Intrapituitary mechanisms underlying the control of fertility: key players in seasonal breeding.

Recent studies have shown that, in conjunction with dynamic changes in the secretion of GnRH from the hypothalamus, paracrine interactions within the pituitary gland play an important role in the regulation of fertility during the annual reproductive cycle. Morphological studies have provided evidence for close associations between gonadotropes and lactotropes and gap junction coupling between these cells in a variety of species. The physiological significance of this cellular interaction was supported by subsequent studies revealing the expression of prolactin receptors in both the pars distalis and pars tuberalis regions of the pituitary. This cellular interaction is critical for adequate gonadotropin output because, in the presence of dopamine, prolactin can negatively regulate the LH response to GnRH. Receptor signaling studies showed that signal convergence at the level of protein kinase C and phospholipase C within the gonadotrope underlies the resulting inhibition of LH secretion. Although this is a conserved mechanism present in all species studied so far, in seasonal breeders such as the sheep and the horse, this mechanism is regulated by photoperiod, as it is only apparent during the long days of spring and summer. At this time of year, the nonbreeding season of the sheep coincides with the breeding season of the horse, indicating that this inhibitory system plays different roles in short- and long-day breeders. Although in the sheep, it contributes to the complete suppression of the reproductive axis, in the horse, it is likely to participate in the fine-tuning of gonadotropin output by preventing gonadotrope desensitization. The photoperiodic regulation of this inhibitory mechanism appears to rely on alterations in the folliculostellate cell population. Indeed, electron microscopic studies have recently shown increased folliculostellate cell area together with upregulation of their adherens junctions during the spring and summer. The association between gonadotropes and lactotropes could also underlie an interaction between the gonadotropic and prolactin axes in the opposite direction. In support of this alternative, a series of studies have demonstrated that GnRH stimulates prolactin secretion in sheep through a mechanism that does not involve the mediatory actions of LH or FSH and that this stimulatory effect of GnRH on the prolactin axis is seasonally regulated. Collectively, these findings highlight the importance of intercellular communications within the pituitary in the control of gonadotropin and prolactin secretion during the annual reproductive cycle in seasonal breeders.

Ovulatory activity and plasma prolactin concentrations in wild and domestic ewes exposed to artificial photoperiods between the winter and summer solstices.

Mouflon and domestic Manchega sheep differ in the timing of their reproductive season under natural photoperiod (NP) conditions. This study examines whether they also differ in their reproductive responses to artificial photoperiod cues. For this, mouflons (n=24) and ewes (n=24) were exposed between the winter and summer solstices to artificial long days (LD: 16 h light/day), to short days (SD) simulated via the use of melatonin implants, or to NP conditions (controls), and their ovulatory activity monitored. The effects of these treatments on annual changes in prolactin concentration were also recorded. In the LD mouflon ewes, the offset and onset (7 March ± 5 and 2 October ± 4, respectively) of cyclic ovulatory activity occurred earlier (P<0.001) than in the NP animals (26 April ± 6 and 20 October ± 2, respectively), but no differences were seen (P>0.05) between the SD and NP mouflon ewes in either the onset of anoestrus (12 May ± 6 and 26 April ± 6, respectively) or the onset of subsequent ovulatory activity (13 October ± 8 and 20 October ± 2, respectively); however the duration of the anoestrus period was significantly reduced in the SD. In LD Manchega ewes, the onset of anoestrus was advanced (2 February ± 5 vs 15 March ± 11), but ovulatory activity started at the same time as in NP Manchega ewes (16 July ± 4 vs 5 July ± 8). In the SD Manchega ewes, two animals showed continuous cyclic ovulatory activity over the course of the experiment while the remainder entered anoestrus two months later (16 May ± 6, P<0.001) than their NP counterparts. In these SD ewes, the onset of cyclic ovarian activity was very variable. An annual rhythm of plasma prolactin concentration was seen in both the mouflon and Manchega ewes under all three photoperiod conditions. However, the amplitudes of the changes seen in prolactin concentration were smaller in both the LD and SD animals than in the corresponding NP animals (P<0.001). In conclusion, the results show that these two types of Mediterranean sheep differ in their ovulatory response when subjected to artificial photoperiods. The results also suggest that refractoriness to SDs may be the most important physiological mechanism regulating the onset of anoestrus in highly seasonal breeds, but not in less seasonal breeds.

Ovarian VEGF(165)b expression regulates follicular development, corpus luteum function and fertility.

Angiogenesis and vascular regression are critical for the female ovulatory cycle. They enable progression and regression of follicular development, and corpora lutea formation and regression. Angiogenesis in the ovary occurs under the control of the vascular endothelial growth factor-A (VEGFA) family of proteins, which are generated as both pro-(VEGF(165)) and anti(VEGF(165)b)-angiogenic isoforms by alternative splicing. To determine the role of the VEGF(165)b isoforms in the ovulatory cycle, we measured VEGF(165)b expression in marmoset ovaries by immunohistochemistry and ELISA, and used transgenic mice over-expressing VEGF(165)b in the ovary. VEGF(165)b was expressed in the marmoset ovaries in granulosa cells and theca, and the balance of VEGF(165)b:VEGF(165) was regulated during luteogenesis. Mice over-expressing VEGF(165)b in the ovary were less fertile than wild-type littermates, had reduced secondary and tertiary follicles after mating, increased atretic follicles, fewer corpora lutea and generated fewer embryos in the oviduct after mating, and these were more likely not to retain the corona radiata. These results indicate that the balance of VEGFA isoforms controls follicle progression and luteogenesis, and that control of isoform expression may regulate fertility in mammals, including in primates.

Experimental jetlag disrupts circadian clock genes but improves performance in racehorses after light-dependent rapid resetting of neuroendocrine systems and the rest-activity cycle.

Abrupt alterations in the 24-h light : dark cycle, such as those resulting from transmeridian air travel, disrupt circadian biological rhythms in humans with detrimental consequences on cognitive and physical performance. In the present study, a jetlag-simulated phase shift in photoperiod temporally impaired circadian peaks of peripheral clock gene expression in racehorses but acutely enhanced athletic performance without causing stress. Indices of aerobic and anaerobic capacities were significantly increased by a phase-advance, enabling prolonged physical activity before fatigue occurred. This was accompanied by rapid re-entrainment of the molecular clockwork and the circadian pattern of melatonin, with no disturbance of the adrenal cortical axis, but a timely rise in prolactin, which is a hormone known to target organs critical for physical performance. Subsequent studies showed that, unlike the circadian pattern of melatonin, and in contrast to other species, the daily rhythm of locomotor activity was completely eliminated under constant darkness, but it was restored immediately upon the reintroduction of a light : dark cycle. Resetting of the rhythm of locomotion was remarkably fast, revealing a rapid mechanism of adaptation and a species dependency on light exposure for the expression of daily diurnal activity. These results show that horses are exquisitely sensitive to sudden changes in photoperiod and that, unlike humans, can benefit from them; this appears to arise from powerful effects of light underlying a fast and advantageous process of adjustment to the phase shift.

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.

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.

Gonadotroph heterogeneity, density and distribution, and gonadotroph-lactotroph associations in the pars distalis of the male equine pituitary gland.

The intrapituitary mechanisms regulating gonadotrophin secretion in the horse remain unclear. Here, we examined seasonal and gonadal effects on the gonadotroph and lactotroph populations of male horses with the aim of defining a possible morphological basis for the differential release of gonadotrophins. Pituitaries were collected from: (i) gonadal-intact horses in the breeding season (GBS); (ii) orchidectomized horses in the breeding season (OBS); and (iii) orchidectomized horses in the nonbreeding season (ONBS). Immunohistochemistry was performed using antibodies to the luteinizing hormone (LH) beta subunit, follicle-stimulating hormone (FSH) beta subunit and prolactin. In all groups, gonadotrophs were distributed throughout the pars distalis, with dense populations detected near the borders with the pars tuberalis and pars intermedia. The numbers of LH-monohormonal, FSH-monohormonal and bihormonal cells/field were greater in GBS than in OBS and ONBS horses. Similarly, the proportion of gonadotrophs in relation to all pituitary cells was larger in gonadal-intact than orchidectomized horses. In the absence of the gonads, no effects of season were observed on these variables. Interestingly, the relative proportions of gonadotroph subtypes and the LH/FSH gonadotroph ratio were similar among groups. Furthermore, while specific gonadotroph-lactotroph associations were identified in all groups, significant gonadal effects within the breeding season and direct effects of season within orchidectomized horses were detected for the number of lactotrophs. This study reveals a gonadal-independent effect of season on the lactotroph, but not the gonadotroph population of the equine pituitary, and a clear gonadal stimulation of both cell types within the breeding season. We suggest that alterations in intercellular arrangements, rather than changes in the incidence of gonadotroph subtypes, may contribute to the differential release of gonadotrophins and, accordingly, to the intrapituitary control of fertility throughout the male equine annual reproductive cycle.

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 microg) 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-microm 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.

Developmental capacity in vitro of prepubertal oocytes.

A 2-year comparative study was carried out to evaluate the effect of ovary size, follicle size and oocyte quality of 3-month-old Simmental calves and the efficiency of using calf ovaries in an in vitro fertilization (IVF) programme. We evaluated the effects of different concentrations of follicle-stimulating hormone (FSH) and oestradiol-17beta (E-17beta) in the maturation medium on the in vitro development of calf oocytes into morula and blastocysts. The proportion of recovered oocytes (62.1%; 42.8%; 25.3%) and the percentage of good quality cumulus oocyte complexes (84.2%: 59.8%; 45.9%) decreased significantly (P < 0.01) with decreasing ovary size (L, M and S). The rates of two or more cells on Day 2 and of blastocysts on Day 7 and Day 9 were significantly lower (P < 0.01) for calf oocytes (61.5%; 18.9%: 15.9%) compared with those from sexually matured females (70.1%: 32.3%; 22.2%). Calf oocytes. matured in medium supplemented with 20 microg/ml or 10 microg/ml FSH plus 2 microg/ml E-17beta had higher rates of cleavage on Day 2 (64.1% and 64.7%) and blastocysts on Day 7 (24.5% and 22.4%) than the control supplemented with 10 microg/ml FSH (55.6% and 19.2%, respectively). Groups supplemented with 20 microg/ml FSH plus 2 microg/ml E-17beta and 10 mg/ml plus 4 mg/ml E-17beta showed a significantly lower developmental rate of blastocysts on Day 7 (14.6% and 14.5%). High concentrations of E-17beta (4 microg/ml) resulted in a significantly lower development of blastocysts on Day 9 (8.1%) and hatched blastocysts on Day 13 (3.5%) (P < 0.01). We conclude that the proportion of calf oocytes obtained from immature animals and their suitability for IVF are lower than those of cows. Thus, the use of oocytcs from sexually immature females would decrease the relative efficiency of IVF programmes. Supplementation with high concentrations of FSH can improve the maturation and developmental capacity of oocytes from prepubertal calves.

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.

Characterization and distribution of gonadotrophs in the pars distalis and pars tuberalis of the equine pituitary gland during the estrous cycle and seasonal anestrus.

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-microm sections using monoclonal antibodies to the LHbeta or FSHbeta subunits and a polyclonal antibody to ovine LHbeta. 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.

Gonadotroph-lactotroph associations and expression of prolactin receptors in the equine pituitary gland throughout the seasonal reproductive cycle.

An interaction between gonadotroph and lactotroph cells of the pituitary gland has long been recognized in several species. The current study was conducted to investigate whether an association between gonadotrophs and lactotrophs occurs in mares and whether prolactin receptors are expressed within the pituitary gland of this species. The effects of both reproductive state and season on these variables were examined in pituitary glands obtained from sexually active mares in July (breeding season), sexually active mares in November (non-breeding season) and anoestrous mares in November. Pituitaries were dissected out immediately after death and immunofluorescent staining was carried out on 6 micrometer sections using specific antibodies to the LHbeta subunit, FSHbeta subunit, prolactin and prolactin receptor. Gonadotrophs were observed in both the pars distalis and pars tuberalis; although they appeared mostly as isolated cells, small groups of gonadotrophs were also identified in the pars distalis. In contrast, lactotrophs were observed only as clusters of cells exclusively in the pars distalis of sexually active and anoestrous mares in November and in most of the sexually active mares in July. A specific gonadotroph-lactotroph association was identified only between large isolated gonadotrophs and lactotroph clusters. Double immunofluorescent staining for FSHbeta and prolactin revealed a similar gonadotroph-lactotroph association to the one detected for LH gonadotrophs. No statistical difference in the gonadotroph:lactotroph ratio was observed as a result of changes in reproductive status or season. However, a tendency for a simultaneous decrease in the number of gonadotrophs and an increase in the number of lactotrophs was detected in anoestrous animals. Prolactin receptor immunoreactivity was found in the pars distalis, but not in the pars tuberalis, of sexually active (July and November) and anoestrous animals for both long and short forms of the receptor. No prolactin receptor co-localization for either form of the receptor was observed in LH or FSH gonadotrophs in either of the reproductive states examined during both summer and winter seasons. Furthermore, no significant difference was apparent in the proportion of cells expressing prolactin receptors between mares of different reproductive state or season. The specific anatomical association between gonadotroph and lactotroph cells and the expression of prolactin receptors in the equine pituitary gland indicate a potential role of prolactin in the regulation of gonadotrophin secretion. However, the absence of evidence for co-localization of prolactin receptors in LH or FSH cells does not support the hypothesis of a direct effect of prolactin on the gonadotroph as reported in a short day breeder. The results raise the possibility that, in horses, an intermediate regulatory cell may mediate the action of prolactin on gonadotroph function.

Interaction between hypothalamic dopaminergic and opioidergic systems in the photoperiodic regulation of pulsatile luteinizing hormone secretion in sheep.

Previous studies in sheep have shown that whereas the inhibitory effects of dopamine (DA) systems on GnRH/gonadotrophin secretion are readily detectable during the sexually inactive phase under long days (LD), the suppressive effects of endogenous opioid peptide (EOP) systems are most evident during the sexually active phase under short days (SD). The hypothesis proposed in this study is that inhibitory DA pathways interact with EOP neurons to regulate GnRH/gonadotropin secretion in sheep and that photoperiod modulates this interaction to relay its effect on the seasonal reproductive cycle. To test this hypothesis, the effects of a DA agonist (bromocriptine) or of a DA antagonist (sulpiride) on the pulsatile LH response to an opioid antagonist (naloxone) were evaluated in sexually active Soay rams exposed to SD, and then reassessed when sexually inactive under LD. The experimental design comprised six treatments: 1) control (vehicle); 2) bromocriptine; 3) sulpiride; 4) naloxone; 5) pretreatment with bromocriptine followed by naloxone; 6) pretreatment with sulpiride followed by naloxone. Under SD, when DA pathways are thought to be quiescent and EOP systems active, bromocriptine suppressed pulsatile LH secretion (P < 0.01), whereas sulpiride had no effect. Under this photoperiod, naloxone induced a conspicuous stimulation of episodic LH release (P < 0.01). This effect was prevented by pretreatment with bromocriptine (P < 0.01), but was not affected by pretreatment with sulpiride. Conversely, under LD, when the activity of DA pathways is thought to be increased and that of EOP systems reduced, bromocriptine was without effect, whereas sulpiride evoked a mild increase in LH pulse frequency (P < 0.05). Under this photoperiod, naloxone induced a smaller stimulation than under SD. This effect was again blocked by pretreatment with bromocriptine but, in contrast to SD, markedly enhanced by pretreatment with sulpiride (P < 0.01). Particularly relevant was that the DA agonist blocked the stimulatory effects of the EOP antagonist under SD, and that the DA antagonist enhanced the effects of the EOP antagonist only under LD. These results are consistent with the hypothesis proposing that, in sheep, DA pathways have a predominant inhibitory effect on both GnRH and EOP neurons, and that changes in day length modulate the interplay between DA and EOP systems as part of the mechanisms involved in the photoperiodic control of the seasonal reproductive cycle.

Prolactin replacement fails to inhibit reactivation of gonadotropin secretion in rams treated with melatonin under long days.

This study tested the hypothesis that prolactin (PRL) inhibits gonadotropin secretion in rams maintained under long days and that treatment with melatonin (s.c. continuous-release implant; MEL-IMP) reactivates the reproductive axis by suppressing PRL secretion. Adult Soay rams were maintained under long days (16L:8D) and received 1) no further treatment (control, C); 2) MEL-IMP for 16 wk and injections of saline/vehicle for the first 8 wk (M); 3) MEL-IMP for 16 wk and exogenous PRL (s.c. 5 mg ovine PRL 3x daily) for the first 8 wk (M+P). The treatment with melatonin induced a rapid increase in the blood concentrations of FSH and testosterone, rapid growth of the testes, an increase in the frequency of LH pulses, and a decrease in the LH response to N-methyl-D,L-aspartic acid. The concomitant treatment with exogenous PRL had no effect on these reproductive responses but caused a significant delay in the timing of the sexual skin color and growth of the winter pelage. These results do not support the hypothesis and suggest that PRL at physiological long-day concentrations, while being totally ineffective as an inhibitor of gonadotropin secretion, acts in the peripheral tissues and skin to maintain summer characteristics.

Detection of prolactin receptor gene expression in the sheep pituitary gland and visualization of the specific translation of the signal in gonadotrophs.

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.

Impairment in follicle-stimulating hormone secretion by different treatments with follicular fluid as a source of inhibin blocks the ability of pulsatile luteinizing hormone to induce preovulatory surges of gonadotropins in prepubertal ewe lambs.

This study was conducted to investigate whether FSH can affect the ability of pulsatile exogenous LH to induce a preovulatory surge of gonadotropins in prepubertal ewe lambs. March-born ewe lambs were randomly assigned in November to the following treatments: 1) control; 2) 1.5 ml charcoal-extracted bovine follicular fluid (bFF) for 240 h; 3) 1.5 ml bFF for 72 h; and 4) 3 ml bFF for 72 h. Follicular fluid was administered as a source of inhibin to reduce secretion of FSH. Starting at 0 h, all animals were given (i.v.) 10 micrograms ovine LH every h for a 48-h period. Lambs in the control group did not receive further treatment. Females in group 2 were administered (s.c.) 1.5 ml bFF every 8 h from -184 h to 56 h. In groups 3 and 4, lambs received (s.c.) 1.5 ml bFF, respectively, every 8 h from -16 h to 56 h. Preovulatory surges of LH and FSH occurred concomitantly in 4 of 6 control lambs at a mean time of 34 h (range 20-42 h). In contrast, none of the females in groups 2, 3, or 4 showed a preovulatory surge of gonadotropins in response to pulsatile LH (p < 0.05). At -16 h, FSH was reduced in the ewe lambs of group 2 (p < 0.01). Thereafter, mean concentrations and area under the curve for FSH were lower (p < 0.001), and variability in the pattern of secretion was reduced (p < 0.05), in lambs of groups 2, 3, and 4 as compared to controls. LH, apart from the preovulatory surge, was not affected. Females in groups 2, 3, and 4 secreted less estradiol than controls (p < 0.01). Mean concentrations and area under the curve for estradiol during the 20 h preceding preovulatory surges (20 h = earliest surge) in controls were greater than those during the 20 h preceding 34 h (34 h = mean time for surges) in lambs that did not show a surge (p < 0.001). The induced luteal phases were of short duration, and no animal continued to cycle. Ovarian follicular development was evident in the majority (8 of 10) of the randomly selected lambs examined by laparoscopy during the expected midluteal phase. In summary, long- and short-term administration of bFF resulted in reduced concentrations of FSH, preventing the ability of pulsatile LH to induce a preovulatory surge of gonadotropins in prepubertal ewe lambs. The decrease in estradiol associated with the bFF-induced reduction in FSH may have been the mechanism responsible for the lack of LH and FSH surges. It is proposed that unimpaired secretion of FSH is required for treatment with LH to induce preovulatory surges of gonadotropins in this model.

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.

Effects of melatonin in the mediobasal hypothalamus on the secretion of gonadotrophins in sheep: role of dopaminergic pathways.

Previous studies have shown that treatment with micro-implants of melatonin in the mediobasal hypothalamus (MBH) of sexually inactive Soay rams exposed to long days induces an increase in the secretion of FSH and reactivation of the testicular axis, as normally occurs in response to short days. The current study was conducted to investigate the possible involvement of hypothalamic dopaminergic (DA) systems in this melatonin-induced effect. At 10 weeks under long days, sexually inactive Soay rams were treated in the MBH with micro-implants containing bromocriptine (DA agonist) or sulpiride (DA antagonist), given alone or in combination with melatonin, to establish whether the DA drugs would mimic or negate the effects of melatonin. All micro-implants were inserted bilaterally and left in place for 14 weeks; the study lasted a total of 28 weeks (14 weeks implant period and 14 weeks post-implant period) while the animals remained under long days. The ability of the micro-implants to release bromocriptine and sulpiride for 14 weeks was confirmed by incubating implants in vitro and testing for the presence of the compounds in the incubate using a pituitary cell bioassay. Profiles of FSH, determined in blood samples collected three times weekly, were significantly different among treatments (time x treatment interaction, P < 0.001, ANOVA). Melatonin in the MBH induced a marked increase in the concentrations of FSH during the implant period, and a decrease during the post-implant period (P < 0.001). Bromocriptine given alone in the MBH induced a decrease in the concentrations of FSH which became statistically different from the control during the post-implant period (P < 0.05). Treatment with sulpiride alone also resulted in a suppressive effect during the post-implant period (P < 0.01). When given in combination with melatonin, bromocriptine or sulpiride significantly reduced the melatonin-induced increase in the concentrations of FSH observed during the implant period (P < 0.001). The results support the view that DA pathways in the MBH play an important role in the inhibitory regulation of gonadotrophin secretion in the ram. The inhibitory effect of bromocriptine is likely to result from the direct activation of the hypothalamic DA receptors linked to GnRH neurones regulating the secretion of FSH. The apparent paradoxical inhibitory effect of sulpiride is thought to be due to enhanced gonadal steroid negative feedback resulting from blockade of the inhibitory DA pathways, as evidenced by significantly increased secretion of testosterone (P < 0.05) in the animals receiving sulpiride in combination with melatonin.(ABSTRACT TRUNCATED AT 400 WORDS)

Photoperiodic modulation of the dopaminergic control of pulsatile LH secretion in sheep.

This study was conducted to investigate whether the photoperiodic regulation of the seasonal changes in pulsatile LH secretion in the ram involves changes in the activity of inhibitory hypothalamic dopaminergic (DA) pathways. To test this hypothesis, a series of experiments was carried out in Soay rams in which the effects of a DA-D2 receptor antagonist (sulpiride) or a DA-D2 receptor agonist (bromocriptine) on the pulsatile secretion of LH were determined under both long and short days. In each experiment blood samples were collected every 10 min for 8 h starting at the time of vehicle, sulpiride or bromocriptine injections to assess concentrations of LH. Sulpiride (0.59 mg/kg, s.c.) administered to rams under long days induced an immediate and sustained increase in the secretion of LH that lasted for approximately 4 h (P < 0.05; ANOVA); this LH response reflected both a rise in mean concentrations (0.247 +/- 0.03 vs. 0.452 +/- 0.1 microgram/l) and an increase in the frequency of LH pulses (0.5 +/- 0.5 vs. 2.33 +/- 0.42 pulses/8 h; P < 0.01). In contrast, under short days sulpiride had no effect. Bromocriptine (0.06 mg/kg, s.c.) administered to rams under long days, when LH concentrations were low, was without effect, but when given to rams under short days significantly (P < 0.05) suppressed mean LH concentrations (0.627 +/- 0.08 vs. 0.320 +/- 0.02 microgram/l) and LH pulse frequency (4.86 +/- 0.46 vs. 2.43 +/- 0.37 pulses/8 h).(ABSTRACT TRUNCATED AT 250 WORDS)