Effects of gonadotropin-releasing hormone pulse-frequency modulation on luteinizing hormone, follicle-stimulating hormone and testosterone secretion in hypothalamo/pituitary-disconnected rams

The effects of changes in pulse frequency of exogenously infused gonadotropin-releasing hormone (GnRH) were investigated in 6 adult surgically hypothalamo/pituitary-disconnected (HPD) gonadal-intact rams. Ten-minute sampling in 16 normal animals prior to HPD showed endogenous luteinizing hormone (LH) pulses occurring every 2.3 h with a mean pulse amplitude of 1.11 +/- 0.06 (SEM) ng/ml. Mean testosterone and follicle-stimulating hormone (FSH) concentrations were 3.0 +/- 0.14 ng/ml and 0.85 +/- 0.10 ng/ml, respectively. Before HPD, increasing single doses of GnRH (50-500 ng) elicited a dose-dependent rise of LH, 50 ng producing a response of similar amplitude to those of spontaneous LH pulses. The effects of varying the pulse frequency of a 100-ng GnRH dose weekly was investigated in 6 HPD animals; the pulse intervals explored were those at 1, 2, and 4 h. The pulsatile GnRH treatment was commenced 2-6 days after HPD when plasma testosterone concentrations were in the castrate range (less than 0.5 ng/ml) in all animals. Pulsatile LH and testosterone secretion was reestablished in all animals in the first 7 days by 2-h GnRH pulses, but the maximal pulse amplitudes of both hormones were only 50 and 62%, respectively, of endogenous pulses in the pre-HPD state. The plasma FSH pattern was nonpulsatile and FSH concentrations gradually increased in the first 7 days, although not to the pre-HPD range. Increasing GnRH pulse frequency from 2- to 1-hour immediately increased the LH baseline and pulse amplitude. As testosterone concentrations increased, the LH responses declined in a reciprocal fashion between Days 2 and 7. FSH concentration decreased gradually over the 7 days at the 1-h pulse frequency. Slowing the GnRH pulse to a 4-h frequency produced a progressive fall in testosterone concentrations, even though LH baselines were unchanged and LH pulse amplitudes increased transiently. FSH concentrations were unaltered during the 4-h regime. These results show that 1) the pulsatile pattern of LH and testosterone secretion in HPD rams can be reestablished by exogenous GnRH, 2) the magnitude of LH, FSH, and testosterone secretion were not fully restored to pre-HPD levels by the GnRH dose of 100 ng per pulse, and 3) changes in GnRH pulse frequency alone can influence both gonadotropin and testosterone secretion in the HPD model.     

Effects of season and testosterone treatment on gonadotrophin secretion and pituitary responsiveness to gonadotrophin-releasing hormone in castrated Romney and Poll Dorset rams.

In castrated rams (Romney and Poll Dorset, n = 8 for each breed), inhibition by testosterone treatment (administered via Silastic capsules) of luteinizing hormone (LH) pulse frequency, basal and mean LH concentrations, mean follicle-stimulating hormone (FSH) concentration, and the peak and total LH responses to exogenous gonadotrophin-releasing hormone (GnRH) were significantly (P less than 0.01) greater during the nonbreeding than during the breeding season. Poll Dorset rams were less sensitive to testosterone treatment than Romney rams. In rams not receiving testosterone treatment, LH pulse frequency was significantly (P less than 0.05) lower during the nonbreeding season than during the breeding season in the Romneys (15.8 +/- 0.9 versus 12.0 +/- 0.4 pulses in 8 h), but not in the Poll Dorsets (13.6 +/- 1.2 versus 12.8 +/- 0.8 pulses in 8 h). It is concluded that, in rams, season influences gonadotrophin secretion through a steroid-independent effect (directly on hypothalamic GnRH secretion) and a steroid-dependent effect (indirectly on the sensitivity of the hypothalamo-pituitary axis to the negative feedback of testosterone). The magnitude of these effects appears to be related to the seasonality of the breed.     

Activin stimulates secretion of follicle-stimulating hormone from pituitary cells desensitized to gonadotropin-releasing hormone

The secretion of follicle-stimulating hormone (FSH) by pituitary cells is stimulated by activin and gonadotropin-releasing hormone, GnRH. To examine the possible interrelationships between the intracellular actions of these secretagogues, responsiveness to activin was tested following pretreatment with 0, 0.1, or 10 nM GnRH. In cells pretreated with 0 or 0.1 nM GnRH, FSH secretion was increased approximately 2-fold during a subsequent challenge with either activin or GnRH. In contrast, in cells pretreated with 10 nM GnRH, FSH secretion became unresponsive to GnRH but could still be stimulated 2-fold by activin. These results demonstrate that activin is able to stimulate FSH secretion in cells that have undergone desensitization to GnRH.     

Influence of photoperiod and protein diet on growth hormone secretion in rams

Studies of sheep were undertaken to determine the effects of photoperiod and protein diet on growth hormone (GH) secretion. Rams were subjected to either a control (RI) or an inverted (R2) 6-month (semestral) light regime. In both light regimes day lengths varied gradually between 8 and 16 hr. Within each light regime group of animals, the rams received either a low (L) or a high (H) protein diet containing the same level of energy. Plasma GH profiles consisting of 13 hourly samples were determined at regular intervals corresponding to known day lengths. Analysis of variance indicated that there was a significant effect of day length (P less than 0.01) and protein diet (P less than 0.05) on GH secretion, the two light regimes R1 and R2 were equivalent with respect to GH secretion, and there were no interactions among the three experimental factors. Although mean GH secretion was consistently higher in L groups than in H groups, there was a similar trend in all the animals of increasing GH secretion as day length increased. GH secretion was maximum when the day length reached 13 hr 20 min in increasing photoperiods in L groups (15.6 +/- 1.6 ng X h X ml-1) and 16 hr in H groups (13.0 +/- 1.2 ng X h X ml-1). From these results we conclude that both an increasing day length and a deficiency in protein diet stimulate GH secretion in rams but the GH response to these two factors may involve different regulatory processes and may have different functions.     

Effects of gonadotropin-releasing hormone on growth hormone secretion and gene expression in common carp pituitary

Using radioimmuno- and ribonuclease protection assays, we examined the effects of gonadotropin-releasing hormone and its analogs on the growth hormone mRNA level and growth hormone secretion in common carp (Cyprinus carpio) pituitary fragments with static incubation. After a 24 h treatment, sGnRH ([Trp(7),Leu(8)]-LHRH) and sGnRH-A ([D-Arg(6),Pro(9)]-LHRH) (0.1 nM-1 microM) elevated the GH mRNA level and stimulated the GH secretion in a dose-dependent manner, with a higher potency for sGnRH-A. In a time-course experiment, the function of sGnRH and sGnRH-A (10 nM) on GH secretion was observed after 6 h incubation, while no action on the GH mRNA level were noted until 12 h after treatment. Comparing mammalian GnRH, avian GnRH and piscine GnRH, sGnRH and sGnRH-A showed the highest potency in increasing GH mRNA level and GH-release, followed by cGnRH-II ([His(5),Tyr(8)]-LHRH), and finally LHRH and LHRH-A([D-Trp(6), Pro(9)]-LHRH). These findings, taken together, suggest that GnRH not only can influence GH release, but also play a role in the regulation of GH synthesis.     

Relationship of testosterone pulses to androgen binding in the pituitary of rams

The effects of testosterone on cytosol and nuclear androgen receptors of ram pituitary were examined in two experiments. In Exp. I, 500 micrograms testosterone were injected intravenously and groups of 4 rams were slaughtered at 0, 15, 30, 45, 90 and 360 min after injection. Cytosolic receptor concentration decreased from 21 +/- 0.9 to 6 +/- 0.9 fmol/mg protein 30 min after the testosterone injection (P less than 0.001), and then returned towards the preinjection level after 90 min. The pattern of nuclear receptor concentration was the opposite; a maximal increase (12 +/- 3.5 to 32 +/- 5.7 fmol/mg protein) was observed 30 min after injection (P less than 0.001), followed by a progressive but incomplete decrease by 360 min. In Exp. II, blood was collected every 20 min for 17 h in three successive series, each of 12 rams, which were then slaughtered. Plasma LH and testosterone concentrations were measured by radioimmunoassay. No changes were observed in cytosol receptor concentration, but nuclear receptor concentration was negatively correlated with the interval elapsed since the beginning of the last testosterone pulse (r = -0.62; P less than 0.001). The highest values for nuclear receptor concentrations were observed at an interval equal to or less than 120 min. These results indicate that natural pulses are associated with androgen binding particularly in the pituitary nuclei.     

Intracellular mechanism for the action of inhibin on the secretion of the follicular-stimulating hormone and luteinizing hormone induced by LH-RH in vitro

The FSH secretion-inhibiting action of inhibin in vitro under basal conditions and also in the presence of LH-RH is suppressed by the addition of MIX, a phosphodiesterase inhibitor. In the presence of LH-RH, inhibin reduces significantly the intracellular level of cAMP in isolated pituitary cells. In contrast, the simultaneous addition of MIX and inhibin raises the cAMP level, and this stimulation is comparable to the increase observed when MIX is added alone. These observations suggest that one mode of action of inhibin could be mediated by a reduction in cAMP within the pituitary gonadotropic cell.     

Novel aspects of gonadotropin-releasing hormone action on inositol polyphosphate metabolism in cultured pituitary gonadotrophs

The hypothalamic neuropeptide gonadotropin-releasing hormone (GnRH) stimulates luteinizing hormone secretion via receptor-mediated activation of phosphoinositide hydrolysis to yield inositol phosphates and diacylglycerol. Application of anion-exchange high-performance liquid chromatography together with absorbance and radiochemical flow detection has enabled both the characterization and quantitative estimation of pituitary cell inositol phosphates and phosphoinositides. In cultured pituitary cells, GnRH caused a rapid and progressive rise in the formation of inositol 1,4,5-trisphosphate and of higher polyphosphoinositols corresponding to inositol tetrakisphosphate, pentakisphosphate, and hexakisphosphate. The inositol 1,4,5-trisphosphate formed during GnRH action was dephosphorylated predominantly via inositol 4-monophosphate rather than the expected metabolite, inositol 1-monophosphate. The catabolism of inositol 4-monophosphate, like that of inositol 1-monophosphate, was inhibited by lithium. For these reasons and because it was the major metabolite of [3H] inositol 1,4,5-trisphosphate in permeabilized gonadotrophs, inositol 4-monophosphate appears to represent a specific marker for ligand-stimulated inositol polyphosphate formation and metabolism. The marked and sustained elevations of inositol 4-monophosphate and inositol 1,4-bisphosphate in GnRH-stimulated gonadotrophs indicate that polyphosphoinositides rather than phosphatidylinositol are the preferred substrates of phospholipase C during GnRH action.     

Studies of hormone secretion in romney rams: Luteinizing hormone,testosterone and prolactin plasma profiles,LH/testosterone interrelationships and the influence of seasons

In a study of hormone secretion patterns in rams remote sampling techniques were utilized for collecting jugular blood samples each 20 min for 24 h from adult Romney rams. Five animals were sampled during the summer, four during the winter, and plasma LH, testosterone and prolactin levels were estimated by specific radioimmunoassays.     

Comparative response of rams and bulls to long-term treatment with gonadotropin-releasing hormone analogs

The objective was to compare the relative response between rams and bulls in characteristics of LH, FSH and testosterone (T) secretion, during and after long-term treatment with GnRH analogs. Animals were treated with GnRH agonist, GnRH antagonist, or vehicle (Control) for 28 days. Serial blood samples were collected on day 21 of treatment, and at several intervals after treatment. Injections of natural sequence GnRH were used to evaluate the capacity of the pituitary to release gonadotropins during and after treatment. Treatment with GnRH agonist increased basal LH and T concentrations in both rams and bulls, with a greater relative increase in bulls. Endogenous LH pulses and LH release after administration of GnRH were suppressed during treatment with GnRH agonist. Treatment with GnRH antagonist decreased mean hormone concentrations, LH and T pulse frequency, and the release of LH and T after exogenous GnRH, with greater relative effects in bulls. Rams previously treated with antagonist had a greater release of LH after administration of GnRH compared with control rams, while rams previously treated with agonist showed a reduced LH response. Bulls previously treated with agonist had reduced FSH concentrations and LH pulse amplitudes compared with control bulls while bulls previously treated with antagonist had greater T concentrations and pulse frequency. The present study was the first direct comparison between domestic species of the response in males to treatment with GnRH analogs. The findings demonstrated that differences do occur between rams and bulls in LH, FSH and testosterone secretion during and after treatment. Also, the consequences of treatment with either GnRH analog can persist for a considerable time after discontinuation of treatment.     

Light entrainment and stimulation of the secretion of prolactin in rams

Light entrainment and stimulation of PRL release were studied in rams submitted to artificial light regimes: In a first experiment four groups of animals were submitted to light regimes with a period of 6, 4, 3 or 2 months respectively (T6 to T2 groups) and amplitude of 8-16 h of daylength. In all four groups PRL level was significantly correlated with daylength (r greater than 0.51; P less than 0.01). However in July, when temperatures were indeed high, an additional PRL increase was observed in the T6, T4 and T3 groups, although the role of temperature appears debatable. In a second experiment, animals were exposed daily to 8 hrs. of light given in two photofractions. The first fraction of 7 hrs. began at the same time as "dawn", and the second of 1 hr ended at the same time as "dusk" of the T6 group in the first experiment which served as control group. Correlation of plasma prolactin in rams receiving 8 h light in one or two photofractions was highly significant (r = 0.66; P less than 0.001). This suggests that rams measured daylength between two limits considered as "dawn" and "dusk" even if lights were turned off during part of this interval.     

A homodimer of the beta-subunits of inhibin A stimulates the secretion of pituitary follicle stimulating hormone

A 24,000 Dalton protein with follicle stimulating hormone (FSH)-releasing activity, named activin, has been characterized previously from porcine follicular fluid as a heterodimer composed of the beta-subunits of inhibins A and B linked by disulfide bond(s) [Ling et al. (1986) Nature, in press]. In this paper we report the isolation of another 24,000 Dalton protein with FSH-releasing activity from porcine follicular fluid, using successive steps of heparin-Sepharose affinity chromatography, gel filtration on Sephacryl S-200, and four steps of reversed-phase HPLC, followed by preparative sodium dodecyl-sulfate-polyacrylamide gel electrophoresis chromatography. Based on the molecular weight of the isolated molecule and its deduced NH2-terminal sequence, we propose that this second FSH-releasing substance present in porcine follicular fluid is a homodimeric protein composed of two beta-subunits of inhibin A joined together by disulfide bond(s). The name homo-activin-A is proposed for this substance.     

Activation of translation in pituitary gonadotrope cells by gonadotropin-releasing hormone

The neuropeptide GnRH is a central regulator of mammalian reproductive function produced by a dispersed population of hypothalamic neurosecretory neurons. The principal action of GnRH is to regulate release of the gonadotropins, LH and FSH, by the gonadotrope cells of the anterior pituitary. Using a cultured cell model of mouse pituitary gonadotrope cells, alphaT3-1 cells, we present evidence that GnRH stimulation of alphaT3-1 cells results in an increase in cap-dependent mRNA translation. GnRH receptor activation results in increased protein synthesis through a regulator of mRNA translation initiation, eukaryotic translation initiation factor 4E-binding protein, known as 4EBP or PHAS (protein, heat, and acid stable). Although the GnRH receptor is a member of the rhodopsin-like family of G protein-linked receptors, we show that activation of translation proceeds through a signaling pathway previously described for receptor tyrosine kinases. Stimulation of translation by GnRH is protein kinase C and Ras dependent and sensitive to rapamycin. Furthermore, GnRH may also regulate the cell cycle in alphaT3-1 cells. The activation of a signaling pathway that regulates both protein synthesis and cell cycle suggests that GnRH may have a significant role in the maintenance of the pituitary gonadotrope population in addition to directing the release of gonadotropins.     

Effect of clomiphene citrate on pituitary responsiveness to gonadotropin releasing hormone in rams and wethers

The objective of this study was to determine the effect of clomiphene citrate (clomid) on pituitary responsiveness to gonadotropin releasing hormone (GnRH) in rams and wethers. Doses of 200 mg clomid per ram and 1 mug GnRH per 50 kg body weight were used in studies on 12 rams and 4 wethers. The experimental design involved bleeding each animal at 15-minute intervals for 6.5 hours. At the end of the first hour, GnRH was injected IV. The second GnRH challenge was administered 0.5 hours after an injection of clomid or vehicle (4.5% sorbitol solution) which was given on the third hour. The relative response to clomid or vehicle was calculated as the mean increase in concentration of LH during the two-hour period after the second GnRH injection. Each treatment (clomid and vehicle) was given to all animals with a 14-day recovery period between treatment days. The relative response for the rams receiving vehicle (1.80 +/- 0.65) was greater (P < 0.05) than the response during clomid treatment (0.34 +/- 0.22). This suppression of LH response by clomid was observed in 10 of the 12 rams. In contrast to the rams, the concentrations of LH in wethers after the second GnRH injection were lower than those observed after the first GnRH injection. Similar to the rams, the relative response following clomid treatment of wethers (0.04 +/- 0.04) was less than the relative response (P > 0.05) following vehicle (0.40 +/- 0.16). The results suggest that clomid at this dosage inhibits GnRH-induced release of LH from the pituitary of rams but not of wethers.