Molecular characterization and phenotypic expression of mutations in genes for gonadotropins and their receptors in humans.

Gonadotropin hormones undergo important dynamic changes during life. Their rise during puberty stimulates gonadal steroid secretion, triggering the development of secondary sexual characteristics and the acquisition of fertility. The full spectrum of possible mutations and polymorphisms in the human gonadotropins and in their receptor genes has been described in recent years. Patients harboring these mutations display a very wide range of phenotypes affecting all aspects of the reproductive axis. An important insight provided by the careful study of these patients lies in the striking gender differences in the phenotypes associated with a given mutation. As a result, the careful study of these rare patients has allowed us to better define the respective roles of luteinizing hormone and follicle-stimulating hormone in normal human pubertal development and in the achievement of full fertility potential in either males or females. In this work, we describe briefly the known mutations in the genes for both gonadotropins and their receptors, and discuss their genotype/phenotype correlations in light of these important gender differences.

Testicular Function in Biotin-Deficient Adult Rats

We have studied testicular function in the biotin- deficient rat biochemically and morphologically. Serum testosterone and luteinizing hormone (LH) levels were decreased significantly in the deficient rats. Administration of biotin or gonadotropins to the deficient rats reversed this decrease in serum testosterone. There was no difference in the serum cholesterol level between the control and biotin-deficient rats. A significant degree of sloughing of seminiferous tubule germinal epithelium was noticed in the biotin-deficient rat testes. Biotin treatment of biotindeficient rats reversed this condition whereas testosterone treatment was without any effect. The development and maintenance of morphological and functional integrity of the seminiferous tubules appears to require a biotin-mediated step in addition to testosterone.

Local roles of TGF-beta superfamily members in the control of ovarian follicle development

Members of the transforming growth factor-beta (TGF-beta) superfamily have wide-ranging influences on many tissue and organ systems including the ovary. Two recently discovered TGF-beta superfamily members, growth/differentiation factor-9 (GDF-9) and bone morphogenetic protein-15 (BMP-15; also designated as GDF-9B) are expressed in an oocyte-specific manner from a very early stage and play a key role in promoting follicle growth beyond the primary stage. Follicle growth to the small antral stage does not require gonadotrophins but appears to be driven by local autocrine/paracrine signals from both somatic cell types (granulosa and theca) and from the oocyte. TGF-beta superfamily members expressed by follicular cells and implicated in this phase of follicle development include TGF-beta, activin, GDF-9/9B and several BMPs. Acquisition of follicle-stimulating hormone (FSH) responsiveness is a pre-requisite for growth beyond the small antral stage and evidence indicates an autocrine role for granulosa-derived activin in promoting granulosa cell proliferation, FSH receptor expression and aromatase activity. Indeed, some of the effects of FSH on granulosa cells may be mediated by endogenous activin. At the same time, activin may act on theca cells to attenuate luteinizing hormone (LH)-dependent androgen production in small to medium-size antral follicles. Dominant follicle selection appears to depend on differential FSH sensitivity amongst a growing cohort of small antral follicles. Activin may contribute to this selection process by sensitizing those follicles with the highest "activin tone" to FSH. Production of inhibin, like oestradiol, increases in selected dominant follicles, in an FSH- and insulin-like growth factor-dependent manner and may exert a paracrine action on theca cells to upregulate LH-induced secretion of androgen, an essential requirement for further oestradiol secretion by the pre-ovulatory follicle. Like activin, BMP-4 and -7 (mostly from theca), and BMP-6 (mostly from oocyte), can enhance oestradiol and inhibin secretion by bovine granulosa cells while suppressing progesterone secretion; this suggests a functional role in delaying follicle luteinization and/or atresia. Follistatin, on the other hand, may favor luteinization and/or atresia by bio-neutralizing intrafollicular activin and BMPs. Activin receptors are expressed by the oocyte and activin may have a further intrafollicular role in the terminal stages of follicle differentiation to promote oocyte maturation and developmental competence. In a reciprocal manner, oocyte-derived GDF-9/9B may act on the surrounding cumulus granulosa cells to attenuate oestradiol output and promote progesterone and hyaluronic acid production, mucification and cumulus expansion.(C) 2003 Elsevier Science B.V. All rights reserved.

Theca cells and the regulation of ovarian androgen production

Theca cells are essential for female reproduction being the source of androgens that are precursors for follicular oestrogen synthesis and also signal through androgen receptors (AR) in the ovary and elsewhere. Theca cells arise from mesenchymal cells around the secondary follicle stage. Their recruitment, proliferation and cytodifferentiation are influenced, directly or indirectly, by paracrine signals from granulosa cells and oocyte although uncertainty remains over which are the critically important signals at particular stages. In a reciprocal manner, theca cells secrete factors that influence granulosa cell proliferation and differentiation at different follicle stages. Differentiated theca interna cells acquire responsiveness to luteinizing hormone (LH) and other endocrine signals and express components of the steroidogenic machinery required for androgen biosynthesis. They also express insulin-like peptide 3 (INSL3) and its receptor (RXFP2), levels of which increase during bovine antral follicle development. INSL3 signaling may play a role in promoting androgen biosynthesis since knockdown of either INSL3 or its receptor (RXFP2) in bovine theca cells inhibits androgen biosynthesis while exogenous INSL3 can raise androgen secretion. Bone morphogenetic proteins (BMPs) of thecal or granulosal origin suppress thecal production of both INSL3 and androgen. Inhibin, produced in greatest amounts by granulosa cells of preovulatory follicles, reverses these BMP actions. Thus, BMP-induced inhibition of thecal androgen production may be mediated by reduced INSL3-RXFP2 signaling. Activins also inhibit androgen production in an inhibin-reversible manner and recent evidence in sheep indicates that theca cells synthesize and secrete activin, implying an autocrine role in suppressing androgen biosynthesis in smaller follicles, akin to that envisaged for BMPs.

Human-animal interaction, stress, and embryo production in Bos indicus embryo donors under tropical conditions

This study investigated the effect of human-animal interaction (HAI) and the stress response on the quality of embryo production in superovulated Nelore (Bos indicus) cattle, under tropical conditions. Thirty-two females underwent a superovulation protocol for 5 days. Cortisol concentrations were determined in blood plasma collected on days 0, 4, and 5. Artificial insemination was performed on days 4 and 5, and nonsurgical embryo flushing on day 11. Embryo production and viability were determined. Human stimulation, animal behaviors, accidents, and handling time were recorded to assess HAI. Cattle age was negatively correlated with accidents, frequency of aversive behaviors, and negative stimuli by stockperson during transit through corral compartments to receive superovulation treatments. The factor analysis revealed two distinct groups. The first group was called stressed and had higher cortisol concentration than the nonstressed group, 16.0 +/- 2.1 and 12.5 +/- 1.0 ng/mL, respectively. Comparisons between these groups showed that the frequency of voice emissions by the stockperson and the number of accidents were higher in the stressed group, and also, the mean handling time was longer in the stressed group than for the nonstressed. As a result, viability rate of the embryos was 19% lower in the stressed group (P < 0.05). This indicates that intensive negative HAI is likely related to stress, which affects embryo production in a superovulation program.

New molecular variants of hypothalamus-pituitary-gonad axis genes and their association with early puberty phenotype in Bos taurus indicus (Nellore)

Endocrine system plays a major role in the control of reproductive functions which are regulated by the hypothalamus-pituitary-gonad axis and its interactions. FSH and LH receptor genes are expressed at the gonads and GnRH receptor gene is expressed at the anterior pituitary gland. Misense mutations of the FSH, LH or GnRH receptors, activating or inactivating their functions in mammals, are potentially useful to allow the understanding of the role of this group of gonadotropins in reproductive phenotypes as early puberty and birth interval length. In the present study, polymorphisms in bovine exon 11 and 3`UTR of LHR, exon 10 and 3`UTR of FSHR and GnRHR genes were characterized with some of them resulting in changes in the aminoacidic chain. These polymorphic sites were found in a Bos taurus indicus (Nellore) female population by means of PCR-SSCP and DNA sequencing. Association between nucleotidic/aminoacidic changes and early puberty were determined by Chi-square analysis. It was found association between FSHR 3`UTR polymorphisms at position 2181, 2248 and 2249 bp and early puberty phenotype (p < 0.05). The presence of these new molecular markers might be considered in further studies to validate its correlation with early puberty or other reproduction associated phenotypes in cattle breeds. (C) 2007 Published by Elsevier B.V.

Obesity induced by high-fat diet promotes insulin resistance in the ovary

Besides the effects on peripheral energy homeostasis, insulin also has an important role in ovarian function. Obesity has a negative effect on fertility, and may play a role in the development of the polycystic ovary syndrome in susceptible women. Since insulin resistance in the ovary could contribute to the impairment of reproductive function in obese women, we evaluated insulin signaling in the ovary of high-fat diet-induced obese rats. Female Wistar rats were submitted to a high-fat diet for 120 or 180 days, and the insulin signaling pathway in the ovary was evaluated by immunoprecipitation and immunoblotting. At the end of the diet period, we observed insulin resistance, hyperinsulinemia, an increase in progesterone serum levels, an extended estrus cycle, and altered ovarian morphology in obese female rats. Moreover, in female obese rats treated for 120 days with the high-fat diet, the increase in progesterone levels occurred together with enhancement of LH levels. The ovary from high-fat-fed female rats showed a reduction in the insulin receptor substrate/phosphatidylinositol 3-kinase/AKT intracellular pathway, associated with an increase in FOXO3a, IL1B, and TNF alpha protein expression. These changes in the insulin signaling pathway may have a role in the infertile state associated with obesity. Journal of Endocrinology (2010) 206, 65-74

Psychosocial stress suppresses attractivity, proceptivity and pulsatile LH secretion in the ewe

Various stressors suppress pulsatile secretion of luteinizing hormone (LH) in ewes and cortisol has been shown to be a mediator of this effect under various conditions. In contrast, little is known about the impact of stress and cortisol on sexual behavior in the ewe. Therefore, we tested the hypothesis that both psychosocial stress and stress-like levels of cortisol will reduce the level of attractivity, proceptivity and receptivity in addition to suppressing LH secretion in the ewe. In Experiment 1, a layered stress paradigm of psychosocial stress was used, consisting of isolation for 4 h with the addition of restraint, blindfold and noise of a barking dog (predator stress) at hourly intervals. This stress paradigm reduced LH pulse amplitude in ovariectomized ewes. In Experiment 2, ovariectomized ewes were artificially induced into estrus with progesterone and estradiol benzoate treatment and the layered stress paradigm was applied. LH was measured and sexual behavior was assessed using T-mazes and mating tests. Stress reduced pulsatile LH secretion, and also reduced attractivity and proceptivity of ewes but had no effect on receptivity. In Experiment 3, ewes artificially induced into estrus were infused with cortisol for 30 h. Cortisol elevated circulating plasma concentrations of cortisol, delayed the onset of estrus and resulted in increased circling behavior of ewes (i.e. moderate avoidance) during estrus and increased investigation and courtship from rams. There was no effect of cortisol on attractivity, proceptivity or receptivity during estrus. We conclude that psychosocial stress inhibits LH secretion, the ability of ewes to attract rams (attractivity) and the motivation of ewes to seek rams and initiate mating (proceptivity), but cortisol is unlikely to be the principal mediator of these effects.

Cortisol disrupts the ability of estradiol-17β to induce the LH surge in ovariectomized ewes

Stress disrupts the preovulatory luteinizing hormone (LH) surge in females, but the mechanisms are unknown. We tested the hypothesis that cortisol compromises the ability of estrogen to induce a preovulatory-like LH surge in ovariectomized ewes in both the breeding and nonbreeding season. Luteinizing hormone surges were induced in ovariectomized ewes by treatment with progesterone followed by a surge-inducing estradiol-17β (E2) stimulus using a crossover design. The experiment was replicated in the breeding and nonbreeding seasons. Cortisol reduced the incidence of LH surges irrespective of season. Cortisol increased the latency from E2 stimulus to the onset of the surge in the breeding season only and suppressed the LH surge amplitude during both seasons (P < 0.01). We conclude that cortisol can interfere with the LH surge in several ways: delay, blunt, and in extreme cases prevent the E2-induced LH surge. Furthermore, the effect of cortisol to delay the E2-induced LH surge is more pronounced in the breeding season. These results show that cortisol disrupts the positive feedback effect of E2 to trigger an LH surge and suggest the involvement of multiple mechanisms.

Estradiol enables cortisol to act directly upon the pituitary to suppress pituitary responsiveness to GnRH in sheep

We have shown that cortisol infusion reduced the luteinizing hormone (LH) response to fixed hourly GnRH injections in ovariectomized ewes treated with estradiol during the non-breeding season (pituitary-clamp model). In contrast, cortisol did not affect the response to 2 hourly invariant GnRH injections in hypothalamo-pituitary disconnected ovariectomized ewes during the breeding season. To understand the differing results in these animal models and to determine if cortisol can act directly at the pituitary to suppress responsiveness to GnRH, we investigated the importance of the frequency of GnRH stimulus, the presence of estradiol and stage of the circannual breeding season. In experiment 1, during the non-breeding season, ovariectomized ewes were treated with estradiol, and pulsatile LH secretion was restored with i.v. GnRH injections either hourly or 2 hourly in the presence or absence of exogenous cortisol. Experiments 2 and 3 were conducted in hypothalamo-pituitary disconnected ovariectomized ewes in which GnRH was injected i.v. every 2 h. Experiment 2 was conducted during the non-breeding season and saline or cortisol was infused for 30 h in a cross-over design. Experiment 3 was conducted during the non-breeding and breeding seasons and saline or cortisol was infused for 30 h in the absence and presence of estradiol using a cross-over design. Samples were taken from all animals to measure plasma LH. LH pulse amplitude was reduced by cortisol in the pituitary clamp model with no difference between the hourly and 2-hourly GnRH pulse mode. In the absence of estradiol, there was no effect of cortisol on LH pulse amplitude in GnRH-replaced ovariectomized hypothalamo-pituitary disconnected ewes in either season. The LH pulse amplitude was reduced in both seasons in experiment 3 when cortisol was infused during estradiol treatment. We conclude that the ability of cortisol to reduce LH secretion does not depend upon the frequency of GnRH stimulus and that estradiol enables cortisol to act directly on the pituitary of ovariectomized hypothalamo-pituitary disconnected ewes to suppress the responsiveness to GnRH; this effect occurs in the breeding and non-breeding seasons.

Cortisol delays the estradiol-induced LH surge : is this dependent on prior ovarian steroid exposure?

Glucocorticoids can inhibit pulsatile LH secretion and can delay or even block the preovulatory LH surge. Previous work in ovariectomized ewes has indicated that cortisol can delay the estradiol-induced LH surge in an artificial follicular phase model but the results suggest this effect may be influenced by prior exposure to ovarian steroids. Here we tested the hypothesis that this disruptive effect of cortisol on the positive feedback action of estradiol is dependent on prior exposure to the ovarian steroidal milieu of the estrous cycle. Using long-term ovariectomized ewes, sequential artificial estrous cycles were created in the anestrous season by treatment and subsequent withdrawal of progesterone (CIDRs inserted for 9 d) followed by estradiol implants simulating the pre-ovulatory estradiol rise that induces the LH surge. Following the first artificial estrous cycle, a second cycle was initiated. Progesterone was again administered for 9 d followed by a second artificial follicular phase two weeks later. Beginning 2 hr prior to estradiol administration and ending at 40 hr, animals received either a cortisol infusion (elevate plasma levels to ∼170 ng/ml) or vehicle. Jugular blood was sampled hourly to assess occurrence and timing of the LH surge. Four different treatment sequences were tested (Cycle 1-Cycle 2): cortisol-cortisol; vehicle-cortisol; cortisol-vehicle; and vehicle-vehicle (n=5-6/sequence). If prior exposure to the ovarian steroidal milieu of the estrous cycle was necessary for cortisol to interfere with the positive feedback action of estradiol, then we would predict that cortisol would only delay the LH surge when it was delivered in Cycle 2 but not Cycle 1. Our results failed to support this prediction. Cortisol delayed the surge in both cycles (p<0.01), and the extent of the delay was the same in both Cycles 1 and 2 (4 hrs). Cortisol did not significantly affect surge amplitude in either cycle. These findings reinforce our previous conclusion that cortisol can delay the estradiol-induced LH surge but they do not support the hypothesis that this action of cortisol is dependent upon exposure to the ovarian steroidal milieu of the previous estrous cycle. (NIH-HD-30773)

Lipid composition of cashmere goat fibres

This study examined the differences in the chemical composition, particularly fatty acids, of the lipid extracted from the fibre of bucks, does and castrated goats. The study provides a more detailed understanding of the chemical composition of buck fibre lipid and how it varies throughout the year, and also details the effect of body region and nutrition on the production and chemical composition of lipid from buck fibre. Lipid was extracted with either petroleum ether (non-polar) or chloroform/methanol azeotrope (polar) and analysed by gas chromatography and gas chromatography-mass spectrometry. The more polar solvent system extracted larger amounts of lipid and more of each individual fatty acid. The following buck specific ethyl branched fatty acids were identified: 2-ethylhexanoic, 4-ethylhexanoic, 2-ethyloctanoic, 4-ethyloctanoic, 6-ethyloctanoic, 2-ethyldecanoic, 4-ethyldecanoic, 2-ethyldodecanoic, 6-ethyldodecanoic, 4-ethyldodecanoic, 2-ethyltetradecanoic, 6-ethyltetradecanoic, 4-ethyltetradecanoic, 2-ethylhexadecanoic and 4-ethyloctadecanoic acids. Of these buck specific fatty acids only 4-ethylhexanoic (T), 4-ethyloctanoic, 4-ethyldecanoic, 4-ethyldodecanoic, 6-ethyldodecanoic (T), 4-ethyltetradecanoic, 2-ethylhexadecanoic (T) and 4-ethylhexadecanoic acids have been previously identified or tentatively identified (T) in buck fibre extracts. This shows that the chemical composition of buck fibre lipid is more complex than previously reported, and that it may be more difficult than previously thought to artificially duplicate the odour of the buck. Buck fibre samples had lower average concentrations of 2-methylpropanoic, 2-methylbutanoic, iso-pentadecanoic, anteiso-pentadecanoic, iso-hexadecanoic, anteiso-heptadecanoic, iso-octadecanoic and anteiso-nonadecanoic acids as compared with fibre samples from does, spayed does, or wethers that were castrated at one month of age. The reduced concentrations of these fatty acids in buck fibre extracts were likely to be due to the synthesis of ethyl branched derivatives of iso and anteiso fatty acids. Buck fibre samples had higher concentrations of benzoic acid as compared with fibre samples from does, spayed does, or wethers that were castrated at one month of age. The significance of these results is that non buck specific fatty acids may also make a contribution to the odour of bucks. When fibre samples were collected at various times throughout the year, it was found that the bucks had increased amounts of lipid and ethyl branched fatty acids in fibre samples shorn from March to September, as compared with fibre samples shorn in November and January. The increase in the amount of lipid and ethyl branched fatty acids corresponded with both the rutting period of the buck and the period when the buck odour was increased. This suggests that ethyl branched fatty acids could be pheromones. The variation in lipid content and fatty acid composition was also examined between fibre samples collected from different body regions of the buck during April, as alterations in sebaceous gland activity around the neck during rutting have been reported. It was found that the average amount of lipid in the neck region of the bucks was not statistically higher than the average amounts in the midside and hind regions. However, the ethyl branched fatty acid concentrations were statistically higher in the fibre from around the neck as compared with the fibre from the other body regions, which is consistent with the odour of the buck being most pronounced around the head and neck region. The lipid content and composition of fibre samples from bucks fed high and low quality diets (lucerne and pangola grass, respectively) was examined to determine the effect of nutrition on buck specific components. The high quality diet increased the amount of lipid and ethyl branched fatty acids in fibre samples collected in April from the neck, midside and hind regions, as compared with fibre samples from the corresponding body regions from bucks fed the low quality diet. Thus it may be possible for the pheromone levels of bucks to be increased by simply providing them with good nutrition. The lipid content and ethyl branched fatty acid concentrations of fibre samples increased earlier in the year for the lucerne fed bucks as compared with the pangola grass fed bucks. The lucerne fed bucks had increased concentrations of ethyl branched fatty acids in fibre samples shorn during December to June (6 months) whereas the pangola grass fed bucks had increased concentrations of ethyl branched fatty acids in fibre samples shorn during April to August (4 months). These observations show that good nutrition can result in both the earlier production of ethyl branched fatty acids and an extended period when ethyl branched fatty acids are produced. This suggests that nutrition can be used to manipulate pheromone levels in the buck. The period when the ethyl branched fatty acids were increased corresponded with the period when the plasma luteinizing hormone (LH) and testosterone concentrations, odour and sebaceous gland volume of the bucks were increased, which supports the assumption that ethyl branched fatty acids are involved in odour production and act as pheromones.

Inhibition of the secretion of LH in ovariectomised pigs by sustained but not repeated acute elevation of cortisol in the absence but not the presence of oestradiol

Prolonged stress is known to impair reproduction. It has been proposed that reproduction will also be impaired when a severe acute stress occurs during a period of elevated plasma concentrations of oestradiol, such as during the follicular phase of the oestrous cycle. In this experiment, we hypothesised that repeated acute and sustained elevation of cortisol would suppress the secretion of LH in ovariectomised pigs and that these effects would be enhanced in the presence of oestradiol negative feedback. Cortisol (or vehicle) was administered 12 hourly to ovariectomised pigs (n=6/treatment) for 8 days in the absence of oestradiol treatment and for a further 8 days during treatment with oestradiol. Vehicle was administered to 'control' pigs, 10 or 20 mg cortisol was administered i.v. to pigs to produce 'repeated acute' elevation of cortisol and 250 mg cortisol was administered i.m. to pigs to give a 'sustained' elevation of cortisol. Both before and during treatment with oestradiol, plasma concentrations of LH were monitored on the day before treatment, on the 4th and 8th days of treatment and following an i.v. injection of GnRH at the end of the 8th day of treatment. The repeated acute elevation of cortisol did not impair any parameters of LH secretion (i.e. mean plasma concentrations of LH, pulse amplitude or frequency, pre-LH pulse nadir or the LH response to GnRH) in the absence or in the presence of oestradiol. In contrast, when the elevation of cortisol was sustained, the mean plasma concentrations of LH and the pre-LH pulse nadir were significantly (P<0.05) lower on the 8th day of treatment than on the day before treatment and on the 4th day of treatment. Nevertheless, no other parameters of LH secretion were affected and these effects only occurred in the absence (not in the presence) of oestradiol. In conclusion, cortisol needed to be elevated for more than 4 days to impair the secretion of LH, and oestradiol did not enhance the impact of cortisol on LH secretion in ovariectomised pigs.

Effects of stress on reproduction in non-rodent mammals : the role of glucocorticoids and sex differences

The means by which stress influences reproduction is not clearly understood, but may involve a number of endocrine, paracrine and neural systems. Stress impacts on the reproductive axis at the hypothalamus (to affect GnRH secretion) and the pituitary gland (to affect gonadotrophin secretion), with direct effects on the gonads being of less importance. Different stressors have different effects and there are differences in response to short- and long-term stress. Many short-term stresses fail to affect reproduction and there are reports of stimulatory effects of some 'stressors'. There are species differences in the way that specific stressors affect reproduction. Sex differences in the effects of a particular stressor have been delineated and these may relate to effects of stress at different levels of the hypothalamo-pituitary axis. The significance of stress-induced secretion of cortisol varies with species. In some instances, there appears to be little impact of short-term increases in cortisol concentrations and protracted increases in plasma concentration seem to be required before any deleterious effect on reproduction is apparent. Issues of sex, sex steroid status, type of stressor and duration of stress need to be considered to improve understanding of this issue.

Progesterone and testosterone in combination act in the hypothalamus of castrated rams to regulate the secretion of LH

We tested the hypotheses that progesterone enhances the negative feedback actions of testosterone in rams and that this occurs through actions at the hypothalamus. In the first part of this study, blood samples were collected every 10 min for 12 h before and after 7 days of treatment (i.m.) of castrated Romney Marsh rams (n=5 per group) with vehicle, progesterone (4 mg/12 h), testosterone (4 mg/12 h) or a combination of progesterone (4 mg/12 h) and testosterone (4 mg/12 h). In the second part of this study the brains of four gonad-intact Romney Marsh rams were collected, the hypothalamus was sectioned and in situ hybridisation of mRNA for progesterone receptors conducted. After 7 days of treatment with vehicle or progesterone or testosterone alone, there were no changes in the secretion of LH. In contrast, treatment with a combination of progesterone and testosterone resulted in a significant (P<0.01, repeated measures ANOVA) decrease in mean plasma concentrations of LH, the number of LH pulses per hour and the pre-LH pulse nadir and a significant (P<0.01) increase in the inter-LH pulse interval. We found cells containing mRNA for progesterone receptors throughout the hypothalamus, including the preoptic area (where most GnRH neurons are located in sheep), the periventricular, ventromedial and arcuate nuclei and the bed nucleus of the stria terminalis. This study shows that progesterone is capable of acting centrally with testosterone to suppress the secretion of LH in castrated rams and that cells containing mRNA for progesterone receptors are located in the hypothalamus of rams in the vicinity of GnRH neurons.