Immunoreactive Gonadotropin-releasing Hormone Research Articles

Common and female-specific roles of protein tyrosine phosphatase receptors N and N2 in mice reproduction

Simultaneous knockout of the neuroendocrine marker genes Ptprn and Ptprn2, which encode the protein tyrosine phosphatase receptors N and N2, causes infertility in female mice while males are fertile. To elucidate the mechanism of the sex-specific roles of Ptprn and Ptprn2 in mouse reproduction, we analyzed the effects of their double knockout (DKO) on the hypothalamic-pituitary–gonadal axis. In DKO females, delayed puberty and lack of ovulation were observed, complemented by changes in ovarian gene expression and steroidogenesis. In contrast, testicular gene expression, steroidogenesis, and reproductive organs development were not significantly affected in DKO males. However, in both sexes, pituitary luteinizing hormone (LH) beta gene expression and LH levels were reduced, as well as follicle-stimulating hormone beta gene and gonadotropin-releasing hormone (GnRH) gene, while the calcium-mobilizing and LH secretory actions of GnRH were preserved. Hypothalamic Gnrh1 and Kiss1 gene expression was also reduced in DKO females and males. In parallel, a significant decrease in the density of immunoreactive GnRH and kisspeptin fibers was detected in the hypothalamic arcuate nucleus of DKO females and males. The female-specific kisspeptin immunoreactivity in the rostral periventricular region of the third ventricle was also reduced in DKO females, but not in DKO males. These data indicate a critical role of Ptprn and Ptprn2 in kisspeptin-GnRH neuronal function and sexual dimorphism in the threshold levels of GnRH required to preserve reproductive functions.

Role of Melatonin in Temperature-Induced Activation of the Neuroendocrine Reproductive Axis in Garter Snakes

An animal’s ability to respond optimally to changing environmental conditions is paramount to successfully reproducing and thus maximizing fitness. Studies on photoperiod-induced changes in neural thyroid hormone metabolism have conclusively linked environmental cues to the neuroendocrine reproductive axis of birds and mammals. Whether this conserved mechanism also transduces changes in environmental temperature, however, has not been fully addressed. We investigated whether the hormone melatonin mediates the effects of low-temperature dormancy on thyroid hormone metabolism within the hypothalamus of red-sided garter snakes (Thamnophis sirtalis parietalis). To address this question, we used immunohistochemistry to assess changes in thyroid-stimulating hormone (TSH) in the infundibulum of the pituitary and deiodinase 3 (Dio3) and gonadotropin-releasing hormone (GnRH) in the hypothalamus. We also asked if changes in TSH, Dio3, and/or GnRH immunoreactivity are associated with changes in male courtship behavior. In contrast to our predictions, 6 weeks of dormancy at 4°C significantly decreased the number of TSH-labeled cells in the infundibulum. It is possible that the observed decrease in TSH is related to the release of snakes from temperature refractoriness, but this idea needs further testing. Treatment of snakes with the melatonin precursor 5-hydroxytryptophan during dormancy at 4°C both reversed the temperature-induced change in TSH immunoreactivity and disrupted the temporal pattern of male courtship behavior. These results suggest that TSH cells within the infundibulum are both modulated by temperature and sensitive to changes in melatonin. As predicted, male snakes hibernated at an elevated temperature of 12°C for 6 weeks and treated with vehicle showed no change in TSH-, Dio3-, or GnRH-immunoreactive cell number. Treatment of snakes with the melatonin receptor antagonist luzindole was not sufficient in rescuing the effects of dormancy at 12°C on TSH immunoreactivity or courtship behavior. However, luzindole-treated snakes showed a significant increase in GnRH-immunoreactive cell number, suggesting that melatonin exerts an inhibitory effect on GnRH in garter snakes. In summary, our results provide critical insights into the mechanisms that mediate the effects of temperature on reproductive physiology and behavior.

Effects of noopept on cognitive functions and pubertal process in rats with diabetes

AimType 1 diabetes (T1DM) is a common chronic disease in childhood. Increasing insulin resistance in puberty gives rise to higher doses of insulin usage in treatment. Of this reason new approaches in treatment are needed. Noopept researches suggest it to have anti-diabetic properties. We tried to determine the effects of noopept on pubertal diabetes. Main methodThe research was made with 60 prepubertal, 28 day-old, male, Sprague Dawley rats. The rats were divided into randomised 6 groups (n = 10/group). i) Control, ii) Diabetes Control, iii) Noopept Control, iv) Diabetes + Noopept, v) Diabetes + Insulin, vi) Diabetes + Insulin + Noopept. T1DM model was induced by streptozotocin on postnatal 28th day. 0.5 mg/kg noopept and 1 IU insulin were administered intraperitoneally for 14 days. Blood glucose and body weight measurements, puberty follow-up and MWM tests were performed. Hippocampus, hypothalamus and testis were evaluated histologically. Hypothalamic GnRH and kisspeptin were studied immunohistochemically. Serum LH, FSH and insulin, hippocampal homogenate NGF and BDNF levels were determined by ELISA. Key findingsDelayed puberty was normalized by noopept (p < 0.05). Blood glucose levels were lower in noopept-administered diabetic groups (p < 0.05). Noopept decreased HOMA-IR in insulin administered diabetic group (p < 0.05). Number of degenerated cells in hippocampus and testis were higher in diabetes control group when compared with other groups (p < 0.05). GnRH immunoreactivity in Diabetes + Noopept group was increased when compared to insulin + noopept group (p = 0.018). There was no difference in kisspeptin, serum LH, FSH, hippocampal NGF-BDNF levels and spatial learning assessment among groups (p > 0.05). SignificanceNoopept may have positive effect in treatment of pubertal diabetes.

Immunohistochemical study of gonadotropin-releasing hormone and somatolactin during induced spawning of Liza ramada

Introduction: Gonadotropin releasing hormone (GnRH) is a crucial regulator of gonadal development and reproduction in fish. In addition, it is a possible secretagogue of somatolactin (SL) in teleosts. Little is known about the possible association of GnRH and SL during reproduction of Liza ramada.Aim of the work: The present study was designed to investigate the GnRH and SL immunoreactivities during inducing the final stages of maturation, ovulation and spawning of Liza ramada; to assess the possible actions of these hormones on sexual maturation and spawning of teleosts.Materials and Methods: Histological and immunohistochemical techniques were generally performed to describe the immunoreactivity of both GnRH and SL during ovarian maturation and spawning of Liza ramada.Results: The mammalian GnRH (mGnRH) cell bodies were observed in the medulla oblongata. However, SL-immunoreactive (-IR) cells were detected in the pars intermedia (PI) of the pituitary gland. The GnRH-IR nerve terminals are in close contact with SL-IR pituitary cells. Importantly, the activity of both mGnRH-IR neurons and SL-IR pituitary cells were increased as reflected with the increased cell number and size during sexual maturation and spawning. The activity of these cells was more pronounced as indicated by the increased cells number and size with more immunoreactivity in pre-spawning female. During final maturation and spawning, mGnRH-IR neurons and SL cells showed an increase in the secretory activity as reflected by their small sizes, vacuolated appearance and weak immunoreactivity.Conclusions: Taken together, these findings suggest that the close contact of mGnRH-IR terminals with SL-IR cells in the pituitary, together with the concomitant changes of GnRH and SL immunoreactivity in response to progress in the reproductive events suggest that GnRH may regulate SL release and the regulation of reproductive activity in L. ramada.

Distribution and regulation of gonadotropin-releasing hormone, kisspeptin, RF-amide related peptide-3, and dynorphin in the bovine hypothalamus.

Recent work has led to the hypothesis that kisspeptin/neurokinin B/dynorphin (KNDy) neurons in the arcuate nucleus (ARC) play a key role in gonadotropin-releasing hormone (GnRH) pulse generation and gonadal steroid feedback, with kisspeptin driving GnRH release and neurokinin B and dynorphin acting as pulse start and stop signals, respectively. A separate cell group, expressing RFamide-related peptide-3 (RFRP-3) has been shown to be a primary inhibitor of GnRH release. Very little is known regarding these cell groups in the bovine. In this study, we examined the relative immunoreactivity of kisspeptin, dynorphin, and RFRP-3 and their possible connectivity to GnRH neurons in the hypothalami of periestrus and diestrus bovine. While GnRH and RFRP-3 immunoreactivity were unchanged, kisspeptin and dynorphin immunoreactivity levels varied in relation to plasma progesterone concentrations and estrous status. Animals with higher plasma progesterone concentrations in diestrus had lower kisspeptin and increased dynorphin immunoreactivity in the ARC. The percentage of GnRH cells with kisspeptin or RFRP-3 fibers in close apposition did not differ between estrous stages. However, the proportions of GnRH cells with kisspeptin or RFRP-3 contacts (∼49.8% and ∼31.3%, respectively) suggest direct communication between kisspeptin and RFRP-3 cells to GnRH cells in the bovine. The data produced in this work support roles for kisspeptin and dynorphin, within the KNDy neural network, in controlling GnRH release over the ovarian cycle and conveying progesterone-negative feedback onto GnRH neurons in the bovine.

Gonadotropin-releasing hormone and gonadotropin-releasing hormone receptor are expressed at tubal ectopic pregnancy implantation sites

To investigate whether gonadotropin-releasing hormone (GnRH) and GnRH receptor (GnRHR) are expressed at tubal ectopic pregnancy sites, and to study the potential role of GnRH signaling in regulating immortalized human trophoblast cell viability. Immunohistochemical and experimental studies. Academic research laboratory. Fallopian tube implantation sites (n = 25) were collected from women with ectopic pregnancy. First-trimester human placenta biopsies (n = 5) were obtained from elective terminations of pregnancy. None. GnRH and GnRHR expression was examined by means of immunohistochemistry and histoscoring. Trophoblastic BeWo choriocarcinoma and immortalized extravillous trophoblast (HTR-8/SVneo) cell viability was examined by means of cell counting after incubation with GnRH and/or GnRH antagonist (Antide). GnRH and GnRHR immunoreactivity was detected in cytotrophoblast, syncytiotrophoblast, and extravillous trophoblast in all women with tubal pregnancy. GnRH immunoreactivity was higher and GnRHR immunoreactivity lower in syncytiotrophoblast compared with cytotrophoblast. GnRH and GnRHR immunoreactivity was detected in adjacent fallopian tube epithelium. Whereas neither GnRH nor Antide altered HTR-8/SVneo cell viability, treatment with GnRH significantly increased the overall cell viability of BeWo cells at 48 and 72hours, and these effects were abolished by pretreatment with Antide. GnRH and GnRHR are expressed in trophoblast cell populations and fallopian tube epithelium at tubal ectopic pregnancy sites. GnRH increases BeWo cell viability, an effect mediated by the GnRHR. Further work is required to investigate the potential role of GnRH signaling in ectopic pregnancy.

Immunohistochemical evidence for the involvement of gonadotropin releasing hormone in neuroleptic and cataleptic effects of haloperidol in mice

Blockade of dopamine D2 receptor by haloperidol is attributed for neuroleptic and cataleptic effects; and also for the release of gonadotropin releasing hormone (GnRH) from the hypothalamus. GnRH agonist is reported to exhibit similar behavioural effects as that of haloperidol, and pre-treatment with GnRH antagonist is shown to attenuate the effects of haloperidol, suggesting a possibility that GnRH might mediate the effects of haloperidol. To substantiate such possibility, the influence of haloperidol on GnRH immunoreactivity (GnRH-ir) in the brain was studied in vehicle/antide pre-treated mice by peroxidase–antiperoxidase method. Initially, an earlier reported antide–haloperidol interaction in rat was confirmed in mice, wherein haloperidol (250μg/kg, i.p.) exhibited suppression of conditioned avoidance response (CAR) on two-way shuttle box, and induced catalepsy in bar test; and pre-treatment with antide (50μg/kg, s.c., GnRH antagonist) attenuated both effects of haloperidol. Immunohistochemical study was carried out to identify GnRH-ir in the brain, isolated 1h after haloperidol treatment to mice pre-treated with vehicle/antide. The morphometric analysis of microphotographs of brain sections revealed that haloperidol treatment increased integrated density units of GnRH-ir in various regions of the limbic system. Considering basal GnRH-ir in vehicle treated group as 100%, the increase in GnRH-ir after haloperidol treatment was by 100.98% in the medial septum; 54.26% in the bed nucleus of the stria terminalis; 1152.85% in the anteroventral periventricular nucleus; 120.79% in the preoptic area–organum vasculosum of the lamina terminalis and 138.82% in the arcuate nucleus. Antide did not influence basal and haloperidol induced increase in GnRH-ir in any of the regions.As significant increase in GnRH-ir after haloperidol treatment was observed in such regions of the brain which are reported to directly or indirectly communicate with the hippocampus and basal ganglia, the regions respectively responsible for neuroleptic and cataleptic effects; and as GnRH antagonist eliminated the effects of haloperidol without affecting GnRH-ir, it appears that GnRH released by haloperidol mediates its neuroleptic and cataleptic effects.

“Brain sex differentiation” in teleosts: Emerging concepts with potential biomarkers

“Brain sex differentiation” in teleosts is a contentious topic of research as most of the earlier reports tend to suggest that gonadal sex differentiation drives brain sex differentiation. However, identification of sex-specific marker genes in the developing brain of teleosts signifies brain–gonadal interaction during early sexual development in lower vertebrates. In this context, the influence of gonadotropin-releasing hormone (GnRH)–gonadotropin (GTH) axis on gonadal sex differentiation, if any requires in depth analysis. Presence of seabream (sb) GnRH immunoreactivity (ir−) in the brain of XY Nile tilapia was found as early as 5days post hatch (dph) followed by qualitative reduction in the preoptic area-hypothalamus region. In contrast, in the XX female brain a steady ir− of sbGnRH was evident from 15dph. Earlier studies using sea bass already implied the importance of hypothalamic gonadotropic axis completion during sex differentiation period. Such biphasic pattern of localization was also seen in pituitary GTHs using heterologous antisera in tilapia. However, more recent analysis in the same species could not detect any sexually dimorphic pattern using homologous antisera for pituitary GTHs. Detailed studies on the development of hypothalamo–hypophyseal–gonadal axis in teleosts focusing on hypothalamic monoamines (MA) and MA-related enzymes demonstrated sex-specific differential expression of tryptophan hydroxylase (Tph) in the early stages of developing male and female brains of tilapia and catfish. The changes in Tph expression was in agreement with the levels of serotonin (5-HT) and 5-hydroxytryptophan in the preoptic area-hypothalamus. Considering the stimulatory influence of 5-HT on GnRH and GTH release, it is possible to propose a network association between these correlates during early development, which may bring about brain sex dimorphism in males. A recent study from our laboratory during female brain sex development demonstrated high expression of tyrosine hydroxylase in correlation with catecholamine levels, brain aromatase and its related transcription factors such as fushi tarazu factor 1, Ftz-f1 and fork head box protein L2, foxl2. Taken together, gender differences in the levels of various transcripts provide new perspectives on brain sex differentiation in lower vertebrates. Sexually dimorphic or differentially expressing genes may play an essential role at the level of brain in response to gonadal differentiation, which might consequentially or causatively respond to gonadal sex.

The effect of intracerebroventricular infusions of ghrelin on the secretory activity of the GnRH/LH system in peripubertal ewes

Ghrelin is a 28-amino acid orexigenic peptide, synthesized mainly by cells located in the stomach mucosa, that can be also engaged in modula- tion of reproductive functions. The aim of this study was to investigate the ef- fect of intracerebroventricular (icv) infusion of ghrelin on the secretory activity of the gonadotropin hormone releasing hormone/luteinizing hormone (GnRH/ LH) system in peripubertal ewes. The animals were randomly divided into three groups: 1. standard fed, 2. starved for 72 h, and 3. standard fed + icv infused with ghrelin. Infusions were performed during three consecutive days; blood samples were collected on day 0 and day 3. Ghrelin infusions and short-term starvation increased the accumulation of immunoreactive (ir) GnRH material in the median eminence (P < 0.001) and ir LH material in the secretory granules of pituitary cells (P < 0.001) in the fasted and ghrelin-infused groups. The short starvation resulted in a decrease of mean LH plasma concentrations (P < 0.05) between days 0 and 3 of infusion, which was related to a lower pulse amplitude. After the icv ghrelin infusion, significant (P < 0.05) changes in LH pulse ampli- tude and frequency, but not in LH concentrations were observed. The obtained results revealed that in peripubertal ewes, exogenous ghrelin administration affects the GnRH/LH axis mainly at the level of neurohormone/protein accu- mulation and LH pulsatility modifications. Ghrelin action at the hypothalamus- pituitary axis is likely involved in the endocrine network that integrates energy balance and fertility in immature sheep.

GnIH and GnRH expressions in the central nervous system and pituitary of Indian major carp, Labeo rohita during ontogeny: An immunocytochemical study

Gonadotropin-releasing hormone (GnRH) is the major hypothalamic neuropeptide stimulating gonadotropin secretion in vertebrates. In 2000, gonadotropin-inhibitory hormone (GnIH) was discovered as a hypothalamic neuropeptide that inhibits gonadotropin secretion in birds. Subsequent studies have shown that GnIH is present in the brain of other vertebrates. We show for the first time GnIH immunoreactivity in the central nervous system and pituitary during development of Indian major carp, Labeo rohita and compare it with the localization of GnRH. GnIH and GnRH immunoreactivities were observed from the olfactory system to spinal cord throughout development. In the brain, both neuropeptides were localized in the telencephalon, diencephalon including the preoptic area and rhombencephalon. The localization of GnIH and GnRH in the pituitary suggests that these neuropeptides are involved in the regulation of pituitary hormones by an autocrine manner during development. In addition, the presence of GnIH and GnRH in several other brain regions including the olfactory system suggests their involvement in the regulation of other physiological functions.

Immunohistochemical detection of GnRH-like peptides in the neural ganglia and testis of Haliotis asinina

Gonadotropin releasing hormone (GnRH) is a peptide that is conserved in both vertebrate and invertebrate species. In this study, we have demonstrated the distribution pattern of two isoforms of GnRH-like peptides in the neural ganglia and testis of reproductively mature male abalone, H. asinina, by immunohistochemistry and whole mount immunofluorescence. We found octopus (oct) GnRH and tunicate-I (t) GnRH-I immunoreactivities (ir) in type 1 neurosecretory cells (NS1) and they were expressed mostly within the ventral horn of the cerebral ganglion, whereas in pleuropedal ganglia they were localized primarily in the dorsal horn. Furthermore, tGnRH-I-ir were strongly detected in fibers at the caudal part of the cerebral ganglia and both ventral and dorsal horns of the pleuropedal ganglia. In the testis, only octGnRH-ir was found primarily in the granulated cell and central capillaries within the trabeculae. These results suggest that multiple GnRH-like peptides are present in the neural ganglia which could be the principal source of their production, whereas GnRH may also be synthesized locally in the testis and act as the paracrine control of testicular maturation.

Gonadotropin releasing hormone (GnRH) and gonadotropin inhibitory hormone (GnIH) in the songbird hippocampus: Regional and sex differences in adult zebra finches

Hypothalamic gonadotropin releasing hormone (GnRH) and gonadotropin inhibitory hormone (GnIH) are vital to reproduction in all vertebrates. These neuropeptides are also present outside of the hypothalamus, but the roles of extra-hypothalamic GnRH and GnIH remain enigmatic and widely underappreciated. We used immunohistochemistry and PCR to examine whether multiple forms of GnRH (chicken GnRH-I (GnRH1), chicken GnRH-II (GnRH2) and lamprey GnRH-III (GnRH4)) and GnIH are present in the hippocampus (Hp) of adult zebra finches (Taeniopygia guttata). Using immunohistochemistry, we provide evidence that GnRH1, GnRH2 and GnRH4 are present in hippocampal cell bodies and/or fibers and that GnIH is present in hippocampal fibers only. There are regional differences in hippocampal GnRH immunoreactivity, and these vary across the different forms of GnRH. There are also sex differences in hippocampal GnRH immunoreactivity, with generally more GnRH1 and GnRH2 in the female Hp. In addition, we used PCR to examine the presence of GnRH1 mRNA and GnIH mRNA in micropunches of Hp. PCR and subsequent product sequencing demonstrated the presence of GnRH1 mRNA and the absence of GnIH mRNA in the Hp, consistent with the pattern of immunohistochemical results. To our knowledge, this is the first study in any species to systematically examine multiple forms of GnRH in the Hp or to quantify sex or regional differences in hippocampal GnRH. Moreover, this is the first demonstration of GnIH in the avian Hp. These data shed light on an important issue: the sites of action and possible functions of GnRH and GnIH outside of the HPG axis.

Prior Experience with Photostimulation Enhances Photo-Induced Reproductive Response in Female House Finches

In vertebrates, reproductive output often increases with age. Unlike older birds, first-year photoperiodic birds lack experience with the reproductively stimulatory effects of long day lengths (photostimulation). We examined whether age-related differences in annual reproductive development could be partially attributed to previous experience with photostimulation in the photoperiodic house finch (Carpodacus mexicanus). By manipulating photoperiod, we generated 2 groups of first-year females: a photo-experienced group that underwent 1 photoperiodically induced cycle of gonadal development and regression and a photo-naïve group exposed to long days since hatch. We transferred both groups from long to short days and then photostimulated and exposed them to male birdsong prior to sacrifice. Following concurrent photostimulation, both groups exhibited similar plasma luteinizing hormone surges and hypothalamic vasoactive intestinal polypeptide immunoreactivity. In contrast, hypothalamic gonadotropin-releasing hormone immunoreactivity and circulating vitellogenin levels were higher in photo-experienced birds, and yolk deposition occurred in only 2 females, both of which were photo-experienced. Our results demonstrate that photo-experience enhances some aspects of early photo-induced reproductive development and raise the hypothesis that photo-experience may account for at least some age-related variation in reproductive output.

Atrazine Inhibits Pulsatile Gonadotropin-Releasing Hormone (GnRH) Release Without Altering GnRH Messenger RNA or Protein Levels in the Female Rat1

Atrazine (ATR) is a commonly used pre-emergence/early postemergence herbicide. Previous work has shown that exposure to high doses of ATR in rats results in blunting of the hormone-induced luteinizing hormone (LH) surge and inhibition of pulsatile LH release without significantly reducing pituitary sensitivity to a gonadotropin-releasing hormone (GnRH) agonist. Accompanying the reduction in the LH surge was an attenuation of GnRH neuronal activation. These findings suggest that ATR exposure may be acting to inhibit GnRH release. In this study, we examined GnRH directly to determine the effect of high doses of ATR on GnRH pulsatile release, gene expression, and peptide levels in the female rat. Ovariectomized adult female Wistar rats were treated with ATR (200 mg/kg) or vehicle for 4 days via gavage. Following the final treatment, GnRH release was measured from ex vivo hypothalamic explants for 3 h. In another experiment, animals were administered either vehicle or ATR (50, 100, or 200 mg/kg) daily for 4 days. Following treatment, in situ hybridization was performed to examine total GnRH mRNA and the primary GnRH heterogeneous nuclear RNA transcript. Finally, GnRH immunoreactivity and total peptide levels were measured in hypothalamic tissue of treated animals. ATR treatment resulted in no changes to GnRH gene expression, peptide levels, or immunoreactivity but a reduction in GnRH pulse frequency and an increased pulse amplitude. These findings suggest that ATR acts to inhibit the secretory dynamics of GnRH pulses without interfering with GnRH mRNA and protein synthesis.

Hypothalamic-pituitary-gonadal endocrine system in the hagfish.

The hypothalamic-pituitary system is considered to be a seminal event that emerged prior to or during the differentiation of the ancestral agnathans (jawless vertebrates). Hagfishes as one of the only two extant members of the class of agnathans are considered the most primitive vertebrates known, living or extinct. Accordingly, studies on their reproduction are important for understanding the evolution and phylogenetic aspects of the vertebrate reproductive endocrine system. In gnathostomes (jawed vertebrates), the hormones of the hypothalamus and pituitary have been extensively studied and shown to have well-defined roles in the control of reproduction. In hagfish, it was thought that they did not have the same neuroendocrine control of reproduction as gnathostomes, since it was not clear whether the hagfish pituitary gland contained tropic hormones of any kind. This review highlights the recent findings of the hypothalamic-pituitary-gonadal endocrine system in the hagfish. In contrast to gnathostomes that have two gonadotropins (GTH: luteinizing hormone and follicle-stimulating hormone), only one pituitary GTH has been identified in the hagfish. Immunohistochemical and functional studies confirmed that this hagfish GTH was significantly correlated with the developmental stages of the gonads and showed the presence of a steroid (estradiol) feedback system at the hypothalamic-pituitary levels. Moreover, while the identity of hypothalamic gonadotropin-releasing hormone (GnRH) has not been determined, immunoreactive (ir) GnRH has been shown in the hagfish brain including seasonal changes of ir-GnRH corresponding to gonadal reproductive stages. In addition, a hagfish PQRFamide peptide was identified and shown to stimulate the expression of hagfish GTHβ mRNA in the hagfish pituitary. These findings provide evidence that there are neuroendocrine-pituitary hormones that share common structure and functional features compared to later evolved vertebrates.

Teasing apart socially‐induced infertility in non‐reproductive female Damaraland mole‐rats, Fukomys damarensis (Rodentia: Bathyergidae)

The Damaraland mole-rat is a subterranean mammal exhibiting extreme reproductive skew with a single reproductive female in each colony responsible for procreation. Non-reproductive female colony members are physiologically suppressed while in the colony, exhibiting reduced concentrations of plasma luteinizing hormone (LH) and a decreased response of the pituitary, as measured by the release of bioactive LH, to an exogenous dose of gonadotrophin releasing hormone (GnRH). Removal of the reproductive female from the colony results in an elevation of LH and an enhanced response of the pituitary to a GnRH challenge in non-reproductive females comparable to reproductive females, implying control of reproduction in these individuals by the reproductive female. The Damaraland mole-rat is an ideal model for investigating the physiological and behavioral mechanisms that regulate the hypothalamo-pituitary-gonadal axis. In contrast, we know less about the control of reproduction at the level of the hypothalamus. The immunohistochemistry of the GnRH system of both reproductive and non-reproductive female Damaraland mole-rats has revealed no significant differences with respect to morphology, distribution or numbers of immunoreactive GnRH perikarya. We examined whether the endogenous opioid peptide beta-endorphin was responsible for the inhibition of the release of the GnRH from the neurons indirectly by measuring LH concentrations in these non-reproductive females following single, hourly and 8 hourly injections of the opioid antagonist naloxone. The results imply that the endogenous opioid peptide, beta-endorphin, is not responsible for the inhibition of GnRH release from the perikarya in non-reproductive females. Preliminary data examining the circulating levels of cortisol also do not support a role for circulating glucocorticoids. The possible role of kisspeptin is discussed.

Developmental Programming: Reproductive Endocrinopathies in the Adult Female Sheep After Prenatal Testosterone Treatment Are Reflected in Altered Ontogeny of GnRH Afferents

The GnRH system represents a useful model of long-term neural plasticity. An unexplored facet of this plasticity relates to the ontogeny of GnRH neural afferents during critical periods when the hypothalamic-pituitary-gonadal axis is highly susceptible to perturbation by sex steroids. Sheep treated with testosterone (T) in utero exhibit profound reproductive neuroendocrine dysfunctions during their lifespan. The current study tested the hypothesis that these changes are associated with alterations in the normal ontogeny of GnRH afferents and glial associations. Adult pregnant sheep (n=50) were treated with vehicle [control (CONT)] or T daily from gestational day (GD)30 to GD90. CONT and T fetuses (n=4-6/treatment per age group) were removed by cesarean section on GD90 and GD140 and the brains frozen at -80°C. Brains were also collected from CONT and T females at 20-23 wk (prepubertal), 10 months (normal onset of puberty and oligo-anovulation), and 21 months (oligo-anovulation in T females). Tissue was analyzed for GnRH immunoreactivity (ir), total GnRH afferents (Synapsin-I ir), glutamate [vesicular glutamate transporter-2 (VGLUT2)-ir], and γ-aminobutyric acid [GABA, vesicular GABA transporter (VGAT)-ir] afferents and glial associations (glial fibrillary acidic protein-ir) with GnRH neurons using optical sectioning techniques. The results revealed that: 1) GnRH soma size was slightly reduced by T, 2) the total (Synapsin-I) GnRH afferents onto both somas and dendrites increased significantly with age and was reduced by T, 3) numbers of both VGAT and VGLUT inputs increased significantly with age and were also reduced by T, and 4) glial associations with GnRH neurons were reduced (<10%) by T. Together, these findings reveal a previously unknown developmental plasticity in the GnRH system of the sheep. The altered developmental trajectory of GnRH afferents after T reinforces the notion that prenatal programming plays an important role in the normal development of the reproductive neuroendocrine axis.

P.4.011 Analogues of cyclo-L-prolylglycine and their anxiolytic, nootropic and antihypoxic activity

Type 1 diabetes (T1DM) is a common chronic disease in childhood. Increasing insulin resistance in puberty gives rise to higher doses of insulin usage in treatment. Of this reason new approaches in treatment are needed. Noopept researches suggest it to have anti-diabetic properties. We tried to determine the effects of noopept on pubertal diabetes.The research was made with 60 prepubertal, 28 day-old, male, Sprague Dawley rats. The rats were divided into randomised 6 groups (n = 10/group). i) Control, ii) Diabetes Control, iii) Noopept Control, iv) Diabetes + Noopept, v) Diabetes + Insulin, vi) Diabetes + Insulin + Noopept. T1DM model was induced by streptozotocin on postnatal 28th day. 0.5 mg/kg noopept and 1 IU insulin were administered intraperitoneally for 14 days. Blood glucose and body weight measurements, puberty follow-up and MWM tests were performed. Hippocampus, hypothalamus and testis were evaluated histologically. Hypothalamic GnRH and kisspeptin were studied immunohistochemically. Serum LH, FSH and insulin, hippocampal homogenate NGF and BDNF levels were determined by ELISA.Delayed puberty was normalized by noopept (p < 0.05). Blood glucose levels were lower in noopept-administered diabetic groups (p < 0.05). Noopept decreased HOMA-IR in insulin administered diabetic group (p < 0.05). Number of degenerated cells in hippocampus and testis were higher in diabetes control group when compared with other groups (p < 0.05). GnRH immunoreactivity in Diabetes + Noopept group was increased when compared to insulin + noopept group (p = 0.018). There was no difference in kisspeptin, serum LH, FSH, hippocampal NGF-BDNF levels and spatial learning assessment among groups (p > 0.05).Noopept may have positive effect in treatment of pubertal diabetes.

Effect of maternal deprivation on the gonadotrophin-releasing hormone (GnRH) and GnRH-associated peptide neurobiology in lambs during the transition from infancy to prepuberty.

Using morphological criteria we describe the effect of maternal deprivation on the gonadotrophin-releasing hormone (GnRH) and GnRH-associated peptide (GAP) of the GnRH prohormone (proGnRH) in the preoptic area (POA)-hypothalamus during the weaning period. The immunohistochemical GnRH- and GAP-neuroanatomy was investigated in female 12-week-old weanling and maternally deprived lambs and 15-week-old weaned lambs. The GnRH-immunoreactive (ir) nerve elements in the POA were more numerous in weanling and weaned lambs in comparison with maternally deprived lambs, whereas the nerve elements ir for GAP were numerous in weanlings and scarce in remaining lambs. In the hypothalamus, GnRH-ir fibers were more numerous in weaned lambs in comparison with others. Immunoreactive GnRH in the median eminence was scarce in weanlings and comparable greater in maternally deprived and weaned lambs. In contrast to ir GnRH, the GAP-ir fibers and nerve terminals in the hypothalamus and median eminence were numerous in weanlings and maternally deprived lambs and scarce in weaned lambs. In conclusion, maternal deprivation affects the intraneuronal locations involved in the maturation of GnRH from proGnRH in the POA-hypothalamus of weanlings. The described effect involves the increase in the GnRH posttranslational processing and terminal accumulation in the median eminence, which reflects the maturational increase from the low infantile terminal storage to the high prepubertal one.

Prenatal Exposure to Vinclozolin Disrupts Selective Aspects of the Gonadotrophin‐Releasing Hormone Neuronal System of the Rabbit

Developmental exposure to the agricultural fungicide vinclozolin can impair reproductive function in male rabbits and was previously found to decrease the number of immunoreactive-gonadotrophin-releasing hormone (GnRH) neurones in the region of the organum vasculosum of the lamina terminalis and rostral preoptic area by postnatal week (PNW) 6. In the present study, in an aim to further examine the disruption of GnRH neurones by foetal vinclozolin exposure, pregnant rabbits were dosed orally with vinclozolin, flutamide or carrot paste vehicle for the last 2 weeks of gestation. Offspring were euthanised at birth (males and females), PNW 6 (females), PNW 26 (adult males) or PNW 30 (adult females) of age. At birth and in adults, brains were sectioned and processed for immunoreactive GnRH. The numbers of immunoreactive GnRH neuronal perikarya were significantly decreased in vinclozolin-treated rabbits at birth and in adult littermates. By contrast, there was an increase in GnRH immunoreactivity in the terminals in the region of the median eminence. Analysis of PNW 6 female brains by radioimmunoassay revealed a two-fold increase in GnRH peptide content in the mediobasal hypothalamus in vinclozolin-treated rabbits. This finding was complemented by immunofluorescence analyses, which revealed a 2.8-fold increase in GnRH immunoreactivity in the median eminence of vinclozolin compared to vehicle-treated females at PNW 30. However, there was no difference between treatment groups in the measures of reproduction that were evaluated: ejaculation latency, conception rates or litter size. These results indicate that sub-acute, prenatal vinclozolin treatment is sufficient to create perdurable alterations in the GnRH neuronal network that forms an important input into the reproductive axis. Finally, the effect of vinclozolin on the GnRH neuronal network was not comparable to that of flutamide, suggesting that vinclozolin was not acting through anti-androgenic mechanisms.