The endocrine control by luteinizing hormone of testosterone secretion from the testis of the Japanese quail

Injections of chicken or ovine luteinizing hormone (LH) into sexually mature male Japanese quail greatly increased plasma levels of testosterone. Maximal responses were obtained within 15 min of an iv injection and between 1 and 2 hr following sc or im injections. Saline treatment had no effect on plasma testosterone. In chronically castrated quail LH was not effective in altering androgen levels. The responses to LH were dose related, significant increases being obtained following sc injections of 5 μg of chicken LH (fraction AE1) or 10 μg of ovine LH (NIH-LH-S19). Chicken LH (AE1) was appropriately 1.8 times as potent as NIH-LH-S19. Ovine FSH (NIH-FSH-S10) stimulated testosterone release in very large doses (1 mg) but was at least 100 times less active than LH-S19. An iv injection of an antiserum raised against chicken LH into mature male quail caused a rapid decrease in plasma testosterone levels. Treatment with FSH-S-10 for up to 1 week failed to facilitate the subsequent response to an injection of LH. The responsiveness of the testis to exogenous LH was tested at various times during a photoinduced gonadal growth cycle. Sexually immature quail showed only a marginal response to an sc injection of 20 μg of NIH-LH-S19. A marked increase in responsiveness occurred after 6 long days. This coincides with the time when plasma testosterone levels increase naturally after transfer to long daylengths and with the period when Leydig cell maturation becomes complete. These in vivo results add further weight to the belief that, in birds, or at least in the quail, peripheral androgens are controlled by pituitary LH and that FSH plays no significant role in the acute release of testosterone from the mature testis.     

Plasma testosterone levels in male turtles, Chrysemys picta, following single injections of mammalian, avian and teleostean gonadotropins.

The response of male turles (Chrysemys picta) to single injections of either mammalian (human, ovine), avian (domestic hen) or teleostean (salmon) gonadotropins was studied at different points of the testicular cycle (May, July, November) by measurement of plasma testosterone. Mammalian FSH's and avian FSH and LH were markedly stimulatory, and the responses were qualitatively and quantitatively similar. Further, the condition of the testis at the time of injection did not appear to affect the response, with the exception that it was quantitatively less in May. The response was characterized by: (a) Rapidity, reaching maximum or near maximum between 1 and 2 hr postinjection. (b) Magnitude, rising from 10 to 20 ng T/100 ml plasma to 1200 ng/100 ml plasma. (c) Duration, not returning to original plasma levels of T until between 96 and 192 hr postinjection. Injections of purified ovine and human FSH at levels calculated to be equivalent in hormone content to 50 μg of ovine FSH standard duplicated the response. The response was dose related, a 50 μg dose of ovine FSH evoking a response about 10-fold that of a 5 μg injection. Injections of up to 50 μg of NIH ovine LH standard, or 25 μg purified ovine LH only doubled plasma T levels and 100 μg of teleostean gonadotropin had no effect.     

In vivo regulation of steroidogenesis by ovine gonadotropins in male and female mudpuppies,Necturus maculosus Rafinesque

Using steroid radioimmunoassay the in vivo steroidogenic responses of male and female mudpuppies (Necturus maculosus) to single injections of ovine FSH and LH were investigated. In males, the major effect of LH was to stimulate testosterone and 5α-dihydrotestosterone secretion within 2 hr of injection, with a lesser effect on estrogen (estrone and estradiol-17β) secretion. In contrast, FSH primarily stimulated secretion of estrogens. The effects of both FSH and LH on steroid secretion in males were dose related. Castration of males reduced plasma androgen, and estrogen to very low or nondetectable levels and abolished the steroidogenic response to LH. Interrenalectomy prevented a postcastration rise in the progesterone level. In females, FSH did not stimulate steroid secretion but LH increased plasma androgens. Homologous pituitary extracts also stimulated plasma levels of all gonadal steroids measured.     

Origin and regulation of plasma dihydrotestosterone and testosterone in the rough-skinned newt, Taricha granulosa.

The testis of the urodele amphibian Taricha granulosa was studied with regard to localization of androgen production and stimulation of androgen secretion by mammalian LH. Plasma testosterone (T) and 5α-dihydrotestosterone (DHT) increased in proportion to the amount of ovine LH injected; this stimulatory effect of ovine LH was essentially abolished by bilateral castration. In vitro androgen secretion from testicular fragments of the mature zone far exceeded that from fragments of the immature zone. These data indicate that in urodeles plasma T and DHT are produced by cells predominantly localized in the mature zone of the testis, even before spermiation has occurred.     

Plasma 5 alpha-dihydrotestosterone and testosterone in the bullfrog, Rana catesbeiana: stimulation by bullfrog LH.

Effects of purified bullfrog (Rana catesbeiana) LH and FSH on plasma levels of the androgens, testosterone (T), and 5α-dihydrotestosterone (DHT), were studied using adult male bullfrogs. Rana LH was considerably more potent than Rana FSH in stimulating increased plasma androgen levels in hypophysectomized and intact animals. Simultaneous injection of Rana FSH or ovine PRL with Rana LH, over a 10-day period, did not alter the LH-induced increase in plasma androgens. More DHT than T was present in plasma after LH injection. Castration abolished plasma DHT and greatly reduced plasma T. Results indicate that DHT is a major testicular steroid, and that testicular androgen secretion is stimulated primarily by LH in the bullfrog.     

Testicular oxytocin: effects of intratesticular oxytocin in the rat

The long-term effects of oxytocin administration on the testis were studied using intratesticular implants. Adult male rats had an Accurel device containing 20 micrograms oxytocin (releasing approximately 200 ng/day) implanted into the parenchyma of each testis; control animals received empty devices. The animals were killed at weekly intervals for 4 weeks. Some animals were perfused and the testes processed for light and electron microscopy. Blood was collected from the remaining animals for the measurement of testosterone, dihydrotestosterone, LH, FSH and oxytocin; epididymal sperm counts were measured and the testes were extracted and radioimmunoassayed for testosterone, dihydrotestosterone and oxytocin. Long-term administration of oxytocin resulted in a significant reduction in testicular and plasma testosterone levels throughout the 4-week period examined and, after 14 days of treatment, lipid droplets were seen in the Leydig cells of treated but not control animals. Concentrations of dihydrotestosterone in the plasma and testes of the oxytocin-treated animals, however, were significantly elevated after 7 and 14 days and at no time fell below control values. Plasma FSH levels were also lower in the oxytocin-treated animals. Intratesticular oxytocin treatment did not affect LH or oxytocin concentrations in the plasma, epididymal sperm counts or the number of Leydig cells in the testis. Empty Accurel devices had no effect on testicular morphology. This study provides the first evidence that oxytocin in vivo can modify steroidogenesis in the testis.     

In vitro stimulationof 5 alpha-dihydrotestosterone and testosterone secretion from bullfrog testis by nonmammalian and mammalian gonadotropins.

In vitro androgen secretion by bullfrog (Rana catesbeiana) testis was studied using mammalian and nonmammalian gonadotropins, including those from the bullfrog. LH from bullfrog, salamander, turtle, alligator, and sheep increased secretion of 5α-dihydrotestosterone (DHT) and testosterone (T) into incubation medium; Rana and ovine FSH preparations were inactive except at relatively high concentrations. Rana FSH, ovine PRL, and bovine GH did not influence LH-induced steroid secretion. Rana LH was about 3–10 times more potent than other nonmammalian LH preparations, and 30–50 times more potent than NIH-ovine LH. Dose-response characteristics were dependent upon incubation time and temperature. More DHT than T was present in incubation medium at all times and temperatures studied, and during all seasons. Contents of DHT and T in incubation medium were highly correlated, and the ratio $\text{DHT}\text{T}$ increased with time and with dose of LH. These data support in vivo observations and indicate that DHT is a major testicular androgen in the bullfrog, stimulated specifically by LH. Control of testicular androgen secretion by LH may represent the ancestral tetrapod condition.     

Chronic GnRH administration in prepubertal male pigs. A model to evaluate the effects of GnRH treatment in cryptorchidism

The effect of chronic pulsatile low-dose GnRH treatment on the juvenile testis and associated structures was evaluated in relation to hormonal parameters in the peripheral blood in the pig. Starting at 8 weeks of age, male pigs (crossbreds of Dutch Landrace and Yorkshire breeds) were injected 6 times daily im with 0, 75 or 250 ng GnRH/kg body weight during 4 weeks. Immediately after the treatment period, a GnRH stimulation test with 750 ng GnRH/kg iv was carried out. Samples for plasma LH, FSH, testosterone and 5 alpha DHT measurement were obtained weekly (basal level) and after GnRH stimulation. The pigs were castrated at 12 weeks of age and the weights and lengths of the testis, epididymis and cremaster muscle were recorded. Intratesticular testosterone and 5 alpha DHT concentrations were determined, and the testis and epididymis were evaluated for histological changes. Basal plasma hormone concentrations, intratesticular androgen concentrations and the response of the pituitary gland to stimulation had not been affected by GnRH treatment. Pigs receiving the higher treatment dose of GnRH showed less increase in testosterone levels in response to the stimulation dose at 12 weeks of age than the other pigs. Morphologically, no changes were observed in the epididymis and cremaster muscle after GnRH treatment and no signs of reactivation of structures that can provoke testicular descent could be seen. The development of the seminiferous epithelium was more advanced in the GnRH-treated groups, apparently in a dose-dependent manner.     

Crystalline dihydrotestosterone implants in the lateral septum of male rats. A positive effect on LH and FSH.

Previous investigations in our laboratory have shown that testosterone implanted into the lateral septum in male rats increases LH and FSH secretion. However, it was unclear whether the effect of testosterone was direct via androgen receptor, or indirect via the estrogen receptor after conversion by aromatization to estradiol. To answer this question, we implanted either testosterone or the non-aromatizable androgen 5 alpha-dihydrotestosterone (DHT), into the lateral septum of adult male rats and measured plasma levels of LH and FSH by radioimmunoassay 2 days after implantation. Both testosterone and DHT significantly increased the plasma LH and FSH concentrations. Mean concentration of LH in control animals was 0.21 +/- 0.06 ng/ml, a figure that increased to 0.7 +/- 0.12 and 0.55 +/- 0.1 ng/ml after DHT or testosterone implantation respectively. Mean concentration of FSH in control animals was 1.5 +/- 0.3 ng/ml; this figure increased to 3 +/- 0.3 and 2.9 +/- 0.3 ng/ml after DHT or testosterone implantation. Neither plasma DHT (64.0 +/- 5.6 vs. 52 +/- 5 ng/100ml) nor plasma testosterone levels (4.1 +/- 0.38 vs. 3.3 +/- 0.18 ng/ml) were significantly affected by the implants. We conclude that androgens independently of conversion to estrogen acting in the lateral septum facilitates the release of LH and FSH.     

Differential effects of FSH and testosterone on the maintenance of spermatogenesis in the adult hypophysectomized rat

In order to clarify further the role of FSH in the maintenance of spermatogenesis, adult rats were treated with purified human FSH (2 X 5 IU/day per rat), testosterone (1.5 cm silicone elastomer implant) or a combination of both hormones for 2 weeks following hypophysectomy. After hypophysectomy alone, no elongate spermatids were observed and the numbers of pachytene spermatocytes and round spermatids observed were reduced when compared with untreated controls. Testosterone supplementation alone qualitatively maintained the formation of elongate spermatids in most seminiferous tubules, whilst in FSH-treated rats increased numbers of round spermatids and pachytene spermatocytes were observed when compared with hypophysectomized animals. Formation of elongate spermatids, however, did not occur under FSH treatment alone. A combination of FSH and testosterone treatment maintained spermatogenesis in an almost quantitative fashion. Numbers of pachytene spermatocytes and round spermatids were maintained at about 80% of levels seen in intact control animals. Treatment with FSH or testosterone alone maintained testis weights at significantly higher levels than those seen in hypophysectomized controls (FSH, 0.79 +/- 0.05 g; testosterone, 0.81 +/- 0.07 g; hypophysectomized, 0.50 +/- 0.04 g). Animals treated with FSH and testosterone showed testis weights 20% below control values (1.22 +/- 0.05 vs 1.51 +/- 0.06 g; P less than 0.05). No increases in intratesticular or intratubular androgen concentrations or in testosterone: dihydrotestosterone ratios were observed in any of the hormone-treated groups when compared with hypophysectomized controls. In all hypophysectomized animals testicular androgen concentrations were reduced to less than 5% of control values. The results obtained in this study suggest that FSH is involved in the maintenance of spermatogenesis in the adult rat and that the effects of FSH are not mediated through changes in intratesticular androgens. Low levels of testosterone in combination with FSH can almost quantitatively maintain spermatogenesis in adult rats.     

In vivo responses of female snakes (Natrix fasciata) and female turtles (Chrysemys picta) to ovine gonadotropins (FSH and LH) as measured by plasma progesterone, testosterone, and estradiol levels

Changes in plasma progesterone, testosterone, and estradiol-17β in serially bled female snakes (Natrix fasciata) and female turtles (Chrysemys picta) in response to single iv injections of ovine LH or FSH were measured by RIA. In snakes, both FSH and LH increased the levels of all three steroids in plasma, though considerable individual variation in response to either hormone was encountered. The apparent differences in the responses to the gonadotropins may have been due to differences in the stage of follicular development in different animals. In contrast, in female turtles, ovine FSH was clearly most active in stimulating steroid (testosterone and estradiol) synthesis, although LH has a consistent, though minor, stimulatory effect on estradiol secretion 30 min after injection. Neither hormone stimulated progesterone secretion at the time of year tested. In snakes, but not in turtles, it was possible to correlate preinjection plasma levels of estradiol with the stage of follicular development, high levels of hormone being found in animals with large follicles and low levels in animals with small follicles.     

Factors determining whether the direct effects of an LHRH agonist on Leydig cell function in vivo are stimulatory or inhibitory

The influence of (1) repeated exposure to LHRH agonist and (2) concomitant exposure to various levels of LH or hCG, on the direct testicular effects of LHRH agonist have been investigated in hypophysectomized and intact rats. In the latter, LHRH agonist was injected intratesticularly into the right testis whilst the left testis was injected with vehicle, and the level of testosterone in interstitial fluid (IF) from left and right testes was compared 2-4 h later. In hypophysectomized rats, a single injection of 50-1000 ng LHRH agonist raised (P less than 0.001) serum levels of testosterone 2 h later to within the normal range (1-8 ng/ml) for intact rats of comparable age. Subsequent daily injections of LHRH agonist elicited progressively smaller responses and by day 5 no response was evident, this decline being unrelated to the increase in time after hypophysectomy. Comparable changes were observed in hypophysectomized rats pretreated with an LH antiserum. Daily injection of hypophysectomized rats with 10 IU hCG for 6 days raised serum levels of testosterone to supraphysiological levels on each day, whilst concomitant injection of LHRH agonist (1 microgram) decreased (P less than 0.001) this response at all times, an effect that became progressively more pronounced. In contrast, daily treatment with 1 microgram ovine LH raised serum levels of testosterone to within the physiological range, and concomitant treatment with LHRH agonist (50 ng) had either no effect (days 1-2) or significantly increased (days 4-6) the testosterone response to LH. In intact rats, a single unilateral intratesticular injection of 1 ng LHRH agonist increased the IF levels of testosterone unilaterally 3 h later, but subsequent injections on days 2 and 3 elicited progressively smaller responses. In rats given a single intratesticular injection of LHRH agonist combined with a peripheral injection of different doses of LH or hCG, the LHRH agonist induced a unilateral increase in IF levels of LH/hCG, whilst in rats treated with high doses of LH (12.5--25 micrograms) or hCG (50 IU). LHRH agonist either had no effect or significantly reduced the IF levels of testosterone unilaterally. However, LHRH agonist also had significant effects on testicular IF volume and, as this may reflect altered transport of LH and hCG to the Leydig cells, the inhibitory effects of LHRH agonist may be related to this change rather than to an effect of steroidogenesis itself.(ABSTRACT TRUNCATED AT 400 WORDS)     

Testosterone-induced inhibition of spermatogenesis is more closely related to suppression of FSH than to testicular androgen levels in the cynomolgus monkey model (Macaca fascicularis)

We have investigated the antigonadotropic and antispermatogenic effects of exposure to a long-acting testosterone ester in the cynomolgus monkey model. Groups of five adult animals were exposed either to vehicle or to 10 mg/kg or 20 mg/kg testosterone buciclate (TB) over a 26-week period with injections given in weeks 0, 11 and 18. In week 26, testicular biopsy tissue was collected. Serum testosterone levels were in the upper normal range with 10 mg/kg TB and were approximately twofold higher with 20 mg/kg TB. The estradiol pattern followed that of testosterone and body weights increased in a testosterone-dependent manner. TB completely abolished serum LH bioactivity. Serum concentrations of FSH and inhibin-alpha were suppressed in a TB dose-dependent manner. During weeks 4-8 after the first injection, a rebound of FSH and inhibin but not bioactive LH secretion occurred. This rebound was followed immediately by a restimulation of testis size and sperm numbers. After the next TB injections these parameters were once again suppressed. Nadir testis size was 30-40% of baseline and animals were severely oligozoospermic or transiently azoospermic. Consistent azoospermia was not achieved. Quantitation of serum inhibin B, proliferating cell-nuclear antigen staining and flow cytometric analysis of germ cell populations revealed pronounced suppression of spermatogenesis in both TB-treated groups whereas androgen receptor expression remained unchanged. Testicular androgens levels, determined in week 26, did not differ among all three groups and did not correlate with sperm numbers, histological and immunocytochemical findings. All suppressive effects were fully reversed during the recovery period. We have concluded that pronounced suppression of primate spermatogenesis seemingly requires inhibition of FSH rather than testicular androgen levels, at least in this preclinical non-human primate model. For the purpose of male contraception, FSH inhibition appears mandatory.     

Effects of FSH on Leydig cell morphology and function in the hypogonadal mouse.

The hypogonadal (hpg) mouse has a congenital deficiency in gonadotrophin-releasing hormone and the gonads consequently lack exposure to endogenous gonadotrophins during development. To determine the effect of FSH on Leydig cell function in these animals adult hpg mice were injected twice daily with FSH (2 micrograms injections) or LH (40 ng injections, the presumed LH contamination of FSH used). Following FSH treatment there was a clear stimulation of the seminiferous epithelium and in animals injected with FSH plus [3H]thymidine, the incorporation of label was largely confined to the germ cells with no apparent uptake by the Sertoli cells. In FSH-treated testes the Leydig cells contained numerous large lipid droplets, similar to the unstimulated hpg testis. There was no evidence of the interstitial hyperplasia which is observed following injection of high doses of LH (2 micrograms twice daily). There was no change in basal androgen content of the testis in vivo following FSH treatment but injection of a maximal dose of human chorionic gonadotrophin (hCG), 1 h before death, markedly increased testicular androgen content only in the FSH-treated group. Testicular androgen production in vitro was significantly increased following FSH treatment both under basal conditions (FSH-treated, 17.4 pmol/testis; control, 1.46 pmol/testis) and during stimulation by hCG (FSH-treated, 940 pmol/testis; control, 81 pmol/testis). Associated with the increased androgen production following FSH treatment there were significant increases in the activities of three steroidogenic enzymes; cholesterol side-chain cleavage (186-fold increase over control), 17 alpha-hydroxylase (103-fold increase) and 17-ketosteroid reductase (177-fold increase).(ABSTRACT TRUNCATED AT 250 WORDS)     

The effects of testosterone propionate and gonadotropins on the bill pigmentation and testes of the house sparrow (Passer domesticus)

Sexually regressed winter house sparrows, Passer domesticus, were treated with exogenous testosterone propionate, luteinizing hormone (LH) or follicle-stimulating hormone (FSH) after first confirming they were no longer photorefractory. Controls at the start and end of the experiment in December had the pale brown beaks typically noted in this species during the early winter. Treatment with relatively large doses of exogenous androgen did not cause the bill to assume a darker coloration than would occur in the wild in early spring. Neither FSH nor LH given alone restored pigmentation. A combination of FSH and LH caused a complete darkening of the bill, and FSH injected in combination with testosterone propionate was also effective. Testosterone propionate exerted a spermatokinetic effect consistent with other reports in the literature. Testicular weight and spermatogenetic condition was most advanced in subjects given FSH plus LH injections.     

Age-related changes in testicular histology and androgen levels of the immature spiny-tailed lizard, Uromastix hardwicki

Age-related changes in testicular histology and androgen levels have been studied during the first 16 weeks of life of the immature spiny-tailed lizard (Uromastix hardwicki). In the young lizards which emerge from eggs in late June to early July, until approximately 3 months of age, the seminiferous tubules contain only two types of cells: gonocytes and pre-Sertoli cells. Subsequently, in the month of October (age: 12–16 weeks) there is a marked proliferation of germ cells accompanied by an increase in the diameter of the seminiferous tubules and weight of the testis. The interstitial cells which are markedly differentiated even in the earliest phase of posthatching development are considerably hypertrophied in early August (age: 4–5 weeks). Plasma titers of testoslerone, as measured by radioimmunoassay, showed an initial rise in August followed by a second major peak at the end of October. Testicular testosterone concentrations expressed per milligram of tissue were lowest in July and maximal in August. A significant increase in testosterone concentration in both the plasma and the testicular tissue was also observed following administration of FSH and LH to immature lizards. It is suggested that, in the immature spiny-tailed lizard, androgenesis resulting in detectable levels of testosterone in peripheral plasma and testicular tissue precedes gamatogenic activity. Furthermore, the steroidogenic tissue in this species has been shown to be responsive to exogenous gonadotropins even in the earliest phase of posthatching development.     

Use of a pulsed infusion of luteinizing hormone releasing hormone to mimic seasonally induced endocrine changes in the ram.

Adult Soay rams were housed indoors under natural lighting during the spring non-mating season when gonadotrophin secretion was low. Four animals received small doses (100 ng or 500 ng) of synthetic LH releasing hormone (LH-RH) infused into the jugular vein by a mechanical device for 60 s every 2 h for 33-57 days: two other rams acted as controls. The prolonged treatment with LH-RH resulted in growth of the testes and the development of the sexual skin flush; these effects were lost when treatment stopped. The plasma concentrations of LH, FSH and testosterone were low at the beginning; each short infusion of LH-RH resulted in a transitory increase in the level of LH and testosterone while the concentration of FSH was only marginally affected. After prolonged treatment with 500 ng pulses of LH-RH the plasma concentrations of all three hormones were permanently raised. The response to the individual injections of LH-RH was also modified, the peak in LH being reduced in amplitude but more prolonged while the FSH and testosterone responses were both enhanced. When the pulsed infusion was stopped the concentration of LH and testosterone declined rapidly while the decline in FSH levels took many days. These endocrine changes induced by the pulsed infusion are comparable to those that occur naturally in the ram during testicular redevelopment before the mating season.     

Synergism between bovine seminal plasma extract and testosterone propionate in suppressing serum concentrations of gonadotrophins in acutely castrated rats: a role for inhibin

The rise in concentrations of FSH and LH in serum seen 24 h after castration was suppressed by the administration of an extract of bull seminal plasma or testosterone propionate at the time of castration. Whereas testosterone propionate preferentially suppressed LH, the seminal plasma extract suppressed FSH and LH equally. Small doses of bull seminal plasma extract and testosterone, that had little effect separately, acted synergistically to supress levels of FSH and LH to those found in intact animals, while combinations of larger doses had little further effect. This selective interaction suggests how inhibin and testosterone might together regulate concentrations of FSH and LH in the blood of the male rat.     

Differential control of luteinizing hormone and follicle-stimulating hormone by luteinizing hormone releasing hormone in the ram.

Adult Soay rams with low concentrations of gonadotrophins in the circulation as a result of 12 weeks of exposure to long daylengths (16 h light : 8 h darkness) were given small doses (100 ng) of synthetic luteinizing hormone releasing hormone (LH-RH) into the jugular vein two, four or seven times/day for 10 days. Each injection of LH-RH induced a transitory increase in the concentration of LH and testosterone in the plasma, whereas the concentration of FSH showed little immediate change. After repeated treatment with pulses of LH-RH, the responses of LH and testosterone became slightly enhanced and the plasma concentration of FSH became permanently raised; these changes were most conspicuous in the animals receiving the most frequent injections. At the end of the study when the injections of LH-RH were stopped, the concentrations of LH and testosterone remained low but the concentrations of FSH continued to be maintained at a high level for at least 24 h.     

The negative feedback effects of testicular steroids are predominantly at the hypothalamus in the ram

This study aimed to delineate the hypothalamic and/or pituitary actions of testosterone and its primary metabolites 5 alpha-dihydrotestosterone and estradiol (E) in adult castrated rams (wethers) during the breeding season. In Exp 1, wethers were treated for a week with twice daily injections (im) of peanut oil, 8, 16 or 32 mg/day testosterone propionate (TP) or dihydrotestosterone benzoate (DHTB) or an sc silastic implant containing 1 or 3 cm E. TP decreased plasma LH concentrations, increased (P less than 0.05) LH interpulse interval, did not have consistent effects on LH pulse amplitude, and had minimal effects on plasma FSH concentrations. DHTB decreased LH and FSH concentrations and increased (P less than 0.05) LH interpulse interval. E reduced (P less than 0.05) plasma LH and FSH concentrations and increased LH interpulse interval but had no effects on LH pulse amplitude. In Exp 2, hypothalamo-pituitary disconnected wethers given 125 ng GnRH every 2 h, were treated with either peanut oil, 32 mg/day TP or DHTB or 3 cm E. None of the treatments affected plasma LH or FSH concentrations or LH pulse amplitude. Exp 3 investigated the effects on GnRH of treatment of wethers either with peanut oil or TP. TP reduced GnRH concentrations (P less than 0.05) and pulse amplitude (P less than 0.01) and increased interpulse interval (P less than 0.05). These data provide evidence that, during the breeding season, the principal site of negative feedback of testicular steroids in the ram is the hypothalamus, resulting in decreased GnRH secretion; feedback effects at the pituitary are minimal.