EGF modulates phosphoinositide levels in ovarian granulosa cells stimulated by luteinizing hormone

Hamster granulosa cells were exposed to epidermal growth factor (EGF) and luteinizing hormone (LH) to study cross-talk between second messenger pathways involving tyrosine kinase, cAMP, and phosphoinositides. Granulosa cells from ovarian preovulatory follicles of PMSG-primed hamsters were incubated with various additives in serum-free medium. LH, but not EGF, stimulated inositol phosphate (IP) accumulation; however, when combined with LH, EGF inhibited IP accumulation in a manner that was concentration dependent for both LH and EGF. The inhibitory effects of EGF were significantly reduced by the tyrosine kinase inhibitor genistein and by pertussis toxin suggesting a role for tyrosine kinase and an inhibitory G-protein (G_i) in this system. EGF stimulated an increase in cAMP, but it does not appear to modulate LH-stimulated IP levels via cAMP. © 1994 Wiley-Liss, Inc.     

Effects of prostaglandins and luteinizing hormone-releasing hormone on phosphatidic acid-phosphatidylinositol labeling in rat granulosa cells

In cultures of rat granulosa cells, luteinizing hormone-releasing hormone (LHRH) increases 32P incorporation into both phosphatidylinositol (PI) and phosphatidic acid (PA). After 20 min, the level of radioactivity was three- to four-fold (p less than 0.01) above control in the PI and PA fractions, respectively. The stimulatory effect of LHRH on 32P incorporation was limited to PI and PA. Similar to the effects of LHRH, a rapid and marked increase of 32P incorporation into both PI and PA is observed upon addition of prostaglandin F2 alpha (PGF2 alpha) (10(-5)M) to rat granulosa cells. Incorporation of radioactivity into PA was already increased (p less than 0.05) by 2 min following PGF2 alpha addition, while the increase in 32P-labeled PI became significant (p less than 0.01) by 5 min. In contrast to PGF2 alpha, the labeling of PI and PA following the addition of PGE2 (10(-5)M) was not significantly different from control levels during the entire 10 min of incubation. The sensitivity of the increased PA-PI labeling induced by LHRH and PGF2 alpha is compared in another experiment. After 20 min incubation 10(-6)M LHRH increased PI and PA labeling by six- and four-fold, respectively. Although the effect of PGF2 alpha is less than that of LHRH, 10(-5)M PGF2 alpha significantly (p less than 0.01) increased PI and PA labeling by three- and two-fold, respectively. By contrast, 10(-6)M PGE2 failed to affect 32P incorporation into the various phospholipid fractions, but a small enhancement (p less than 0.05) of PI and PA labeling was observed only at 10(-5)M PGE2.(ABSTRACT TRUNCATED AT 250 WORDS)     

Gonadotropin-releasing hormone rapidly alters polyphosphoinositide metabolism in rat granulosa cells

This report describes the rapid effects of GnRH and an agonist [D-Ala6, des-Gly10] GnRH ethylamide (GnRHa) on polyphosphoinositide metabolism in rat granulosa cells. As indicated by the depletion of cellular levels of 32P-prelabeled triphosphoinositide (TPI) and diphosphoinositide (DPI), GnRHa rapidly stimulated the hydrolysis of TPI and DPI. The effect of GnRHa was maximal at the earliest time point examined (30 sec) and preceded GnRHa-induced increases in labeling of phosphatidylinositol. A specific GnRH antagonist had no effect on TPI or DPI levels, but prevented the polyphosphoinositide depletion induced by GnRH. LH did not stimulate depletion of 32P-polyphosphoinositides. The rapid and specific effects of GnRH on polyphosphoinositide depletion may represent an early and possibly initiating event in the action of GnRH.     

In vitro responses of luteinizing rat granulosa cells to human thyroid-stimulating hormone

The effect of human thyroid-stimulating hormone (hTSH) on progesterone (P4) secretion during initial luteinization and subsequent prolactin (Prl)-mediated steroidogenesis by cultured rat granulosa cells was studied. Granulosa cells, obtained from pregnant mare's serum gonadotropin (PMSG)-treated immature female rats, were preincubated for 1, 3, 6, 12, or 24 h in control medium lacking added hormones or in medium containing 1.0 microgram/ml human chorionic gonadotropin (hCG) or hTSH, and maintained subsequently for 6 days in medium containing 1.0 microgram/ml bovine (bPrl). Indices of luteotropic stimulation were provided by: 1) elevated P4 concentrations determined by radioimmunoassay of spent media samples; and 2) cytoplasmic lipid accumulation assessed by osmium tetroxide staining following fixation after 7 days of culture. Progesterone levels in media from cultures exposed to hCG for 24 h were twofold higher than control cultures, whereas those in media from cultures preincubated in hTSH for 24 h were fourfold higher than control levels. Cultures preincubated in 1.0 microgram/ml hCG for as little as 1 h and then maintained for 6 days in Prl secreted significantly more P4 than did control cultures also maintained with Prl for 6 days. Cultures preincubated in hTSH required a 24-h exposure before a significant increase in Prl-mediated P4 secretion was observed. Intensity of cytoplasmic osmiophilia correlated directly with P4 concentration. These results suggest that: 1) hTSH has the ability to promote P4 secretion during initial luteinization and to regulate subsequent Prl-mediated steroidogenesis by cultured rat granulosa cells; and 2) the mechanism by which hTSH stimulates Prl-mediated P4 secretion in this model system may differ from that of hCG.     

Interleukin 1: an inhibitor of luteinizing hormone receptor formation in cultured rat granulosa cells

We have shown that interleukin 1 (IL 1) suppresses follicle-stimulating hormone (FSH)-induced progesterone secretion and 125I-labeled human chorionic gonadotropin (hCG) binding (a measurement of LH receptors) in cultured rat granulosa cells. The present study was designed to examine if the reduction of FSH-stimulated 125I-labeled hCG binding by IL 1 was caused by a decline in the binding capacity or by an alteration in the affinity of the LH receptor and, further, to determine the minimum period of exposure of the granulosa cells to IL 1 necessary to suppress 125I-labeled hCG binding. IL 1 produced a dose-dependent inhibition of 125I-labeled hCG binding in FSH-stimulated granulosa cells. Scatchard analysis revealed that this effect resulted from a reduction of the binding capacity of the LH receptor with no change in affinity. Also, a minimum of 12-24 h of exposure to IL 1 is necessary to significantly inhibit FSH-induced LH receptor formation. These results suggest that IL 1 decreases the number of LH receptors and that protein synthesis may be necessary for IL 1's action. However, a physiological/pathological role for IL 1 in ovarian regulation has yet to be established.     

Fibroblast growth factor regulates the expression of luteinizing hormone receptors in cultured rat granulosa cells

We have investigated the effects of bFGF on both the FSH-induced LH receptor expression and cAMP production in cultured rat granulosa cells. Concentrations of pure FGF, from 10(-12) M to 10(-10) M, progressively inhibit the stimulatory actions of FSH with an ED50 of approximately 4 x 10(-12) M for both parameters. Higher FGF concentrations, from 4 x 10(-10) M to 10(-8) M, lead to a gradual reduction of the growth factor inhibitory effect. The effects of FGF are more prominent on the modulation of LH receptors than on the FSH-induced cAMP production. Moreover, FGF impairs the LH receptor formation induced by cholera toxin or 8-Bromo-cAMP, indicating that the growth factor also acts at a step distal to cAMP formation. The inhibitory effect of FGF on LH receptor expression increases during the entire course of granulosa cell differentiation, from 24 to 96 h, and is not due to variations in cell number or viability, but rather to a change in the content of LH receptors with no significant modification of binding affinity (KD congruent to 0.8 x 10(-10) M). These results suggest that bFGF may acutely regulate the capacity of granulosa cells to differentiate upon FSH stimulation and to respond to LH during the ovarian follicular maturation.     

Control of the expression of luteinizing hormone receptor by local factors in rat granulosa cells.

To identify the mechanisms underlying the hormone-dependent induction and maintenance of luteinizing hormone receptor (LH-R) in rat granulosa cells, the effect of follicle-stimulating hormone (FSH) and local factors on the LH-R mRNA levels were studied. LH-R mRNA levels of the cells incubated with FSH decreased rapidly after medium removal, and readdition of FSH with the fresh medium did not restore these levels. On the other hand, 8-bromoadenosine 3,5-cyclic monophosphate significantly enhanced the expression of LH-R mRNA after medium removal, while the level of LH-R mRNA was lower than that of the cells replaced by original medium including FSH. In addition, the incubation with 8-Br-cAMP produced dose-dependent responses for LH-R mRNAs and enhanced the activity of 1379 bp of the LH-R 5'-flanking region, while the level of LH-R mRNA decreased 3 days after medium removal. Further studies were undertaken to assess the role of factors in maintaining the LH receptor once induced by FSH. Since FSH and cAMP increase follistatin production in granulosa cells, we examined the effect of follistatin on LH-R induction in the presence of activin and FSH. Activin induced LH-R in the presence of FSH significantly, and follistatin antagonized this effect in a dose-dependent manner. However, insulinlike growth factor-I (IGF-I) induced LH-R mRNA in the presence of FSH even after medium change. IGF-I might be one of the important factors that act in the medium to maintain LH-R levels in granulosa cells.     

Autocrine role of estrogens in the augmentation of luteinizing hormone receptor formation in cultured rat granulosa cells

The effects of estrogens on gonadotropin-stimulated luteinizing hormone (LH) receptor formation were examined in primary cultures of rat granulosa cells. Granulosa cells were cultured for 3 days with increasing concentrations of follicle-stimulating hormone (FSH) in the presence or absence of native and synthetic estrogens. Follicle-stimulating hormone stimulated LH receptor formation in a dose-dependent fashion, and estrogens enhanced the FSH-stimulated LH receptor content by decreasing the apparent ED50 of FSH. At 6.25 ng/ml FSH, the enhancement in LH receptor was estrogen dose dependent, with an ED50 value of about 3 X 10(-9) M for 17 beta-estradiol. The increased LH receptor content seen in cells treated with FSH and estrogen was correlated with increased cAMP production by these cells in response to LH stimulation. Time course studies revealed enhancement of FSH-stimulated LH receptor induction at 48 and 72 h of culture. Granulosa cells were also cultured with FSH for 2 days to induce functional LH receptors, then further cultured for 3 days with LH in the presence or absence of estrogens. At 30 ng/ml LH, increasing concentrations of estrogens maintained LH receptor content in a dose-dependent fashion, with their relative estrogenic potencies in keeping with reported binding affinities to estrogen receptors. An autocrine role of estrogens on LH receptor formation was further tested in granulosa cells treated with FSH and an aromatase substrate (androstenedione) to increase estrogen biosynthesis. Cotreatment with semipurified estrogen antibodies partially blocked the FSH stimulation of LH receptors, whereas nonimmune serum was ineffective. Also, inclusion of diethylstilbestrol prevented the inhibitory effect of the estrogen antibodies. Thus, local estrogens in ovarian follicles may play an autocrine role in granulosa cells to enhance LH receptor formation and to increase granulosa cell responsiveness to the LH surge, with subsequent ovulation and adequate corpus luteum formation.     

Effects of Prostaglandins on the action of luteinizing hormone in dispersed rat interstitial cells

Prostaglandins E1, E2, A1 and A2 at 10⁻⁵ and 10⁻⁴M stimulated basal testosterone production in dispersed rat interstitial cells in vitro. They effectively inhibited steroidogenesis induced by ovine pituitary luteinizing hormone (LH) (0.2 nM), dibutyryl cyclic AMP-(DBC 0.5 mM), and cholera toxin (100 ng). PGF2α (10⁻³ to 10⁻¹²M) had no effect on either basal or hormonal or non-hormonal stimulated steroidogenesis in the cells. PGA1 and PGA2 were more effective inhibitors than PGE1 and E2. None of the PG's had any influence on ¹²⁵I LH-receptor interaction. In view of this and the inhibition of DBC stimulated testosterone production, it may be suggested that the PG inhibition lies beyond the receptor-cyclic AMP formation step.     

Effects of prostaglandins on the action of luteinizing hormone in dispersed rat intestitial cells.

Prostaglandins E1, E2, A1 and A2 at 10(-5) and 10(-4)M stimulated basal testosterone production in dispersed rat interstitial cells in vitro. They effectively inhibited steroidogenesis induced by ovine pituitary luteinizing hormone (LH) (0.2 nM), dibutyryl cyclic AMP-(DBC 0-5 mM), and cholera toxin (100 ng). PGF2 alpha (10(-3) to 10(-12)M) had no effect on either basal or hormonal or non-hormonal stimulated steroidogenesis in the cells. PGA1 and PGA2 were more effective inhibitors than PGE1 and E2. None of the PG's had any influence on 125I LH-receptor interaction. In view of this and the inhibition of DBC stimulated testosterone production, it may be suggested that the PG inhibition lies beyond the receptor-cyclic AMP formation step.     

Transforming growth factor-beta regulates the expression of luteinizing hormone receptors in ovarian granulosa cells

In cultured granulosa cells, addition of 1 to 50 ng follicle-stimulating hormone induced a 350-fold rise in luteinizing hormone receptors, while larger amounts of gonadotropin up to 200 ng reduced these receptors to approximately 50% of peak levels. Transforming growth factor-beta (16 pM) enhanced the stimulatory actions of low levels of gonadotropin (2.5-10 ng) by 2 to 3-fold, and inhibited the induction of luteinizing hormone receptors by higher levels of follicle-stimulating hormone (greater than or equal to 50 ng) by 30-50%. The actions of the growth factor were concentration-dependent over the range from 0.8 to 16 pM and included a similar biphasic effect upon gonadotropin-induced cAMP production. Modulation of cAMP formation and luteinizing hormone receptor expression by transforming growth factor-beta could influence the ability of the granulosa cell to respond to luteinizing hormone during ovarian follicular maturation and ovulation.     

Luteinizing hormone-releasing hormone enhances polyphosphoinositide breakdown in rat granulosa cells

A 2-min addition of LHRH to [3H]inositol-prelabeled rat granulosa cells in primary culture evoked significant increases in the accumulation of [3H]inositol phosphates, i.e. radiolabeled inositol monophosphate (IP), inositol diphosphate (IP2), and inositol triphosphate (IP3) levels increased to 210, 590 and 520%, respectively, when compared to control cultures. By contrast, addition of FSH failed to elicit such a response. The effect of LHRH was completely blocked by the concomitant presence of a specific LHRH antagonist. LHRH evoked increase in [3H]IP3 and [3H]IP2 accumulation as early as 30 sec, while the increase in [3H]IP became significant at 2 min. These data support the hypothesis that polyphosphoinositide breakdown may be an early step in the intracellular signal mechanism which mediates the action of LHRH.     

The effects of gonadotropins on metabolism of isolated rat granulosa cells

FSH $\text{in vitro}$, but not LH, increased the O₂ uptake of isolated granulosa cells from 23 day old rats previously treated with DES or with DES and FSH. Dose response studies showed that the cells were most sensitive to FSH when the cellular binding of FSH was highest. LH increased the O₂ uptake of granulosa cells of untreated 30 day old rats. DES treatment inhibited the LH induced rise in O₂ uptake when the rats were implanted with DES capsules unless FSH was injected to induce LH receptors. Addition of dbcAMP $\text{in vitro}$ increased O₂ uptake of granulosa cells from 30 day old rats at concentrations 10X lower than those required to stimulate O₂ uptake in cells from 23 day old rats treated with DES alone.FSH $\text{in vitro}$ increased lactate formation in the absence of added substrates but did not do so when glucose was added to the media. In contrast, LH greatly increased lactate formation with added glucose. Dose response studies showed that less than 0.6 ug/ml LH S21 was effective in increasing lactate above control levels. These data suggest that FSH affects aerobic pathways while LH affects anaerobic pathways in the process of the differentiation of granulosa cells toward luteal cells.It is well known that FSH and LH interact with their target cells in the ovary by binding to specific receptors and that FSH stimulates LH-receptor production (1). Receptor binding by either hormone activates adenylate cyclase (2) raising cyclic adenosine monosphosphate (cAMP) levels (3) and increasing protein kinase activity (4). Such changes probably trigger changes in the major metabolic pathways that support follicular development because cells of corpora lutea have glycogen (5) which is not present in follicular granulosa cells (6–9). Several studies suggest that FSH and LH may regulate metabolic processes in the ovary. LH increases lactate in whole prepuberal ovaries (10,11,12) and also increases the uptake of glucose (13). FSH increases oxygen uptake in chick ovaries (14), rat ovaries (15) and prairie dog ovaries (16). However, only one study has been done using isolated ovarian cells. Hamberger (17) has reported that FSH increased the oxygen uptake of thecal cells of immature rats while LH increased the oxygen uptake of granulosa cells. Since granulosa cells from immature rats are reported to have FSH receptors while theca cells have LH receptors the effects of these hormones appear unclear.The present studies were undertaken to more accurately characterize the actions of FSH, LH, and dibutyryl cAMP (dbcAMP) on the oxygen uptake of isolated granulosa cells and remaining tissues of immature ovaries and to determine the effects of FSH and LH on the production of lactate by granulosa cells.     

Mechanism of action of luteinizing hormone-releasing hormone in rat ovarian cells

The initial step in the signal transduction of luteinizing hormone-releasing hormone (LHRH) in rat ovarian cells is the hydrolysis of membrane polyphosphoinositides into inositol phosphates and 1,2-diacylglycerol. The former compounds, especially inositol 1,4,5-triphosphate, are known to cause the release of calcium from intracellular stores, while diacylglycerol is a potent activator of protein kinase C. LHRH causes a rapid and transient increase in intracellular concentrations of free calcium ions, by approximately 4.5-fold, in the majority of granulosa cells as assessed by fura-2 microspectrofluorimetry. Like LHRH, a calcium ionophore (A23187) and activators of protein kinase C attenuate the steroidogenic response of the cells to follicle-stimulating hormone, but enhance the formation of gonadotropin-induced prostaglandin formation. These results support the concept that stimulation of polyphosphoinositide hydrolysis is intimately involved in the direct action of LHRH at the level of the ovary.