In this study we used an isolation/restraint stress to test the hypothesis that stress will affect the secretion of LH differently in gonadectomised rams and ewes treated with different combinations of sex steroids. Romney Marsh sheep were gonadectomised two weeks prior to these experiments. In the first experiment male and female sheep were treated with vehicle or different sex steroids for 7 days prior to the application of the isolation/restraint stress. Male sheep received either i.m. oil (control rams) or 6 mg testosterone propionate injections every 12 h. Female sheep were given empty s.c. implants (control ewes), or 2 1 cm s.c. implants containing oestradiol, or an intravaginal controlled internal drug release device containing 0·3 g progesterone, or the combination of oestradiol and progesterone. There were four animals in each group. On the day of application of the isolation/ restraint stress, blood samples were collected every 10 min for 16 h for the subsequent measurement of plasma LH and cortisol concentrations. After 8 h the stress was applied for 4 h. Two weeks later, blood samples were collected for a further 16 h from the control rams and ewes, but on this day no stress was imposed. In the second experiment, separate control gonadectomised rams and ewes (n=4/ group) were studied for 7 h on 3 consecutive days, when separate treatments were applied. On day 1, the animals received no treatment; on day 2, isolation/restraint stress was applied after 3 h; and on day 3, an i.v. injection of 2 µg/kg ACTH 1-24 was given after 3 h. On each day, blood samples were collected every 10 min and the LH response to the i.v. injection of 500 ng GnRH administered after 5 h of sampling was measured. In Experiment 1, the secretion of LH was suppressed during isolation/ restraint in all groups but the parameters of LH secretion (LH pulse frequency and amplitude) that were affected varied between groups. In control rams, LH pulse amplitude, and not frequency, was decreased during isolation/restraint whereas in rams treated with testosterone propionate the stressor reduced pulse frequency and not amplitude. In control ewes, isolation/restraint decreased LH pulse frequency but not amplitude. Isolation/restraint reduced both LH pulse frequency and amplitude in ewes treated with oestradiol, LH pulse frequency in ewes treated with progesterone and only LH pulse amplitude in ewes treated with both oestradiol and progesterone. There was no change in LH secretion during the day of no stress. Plasma concentrations of cortisol were higher during isolation/restraint than on the day of no stress. On the day of isolation/restraint maximal concentrations of cortisol were observed during the application of the stressor but there were no differences between groups in the magnitude of this response. In Experiment 2, isolation/restraint reduced the LH response to GnRH in rams but not ewes and ACTH reduced the LH response to GnRH both in rams and ewes. Our results show that the mechanism(s) by which isolation/restraint stress s...

This minireview considers the state of knowledge regarding the interactions of testicular hormones to regulate the secretion and actions of GnRH in males, with special focus on research conducted in rams and male rhesus monkeys. In these two species, LH secretion is under the negative feedback regulation of testicular steroids that act predominantly within the central nervous system to suppress GnRH secretion. The extent to which these actions of testicular steroids result from the direct actions of testosterone or its primary metabolites, estradiol or dihydrotestosterone, is unclear. Because GnRH neurons do not contain steroid receptors, the testicular steroids must influence GnRH neurons via afferent neurons, which are largely undefined. The feedback regulation of FSH is controlled by inhibin acting directly at the pituitary gland. In male rhesus monkeys, the feedback regulation of FSH secretion is accounted for totally by the physiologically relevant form of inhibin, which appears to be inhibin B. In rams, the feedback regulation of FSH secretion involves the actions of inhibin and testosterone and interactions between these hormones, but the physiologically relevant form of inhibin has not been determined. The mechanisms of action for inhibin are not known.

Plasma follistatin (FS) concentrations were determined after castration (n = 5) or sham castration (n = 4) of mature rams. Both treatments resulted in a prolonged increase in FS between 7 and 19 h after surgery, which returned to pretreatment concentrations by 24 h. Tumour necrosis factor-alpha (TNF-alpha), a sensitive maker of an acute-phase response, was undetectable in plasma, indicating that the FS response was not induced by trauma due to surgery. In a second experiment, injection of castrated rams (n = 4) with ovine recombinant interleukin-1 beta, an acute-phase mediator, resulted in a sustained rise in FS concentrations within 4 h of injection. Plasma TNF-alpha concentrations increased transiently within 1 h of interleukin-1 beta injection, indicating that an acute-phase response had been initiated. Plasma follicle-stimulating hormone (FSH) concentrations were significantly decreased at 8 and 24 h after interleukin-1 beta injection, strongly suggestive of an inhibitory effect of increased FS concentrations on the secretion of FSH. Injection of castrated rams (n = 2) with a control preparation of recombinant interleukin-2 did not induce an acute-phase response, and plasma FS and FSH concentrations were unaffected. These data show that the testis is not a major source of circulating FS, that the increase in circulating FS following sham castration/castration is not due to an acute-phase response, but that conversely FS concentrations are modulated by the acute-phase mediator, interleukin-1 beta.

This study tested the hypothesis that inhibin is a major negative feedback regulator of FSH secretion but has minimal effects on LH secretion in rams. In experiment 1, castrated rams (wethers) were given either vehicle or human recombinant inhibin A (hr-inhibin) as three s.c. or three i.v. 50-micrograms injections 6 h apart or as one 50-micrograms i.v. injection followed by 100-micrograms infusion over 12 h. Human recombinant inhibin suppressed plasma FSH while the vehicle had no effect. The greatest suppression in plasma FSH was achieved following i.v. administration of hr-inhibin given either by repeated injection or by infusion. In experiment 2, wethers were given vehicle or a 50-micrograms i.v. injection followed by 800-micrograms infusion of hr-inhibin over 12 h. Infusion of hr-inhibin suppressed plasma FSH with a maximal suppression of 53.3% occurring between 15 and 24 h after the start of treatment. During this period, the plasma concentrations of FSH and inhibin were in the range of values for intact rams. Human recombinant inhibin did not influence plasma LH in either experiment. This study demonstrated that physiological treatment with inhibin, in the absence of testosterone, has the capacity to suppress plasma concentrations of FSH in wethers to the levels found in intact rams.

Three groups of mature rams were maintained on diets of hay, hay + 2% lupin or hay + 2% cowpea for 11 weeks. Serial blood samples were taken at 15-min intervals for 12 h for the determination of GH and IGF-I content by radioimmunoassay and for IGF-binding protein-3 (IGFBP-3) levels by Western blotting. The rams were killed after 77 days of supplementary feeding and their pituitary glands analysed for content of GH and GH mRNA. Mean plasma GH and baseline GH levels were significantly (P < 0.01) decreased in the rams fed lupin and cowpea compared with controls fed hay and GH pulse amplitude was significantly (P < 0.001) decreased in the group fed the cowpea diet. The frequency of GH pulses was not significantly altered by either treatment. Plasma concentrations of IGF-I were elevated in rams fed lupin (P < 0.001) or cowpea (P < 0.05). IGFBP-3 levels were not significantly (P > 0.05) altered by either treatment. There were no significant differences in pituitary content of GH mRNA but pituitary content of GH was increased in rams fed lupin (P < 0.05) and cowpea (P = 0.07). In conclusion, a high-protein diet decreases plasma GH levels and increases IGF-I without changing plasma IGFBP-3 levels in rams. Thus ongoing synthesis of GH, as indicated by the mRNA levels, may cause a build up of GH stores in the pituitary gland.

This experiment determined if the degree of stimulation of the pituitary gland by GnRH affects the suppressive actions of inhibin and testosterone on gonadotropin secretion in rams. Two groups (n = 5) of castrated adult rams underwent hypothalamopituitary disconnection and were given two i.v. injections of vehicle or 0.64 microg/kg of recombinant human inhibin A (rh-inhibin) 6 h apart when treated with i.m. injections of oil and testosterone propionate every 12 h for at least 7 days. Each treatment was administered when the rams were infused i.v. with 125 ng of GnRH every 4 h (i.e., slow-pulse frequency) and 125 ng of GnRH every hour (i.e., fast-pulse frequency). The FSH concentrations and LH pulse amplitude were lower and the LH concentrations higher during the fast GnRH pulse frequency. The GnRH pulse frequency did not influence the ability of rh-inhibin and testosterone to suppress FSH secretion. Testosterone did not affect LH secretion. Following rh-inhibin treatment, LH pulse amplitude decreased at the slow, but not at the fast, GnRH pulse frequency, and LH concentrations decreased at both GnRH pulse frequencies. We conclude that the degree of stimulation of the pituitary by GnRH does not influence the ability of inhibin or testosterone to suppress FSH secretion in rams. Inhibin may be capable of suppressing LH secretion under conditions of low GnRH.

To determine whether Leydig cells produce inhibin in the ram, Leydig cells were stimulated by administering human chorionic gonadotrophin (hCG) or raising the levels of endogenous LH by an injection of gonadotrophin releasing hormone (GnRH). Plasma concentrations of testosterone increased in the 72 h after either a single injection (P less than 0.05) or two injections (P less than 0.01) of hCG. Plasma concentrations of inhibin were not significantly influenced by either one or two injections of hCG. Administration of GnRH (1 microgram) caused an 11-fold increase in plasma concentrations of LH but did not influence concentrations of inhibin in either the jugular or testicular veins (pampiniform plexus). In contrast, concentrations of testosterone were increased by about fourfold in both jugular (P less than 0.01) and testicular (P less than 0.05) veins. The concentrations of inhibin in the testicular vein were 1.3-fold higher than in the peripheral plasma (P less than 0.05) both before and following treatment with GnRH whereas the concentrations of testosterone were 18- to 21-fold greater than in peripheral concentrations. In view of the difference in concentrations of inhibin between testicular and jugular veins, in a further experiment a sample was taken from the jugular vein, a vein located in the tunica vasculosa of the testis (testicular vein) and from a vein (spermatic vein) and lymph vessels located in the spermatic cord. The mean (+/- S.E.M.) concentrations of inhibin were highest in the testicular lymph (45.93 +/- 4.21 micrograms/l; P less than 0.001) compared with the peripheral (4.14 +/- 0.31 micrograms/l), spermatic (8.0 +/- 1.17 micrograms/l) or testicular (6.4 +/- 0.82 micrograms/l) plasma.(ABSTRACT TRUNCATED AT 250 WORDS)

The roles of testicular hormones in the negative feedback regulation of the secretion and actions of GnRH in male domestic ruminants are reviewed, concentrating mainly on research conducted with rams. Testicular steroids have major feedback actions directly at the hypothalamus to inhibit the secretion of GnRH, although it is apparent that, under certain circumstances, the steroids also have actions directly at the pituitary gland. Further research is necessary to delineate these actions and to determine the contribution of testosterone and its primary metabolites, dihydrotestosterone and oestradiol, to negative feedback on the hypothalamo-pituitary unit. Since GnRH neurones do not possess receptors for steroids, testicular steroids must evoke other neuronal pathways to influence GnRH producing neurones. While the opioids may be important in this regard, it is necessary to determine which other neuronal pathways may also be involved to understand fully the mechanism of action of testicular steroids. It appears that the feedback regulation of the secretion of LH can be accounted for by testicular steroids, whereas the secretion of FSH is influenced by inhibin and steroids, and possibly the recently isolated proteins follistatin and activin. The actions of inhibin to suppress the secretion of FSH occur at the pituitary gland and not on the synthesis or secretion of GnRH. There is a complex interaction between testosterone and inhibin in the control of FSH secretion that results in synergistic effects during the non-breeding season but not during the breeding season. Activin has been shown to have FSH-stimulating properties and follistatin has been shown to have FSH-inhibiting properties, but it is unknown if these proteins play a physiological role in the feedback regulation of FSH in male domestic ruminants.