Early maturity in the male striped bass, Morone saxatilis: follicle-stimulating hormone and luteinizing hormone gene expression and their regulation by gonadotropin-releasing hormone analogue and testosterone

Striped bass are seasonal breeding fish, spawning once a year during the spring. All 3-yr-old males are sexually mature; however, 60-64% of the fish mature earlier as 1- or 2-yr-old animals. The endocrine basis underlying early maturity in 2-yr-old males was studied at the molecular level by monitoring changes in pituitary beta FSH and beta LH mRNA levels by ribonuclease protection assay, and correlating these changes to stages of testicular development. In maturing males, the mRNA levels of beta FSH were elevated during early spermatogenesis, whereas beta LH mRNA levels peaked during spermiation. The appearance of spermatozoa in the testis was associated with a decrease in beta FSH mRNA and a rise in beta LH mRNA abundance. Immature males had lower levels of beta LH mRNA than maturing males, but there were no differences in beta FSH mRNA levels between immature and maturing males. The regulation of gonadotropin gene expression in 2-yr-old males was studied by the chronic administration of GnRH analogue (GnRHa) and testosterone (T), with or without pimozide (P) supplementation. In immature males, the combination of T and GnRHa stimulated a three- to fivefold increase in beta FSH and beta LH mRNA levels, but the same treatment had no effect on gonadotropin gene expression in maturing males. In addition, the coadministration of P to immature males suppressed the stimulatory effect of GnRHa and T on beta FSH and beta LH mRNA levels, suggesting that dopamine may have a novel role in regulating gonadotropin gene expression.     

The effect of a GnRH agonist on follicular dynamics and response to FSH stimulation in prepubertal calves

Endocrine control of follicular growth was determined by observing the left ovary of prepubertal calves previously treated with a potent GnRH agonist for 13 days. The ovarian response to hormonal stimulation was determined using the right ovaries of the same animals. Three-month-old crossbred calves were assigned to one of the two following treatment groups: 1) saline control for 13 days, with purified porcine FSH for the last 3 days (n = 5); and 2) GnRHa for 13 days, with purified porcine FSH for the final 3 days (n = 5). The left ovaries were removed from all calves after 10 days, and the right ovaries were removed at the end of treatment. Plasma concentrations of FSH, LH and oestradiol-17 beta were followed up during the GnRHa and pFSH treatments. The maximum macroscopic diameter of the F1 follicle, as determined by daily ultrasonography, did not differ between GnRHa-treated calves (from 6.6 to 10.4 mm) and the saline control calves (from 6.7 to 10.3 mm). Histological analysis of the ovaries showed that the number of follicles > 0.40 mm in diameter varied greatly for calves of the two groups (from 11 to 220 at 10 days). GnRHa significantly increased the mean number of follicles (total and nonatretic) of size class > 5.4 mm as compared to saline control calves (P < 0.05). The FSH treatment significantly increased the mean number of follicles 3.00-5.4 and > 5.4 mm in diameter (P < 0.05), with no change in the number of follicles smaller than 3.00 mm. The rate of atresia of large follicles (3.01-5.40 mm) was significantly reduced by purified porcine FSH treatment in both groups (P < 0.05). In no case did the GnRHa induce ovulation or luteinization of follicles. The LH and FSH concentrations increased transiently after GnRHa treatment on the first day, but afterwards, both hormones increased to only one sixth of what was observed after the initial GnRHa injection treatment. This increase in LH and FSH was observed 1 h after GnRHa treatment on each consecutive day of the experiment and were significantly different in the control group (0 h versus 1 h versus 2 h x saline control versus GnRH agonists groups; P < 0.01). During the superovulatory treatment, FSH concentrations peaked at around 0.70 ng.mL-1 in both saline- and GnRHa-treated groups on the first day but on the last day of surovulatory treatment, FSH concentrations were higher in GnRHa agonist-treated calves than in the control calves (day 11 versus day 12 versus day 13 x saline control versus GnRH agonist treatment groups; P < 0.01). LH profiles were unchanged by surovulatory treatment. Concentrations of oestradiol-17 beta increased significantly over the three days (P < 0.001) of the superovulatory treatments in both groups (P < 0.01). These results indicate that GnRH agonist treatment allows recruited antral follicles to pursue their growth during the early selection process via sustained FSH and LH secretion allowing more than a single large follicle to maintain their growth without going to atresia.     

Reversible,long-term inhibition of ovulation with a gonadotropin-releasing hormone antagonist in the marmoset monkey (Callithrix jacchus)

The effects of weekly injections of a gonadotropin-releasing hormone (GnRH) antagonist (GnRHa) ([N-acetyl-DβNal1-D-pCl-Phe2-D-Phe3-D-Arg6-Phe7-Arg8D-Ala10] NH2 GnRH) on pituitary and ovarian function were examined in the marmoset monkey, Callithrix jacchus. In experiment 1, five cyclic females were given weekly injections of vehicle (50% propylene glycol in saline) for 6 weeks followed by GnRHa for 20 weeks, animals receiving either 200 μg GnRHa/injection (n = 2) or 67 μg GnRHa/injection (n = 3) for 10 weeks, after which the treatment was reversed. Bioactive luteinizing hormone (LH) and progesterone (Po) were measured in blood samples (0.2–0.4 ml) collected twice weekly until at least 8 weeks after the last GnRHa injection. GnRHa treatment, timed to begin in the midluteal phase, caused a rapid decline in LH and Po and luteal regression after a single injection (both doses). Po levels were consistently low (<10 ng/ml), and ovulation was inhibited throughout 200 μg treatment in all animals. Short periods of elevated Po (>10 ng/ml) were, however, occasionally seen during 67 μg treatment, indicating incomplete ovarian suppression. Mean LH levels were significantly lower during GnRHa treatment compared with the period of vehicle injection (all animals 200 μg; three animals 67 μg), and there were significant differences in LH levels between GnRHa treatments (200 μg vs. 67 μg) in four animals. Four animals resumed normal ovarian cycles after the end of GnRHa treatment (15/16 days, three animals; 59 days, one animal); the fifth animal died of unknown causes 32 days after the last GnRHa injection. In a second experiment, pituitary responsiveness to exogenous GnRH was tested 1 day after a single injection of vehicle or antagonist (200 or 67 μg). Measurement of bioactive LH indicated that pituitary response to 200 ng native GnRH was significantly suppressed in animals receiving the antagonist, the degree of suppression being dose related. A third experiment examined the effect of four weekly injections of 200 μg GnRHa on follicular size and granulosa cell responsiveness to human follicle-stimulating hormone (hFSH) in vitro. Follicular development beyond 1 mm was inhibited by GnRHa treatment (preovulatory follicles normally 2-4 mm) although granulosa cell responsiveness to FSH during 48 hr of culture was not impaired. These results suggest that the GnRHa-induced suppression of follicular development and ovulation was mediated primarily by an inhibition of pituitary gonadotropin secretion and not by a direct action at the level of the ovary.     

Divergent responses of gonadotropin subunit messenger RNAs to continuous versus pulsatile gonadotropin-releasing hormone in vitro

Episodic GnRH input is necessary for the maintenance of LH and FSH secretion. In the current study we have assessed the requirement of a pulsatile GnRH signal for the regulation of gonadotropin alpha- and beta-subunit gene expression. Using a dispersed rat pituitary perifusion system, GnRH (10 nM) was administered as a continuous infusion vs. hourly pulses. Secretion of free alpha-subunit, LH, and FSH were monitored over 5-min intervals for the entire 12-h treatment period before the responses of alpha, LH beta, and FSH beta mRNAs were assessed. Basal release of all three glycoproteins declined slowly over 6-8 h before reaching a plateau. The cells were responsive to each pulse of GnRH, but continuous GnRH elicited only a brief episode of free alpha-subunit, LH, and FSH release, followed by a return to unstimulated levels. Despite the similar patterns of secretion, differences were observed in the responses of gonadotropin mRNAs to the two modes of GnRH. alpha mRNA increased in response to continuous (1.6-fold) or pulsatile (1.7-fold) GnRH. FSH beta mRNA was suppressed to 48% of the control value after continuous GnRH, but was stimulated over 4-fold by the pulses. LH beta mRNA was unresponsive to either treatment paradigm. We conclude that in vitro 1) alpha mRNA levels are increased in response to GnRH independent of the mode of stimulation; 2) under the conditions studied, LH beta mRNA levels are unresponsive to either mode of GnRH input; and 3) the response of FSH beta mRNA to GnRH is highly dependent on the mode of administration, with levels depressed in response to continuous GnRH, but stimulated by pulsatile GnRH.(ABSTRACT TRUNCATED AT 250 WORDS)     

Disruption of neuroendocrine control of luteinizing hormone secretion by aroclor 1254 involves inhibition of hypothalamic tryptophan hydroxylase activity

Mechanisms governing the effect of polychlorinated biphenyl (PCB) toxicity on hypothalamic serotonergic function and the neuroendocrine system controlling LH secretion were investigated in Atlantic croaker (Micropogonias unulatus) exposed to the PCB mixture Aroclor 1254 (1 microg x g body weight(-1) x day(-1)) in the diet for 30 days. PCB treatment caused a decrease in hypothalamic 5-hydroxytryptamine (5-HT) concentrations and significant inhibition of hypothalamic tryptophan hydroxylase (TPH), the rate-limiting enzyme in 5-HT synthesis, but did not alter the activity of monoamine oxidase, the catabolic enzyme. Further, PCB treatment caused significant decreases in GnRH content in the preoptic-anterior hypothalamic area. Significant decreases in pituitary GnRH receptor concentrations and the LH response to the GnRH analogue (GnRHa) were also observed in PCB-exposed fish, possibly as a consequence of a decline in GnRH release. The possible association between impaired serotonergic and neuroendocrine functions after PCB treatment was explored using serotonergic drugs. Treatment of croaker with p-chlorophenylalanine, an irreversible TPH inhibitor, mimicked the effects of PCB on the GnRH system and the LH response to GnRHa. Bypassing the TPH-dependent hydroxylation step with the administration of 5-hydroxytryptophan restored 5-HT to control levels and prevented the deleterious effects of PCB on the neuroendocrine parameters. Moreover, slow-release GnRH implants prevented the PCB-induced decline in GnRH receptors and restored the LH response to GnRHa, suggesting that GnRH therapy can reverse PCB-induced disruption of LH secretion. These results demonstrate that TPH is one of the targets of PCB neurotoxicity and indicate that a decrease in 5-HT availability in PCB-exposed croaker results in disruption of the stimulatory 5-HT/GnRH pathway controlling LH secretion.     

Phorbol ester-induced LH release in pituitary gonadotrophs: effects of antagonists of calmodulin and GnRH.

The phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA), a potent activator of Ca(2+)- and phospholipid-dependent protein kinase (C kinase), stimulates luteinizing hormone (LH) release from rat pituitary cells. The actions of TPA upon LH release were compared with those of the GnRH superagonist [D-Ala6] des-Gly10-GnRH N-ethylamide (GnRHa) in cultured pituitary cells. LH release was stimulated by 0.1 nM TPA and the maximum response at 10 nM TPA was 50% of the LH response to GnRHa. The ED50 values for TPA and GnRHa were 1.2 and 0.037 nM, respectively, and the maximum stimulatory effects of TPA and GnRHa on LH release were not additive. GnRHa-stimulated LH release was decreased by calmodulin (CaM) antagonists including pimozide, trifluoperazine, W5 and W7, being most effectively reduced (by 70%) by 10 microM pimozide. In contrast to their inhibition of GnRH action, these antagonists enhanced TPA-stimulated LH release, so that 10 microM pimozide and W7 doubled the maximum LH response. The potent GnRH antagonist [Ac-D-p-Cl-Phe1.2, D-Trp3, D-Lys6, D-Ala10]GnRH, which completely inhibited GnRHa-stimulated LH release with ID50 of 6.8 nM, also reduced maximum TPA-stimulated LH release by about 50%. These results suggest that both Ca2+/CaM and C kinase pathways are involved in the LH release mechanism, and indicate that C kinase plays a major role in the action of GnRH upon gonadotropin secretion. The synergism between CaM antagonists and TPA suggests that blockade of CaM-mediated processes leads to enhanced activation of the C kinase pathway, possibly by removal of an inhibitory influence. Furthermore, the partial inhibition of TPA-stimulated LH release by a GnRH antagonist suggests that the pathway(s), specifically connected with LH release in the diverse effects of C kinase, might be locked by the continuous receptor inactivation by antagonist and indicates the complicated pathways which diverge from the receptor and converge into specific cellular response.     

Differential regulation of gonadotropin subunit messenger ribonucleic acids by gonadotropin-releasing hormone pulse frequency in ewes

Changes in the frequency of GnRH and LH pulses have been shown to occur between the luteal and preovulatory periods in the ovine estrous cycle. We examined the effect of these different frequencies of GnRH pulses on pituitary concentrations of LH and FSH subunit mRNAs. Eighteen ovariectomized ewes were implanted with progesterone to eliminate endogenous GnRH release during the nonbreeding season. These animals then received 3 ng/kg body weight GnRH in frequencies of once every 4, 1, or 0.5 h for 4 days. These frequencies represent those observed during the luteal and follicular phases, and the preovulatory LH and FSH surge of the ovine estrous cycle, respectively. On day 4, the ewes were killed and their anterior pituitary glands were removed for measurements of pituitary LH, FSH, and their subunit mRNAs. Pituitary content of LH and FSH, as assessed by RIA, did not change (P greater than 0.10) in response to the three different GnRH pulse frequencies. However, subunit mRNA concentrations, assessed by solution hybridization assays and expressed as femtomoles per mg total RNA, did change as a result of different GnRH frequencies. alpha mRNA concentrations were higher (P less than 0.05) when the GnRH pulse frequency was 1/0.5 h and 1 h, whereas LH beta and FSH beta mRNA concentrations were maximal (P less than 0.05) only at a pulse frequency of 1/h. Additionally, pituitary LH and FSH secretory response to GnRH on day 4 was maximal (P = 0.05) when the pulse infusion was 1/h.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Activation of the luteinizing hormone beta promoter by gonadotropin-releasing hormone requires c-Jun NH2-terminal protein kinase

Regulation of the mitogen-activated protein kinase (MAPK) family by gonadotropin-releasing hormone (GnRH) in the gonadotrope cell line LbetaT2 was investigated. Treatment with gonadotropin-releasing hormone agonist (GnRHa) activates extracellular signal-regulated kinase (ERK) and c-Jun NH(2)-terminal kinase (JNK). Activation of ERK by GnRHa occurred within 5 min, and declined thereafter, whereas activation of JNK by GnRHa occurred with a different time frame, i.e. it was detectable at 5 min, reached a plateau at 30 min, and declined thereafter. GnRHa-induced ERK activation was dependent on protein kinase C or extracellular and intracellular Ca(2+), whereas GnRHa-induced JNK activation was not dependent on protein kinase C or on extracellular or intracellular Ca(2+). To determine whether a mitogen-activated protein kinase family cascade regulates rat luteinizing hormone beta (LHbeta) promoter activity, we transfected the rat LHbeta (-156 to +7)-luciferase construct into LbetaT2 cells. GnRH activated the rat LHbeta promoter activity in a time-dependent manner. Neither treatment with a mitogen-activated protein kinase/ERK kinase (MEK) inhibitor, PD98059, nor cotransfection with a catalytically inactive form of a mitogen-activated protein kinase construct inhibited the induction of the rat LHbeta promoter by GnRH. Furthermore, cotransfection with a dominant negative Ets had no effect on the response of the rat LHbeta promoter to GnRH. On the other hand, cotransfection with either dominant negative JNK or dominant negative c-Jun significantly inhibited the induction of the rat LHbeta promoter by GnRH. In addition, GnRH did not induce either the rat LHbeta promoter activity in LbetaT2 cells transfected stably with dominant negative c-Jun. These results suggest that GnRHa differentially activates ERK and JNK, and a JNK cascade is necessary to elicit the rat LHbeta promoter activity in a c-Jun-dependent mechanism in LbetaT2 cells.     

A gonadotropin-releasing hormone (GnRH) antagonist distinguishes three populations of GnRH analog-responsive cells in human and rat pituitary in vitro and produces an acute increase in intracellular Ca2+ concentration without inducing gonadotropin secretion

We have examined the actions of the potent GnRH antagonist [N-acetyl-D beta Na11-D-pCl-Phe2-D-Phe3-D-Arg6,Phe7,Arg8-D-Ala10]NH2GnRH++ + (GnRHa) on basal and GnRH-stimulated LH secretion, inositol phospholipid turnover, and intracellular Ca2+ levels in dispersed rat anterior pituitary tissue. As expected, GnRHa was found to be a pure antagonist of secretion, but was paradoxically equipotent with GnRH in stimulating inositol phospholipid turnover. Examination of intracellular Ca2+ changes at the single cell level using digital video-enhanced fluorescence imaging demonstrated that dispersed rat pituitary cells appeared to contain three GnRH analog-responsive cell populations: those that increased intracellular Ca2+ in response to both GnRH and GnRHa, and those that responded to either GnRH or GnRHa only. These observations were extended to studies of the relatively homogeneous gonadotroph cell populations of endocrinologically inactive pituitary adenomas. Of five adenomas examined, one increased inositol phospholipid turnover in response to GnRHa plus GnRH, but not GnRH alone, three responded to GnRH only, and one responded to both GnRH and GnRHa. Our findings, therefore, suggest that three GnRH analog-responsive cell types are also present in human pituitary and that clonal expansion of any of these cell types may be responsible for tumor formation.     

The effect of estradiol-17beta and estrone administration on GnRH-induced LH release during the early postpartum in dairy cattle

The effect of high plasma concentrations of estradiol-17beta or estrone, similar to those observed in late gestation, on the gonadotropin releasing hormone (GnRH)-induced luteinizing hormone (LH) release was studied in early postpartum dairy cows. Twenty dairy cows in late gestation were assigned to four groups of five cows each. Treatment groups were 1) no exogenous estrogens, 2) 20 mg estradiol-17beta (E(2)beta) daily, 3) 30 mg estrone (E(1)) daily and 4) 20 mg E(2)beta and 30 mg E(1) daily. Steroids were dissolved in ethanol (vehicle). Injections of the vehicle or steroids were given in two daily subcutaneous injections for seven consecutive days starting immediately following parturition. All cows (Groups 1-4) were given 100 mug GnRH intramuscularly on days 2, 10, 18 and 26 postpartum. Blood for plasma determination of E(2)beta, E(1), progesterone (P) and LH was collected daily from parturition to completion of vehicle or steroid injection and on alternate days thereafter. In addition, blood was collected on GnRH treatment days prior to GnRH and at 30-min intervals thereafter for four hours. Concentrations of hormones were determined by validated radioimmunoassays (RIA's). Effects of treatment (T), days postpartum (D) and the interaction between T and D (T x D) on the amount of LH released (area under the curve) in response to GnRH were significant (P < 0.01). More LH was released over all days combined in Group 1 compared to the other groups. LH release to GnRH increased as time postpartum increased in Groups 1 and 3, but at a ratelower for Group 3 than Group 1 (P < 0.05). In contrast, LH release to GnRH was greater (P < 0.05) on day 2 postpartum for Groups 2 and 4 compared to Groups 1 and 3, but less on days 10 and 18 postpartum. Average LH release was less (P < 0.05) on day 10 for Groups 2 and 4 than for day 2 postpartum. By day 26 postpartum, however, LH release in Groups 2 and 4 was greater than in Group 3. In summary, E(2)beta appeared to stimulate LH release early postpartum with a subsequent inhibition of LH release after prolonged E(2)beta administration, and E(1) administration did not stimulate LH release early postpartum.     

Influence of GnRH infusion on endocrine parameters and duration of postpartum anestrus in beef cows

The effect of an intravenous infusion of gonadotrophin releasing hormone (GnRH) on the duration of postpartum anestrus in suckled beef cows was studied. Twenty-eight, mature, suckled beef cows were assigned in equal numbers to one of four treatment groups which were based on infusion with saline or GnRH (15ug/hour for 12 hours) and stage postpartum (pp) (20 or 35 days). Serum LH and progesterone were determined by radioimmunoassay for the period which began 5 days pre-infusion and ended at 55 days postpartum (ie: 35 or 20 days post-infusion). Serum LH remained below 5ng/ml during infusion in all control cows. Peak serum LH values, times of LH peaks, and duration of LH responses (means +/- SE) during infusion were 49 +/- 12 ng/ml, 162 +/- 42 minutes and 7.8 +/- 1.3 hours for the 20 day group and 44 +/- ng/ml, 144 +/- 6 minutes, and 8.2 +/- 1.1 hours for the 35 day group respectively. Serum progesterone levels indicated that the proportion of cows showing the onset of estrous cycles within 10 days of infusion was greater in the 20 day pp GnRH group (4/7) than the 20 day pp saline group (0/7) (p < .05) but was not significantly different between the 35 day pp GnRH (4/7) and 35 day pp saline (2/6) groups. The incidence of estrus was not affected by GnRH treatment and was 37% in all cows prior to 55 days pp. It was concluded that infusions of GnRH for 12 hours at a rate of 15 ug/hour could induce estrous cycles in suckled beef cows treated at 20 days postpartum.     

Effect of stress-like concentrations of cortisol on the feedback potency of oestradiol in orchidectomized sheep

The effect of stress-like concentrations of cortisol on oestradiol-induced change in LH secretion and GnRH receptor expression was evaluated in orchidectomized sheep (wethers). Twenty-four wethers were assigned at random to one of the four treatment groups in a 2x2 factorial design (n=6 wethers/group). Wethers received cortisol (90 microg/kg/h; groups 2 and 4) or a comparable volume of cortisol delivery vehicle (groups 1 and 3) by continuous infusion for 48 h. During the final 24 h of infusion, wethers received oestradiol (6 ng/kg/h; groups 3 and 4) or oestradiol delivery vehicle (groups 1 and 2). The pattern of LH secretion was assessed during a 3-h period of intensive blood collection beginning 21 h after initiation of oestradiol infusion. Although neither cortisol nor oestradiol alone affected (P>0.05) mean serum concentration of LH or LH pulse frequency, serum LH and the frequency of secretory episodes of LH were significantly reduced (P<0.05) in wethers receiving cortisol and oestradiol in combination. Anterior pituitary tissue was collected at the end of the infusion period. Oestradiol increased (P<0.05) tissue concentrations of GnRH receptor and GnRH receptor mRNA. Although cortisol alone did not affect (P>0.05) basal concentrations of receptor or receptor mRNA, the magnitude of oestradiol-induced increase in GnRH receptor and GnRH receptor mRNA was significantly reduced in wethers receiving cortisol and oestradiol concurrently. Conversely, steady-state concentrations of mRNA encoding the LHbeta and FSHbeta subunits were increased (P<0.05) in wethers receiving cortisol. These observations demonstrate that stress-like concentrations of cortisol act in concert with oestradiol to suppress LH secretion. In addition, cortisol blocks oestradiol-dependent increase in pituitary tissue concentrations of GnRH receptor and GnRH receptor mRNA.     

Comparable testosterone responses are produced by constant infusion of LH or GnRH in normal men

Luteinizing hormone (LH) was infused continuously at a rate of 1.3 IU/min to 4 normal adult men. A 4 to 5-fold increase in serum LH was noted by 8 hours. Serum FSH declined steadily throughout the infusion period in the face of rising concentrations of gonadal steroids. Basal plasma testosterone of 4.7 +/- 0.4 ng/ml rose progressively to a peak of 11.1 +/- 0.9 ng/ml at hour 56 (p less than 0.005). A similar pattern was demonstrated by plasma androstenedione. Plasma 17 alpha-hydroxyprogesterone rose from a basal concentration of 0.81 +/- 0.14 ng/ml to a peak concentration of 2.6 +/- 0.3 ng/ml at hour 36 of the infusion and subsequently declined. A similar course was followed by serum estradiol-17 beta, which achieved a maximal concentration of 70.0 +/- 10.4 pg/ml at hour 36. Results are compared to those obtained with continuous infusion of GnRH in normal adult men. Testosterone responses were similar, whereas elevations in 17 alpha-hydroxyprogesterone and estradiol were higher following GnRH infusion. This difference may be consequent upon a direct gonadal effect of GnRH, or may be secondary to local regulation of testicular steroidogenesis by estradiol-17 beta.     

Progesterone does not affect the amount of mRNA for gonadotropins in the anterior pituitary gland of ovariectomized ewes

To evaluate the effect of progesterone on the synthesis and secretion of gonadotropins, ovariectomized ewes either were treated with progesterone (n = 5) for 3 wk or served as controls (n = 5) during the anestrous season. After treatment for 3 wk, blood samples were collected from progesterone-treated and ovariectomized ewes. After collection of blood samples, hypothalamic and hypophyseal tissues were collected from all ewes. Half of each pituitary was used to determine the content of luteinizing hormone (LH) and follicle-stimulating hormone (FSH), and the number of receptors for gonadotropin-releasing hormone (GnRH). The amounts of mRNA for LH beta subunit, FSH beta subunit, alpha subunit, growth hormone, and prolactin were measured in the other half of each pituitary. Treatment with progesterone reduced mean serum concentrations of LH (p less than 0.001) but ot FSH (p greater than 0.05). Further, progesterone decreased (p less than 0.05) the total number of pulses of LH. We were unable to detect pulsatile release of FSH. Hypothalamic content of GnRH, number of receptors for GnRH, pituitary content of gonadotropins and mRNA for LH beta subunit, FSH beta subunit, alpha subunit, growth hormone, and prolactin were not affected (p greater than 0.05) by treatment with progesterone. Thus, after treatment with progesterone, serum concentrations of LH (but not FSH) are decreased. This effect, however, is not due to a decrease in the steady-state amount of mRNA for LH beta or alpha subunits.     

Epidermal growth factor and gonadotropin-releasing hormone inhibit cyclic AMP–dependent luteinizing hormone receptor formation in ovarian granulosa cells

The induction of luteinizing hormone (LH) receptors was studied in granulosa cells prepared from the ovaries of hypophysectomized diethylstilbestrol-treated immature rats. Incubation of granulosa cells for 48 h with increasing concentrations of follicle-stimulating hormone (FSH) or choleragen caused parallel rises in cAMP levels and LH receptors. These observations, with the finding that 8-Bromo-cAMP also induced LH receptor formation, indicate that hormonal stimulation of LH binding sites is mediated by cAMP. Peptide hormones that inhibited FSH-stimulated cAMP production, such as epidermal growth factor (EGF) and a gonadotropin-releasing hormone agonist (GnRHa), also prevented LH receptor formation. GnRHa and EGF had negligible effects on FSH-stimulated cAMP production from 0 to 24 h of culture, but reduced cAMP accumulation by 80% and 90%, respectively, from 24 to 48 h when the majority of LH receptors appeared. FSH-sensitive adenylate cyclase activity, as measured by the conversion of (³H)-ATP to (³H)-cAMP, was inhibited by GnRHa and EGF at 48 h of culture. EGF and GnRHa also reversed the inhibition of ectophosphodiesterase activity caused by FSH in granulosa cells between 48 and 72 h of culture. Both EGF and GnRHa inhibited induction of LH receptors by 8-Bromo-cAMP, suggesting that their effects are also on cAMP action. Addition of GnRHa, but not EGF, between 36 and 48 h of culture completely prevented further increases in LH receptors induced by 8-Bromo-cAMP, indicating that the inhibitory action of GnRHa can be initiated at later times during granulosa cell differentiation, whereas full expression of EGF action requires a longer period. These results demonstrate that EGF and GnRH inhibit FSH-induced LH receptor formation in the granulosa cell by reducing hormone-dependent cAMP production and also by impairing the ability of cAMP to stimulate LH receptor formation.     

Passive immunization of the pig against gonadotropin releasing hormone during the follicular phase of the estrous cycle

Three experiments were conducted to determine the effects of passively immunizing pigs against gonadotropin releasing hormone (GnRH) during the follicular phase of the estrous cycle. In Experiment 1, sows were given GnRH antibodies at weaning and they lacked estrogen secretion during the five days immediately after weaning and had delayed returns to estrus. In Experiment 2, gilts passively immunized against GnRH on Day 16 or 17 of the estrous cycle (Day 0 = first day of estrus) had lower (P<0.03) concentrations of estradiol-17beta than control gilts, and they did not exhibited estrus at the expected time (Days 18 to 22). When observed three weeks after passive immunization, control gilts had corpora lutea present on their ovaries, whereas GnRH-immunized gilts had follicles and no corpora lutea. The amount of GnRH antiserum given did not alter (P<0.05) serum concentrations of LH or pulsatile release of LH in sows and gilts. In Experiment 3, prepuberal gilts were given 1,000 IU PMSG at 0 h and GnRH antiserum at 72 and 120 h. This treatment lowered the preovulatory surge of LH and FSH, but it did not alter serum estradiol-17beta concentrations, the proportion of pigs exhibiting estrus, or the ovulation rate. These results indicate that passive immunization of pigs against GnRH before initiation of or during the early part of the follicular phase of the estrous cycle retards follicular development, whereas administration of GnRH antibodies during the latter stages of follicular development does not have an affect. Since the concentration of antibodies was not high enough to alter basal or pulsatile LH secretion, the mechanism of action of the GnRH antiserum may involve a direct ovarian action.     

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.