alpha-hydroxypregn-4-en-3-one (20 alpha-OH-progesterone) during 24 h of culture. In addition, the influences of colchicine and nocodazole, two agents known to induce microtubule depolymerization, and of taxol, an agent that stabilizes tubulin polymers, on calcium-dependent regulation of granulosa cell progestin production in vitro were examined. Cells cultured as controls were flattened, highly irregular in outline, and associated with a complexly organized, well-spread cytoplasmic network of microtubules. In contrast, those maintained in the presence of increasing concentrations of ionophore were progressively more circular and smooth in outline, occupied less area on the growth surface, and contained cytoplasmic arrays of microtubules considerably less extensive than those of the controls and occupying areas defined by the more regular cellular perimeters. While progestin production in the absence or presence of a submaximally stimulatory concentration of A23187 was increased by both colchicine and nocodazole, the microtubule-depolymerizing agents had little to no effect on the production of the steroids by granulosa cells maximally stimulated by the ionophore. However, both basal and ionophore-induced progestin production were unaltered by taxol except at a concentration of 10 microM in the presence of 0.25 micrograms/ml A23187.(ABSTRACT TRUNCATED AT 250 WORDS)     

Microfilaments and FSH stimulation of rat granulosa cell steroidogenesis in vitro

The secretion of progesterone and 20 alpha-hydroxypregn-4-en-3-one (20 alpha-dihydroprogesterone) by granulosa cells from 30-day-old rats pretreated with PMSG (4 i.u.; i.p.) was significantly increased in a time- and concentration-dependent manner by FSH or cytochalasin B. Whereas FSH markedly stimulated progestagen secretion during 3 h of incubation, a significant enhancement of the steroidogenic response was not noted until 12 h of exposure to the inhibitor in vitro. Although cytochalasin B also enhanced the submaximal stimulation of progestagen production by FSH (15 ng/ml), it was ineffective in the presence of maximal stimulatory concentration of the gonadotrophin (150 ng/nl). With increasing concentrations of cytochalasin B, the ability of FSH to further stimulate progestagen secretion was progressively reduced. Granulosa cells cultured in medium alone contained a prominent cytoplasmic array of microfilaments which was markedly reduced by FSH or cytochalasin B. FSH and, to a greater extent, cytochalasin B elicited concentration-dependent reductions in the mean area occupied by the cells on the culture surface, the contour index (a size-independent representation of cell profile irregularity) and cell perimeter, indicating that the cells underwent less spreading and were more spherical and regular in outline in the presence of either agent. The FSH-induced reductions in the three shape-related parameters were augmented by cytochalasin B although the influence of the FSH on the mean area and perimeter was progressively reduced in the presence of higher concentrations of cytochalasin B.(ABSTRACT TRUNCATED AT 250 WORDS)     

Hypertonicity-induced projections reflect cell polarity in mouse metaphase II oocytes: involvement of microtubules,microfilaments, and chromosomes

A previous study showed that with hypertonic sucrose treatment, a projection is formed in mouse metaphase II (MII) oocytes in proximity to the spindle and chromosomes, where a polarized cortical domain is located. However, little is known about the mechanisms involved in this process. Here, we designed a series of experiments to test the hypothesis that hypertonicity is the induction factor for the formation of projections in mouse MII oocytes. Our hypothesis was supported by the following evidence: 1) different concentrations of sucrose affected the formation and shape of projections, whereas serum or basic media had little effect; 2) other hypertonic sugar solutions could also induce projection formation; and 3) projections could also be induced by hypertonic NaCl solution. We then tested the hypothesis that the cytoskeleton was involved in the formation of hypertonicity-induced projections. This was investigated by culturing MII- and germinal vesicle-stage mouse oocytes in the presence or absence of cytoskeletal inhibitors, including cytochalasin B (disruption of actin filaments), nocodazole (disruption of microtubules), and taxol (polymerization of tubulin molecules). We found that none of the cytoskeletal inhibitors alone could prevent hypertonicity-induced projection formation, whereas the combination of cytochalasin B with nocodazole or with taxol blocked the formation of these projections in most matured oocytes. When immature oocytes were incubated in cytochalasin B, but not in nocodazole or taxol, the formation of an actin-rich domain and the peripheral positioning of the spindle were blocked during maturation; hence, no projections were formed, even after hypertonic sucrose treatment. Based on these observations, we propose that three components are necessary for projection formation: 1) a polarized cortical patch (e.g., an actin-rich domain), 2) rigid submembrane structures (e.g., a spindle and/or chromosomes), and 3) solid connections between the above. Any disturbance of one of these factors will affect the hypertonicity-induced projection formation. Hypertonicity-induced projection in mouse oocytes thus provides an experimental model for studies regarding cell polarity and the interaction between membrane and submembrane components.     

Mononuclear cell-modulation of granulosa cell steroidogenesis in rat: influence of ovarian cycle.

The mononuclear cells exert a paracrine influence on the ovarian function, including steroidogenesis. This study examines the ability of the conditioned medium from the cultures of splenic mononuclear cells, obtained during various phases of the ovarian cycle, on progesterone accumulation by the granulosa cells in the culture medium. Female Wistar rats, aged twenty-five days, were made pseudopregnant by an injection of pregnant mare's serum gonadotropin. The splenic mononuclear cells were isolated at follicular phase, early luteal phase, mid luteal phase and late luteal phase and cultured for 48 h. The ammonium sulphate precipitated fraction of the conditioned medium was added to the granulosa cells obtained from immature rats treated with diethylstillboestrol. The granulosa cells were cultured for 48 h, and the progesterone accumulated in the medium was assayed. The conditioned medium from the cultures of the mononuclear cells obtained during follicular phase and late luteal phase inhibited FSH-induced progesterone secretion, whereas conditioned medium obtained from mid luteal phase mononuclear cells enhanced the effect of FSH. The stimulatory effect of db-cAMP on progesterone accumulation in the culture medium is inhibited by conditioned medium obtained from all the phases of the ovarian cycle. This study demonstrates a cyclicity in the behaviour of the splenic mononuclear cells on ovarian steroidogenesis, suggesting a bi-directional paracrine and/or endocrine relationship between ovary and the mononuclear cells.     

Three-dimensional organization of microfilaments and microtubules in the cytoskeleton : Immunoperoxidase labelling and stereo-electron microscopy of detergent-extracted cells

Stereo-electron microscopy has been combined with indirect immunoperoxidase labelling to describe the three-dimensional organization of microfilaments and microtubules in spreading cells of a cultured cell line with fibroblastic morphology. Labelling was carried out after extraction of the cells with a non-ionic detergent in a buffer system allowing retention of many of the cytoskeletal elements. Preservation of the three-dimensional organization was ensured by critical point drying. The peroxidase reaction product is readily detectable in electron micrographs both at high and low magnification. Thus, visualization of the three-dimensional organization of the labelled cytoskeletal elements is possible at a magnification where entire cells or large parts of them can be examined in whole mounts. The microfilament system is shown to constitute a continuous, three-dimensional sheath enclosing the bulk of the cytoplasm and most of the microtubular system. In cytoskeletons labelled with actin antibodies, the unlabelled intermediate filaments (10 nm filaments) can be identified by their size and morphology. They constitute a network throughout the cytoplasm which is in part interwoven with the large actin cables located near the lower surface of the cell.     

Effect of activin-a on goat granulosa cell steroidogenesis

Growth factors are said to play a significant role in the development of ovarian follicles. We wished to measure the content of one growth factor, activin-A in goat ovarian follicles, and study its effect on goat granulosa cells steroidogenesis. The follicular fluid content of activin-A from small, medium and large antral follicles was determined by two-site enzyme immunoassay. The results showed that activin-A concentration in the follicular fluid increased as the size of the follicle increased and, thus, may act as a local regulator of follicle development. To examine this possibility, the effect of increasing concentration of activin-A (0, 1, 10, 100 ng/mL) on differentiated goat granulosa cells steroidogenesis was evaluated in vitro for 48 hours in a chemically defined medium. Activin-A treatment resulted in a significant inhibition of progesterone production concomitant with a significant stimulation of estradiol production. These results were confirmed by time-effect of 50 ng/mL activin-A on goat granulosa cells steroidogenesis for 24, 48 and 72 hours. Granulosa cells displayed differential steroidogenic responses to activin-A, estradiol production becoming enhanced and progesterone production suppressed. Based on these findings, it appears that activin-A is a local regulator of goat granulosa cell steroidogenesis, and may act to promote granulosa cell differentiation and inhibit its luteinization.     

Effects of metformin on bovine granulosa cells steroidogenesis: possible involvement of adenosine 5' monophosphate-activated protein kinase (AMPK)

In mammals, IGFs are important for the proliferation and steroidogenesis of ovarian cells. Metformin is an insulin sensitizer molecule used for the treatment of the infertility of women with polycystic ovary syndrome. It is, however, unclear whether metformin acts on ovarian cells. Adenosine 5' monophosphate-activated protein kinase (AMPK) is involved in metformin action in various cell types. We investigated the effects of metformin on bovine granulosa cell steroidogenesis in response to IGF1 and FSH, and studied AMPK in bovine ovaries. In granulosa cells from small follicles, metformin (10 mM) reduced production of both progesterone and estradiol and decreased the abundance of HSD3B, CYP11A1, and STAR proteins in presence or absence of FSH (10(-8) M) and IGF1 (10(-8) M). In cows, the different subunits of AMPK are expressed in various ovarian cells including granulosa and theca cells, corpus luteum, and oocytes. In bovine granulosa cells from small follicles, metformin, like AICAR (1 mM) a pharmaceutical activator of AMPK, increased phosphorylation of both Thr172 of AMPK alpha and Ser 79 of ACACA (Acetyl-CoA Carboxylase). Both metformin and AICAR treatment reduced progesterone and estradiol secretion in presence or absence of FSH and IGF1. Metformin decreased phosphorylation levels of MAPK3/MAPK1 and MAPK14 in a dose- and time-dependent manner. The adenovirus-mediated production of dominant negative AMPK abolished the effects of metformin on secretion of progesterone and estradiol and on MAPK3/MAPK1 phosphorylation but not on MAPK14 phosphorylation. Thus, in bovine granulosa cells, metformin decreases steroidogenesis and MAPK3/MAPK1 phosphorylation through AMPK activation.     

The role of microfilaments and of microtubules in taurocholate uptake by isolated rat liver cells

The role of microfilaments and microtubules on bile salt transport was studied by investigating the influence of a microfilament and a microtubule inhibitor, cytochalasin B and colchicine, respectively, on taurocholate uptake by isolated hepatocytes in vitro. Hepatocytes were prepared by the enzyme perfusion method and [¹⁴C]taurocholate uptake velocity was determined by a filtration assay. Taurocholate uptake obeyed Michaelis-Menten kinetics, maximal uptake velocity and apparent half-saturation constants averaging $\text{0.87 ±}\text{SD}\text{0.05}\text{nmol}\text{· s}{}^{\mathrm{?1}}\text{· 10}{}^{\mathrm{?6}}\text{cells}$ and $\text{10.9 ± 1.8 μ}\text{M}$, respectively. Cytochalasin B ($\text{4.2–420 μ}\text{M}$) inhibited taurocholate uptake in a competitive fashion; $\text{K}{}_{\mathrm{<mtext>i</mtext>}}$ being $\text{33 ± 7 μ}\text{M}$. At concentrations above 100 μM the compound decreased ³⁶Cl membrane potential and intracellular K⁺ concentration. Other parameters of cell viability were not affected by cytochalasin B. Colchicine (0.1–1.0 mM), by contrast, inhibited taurocholate uptake non-competitively, $\text{K}{}_{\mathrm{<mtext>i</mtext>}}$ being $\text{0.47 ± 0.07}\text{mM}$. The inhibition brought about by colchicine was considerably smaller than that induced by cytochalasin B. None of the parameters of cell viability tested was affected by colchicine. These results suggest that microfilaments may be involved in the carrier-mediated hepatocellular transport of bile salts. This could, at least in part, account for cytochalasin B-induced cholestasis. The contribution of the microtubular system, if any, is less important quantitatively. The mechanisms whereby these two components of the cytoskeleton partake in bile salt transport remain to be elucidated.     

The reorganization of microtubules and microfilaments in differentiating keratinocytes

Using immunofluorescence techniques, we have examined the microtubules and microfilaments in colonies of terminally differentiating human keratinocytes in tissue culture. The undifferentiated keratinocytes contained numerous microtubules, which radiated from a centrosomal organization center (MTOC). Differentiating keratinocytes, which leave the basal layer and begin to synthesize involucrin, displayed an altered cytoskeleton. Thick mats and coils of microtubules formed throughout the cytoplasm of the differentiated squames, and microfilaments were no longer visible after staining with phalloidin. Instead, only scattered stipples of phalloidin-stained material were observed. The results suggest that the terminal differentiation of epidermal cells involves a reorganization not only of the keratin filaments but of the entire cytoskeleton.     

Possible involvement of microtubules and microfilaments of the epididymal epithelial cells in 17beta-estradiol synthesis

The rat epididymal epithelial cells revealed features of steroidogenic cells and released 17beta-estradiol (E2) into the culture medium. In steroidogenic cells, elements of the cytoskeleton due to their influence on organelle distribution are implicated in the regulation of steroidogenesis. In the present study, the morphology of cultured epididymal epithelial cells in light, scanning and transmission electron microscopes was evaluated. The organization of microtubules and microfilaments revealed by fluorescence microscopy, and the concentration of E2 in cultured medium were also studied. The epididymal epithelial cells were cultured in different conditions: in the medium with or without exogenous testosterone (T) and in the co-culture with Leydig cells as a source of androgens. The cells in co-culture located close to Leydig cells were rich in glycogen, PAS-positive substances and lipid droplets, in higher amount than the cells cultured with addition of exogenous testosterone. Stress fibers and microtubules of epididymal epithelial cells cultured with exogenous T and in co-culture with Leydig cells presented typical structure, and numerous granular protrusions appeared on the surface of the cells. Disorganization of microtubules and shortening of stress fibers as well as the smooth cell surface deprived of granular protrusions were observed in the epididymal epithelial cells cultured without T. Change of the cytoskeleton organization caused by the absence of androgen in culture medium resulted in an increased E2 secretion.     

Bone morphogenetic protein 5 expression in the rat ovary: biological effects on granulosa cell proliferation and steroidogenesis

Recently, the role of several elements of the bone morphogenetic protein (BMP) family has been studied in the ovary, some of them being crucial for ovarian function. In the present work, we have studied bone morphogenetic protein 5 (BMP5) expression and its biological role in the rat ovary. BMP5 is expressed by rat granulosa cells (GCs) and exerts specific biological effects on proliferation and steroidogenesis of these cells in an autocrine manner. These effects were shown to be associated with an increase in cyclin D2 protein level and a decrease in steroidogenic acute regulatory (StAR) protein expression in GCs in vitro. Ultimately, BMP5 actions were inhibited by follistatin. Overall, these data show that BMP5 is a novel element of the BMP family that might play a fully paracrine role in rodent ovarian folliculogenesis.     

Roles of microfilaments and microtubules in paxillin dynamics

We investigated the roles of microfilaments and microtubules in the localization and tyrosine phosphorylation of paxillin, a focal adhesion-associated signaling molecule, in bovine aortic endothelial cells (BAECs). Paxillin tyrosine phosphorylation is inhibited by cytochalasin D (CD), but slightly increased by colchicine and paclitaxol (taxol). CD also caused an overall disassembly of paxillin-containing focal adhesions (paxillin-FAs) and translocation of paxillin to the cytoplasm and perinuclear region with a diffuse distribution. Meanwhile, colchicine and taxol caused a disassembly of paxillin-FAs from cell periphery and lamellipodia, and their assembly in cell center. These results indicate that actin filaments are important in paxillin assembly in the FAs of the whole ECs and that microtubules are critical in paxillin assembly in cell periphery and lamellipodia; thus the microfilaments and microtubules play differential roles in the dynamics of paxillin assembly/disassembly. Our findings also suggest that tyrosine phosphorylation is an important element in paxillin dynamics at FAs.     

A possible complementary role of actin microfilaments and microtubules in triacylglycerol secretion by isolated rate hepatocytes

Incubation of isolated rat hepatocytes with phalloidin, cytochalasins (which, respectively, stabilize and destabilize actin microfilaments), or colchicine (which inhibits polymerization of microtubules), resulted in a dose-dependent inhibition of triacyglycerol secretion (an index of very low density lipoprotein secretion). Upon removal of drugs from incubation media, the inhibitory effect of cytochalasin D on triacylglycerol secretion was reversible, while such was not the case for phalloidin. When used at maximal concentrations, the combined presence of phalloidin + colchicine or cytochalasin D + colchicine had additive inhibitory effects upon hepatic triacylglycerol secretion, which was virtually blocked; this was not the case for phalloidin + cytochalasin D. These experiments support the concept that microfilaments and microtubules may have complementary functions for the hepatic secretion of very low density lipoproteins.     

Ooplasmic segregation in theTubifex egg: Mode of pole plasm accumulation and possible involvement of microfilaments

Summary Ooplasmic segregation, i.e. the accumulation of pole plasm in theTubifex egg, consists of two steps: (1) Cytoplasm devoid of yolk granules and lipid droplets migrates toward the egg periphery and forms a continuous subcortical layer around the whole egg; (2) the subcortical cytoplasm moves along the surface toward the animal pole in the animal hemisphere and toward the vegetal pole in the vegetal hemisphere, and finally accumulates at both poles of the egg to form the animal and vegetal pole plasms. Whereas the subcortical layer increases in volume during the first step, it decreases during the second step. This is ascribed to the compact rearrangement in the subcortical layer of membraneous organelles such as endoplasmic reticulum and mitochondria. The number of membraneous organelles associated with the cortical layer increases during the second step. Electron microscopy reveals the presence of microfilaments not only in the cortical layer but also in the subcortical layer. Subcortical microfilaments link membraneous organelles to form networks; some are associated with bundles of cortical microfilaments. The thickness of the cortical layer differs regionally. The pattern of this difference does not change during the second step. On the other hand, the subcortical cytoplasm moves ahead of the stationary cortical layer. The accumulation of pole plasm is blocked by cytochalasin B but not by colchicine. The first step of this process is less sensitive to cytochalasin B than the second step, suggesting that these two steps are controlled by differnt mechanisms. The mechanical aspects of ooplasmic segregation in theTubifex egg are discussed in the light of the present observations.     

Effects of cytokines on porcine granulosa cell steroidogenesis in vitro

Recent evidence indicates that factors produced by immune cells (cytokines) may play a role in ovarian function. To explore this possibility, we examined the effects of conditioned medium obtained from cultures of either unstimulated splenocytes (splenocyte-conditioned medium; SCM) or concanavalin A-stimulated splenocytes (CAS) on estrogen and progesterone production by porcine granulosa cells. Granulosa cells were obtained from small (less than 3 mm) or large (greater than 7 mm) follicles and treated with increasing doses of SCM or CAS in the presence or absence of pFSH (100 ng/ml) for 24 h at 37 degrees C. In granulosa cells obtained from small follicles it was found that both SCM and CAS evoked a dose-dependent increase in estrogen but not progesterone production. Estrogen production was no further enhanced by the presence of FSH. Additionally, SCM was able to augment FSH-stimulated progesterone production by these cells, whereas CAS had no effect. Identical treatment of granulosa cells obtained from large follicles demonstrated that both SCM and CAS caused dose-dependent increases in estrogen as well as progesterone production. In response to CAS, FSH augmented progesterone production but exerted a biphasic on estrogen production (inhibiting at lower doses while stimulating at higher doses). In contrast, SCM had no effect on FSH-stimulated estrogen production. Additional controls indicated that the above results could not be attributed to either concanavalin A or serum. Taken together, these findings suggest that cytokines can exert significant effects over granulosa cell steroidogenesis and further imply that these factors may play an important role in the differentiation and developmental regulation of granulosa cell function.     

Medroxyprogesterone acetate: receptor binding and correlated effects on steroidogenesis in rat granulosa cells

Medroxyprogesterone acetate (MPA), a widely used synthetic steroid, was studied to determine both its effects on steroid receptors and steroidogenesis in the well-characterized rat ovarian granulosa cell model. Initial receptor binding studies showed MPA was as potent as progesterone and 10-fold less potent than R-5020 (an active synthetic progestin) in binding to progesterone cytosolic receptors in rat ovarian granulosa cells. MPA was 20-fold less potent than testosterone, and 10-fold less potent than dexamethasone in binding to the androgen and glucocorticoid cytosolic receptors, respectively. The binding of MPA to progestrone, androgen and glucocorticoid receptors predicted direct effects of MPA on FSH-stimulated estrogen (E), progesterone (P), and 20 alpha-dihydroprogesterone (DHP) production by cultured rat ovarian granulosa cells. MPA at 10(-7) to 10(-6) M significantly augmented FSH-stimulated P and DHP production (a previously documented progestin, androgen and glucocorticoid effect). This augmentation was blocked by the concurrent addition to cell culture of 10-fold excess RU-486 (a potent anti-progestin and anti-glucocorticoid). At concentrations greater than 10(-6) M, MPA inhibited the production of P and DHP (a progestin effect), and the production of E (a progestin and glucocorticoid effect). MPA, structurally a progestin, has complex steroid hormone effects predicted by its interaction with progesterone, androgen and glucocorticoid receptors.