Photoperiod, but not progesterone, has a strong impact upon the transcriptome of the medio-basal hypothalamus in female goats and ewes.

Photoperiod is the main environmental driver of seasonal responses in organisms living at temperate and polar latitudes. Other external cues such as food and temperature, and internal cues including hormones, intervene to fine-tune phasing of physiological functions to the solar year. In mammals, the medio-basal hypothalamus (MBH) is the key integrator of these cues, which orchestrates a wide array of seasonal functions, including breeding. Here, using RNAseq and RT-qPCR, we demonstrate that molecular components of the photoperiodic response previously identified in ewes are broadly conserved in does (female goats, Capra hircus), with a common core of ∼50 genes. This core group can be defined as the "MBH seasonal trancriptome", which includes key players of the pars tuberalis-tanycytes neuroendocrine retrograde pathway that governs intra-MBH photoperiodic switches of triiodothyronine (T3) production (Tshb, Eya3, Dio2 and SlcO1c1), the two histone methyltransferases Suv39H2 and Ezh2 and the secreted protein Vmo1. Prior data in ewes revealed that T3 and estradiol (E2), both key hormones for the proper timing of seasonal breeding, differentially impact the MBH seasonal transcriptome, and identified cellular and molecular targets through which these hormones might act. In contrast, information regarding the potential impact of progesterone (P4) upon the MBH transcriptome was nonexistent. Here, we demonstrate that P4 has no discernible transcriptional impact in either does or ewes. Taken together, our data show that does and ewes possess a common core set of photoperiod-responsive genes in the MBH and conclusively demonstrate that P4 is not a key regulator of the MBH transcriptome.

Highly-Sensitive In Vitro Bioassays for FSH, TSH, PTH, Kp, and OT in Addition to LH in Mouse Leydig Tumor Cell.

We demonstrate here that highly sensitive in vitro bioassays for FSH, TSH, and PTH can be set up in mouse Leydig Tumor Cells (mLTC), in addition to the normal LH/CG bioassay, after they were transfected with expression vectors encoding the corresponding Gs Protein-Coupled Receptors (GsPCR), such as FSHR, TSHR, or PTHR. Although the ß2 adrenergic receptor is also a GsPCR, its expression in mLTC led to a significant but very low cAMP response compared to those observed with FSH, TSH, or PTH. Similarly, after transfection of the GiPCR MT1 melatonin receptor, we did not observe any inhibitory effect by melatonin of the LH or hCG stimulation. Interestingly, after transfection of mLTC with the human kisspeptin receptor (hKpR), which is a GqPCR, we observed a dose-dependent synergy of 10-12-10-7 M kisspeptin variants with a fixed concentration of 0.3 nM LH or hCG. Without any exogenous receptor transfection, a 2 h preincubation with OT or AVP led to a dose-dependent cAMP response to a fixed dose of LH or hCG. Therefore, highly sensitive in vitro bioassays for various hormones and other GPCR ligands can be set up in mLTC to measure circulating concentrations in only 3-10 µL of blood or other body fluids. Nevertheless, the development of an LHRKO mLTC cell line will be mandatory to obtain strict specificity for these bioassays to eliminate potential cross-reaction with LH or CG.

Interactions between ß-endorphin and kisspeptin neurons of the ewe arcuate nucleus are modulated by photoperiod.

Opioid peptides are well-known modulators of the central control of reproduction. Among them, dynorphin coexpressed in kisspeptin (KP) neurons of the arcuate nucleus (ARC) has been thoroughly studied for its autocrine effect on KP release through κ opioid receptors. Other studies have suggested a role for ß-endorphin (BEND), a peptide cleaved from the pro-opiomelanocortin precursor, on food intake and central control of reproduction. Similar to KP, BEND content in the ARC of sheep is modulated by day length and BEND modulates food intake in a dose-dependent manner. Because KP levels in the ARC vary with photoperiodic and metabolic status, a photoperiod-driven influence of BEND neurons on neighboring KP neurons is plausible. The present study aimed to investigate a possible modulatory action of BEND on KP neurons located in the ovine ARC. Using confocal microscopy, numerous KP appositions on BEND neurons were found but there was no photoperiodic variation of the number of these interactions in ovariectomized, estradiol-replaced ewes. By contrast, BEND terminals on KP neurons were twice as numerous under short days, in ewes having an activated gonadotropic axis, compared to anestrus ewes under long days. Injection of 5 µg BEND into the third ventricle of short-day ewes induced a significant and specific increase of activated KP neurons (16% vs. 9% in controls), whereas the percentage of overall activated (c-Fos positive) neurons, was similar between both groups. These data suggest a photoperiod-dependent influence of BEND on KP neurons of the ARC, which may influence gonadotropin-releasing hormone pulsatile secretion and inform KP neurons about metabolic status.

Impact of food restriction on the medio-basal hypothalamus of intact ewes as revealed by a large-scale transcriptomics study.

In mammals, the medio-basal hypothalamus (MBH) integrates photoperiodic and food-related cues to ensure timely phasing of physiological functions, including seasonal reproduction. The current human epidemics of obesity and associated reproductive disorders exemplifies the tight link between metabolism and reproduction. Yet, how food-related cues impact breeding at the level of the MBH remains unclear. In this respect, the sheep, which is a large diurnal mammal with a marked dual photoperiodic/metabolic control of seasonal breeding, is a relevant model. Here, we present a large-scale study in ewes (n = 120), which investigated the impact of food restriction (FRes) on the MBH transcriptome using unbiased RNAseq, followed by RT-qPCR. Few genes (~100) were impacted by FRes and the transcriptional impact was very modest (<2-fold increase or < 50% decrease for most genes). As anticipated, FRes increased expression of Npy/AgRP/LepR and decreased expression of Pomc/Cartpt, while Kiss1 expression was not impacted. Of particular interest, Eya3, Nmu and Dio2, genes involved in photoperiodic decoding within the MBH, were also affected by FRes. Finally, we also identified a handful of genes not known to be regulated by food-related cues (e.g., RNase6, HspA6, Arrdc2). In conclusion, our transcriptomics study provides insights into the impact of metabolism on the MBH in sheep, which may be relevant to human, and identifies possible molecular links between metabolism and (seasonal) reproduction.

The Kisspeptin analogue C6 induces ovulation in jennies.

Kisspeptins (KPs) are the most potent stimulating neurotransmitters of GnRH release, and consequently KP administration triggers LH and/or FSH release. In small ruminants, KP or its analogs induced an LH surge followed by ovulation in both cyclic and acyclic animals, while in the mare KP only increased LH plasma levels but failed to induce ovulation. This study in jennies compares the endocrinological effects, ovulatory and pregnancy rates of the KP analog C6 and the GnRH analog buserelin acetate. The ovarian activity of nine Amiata jennies was monitored daily by transrectal ultrasound for three complete estrous cycles. Jennies in estrus were assigned, to one of three treatment groups: 50 nmol of the KP analog C6 (injected twice, 24 h apart, C6 group); 0.4 mg buserelin acetate (injected once, Bu group); and 2 mL of saline (injected once, CTRL group). Blood samples were collected at Day-1 (-24 h) Day0 (h0, before treatment), h2, h4, h6, h8, h10, h24 (before second treatment with C6), h26, h28, h30, h32, h34, h48 and every 24 h until ovulation. Jennies were inseminated once at h24 with fresh extended semen from a donkey stallion. Pregnancy diagnoses were performed 14 days after ovulation. On days 5, 10, and 14 after ovulation, for every CL the cross-sectional area (CSA) and the vascularized area (VA) were recorded by color doppler ultrasound and measured. Significantly higher plasma LH levels were found after induction between the Bu and CTRL groups at h6 and h8 (P < 0.05), while tendentially higher differences were found between the Bu/C6 groups and CTRL at h10. Five/9, 4/9, and 2/9 jennies ovulated between 24 and 48 h after induction from the Bu, C6, and CTRL groups respectively, (P > 0.05). Correlations between corpora lutea CSA and VA with serum progesterone concentration were r = 0.31, P = 0.01, r = 0.38, P = 0.01, respectively. Pregnancy rates after artificial insemination did not differ among groups (CTRL: 6/9, 66.7%; C6: 7/9, 77.8%; Bu: 6/9, 66.7%; P > 0.05). Ovulation rates after C6 treatment were comparable to that of Bu, although not different from the CTRL. Pregnancy rates were comparable to the literature in terms of fresh extended donkey semen in every group. This study suggests that stimulation of the Kp system in jennies, in contrast to findings observed in mares, induces ovulation. Further studies using higher doses and/or more animals are needed to better characterize the efficacy of C6 in jennies.

A customized long acting formulation of the kisspeptin analog C6 triggers ovulation in anestrus ewe.

The modulation of the kisspeptin system holds promise as a treatment for human reproductive disorders and for managing livestock breeding. The design of analogs has overcome some unfavorable properties of the endogenous ligands. However, for applications requiring a prolongation of drug activity, such as ovulation induction in the ewe during the non-breeding season, additional improvement is required. To this aim, we designed and tested three formulations containing the kisspeptin analog C6. Two were based on polymeric nanoparticles (NP1 and NP2) and the third was based on hydrogels composed of a mixture of cyclodextrin polymers and dextran grafted with alkyl side chains (MD/pCD). Only the MD/pCD formulation prolonged C6 activity, as shown by monitoring luteinizing hormone (LH) plasma concentration (elevation duration 23.4 ± 6.1, 13.7 ± 4.7 and 12.0 ± 2.4 h for MD/pCD, NP1 and NP2, respectively). When compared with the free C6 (15 nmol/ewe), the formulated (MD/pCD) doses of 10, 15 and 30 nmol/ewe, but not the 90 nmol/ewe dose, provided a more gradual release of C6 as shown by an attenuated LH release during the first 6 h post-treatment. When tested during the non-breeding season without progestogen priming, only, the formulated 30 nmol/ewe dose triggered ovulation (50% of ewes). Hence, we showed that a formulation with an adapted action time would improve the efficacy of C6 with respect to inducing ovulation during the non-breeding season. This result suggests that formulations containing a kisspeptin analog might find applications in the management of livestock reproduction but also point to the possibility of their use for the treatment of some human reproductive pathologies.

No evidence that Spexin impacts LH release and seasonal breeding in the ewe.

Spexin (SPX) is a recently identified peptide hormone of 14 amino acids. Interestingly, Spx and Kiss1 genes share a common ancestor gene. Considering that KISS1 peptides are key controllers of breeding in mammals and circumstantial evidence that SPX regulates gonadotropins in some fish species, we hypothesized that SPX may play a KISS1-related role in sheep. Here, we cloned the ovine Spx cDNA, performed in vivo injection and infusion of SPX (i.c.v. route, with or without concomittant KISS1 presence) and assessed a potential regulation of Spx expression by season, thyroid hormone and estradiol in the medio-basal hypothalamus of the ewe. Our data do not provide support for a role of SPX in the control of the gonadotropic axis in the ewe.

Circuit-level analysis identifies target genes of sex steroids in ewe seasonal breeding.

Thyroid hormone (TH) and estradiol (E2) direct seasonal switches in ovine reproductive physiology. In sheep, as in other mammals and birds, control of thyrotropin (TSH) production by the pars tuberalis (PT) links photoperiod responsiveness to seasonal breeding. PT-derived TSH governs opposite seasonal patterns of the TH deiodinases Dio2/Dio3 expression in tanycytes of the neighboring medio-basal hypothalamus (MBH), which explain the key role of TH. We recently used RNA-Seq to identify seasonal markers in the MBH and define the impact of TH. This impact was found to be quite limited, in terms of number of target genes, and very restricted with regards to neuroanatomical location, as TH specifically impacts genes expressed in tanycytes and hypothalamus, not in the PT. Here we address the impact of E2 on these seasonal markers, which are specifically expressed in either PT, tanycytes or hypothalamus. We also investigate if progesterone (P4) may be involved in timing the seasonal transition to anestrus. Our analysis provides circuit-level insights into the impact of sex steroids on the ewe seasonal breeding cycle. First, seasonal gene expression in the PT is independent of the sex steroid status. The fact that seasonal gene expression in the PT is also TH-independent strengthens the view that the PT is a circannual timer. Second, select tanycytic markers display some level of responsiveness to E2 and P4, which indicates another potential level of feedback control by sex steroids. Third, Kiss1 neurons of the arcuate nucleus are responsive to both TH and E2, which places them at the crossroads of photoperiodic transduction pathway and sex steroid feedback. This provides strong support to the concept that these Kiss1 neurons are pivotal to the long-recognized "seasonal switch in the ability of E2 to exert negative feedback", which drives seasonal breeding.

Discontinuity in the molecular neuroendocrine response to increasing daylengths in Ile-de-France ewes: Is transient Dio2 induction a key feature of circannual timing?

In mammals, melatonin is responsible for the synchronisation of seasonal cycles to the solar year. Melatonin is secreted by the pineal gland with a profile reflecting the duration of the night and acts via the pituitary pars tuberalis (PT), which in turn modulates hypothalamic thyroid hormone status via seasonal changes in the production of locally-acting thyrotrophin. Recently, we demonstrated that, in the Soay sheep, photoperiodic induction of Tshb expression and consequent downstream hypothalamic changes occur over a narrow range of photoperiods between 12 and 14 hours in duration. In the present study, we aimed to extend our molecular characterisation of this pathway, based on transcriptomic analysis of photoperiodic changes in the pituitary and hypothalamus of ovariectomised, oestradiol-implanted Ile-de-France ewes. We demonstrate that photoperiodic treatments applied before the winter solstice elicit two distinctive modes of accelerated reproductive switch off compared to ewes held on a simulated natural photoperiod, with shut-down occurring markedly faster on photoperiods of 13 hours or more than on photoperiods of 12 hours and less. This pattern of response was reflected in gene expression profiles of photoperiodically sensitive markers, both in the PT (Tshb, Fam150b, Vmo1, Ezh2 and Suv39H2) and in tanycytes (Tmem252 and Dct). Unexpectedly, the expression of Dio2 in tanycytes did not show any noticeable increase in expression with lengthening photoperiods. Finally, the expression of Kiss1, the key activator of gonadotrophin-releasing hormone release, was proportionately decreased by lengthening photoperiods, in a pattern that correlated strongly with gonadotrophin suppression. These data show that stepwise increases in photoperiod lead to graded molecular responses at the level of the PT, a progressive suppression of Kiss1 in the hypothalamic arcuate nucleus and luteinising hormone/follicle-stimulating hormone release by the pituitary, despite apparently unchanged Dio2 expression in tanycytes. We hypothesise that this apparent discontinuity in the seasonal neuroendocrine response illustrates the transient nature of the thyroid hormone-mediated response to long days in the control of circannual timing.

The kisspeptin analog C6 is a possible alternative to PMSG (pregnant mare serum gonadotropin) for triggering synchronized and fertile ovulations in the Alpine goat.

In temperate regions goat's reproduction is seasonal. To obtain year-round breeding, hormonal treatments are currently applied. These treatments usually combine a progesterone analog with the pregnant mare serum gonadotropin (PMSG). However, their use has significant ethical and environmental drawbacks. Therefore, alternative methods to manage reproduction are needed. The discovery that in mammals the neuropeptide kisspeptin is a major positive regulator of hypothalamo-pituitary gonadal axis offered an attractive alternative strategy to control reproduction. We have previously designed a kisspeptin analog, called C6, which offers pharmacological advantages over endogenous kisspeptin. These include a longer lasting effect and enhanced activity following intramuscular injection. In the present work, we evaluated C6 effect on LH and FSH plasma concentrations in the Alpine goat breed and tested whether C6 could replace PMSG to trigger ovulation. An intramuscular injection of C6 (15 nmol/doe) given 24 hours after the end of progestogen treatment induced a surge-like peak of both LH and FSH. This was followed by an increase of progesterone, a hallmark of ovulation induction and corpus luteus formation. These results were obtained at three different time of the year: during the breeding season, the non-breeding season and at the onset of the breeding season. Furthermore, we compared the efficacy of C6 and PMSG to induce fertile ovulations when these treatments are given at the onset of the breeding season and are followed by artificial insemination. The results of this first attempt were extremely promising with gestation rates of 45% and 64% for C6 and PMSG respectively. Pending optimization of the treatment procedure in order to improve efficacy, kisspeptin analogs could be the long sought-after alternative to PMSG.

Brain mapping of the gonadotropin-inhibitory hormone-related peptide 2 with a novel antibody suggests a connection with emotional reactivity in the Japanese quail (Coturnix japonica, Temminck & Schlegel, 1849).

Gonadotropin-inhibitory hormone (GnIH) is a neuropeptide first discovered in the quail brain that is involved in the control of reproductive physiology and behaviors, and stress response. GnIH gene encodes a second peptide, GnIH-related peptide-2 (RP2), the distribution and function of which remain unknown. We therefore studied GnIH-RP2 distribution by immunohistochemistry using a novel antibody capable of discriminating between GnIH and GnIH-RP2. The overall distribution of GnIH-RP2 is similar to that of GnIH. The vast majority of labeled neurons is located in the paraventricular nucleus (PVN) of the hypothalamus. Labeling of fibers is conspicuous in the diencephalon, but present also in the mesencephalon and telencephalon. Several regions involved in the control of reproduction and stress response (the PVN, septum, bed nucleus of the stria terminalis and nucleus commissura pallii) showed a dense network of immunolabeled fibers. To investigate the potential function of GnIH-RP2 we compared its expression in two quail lines genetically selected for divergence in their emotional reactivity. A quantitative analysis in the above-mentioned brain regions showed that the density of fibers was similar in the two lines. However, the number of GnIH-RP2 labeled neurons was higher in the median portion of the PVN in birds with higher emotional reactivity. These results point to a possible involvement of GnRH-RP2 in modulating stress response and/or emotional reactivity.

Photoperiod and thyroid hormone regulate expression of l-dopachrome tautomerase (Dct), a melanocyte stem-cell marker, in tanycytes of the ovine hypothalamus.

The pars tuberalis (PT) of the pituitary is central to the control of seasonal breeding. In mammals, the PT translates the photoperiodic message carried by melatonin into an endocrine thyroid-stimulating hormone output, which controls local thyroid hormone (TH) signalling in tanycytes of the neighbouring hypothalamus. In the present study, we identify l-dopachrome tautomerase (Dct) as a novel marker of ovine tanycytes and show that Dct displays marked seasonal variations in expression, with higher levels during spring and summer. This seasonal profile is photoperiod-dependent because an acute exposure to long days induces Dct expression. In addition, we find that TH also modulates Dct expression. DCT functions as an enzyme in the melanin synthesis pathway within skin melanocytes, whereas expression in other tissues is comparatively low. We demonstrate that both Tyr and Tyrp1, which are enzymes that intervene upstream and downstream of Dct in the melanin synthesis pathway, respectively, are expressed at very low levels in the ovine hypothalamus. This suggests that Dct in tanycytes may not be involved in melanin synthesis. We speculate that DCT function is linked to its protective role towards oxidative stress and/or its function in the control of neural progenitor cell proliferation.

Neuroendocrine correlates of the critical day length response in the Soay sheep.

In mammals, melatonin is the hormone responsible for synchronisation of seasonal physiological cycles of physiology to the solar year. Melatonin is secreted by the pineal gland with a profile reflecting the duration of the night and acts via melatonin-responsive cells in the pituitary pars tuberalis (PT), which in turn modulate hypothalamic thyroid hormone status. Recent models suggest that the actions of melatonin in the PT depend critically on day length-dependent changes in the expression of eyes absent 3 (Eya3), which is a coactivator for thyrotrophin ß-subunit (Tshß) gene transcription. According to this model, short photoperiods suppress Eya3 and hence Tshß expression, whereas long photoperiods produce the inverse effect. Studies underpinning this model have relied on step changes in photoperiod (from 8 to 16 hours of light/24 hours) and have not compared the sensitive ranges of photoperiods for changes in Eya3 and Tshß expression with those for relevant downstream molecular and endocrine responses. We therefore performed a "critical day length" experiment in Soay sheep, in which animals acclimated to 8 hours of light/24 hours (SP) were exposed to a range of increased photoperiods spanning the range 11.75 to 16 hours (LP) and then responses at the level of the PT, hypothalamus and hormonal output were assessed. Although Eya3 and Tshß both showed the predicted SP vs LP differences, they responded quite differently to intermediate photoperiods within this range and, at the individual animal level, no clear Eya3-Tshß relationship could be seen. This result is inconsistent with a simple coactivator model for EYA3 action in the PT. Further downstream layers of nonlinearity were also seen in terms of the Tshß-dio2 and the dio2-testosterone relationships. We conclude that the transduction of progressive changes in photoperiod into transitions in endocrine output is an emergent property of a multistep signalling cascade within the mammalian neuroendocrine system.

Anti-angiogenic VEGFAxxxb transcripts are not expressed in the medio-basal hypothalamus of the seasonal sheep.

This study investigated Vegfa expression in the pars tuberalis (PT) of the pituitary and medio-basal hypothalamus (MBH) of sheep, across seasons and reproductive states. It has recently been proposed that season impacts alternative splicing of Vegfa mRNA in the PT, which shifts the balance between angiogenic VEGFAxxx and anti-angiogenic VEGFAxxxb isoforms (with xxx the number of amino acids of the mature VEGFA proteins) to modulate seasonal breeding. Here, we used various RT-PCR methodologies and analysis of RNAseq datasets to investigate seasonal variation in expression and splicing of the ovine Vegfa gene. Collectively, we identify 5 different transcripts for Vegfa within the ewe PT/MBH, which correspond to splicing events previously described in mouse and human. All identified transcripts encode angiogenic VEGFAxxx isoforms, with no evidence for alternative splicing within exon 8. These findings led us to investigate in detail how "Vegfaxxxb-like" PCR products could be generated by RT-PCR and misidentified as endogenous transcripts, in sheep and human HEK293 cells. In conclusion, our findings do not support the existence of anti-angiogenic VEGFAxxxb isoforms in the ovine PT/MBH and shed new light on the interpretation of prior studies, which claimed to identify Vegfaxxxb isoforms by RT-PCR.

The impact of thyroid hormone in seasonal breeding has a restricted transcriptional signature.

Thyroid hormone (TH) directs seasonal breeding through reciprocal regulation of TH deiodinase (Dio2/Dio3) gene expression in tanycytes in the ependymal zone of the medio-basal hypothalamus (MBH). Thyrotropin secretion by the pars tuberalis (PT) is a major photoperiod-dependent upstream regulator of Dio2/Dio3 gene expression. Long days enhance thyrotropin production, which increases Dio2 expression and suppresses Dio3 expression, thereby heightening TH signaling in the MBH. Short days appear to exert the converse effect. Here, we combined endocrine profiling and transcriptomics to understand how photoperiod and TH control the ovine reproductive status through effects on hypothalamic function. Almost 3000 genes showed altered hypothalamic expression between the breeding- and non-breeding seasons, showing gene ontology enrichment for cell signaling, epigenetics and neural plasticity. In contrast, acute switching from a short (SP) to a long photoperiod (LP) affected the expression of a much smaller core of 134 LP-responsive genes, including a canonical group previously linked to photoperiodic synchronization. Reproductive switch-off at the end of the winter breeding season was completely blocked by thyroidectomy (THX), despite a very modest effect on the hypothalamic transcriptome. Only 49 genes displayed altered expression between intact and THX ewes, including less than 10% of the LP-induced gene set. Neuroanatomical mapping showed that many LP-induced genes were expressed in the PT, independently of the TH status. In contrast, TH-sensitive seasonal genes were principally expressed in the ependymal zone. These data highlight the distinctions between seasonal remodeling effects, which appear to be largely independent of TH, and TH-dependent localised effects which are permissive for transition to the non-breeding state.

Rostro-caudal maturation of glial cells in the accessory olfactory system during development: involvement in outgrowth of GnRH neurites.

During mammalian embryonic development, GnRH neurones differentiate from the nasal placode and migrate through the nasal septum towards the forebrain. We previously showed that a category of glial cells, the olfactory ensheathing cells (OEC), forms the microenvironment of migrating GnRH neurones. Here, to characterize the quantitative and qualitative importance of this glial, we investigated the spatiotemporal maturation of glial cells in situ and the role of maturing glia in GnRH neurones development ex vivo. More than 90% of migrating GnRH neurones were found to be associated with glial cells. There was no change in the cellular microenvironment of GnRH neurones in the regions crossed during embryonic development as glial cells formed the main microenvironment of these neurones (53.4%). However, the phenotype of OEC associated with GnRH neurones changed across regions. The OEC progenitors immunoreactive to brain lipid binding protein formed the microenvironment of migrating GnRH neurones from the vomeronasal organ to the telencephalon and were also present in the diencephalon. However, during GnRH neurone migration, maturation of OEC to [GFAP+] state (glial fibrillary acid protein) was only observed in the nasal septum. Inducing depletion of OEC in maturation, using transgenic mice expressing herpes simplex virus thymidine kinase driven by the GFAP promoter, had no impact on neurogenesis or on triggering GnRH neurones migration in nasal explant culture. Nevertheless, depletion of [GFAP+] cells decreased GnRH neurites outgrowth by 57.4%. This study suggests that specific maturation of OEC in the nasal septum plays a role in morphological differentiation of GnRH neurones.

The Two Populations of Kisspeptin Neurons Are Involved in the Ram-Induced LH Pulsatile Secretion and LH Surge in Anestrous Ewes.

Exposure to a ram during spring stimulates luteinizing hormone (LH) secretion and can induce ovulation in sexually quiescent ewes ("ram effect"). Kisspeptin (Kiss) present in the arcuate nucleus (ARC) and the preoptic area (POA) is a potent stimulators of LH secretion. Our aim was to investigate whether Kiss neurons mediate the increase in LH secretion during the ram effect. With double immunofluorescent detection, we identified Kiss neurons (Kiss IR) activated (Fos IR) by exposure to a ram for 2 hours (M2) or 12 hours (M12) or to ewes for 2 hours (C). The density of cells Kiss + Fos IR and the proportion of Kiss IR cells that were also Fos IR cells were higher in M2 and M12 than in C in ARC (P < 0.002) and POA (P < 0.02). In ARC, these parameters were also higher in M12 than in M2 (P < 0.02 and P < 0.05). Kiss antagonist (P234 10-6M) administered by retrodialysis in POA for 3 hours at the time of introduction of the ram reduced the amplitude of the male-induced increase in LH concentration compared with solvent (P < 0.02). In ARC, P234 had a more limited effect (P < 0.038 1 hour after P234) but pulse frequency increased less than after solvent (P = 0.07). In contrast, Kiss antagonist (P271 10-4M) infused in ARC but not POA 6 to 18 hours after introduction of the ram prevented the LH surge in the ewe (0/6 vs 4/5 and 4/6 in C). These results suggest that both populations of Kiss neurons are involved in the ram-induced pulsatile LH secretion and in the LH surge.

Crosstalks between kisspeptin neurons and somatostatin neurons are not photoperiod dependent in the ewe hypothalamus.

Seasonal reproduction is under the control of gonadal steroid feedback, itself synchronized by day-length or photoperiod. As steroid action on GnRH neurons is mostly indirect and therefore exerted through interneurons, we looked for neuroanatomical interactions between kisspeptin (KP) neurons and somatostatin (SOM) neurons, two populations targeted by sex steroids, in three diencephalic areas involved in the central control of ovulation and/or sexual behavior: the arcuate nucleus (ARC), the preoptic area (POA) and the ventrolateral part of the ventromedial hypothalamus (VMHvl). KP is the most potent secretagogue of GnRH secretion while SOM has been shown to centrally inhibit LH pulsatile release. Notably, hypothalamic contents of these two neuropeptides vary with photoperiod in specific seasonal species. Our hypothesis is that SOM inhibits KP neuron activity and therefore indirectly modulate GnRH release and that this effect may be seasonally regulated. We used sections from ovariectomized estradiol-replaced ewes killed after photoperiodic treatment mimicking breeding or anestrus season. We performed triple immunofluorescent labeling to simultaneously detect KP, SOM and synapsin, a marker for synaptic vesicles. Sections from the POA and from the mediobasal hypothalamus were examined using a confocal microscope. Randomly selected KP or SOM neurons were observed in the POA and ARC. SOM neurons were also observed in the VMHvl. In both the ARC and POA, nearly all KP neurons presented numerous SOM contacts. SOM neurons presented KP terminals more frequently in the ARC than in the POA and VMHvl. Quantitative analysis failed to demonstrate major seasonal variations of KP and SOM interactions. Our data suggest a possible inhibitory action of SOM on all KP neurons in both photoperiodic statuses. On the other hand, the physiological significance of KP modulation of SOM neuron activity and vice versa remain to be determined.

Visfatin and resistin in gonadotroph cells: expression, regulation of LH secretion and signalling pathways.

Visfatin and resistin appear to interfere with reproduction in the gonads, but their potential action at the hypothalamic-pituitary level is not yet known. The aim of the present study was to investigate the mRNA and protein expression of these adipokines in murine gonadotroph cells and to analyse the effects of different concentrations of recombinant mouse visfatin and resistin (0.01, 0.1, 1 and 10ngmL-1) on LH secretion and signalling pathways in LßT2 cells and/or in primary female mouse pituitary cells. Both visfatin and resistin mRNA and protein were found in vivo in gonadotroph cells. In contrast with resistin, the primary tissue source of visfatin in the mouse was the skeletal muscle, and not adipose tissue. Visfatin and resistin both decreased LH secretion from LßT2 cells after 24h exposure of cells (P<0.03). These results were confirmed for resistin in primary cell culture (P<0.05). Both visfatin (1ngmL-1) and resistin (1ngmL-1) increased AMP-activated protein kinase α phosphorylation in LßT2 cells after 5 or 10min treatment, up to 60min (P<0.04). Extracellular signal-regulated kinase 1/2 phosphorylation was transiently increased only after 5min resistin (1ngmL-1) treatment (P<0.01). In conclusion, visfatin and resistin are expressed in gonadotroph cells and they may affect mouse female fertility by regulating LH secretion at the level of the pituitary.

The "Ram Effect": A "Non-Classical" Mechanism for Inducing LH Surges in Sheep.

During spring sheep do not normally ovulate but exposure to a ram can induce ovulation. In some ewes an LH surge is induced immediately after exposure to a ram thus raising questions about the control of this precocious LH surge. Our first aim was to determine the plasma concentrations of oestradiol (E2) E2 in anoestrous ewes before and after the "ram effect" in ewes that had a "precocious" LH surge (starting within 6 hours), a "normal" surge (between 6 and 28h) and "late¼ surge (not detected by 56h). In another experiment we tested if a small increase in circulating E2 could induce an LH surge in anoestrus ewes. The concentration of E2 significantly was not different at the time of ram introduction among ewes with the three types of LH surge. "Precocious" LH surges were not preceded by a large increase in E2 unlike "normal" surges and small elevations of circulating E2 alone were unable to induce LH surges. These results show that the "precocious" LH surge was not the result of E2 positive feedback. Our second aim was to test if noradrenaline (NA) is involved in the LH response to the "ram effect". Using double labelling for Fos and tyrosine hydroxylase (TH) we showed that exposure of anoestrous ewes to a ram induced a higher density of cells positive for both in the A1 nucleus and the Locus Coeruleus complex compared to unstimulated controls. Finally, the administration by retrodialysis into the preoptic area, of NA increased the proportion of ewes with an LH response to ram odor whereas treatment with the α1 antagonist Prazosin decreased the LH pulse frequency and amplitude induced by a sexually active ram. Collectively these results suggest that in anoestrous ewes NA is involved in ram-induced LH secretion as observed in other induced ovulators.