In sheep, as in other mammalian species, the pronounced reduction in GnRH and gonadotropin secretion that characterizes stages of infertility is normally associated with a conspicuous increase in the secretion of PRL. A possible role of PRL in modulating gonadotropin release implies the presence and activation of specific receptors in target tissues (i.e. pituitary, hypothalamus). In this study, we investigated the expression of PRL receptor (PRL-R) messenger RNA (mRNA) in the sheep pituitary and the distribution of the translated product in specific pituitary cell types. Using primers designed to flank different regions of the extracellular and cytoplasmic domains of the PRL-R, two complementary DNA (cDNA) fragments, one of which was specific for the long-form PRL-R, were amplified by reverse transcriptase-PCR. Sequencing revealed more than 95% identity with nucleotides 267-1272 of the bovine PRL-R cDNA. When these cDNA fragments were used as probes for the detection of PRL-R mRNA expression by Northern analysis, three major transcripts of approximately 13, 10, and 3.5 kb were identified in the pituitary. Both probes detected identical transcripts, suggesting that primarily the long form of PRL-R is expressed in the sheep pituitary gland. No difference in the abundance of pituitary PRL-R mRNA transcripts was observed between anestrous and breeding season ewes (P > 0.05). Additional RT-PCR studies revealed the existence of a cDNA variant bearing a 39-bp insert with a premature stop codon. Translation of the PRL-R mRNA was confirmed by Western blot analysis. The identification of PRL-R in specific pituitary cell types was carried out by immunocytochemistry. Double immunofluorescent staining, using antibodies to the rat liver PRL-R and specific monoclonal antibodies to the LHbeta-subunit, FSHbeta-subunit, free alpha-subunit, PRL, or GH, revealed that in both the pars distalis and pars tuberalis, all pituitary cells expressing PRL-R immunoreactivity were positive for LHbeta, although only 53% of LHbeta-positive cells expressed PRL-R. A small proportion (2%) of gonadotrophs expressing PRL-R immunoreactivity were negative for FSHbeta, indicating the specific localization of PRL-R in LH (or LH/FSH) secreting cells. Further, a selective cytological association was detected in the pars distalis where LH gonadotrophs appeared surrounded by lactotrophs. In contrast to these observations, PRL-R immunoreactivity was completely absent in lactotrophs and in the vast majority (>98%) of somatotrophs. In conclusion, here we show the expression of PRL-R mRNA in the sheep pituitary and the specific translation of the signal in LH (or LH/FSH) gonadotrophs. These results support the hypothesis that PRL may be involved in the regulation of gonadotropin secretion through a paracrine mechanism within the pituitary gland and that this action does not seem to be mediated by changes in PRL-R mRNA expression.

Seasonal changes in mammalian physiology, such as those affecting reproduction, hibernation, and metabolism, are controlled by pituitary hormones released in response to annual environmental changes. In temperate zones, the primary environmental cue driving seasonal reproductive cycles is the change in day length (i.e., photoperiod), encoded by the pattern of melatonin secretion from the pineal gland. However, although reproduction relies on hypothalamic gonadotrophin-releasing hormone output, and most cells producing reproductive hormones are in the pars distalis (PD) of the pituitary, melatonin receptors are localized in the pars tuberalis (PT), a physically and functionally separate part of the gland. How melatonin in the PT controls the PD is not understood. Here we show that melatonin time-dependently acts on its receptors in the PT to alter splicing of vascular endothelial growth factor (VEGF). Outside the breeding season (BS), angiogenic VEGF-A stimulates vessel growth in the infundibulum, aiding vascular communication among the PT, PD, and brain. This also acts on VEGF receptor 2 (VEGFR2) expressed in PD prolactin-producing cells known to impair gonadotrophin secretion. In contrast, in the BS, melatonin releases antiangiogenic VEGF-Ab from the PT, inhibiting infundibular angiogenesis and diminishing lactotroph (LT) VEGFR2 expression, lifting reproductive axis repression in response to shorter day lengths. The time-dependent, melatonin-induced differential expression of VEGF-A isoforms culminates in alterations in gonadotroph function opposite to those of LTs, with up-regulation and down-regulation of gonadotrophin gene expression during the breeding and nonbreeding seasons, respectively. These results provide a mechanism by which melatonin can control pituitary function in a seasonal manner.

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.

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.