Vasoactive intestinal peptide indirectly elicits pituitary LH secretion independent of GnRH in female zebrafish

Vasoactive intestinal peptide (Vip) regulates luteinizing hormone (LH) release through the direct regulation of gonadotropin-releasing hormone (GnRH) neurons at the level of the brain in female rodents. However, little is known regarding the roles of Vip in teleost reproduction. Although GnRH is critical for fertility through the regulation of LH secretion in vertebrates, the exact role of the hypophysiotropic GnRH (GnRH3) in zebrafish is unclear since GnRH3 null fish are reproductively fertile. This phenomenon raises the possibility of a redundant regulatory pathway(s) for LH secretion in zebrafish. Here, we demonstrate that VipA (homologues of mammalian Vip) both inhibits and induces LH secretion in zebrafish. Despite the observation that VipA axons may reach the pituitary proximal pars distalis including LH cells, pituitary incubation with VipA in vitro, and intraperitoneal injection of VipA, did not induce LH secretion and lhβ mRNA expression in sexually mature females, respectively. On the other hand, intracerebroventricular administration of VipA augmented plasma LH levels in both wild type and gnrh3-/- females at 1 hour post-treatment, with no observed changes in pituitary GnRH2 and GnRH3 contents and gnrh3 mRNA levels in the brains. While VipA’s manner of inhibition of LH secretion has yet to be explored, the stimulation seems to occur via a different pathway than GnRH3, dopamine, and E2 in regulating LH secretion. The results indicate that VipA induces LH release possibly by acting with or through a non-GnRH factor(s), providing proof for the existence of functional redundancy of LH release in sexually mature female zebrafish.

The GnRH Antagonist Degarelix Suppresses Gonadotropin Secretion and Pituitary Sensitivity in Midgestation Sheep Fetuses

The specific role of GnRH on brain sexual differentiation remains unclear. To investigate whether gonadotropin and, in turn, testosterone (T) secretion is regulated by GnRH during the critical period for brain differentiation in sheep fetuses, we attempted to selectively suppress pituitary-testicular activation during midgestation with the long-acting GnRH antagonist degarelix. Fetuses received subcutaneous injections of the antagonist or vehicle on day 62 of gestation. After 2 to 3 weeks we examined consequences of the intervention on baseline and GnRH-stimulated plasma LH and T levels. In addition, we measured the effect of degarelix-treatment on mRNA expression for the pituitary gonadotropins and key gonadal steroidogenic enzymes. Baseline and GnRH-stimulated plasma LH levels were significantly suppressed in degarelix-treated male and female fetuses compared to control values. Similarly, T concentrations were suppressed in degarelix-treated males. The percentage of LHβ-immunoreactive cells colocalizing c-fos was significantly reduced by degarelix treatment indicating that pituitary sensitivity was inhibited. Degarelix treatment also led to the significant suppression of mRNA expression coding for the pituitary gonadotropin subunits and for the gonadal enzymes involved in androgen synthesis. These findings demonstrate that pharmacologic inhibition of GnRH early in gestation results in suppression of LH secretion and deficits in the plasma T levels of male lamb fetuses. We conclude that GnRH signaling plays a pivotal role for regulating T exposure during the critical period of sheep gestation when the brain is masculinized. Thus, disturbance to gonadotropin secretion during this phase of gestation could have long-term consequence on adult sexual behaviors and fertility.

Effects of melatonin on the production of GnRH and LH in luteal cells of pregnant sows

Effects of melatonin on the release and synthesis of gonadotropin releasing hormone (GnRH) and luteinizing hormone (LH) at the hypothalamus and pituitary levels have been explored in some species, but a similar study in the corpora lutea (CL) has not yet been conducted. In this study, the immunostaining for GnRH and LH was observed in luteal cells of porcine CL during pregnancy, and a significant effect of pregnant stage on the level of GnRH and LH was found; higher values for GnRH and LH immunostaining and mRNA were detected in the early- and mid- stages CL than in the later-stage CL (P < 0.01). Furthermore, the patterns of melatonin membrane receptors (MT1 and MT2) expression were consistent with those of GnRH and LH expression in the CL of pregnant sows; the relative levels of MT1 and MT2 in the early- and mid- stages were significantly higher than those in the later-stage (P < 0.01). In luteal cells, melatonin dose-dependently increased in GnRH and LH secretion and mRNA expression. Melatonin also increased the GnRH–induced accumulation of LH, and the LH–induced secretion of P4 in luteal cells. Additionally, the effects of melatonin on luteal GnRH and LH production, were blocked by luzindole, a nonselective MT1 and MT2 receptor antagonist. Our results demonstrate the stimulatory effects of melatonin on GnRH and LH production in luteal cells of pregnant sows, suggesting a potential role for melatonin in luteal function through regulating the release and synthesis of GnRH and LH in luteal cells.

The Effects of Ram Exposure, Progestogen and eCG on Reproductive Performance of Fat Tail Ewes in Non-Breeding Season

Sheep are considered short-day breeders. Exposure of rams to anestrus ewes results in an increase in LH secretion. The aims of this study was to determine whether the presence and exposure of rams can effect on reproductive performance such as: pregnancy rate, litter size, sex of lamb, fecundity rate and prolificacy rate of fat tail Iranian Shaal breed ewes in non-breeding season using progesterone (p4) analogue with or without equine chorionic gonadotropin (eCG). Totally, 100 ewes out of 138 non-cyclic ewes with <0.5ng/ml P4 were selected for this study. They were aged 2 to >7 years old. They were stratified to two groups (control and treatment). The ewes in treatment and control groups received 13 days a sponge containing 60 mg medroxyprogesterone acetate (MPA). The treatment group consists of 3 subgroups and each group contains 25 ewes equally. They were included: 1- P4, eCG and Ram exposure. 2- P4 and ram exposure. 3- P4 and eCG. Ewes in control group received only P4. The ewes were located in isolated corrals for 40 days. The ram to ewe ratio was 1 to 5. Twenty healthy rams were housed in an isolated pen from ewes with distance 1500 m during 40 days. Rams exposed three days before sponge withdraw (day 10). They were separated by considerable distance (2 meters) using fence. The related groups received 500 IU eCG on the day of sponge removal (day 13). The rams were released into the ewe flock after removing the sponge in treatment and control groups. Pregnancy diagnosis performed using transabdominal ultrasonography. The obtained data analyzed using SPSS version 16. The conception rate in treatment and control groups were differed significantly (P<0.01). The conception rate in P4+ram exposure+ eCG, P4+ramexposure and P4+ eCG were 96%(no.24), 80%(no.20) and 88%(no.22), respectively. However, the conception rate in control group was 60% (no. 15). The fecundity rates were 136%, 100% and 124% in treatment subgroups, however, it was 84% in control group, respectively. There were no significant differences among genus and weight of lambs in treatments subgroups and control group (P>0.05). It is concluded that ram exposure can increase significantly reproductive performance in non-breeding season fat tailed ewes Shaal breed.

The Hippo Pathway Effectors YAP and TAZ Regulate LH Release by Pituitary Gonadotrope Cells in Mice

The Hippo transcriptional coactivators YAP and TAZ exert critical roles in morphogenesis, organ size determination and tumorigenesis in many tissues. Although Hippo kinase cascade activity was recently reported in the anterior pituitary gland in mice, the role of the Hippo effectors in regulating gonadotropin production remains unknown. The objective of this study was therefore to characterize the roles of YAP and TAZ in gonadotropin synthesis and secretion. Using a conditional gene targeting approach (cKO), we found that gonadotrope-specific inactivation of Yap and Taz resulted in increased circulating levels of follicle-stimulating hormone (FSH) and luteinizing hormone (LH) in adult male mice, along with increased testosterone levels and testis weight. Female cKO mice had increased circulating LH (but not FSH) levels, which were associated with a hyperfertility phenotype characterized by higher ovulation rates and larger litter sizes. Unexpectedly, the loss of YAP/TAZ did not appear to affect the expression of gonadotropin subunit genes, yet both basal and GnRH-induced LH secretion were increased in cultured pituitary cells from cKO mice. Likewise, pharmacologic inhibition of YAP binding to the TEAD family of transcription factors increased both basal and GnRH-induced LH secretion in LβT2 gonadotrope-like cells in vitro without affecting Lhb expression. Conversely, mRNA levels of ChgA and SgII, which encode key secretory granule cargo proteins, were decreased following pharmacologic inhibition of YAP/TAZ, suggesting a mechanism whereby YAP/TAZ regulate the LH secretion machinery in gonadotrope cells. Together, these findings represent the first evidence that Hippo signaling may play a role in regulating pituitary LH secretion.

Reproductive Regulation of PrRPs in Teleost: The Link Between Feeding and Reproduction

Prolactin-releasing peptide (PrRP), a sort of vital hypothalamic neuropeptide, has been found to exert an enormous function on the food intake of mammals. However, little is known about the functional role of PrRP in teleost. In the present study, two PrRP isoforms and four PrRP receptors were isolated from grass carp. Ligand-receptor selectivity displayed that PrRP1 preferentially binds with PrRP-R1a and PrRP-R1b, while PrRP-R2a and PrRP-R2b were special receptors for PrRP2. Tissue distribution indicated that both PrRPs and PrRP-Rs were highly expressed in the hypothalamus-pituitary-gonad axis and intestine, suggesting a latent function on food intake and reproduction. Using grass carp as a model, we found that food intake could significantly induce hypothalamus PrRP mRNA expression, which suggested that PrRP should be also an anorexigenic peptide in teleost. Interestingly, intraperitoneal (IP) injection of PrRPs could significantly induce serum luteinizing hormone (LH) secretion and pituitary LHβ and GtHα mRNA expression in grass carp. Moreover, using primary culture grass carp pituitary cells as a model, we further found that PrRPs could directly induce pituitary LH secretion and synthesis mediated by AC/PKA, PLC/IP3/PKC, and Ca2+/CaM/CaMK-II pathways. Finally, estrogen treatment of prepubertal fish elicited increases in PrRPs and PrPR receptors expression in primary cultured grass carp hypothalamus cells, which further confirmed that the PrRP/PrRPR system may participate in the neuroendocrine control of fish reproduction. These results, taken together, suggest that PrRPs might act as a coupling factor in feeding metabolism and reproductive activities in teleost.

Optogenetic Activation of Arcuate Kisspeptin Neurons Generates a Luteinizing Hormone Surge-Like Secretion in an Estradiol-Dependent Manner

Traditionally, the anteroventral periventricular (AVPV) nucleus has been the brain area associated with luteinizing hormone (LH) surge secretion in rodents. However, the role of the other population of hypothalamic kisspeptin neurons, in the arcuate nucleus (ARC), has been less well characterized with respect to surge generation. Previous experiments have demonstrated ARC kisspeptin knockdown reduced the amplitude of LH surges, indicating that they have a role in surge amplification. The present study used an optogenetic approach to selectively stimulate ARC kisspeptin neurons and examine the effect on LH surges in mice with different hormonal administrations. LH level was monitored from 13:00 to 21:00 h, at 30-minute intervals. Intact Kiss-Cre female mice showed increased LH secretion during the stimulation period in addition to displaying a spontaneous LH surge around the time of lights off. In ovariectomized Kiss-Cre mice, optogenetic stimulation was followed by a surge-like secretion of LH immediately after the stimulation period. Ovariectomized Kiss-Cre mice with a low dose of 17β-estradiol (OVX+E) replacement displayed a surge-like increase in LH release during period of optic stimulation. No LH response to the optic stimulation was observed in OVX+E mice on the day of estradiol benzoate (EB) treatment (day 1). However, after administration of progesterone (day 2), all OVX+E+EB+P mice exhibited an LH surge during optic stimulation. A spontaneous LH surge also occurred in these mice at the expected time. Taken together, these results help to affirm the fact that ARC kisspeptin may have a novel amplificatory role in LH surge production, which is dependent on the gonadal steroid milieu.

Evidence that pubertal status impacts KNDy neurons in the gilt

Puberty onset is a complex physiological process which enables the capacity for reproduction through increased gonadotropin-releasing hormone (GnRH), and subsequently luteinizing hormone (LH), secretion. While cells that coexpress kisspeptin, neurokinin B (NKB), and dynorphin in the hypothalamic arcuate nucleus (ARC) are believed to govern the timing of puberty, the degree to which KNDy neurons exist and are regulated by pubertal status remains to be determined in the gilt. Hypothalamic tissue from prepubertal and postpubertal, early follicular phase gilts was used to determine the expression of kisspeptin, NKB, and dynorphin within the ARC. Fluorescent in situ hybridization revealed that the majority (&gt; 74%) of ARC neurons that express mRNA for kisspeptin coexpressed mRNA for NKB and dynorphin. There were fewer ARC cells that expressed mRNA for dynorphin in postpubertal gilts compared to prepubertal gilts (P &lt; 0.05), but the number of ARC cells expressing mRNA for kisspeptin or NKB was not different between groups. Within KNDy neurons, mRNA abundance for kisspeptin, NKB, and dynorphin of postpubertal gilts was the same as, less than, and greater than, respectively, prepubertal gilts. Immunostaining for kisspeptin did not differ between prepubertal and postpubertal gilts, but there were fewer NKB immunoreactive fibers in postpubertal gilts compared to prepubertal gilts (P &lt; 0.05). Together, these data reveal novel information about KNDy neurons in gilts and supports the idea that NKB and dynorphin play a role in puberty onset in the female pig.

242 Divergent Selection for Early Puberty Impacts KNDy Neuron Gene Expression in Gilts

Advancing gilt puberty onset is financially desirable for swine production. Neurons in the hypothalamic arcuate nucleus (ARC) that co-express kisspeptin, neurokinin B (NKB), and dynorphin (i.e. KNDy cells) are believed to control gonadotropin-releasing hormone (GnRH) and luteinizing hormone (LH) secretion, but their role in gilt pubertal development is unknown. We hypothesized that puberty onset in gilts would coincide with greater expression of mRNA for kisspeptin and NKB, and less expression of dynorphin. Using fluorescent in situ hybridization (RNAscope), we examined expression of kisspeptin, NKB, and dynorphin in pre- and postpubertal gilts from two genetic lines divergently selected for age at puberty. Prepubertal (n = 6/line) and postpubertal (n = 6/line) gilts were used, and postpubertal animals all received Matrix (0.22% altrenogest) orally for 14 days with tissue collection two days after the final dose. Gilts were euthanized and heads were perfused with 8 L of 4% paraformaldehyde (PFA). Hypothalamic brain tissue was removed, placed in 4% PFA for 24 hrs, and then in 20% sucrose until sectioning (50 µm). Sectioned tissue was stored in cryopreservative at -20°C until RNAscope. Data were analyzed using SAS software (Version 9.4, SAS Institute, Cary NC) with significance declared at P &lt; 0.05. We determined mRNA expression for kisspeptin was not different between groups (P &gt; 0.05). In addition, we found that mRNA expression for NKB was higher in prepubertal gilts compared to postpubertal gilts (P &lt; 0.05) but was not different between lines; mRNA expression was lowest in postpubertal late puberty gilts. Furthermore, total number of dynorphin cells were higher in prepubertal gilts compared to postpubertal gilts (P &lt; 0.05), while individual cell mRNA expression for dynorphin was greatest in postpubertal early puberty gilts (P &lt; 0.05). Taken together, we suggest puberty onset in gilts is more dependent on NKB and dynorphin than kisspeptin.

Kisspeptin, Neurokinin B, and Dynorphin Expression during Pubertal Development in Female Sheep

The neural mechanisms underlying increases in gonadotropin-releasing hormone (GnRH) and luteinizing hormone (LH) secretion that drive puberty onset are unknown. Neurons coexpressing kisspeptin, neurokinin B (NKB), and dynorphin, i.e., KNDy neurons, are important as kisspeptin and NKB are stimulatory, and dynorphin inhibitory, to GnRH secretion. Given this, we hypothesized that kisspeptin and NKB expression would increase, but that dynorphin expression would decrease, with puberty. We collected blood and hypothalamic tissue from ovariectomized lambs implanted with estradiol at five, six, seven, eight (puberty), and ten months of age. Mean LH values and LH pulse frequency were the lowest at five to seven months, intermediate at eight months, and highest at ten months. Kisspeptin and NKB immunopositive cell numbers did not change with age. Numbers of cells expressing mRNA for kisspeptin, NKB, or dynorphin were similar at five, eight, and ten months of age. Age did not affect mRNA expression per cell for kisspeptin or NKB, but dynorphin mRNA expression per cell was elevated at ten months versus five months. Thus, neither KNDy protein nor mRNA expression changed in a predictable manner during pubertal development. These data raise the possibility that KNDy neurons, while critical, may await other inputs for the initiation of puberty.