Variable duration of reproductive suppression in male coyotes (Canis latrans) treated with a high dose of the gonadotrophin-releasing hormone agonist deslorelin.

Effective and humane management strategies for coyotes (Canis latrans) remain elusive. We hypothesised that exposure to a high dose of a gonadotrophin-releasing hormone (GnRH) agonist would cause prolonged suppression of the reproductive axis. Two groups of male coyotes were administered 47mg deslorelin in the form of either five 9.4-mg controlled-release Suprelorin (Peptech Animal Health, Macquarie Park NSW, Australia) implants (n=3) or 10 4.7-mg implants (n=5). In the first group, deslorelin suppressed plasma LH, testosterone and testes volume in two of three coyotes for three breeding seasons. In the second group, two of five deslorelin-treated coyotes had no sperm production after 1 year and plasma LH, FSH, testosterone and testes volume were suppressed. Although plasma gonadotropins and testosterone were suppressed in three treated coyotes in group two, testes volume and sperm production were evident. Because the duration of suppression differed among individual coyotes, we further hypothesised that a variation in deslorelin release underlay the variability. To test this, we analysed in vivo plasma profiles of deslorelin concentrations. These profiles suggested that deslorelin concentrations >100 pg mL-1 are required to maintain suppression in male coyotes. For field implementation, the development of an implant capable of releasing deslorelin for the life of the coyote is necessary.

Developmental Programming: Insulin Sensitizer Prevents the GnRH-Stimulated LH Hypersecretion in a Sheep Model of PCOS.

Prenatal testosterone (T) treatment recapitulates the reproductive and metabolic phenotypes of polycystic ovary syndrome in female sheep. At the neuroendocrine level, prenatal T treatment results in disrupted steroid feedback on gonadotropin release, increased pituitary sensitivity to GnRH, and subsequent LH hypersecretion. Because prenatal T-treated sheep manifest functional hyperandrogenism and hyperinsulinemia, gonadal steroids and/or insulin may play a role in programming and/or maintaining these neuroendocrine defects. Here, we investigated the effects of prenatal and postnatal treatments with an androgen antagonist (flutamide [F]) or an insulin sensitizer (rosiglitazone [R]) on GnRH-stimulated LH secretion in prenatal T-treated sheep. As expected, prenatal T treatment increased the pituitary responsiveness to GnRH leading to LH hypersecretion. Neither prenatal interventions nor postnatal F treatment normalized the GnRH-stimulated LH secretion. Conversely, postnatal R treatment completely normalized the GnRH-stimulated LH secretion. At the tissue level, gestational T increased pituitary LHß, androgen receptor, and insulin receptor-ß, whereas it reduced estrogen receptor (ER)α protein levels. Although postnatal F normalized pituitary androgen receptor and insulin receptor-ß, it failed to prevent an increase in LHß expression. Contrarily, postnatal R treatment restored ERα and partially normalized LHß pituitary levels. Immunohistochemical findings confirmed changes in pituitary ERα expression to be specific to gonadotropes. In conclusion, these findings indicate that increased pituitary responsiveness to GnRH in prenatal T-treated sheep is likely a function of reduced peripheral insulin sensitivity. Moreover, results suggest that restoration of ERα levels in the pituitary may be one mechanism by which R prevents GnRH-stimulated LH hypersecretion in this sheep model of polycystic ovary syndrome-like phenotype.

Does salt have a permissive role in the induction of puberty?

Puberty is starting earlier than ever before and there are serious physiological and sociological implications as a result of this development. Current research has focused on the potential role of high caloric, and commensurate high adiposity, contributions to early puberty. However, girls with normal BMI also appear to be initiating puberty earlier. Westernized diets, in addition to being high in fat and sugar, are also high in salt. To date, no research has investigated a link between elevated salt and the reproductive axis. We hypothesize that a high salt diet can result in an earlier onset of puberty through three mechanisms that are not mutually exclusive. (1) High salt activates neurokinin B, a hormone that is involved in both the reproductive axis and salt regulation, and this induces kisspeptin release and ultimate activation of the reproductive axis. (2) Vasopressin released in response to high salt acts on vasopressin receptors expressed on kisspeptin neurons in the anteroventral periventricular nucleus, thereby stimulating gonadotropin releasing hormone and subsequently luteinizing hormone secretion. (3) Salt induces metabolic changes that affect the reproductive axis. Specifically, salt acts indirectly to modulate adiposity, ties in with the obesity epidemic, and further compounds the pathologic effects of obesity. Our overall hypothesis offers an additional cause behind the induction of puberty and provides testable postulates to determine the mechanism of potential salt-mediated affects on puberty.

Contraception has gone to the coyotes (Canis latrans).

Coyotes (Canis latrans) are predators of livestock. Current management programs, primarily lethal control, are ineffective for long-term management of predation. Controlling reproduction of coyotes may reduce depredations if territory fidelity is maintained by breeding pairs. Surgical sterilization is successful in altering predatory behaviors of coyotes but may provide a challenge for field implementation. An alternative approach is the development of a one-time non-transferable chemical contraceptive. This research is investigating the efficacy of a single high dose treatment of a sustained release gonadotropin-releasing hormone agonist, deslorelin, on coyotes as a long term contraceptive. Male coyotes were administered 47 mg deslorelin subcutaneously. Preliminary data show full suppression of the reproductive axis for over 12 mo as indicated by complete absence of sperm.

Dose and durational effects of the gonadotropin-releasing hormone agonist, deslorelin: the male rat (Rattus norvegicus) as a model.

Gonadotropin-releasing hormone (GnRH) agonists are routinely used to suppress the reproductive axis of many mammals, especially in zoos. Current treatments are reversible. There is a need to develop nonreversible agents, and this study investigates the effects of high-dose and long-duration exposure to the GnRH agonist, deslorelin, in the rat model. Studies indicate that the follicle-stimulating hormone (FSH) gonadotropin is predominantly affected, and following high-dose exposure to deslorelin for a long duration, the ability of gonadotropes to synthesize FSH may be compromised, perhaps permanently. Understanding the mechanisms by which such persistent suppression of FSH occurs may facilitate the development of novel next-generation contraceptives. It is hypothesized that direct testicular effects of GnRH agonists may play a critical role in the efficacy of GnRH agonists in male contraception.

Intra-pituitary administration revisited: development of a novel in vivo approach to investigate the ovine hypophysis.

The anterior pituitary gland regulates physiological processes via the secretion of hormones, which are under the control of factors produced either in the hypothalamus or the pituitary gland itself. Studies investigating how the pituitary gland functions have employed both in vitro and in vivo approaches. Although in vitro analysis has the advantage that it is pituitary specific, the results may be incomplete because the tissue is isolated from other physiological inputs that could affect function under natural conditions. Without vascular input, such studies are inherently of short duration. Conversely, in vivo experiments that rely upon systemic hormone injections require high doses, are non-target specific and the precise hormone concentrations reaching the pituitary gland are difficult to control. Intracerebroventricular hormone infusions are reliant on assumptions that factors are transported to the pituitary gland from the cerebrospinal fluid and are without cerebral effects. Here we describe an innovative method to investigate anterior pituitary function in conscious sheep by direct infusion of peptides into the pituitary tissue surrounding the hypophyseal portal blood vessels. This approach is an adaptation of the hypophyseal portal cannulation technique whereby an indwelling cannula provides direct access to the rostral aspect of the adenohypophysis. Peptide infusions were achieved by insertion of a needle through the implanted cannula such that it penetrated the pituitary. Using this technique, infusion of TRH (17 ng/1 µl/min for up to 6h) induced a sustained rise in systemic prolactin levels that lasted for the duration of the infusion.

Wired on steroids: sexual differentiation of the brain and its role in the expression of sexual partner preferences.

The preference to seek out a sexual partner of the opposite sex is robust and ensures reproduction and survival of the species. Development of female-directed partner preference in the male is dependent on exposure of the developing brain to gonadal steroids synthesized during critical periods of sexual differentiation of the central nervous system. In the absence of androgen exposure, a male-directed partner preference develops. The development and expression of sexual partner preference has been extensively studied in rat, ferret, and sheep model systems. From these models it is clear that gonadal testosterone, often through estrogenic metabolites, cause both masculinization and defeminization of behavior during critical periods of brain development. Changes in the steroid environment during these critical periods result in atypical sexual partner preference. In this manuscript, we review the major findings which support the hypothesis that the organizational actions of sex steroids are responsible for sexual differentiation of sexual partner preferences in select non-human species. We also explore how this information has helped to frame our understanding of the biological influences on human sexual orientation and gender identity.

A novel animal model to study hot flashes: no effect of gonadotropin-releasing hormone.

OBJECTIVE: Menopausal hot flashes compromise the quality of life for most women. The physiological mechanisms underlying hot flashes remain poorly understood, and the absence of an animal model to investigate hot flashes hinders investigations in this field. METHODS: We first developed the sheep as a model to study peripheral skin temperature changes using fever-inducing lipopolysaccharide (LPS; 200 microg/kg) administered to ovary-intact ewes. Because a strong correlation between luteinizing hormone pulses and hot flashes has previously been reported, we then determined whether intravenous gonadotropin-releasing hormone (GnRH; 1 mg), a dose sufficient to elevate cerebrospinal fluid-GnRH concentrations, could modulate ear skin temperature in both ovariectomized and low-estrogen-replaced ovariectomized ewes. RESULTS: Some ewes responded to LPS in heart rate and abdominal temperature, but there was no significant effect on either parameter or cheek temperature for the group. In contrast, LPS injection caused a significant (P < 0.001) change in skin temperature at the ear. Ear temperature showed no significant change in response to GnRH relative to control injections in both ovariectomized and low estrogen ewes. CONCLUSIONS: We developed a model animal system in the ewe that can accurately detect small changes in peripheral skin temperature. This system has the potential to be extremely useful in future studies investigating the pathology of hot flashes and holds several advantages over previous model systems developed for this research. GnRH per se does not seem to be involved in thermoregulatory events.

Substance P-immunoreactive cells in the ovine pars tuberalis.

The pars tuberalis (PT) is a distinct subdivision of the anterior pituitary gland that plays a central role in regulating seasonal prolactin release. In sheep, there is compelling evidence that seasonal changes in light, transformed into a melatonin signal, are interpreted by the PT to modulate the release of a factor which affects prolactin release. The identity of this factor(s) is unknown but has been preemptively called 'tuberalin'. In the present study, we report on an initial immunocytochemical investigation where we have identified that many ovine PT cells are immunoreactive for the tachykinin substance P (SP). Few cells in the pars distalis immunoreact for SP. The SP-immunoreactive cells did not colocalize with beta-luteinizing hormone. RT-PCR confirmed the presence of preprotachykinin A mRNA in the PT. We hypothesize that SP, and possibly other preprotachykinin A-derived tachykinins, may play a role in the seasonal regulation of prolactin secretion in sheep.

Rethinking the stalk effect: a new hypothesis explaining suprasellar tumor-induced hyperprolactinemia.

The pars tuberalis is a distinct subdivision of the pituitary gland but its function remains poorly understood. Suprasellar tumors in this pars tuberalis region are frequently accompanied by hyperprolactinemia. As these tumors do not immunoreact for any of the established pituitary hormones, they are classified as nonsecretory. It has been postulated that these suprasellar tumors induce hyperprolactinemia by compressing the pituitary stalk, resulting in impaired dopamine delivery to the pituitary and, consequently, disinhibition of the lactotropes. An alternative hypothesis proposed is that suprasellar tumors secrete a specific pars tuberalis factor that stimulates prolactin secretion. Hypothesized candidates are the preprotachykinin A derived tachykinins, substance P and/or neurokinin A.

The gonadotropin-releasing hormone type I receptor is expressed in the mouse cerebellum.

Gonadotropin-releasing hormone (GnRH) is a decapeptide hypothalamic hormone that was named according to its first discovered function--at the head of the neuroendocrine reproductive axis. Numerous other organ systems express GnRH and/or its receptor, although a specific physiological role for GnRH outside of the reproductive axis has yet to be established. Several studies in lower vertebrates have reported GnRH and/or its receptor in the cerebellum. Here, we describe the presence of immunoreactive GnRH receptors in the Purkinje cells of the mammalian cerebellum for the first time. This study provides compelling anatomical evidence for a common link between the cerebellum and the hypothalamo-pituitary axis. Dysfunction of this link occurs in the rare genetic ataxia disorders--Gordon Holmes syndrome and Boucher-Neuhauser syndrome.

Gonadotropin-releasing hormone in third ventricular cerebrospinal fluid: endogenous distribution and exogenous uptake.

GnRH is detectable in the cerebrospinal fluid (CSF), but its source remains unidentified. Previous studies have harvested CSF for GnRH analysis from the median eminence region, but it is unknown whether GnRH in CSF is restricted to this region. If CSF-GnRH plays a physiological role, through volume transmission, to communicate with brain regions that express GnRH receptors but are not evidently innervated by GnRH neurons, then it is essential to establish whether GnRH is more pervasive throughout the cerebroventricular system. Three cannulae were placed in the supraoptic, infundibular, and pineal recesses of the third ventricle. GnRH was undetectable in lateral ventricle CSF. GnRH pulses were detected in all ewes in infundibular recess CSF, but at sites more rostral (supraoptic) and caudal (pineal), GnRH pulse frequency and amplitude significantly (P<0.05) decreased. A GnRH surge was evident in CSF collected simultaneously from all cannulae, but the amplitude was greatest (P<0.05) at the infundibular recess. A final study established whether iv administered GnRH enters the CSF. A 250-ng GnRH dose did not affect CSF-GnRH concentrations (1.6+/-0.3 pg/ml), but 2.5 microg (2.7+/-0.2 pg/ml; P<0.001) and 1 mg (38.5+/-10.6 pg/ml; P<0.05) significantly increased CSF-GnRH concentrations. The present study shows: 1) the median eminence region is likely to be the major, if not only, source of GnRH entering the cerebroventricular system; and 2) exogenous GnRH crosses the blood-brain barrier, but extremely high doses are required to elevate CSF concentrations to physiological levels. Thus, CSF-GnRH may affect sites that are closer in proximity to the infundibular recess region than previously thought.

Immunoreactive GnRH type I receptors in the mouse and sheep brain.

Gonadotropin Releasing Hormone-I (GnRH) has been implicated in an array of functions outside the neuroendocrine reproductive axis. Previous investigations have reported extensive GnRH binding in numerous sites and this has been supported by in situ hybridization studies reporting GnRH receptor mRNA distribution. The present study on mice and sheep supports and extends these earlier investigations by revealing the distribution of cells immunoreactive for the GnRH receptor. In addition to sites previously shown to express GnRH receptors such as the hippocampus, amygdala and the arcuate nucleus, the improved resolution afforded by immunocytochemistry detected cells in the mitral cell lay of the olfactory bulb as well as the central grey of the mesencephalon. In addition, GnRH receptor immunoreactive neurons in the hippocampus and mesencephalon of the sheep were shown to colocalize with estrogen receptor beta. Although GnRH may act at some of these sites to regulate reproductive processes, evidence is accumulating to support an extra-reproductive role for this hypothalamic decapeptide.

No effect of nutrient restriction from gestational days 28 to 78 on immunocytochemically detectable growth hormone-releasing hormone (GHRH) neurons and GHRH receptor colocalization in somatotropes of the ovine female fetus.

The maternal environment affects fetal development and may permanently affect the physiology of the adult. Fetal growth hormone (GH) secretion is increased by maternal undernutrition but the physiological mechanisms responsible for this increase are unknown. We have recently found evidence suggesting that the GHRH component of the fetal neuroendocrine GH axis may be perturbed by undernutrition. This study sought to determine the effect of maternal undernutrition on immunocytochemically detectable GHRH neurons and the expression of GHRH receptors by somatotropes in the pituitary gland. Ewes were grouped (n=12 per group) randomly into control (fed 100% of requirements) or nutrient restricted (fed 50% of requirements) from days 28 to 78 of gestation, corresponding to the period from implantation to the end of placentation. At day 78, half the ewes were killed and the fetal brains were perfused. The remaining ewes were re-alimented to 100% of nutritional requirements and killed at day 135. There was no effect of nutrition restriction or age on the number of GHRH neurons. Similarly, the mean density and percentage of somatotropes expressing GHRH receptors was not significantly different between treatment groups at either age. This study found no effect, as determined by immunocytochemistry, of nutrient restriction on the GHRH component of the fetal neuroendocrine GH axis. It remains to be established if the release of GHRH and responsiveness of somatotropes to GHRH in the fetus are affected by undernutrition.

Signaling complexes associated with the type I gonadotropin-releasing hormone (GnRH) receptor: colocalization of extracellularly regulated kinase 2 and GnRH receptor within membrane rafts.

Our previous work demonstrated that the type I GnRH receptor (GnRHR) resides exclusively and constitutively within membrane rafts in alphaT3-1 gonadotropes and that this association was necessary for the ability of the receptor to couple to the ERK signaling pathway. G(alphaq), c-raf, and calmodulin have also been shown to reside in this compartment, implicating a raft-associated multiprotein signaling complex as a functional link between the GnRHR and ERK signaling. In the studies reported here, we used subcellular fractionation and coimmunoprecipitation to analyze the behavior of ERKs with respect to this putative signaling platform. ERK 2 associated partially and constitutively with low-density membranes both in alphaT3-1 cells and in whole mouse pituitary. Cholesterol depletion of alphaT3-1 cells reversibly blocked the association of both the GnRHR and ERKs with low-density membranes and uncoupled the ability of GnRH to activate ERK. Analysis of the kinetics of recovery of ERK inducibility after cholesterol normalization supported the conclusion that reestablishment of the association of the GnRHR and ERKs with the membrane raft compartment was not sufficient for reconstitution of signaling activity. In alphaT3-1 cells, the GnRHR and ERK2 coimmunoprecipitated from low-density membrane fractions prepared either in the presence or absence of detergent. The GnRHR also partitioned into low-density, detergent-resistant membrane fractions in mouse pituitary and coimmunoprecipitated with ERK2 from these fractions. Collectively, these data support a model in which coupling of the GnRHR to the ERK pathway in gonadotropes involves the assembly of a multiprotein signaling complex in association with specialized microdomains of the plasma membrane.

Effect of nutrient restriction on the somatotropes and substance P-immunoreactive cells in the pituitary of the female ovine fetus.

The maternal environment affects fetal development and may influence the physiology of the adult. Fetal growth hormone (GH) is increased by maternal undernutrition but the mechanisms responsible are unknown. This study determined the effect of maternal undernutrition on the development of fetal pituitary somatotropes in the female. Ewes were grouped randomly into control (fed 100% of requirements) or nutrient restricted (fed 50%) from Days 28 to 78 of gestation. At Day 78, the ewes were killed and fetuses collected (Day 78 NR (nutrient restricted): n=6; Day 78C (control): n=6). Remaining ewes were realimented to 100% of nutritional requirements and were killed at Day 135 (Day 135 NR (nutrient restricted): n=6; Day 135 C (control): n=6). Somatotropes were visualized immunocytochemically and the size, mean density, total percentage and proportion colocalized with substance P were determined for each group. Nutrient restriction increased (p<0.01) the density of pituitary cells in Day 78 fetuses but this difference was no longer apparent by Day 135 after realimentation. The density and proportion of somatotropes were not different between treatment groups at Day 78 but were significantly (p<0.05) lower in the nutrient restricted Day 135 fetuses as compared to the Day 135 control animals. Somatotropes from restricted fetuses were significantly (p<0.001) larger at Day 78. Nutrient restriction increased the density (p<0.001) and percentage (p<0.05) of substance P-immunoreactive cells Day 135 fetuses. Similarly, the proportion of somatotropes that expressed substance P was significantly (p<0.05) increased by nutrient restriction in the Day 135 fetuses. Although nearly two thirds of substance P-immunoreactive cells co-expressed GH, there was no significant effect of treatment on this co-expression. Additional studies are required to determine if other components of the neuroendocrine GH axis are affected by this nutritional insult, if the alterations that we have observed, particularly in the tachykinin system, persist into adulthood and, importantly, what are the long-term consequences of an altered GH axis.

Progesterone-receptive dopaminergic and neuropeptide Y neurons project from the arcuate nucleus to gonadotropin-releasing hormone-rich regions of the ovine preoptic area.

Progesterone inhibits gonadotropin-releasing hormone (GnRH) secretion in sheep through an interneuronal system located in the mediobasal hypothalamus. This study focused on known inhibitors of GnRH secretion in sheep, dopamine and neuropeptide Y (NPY). As the distributions of tyrosine hydroxylase (TH)- and NPY-immunoreactive neurons overlap with progesterone receptors (PR) in the arcuate nucleus, we hypothesized that, if these neurons mediate, at least partially, the inhibitory feedback signal of progesterone, then they should co-express PRs. Fluorogold (FG), a retrograde tracer, was injected into the preoptic area of ovariectomized ewes pretreated with estrogen and progesterone. When the FG injection site encompassed at least 80 GnRH neurons, sections from the arcuate nucleus were processed using dual immunocytochemistry for PR and either TH or NPY. We found that 30% of PR-immunoreactive, 12% of TH-containing and 21% of NPY-synthesizing neurons project toward this GnRH-rich region. Of the PR/TH dual-labeled cells, which represent 21% of PR and 31% of TH cells, respectively, 22% displayed FG labeling. Of the PR/NPY neurons, which account for 19% of PR and 67% of NPY neurons, respectively, 26% were FG fluorescent. This study suggests that subsets of arcuate nucleus dopaminergic and NPY neurons may transduce, at least in part, the progesterone-mediated inhibition of GnRH secretion.

Distribution of galanin receptor 1-immunoreactive neurons in the ovine hypothalamus: colocalization with GnRH.

Galanin is implicated in numerous physiological functions, including reproduction. Where and how galanin acts in the brain is poorly understood, but recent evidence suggests that it is predominantly through the GAL-R1 receptor. Using an antibody raised against the third intracellular loop of rat GAL-R1, a region that is highly conserved among species, our first objective was to determine the distribution of cells expressing immunoreactive GAL-R1 in the hypothalamus of the sheep. GAL-R1-immunoreactive cells were spread widely in the ovine diencephalon and overlapped with the known distribution of GnRH neurons. Galanin has been shown to enhance GnRH secretion, but it is not known whether this effect is transduced at the level of the GnRH neuron or is indirect. Thus, our second objective was to establish if GnRH neurons throughout the hypothalamus expressed GAL-R1 receptors and, if so, whether GAL-R1 expression in GnRH neurons was influenced by season, gender and/or stage of the estrous cycle. In rams and ewes during the non-breeding season, only a tenth of the GnRH neurons expressed immunocytochemically detectable GAL-R1 receptors. In contrast, a fifth of the GnRH neurons expressed immunocytochemically detectable GAL-R1 in the luteal phase, whereas only a twentieth expressed GAL-R1 in the follicular phase. These data suggest that galanin may affect a subpopulation of GnRH neurons through the GAL-R1 receptor and that this affect may be modulated by steroids.

Progesterone-receptive beta-endorphin and dynorphin B neurons in the arcuate nucleus project to regions of high gonadotropin-releasing hormone neuron density in the ovine preoptic area.

Progesterone inhibits gonadotropin-releasing hormone (GnRH) secretion through interneuronal systems located in the mediobasal hypothalamus in ewes. Endogenous opioid peptides are implicated in this inhibition of GnRH secretion. The distributions of endogenous opioid peptides are known to overlap with progesterone receptors (PR) in the arcuate nucleus. We investigated whether PR is expressed by beta-endorphin and dynorphin B neurons in the arcuate nucleus and if a subset of double-labeled cells projects to the preoptic area where most GnRH neurons are detected. Injection of a retrograde tracer, Fluorogold, into the rostral preoptic area was performed in ovariectomized ewes pretreated with estrogen and progesterone. Brain sections were processed using double immunocytochemistry. Only brains of ewes with an injection site encompassing at least 80 GnRH neurons were processed for PR and then either beta-endorphin or dynorphin B immunocytochemistry. Antigen retrieval is essential for PR detection but causes Fluorogold to fade. Thus, quantitative analysis was performed on photographs taken before and after antigen retrieval. We found that 25-30% of PR-containing neurons, 20% of beta-endorphin cells and 22% of dynorphin B neurons in the arcuate nucleus project toward the preoptic area. From the PR/beta-endorphin double-labeled cells that represent 25 and 36% of PR and beta-endorphin cells, respectively, 35% were labeled with Fluorogold. From the PR/dynorphin B double-labeled cells that account for 39 and 62% of PR and dynorphin B neurons, respectively, 26% contained Fluorogold. These data strongly support the hypothesis that progesterone acts in the arcuate nucleus through beta-endorphin and dynorphin B neurons to affect preoptic area GnRH neurons.

Colocalization of GH, TSH and prolactin, but not ACTH, with betaLH-immunoreactivity: evidence for pluripotential cells in the ovine pituitary.

Increasing evidence suggests that multihormonal cells in the pituitary gland may be more commonplace than previously thought. This has forced us to reconsider our classical view of cell populations in the pituitary gland. Studies so far have focused almost exclusively on the rat, and there is a dearth of information on other species. Our first objective was to determine whether a subpopulation of gonadotropes also express somatotropin in the ewe, as reported in the rat. In addition, we sought to determine whether gonadotropes express any of the other known pituitary hormones. Finally, we investigated whether the stage of the estrous cycle influenced the occurrence of these pluripotential gonadotropes. We found that a small population of betaLH-immunoreactive cells also expresses immunoreactive GH, prolactin and TSH. No gonadotropes colocalized with ACTH. Significantly (P<0.001) more gonadotropes expressed GH during the luteal (10.7+/-0.4%) than the late follicular (5.4+/-0.3%) phase but there was no difference between the luteal and follicular phases in the proportion of gonadotropes expressing prolactin (follicular: 5.7+/-0.7%; luteal: 5.5+/-0.6%) or TSH (follicular: 3.1+/-0.7%; luteal: 4.2+/-0.5%). Similarly, there was a significant (P<0.05) difference in the proportion of GH-immunoreactive cells expressing betaLH immunoreactivity in the luteal (5.9+/-0.3%) and follicular (3.4+/-0.5%) phases but no difference in the proportion of prolactin- (follicular: 2.2+/-0.7%; luteal: 2.0+/-0.8%) or TSH-immunoreactive cells (follicular: 9.6+/-3.7%; luteal: 10.8+/-2.9%) expressing betaLH. The specific function of these multihormonal gonadotropes in sheep remains to be determined.