Inhibition Of LH Secretion Research Articles

30 Central Regulation of Kndy Neurons During Undernutrition in Sheep

Abstract Adequate energy intake is essential for reproduction. While we have known for decades that feed restriction inhibits gonadotropin-releasing hormone/luteinizing hormone (GnRH/LH) secretion, the central mechanisms that govern this reduction during undernutrition remain to be fully elucidated. The past few years, we have focused on examining central mechanisms whereby chronic feed restriction in young sheep results in the inhibition of LH secretion. This presentation will focus on our current understanding of how feed restriction impacts kisspeptin/neurokinin B/dynorphin (KNDy) neurons in young sheep and will give a glimpse into our next few years as we examine how input to KNDy neurons could play a key role in regulating reproduction during undernutrition.

Presence and localization of apelin and its cognate receptor in canine testes using immunohistochemical and RT-PCR techniques

Apelin, a member of the adipokine family, is a novel endogenous peptide which regulates the male reproductive system of mammals by interacting with a specific receptor. Recent studies have highlighted that apelin may play a role in the regulation of reproduction by reducing testosterone production and inhibiting LH secretion. To the best of our knowledge, there is no available data on the presence of the apelin and its receptor in canine testes. Therefore, the aim of this study was to reveal the presence of apelin and evaluate its distribution in the canine testes using immunohistochemical and RT-PCR techniques. For this purpose, five fertile and healthy male dogs were subjected to elective orchiectomy. The immunohistochemical reaction revealed the presence of apelin and its receptor in the canine testes. Apelin was localized in spermatids and spermatozoa with a positive signal in the “acrosomal bodies”. As regards the apelin receptor, a positive immunoreaction was detected in the cytoplasm of the cells localized near to the basal membrane of the seminiferous tubules and in the cytoplasm of Leydig cells. The RT-PCR analysis showed the presence of transcripts for apelin and apelin receptor in all of the samples under study. A 35kDa band confirmed apelin receptor protein expression in all of the samples analysed. In conclusion, the paracrine and endocrine role of apelin and its cognate receptor on male reproduction reported in humans and laboratory animals could also be hypothesized in dogs.

Comparative Transcriptomic Analysis of Hu Sheep Pituitary Gland Prolificacy at the Follicular and Luteal Phases.

The pituitary gland directly regulates the reproduction of domestic animals. Research has increasingly focused on the potential regulatory mechanism of non-coding RNA in pituitary development. Little is known about the differential expression pattern of lncRNAs in Hu sheep, a famous sheep breed with high fecundity, and its role in the pituitary gland between the follicular phase and luteal phase. Herein, to identify the transcriptomic differences of the sheep pituitary gland during the estrus cycle, RNA sequencing (RNA-Seq) was performed. The results showed that 3529 lncRNAs and 16,651 mRNAs were identified in the pituitary gland. Among of them, 144 differentially expressed (DE) lncRNA transcripts and 557 DE mRNA transcripts were screened in the follicular and luteal phases. Moreover, GO and KEGG analyses demonstrated that 39 downregulated and 22 upregulated genes interacted with pituitary functions and reproduction. Lastly, the interaction of the candidate lncRNA XR_001039544.4 and its targeted gene LHB were validated in sheep pituitary cells in vitro. LncRNA XR_001039544.4 and LHB showed high expression levels in the luteal phase in Hu sheep. LncRNA XR_001039544.4 is mainly located in the cytoplasm, as determined by FISH analysis, indicating that XR_001039544.4 might act as competing endogenous RNAs for miRNAs to regulate LHB. LncRNA XR_001039544.4 knockdown significantly inhibited LH secretion and cell proliferation. LncRNA XR_001039544.4 may regulate the secretion of LH in the luteal-phase pituitary gland via affecting cell proliferation. Taken together, these findings provided genome-wide lncRNA- and mRNA-expression profiles for the sheep pituitary gland between the follicular and luteal phases, thereby contributing to the elucidation of the molecular mechanisms of pituitary function.

GH-induced LH hyporesponsiveness as a potential mechanism for hypogonadism in male patients with acromegaly.

Male patients with acromegaly frequently have hypogonadism. However, whether excess GH affects gonadal function remains unclear. We retrospectively compared clinical features affecting total testosterone (TT) and free testosterone (FT) levels between 112 male patients with acromegaly and 100 male patients with non-functioning pituitary adenoma (NFPA) without hyperprolactinemia. Median maximum tumor diameter (14.4 vs. 26.5 mm) and suprasellar extension rate (33 vs. 100%) were lower in acromegaly, but LH, FSH, TT, and FT were not significantly different. In acromegaly, TT was less than 300 ng/dL in 57%, and FT was below the age-specific reference range in 77%. TT and FT were negatively correlated with GH, IGF-1, and the tumor size, and positively correlated with LH. In NFPA, they were positively correlated with IGF-1, LH, FSH, ACTH, cortisol, and free T4, reflecting hypopituitarism. Multiple regression analysis showed that TT and FT had the strongest correlation with GH in acromegaly, and with LH in NFPA. Surgical remission was achieved in 87.5% of 56 follow-up patients with acromegaly. TT and FT increased in 80.4 and 87.5%, respectively, with a significant increase in LH. In acromegaly, the degree of postoperative increase in TT(FT) correlated with the fold increase of TT(FT)/LH ratio, a potential parameter of LH responsiveness, but not with fold increase of LH, whereas in NFPA it correlated with both. These results suggest that excessive GH is the most relevant factor for hypogonadism in male acromegaly, and may cause impaired LH responsiveness as well as the suppression of LH secretion.

The effects of LH inhibition with cetrorelix on cumulus cell gene expression during the luteal phase under ovarian coasting stimulation in cattle

Cumulus cells have an important role to play in the final preparation of the oocyte before ovulation. During the final phase of follicular differentiation, FSH levels are low and LH maintains follicular growth; however, it is not known if at that time LH has an influence on cumulus cells inside the follicle. In humans, LH is often inhibited to avoid a premature ovulatory LH surge. This procedure provides a tool to investigate the role of LH in follicular development. In this study, we investigated the impact of suppressing LH using the GnRH antagonist cetrorelix during an ovarian coasting stimulation protocol on the transcriptome of bovine cumulus cells (CC). Oocytes were collected twice from 6 dairy cows. For the first collection, the cows received FSH twice daily for 3 d, followed by FSH withdrawal for 68 h as a control protocol. For the second collection, the same stimulation protocol was used, but the cows also received, starting on day 2 of FSH stimulation, a GnRH antagonist once a day until recovery of the cumulus-oocyte complexes (COC). Half of the COC were subjected to in vitro maturation, fertilization, and culture to assess blastocyst rates. The other half of the COC underwent microarray analysis (n = 3 cows, 2 treatments, 6 oocyte collections) and qRT-PCR (n = 6 cows: 3 microarray cows +3 other cows, 2 treatments, 12 oocyte collections). The differential expression of specific genes was confirmed by RT-qPCR: decrease of ATP6AP2, SC4MOL, and OSTC and increase of PTGDS in the LH-inhibited condition. The global transcriptomic analysis of cumulus cells demonstrated that the inhibition of LH secretion may decrease survival and growth of the follicle. Moreover, the results suggested that LH may be important to cumulus for the maintenance of cellular mechanisms such as global RNA expression, protein and nucleic acid metabolism, and energy production. These results support the hypothesis that LH support is important during the final part of follicle maturation through its influence on the cumulus cells.

SAT-423 The Involvement of GnRH Neurons in Mediating the Effects of RFRP-3 on the Mouse Reproductive Axis

In 2000, gonadotrophin-inhibitory hormone was discovered in birds and shown to inhibit gonadotrophin secretion. The mammalian ortholog was concurrently discovered in humans and rats and termed RFamide-related peptide-3 (RFRP-3). Recent results have shown that the effects of centrally-administered RFRP-3 on gonadotrophin secretion are sex- and cycle stage-dependent in mice (Ancel et al., 2017). Indeed, intracerebroventricular injections of RFRP-3 stimulated LH secretion in males, and inhibited LH secretion in females at the time of the preovulatory LH surge. In addition, the stimulatory effect observed in males was shown to be mediated in part by GPR54, the receptor for Kisspeptins, suggesting the involvement of other pathways. In order to further our understanding of the ways in which RFRP neurons modulate the reproductive axis, we have developed a novel transgenic mouse line. Using a Cre-loxP conditional transgenic method, we knocked the receptor for RFRP-3 (GPR147) out of GnRH neurons and analysed puberty onset in these mice. While the absence of GPR147 on GnRH neurons advanced puberty onset in male mice, it resulted in a delay in female puberty, once again indicating a sex-specific role of the RFRP system in the regulation of the reproductive function. Current work is aimed at dissecting the potential involvement of GnRH neurons in the pathways mediating RFRP-3 effects on LH secretion in mice carrying a deletion of GPR147 in GnRH neurons. Both male and female mice received intracerebroventricular injections of RFRP-3 and LH levels were subsequently assayed in tail-tip blood samples. This work is still ongoing. Taken together, these new tools provide us with the possibility to advance our understanding of the structure and the functions of the RFRP neuronal system in mice.

Stress and reproduction in farm animals

Transport of post-partum cows or sheep before an oestradiol-induced LH surge delayed gonadotrophin secretion possibly by affecting hypothalamic activity but not via an opioid mediated mechanism as the effect could not be reversed by naloxone. In addition, reduced LH responses to GnRH were observed in cattle during transport. In sheep, adrenocorticotrophic hormone (ACTH) also diminished the LH response to GnRH, but only when GnRH was administered 3 h after ACTH, not after 0.5 h. This finding suggests that very early suppression of LH secretion by stressors is not mediated by ACTH action at the pituitary but that immediate activation of the sympathetic nervous system may be involved. In ewes during the breeding season, repeated exposure to GnRH at intervals of 2 h during transport resulted in lower LH responses to the second and third injections. When anoestrous ewes were treated with oestradiol and GnRH while being restrained and isolated, the onset of the LH surge was delayed. The effects of hypothalamus-pituitary-adrenal hyperactivity on LH release may involve suppression of GnRH receptor activity, a reduction in releasable LH, or both factors. Studies in vitro with perifused ovine pituitaries showed that ACTH or corticotrophin releasing hormone markedly suppressed LH secretion in response to the second of two exposures to GnRH. This occurred with pituitaries obtained from anoestrous ewes irrespective of prior treatment with oestradiol, suggesting that compounds from the hypothalamus-pituitary-adrenal do not exert effects on the oestradiol-sensitizing mechanisms on the pituitary. In conclusion, stressors affect reproductive function via actions at the hypothalamus as well as impairing pituitary LH release induced by GnRH.

Neurokinin B receptor agonist and Dynorphin receptor antagonist stimulated luteinizing hormone secretion in fasted male rodents.

Kisspeptin/neurokinin B (NKB)/dynorphin (Dyn) (KNDy) neuron in hypothalamic arcuate nucleus plays a key role in GnRH/LH pulsatile secretion. We aimed to determine whether stimulation of NKB/neurokinin 3 receptor (NK3R) signaling and inhibition of Dyn/kappa-opioid receptor (KOR) signaling recover LH secretion that is suppressed by acute fasting in male rats. Furthermore, we determined dose dependent effect of NKB/NK3R signaling on serum LH level under acute fasting condition in male mice. Mature male rats were injected saline (0.1 mL) and senktide (20 μg/kg), a NK3R agonist, or nor-BNI (800 μg/kg), a KOR antagonist intraperitoneally (ip) after 72 h fasting. And mature male mice were injected multiple doses of senktide, ip after 48 h fasting. Blood and brain sample were collected 90 min after injections for LH measurement and hypothalamic mRNA expressions. All three studies showed significantly lower LH concentration in fasted groups than non-fasted groups. Senktide did not recover LH suppressed by acute fasting in male rats, whereas nor-BNI injected male rats showed significantly higher LH than 72 h fasted male rats (p < 0.05). Mice study showed significantly higher LH concentration in higher doses senktide groups than 48 h fasted group and one of lower doses senktide group. These results suggest that stimulation of NKB/NK3R signaling and attenuation of Dyn/KOR signaling could recover suppressed LH secretion under acute fasting condition in male rodents.

Effect of gonadotropin-inhibitory hormone on luteinizing hormone secretion in humans.

Gonadotropin-inhibitory hormone (GnIH, human homologue of RFRP-3) suppresses gonadotropin secretion in animal models, but its effects have not been studied in the human. We tested the hypotheses that exogenous GnIH inhibits LH secretion (i) in postmenopausal women and (ii) in men concurrently administered exogenous kisspeptin. Following in vitro and in vivo preclinical studies to functionally characterize the GnIH peptide, a dose-finding study (human GnIH: 1·5-150 μg/kg/h, iv for 3 h) was undertaken, and 50 μg/kg/h selected for further evaluation. Five postmenopausal women were administered 50 μg/kg/h iv infusion for 3 h or vehicle on two separate days. Four men were administered kisspeptin-10 (0·3 μg/kg iv bolus) with simultaneous infusion of GnIH (50 μg/kg/h, iv for 3 h) or vehicle. Healthy postmenopausal women (mean age 58 ± 2 years, LH: 30·8 ± 2·9 IU/l, FSH: 78·7 ± 6·4 IU/l, oestradiol: <50 pmol/l) and men (39·8 ± 2·1 years, mean total testosterone 12·1 ± 1·8 nmol/l, LH 2·2 ± 0·2 IU/l). Change in area under curve (AUC) of LH during GnIHvs vehicle. During GnIH administration in postmenopausal women, LH secretion decreased (ΔAUC: -9·9 ± 1·8 IU/3 h) vs vehicle (ΔAUC: -0·5 ± 1·7 IU/3 h; P = 0·02). Kisspeptin-10-stimulated LH responses in men were not affected by GnIH co-administration (60-min AUC of LH 6·2 ± 0·8 IU/h with kisspeptin-10 alone, 6·3 ± 1·0 IU/h, kisspeptin-10 with GnIH, P = 0·72). Exogenous GnIH was well tolerated, with no adverse events reported. Gonadotropin-inhibitory hormone decreased LH secretion in postmenopausal women in this first-in-human study. Kisspeptin-stimulated LH secretion in men was not inhibited during concomitant administration of GnIH.

The nonsteroidal mycoestrogen zearalenone and its five metabolites suppress LH secretion from the bovine anterior pituitary cells via the estradiol receptor GPR30 in vitro

Picomolar concentrations of estradiol produce nongenomic suppression of GnRH-induced LH secretion from the anterior pituitary (AP) of cattle via G-protein–coupled receptor 30 (GPR30). Zearalenone (ZEN) is the nonsteroidal mycoestrogen produced by Fusarium fungi and has been detected in cereal grains, animal feed, and ruminant urine worldwide. Zearalenone has a prolonged blood half-life that results from enterohepatic cycling. There are five metabolites of ZEN: α-zearalanol (α-ZAL), β-zearalanol (β-ZAL), α-zearalenol (α-ZOL), β-zearalenol (β-ZOL), and zearalanone, which may persist for long periods in animals and humans after consumption of ZEN-contaminated feed. We recently reported that GPR30 bound with α-ZAL decreases cytoplasmic cAMP but not gene expression of LHα and LHβ subunits, and GPR30 bound with α-ZAL suppresses GnRH-induced LH release in bovine AP cells in vitro. We tested the hypothesis that GPR30 bound with ZEN or one of the four previously untested metabolites suppresses GnRH-induced LH release from the bovine AP cells in vitro. Anterior pituitary cells were cultured for 3 days under steroid-free conditions and were then incubated with various concentrations (0.001–10 nM) of estradiol or one of the ZEN analogs for 5 minutes before GnRH stimulation. Gonadotropin-releasing hormone–stimulated LH secretion from AP cells was inhibited by all of the test concentrations of ZEN, 0.001 to 1 nM of α-ZAL, and 0.001 to 0.1 nM of the remaining four analogs. Pretreatment for 5 minutes with a GPR30-specific antagonist, G36, inhibited estradiol- and the ZEN analog–induced suppression of LH secretion from cultured AP cells. G36 alone had no significant effect on LH secretion. The estimated order of the nongenomic inhibiting effect was ZEN, α-ZAL, zearalanone, α-ZOL, β-ZOL, and β-ZAL, which is quite different from the reported order for their genomic effects. Therefore, ZEN and all of its metabolites suppress LH secretion from the bovine AP cells via GPR30 in vitro.

The effects of intracerebroventricular infusion of apelin-13 on reproductive function in male rats

Apelin is a novel bioactive peptide as the endogenous ligand for APJ. Apelin and APJ have also been identified in the testis, hypothalamic nuclei such as arcuate, supraoptic and paraventricular nuclei, implicating roles in the control of reproduction.Therefore, the present study was designed to investigate the effects of chronic central infusion of apelin-13 on LH, FSH and testosterone levels and testis morphology. 21 Wistar–Albino male rats received continuous intracerebroventricular infusion via Alzet osmotic mini pumps filled artificial cerebrospinal fluid (vehicle) or apelin-13 at concentrations of 1 or 10nmol (10μl/h) for seven days. At the last 90min of the infusion period, the blood samples were collected at 15min intervals (0–90min) for LH and FSH analyses. At the last sampling point, the blood samples were analyzed for testosterone levels.Infusion of high dose apelin-13 significantly suppressed LH release compared with the vehicle values at 30, 60 and 75min (p<0.05). However, FSH levels did not significantly differ among the groups. Serum testosterone levels in high dose apelin-13 group were statistically lower than the control group (p<0.05). In addition, histological examination showed that infusion of high dose apelin-13 significantly decreased the number of Leydig cells compared with the control and lower dose apelin-13 groups (p<0.05, p<0.01).Our results suggest that apelin-13 may play a role in the central regulation and decreases testosterone release by suppressing LH secretion. Thus, antagonists of the apelin receptor may, therefore, be useful for pharmaceuticals in the treatment of infertility.

Hypothalamic Prolactin Regulation of Luteinizing Hormone Secretion in the Female Rat.

Prolactin (PRL) levels increase in response to long-term antipsychotic treatment that disrupts reproductive function. Recent evidence suggests that activation of central PRL receptors (PRLR) inhibits LH secretion and in ovariectomized rats. However, the mechanisms involved, the mode of LH secretion affected and relevance to hyperprolactinemia remain unknown. We therefore investigated the contribution of central PRL/PRLR signaling to the control of estradiol-induced surges of LH and PRL and pulsatile LH secretion under basal and hyperprolactinemic conditions. First, by subjecting ovariectomized estradiol-primed rats intracerebroventricularly administered with PRL to frequent blood sampling, we demonstrated that acute activation of hypothalamic PRLR disrupts pulsatile LH secretion. Pretreatment (intracerebroventricularly) with the pure PRLR antagonist, Δ1-9-G129R-hPRL, or the γ-aminobutyric acid receptor type A antagonist, bicuculline, blocked this effect. Next, we revealed that sustained blockade of hypothalamic PRLR using Δ1-9-G129R-hPRL augmented the magnitude of LH surges induced by estradiol benzoate and progesterone treatment and suppressed the concomitant surges of PRL. Finally, we determined that acute antagonism of central PRLR is insufficient to normalize the duration of the LH pulse interval prolonged as a result of hyperprolactinemia induced by chronic exposure to the atypical antipsychotic sulpiride. These data serve as the first evidence to suggest that PRL signaling through hypothalamic PRLR inhibits pulsatile secretion of LH in a γ-aminobutyric acid receptor type A-dependent fashion and tonically restrains the magnitude of the LH surge. Furthermore, our results indicate that transient blockade of hypothalamic PRL/PRLR signaling is not an effective strategy for restoring LH pulsatility perturbed by chronic hyperprolactinemia.

Gene expression analysis of bovine oocytes at optimal coasting time combined with GnRH antagonist during the no-FSH period

Ovarian stimulation with FSH combined with an appropriate period of FSH withdrawal (coasting) before ovum pick-up now appears to be a successful way to obtain oocytes with high developmental competence in bovine. Recent results showed that extending follicular growth by only 24 hours has a detrimental effect on oocyte quality as shown by the reduced blastocyst formation rate. Although these treatments are initiated during the luteal phase with low LH level, the small LH pulsatility present at that time could potentially impact follicular development as well as oocyte quality. In this study, a GnRH antagonist (Cetrotide) was used to suppress LH secretion during follicular differentiation to get a better insight into the physiological importance of the LH support during that period. Oocytes were collected by ovum pick-up, and quality was assessed by measuring the blastocyst formation rate obtained after IVM-IVF. The oocyte transcriptome from GnRH antagonist–treated animals was also compared with that from a control group (coasting duration of 68 hours) to detect possible alterations at the messenger RNA (mRNA) level. The oocyte quality was not statistically affected by the treatment as shown by the blastocyst formation rate obtained. However, microarray analysis showed that a total of 226 genes had a significant difference (fold change > 2; P < 0.05) at the mRNA level, with the majority being in overabundance in the treated group. Many genes related to RNA posttranscriptional modifications presented different abundance at the mRNA level significant differences in the control group (68 hours), whereas translation function appeared to be affected, with many genes related to structural constituents of the ribosome presenting a overabundance in the GnRH antagonist-treated group. Specific mRNAs with crucial roles in chromosome segregation control also showed significant difference at the mRNA level after Cetrotide treatment. The results presented here indicated that the suppression of the LH secretion in an optimal stimulated context would have an impact on the oocyte, with the possible alteration of critical functions related to translation capacity and chromosome segregation control.

Effects of Neuron-Specific Estrogen Receptor (ER) α and ERβ Deletion on the Acute Estrogen Negative Feedback Mechanism in Adult Female Mice

The negative feedback mechanism through which 17β-estradiol (E2) acts to suppress the activity of the GnRH neurons remains unclear. Using inducible and cell-specific genetic mouse models, we examined the estrogen receptor (ER) isoforms expressed by neurons that mediate acute estrogen negative feedback. Adult female mutant mice in which ERα was deleted from all neurons in the neonatal period failed to exhibit estrous cycles or negative feedback. Adult mutant female mice with neonatal neuronal ERβ deletion exhibited normal estrous cycles, but a failure of E2 to suppress LH secretion was seen in ovariectomized mice. Mutant mice with a GnRH neuron-selective deletion of ERβ exhibited normal cycles and negative feedback, suggesting no critical role for ERβ in GnRH neurons in acute negative feedback. To examine the adult roles of neurons expressing ERα, an inducible tamoxifen-based Cre-LoxP approach was used to ablate ERα from neurons that express calmodulin kinase IIα in adults. This resulted in mice with no estrous cycles, a normal increase in LH after ovariectomy, but an inability of E2 to suppress LH secretion. Finally, acute administration of ERα- and ERβ-selective agonists to adult ovariectomized wild-type mice revealed that activation of ERα suppressed LH secretion, whereas ERβ agonists had no effect. This study highlights the differences in adult reproductive phenotypes that result from neonatal vs adult ablation of ERα in the brain. Together, these experiments expand previous global knockout studies by demonstrating that neurons expressing ERα are essential and probably sufficient for the acute estrogen negative feedback mechanism in female mice.

Prolactin Regulates Kisspeptin Neurons in the Arcuate Nucleus to Suppress LH Secretion in Female Rats

Prolactin (PRL) is known to suppress LH secretion. Kisspeptin neurons regulate LH secretion and express PRL receptors. We investigated whether PRL acts on kisspeptin neurons to suppress LH secretion in lactating (Lac) and virgin rats. Lac rats displayed high PRL secretion and reduced plasma LH and kisspeptin immunoreactivity in the arcuate nucleus (ARC). Bromocriptine-induced PRL blockade significantly increased ARC kisspeptin and plasma LH levels in Lac rats but did not restore them to the levels of non-Lac rats. Bromocriptine effects were prevented by the coadministration of ovine PRL (oPRL). Virgin ovariectomized (OVX) rats treated with either systemic or intracerebroventricular oPRL displayed reduction of kisspeptin expression in the ARC and plasma LH levels, and these effects were comparable with those of estradiol treatment in OVX rats. Conversely, estradiol-treated OVX rats displayed increased kisspeptin immunoreactivity in the anteroventral periventricular nucleus, whereas oPRL had no effect in this brain area. The expression of phosphorylated signal transducer and activator of transcription 5 was used to determine whether kisspeptin neurons in the ARC were responsive to PRL. Accordingly, intracerebroventricular oPRL induced expression of phosphorylated signal transducer and activator of transcription 5 in the great majority of ARC kisspeptin neurons in virgin and Lac rats. We provide here evidence that PRL acts on ARC neurons to inhibit kisspeptin expression in female rats. During lactation, PRL contributes to the inhibition of ARC kisspeptin. In OVX rats, high PRL levels suppress kisspeptin expression and reduce LH release. These findings suggest a pathway through which hyperprolactinemia may inhibit LH secretion and thereby cause infertility.

Progesterone Receptor Isoforms in the Mammary Gland of Cats and Dogs

Progesterone exerts its effect by binding to specific progesterone receptors (PR) within the cell. In dogs and cats, no data are available on PR isoforms as found in other species. We therefore investigated the sequence of the PR gene and encoded protein in dogs and cats, the expression of PR isoforms in mammary tissue using Western blots and the presence of PR in mammary tissue using immunohistochemistry. Comparison of the amino acid sequence of the canine and feline PR with human PR revealed major differences in the PR-B-specific upstream segment (BUS). However, the essential activation function 3 (AF3) domain was intact in the cat but mutated in the dog. The DNA and ligand-binding domains were highly similar among the species. In cats with fibroadenomatous hyperplasia (FAH), high expression of PR mRNA together with growth hormone (GH), GH receptor (GHR) and IGF-I mRNA was found in comparison with feline mammary carcinomas. Immunohistochemical analysis showed strong nuclear as well as cytoplasmic staining for PR in FAH. Western blot analysis revealed expression of the PR-A and PR-B isoforms in the feline mammary gland. In canine mammary tissue, the most abundant PR staining was found in proliferative zones of the mammary gland. Western blot analyses showed mainly staining for PR-A with lower PR-B staining. It is concluded that in dogs and cats both PR isoforms are expressed. The role of mutations found in the canine PR-B is discussed.

GPR54-Dependent Stimulation of Luteinizing Hormone Secretion by Neurokinin B in Prepubertal Rats

Kisspeptin, neurokinin B (NKB) and dynorphin A (Dyn) are coexpressed within KNDy neurons that project from the hypothalamic arcuate nucleus (ARC) to GnRH neurons and numerous other hypothalamic targets. Each of the KNDy neuropeptides has been implicated in regulating pulsatile GnRH/LH secretion. In isolation, kisspeptin is generally known to stimulate, and Dyn to inhibit LH secretion. However, the NKB analog, senktide, has variously been reported to inhibit, stimulate or have no effect on LH secretion. In prepubertal mice, rats and monkeys, senktide stimulates LH secretion. Furthermore, in the monkey this effect is dependent on kisspeptin signaling through its receptor, GPR54. The present study tested the hypotheses that the stimulatory effects of NKB on LH secretion in intact rats are mediated by kisspeptin/GPR54 signaling and are independent of a Dyn tone. To test this, ovarian-intact prepubertal rats were subjected to frequent automated blood sampling before and after intracerebroventricular injections of KNDy neuropeptide analogs. Senktide robustly induced single LH pulses, while neither the GPR54 antagonist, Kp-234, nor the Dyn agonist and antagonist (U50488 and nor-BNI, respectively) had an effect on basal LH levels. However, Kp-234 potently blocked the senktide-induced LH pulses. Modulation of the Dyn tone by U50488 or nor-BNI did not affect the senktide-induced LH pulses. These data demonstrate that the stimulatory effect of NKB on LH secretion in intact female rats is dependent upon kisspeptin/GPR54 signaling, but not on Dyn signaling.

Kisspeptin-10 stimulation of gonadotrophin secretion in women is modulated by sex steroid feedback

Does sex-steroid feedback influence gonadotrophin responses to kisspeptin-10? Gonadotrophin response to kisspeptin-10 is enhanced in sex-steroid deficient post-menopausal women and suppressed in women taking pharmacological doses of exogenous estrogen and progestogen. Kisspeptin, a novel hypothalamic neuropeptide, stimulates gonadotrophin secretion by stimulating GnRH secretion and has been shown in animal models to play a pivotal role in mediating sex steroid feedback. As estrogen feedback occurs at both the hypothalamus and the pituitary levels, we hypothesized that the stimulatory effect of kisspeptin-10 in women would be dependent on prevailing sex steroid milieu. An experimental study of a novel neuropeptide in women-10 in the early follicular phase, 6 post-menopausal and 8 taking sex-steroid contraceptives (combined pill, n = 4; progestogen implants, n = 4) with suppressed LH secretion. Gonadotrophin secretion was followed for 60 min after kisspeptin administration. The gonadotrophin response to intravenous kisspeptin-10 (0.3 µg/kg) in women in the early follicular phase was compared with that in the presence of low endogenous sex steroids/high gonadotrophin secretion (post-menopausal women) and in women taking sex-steroid contraceptives (combined pill, n = 4; progestogen implants, n = 4) with suppressed LH secretion. Area under the curve (AUC) of gonadotrophin secretion sampled at 15 min intervals over 60 min before and after kisspeptin-10 was analysed. Kisspeptin-10 stimulated LH secretion in follicular (ΔAUC 2.3 ± 0.8 IU/l h, P = 0.009), post-menopausal (5.3 ± 0.9 IU/l h P 0.002) and progestogen (2.6 ± 0.8 IU/l h P 0.05) groups but not in women taking combined pill (0.9 ± 0.4 IU/l h P 0.13). FSH secretion was significantly increased only in post-menopausal women (ΔAUC 2.6 ± 0.8 IU/l h P = 0.03) with changes of <0.5 IU/l h observed in the other three groups. Both LH and FSH responses in post-menopausal women were significantly larger than the other groups (one-way ANOVA analysis of ΔAUC; LH (P = 0.012) and FSH (P = 0.001)]. This study only assessed acute responses to an intravenous bolus of kisspeptin-10 administration, and the impact of continuous exposure to kisspeptin-10 on LH pulse frequency in women remains to be studied to fully understand the translational potential. Gonadotrophin secretion in women is stimulated by kisspeptin-10. These results suggest that the pituitary gonadotrope is a functionally important locus of estrogen feedback in women and also inform potential translational applications of kisspeptin in reproductive endocrine disorders. Medical Research Council (UK). None.

The effect of nutrition on the neural mechanisms potentially involved in melatonin-stimulated LH secretion in female Mediterranean goats

This research examines which neural mechanisms among the endogenous opioid, dopaminergic, serotonergic and excitatory amino acid systems are involved in the stimulation of LH secretion by melatonin implantation and their modulation by nutritional level. Female goats were distributed to two experimental groups that received either 1.1 (group H; n=24) or 0.7 (group L; n=24) times their nutritional maintenance requirements. Half of each group was implanted with melatonin after a long-day period. Plasma LH concentrations were measured twice per week. The effects of i.v. injections of naloxone, pimozide, cyproheptadine and N-methyl-d,l-aspartate (NMDA) on LH secretion were assessed the day before melatonin implantation and again on days 30 and 45. The functioning of all but the dopaminergic systems was clearly modified by the level of nutrition, melatonin implantation and time elapsed since implantation. Thirty days after implantation, naloxone increased LH concentrations irrespective of the level of nutrition (P<0.05), similar to NMDA in the melatonin-implanted H goats (HM; P<0.01). On day 45, naloxone increased LH concentrations in the HM animals (P<0.05), similar to cyproheptadine in both the non-implanted H (HC) and the HM animals (P<0.01). Finally, at 45 days, NMDA increased the LH concentration in all subgroups (P<0.01). These results provide evidence that the effects of different neural systems on LH secretion are modified by nutritional level and melatonin implantation. Endogenous opioids seem to be most strongly involved in the inhibition of LH secretion on days 30 and 45 after melatonin implantation. However, the serotonergic mechanism appears to be most influenced by nutritional level.

Effects of RF-Amide Related Peptide 3 (RFRP-3), an Equine Homolog of Avian Gonadotropin-Inhibiting Hormone, on Adenohypophyseal Responsiveness to GnRH in Mares.

Recent evidence from experiments conducted in our laboratories indicate that the 6- to 10-fold reduction in adenohypophyseal content and release of LH during the winter anovulatory period of mares may be attributed in part to a decline in gonadotrope responsiveness to GnRH. Mechanisms explaining such a phenomenon have not been identified, but could involve a recently-discovered neuropeptide, part of the arginine phenylalanine-amide related peptide (RFRP) family. One member of the RFRP family, RFRP-3, profoundly influences reproductive seasonality in seasonal breeding birds and is thus termed gonadotropin-inhibiting hormone (GnIH). Members of the RFRP family, under photoperiodic influence, have altered gene expression and peptide accumulation in the hypothalamus of seasonal breeding species with immunoreactive cellular projections to the neurosecretory zone of the median eminence. Further, RFRP-3 reduces adenohypophyseal responsiveness to GnRH and suppresses LH secretion. Herein, we examined the potential role of equine RFRP-3 in regulating GnRH-mediated release of LH using primary adenohypophyseal cell culture. We hypothesized that RFRP-3 would reduce the responsiveness of gonadotropes to GnRH in a dose-dependent manner. Alzet osmotic pumps were inserted subcutaneously in winter anovulatory mares and delivered GnRH at a rate of 100 µg/h for 12-18 d to induce synthesis of LH. Following euthanasia, adenohypophyses (n = 4) were collected and enzymatically dispersed. Dispersed cells were plated in 6-well plates (300,000 cells/well) in Dulbecco's Modified Eagle Medium with fetal bovine serum for 72 h. Cells were then incubated in media without fetal bovine serum for 24 h, followed by a 4-h treatment period in which cells were incubated with media alone, GnRH alone (10-8 M; positive control), or media containing GnRH plus equine RFRP-3 (10-6, 10-7, 10-8, 10-9, and 10-10 M). Treatment with GnRH increased (P < 0.0001) media concentrations of LH 5-fold (14.4 ± 1.2 ng/ml) compared to vehicle (2.8 ± 1.2 ng/ml). Concentrations of LH in RFRP-3-treated media (14.8 ± 1.2, 16.5 ± 1.2, 15.1 ± 1.2, 16.6 ± 1.2, and 14.0 ± 1.3 ng/ml for 10-6, 10-7, 10-8, 10-9, and 10-10 M, respectively) did not differ (P > 0.15) from the positive control (10-8 M GnRH). Therefore, these results fail to support the hypothesis that RFRP-3 reduces the responsiveness of gonadotropes to GnRH in the mare, and support our recent observations in cyclic and anovulatory mares demonstrating a lack of measurable effect of RFRP-3 on the release of LH. Supported by Texas H-9137 and the Link Equine Research Fund. (poster)