Domingo J. Tortonese

Seasonal Changes in Circadian Peripheral Plasma Concentrations of Melatonin, Serotonin, Dopamine and Cortisol in Aged Horses with Cushing’s Disease under Natural Photoperiod

Equine pituitary pars intermedia dysfunction (PPID) is a common and serious condition that gives rise to Cushing’s disease. In the older horse, it results in hyperadrenocorticism and disrupted energy metabolism, the severity of which varies with the time of year. To gain insight into the mechanism of its pathogenesis, 24‐h profiles for peripheral plasma melatonin, serotonin, dopamine and cortisol concentrations were determined at the winter and summer solstices, and the autumn and spring equinoxes in six horses diagnosed with Cushing’s disease and six matched controls. The nocturnal rises in plasma melatonin concentrations, although different across seasons, were broadly of the same duration and similar amplitude in both groups of animals (P > 0.05). The plasma concentrations of cortisol did not show seasonal variation and were different in diseased horses only in the summer when they were higher across the entire 24‐h period (P < 0.05). Serotonin concentrations were not significantly affected by time of year but tended to be lower in Cushingoid horses (P = 0.07). By contrast, dopamine output showed seasonal variation and was significantly lower in the Cushing’s group in the summer and autumn (P < 0.05). The finding that the profiles of circulating melatonin are similar in Cushingoid and control horses reveals that the inability to read time of year by animals suffering from Cushing’s syndrome is an unlikely reason for the disease. In addition, the results provide evidence that alterations in the dopaminergic and serotoninergic systems may participate in the pathogenesis of PPID.

Endogenous Circannual Cycles of Ovarian Activity and Changes in Prolactin and Melatonin Secretion in Wild and Domestic Female Sheep Maintained under a Long-Day Photoperiod

Abstract The present study examines the ovulatory activity of wild and domesticated ewes subjected to either a constant photoperiod of long days (16L:8D) or natural changes in daily photoperiod for 16 mo. The aim was to determine whether an endogenous reproductive rhythm controls seasonal reproductive activity in these sheep, and how the photoperiod might affect this. The effects of long-day photoperiods on long-term changes in prolactin and melatonin secretion were also evaluated. The two species showed changes in reproductive activity under the constant photoperiod conditions, suggesting the existence of an endogenous rhythm of reproduction. This rhythm was differently expressed in the two types of ewe (P < 0.05), with the domestic animals exhibiting much greater sensitivity to the effects of long days. A circannual rhythm of plasma prolactin concentration was also seen in both species and under both photoperiod conditions, although in both species the amplitude was always lower in the long-day animals (P < 0.01). The duration of the nocturnal melatonin plasma concentrations reflected the duration of darkness in both species and treatments. The peak melatonin concentration did not differ between seasons either under natural or long-day photoperiods.

Direct effects of prolactin and dopamine on the gonadotroph response to GnRH

The intrapituitary mechanisms underlying the inhibitory actions of hyperprolactinaemia on the reproductive axis remain unclear. Previous work on primary pituitary cultures revealed combined suppressive effects of prolactin (PRL) and dopamine on the gonadotrophin response to GnRH. However, whether these effects occur directly at the level of the gonadotroph and are accompanied by changes in gene expression is still unresolved. Here, alphaT(3)-1 and LbetaT2 cells were used to investigate the effects of PRL and dopamine on gonadotrophin synthesis and release in gonadotroph monocultures under basal and GnRH-stimulated conditions. PRL receptor and dopamine receptor mRNA expressions were first determined by RT-PCR in both cell lines. Then, PRL and the dopamine agonist bromocriptine (Br), alone or in combination, were shown to block the maximal alpha-subunit and LHbeta-subunit mRNA responses to a dose-range of GnRH. The LH secretory response was differentially affected by treatments. GnRH dose-dependently stimulated LH release, with a 4-5 fold increase at 10(-8) M GnRH. Unexpectedly, PRL or Br stimulated basal LH release, with PRL, but not Br, enhancing the LH secretory response to GnRH. This effect was, however, completely blocked by Br. These results reveal direct effects of PRL and dopamine at the level of the gonadotroph cell, and interactions between these two hormones in the regulation of gonadotrophin secretion. Moreover, uncoupling between LH synthesis and release in both the basal and the GnRH-stimulated responses to PRL and dopamine was clearly apparent.

Gonadotropin-Releasing Hormone Stimulates Prolactin Release from Lactotrophs in Photoperiodic Species Through a Gonadotropin-Independent Mechanism

Abstract Previous studies have provided evidence for a paracrine interaction between pituitary gonadotrophs and lactotrophs. Here, we show that GnRH is able to stimulate prolactin (PRL) release in ovine primary pituitary cultures. This effect was observed during the breeding season (BS), but not during the nonbreeding season (NBS), and was abolished by the application of bromocriptine, a specific dopamine agonist. Interestingly, GnRH gained the ability to stimulate PRL release in NBS cultures following treatment with bromocriptine. In contrast, thyrotropin-releasing hormone, a potent secretagogue of PRL, stimulated PRL release during both the BS and NBS and significantly enhanced the PRL response to GnRH during the BS. These results provide evidence for a photoperiodically modulated functional interaction between the GnRH/gonadotropic and prolactin axes in the pituitary gland of a short day breeder. Moreover, the stimulation of PRL release by GnRH was shown not to be mediated by the gonadotropins, since immunocytochemical, Western blotting, and PCR studies failed to detect pituitary LH or FSH receptor protein and mRNA expressions. Similarly, no gonadotropin receptor expression was observed in the pituitary gland of the horse, a long day breeder. In contrast, S100 protein, a marker of folliculostellate cells, which are known to participate in paracrine mechanisms within this tissue, was detected throughout the pituitaries of both these seasonal breeders. Therefore, an alternative gonadotroph secretory product, a direct effect of GnRH on the lactotroph, or another cell type, such as the folliculostellate cell, may be involved in the PRL response to GnRH in these species.

Effects of Prolactin on the Luteinizing Hormone Response to Gonadotropin- Releasing Hormone in Primary Pituitary Cell Cultures During the Ovine Annual Reproductive Cycle

Abstract In the sheep pituitary, the localization of prolactin (PRL) receptors in gonadotrophs and the existence of gonadotroph-lactotroph associations have provided morphological evidence for possible direct effects of PRL on gonadotropin secretion. Here, we investigated whether PRL can readily modify the LH response to GnRH throughout the ovine annual reproductive cycle. Cell populations were obtained from sheep pituitaries during the breeding season (BS) and the nonbreeding season (NBS), plated to monolayer cultures for 7 days, and assigned to receive one of the following treatments: 1) nil (control), 2) acute (90- min) bromocriptine (ABr), 3) chronic (7-day) bromocriptine (CBr), 4) ABr and PRL, 5) CBr and PRL, 6) PRL alone, or 7) thyrotropin-releasing hormone. Cells were treated as described above, with the aim of decreasing or increasing the concentrations of PRL in the culture, and simultaneously treated with GnRH for 90 min. The LH concentrations in the medium were then determined by RIA. GnRH stimulated LH in a dose-dependent manner during both stages of the annual reproductive cycle. During the NBS, single treatments did not significantly affect the LH response to GnRH. However, when PRL was combined with bromocriptine, either acutely or chronically, GnRH failed to stimulate LH release at all doses tested (P < 0.01). In contrast, during the BS, the LH response to GnRH was not affected by any of the experimental treatments. These results reveal no apparent effects of PRL alone, but an interaction between PRL and dopamine in the regulation of LH secretion within the pituitary gland, and a seasonal modulation of this mechanism.

Hypothalamic Targets for Prolactin: Assessment of c-Fos Induction in Tyrosine Hydroxylase- and Proopiomelanocortin-Containing Neurones in the Rat Arcuate Nucleus following Acute Central Prolactin Administration

Prolactin (PRL) has been implicated in central actions including those that result in its own regulation and/or the suppression of gonadotropin secretion. It is not clear, however, which neuronal systems may mediate the central effects of PRL. Here, using dual immunohistochemistry for c-Fos and either tyrosine hydroxylase (TH) or proopiomelanocortin (POMC), we have assessed neuronal activation, following centrally administered PRL, within two neuronal networks that have been shown to participate in the inhibitory regulation of reproductive function. Male rats received one intracerebroventricular injection of either PRL (5 µg) or saline (vehicle control) 5 days after cannulae were inserted into the lateral ventricles. Ninety minutes after treatment, animals were perfused with 4% paraformaldehyde, the brains were removed and 30-µm frozen sections were cut throughout the entire hypothalamic region. Parallel sets of sections were processed for both c-Fos immunoreactivity (ir) and either TH-ir or POMC-ir. PRL increased the mean number of c-Fos-ir neurons within the rostral arcuate nucleus (9.3 ± 2.0 vs. 5.0 ± 1.2 cells/section, for PRL and control rats, respectively; p < 0.05). Within the TH-ir neurones, PRL induced a significant increase in c-Fos in the dorsomedial portion of the mid-arcuate nucleus (p < 0.05). In contrast, there was no significant increase in the expression of c-Fos within the POMC neurones of the arcuate nucleus. PRL also induced c-Fos expression in the supraoptic nucleus (SON) (11.7 ± 3.2 vs. 3.0 ± 1.4 cells/section for PRL and control rats, respectively; p < 0.05), but not in the medial preoptic nucleus, ventromedial nucleus or the dorsomedial nucleus, areas reported to either contain gonadotropin-releasing hormone neurones or express PRL receptors. The results from this study show immediate early gene activation within both the arcuate nucleus and the SON of the hypothalamus following acute PRL administration. While the role of PRL-responsive neurones in the SON remains to be elucidated, these findings support the notion that the central actions of PRL could be mediated via the TH neurones of the dorsomedial arcuate nucleus and/or by a population of neurones in the rostral arcuate nucleus that contain neither TH nor POMC.

Characterization and Distribution of Gonadotrophs in the Pars Distalis and Pars Tuberalis of the Equine Pituitary Gland During the Estrous Cycle and Seasonal Anestrus

Abstract Little is known about the neuroendocrine control of fertility in the horse. In this species, unusual features characterize the normal estrous cycle such as a prolonged preovulatory LH surge during the follicular phase and a distinctive FSH surge during the midluteal phase. This study investigated the distribution and hormonal identity of gonadotrophs in the pars distalis (PD) and pars tuberalis (PT) of the equine pituitary gland as possible morphological bases for the referred unusual endocrine characteristics. In addition, the proportion of gonadotrophs in relation to other pituitary cell types during both the estrous cycle and anestrus were investigated. Pituitary glands were collected from sexually active (n = 5) and seasonally anestrous (n = 5) mares in November, and single or double immunofluorescent staining was carried out on 6-μm sections using monoclonal antibodies to the LHβ or FSHβ subunits and a polyclonal antibody to ovine LHβ. Gonadotrophs were densely distributed around the pars intermedia in the PD and in the caudal ventral region of the PT. In addition to isolated cells, clusters of gonadotrophs were found surrounding the capillaries. No significant differences were detected in the number of gonadotrophs between sexually active and anestrous mares in either the PD or PT. In the PD, gonadotrophs represented 22.7 ± 5.8% and 19.1 ± 2.1% of the total cell density in sexually active and anestrous animals, respectively (P > 0.05). However, in the PT, gonadotrophs accounted for a higher proportion of the total cell population in sexually active (6 ± 0.1%) than in anestrous (1.2 ± 0.05%) mares (P < 0.02). Double immunofluorescence revealed that the majority of gonadotrophs were bihormonal (i.e., positive for LH and FSH); however, in the sexually active mare, a larger proportion of gonadotrophs (22.5 ± 3.6%) were monohormonal for either LH or FSH, when compared to anestrous animals (9.7 ± 1.2%; P < 0.02). Based on these findings we conclude that: 1) although the relative distribution of gonadotrophs is similar to those reported for other species, a significantly larger proportion of gonadotroph cells is present in the equine pituitary gland; 2) gonadotroph density does not appear to differ between sexually active and anestrous mares in the PD; 3) a larger proportion of gonadotrophs is apparent in the PT of sexually active animals; and 4) although a large incidence of bihormonal gonadotrophs is present in the horse, specific LH or FSH cells differentiate predominantly during the sexually active phase.

Does melatonin act on dopaminergic pathways in the mediobasal hypothalamus to mediate effects of photoperiod on prolactin secretion in the ram

Previous studies have shown that the chronic administration of melatonin in the mediobasal hypothalamus (MBH) using micro-implants in Soay rams housed under long days causes a sustained decrease in the secretion of prolactin as occurs in response to short days. The purpose of this study was to investigate whether hypothalamic dopaminergic (DA) systems acting through D2 receptors may be involved in this melatonin-induced effect. Groups of Soay rams living under long days were treated in the MBH with micro-implants containing bromocriptine (BROM, DA D2 receptor agonist), or sulpiride (SULP, DA D2 receptor antagonist), given alone or in combination with melatonin (MEL), to establish whether the DA drugs would mimic or negate the effects of melatonin. A control group (C) received empty micro-implants or no treatment. The micro-implants were bilateral and were left in place for 14 weeks; the trial continued for a total of 28 weeks (14-week implant period and 14-week post-implant period) while the animals remained under long days. The ability of the micro-implants to release BROM and SULP for 14 weeks was confirmed by incubating implants in vitro and testing for the presence of the compounds using a pituitary cell bioassay. MEL in the MBH induced a marked decrease in the blood plasma concentrations of prolactin during the implant period and an increase during the postimplant period (MEL vs. C, p < 0.001). BROM given alone induced a sustained decrease in the plasma concentrations of prolactin (less marked than MEL), while SULP caused an increase (BROM and SULP vs. C, p < 0.001); the effects were restricted to the implant period. BROM given in combination with MEL produced the same effect as MEL alone during both the implant and postimplant periods, while SULP given with MEL produced the same effect as MEL during the implant period, but impaired the increase in plasma concentrations of prolactin during the postimplant period (MEL + SULP vs. MEL, p < 0.001). There were changes in growth and moulting of the pelage correlated with the marked changes in the secretion of prolactin induced by MEL, but not related to the lesser effects of BROM and SULP. In conclusion, the long-term effects of the D2 agonist and antagonist are consistent with the inhibitory role of hypothalamic DA pathways in the homeostatic regulation of prolactin secretion. The inhibitory effect of the D2 agonist did not mimic that of MEL in the MBH, thus it is unlikely that the short day MEL signal operates primarily through a hypothalamic DA system to inhibit the secretion of prolactin. However, since the administration of the D2 antagonist in the MBH did influence the response to MEL, it is probable that DA pathways are involved in relaying the effects of MEL on the long-term cycle in the secretion of prolactin in the ram.

Role of prolactin in the gonadotroph responsiveness to gonadotrophin-releasing hormone during the equine annual reproductive cycle.

A combined suppressive effect of prolactin (PRL) and dopamine on the secretion of luteinising hormone (LH) at the level of the pituitary gland has been identified in sheep, a short‐day breeder. However, little is known about the role of PRL in the intra‐pituitary regulation of the gonadotrophic axis in long‐day breeders. In the present study, we investigated the effects of PRL on LH and follicle‐stimulating hormone (FSH) secretion during the equine annual reproductive cycle. Horse pituitaries were obtained during the breeding season (BS) and nonbreeding season (NBS). Cells were dispersed, plated to monolayer cultures and assigned to one of the following specific treatments: (i) medium (Control); (ii) rat PRL (rPRL); (iii) thyrotrophin‐releasing hormone (TRH); (iv) bromocriptine (Br); and (v) Br + rPRL. Gonadotrophin‐releasing hormone (GnRH) dose‐dependently stimulated LH release during the BS and NBS. During the BS, neither rPRL nor TRH affected the LH response to GnRH, but Br significantly (P < 0.01) enhanced both basal and GnRH‐stimulated LH release through a mechanism that did not involve alterations in the concentrations of PRL. However, rPRL prevented the Br‐induced increase in basal and GnRH‐stimulated LH output, and suppressed LH below basal values (P < 0.05). Conversely, during the NBS, no significant effects of treatments were observed. Interestingly, at this time of year, the incidence of pituitary gap junctions within the pars distalis decreased by 50% (P < 0.01). By contrast to the effects on LH, no treatment effects were detected on the FSH response to GnRH, which was only apparent during the NBS. These results reveal no direct effects of PRL but an interaction between PRL and dopamine in the inhibitory regulation of LH, but not FSH, release at the level of the pituitary in the horse, and a modulatory role of season/photoperiod associated with alterations in folliculostellate cell‐derived gap junctions.

The equine hypophysis: a gland for all seasons

The intrahypophysial mechanisms involved in the control of gonadotrophin secretion remain unclear. In the horse, a divergent pattern of gonadotrophins is observed at different stages of the reproductive cycle in response to a single secretagogue (gonadotrophin-releasing hormone), and dramatic changes in fertility take place throughout the year in response to photoperiod. This species thus provides a useful model to investigate the regulation of fertility directly at the level of the hypophysis. A series of studies were undertaken to examine the cytological arrangements and heterogeneity of gonadotrophin storage in the pars distalis (PD) and pars tuberalis (PT) of the hypophysis of male and female horses. Specifically, the seasonal and gonadal effects on distribution, density and hormonal identity of gonadotrophs, the existence of gonadotroph-lactotroph associations and the expression of prolactin receptors (PRL-R) as possible morphological bases for the differential control of gonadotrophin secretion were investigated. It became apparent that both isolated and clustered gonadotrophs are normally distributed around the pars intermedia and surrounding capillaries in the PD, and in the caudal ventral region of the PT. In the PD, no effects of season or of reproductive state on the density or number of gonadotrophs could be detected in either male or female animals. In contrast, a fivefold increase in gonadotroph density was observed in the PT during the sexually active stage. In males, robust gonadal effects were detected on the gonadotroph population; orchidectomy significantly reduced both the number and proportion of gonadotrophs, in relation to other hypophysial cell types, in both the PD and PT regions. Luteinizing hormone (LH) monohormonal, follicle-stimulating hormone (FSH) monohormonal and bihormonal gonadotrophs were identified in the PD and PT of male and female horses. Interestingly, in males, the relative proportions of gonadotroph subtypes and the LH/FSH monohormonal gonadotroph ratio were not affected by either season or the presence of the gonads. In contrast, a larger proportion of monohormonal gonadotrophs was clearly observed in sexually active females. Specific gonadotroph-lactotroph associations and expression of PRL-R in cells other than gonadotrophs were detected in the PD throughout the annual reproductive cycle. In addition to a stimulatory gonadal effect on lactotroph density, a substantial gonadal-independent effect of season was apparent on this variable. The findings have revealed important seasonal and gonadal effects on the cytological configuration of the equine hypophysis, which may provide the morphological basis for the intrahypophysial control of fertility.