In vitro effect of leptin on steroids' secretion by FSH- and LH-treated porcine small, medium and large preovulatory follicles

The role of exogenous leptin in the follicular steroidogenesis in pigs has not been fully elucidated and available data are controversial. In the current study porcine follicles were recovered from ovaries during early, middle, and preovulatory stages of the follicular phase of the estrous cycle. Follicles were cultured in the presence of the recombinant ovine leptin (oLEP) with or without LH (100 ng/ml) or FSH (100 ng/ml). Medium estradiol (E(2)), testosterone (T) and progesterone (P(4)) concentrations were determined after 48h of culture. Leptin at a dose of 2 ng/ml had no effect on basal E(2) and T secretion by small and medium follicles but decreased E(2) secretion by large follicles. Significant synergistic action of FSH and leptin resulting in a 2 - 5 fold stimulation of E(2) secretion by small and medium follicles was observed. The aromatase inhibitor, CGS 16949A augmented T secretion and inhibited E(2) secretion by control and FSH-treated medium follicles. In FSH and leptin-treated follicles, the inhibitory action of CGS 16949A on E(2) secretion was observed. However, there was no augmentation of T secretion. In leptin-treated follicles the stimulatory action on P(4) secretion was observed only during the preovulatory stage. In these follicles, significant synergistic action of leptin with LH on P(4) secretion was also noted. These results indicate that there is a maturation-dependent action of leptin on both E(2) and P(4) secretion. They also suggest a synergistic action of leptin and FSH on E(2) secretion by small and medium follicles as well as leptin and LH on P(4) secretion by large follicles in pigs.     

Action of GnRH on steroid secretion by luteal cells of cyclic and early pregnant sows in vitro

The effect of GnRH was studied on progesterone (P4), oestradiol-17 beta (E2) and testosterone (T) secretion by porcine luteal cells from the 13th day of the oestrous cycle and the 18th day of pregnancy. Trypsin-dispersed luteal cells (5 X 10(4) cells/ml) were incubated in medium 199 with 10% calf serum with or without GnRH in doses of 0.1, 1, 10 and 100 mg/ml and with 1 microgram LH and 50 U/ml hCG. The concentration of P4, E2 and T in the medium was estimated by radioimmunological method after 6 hours of incubation. The results showed that GnRH had no effect on the secretion of the investigated steroid hormones by luteal cells from cyclic sows. GnRH at a dose of 10 g inhibited E2 secretion and at a dose of 1 ng T secretion by cells from pregnant sows. LH and hCG stimulated release of P4 by luteal cells in both physiological stages. The conclusion drawn was that GnRH does not act directly on luteal cells of cyclic sows but may inhibit E2 and T secretion by cells of pregnant sows.     

Pulsatile patterns of gonadotropins and ovarian steroids during estrus and pseudopregnancy in the rabbit

Consecutive daily plasma levels of luteinizing hormone (LH), follicle-stimulating hormone (FSH), estradiol-17 beta (E2), progesterone (P4) and 20 alpha-hydroxypregn-4-en-3-one (20 alpha-OHP) were monitored in estrous rabbits and in these same doses during pseudopregnancy (PSP); these daily hormone levels, as well as the immediate post-coital changes in gonadotropin secretion, were similar to those in previous reports. To examine the pulsatile patterns of the gonadotropins and ovarian steroids, sequential, 10-min plasma samples were collected for 6 h from estrous does and on Days 3, 10, and 17 of PSP. All five hormones were measured in the serial samples from estrous and PSP Day 10 does; LH and FSH only were assayed in the remaining sequential samples. The amplitude and frequency of FSH pulses did not differ between any of these stages. In marked contrast, LH pulse amplitudes, and even pulse frequencies in Day 17 does, were profoundly increased during PSP above those in estrous does. Progestin secretions, both P4 and 20 alpha-OHP, also were sharply elevated in PSP Day 10 does as compared with those in estrous rabbits; the pulse amplitudes of both progestins were severalfold higher during PSP. P4 pulse frequencies were also increased at this time. Conversely, the parameters of E2 secretion did not differ between estrous and PSP Day 10 animals. In PSP Day 10 does, high amplitude pulses of both P4 and 20 alpha-OHP occurred simultaneously with high amplitude LH pulses. Simultaneous E2 and P4 pulses were evident in these same sequential plasma samples, and this E2-P4 pulse association was greater than that of 20 alpha-OHP pulses with E2 pulses. Our findings failed to identify conclusively the trophic stimulus for the progestin pulse patterns, but the mechanism may involve the coordinated action of LH and E2. The results do demonstrate that each gonadotropin and ovarian steroid is secreted in a pulsatile manner in both estrous and pseudopregnant rabbits. There are altered profiles in LH and progestin pulses, without major changes in FSH and E2 patterns, between the stages of estrus and PSP. The causes and consequences of these divergent endocrine shifts cannot be deduced from these data.     

Splenic macrophages enhance prolactin and luteinizing hormone action in rat luteal cell cultures.

We have reported that splenic macrophages play a role in the regulation of progestin secretion in rats. In this study, splenic macrophages were obtained from cycling rats at different estrous cycle stages and co-cultured with luteal cells from mid-pseudopregnant rats in the absence/presence of prolactin (PRL) or luteinizing hormone (LH). The effect of macrophages on the luteotropic action of PRL and LH was evaluated with 2 parameters, i.e. an increase in total progestin output (progesterone plus 20 alpha-hydroxyprgn-4-en-one [20 alpha-OHP]), and an increase in the progesterone to 20 alpha-OHP (P/20 alpha-OHP) secretion ratio. Splenic macrophages obtained from proestrous or metestrous rats enhanced the PRL action to increase the P/20 alpha-OHP secretion ratio, but those from estrous or diestrous donors did not. Only macrophages from proestrous donors enhanced the PRL action to increase the total progestin output. In contrast, the LH action increasing the P/20 alpha-OHP secretion ratio was enhanced by splenic macrophages regardless of the donors' estrous cycle stages. The LH action increasing the total progestin output was enhanced only by proestrous or metestrous macrophages. Therefore, if luteal cells are co-cultured with proestrous macrophages, the luteotropic actions of PRL and LH can be fully expressed. These results indicate that splenic macrophages directly act on luteal cells and enhance the luteotropic action of PRL and LH, and that this function of splenic macrophages is modified somehow according to the donors' estrous cycle stages.     

Episodic luteinizing-hormone release in the ovariectomized female guinea pig: rapid inhibition by estrogen

Negative feedback of estrogen was investigated in ovariectomized female guinea pigs. Two weeks after ovariectomy, indwelling catheters were inserted into the jugular vein, and 3 days later, blood samples were taken every 10 min to determine the pattern of luteinizing hormone (LH) secretion. LH secretion in these guinea pigs was episodic, with a mean pulse period of 32 min. The mean pulse amplitude was 2.1 ng/ml, with mean plasma LH levels of 1.8 ng/ml. Twenty-five micrograms 17 beta-estradiol (E2), given i.v., caused a pronounced inhibition of pulsatile LH release. Twenty-five microliters of 100% ethanol (vehicle) had no effect on plasma LH values. In a second set of experiments, ovariectomized female guinea pigs were given two injections of luteinizing hormone-releasing hormone (LHRH) (1 microgram/kg BW, i.v.) separated by 30 min. Sharp rises in serum LH values were detected after each injection. A third injection of LHRH was administered after an injection of either 25 micrograms E2 or 25 microliters vehicle. In the presence of E2, the LH response was significantly (p less than 0.005) diminished, whereas the vehicle did not change the LH response to LHRH. These rapid effects of E2 on LH secretion and the pituitary responsiveness to LHRH infusion indicate that in the ovariectomized guinea pig E2 can directly block gonadotropin secretion. These findings are consistent with the hypothesis that negative feedback actions of E2 are directly on the membrane of the gonadotrope.     

Effect of alterations in pulsatile luteinizing hormone release on ovarian follicular atresia and steroid secretion on diestrus 1 in the rat estrous cycle

This study examined the importance of pulsatile luteinizing hormone (LH) release on diestrus 1 (D1; metestrus) in the rat estrous cycle to ovarian follicular development and estradiol (E2) secretion. Single injections of a luteinizing hormone-releasing hormone (LHRH) antagonist given at -7.5 h prior to the onset of a 3-h blood sampling period on D1 reduced mean blood LH levels by decreasing LH pulse amplitude, while frequency was not altered. Sequential injections at -7.5 and -3.5 h completely eliminated pulsatile LH secretion. Neither treatment altered the total number of follicles/ovary greater than 150 mu in diameter, the number of follicles in any size group between 150 and 551 mu, or plasma E2, progesterone, or follicle-stimulating hormone (FSH) levels. However, both treatments with LHRH antagonist significantly increased the percentage of atretic follicles in the ovary. These data indicate that: 1) pulsatile LH release is an important factor in determining the rate at which follicles undergo atresia on D1; 2) reductions in LH pulse amplitude alone are sufficient to increase the rate of follicular atresia on D1; 3) an absence of pulsatile LH release for a period of up to 10 h on D1 is not sufficient to produce a decline in ovarian E2 secretion, most likely because the atretic process was in its early stages and had not yet affected a sufficient number of E2-secreting granulosa cells to reduce the follicle's capacity to secrete E2; and 4) suppression or elimination of pulsatile LH release on D1 is not associated with diminished FSH secretion.     

Development of steroidogenic activity in the ovary of the prepubertal hamster: II. Production of steroids from steroidal precursors and response in vitro to cyclic adenosine monophosphate and luteinizing hormone

In vitro exposure for 2 h to 250 ng/ml of pregnenolone led to increased production of progesterone and 17 alpha-hydroxyprogesterone (17 alpha-OHP) by hamster ovaries on Days 5, 10 and 15 of age. Similar incubations with 250 ng/ml progesterone or androstenedione caused significant increases in 17 alpha-OHP or testosterone, respectively. When testosterone was added in doses of 32.5, 250 and 500 ng/ml to ovaries on Days 5-30, as early as Day 5 the ovaries aromatized the androgen to estradiol. Day 30 ovaries were the most efficient in the conversion because antral follicles, the principal site for aromatization, were then present. In terms of progesterone production, 400 ng/ml of luteinizing hormone (LH) during 4 h of in vitro incubation stimulated ovaries on Days 5, 10 and 15. Cyclic adenosine 3':5' monophosphate (cAMP) at a dose of 1 mM and 5 mM stimulated progesterone production by Days 5 and 10 ovaries more efficiently than LH. However, Day 15 ovaries produced more progesterone in response to LH compared to cAMP. These experiments establish that the steroidogenic enzymes differentiate at a very early age in the hamster ovary, even before the appearance of gonadotropin receptors. The inability of the early postnatal ovary to produce steroids is apparently attributable to lack of precursors such as cholesterol or cholesterol side chain cleavage enzymes.     

Further studies on in vitro steroidogenesis by luteal cells from long-term hypophysectomized rats

Immature pregnant mare's serum gonadotropin-treated rats were hypophysectomized on the day of ovulation (Day 1 of luteal function), and luteal steroidogenesis and human chorionic gonadotropin (hCG) and prolactin (Prl) binding sites were determined on Days 5, 10, 20 and 30 (H5- H30 ) compared with intact rats on Days 5 or 10 (C5 or C10). On H5, dispersed luteal cells secreted large amounts of progesterone (P), 20 alpha-dihydroprogesterone (20 alpha-DHP), 17 alpha-hydroxyprogesterone (17 alpha-OHP), and small amounts of testosterone (T) and estradiol-17 beta (E2), but between H10 and H30 , reduced levels of all steroids were produced except for 20 alpha-DHP. Addition of large amount of pregnenolone (P5) or P (100-1000 ng) to dispersed luteal cells increased production of P and 20 alpha-DHP in C5 and H5 rats. The higher serum levels and basal in vitro production of 20 alpha-DHP from H5 to H30 indicates that 20 alpha-oxidoreductase persists in the corpora lutea (CL) at high levels and that 3 beta-ol-dehydrogenase is also present but with P rapidly shunted into its principal metabolite. From H5 to H30 , addition of 10 ng P to luteal cells increased the production of 17 alpha-OHP and addition of 10 ng androstenedione (A) or T enhanced production of T and E2, indicating that 17 alpha-oxidoreductase, 17 beta-hydroxysteroid dehydrogenase and aromatase also persist in the CL. In vitro addition of 10 ng LH significantly stimulated production of P from luteal cells on C5 and H5, whereas on C10 and H10, 100 ng LH was required and on H20 and H30 , 1 microgram LH produced a minimal increase in P.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cyclic AMP changes in the component cells of Graafian follicles: possible influences on maturation in the follicle-enclosed oocytes of hamsters

Mature antral follicles were removed from the ovaries of pregnant mare serum gonadotropin (PMSG)-primed hamsters at proestrus prior to the LH surge. Following various incubation times with either LH (ovine) or FSH (rat), cAMP levels were determined in whole follicles, cumulus-oocyte complexes (COCs), and zona-intact or zona-free oocytes. LH produced a dose- and time-dependent change in follicle cAMP but had a minimal effect on the COCs and caused no change in cAMP in zona-free oocytes. By contrast, rFSH stimulated a small rise in follicular cAMP but significantly increased levels in COCs and zona-free oocytes. In a second series of experiments follicles were exposed for short periods to various additives after which they were washed and returned to hormone-free medium for a 6-hr total incubation period. LH (1 microgram/ml) initiated maturation in follicle-enclosed oocytes after a 5- to 15-min exposure period while groups incubated with 100 ng/ml required 60 min. FSH did not stimulate maturation after a 60-min exposure and when combined with 1 microgram or 100 ng/ml of LH negated the maturational effects seen with LH alone. It was postulated that the reason that lower concentrations of LH did not stimulate maturation following short-term incubations was due to an insufficient rise in cAMP. However, neither dbcAMP nor forskolin augmented the capacity of LH to initiate maturation following short-term exposure. By contrast dbcGMP and the guanylate cyclase activator, sodium nitroprusside (NP) did augment the maturation-inducing effects of LH. NP + LH raised cGMP concentrations in the follicle and oocyte and decreased follicular cAMP at 30 and 120 min. The results of this study indicate that the component cells within a follicle respond selectively with cAMP changes, depending on the gonadotropin, in a variable time- and dose-dependent manner. While LH is the more potent activator of cAMP in whole follicles, cAMP levels in the cumulus oophorus and oocyte show the greatest increase following exposure to FSH. LH was the more potent initiator of maturation, possibly through its effects on the mural granulosa cells. FSH appears to exert a more inhibitory role which may be due in part to elevated cAMP levels and/or a putitative inhibitor in the COC and oocyte.     

Second messenger systems and progesterone secretion in the small cells of the bovine corpus luteum: effects of gonadotropins and prostaglandin F2a

The present studies were conducted to determine the effects of gonadotropins (LH and hCG) and prostaglandin F2a (PGF2a) on the production of "second messengers" and progesterone synthesis in purified preparations of bovine small luteal cells. Corpora lutea were removed from heifers during the luteal phase of the normal estrous cycle. Small luteal cells were isolated by unit-gravity sedimentation and were 95-99% pure. LH provoked rapid and sustained increases in the levels of [3H]inositol mono-, bis-, and trisphosphates (IP, IP2, IP3, respectively), cAMP and progesterone in small luteal cells. LiCl (10 mM) enhanced inositol phosphate accumulation in response to LH but had no effect on LH-stimulated cAMP or progesterone accumulation. Time course studies revealed that LH-induced increases in IP3 and cAMP occurred simultaneously and preceded the increases in progesterone secretion. Similar dose-response relationships were observed for inositol phosphate and cAMP accumulation with maximal increases observed with 1-10 micrograms/ml of LH. Progesterone accumulation was maximal at 1-10 ng/ml of LH. LH (1 microgram/ml) and hCG (20 IU/ml) provoked similar increases in inositol phosphate, cAMP and progesterone accumulation in small luteal cells. 8-Bromo-cAMP (2.5 mM) and forskolin (1 microM) increased progesterone synthesis but did not increase inositol phosphate accumulation in 30 min incubations. PGF2a (1 microM) was more effective than LH (1 microgram/ml) at stimulating increases in inositol phosphate accumulation (4.4-fold vs 2.2-fold increase for PGF2a and LH, respectively). The combined effects of LH and PGF2a on accumulation of inositol phosphates were slightly greater than the effects of PGF2a alone. In 30 min incubations, PGF2a had no effect on cAMP accumulation and provoked small increases in progesterone secretion. Additionally, PGF2a treatment had no significant effect on LH-induced cAMP or progesterone accumulation in 30 min incubations of small luteal cells. These findings provide the first evidence that gonadotropins stimulate the cAMP and IP3-diacylglycerol transmembrane signalling systems in bovine small luteal cells. PGF2a stimulated phospholipase C activity in small cells but did not reduce LH-stimulated cAMP or progesterone accumulation. These results also demonstrate that induction of functional luteolysis in vitro requires more than the activation of the phospholipase C-IP3/calcium and -diacylglycerol/protein kinase C transmembrane signalling system.     

LH responses of female naked mole-rats, Heterocephalus glaber, to single and multiple doses of exogenous GnRH

To investigate possible differential pituitary secretion of LH in breeding and non-breeding female naked mole-rats, the LH responses to administration of exogenous GnRH were measured in 55 females from 20 captive colonies. Single doses of 0.1, 0.5 or 1.0 micrograms GnRH produced a significant rise in plasma LH concentrations 20 min after s.c. injection in breeding and non-breeding females at all doses (P less than 0.001). While at the highest dose of 1.0 microgram there was no difference in the LH response between breeding and non-breeding females, as the dose was lowered there was a progressive decline in the LH response in non-breeding females such that, at the 0.1 microgram dose, GnRH produced only a small, but significant, increase in plasma LH (1.3 +/- 0.2 to 2.9 +/- 0.5 mi.u./ml, N = 5) compared with breeding females (3.4 +/- 0.8 to 9.6 +/- 2.0 mi.u./ml, N = 6). The LH responses of the latter were not significantly reduced at the lower doses of GnRH. The apparent lack of sensitivity to low doses of exogenous GnRH in non-breeding females was reversed by 4 consecutive 1-h injections of 0.1 microgram, which produced a rise in LH from 1.2 +/- 0.2 to 9.0 +/- 0.2 mi.u./ml (N = 4), comparable to that of breeding females given a single injection of 0.1 microgram GnRH. These results suggest that the anterior pituitary in non-breeding female naked mole-rats is less sensitive to low doses of exogenous GnRH than in breeding females, possibly due to a lack of priming by endogenous GnRH. Therefore, the socially-induced block to ovulation in non-breeding female naked mole-rats may be due to inhibition of hypothalamic GnRH secretion.     

Biphasic effect of gonadotropin releasing hormone on progestin secretion by rat granulosa cells

The effect of an agonistic gonadotropin releasing hormone (GnRH)-analog (D-Ala6, des-Gly10-NH2-GnRH-ethylamide, GnRHa) on granulosa cell steroidogenesis in the presence or absence of follicle-stimulating hormone (FSH) or luteinizing hormone (LH) was studied. Granulosa cells, isolated from preovulatory follicles of pregnant mare's serum gonadotropin (PMSG)-treated immature rats or from the less mature follicles of untreated immature rats, were cultured for a period of 72 h with daily changes of medium, and progesterone and its metabolite, 20 alpha-dihydro-progesterone (20 alpha-OHP), were assayed in the medium. In granulosa cells from preovulatory follicles, LH and FSH caused a much greater stimulation of steroidogenesis than did GnRHa. There appeared to be no interaction between GnRHa and FSH during the first 10 h, but at 24 h and later the presence of GnRHa clearly inhibited the steroidogenic response to LH and FSH. Steroidogenesis in granulosa cells from immature rats was considerably lower and the effects of GnRHa and FSH alone less pronounced. In these cells, FSH-stimulated progesterone secretion was inhibited by GnRHa only at 72 h. In contrast, 20 alpha-OHP secretion in the same cultures was potentiated by the combined presence of FSH and GnRHa. In conclusion, it seems as though the effects of GnRHa on granulosa cell steroidogenesis varies with exposure time, the initial response being stimulatory and the later inhibitory. Furthermore, the response is also to some extent determined by the maturational stage of the granulosa cells.     

The regulation of steroidogenesis by opioid peptides in porcine theca cells

The present study was designed to investigate basal and LH-induced steroidogenesis in porcine theca cells from large follicles in response to various concentrations (1-1000 nM) of mu opioid receptor agonists (beta-endorphin, DAMGO, FK 33-824), delta receptor agonists (met-enkephalin, leu-enkephalin, DPLPE) and kappa receptor agonists (dynorphin A, dynorphin B, U 50488). Agonists of mu opioid receptors suppressed basal androstenedione (A4), testosterone (T) and oestradiol-17beta (E2) secretion and enhanced LH-induced A4 and T release by theca cells. The inhibitory effect of the agonists on E2 secretion was abolished in the presence of LH. All delta receptor agonists depressed basal progesterone (P4) output. However, the influence of these agents on LH-treated cells was negligible. Among delta receptor agonist used only leu-enkephalin and DPLPE at the lowest concentrations inhibited basal A4 release. The presence of LH in culture media changed the influence of these opioids from inhibitory to stimulatory. Similarly, DPLPE reduced T secretion by non-stimulated theca cells and enhanced T secretion of stimulated cells. All of delta agonists inhibited basal E2 secretion and unaffected its release from LH-treated theca cells. Agonists of kappa receptors inhibited basal, non-stimulated, P4 secretion and two of them (dynorphin B, U 50488) potentiated LH-induced P4 output. Basal A4 and T release remained unaffected by kappa agonist treatment, but the cells cultured in the presence of LH generally increased both androgen production in response to these opioids. Basal secretion of E2 was also suppressed by kappa agonists. This inhibitory effect was not observed when the cells were additionally treated with LH. In view of these findings we suggest that opioid peptides derived from three major opioid precursors may directly participate in the regulation of porcine theca cell steroidogenesis.     

Feedback effects of ovarian steroids on the hypothalamic-hypophyseal axis in the rabbit

The feedback effects of two ovarian steroids, estradiol-17 beta (E2) and 20 alpha-hydroxypregn-4-en-3-one (20 alpha OH), were examined in both intact (INT) and ovariectomized (OVEX) does. We measured steroid-induced alterations in endogenous gonadotropin-releasing hormone (GnRH) from sequential 10-min samples of hypothalamic perfusates, simultaneous changes in peripheral plasma luteinizing hormone (LH) and follicle-stimulating hormone (FSH), and the modification of pituitary responsiveness, i.e., increments in plasma LH (delta LH) and plasma FSH (delta FSH), after 50 ng, 250 ng, and 1 microgram of exogenous GnRH in individual does of 6 treatment groups. The groups were: INT does, OVEX does, OVEX does receiving either one (1 E2) or two (2 E2) E2-filled Silastic capsules, OVEX does receiving a 20 alpha OH-filled capsule (20 alpha OH), and OVEX does receiving both capsules of E2 and 20 alpha OH (1 E2 + 20 alpha OH). Ovariectomy enhanced the pulsatile release of hypothalamic GnRH and pituitary LH and FSH, and increased the LH response (delta LH) to exogenous GnRH (OVEX vs. INT, p less than 0.05). Replacement of E2 at the time of ovariectomy prevented the increased GnRH and gonadotropin secretion as well as the enhanced delta LH that were observed in untreated OVEX does. The release of hypothalamic GnRH in the 20 alpha OH group was lower (p less than 0.05) than that in the OVEX group and not different from that in the INT group. The release of pituitary LH and FSH and the delta LH in the 20 alpha OH group was not different from that in the OVEX group, but these parameters were greater (p less than 0.05) than those in the INT group. The hypothalamic GnRH pulse frequency in the 1 E2 + 20 alpha OH group was lower (p less than 0.05) than that in either the 1 E2 or the 20 alpha OH group, but the delta LH in the 1 E2 + 20 alpha OH group was not different from that in either the 1 E2 or the 20 alpha OH group. The highest dose (1 microgram) of exogenous GnRH stimulated a modest increase in FSH in the OVEX, 20 alpha OH, 1 E2 + 20 alpha OH, and 1 E2 groups; but a steroid effect on delta FSH among these 4 groups was not apparent.(ABSTRACT TRUNCATED AT 250 WORDS)     

Comparison of cyclic AMP production in response to LH by granulosa and theca cells from Meishan and Large-White hybrid gilts.

Previous experiments have demonstrated differences in various follicular characteristics between the prolific Chinese Meishan (MS) pig and European Large-White (LW) hybrids and the present experiment was designed to compare the cAMP response to LH by granulosa and theca cells in vitro between the two breeds. Ovaries were recovered from MS (n = 7) and LW hybrid (n = 8) gilts on the day before predicted oestrus and the 12 largest follicles dissected. Quadruplicate aliquots of granulosa cells or minced theca tissue were incubated for 1 h in the presence of 0, 0.1, 1.0, 10, 100 or 1000 ng/ml LH and cAMP production measured. Follicles from MS gilts were smaller (5.9 vs. 7.7 mm; P < 0.001), contained less fluid (81.5 vs. 177.4 microl; P < 0.001), had fewer granulosa cells (3.8 vs. 5.3 x 10(6); P < 0.01) and less theca tissue (30.3 vs. 50.5 mg; P < 0.05) than those from LW hybrid animals. Mean follicular fluid oestradiol concentration was > or = 149 ng/ml in all animals and tended to be higher in the MS follicles (P = 0.07). LH stimulated cAMP production by granulosa and theca cells from both breeds (P < 0.001). Although there was no overall breed effect for the granulosa cells, there was a significant (P < 0.001) interaction between LH dose and breed in the granulosa cells, whether cAMP production was expressed per 10(6) cells or per follicle. In the theca incubations, cAMP production by MS tissue was higher (P < 0.01) when results were expressed per mg tissue and again there was an interaction (P < 0.001) between LH dose and breed whether cAMP production was expressed per mg tissue or per follicle. For both tissue types, MS follicles produced more cAMP at the higher LH doses. In conclusion, this study has shown that MS granulosa and theca tissue respond differently to increasing doses of LH in terms of cAMP production in vitro compared to LW hybrid tissue and this supports previous suggestions of enhanced maturity of MS follicles in the late follicular phase.     

Estrogen and leptin have differential effects on FSH and LH release in female rats

Prior experiments have shown that the adipocyte hormone leptin can advance puberty in mice. We hypothesized that it would also stimulate gonadotrophin secretion in adults. Since the secretion of follicle stimulating hormone (FSH) and luteinizing hormone (LH) is drastically affected by estrogen, we hypothesized that leptin might have different actions dependent on the dose of estrogen. Consequently in these experiments, we tested the effect of injection of leptin into the third cerebral ventricle of ovariectomized animals injected with either the oil diluent, 10 microg or 50 microg of estradiol benzoate 72 hr prior to the experiment. The animals were ovariectomized 3-4 weeks prior to implantation of a cannula into the third ventricle 1 week before the experiments. The day after implantation of an external jugular catheter, blood samples (0. 3 ml) were collected just before and every 10 min for 2 hr after 3V injection of 5 microl of diluent or 10 microg of leptin. Both doses of estradiol benzoate equally decreased plasma LH concentrations and pulse amplitude, but there was a graded decrease in pulse frequency. In contrast, only the 50-microg dose of estradiol benzoate significantly decreased mean plasma FSH concentrations without significantly changing other parameters of FSH release. The number of LH pulses alone and pulses of both hormones together decreased as the dose of estrogen was increased, whereas the number of pulses of FSH alone significantly increased with the higher dose of estradiol benzoate, demonstrating differential control of LH and FSH secretion by estrogen, consistent with alterations in release of luteinizing hormone releasing hormone (LHRH) and the putative FSH-releasing factor (FSHRF), respectively. The effects of intraventricularly injected leptin were drastically altered by increasing doses of estradiol benzoate. There was no significant effect of intraventricular injection of leptin (10 microg) on the various parameters of either FSH or LH secretion in ovariectomized, oil-injected rats, whereas in those injected with 10 microg of estradiol benzoate there was an increase in the first hr in mean plasma concentration, area under the curve, pulse amplitude, and maximum increase of LH above the starting value (Deltamax) on comparison with the results in the diluent-injected animals in which there was no alteration of these parameters during the 2 hr following injection. The pattern of FSH release was opposite to that of LH and had a different time-course. In the diluent-injected animals, probably because of the stress of injection and frequent blood sampling, there was an initial significant decline in plasma FSH at 20 min after injection, followed by a progressive increase with a significant elevation above the control values at 110 and 120 min. In the leptin-injected animals, mean plasma FSH was nearly constant during the entire experiment, coupled with a significant decrease below values in diluent-injected rats, beginning at 30 min after injection and progressing to a maximal difference at 120 min. Area under the curve, pulse amplitude, and Deltamax of FSH was also decreased in the second hour compared to values in diluent-injected rats. In contrast to the stimulatory effects of intraventricular injection of leptin on pulsatile LH release manifest during the first hour after injection, there was a diametrically opposite, delayed significant decrease in pulsatile FSH release. This differential effect of leptin on FSH and LH release was consistent with differential effects of leptin on LHRH and FSHRF release. Finally, the higher dose of E2 (50 microg) suppressed release of both FSH and LH, but there was little effect of leptin under these conditions, the only effect being a slight (P < 0.04) increase in pulse amplitude of LH in this group of rats. The results indicate that the central effects of leptin on gonadotropin release are strongly dependent on plasma estradiol levels. These effects are consistent w     

Fluctuation in responsiveness of LH and lack of responsiveness of FSH to prolonged infusion of morphine and naloxone in the ewe

In ewes in the mid-luteal phase, LH pulse frequency (P less than 0.01) and amplitude (P less than 0.05) increased during a 24 h infusion of naloxone (0.5 mg/kg/h) compared to a 24 h infusion of vehicle (mean +/- s.e.m.; 0.25 +/- 0.03 vs 0.14 +/- 0.01 pulses/h and 0.84 +/- 0.08 vs 0.55 +/- 0.08 ng/ml serum, respectively). The increase in pulse amplitude was immediate, but was less (P less than 0.05) during the second 12 h, compared to the first 12 h, of naloxone infusion (0.52 +/- 0.14 vs 0.98 +/- 0.08 ng/ml serum). Oestradiol concentrations were higher (P less than 0.01) during naloxone than during control infusion (5.63 +/- 0.26 vs 4.13 +/- 0.15 pg/ml serum). In ovariectomized ewes in the breeding season, LH pulse frequency was lower (P less than 0.01) during a 24 h infusion of morphine (0.5 mg/kg/h) than during a 24 h infusion of vehicle (mean +/- s.e.m.; 1.17 +/- 0.08 vs 1.71 +/- 0.06 pulses/h). We conclude that long-term infusion of naloxone results in a sustained increase in LH pulse frequency but only a transient elevation in pulse amplitude. No effects on FSH secretion were noted. LH secretion was sensitive to morphine in the absence of ovarian steroids, suggesting that ovarian steroids are not required for the presence of functional opioid receptors capable of modulating LH release.     

Regulation of thyroid hormones on the production of testosterone in rats

The effects of a thyroidectomy and thyroxine (T4) replacement on the spontaneous and human chorionic gonadotropin (hCG)-stimulated secretion of testosterone and the production of adenosine 3',5'-cyclic monophosphate (cAMP) in rat testes were studied. Thyroidectomy decreased the basal levels of plasma luteinizing hormone (LH) and testosterone, which delayed the maximal response of testosterone to gonadotropin-releasing hormone (GnRH) and hCG in male rats. T4 replacement in thyroparathyroidectomized (Tx) rats restored the concentrations of plasma LH and testosterone to euthyroid levels. Thyroidectomy decreased the basal release of hypothalamic GnRH, pituitary LH, and testicular testosterone as well as the LH response to GnRH and testosterone response to hCG in vitro. T4 replacement in Tx rats restored the in vitro release of GnRH, GnRH-stimulated LH release as well as hCG-stimulated testosterone release. Administration of T4 in vitro restored the release of testosterone by rat testicular interstitial cells (TICs). The increase of testosterone release in response to forskolin and androstenedione was less in TICs from Tx rats than in that from sham Tx rats. Administration of nifedipine in vitro resulted in a decrease of testosterone release by TICs from sham Tx but not from Tx rats. The basal level of cAMP in TICs was decreased by thyroidectomy. The increased accumulation of cAMP in TICs following administration of forskolin was eliminated in Tx rats. T4 replacement in Tx restored the testosterone response to forskolin. But the testosterone response to androstenedione and the cAMP response to forskolin in TICs was not restored by T4 in Tx rats. These results suggest that the inhibitory effect of a thyroidectomy on the production of testosterone in rat TICs is in part due to: 1) the decreased basal secretion of pituitary LH and its response to GnRH; 2) the decreased response of TICs to gonadotropin; and 3) the diminished production of cAMP, influx of calcium, and activity of 17beta-HSD. T4 may enhance testosterone production by acting directly at the testicular interstitial cells of Tx rats.     

Effects of follicle stimulating hormone, cholera toxin, pertussis toxin and forskolin on adenosine cyclic 3',5'-monophosphate output by granulosa cells from Booroola ewes with or without the F gene

The cAMP outputs by granulosa cells from 3-4.5 mm diameter (medium) follicles of Booroola FF ewes were similar to those by cells from greater than or equal to 5 mm diameter (large) follicles of ++ ewes with respect to time or dose of FSH, cholera toxin or forskolin. Likewise, the cAMP outputs by cells from 1-2.5 mm diameter (small) FF follicles were similar to those by cells from small and medium ++ follicles with respect to time or dose of FSH, cholera toxin or forskolin. At FSH, cholera toxin or forskolin doses of 1 microgram/ml, 0.5 microgram/ml and 10(-4) M respectively, the granulosa cell cAMP outputs of medium FF or large ++ follicles were approximately 2-fold (P less than 0.05) higher than in the respective small FF and medium ++ follicles. The effects of cholera toxin plus forskolin or FSH plus forskolin were additive irrespective of genotype or follicle size, with significant differences (P less than 0.05) observed between follicle sizes but not genotype. No differences were noted between cholera toxin plus forskolin or FSH plus forskolin on granulosa cell cAMP output. For the FSH and forskolin treatments, increased mean cAMP outputs were evident after 10 min, whereas after cholera toxin treatment they were not evident until after 20 min incubation. For all treatments the rate of cAMP production tended to slow down after 40-60 min. Pre-incubation of granulosa cells with pertussis toxin subsequently resulted in a significantly greater (P less than 0.05) FSH-induced output of cAMP relative to the untreated controls irrespective of follicle size. However, no gene-specific differences were noted when the cAMP outputs of cells from medium or small FF follicles were compared with cells from large or small-medium ++ follicles respectively. These results indicate that the activity (or composition) of the regulatory and catalytic components of adenylate cyclase in the FF granulosa cells change in a manner similar to those observed in ++ cells with the only difference being that the increases in cyclase in FF ewes occurs as follicles enlarge from 1-2.5 to 3-4.5 mm in diameter, whereas in ++ ewes they occur as follicles enlarge from 3-4.5 to greater than or equal to 5 mm in diameter. No evidence was found to link the F gene to the granulosa cell cAMP response independently of follicle size. It is suggested that the association between the F gene and the size-specific difference in follicle maturation may be unrelated to the FSH receptor/cAMP generating system.     

Follicular fluid modulation of functional LH receptor induction in pig granulosa cells

Follicular fluid was collected from small (1-2 mm), medium (3-5 mm) and large (6-12 mm) follicles of pigs, treated with charcoal to remove steroids, and tested for effects on the induction of functional LH/hCG receptors in cultures of granulosa cells from small antral pig follicles. Granulosa cells were cultured for 2, 4 or 6 days in Medium 199 + 10% pig serum. Granulosa cells cultured in the presence of purified human FSH (0.1 microgram/ml, LER 8/117), insulin (1 mU/ml), cortisol (0.01 microgram/ml) and thyroxine (10(-7) M) accumulated a 4- to 8-fold increase in LH/hCG receptors compared to control cultures. The amounts of cyclic AMP and progesterone secreted after exposure to ovine LH (1 microgram/ml: NIH-S19) were also increased 2-3-fold and 80-100-fold, respectively. Exposure to FSH alone resulted in lower amounts of LH/hCG receptors with a concomitant decrease in optimum LH responses. Addition of 12.5-50% follicular fluid obtained from small (1-2 mm) follicles led to a dose-dependent inhibition of the FSH plus insulin, cortisol and thyroxine induction of LH/hCG receptors after 4 days of culture. Fluid from medium follicles showed reduced ability to inhibit LH/hCG receptor induction, and fluid from large follicles exerted only a slight inhibition or no inhibition of receptor induction. Fluid from medium-sized and large follicles exerted a progressive dose-dependent stimulation of progesterone secretion by the granulosa cell cultures. The inhibitory activity was precipitated primarily with 70% ethanol and to a lesser degree by 36 and 90% ethanol. These studies demonstrate that induction of functional LH/hCG receptors in cultures of pig granulosa cells from immature follicles is enhanced by including insulin, cortisol and thyroxine, in addition to FSH, in the culture medium, and that follicular fluid modulates both receptor induction and progesterone secretion as a function of follicular maturation.