Transmembrane regulation of intracellular calcium by a plasma membrane sodium/calcium exchanger in mouse ova.

Regulation of cytoplasmic free calcium concentration ([Ca2+)]i) is a key factor for maintenance of viability of cells, including oocytes. Indeed, during fertilization of an ovum, [Ca2+]i is known to undergo oscillations, but it is unknown how basal [Ca2+]i or calcium oscillations are regulated. In the present study we investigated the role of the plasma membrane in regulating [Ca2+]i of metaphase II-arrested mouse oocytes (ova). Ova were collected from B6C3F1 mice treated with eCG (10 IU) and hCG (5 IU), and intracellular calcium was determined by means of fura-2. Extracellular calcium flux across the zona pellucida was detected noninvasively by a calcium ion-selective, self-referencing microelectrode that was positioned by a computer-controlled micromanipulator. Under basal conditions ova exhibited a calcium net efflux of 20.6 +/- 5.2 fmol/cm2 per sec (n = 69). Treatment of ova with ethanol (7%) or thapsigargin (25 nM-2.5 microM) transiently increased intracellular calcium and stimulated calcium efflux that paralleled levels of [Ca2+]i. The presence of a Na+/Ca2+ exchanger was indicated by experiments employing both bepridil, an inhibitor of Na+/Ca2+ exchange, and sodium-depleted media. In the presence of bepridil, a net influx of calcium was revealed across the zona pellucida, which was reflected by an increase in the [Ca2+]i. In addition, replenishment of extracellular sodium to ova that had been incubated in sodium-depleted media induced a large calcium efflux, consistent with the actions of Na+/Ca2+ exchange. Sodium/calcium exchange in mouse ova may be an important mechanism that regulates [Ca2+]i.

Dissociation between intracellular calcium elevation and development of human oocytes treated with calcium ionophore.

OBJECTIVE: To develop an acceptable model system to study calcium activation of human oocytes. DESIGN: Study of oocyte development and intracellular calcium [Ca]i dynamics of activated oocytes. SETTING: Research center affiliated with infertility service. MAIN OUTCOME MEASURE: Morphologic evidence of meiotic maturation and cell division under high-power Hoffman optics with an inverted microscope. Meiotic maturation was determined by the number of polar bodies or the presence of a pronucleus, and cell division was determined by evidence of a cleavage furrow or presence of blastomeres. To monitor the effect of calcium ionophore on [Ca]i levels, oocytes were incubated with fura-2 (2 microM) for 30 minutes and [Ca]i was determined by rationing the emission fluorescence (510-nm long-pass filter) during simultaneous excitation at 340 and 380 nm with a microspectrofluorimeter. RESULT(S): All oocytes loaded with fura-2 and then exposed to ionophore exhibited an isolated elevation of [Ca]i, followed by prompt return to baseline levels. None of the oocytes showed signs of cleavage or of meiotic maturation after treatment with calcium ionophore. CONCLUSION(S): Human oocytes activated with calcium ionophore A23187 or ionomycin exhibited elevated [Ca]i but remained resistant to subsequent meiotic maturation and cleavage. Our results differ from some reports of parthenogenetic activation of human oocytes. These differences may result from different activation protocols or culture conditions. Because none of the 126 oocytes cleaved after the activation protocols used in these experiments, this approach should provide an ethically acceptable model system to study calcium dynamics in human oocytes.

The ovarian renin-angiotensin system. A paracrine-intracrine regulator of ovarian function.

We present a review that considers the regulation of ovarian function by the ovarian renin-angiotensin system. Evidence is presented that shows the ovarian renin-angiotensin system can regulate normal ovarian function by paracrine and intracrine mechanisms. In addition, the complexity that is inherent in the renin-angiotensin system conferred by multiple biological angiotensin peptides and multiple receptors is discussed. Evidence that a dysfunctional OVRAS induces ovarian pathology such as polycystic ovarian disease is also presented.

The basis and evidence of a role for the ovarian renin-angiotensin system in health and disease.

OBJECTIVE: We reviewed the evidence for an intrinsic ovarian renin-angiotensin system (OVRAS), highlighting potential diverse signaling in this system through different bioactive angiotensin peptides, their specific receptors, and second messengers. In addition, sites of action for OVRAS in the regulation of ovarian function in health and disease were reviewed. DATA SOURCES: We used published journals and abstracts from national scientific meetings. Current developments in the renin-angiotensin field are historically set. STUDY SELECTION: One hundred referenced articles provided studies on renin-angiotensin systems in mammalian species, including humans. DATA ABSTRACTION: Interpretation of the reviewed publication was in line with the original authors' conclusions and statistical analysis. DATA SYNTHESIS: Techniques in molecular biology, biochemistry, and immunohistochemistry have identified an OVRAS in mammalian species. Ovarian tissues contain all the elements for the production of angiotensin, including prorenin/renin, angiotensinogen, and angiotensin-converting enzyme. In addition, angiotensin II is present in ovarian compartments, and receptors for angiotensin II are demonstrated on specific ovarian cells. Angiotensin II is implicated to play a role in ovulation, steroidogenesis, follicular atresia, and hyperandrogenic syndromes. CONCLUSIONS: The newly identified OVRAS may have important actions in the ovary that range from regulation of ovulation to ovarian dysfunction, such as hyperandrogenic syndromes in women. In this respect, the OVRAS is a putative paracrine/autocrine regulator in the ovary, and pharmacologic regulation of the OVRAS may provide new methods for the management of fertility and reproduction.

The type 1 angiotensin-II receptor mediates intracellular calcium mobilization in rat luteal cells.

The intracellular cytosolic calcium concentration ([Ca]i) was determined in cultured rat luteal cells using the calcium-chelating dye fura-2 and microspectrofluorimetry. Angiotensin-II (Ang-II) induced a dose-dependent transient increase in [Ca]i (ED50, 9.0 +/- 6.5 nM). After the initial peak in [Ca]i, cytosolic calcium returned to a secondary elevated basal level that was dependent upon the presence of extracellular calcium. Pretreatment of rat luteal cells with Ang-II (100 nM) desensitized a subsequent response to a higher concentration (1 microM), but did not desensitize a prostaglandin F2 alpha (PGF2 alpha)-induced calcium flux. Although the peak increases in [Ca]i induced by Ang-II (1 microM) and PGF2 alpha (10 microM) were not significantly different, the plateau phase stimulated by PGF2 alpha was significantly higher (P < 0.05) than that stimulated by Ang-II (1 microM). Pretreatment of luteal cells with the type 2 Ang-II receptor antagonist PD 123319 (10 microM) did not inhibit calcium mobilization; however, Ang-II (1 microM)-induced calcium mobilization was dose dependently blocked by the type 1 Ang-II receptor antagonist Losartan (DuP 753). The ID50 for Losartan was 5.2 +/- 1.8 nM. Pretreatment of the luteal cells with the endoplasmic reticulum calcium ATPase inhibitor thapsigargin (1 microM) also blocked Ang-II-induced calcium mobilization. These data demonstrate the presence of the type 1 Ang-II receptor in rat luteal cells, through which Ang-II dose dependently mobilizes calcium from an intracellular source, probably the endoplasmic reticulum.

The in vitro perifused rat ovary: III. Interrelationship of the follicular and stromal compartments on steroid release.

The preovulatory ovary is composed of two primary tissue components, stroma and follicles. To assess the role of these tissue compartments in ovarian steroidogenesis, stromal tissue, follicular tissue, and a mixture of both tissues from pregnant mare serum gonadotropin (PMSG)-treated, prepubertal rats were perifused simultaneously for 8 h. The basal level of estradiol secretion by stromal tissue was lower (24 +/- 3 pg/mg/30 min), than that secreted by follicles (64 +/- 5.6 pg/mg/30 min, n = 6; p < 0.05). On the other hand, the mean basal levels of progesterone and testosterone secreted by stromal tissue (252 +/- 14 pg/mg/30 min and 162 +/- 18 pg/mg/30 min, respectively) were greater than those secreted by follicular tissue (84 +/- 3 pg/mg/30 min and 81 +/- 4 pg/mg/30 min, respectively). When stromal and follicular tissue were combined the secretion of progesterone, testosterone and estradiol was intermediate to that of the separate tissues. Under gonadotropin stimulation (human menopausal gonadotropins plus follicle stimulating hormone), the follicular tissue secreted greater amounts of steroids than did the stromal tissue, or stromal plus follicular tissue. When stromal tissue and follicular tissue were combined, the levels of basal progesterone and testosterone secreted by both tissues were significantly lower than those of stromal tissue alone. However, the reduction in follicular estradiol secretion induced by stromal tissue under basal conditions, was in large part overcome during gonadotropin perifusion. These observations suggest that locally produced factors may play an inhibitory, paracrine role in the regulation of ovarian steroidogenesis.

The in vitro perifused rat ovary: V. The significance of the follicle stimulating hormone and luteinizing hormone ratio on steroid release.

In these studies, an in vitro perifusion model was used to compare stimulation of ovarian tissue with either human menopausal gonadotropin (hMG), which is an equal mixture of luteinizing hormone (LH) and follicle stimulating hormone (FSH), or with hMG plus added human FSH. Eight-hour perifusion studies were conducted on either whole, or dissected clusters of follicles from pregnant mare serum gonadotropin (PMSG)-treated rats. In the two groups, similar stimulatory protocols were used, consisting of a ramp stimulation over 60 min with either hMG (0-8 mIU/ml) or hMG plus FSH (0-8 mIU/ml hMG + 0-8 mIU/ml FSH), followed by hourly pulse stimulation with hMG (8-18 mIU/ml) or hMG plus FSH (8-18 mIU/ml hMG + 8-18 mIU/ml FSH), respectively. In the whole ovaries, no differences were detected in progesterone, testosterone, or estradiol secretion. However, in the cluster of follicles, an elevated hFSH/hMG ratio resulted in a significantly higher secretion of progesterone, testosterone and estradiol (n = 8; p < 0.05) than the steroids secreted by follicles perifused with hMG alone. In conclusion, an elevated FSH: LH ratio led to greater steroidogenic responses by the perifused cluster of follicles, but not by the whole ovary.

The in vitro perifused rat ovary: IV. Modulation of ovarian steroid secretion by insulin.

Insulin has been implicated as a regulatory factor in ovarian steroidogenesis. To assess this issue, we examined the role of insulin on steroid secretion by whole ovaries from pregnant mare serum gonadotropin (PMSG)-pretreated rats, using an in vitro perifusion ovarian model. Three concentrations of insulin were perifused in the absence and presence of gonadotropin pulse-stimulation. Bioactive insulin concentrations after perifusion of ovarian tissue were 4, 40 and 400 mIU/ml. In the absence of gonadotropin-pulse stimulation, acute perifusion with insulin had no effect on ovarian steroid secretion. During stimulation with luteinizing hormone (LH) plus follicle stimulating hormone (FSH), insulin significantly decreased the secretion of testosterone and estradiol but increased the output of progesterone. This effect was only evident after 180 min perifusion and with the highest concentration of insulin. We conclude that acute elevation of insulin concentration has no effect on basal ovarian secretion of progesterone, testosterone, or estradiol in gonadotropin-pretreated rats. However, insulin has a moderate effect on steroid secretion in ovaries exposed to LH/FSH pulsatile stimulation, resulting in a decrease in estradiol and testosterone release, and stimulation of progesterone secretion.

The in vitro perifused rat ovary: I. Steroid secretion in response to ramp and pulsatile stimulation with luteinizing hormone and follicle stimulating hormone.

A computer-controlled perifusion apparatus has been used to investigate the effects of different patterns of hormonal stimulation on secretion of steroids by ovaries from untreated, or pregnant mare serum gonadotropin (PMSG)-pretreated, immature rats. With ovaries from untreated rats, a low rate of increasing concentration of gonadotropins (luteinizing hormone (LH) plus follicle stimulating hormone (FSH)) induced a maximum secretion of estradiol within 60 min (22.6 +/- 1.4 pg/mg/30 min). An intermediate and a high rate of increasing gonadotropin concentration stimulated maximum secretion (26.0 +/- 1.2 pg/mg/30 min) and 28.1 +/- 2.8 pg/mg/30 min), respectively) within 30 min. Peak secretion, however, was not maintained and was reduced despite continued LH/FSH pulses. Progesterone secretion increased during, and subsequent to, the decreasing estradiol output. Increasing amplitudes of LH/FSH or constant perifusion with LH/FSH did not change the profile, or the concentration of estradiol, but these measures increased progesterone release. An occasional transient increase in estradiol secretion was observed when ovaries from unstimulated rats were perifused with low LH/FSH pulse frequency. Thus, these studies support the hypothesis that in the prepubertal rat ovary, elements of pulse characteristics, such as rate of increasing LH/FSH concentration, and amplitude are important in differentially regulating steroid output. The steroid secretory pattern of ovaries from PMSG-treated prepubertal rats was different from that of untreated rats. With ovaries from PMSG-treated rats, an acute increase in secretion of progesterone, testosterone or estradiol was not observed, whether low or high rates of increasing gonadotropin concentration were used. Rather, the concentration of these steroids continued to rise following LH/FSH pulses. Thus, in contrast to the untreated ovary, the PMSG-treated ovary did not show differential regulation of steroid secretion in response to LH/FSH. In conclusion, we have shown the in vitro perifusion model to be a useful tool for studying the effects of different patterns of hormonal stimulation on ovarian steroidogenesis. In addition, differential effects of gonadotropins on steroid output were shown, depending upon prior maturation of the ovary.

The in vitro perifused rat ovary: II. Role of oxygen tension in the whole and quartered ovary.

In vitro perifusion of whole ovaries raises questions about tissue viability and its effects on the observed ovarian steroid secretion. To assess effects of tissue degeneration, whole and quartered ovaries from pregnant mare serum gonadotropin (PMSG-) treated immature rats were perifused for 8 h, employing various levels of pO2. Histological examination of both the whole and quartered ovary showed signs of degeneration in centrally located follicles but not in follicles located near the surface. However, basal and gonadotropin-stimulated (luteinizing hormone plus follicle stimulating hormone) secretion of estradiol, testosterone and progesterone were not significantly different in the whole ovary whether in the presence of high or low pO2 (n = 8; p > 0.05). Similarly, although the quartered ovary secreted greater amounts of steroids than did the whole ovaries (p < 0.05, n = 8), pO2 levels did not affect the steroid output. We conclude that during in vitro perifusion, minimal oxygen supply is sufficient for ovarian steroidogenesis to proceed. In addition, although quartered ovaries displayed some evidence of tissue degeneration, they were more responsive in terms of steroid output per mg ovary than were whole ovaries.

Effects of neutrophils in rat luteal cells.

Ovarian function may be modulated by cells of the immune system. We have investigated the role of neutrophils (polymorphonuclear leukocytes) on rat luteal cell function. Activated neutrophils inhibited LH-sensitive cAMP accumulation, which was dependent on neutrophil cell number. At a concentration of 10(6) neutrophils/ml and 10(5) luteal cells/ml, LH-stimulated cAMP accumulation was inhibited by 50%. The inhibitory effect of activated neutrophils was reversed by superoxide dismutase (SOD) and catalase. LH-stimulated progesterone production was also inhibited by activated neutrophils. Progesterone production by 10(5) luteal cells was inhibited approximately 20% in the presence of 10(6) activated neutrophils, and this inhibition was blocked by SOD and catalase. Conditioned medium from activated neutrophils also produced inhibitory effects on LH-stimulated cAMP accumulation and progesterone production, which could be reversed by SOD and catalase. The phosphodiesterase inhibitor isobutylmethylxanthine had no significant effect on the inhibition of cAMP accumulation by conditioned medium from activated neutrophils. Luteal cells loaded with a fluorescent indicator for determining intracellular reactive oxygen species (dichlorofluorescein diacetate) showed increased fluorescence in the presence of activated neutrophils. No increase in fluorescence occurred in the absence of neutrophils or in the presence of SOD and catalase. These studies demonstrate that reactive oxygen species produced by activated neutrophils can enter the luteal cell and cause antigonadotropic effects. Although the experimental model used in the present studies may not be truly physiological, the data demonstrate that neutrophils may play a role in functional and structural regression of the corpus luteum in the rat.

The calcium-mobilizing agent, thapsigargin, inhibits progesterone production in rat luteal cells by a calcium-independent mechanism.

In rat luteal cells, an increase in intracellular [Ca]i impairs luteal function similar to that of prostaglandin F2a (PGF2a). However, calcium per se is not the mediator of the antigonadotropic action of PGF2a. Thapsigargin, a plant sesquiterpene lactone, increases intracellular calcium concentration concentration ([Ca]i) in several cell types by a mechanism that involves specific inhibition of the endoplasmic reticulum Ca2(+)-ATPase. To further investigate the antigonadotropic role of [Ca]i and the mechanism of action of PGF2a in rat luteal cells, the action of thapsigargin on cellular functional responses was examined in the absence and presence of PGF2a. Thapsigargin dose dependently increased [Ca]i and inhibited cAMP accumulation and progesterone production in response to LH. The inhibitory effect of thapsigargin on cAMP accumulation was calcium dependent but in contrast, inhibition of LH-stimulated progesterone production was independent of calcium mobilization by thapsigargin. Steroidogenesis stimulated by (Bu)2cAMP was also inhibited by thapsigargin. Thus, thapsigargin mimicked some effects of PGF2a with inhibitory sites of action on both cAMP accumulation and progesterone production. Thapsigargin also blocked the mobilization of [Ca]i by PGF2a, but when coincubated with PGF2a an additive effect on inhibition of LH-stimulated progesterone production occurred. However, no additive effects of thapsigargin and PGF2a on gonadotropin-sensitive cAMP accumulation were evident. In conclusion, although thapsigargin and PGF2a may share some similar actions, their antigonadotropic effects are mediated differently.

The antigonadotropic action of prostaglandin F2 alpha is not mediated by elevated cytosolic calcium levels in rat luteal cells.

We have investigated the role of intracellular calcium in the mechanism of action of prostaglandin F2 alpha (PGF2 alpha) in cultured rat luteal cells. PGF2 alpha (1 microM) maximally inhibited LH-stimulated cAMP accumulation and also initiated a transient release of intracellular calcium. Low doses of the calcium ionophore ionomycin also increased intracellular calcium to a similar extent as PGF2 alpha (1 microM), but did not inhibit LH-stimulated cAMP accumulation. Chelation of intracellular calcium with dimethyl bis-(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) (10 microM) attenuated the transient calcium rise stimulated by PGF2 alpha, but did not affect the inhibitory characteristics of PGF2 alpha on LH-stimulated cAMP accumulation. Treatment of luteal cells with EGTA (1 mM) and ionomycin (500 nM) resulted in depletion of intracellular calcium to such an extent that a subsequent exposure of the luteal cells to PGF2 alpha (1 microM) did not elicit any change in intracellular calcium. Depletion of intracellular calcium and ablation of the calcium response to PGF2 alpha, however, did not affect either the dose response or the time course of inhibition of LH-stimulated cAMP accumulation. We conclude that although intracellular calcium is mobilized by PGF2 alpha in cultured rat luteal cells, the antigonadotropic action of PGF2 alpha on LH-stimulated cAMP accumulation is not mediated by this mechanism.