Inhibits Gonadotropin Release Research Articles

LPXRFa and LPXRFa-R in the female swamp eel (Monopterus albus): Molecular cloning, tissue distribution and gonadal function

Gonadotropin-inhibitory hormone (GnIH) is a hypothalamic neuropeptide known for inhibiting gonadotropin release. In teleosts, it is also referred to as LPXRFa. While the effects of LPXRFa on gonadotropin synthesis and release, and its impact on pituitary function in teleosts have been widely studied, its specific mechanism in fish reproduction remains relatively unexplored. In this paper, LPXRFa and LPXRFa-R genes were isolated from the swamp eel brain to assess the roles of LPXRFa in the swamp eel. Their ORFs were 612 bp and 1284 bp, encoding proteins of 203 and 427 amino acids, respectively. The LPXRFa gene encodes a polypeptide possibly cleaved into three mature peptides (LPXRFa-1, −2, and −3). The LPXRFa-R receptor protein comprises seven conserved transmembrane domains. Tissue distribution analysis revealed that LPXRFa-R expression was the highest in the hypothalamus, while LPXRFa showed the highest expression in the rest of brain, with little to no expression in other tissues. The role of LPXRFa in the reproduction of the swamp eel was investigated by studying the effect of intraperitoneal injection with each of the three peptides. The intraperitoneal injection of LPXRFa-2 down-regulated serum FSH and LH levels, while that of LPXRFa-3 down-regulated serum FSH but up-regulated LH levels. Additionally, both LPXRFa-2 and LPXRFa-3 increased the expression levels of fshr, cbx3, dmrt1, gsdf, sox9al, amh, foxl2, and rab37 in the ovaries. Moreover, LPXRFa-2 also increased the ovarian expression of lhr, while LPXRFa-3 had no effect on it. Except upregulating cbx3 and foxl2 expression, LPXRFa-1 neither impacted the serum FSH and LH levels, nor did it alter the expression of other gonadal and reproductive-related genes. In summary, our findings provide the first evidence of a LPXRFa/LPXRFa-R system in the order Synbranchiformes and suggest its involvement in sex differentiation, gonadal development, and sexual reversal in the swamp eel.

RFRP3 influences basal lamina degradation, cellular death, and progesterone secretion in cultured preantral ovarian follicles from the domestic cat.

The hypothalamic neuropeptide RFRP3 can suppress hypothalamic GnRH neuron activation and inhibit gonadotropin release from the anterior pituitary. RFRP3 is also produced locally in the ovary and can inhibit steroidogenesis and follicle development in many vertebrates. However, almost nothing is known about the presence and regulatory action of RFRP3 in gonads of any carnivore species. Such knowledge is important for developing captive breeding programs for endangered carnivores and for inhibiting reproduction in feral species. Using the domestic cat as a model, our objectives were to (1) demonstrate the expression of feline RFRP3 (fRFRP3) and its receptor in the cat ovary and (2) assess the influence of fRFRP3 on ovarian follicle integrity, survival, and steroidogenesis in vitro. We first confirmed that fRFRP3 and its receptors (NPFFR1 and NPFFR2) were expressed in cat ovaries by sequencing PCR products from ovarian RNA. We then isolated and cultured preantral ovarian follicles in the presence of 10 or 1 µM fRFRP3 + FSH (1 µg/mL). We recorded the percentage of morphologically viable follicles (basal lamina integrity) over 8 days and calculated percentage survival of follicles on Day 8 (using fluorescent markers for cell survival and death). Last, we quantified progesterone accumulation in media. 10 µM fRFRP3 had no observable effect on viability, survival, or steroid production compared to follicles exposed to only FSH. However, 1 µM fRFRP3 decreased the percentage of morphologically viable follicles and the percentage of surviving follicles on Day 8. At the same time, 1 µM fRFRP3 increased the accumulation of progesterone in media. Our study shows, for the first time, direct action of RFRP3 on the follicle as a functional unit, and it is the first in a carnivore species. More broadly, our results support a conserved, inhibitory action of RFRP3 on ovarian follicle development and underscore the importance of comparative functional studies.

Direct Effects of Melatonin and Serotonin on the Biosynthesis of Testosterone in Rat Leydig Cells

Previous studies indicate that melatonin (MLT) has anti-gonadal effect by inhibiting gonadotropin release in vertebrates. Serotonin is the precursor of MLT biochemically derived from tryptophan. Serotonin could regulate neuron activity and improve sleeping quality, making people calm, and happy. Both MLT and serotonin might inhibit testosterone (T) release. Whereas the action mechanisms of MLT and serotonin on T production by Leydig cells are still unclear. Our aim was to investigate the direct effects and action mechanisms of MLT and serotonin on T release. Leydig cells were prepared from adult male rats by continuous percoll gradient method. MLT at concentrations of 10^(-8)-10^(-6) M inhibited basal release of T after pre-incubation of MLT for 1 h. Besides, after pre-incubation with MLT (10^(-8)-10^(-6) M) for 1 h, the human chorionic gonadotropin (hCG)-evoked T release was significantly inhibited by the co-incubation of MLT (10^(-8)-10^(-6) M) in vitro. Although serotonin had similar effects, in contrast, tryptophan had no effect on the steroidogenesis in rat Leydig cells. Furthermore, MLT at 10^(-7) M and 10^(-6) M inhibited forskolin- and 8-Br-cAMP-evoked release of T in vitro by rat Leydig cells. MLT at 10^(-8) M also significantly inhibited T release in response to androstenedione. Serotonin had weak effects on T release in response to forskolin and 8-Br-cAMP androstenedione in vitro as compared with MLT. These results suggested that both MLT and serotonin inhibited T release in rat Leydig cells via cAMP-associated pathway and the reduced activity of 17-ketoreductase.

Molecular Mechanisms of Gonadotropin-Inhibitory Hormone (GnIH) Actions in Target Cells and Regulation of GnIH Expression.

Since gonadotropin-inhibitory hormone (GnIH) was discovered in 2000 as the first hypothalamic neuropeptide that actively inhibits gonadotropin release, researches conducted for the last 18 years have demonstrated that GnIH acts as a pronounced negative regulator of reproduction. Inhibitory effect of GnIH on reproduction is mainly accomplished at hypothalamic-pituitary levels; gonadotropin-releasing hormone (GnRH) neurons and gonadotropes are major targets of GnIH action based on the morphological interaction with GnIH neuronal fibers and the distribution of GnIH receptor. Here, we review molecular studies mainly focusing on the signal transduction pathway of GnIH in target cells, GnRH neurons, and gonadotropes. The use of well-defined cellular model systems allows the mechanistic study of signaling pathway occurring in target cells by demonstrating the direct cause-and-effect relationship. The insights gained through studying molecular mechanism of GnIH action contribute to deeper understanding of the mechanism of how GnIH communicates with other neuronal signaling systems to control our reproductive function. Reproductive axis closely interacts with other endocrine systems, thus GnIH expression levels would be changed by adrenal and thyroid status. We also briefly review molecular studies investigating the regulatory mechanisms of GnIH expression to understand the role of GnIH as a mediator between adrenal, thyroid and gonadal axes.

How to Contribute to the Progress of Neuroendocrinology: Discovery of GnIH and Progress of GnIH Research.

It is essential to discover novel neuropeptides that regulate the functions of pituitary, brain and peripheral secretory glands for the progress of neuroendocrinology. Gonadotropin-releasing hormone (GnRH), a hypothalamic neuropeptide stimulating gonadotropin release was isolated and its structure was determined by Schally's and Guillemin's groups at the beginning of the 1970s. It was subsequently shown that GnRH is highly conserved among vertebrates. GnRH was assumed the sole hypothalamic neuropeptide that regulates gonadotropin release in vertebrates based on extensive studies of GnRH over the following three decades. However, in 2000, Tsutsui's group isolated and determined the structure of a novel hypothalamic neuropeptide, which inhibits gonadotropin release, in quail, an avian species, and named it gonadotropin-inhibitory hormone (GnIH). Following studies by Tsutsui's group demonstrated that GnIH is highly conserved among vertebrates, from humans to agnathans, and acts as a key neuropeptide inhibiting reproduction. Intensive research on GnIH demonstrated that GnIH inhibits gonadotropin synthesis and release by acting on gonadotropes and GnRH neurons via GPR147 in birds and mammals. Fish GnIH also regulates gonadotropin release according to its reproductive condition, indicating the conserved role of GnIH in the regulation of the hypothalamic-pituitary-gonadal (HPG) axis in vertebrates. Therefore, we can now say that GnRH is not the only hypothalamic neuropeptide controlling vertebrate reproduction. In addition, recent studies by Tsutsui's group demonstrated that GnIH acts in the brain to regulate behaviors, including reproductive behavior. The 18 years of GnIH research with leading laboratories in the world have significantly advanced our knowledge of the neuroendocrine control mechanism of reproductive physiology and behavior as well as interactions of the HPG, hypothalamic-pituitary-adrenal and hypothalamic-pituitary-thyroid axes. This review describes how GnIH was discovered and GnIH research progressed in this new research era of reproductive neuroendocrinology.

Distribution of gonadotropin-inhibitory hormone (GnIH) in male Luchuan piglets

Gonadotropin inhibitory hormone (GnIH) has emerged as a novel hypothalamic neuropeptide that actively inhibits gonadotropin release in birds and mammals. Recent evidence indicates that GnIH not only acts as a key neurohormone that controls vertebrate reproduction but is also involved in stress response, food intake, and aggressive and sexual behaviors, suggesting a broad physiological role for this neuropeptide. To elucidate its multiple sites of action and potential functions, studying the detailed distribution of GnIH in different organs, except for the hypothalamus-pituitary-ovary/testis axis, is necessary. Therefore, in the present study, in different central nervous system (CNS) and peripheral organs of male Luchuan piglets, the distribution of GnIH was systemically determined using immunohistochemistry, and the expression of GnIH mRNA was investigated using semi-quantitative reverse transcription polymerase chain reaction (RT-PCR). Our results demonstrate that GnIH immune reactive (GnIH-ir) neurons were widely distributed in the pig CNS, but the number and size of the GnIH-ir neurons varied and exhibited morphological diversity. In the peripheral organs, GnIH immunoreactive cells were observed in the respiratory tract, alimentary tract, endocrine organs, genitourinary tract and lymphatic organs. GnIH mRNA was highly expressed in the CNS, with the highest expression in the hypothalamus. In the peripheral organs, high GnIH mRNA levels were detected in the testis, while no GnIH expression was observed in the liver, lungs and heart et al. These results demonstrated that GnIH might play an important role in modulating a variety of physiological functions and provided the morphological data for further study of GnIH in pigs.

Estrogen-induced inhibition of spermatogenesis in zebrafish is largely reversed by androgen.

The hormonal regulation of spermatogenesis involves both gonadotropins and steroid hormones. Long-term in vivo exposure of adult zebrafish to estrogen impaired spermatogenesis associated with an androgen insufficiency, possibly induced by inhibiting gonadotropin release. Using this experimental model, we investigated if androgen treatment could enhance spermatogenesis, while maintaining the inhibition of gonadotropin release through continued estrogen exposure. Moreover, we also exposed animals to androgen alone, in order to examine androgen effects in the absence of estrogen-induced gonadotropin inhibition. Estrogen exposure depleted type B spermatogonia, meiotic and postmeiotic germ cells from the adult testis, but promoted the proliferation of type A undifferentiated spermatogonia, which accumulated in the testis. This change in germ cell composition was accompanied by reduced mRNA levels of those growth factors (e.g. insl3 and igf3) expressed by testicular somatic cells and known to stimulate spermatogonial differentiation in zebrafish. Additional androgen (11-ketoandrostenedione, which is converted to 11-ketotestosterone) treatment in vivo reversed most of the effects of estrogen exposure on spermatogenesis while insl3 and igf3 transcript levels remained suppressed. When androgen treatment was given alone, it promoted the production of haploid cells at the expense of spermatogonia, and increased transcript levels of some growth factor and hormone receptor genes, but not those of insl3 or igf3 We conclude that estrogen exposure efficiently inhibits spermatogenesis because it induces androgen insufficiency and suppresses gonadotropin-regulated growth factors known to stimulate germ cell differentiation. Moreover, our results suggest that androgens and the growth factors Insl3 and Igf3 stimulate spermatogenesis via independent pathways.

Developmental changes and day-night expression of the gonadotropin-inhibitory hormone system in the European sea bass: Effects of rearing temperature

The role of rearing temperature on fish development, sex differentiation and puberty has been largely addressed, but the impact of water temperature on the ontogeny of the main neuroendocrine systems controlling reproduction has received little attention. Gonadotropin-inhibitory hormone (GnIH) has been shown to act on gonadotropin-releasing hormone (GnRH) neurons and on the pituitary to inhibit gonadotropin release and synthesis in vertebrates, including sea bass, Dicentrarchus labrax. In the present study we investigated the effects of rearing temperature during the thermosensitive period (5–60days post-fertilization, dpf) on the expression of the GnIH gene (gnih) and its receptor (gnihr). Animals were maintained under two different conditions, low temperature (LT, 15°C) or high temperature (HT, 21°C), throughout the thermosensitive period and sampled from 5 to 360dpf at mid-light (ML) and mid-dark (MD). Our results showed significant effects of temperature on gnih and gnihr expression during the thermosensitive period, with higher transcript levels under LT condition. Some differences were also evident after the completion of the sex differentiation process. Moreover, we revealed daily variations in the developmental expression of gnih and gnihr, with higher diurnal mRNA levels at early stages (until 25dpf), and a shift to higher nocturnal expression levels at 300–360dpf, which corresponded with the beginning of the winter (reproductive season). To the best of our knowledge, this work represents the first study reporting the effects of rearing temperature on the transcription of gnih system genes, as well as its daily variations during the development of a fish species.

Kisspeptin expression is decreased in the arcuate nucleus of hypothyroid female rats with irregular estrus cycles

Insufficiency of thyroid hormones inhibits gonadotropin release and results in dysregulation of reproductive function, although the precise mechanism of this disrupted gonadotropin secretion remains unclear. Kisspeptin is a neuropeptide that strongly stimulates gonadotropin secretion and plays an important role in reproductive function. To examine the involvement of kisspeptin in the dysregulation of gonadotropin secretion in hypothyroidism, we investigated Kiss1 mRNA expression and kisspeptin immunoreactivity in the hypothalamus of female rats treated with propylthiouracil (PTU). In the PTU-treated rats, serum thyroxine (T4) was significantly decreased, whereas thyroid stimulating hormone (TSH) levels were significantly increased. In addition, irregular estrus cycles were observed in PTU-treated rats. In situ hybridization and immunohistochemistry revealed significant reductions in the number of Kiss1 mRNA-expressing neurons and kisspeptin-immunoreactive neurons in the arcuate nucleus (ARC) but not in the anteroventral periventricular nucleus (AVPV) of the PTU-treated rats. Although the serum levels of luteinizing hormone (LH) and estradiol (E2) were unaffected, serum prolactin levels were significantly increased after PTU treatment. These data indicate that kisspeptin expression in the ARC is suppressed under thyroid hormone insufficiency, suggesting that the dysregulation of reproductive function in hypothyroidism is caused by inhibition of kisspeptin neurons in the ARC.

Reproductive Neuroendocrine Pathways of Social Behavior.

Social behaviors are key components of reproduction, because they are essential for successful fertilization. Social behaviors, such as courtship, mating, and aggression, are strongly associated with sex steroids, such as testosterone, estradiol, and progesterone. Secretion of sex steroids from the gonads is regulated by the hypothalamus–pituitary–gonadal (HPG) axis in vertebrates. Gonadotropin-releasing hormone (GnRH) is a pivotal hypothalamic neuropeptide that stimulates gonadotropin release from the pituitary. In recent years, the role of neuropeptides containing the C-terminal Arg–Phe–NH2 (RFamide peptides) has been emphasized in vertebrate reproduction. In particular, two key RFamide peptides, kisspeptin and gonadotropin-inhibitory hormone (GnIH), emerged as critical accelerator and suppressor of gonadotropin secretion. Kisspeptin stimulates GnRH release by directly acting on GnRH neurons, whereas GnIH inhibits gonadotropin release by inhibiting kisspeptin, GnRH neurons, or pituitary gonadotropes. These neuropeptides can regulate social behavior by regulating the HPG axis. However, distribution of neuronal fibers of GnRH, kisspeptin, and GnIH neurons is not limited within the hypothalamus, and the existence of extrahypothalamic neuronal fibers suggests direct control of social behavior within the brain. It has traditionally been shown that central administration of GnRH can stimulate female sexual behavior in rats. Recently, it was shown that Kiss1, one of the paralogs of kisspeptin peptide family, regulates fear responses in zebrafish and GnIH inhibits sociosexual behavior in birds. Here, we highlight recent findings regarding the role of GnRH, kisspeptin, and GnIH in the regulation of social behaviors in fish, birds, and mammals and discuss their importance in future biological and biomedical research.

Contribution of GnIH Research to the Progress of Reproductive Neuroendocrinology.

Since the discovery of gonadotropin-releasing hormone (GnRH) in mammals at the beginning of the 1970s, it was generally accepted that GnRH is the only hypothalamic neuropeptide regulating gonadotropin release in mammals and other vertebrates. In 2000, however, gonadotropin-inhibitory hormone (GnIH), a novel hypothalamic neuropeptide that actively inhibits gonadotropin release, was discovered in quail. Numerous studies over the past decade and a half have demonstrated that GnIH serves as a key player regulating reproduction across vertebrates, acting on the brain and pituitary to modulate reproductive physiology and behavior. In the latter case, recent evidence indicates that GnIH can regulate reproductive behavior through changes in neurosteroid, such as neuroestrogen, biosynthesis in the brain. This review summarizes the discovery of GnIH, and the contributions to GnIH research focused on its mode of action, regulation of biosynthesis, and how these findings advance our understanding of reproductive neuroendocrinology.

How to contribute to the progress of neuroendocrinology: New insights from discovering novel neuropeptides and neurosteroids regulating pituitary and brain functions

Obtaining new insights by discovering novel neuropeptides and neurosteroids regulating pituitary and brain functions is essential for the progress of neuroendocrinology. At the beginning of 1970s, gonadotropin-releasing hormone (GnRH) was discovered in mammals. Since then, it was generally accepted that GnRH is the only hypothalamic neuropeptide regulating gonadotropin release in vertebrates. In 2000, however, gonadotropin-inhibitory hormone (GnIH), a novel hypothalamic neuropeptide that actively inhibits gonadotropin release, was discovered in quail. The follow-up studies demonstrated that GnIH acts as a new key player for regulation of reproduction across vertebrates. It now appears that GnIH acts on the pituitary and the brain to serve a number of behavioral and physiological functions. On the other hand, a new concept has been established that the brain synthesizes steroids, called neurosteroids. The formation of neurosteroids in the brain was originally demonstrated in mammals and subsequently in other vertebrates. Recently, 7α-hydroxypregnenolone was discovered as a novel bioactive neurosteroid inducing locomotor behavior of vertebrates, indicating that neurosteroidogenesis in the brain is still incompletely elucidated in vertebrates. At the beginning of 2010s, it was further found that the pineal gland actively produces neurosteroids. Pineal neurosteroids act on the brain to regulate locomotor rhythms and neuronal survival. Furthermore, the interaction of neuropeptides and neurosteroids is becoming clear. GnIH decreases aggressive behavior by regulating neuroestrogen synthesis in the brain. This review summarizes these new insights by discovering novel neuropeptides and neurosteroids in the field of neuroendocrinology.

Identification and localization of gonadotropin-inhibitory hormone (GnIH) orthologs in the hypothalamus of the red-eared slider turtle, Trachemys scripta elegans

Gonadotropin-inhibitory hormone (GnIH) was discovered in 2000 as a novel hypothalamic neuropeptide that inhibited gonadotropin release in the Japanese quail. GnIH and its orthologs have a common C-terminal LPXRFamide (X=L or Q) motif, and have been identified in vertebrates from agnathans to humans, apart from reptiles. In the present study, we characterized a cDNA encoding GnIH orthologs in the brain of the red-eared slider turtle. The deduced precursor protein consisted of 205 amino-acid residues, encoding three putative peptide sequences that included the LPXRFamide motif at their C-termini. In addition, the precursor sequence was most similar to those of avian species. Immunoaffinity purification combined with mass spectrometry confirmed that three mature peptides were produced in the brain. In situ hybridization and immunohistochemistry showed that turtle GnIH-containing cells were restricted to the periventricular hypothalamic nucleus. Immunoreactive fibers were densely distributed in the median eminence. Thus, GnIH and related peptides may act on the pituitary to regulate pituitary hormone release in turtles as well as other vertebrates.

Structural and functional divergence of gonadotropin-inhibitory hormone from jawless fish to mammals.

Gonadotropin-inhibitory hormone (GnIH) was discovered as a novel hypothalamic peptide that inhibits gonadotropin release in the quail. The presence of GnIH-homologous peptides and its receptors (GnIHRs) have been demonstrated in various vertebrate species including teleosts, suggesting that the GnIH-GnIHR family is evolutionarily conserved. In avian and mammalian brain, GnIH neurons are localized in the hypothalamic nuclei and their neural projections are widely distributed. GnIH acts on the pituitary and gonadotropin-releasing hormone neurons to inhibit reproductive functions by decreasing gonadotropin release and synthesis. In addition, GnIH-GnIHR signaling is regulated by various factors, such as environmental cues and stress. However, the function of fish GnIH orthologs remains inconclusive because the physiological properties of fish GnIH peptides are debatable. This review summarizes the current research progress in GnIH-GnIHR signaling and their physiological functions in vertebrates with special emphasis on non-mammalian vertebrate species.

Review: evolution of GnIH and related peptides structure and function in the chordates.

Discovery of gonadotropin-inhibitory hormone (GnIH) in the Japanese quail in 2000 was the first to demonstrate the existence of a hypothalamic neuropeptide inhibiting gonadotropin release. We now know that GnIH regulates reproduction by inhibiting gonadotropin synthesis and release via action on the gonadotropin-releasing hormone (GnRH) system and the gonadotrope in various vertebrates. GnIH peptides identified in birds and mammals have a common LPXRF-amide (X = L or Q) motif at the C-terminus and inhibit pituitary gonadotropin secretion. However, the function and structure of GnIH peptides are diverse in fish. Goldfish GnIHs possessing a C-terminal LPXRF-amide motif have both stimulatory and inhibitory effects on gonadotropin synthesis or release. The C-terminal sequence of grass puffer and medaka GnIHs are MPQRF-amide. To investigate the evolutionary origin of GnIH and its ancestral structure and function, we searched for GnIH in agnathans, the most ancient lineage of vertebrates. We identified GnIH precursor gene and mature GnIH peptides with C-terminal QPQRF-amide or RPQRF-amide from the brain of sea lamprey. Lamprey GnIH fibers were in close proximity to GnRH-III neurons. Further, one of lamprey GnIHs stimulated the expression of lamprey GnRH-III peptide in the hypothalamus and gonadotropic hormone β mRNA expression in the pituitary. We further identified the ancestral form of GnIH, which had a C-terminal RPQRF-amide, and its receptors in amphioxus, the most basal chordate species. The amphioxus GnIH inhibited cAMP signaling in vitro. In sum, the original forms of GnIH may date back to the time of the emergence of early chordates. GnIH peptides may have had various C-terminal structures slightly different from LPXRF-amide in basal chordates, which had stimulatory and/or inhibitory functions on reproduction. The C-terminal LPXRF-amide structure and its inhibitory function on reproduction may be selected in later-evolved vertebrates, such as birds and mammals.

Effect of RFRP-3 on reproduction is sex- and developmental status-dependent in the striped hamster (Cricetulus barabensis)

RFamide-related peptides (RFRPs) are orthologous to gonadotropin-inhibitory hormone (GnIH) inhibiting gonadotropin release. There are only two RFRP sequences (RFRP-1 and RFRP-3) encoded in rodents. RFRP-3, which was considered as a hypothetical inhibitor on GnRH, shows a stimulatory effect on the male Syrian and male Siberian hamster in short days. As a dominant rodent pest in northern China farmland, the striped hamster (Cricetulus barabensis) has higher reproductive activities and could act as a model to study the mechanism of reproduction. However, the effect of RFRP-3 on the reproductive activity for the striped hamster is less understood. In the study, we cloned 643 bp RFRP cDNA from the striped hamster hypothalamus, which contained an ORF of 570 bp encoding two RFamide-related peptide (RFRP) sequences: SPAPANKVPHSAANLPLRF-NH2 (C. barabensis RFRP-1) and TLSRVPSLPQRF-NH2 (C. barabensis RFRP-3). We also investigated the expression variation of RFRP mRNA and GnRH mRNA in the hypothalamus from hamsters with different developmental statuses (7-week-, 13-week- and 1.5-year-olds) using FQ-PCR, in which the 13-week-old female individuals were in estrous. The striped hamsters that are 7 weeks and 1.5 years old are non-breeding individuals, and those that are 13-week hamsters have breeding phenomena. The highest hypothalamus RFRP mRNA level was found in breeding males as compared to non-breeding males. Conversely, the lowest RFRP mRNA level in the hypothalamus was observed in breeding females, with no significant level when the breeding females were compared to the 7-week-old individuals. Additionally, the investigation of GnRH expression level showed a declining expression trend across the developmental stages (7-week-, 13-week- and 1.5-year-olds) in both sexes. Significant negative and positive relationships were detected in the 13-week estrous female (r=-0.997, P=0.035) and the 13-week male (r=0.998, P=0.029) striped hamsters respectively, which suggest that RFRP-3 has inhibitory and stimulatory effects on female and male adults respectively. Our results suggest that the effects of RFRP-3 on reproduction are sex- and developmental status-dependent in the striped hamster.

Leptin-responsive GABAergic neurons regulate fertility through pathways that result in reduced kisspeptinergic tone.

The adipocyte-derived hormone leptin plays a critical role in the central transmission of energy balance to modulate reproductive function. However, the neurocircuitry underlying this interaction remains elusive, in part due to incomplete knowledge of first-order leptin-responsive neurons. To address this gap, we explored the contribution of predominantly inhibitory (GABAergic) neurons versus excitatory (glutamatergic) neurons in the female mouse by selective ablation of the leptin receptor in each neuronal population: Vgat-Cre;Lepr(lox/lox) and Vglut2-Cre;Lepr(lox/lox) mice, respectively. Female Vgat-Cre;Lepr(lox/lox) but not Vglut2-Cre;Lepr(lox/lox) mice were obese. Vgat-Cre;Lepr(lox/lox) mice had delayed or absent vaginal opening, persistent diestrus, and atrophic reproductive tracts with absent corpora lutea. In contrast, Vglut2-Cre;Lepr(lox/lox) females exhibited reproductive maturation and function comparable to Lepr(lox/lox) control mice. Intracerebroventricular administration of kisspeptin-10 to Vgat-Cre;Lepr(lox/lox) female mice elicited robust gonadotropin responses, suggesting normal gonadotropin-releasing hormone neuronal and gonadotrope function. However, adult ovariectomized Vgat-Cre;Lepr(lox/lox) mice displayed significantly reduced levels of Kiss1 (but not Tac2) mRNA in the arcuate nucleus, and a reduced compensatory luteinizing hormone increase compared with control animals. Estradiol replacement after ovariectomy inhibited gonadotropin release to a similar extent in both groups. These animals also exhibited a compromised positive feedback response to sex steroids, as shown by significantly lower Kiss1 mRNA levels in the AVPV, compared with Lepr(lox/lox) mice. We conclude that leptin-responsive GABAergic neurons, but not glutamatergic neurons, act as metabolic sensors to regulate fertility, at least in part through modulatory effects on kisspeptin neurons.

Expression of gonadotropin-inhibitory hormone receptors in mouse pituitary gonadotroph LβT2 cells and hypothalamic gonadotropin-releasing hormone-producing GT1-7 cells

Gonadotropin-inhibitory hormone (GnIH) was first identified in quail as a novel neurohormone that acts directly on the anterior pituitary to inhibit gonadotropin release. GnIH inhibits not only gonadotropin release from the pituitary gland but also inhibits the release of gonadotropin-releasing hormone (GnRH) from the hypothalamus. In this study, we examined how GnIH receptors were regulated in pituitary gonadotroph cells and GnRH-producing neurons in the hypothalamus. In the mouse pituitary gonadotroph cell line LβT2, GnRH increased expression of the GnIH receptor, G-protein coupled receptor 74 (GPR74). GnRH also stimulated the expression of GPR74 and GPR147 in primary cultures of rat anterior pituitary cells. In addition, when GnRH was administered to LβT2 cells in a pulsatile manner, low frequency GnRH pulse stimulation stimulated GPR74 and GPR147 expression more than did high frequency GnRH pulses. In the mouse hypothalamic GnRH-producing cell line GT1-7, hypothalamic kisspeptin did not significantly increase the expression of GnIH receptors. However, the intermittent administration of kisspeptin to GT1-7 cells significantly increased GPR74 and GPR147 mRNA expression. The overexpression of either constitutively active MEK kinase (MEKK) or protein kinase A (PKA) in LβT2 cells increased the expression of GPR74 mRNA. Conversely, in GT1-7 cells, although the overexpression of either MEKK or PKA failed to stimulate GnIH receptor expression, the combined overexpression of both kinases together increased GPR74 and GPR147 mRNA levels. Our current observations suggest that two central controllers of reproductive function, GnRH and kisspeptin, stimulate the expression of GnIH receptors in pituitary gonadotroph cells and hypothalamic GnRH neurons.

Possible roles of photoperiod and melatonin in reproductive activity via changes in dopaminergic activity in the brain of a tropical damselfish, Chrysiptera cyanea

The perception of periodical change in photoperiodic conditions by photosensory organs is the first step to initiating reproductive activity in fish. To date, it is not known how photoperiodic cues are endogenously transduced to the endocrine network of the brain–pituitary–gonad axis after the perception of photoperiodic stimuli. Because dopamine (DA) inhibits gonadotropin release from the pituitary in certain teleosts, it is likely that it is a key mediator of photoperiodic stimulation. We examined the relationship between photoperiodicity and dopaminergic activity in the brain of the sapphire devil Chrysiptera cyanea, which is a reef-associated damselfish and uses long-day conditions for triggering gonadal development. DA and 3,4-dihydroxyphenylacetic acid (DOPAC; DA metabolite) were measured electrochemically with a high-performance liquid chromatography (HPLC) system, and then DOPAC/DA (metabolic rate of DA activity) was determined. Daily fluctuations in DA, DOPAC, and DOPAC/DA were observed under LD 12:12. Light–dark cycles, but not constant conditions, influenced DA and DOPAC contents as well as DOPAC/DA in the brain; DOPAC content and DOPAC/DA increased under LD 14:10, while DA increased under LD 10:14. When fish were reared in melatonin-containing water, DOPAC and DOPAC/DA, but not DA, decreased within 6h after treatment. Culturing the isolated brain with melatonin stimulated DA release into the medium. Active vitellogenesis in females during the reproductive season was suppressed by melatonin treatment. These results suggest that day length alters DA metabolism in the brain of the sapphire devil through fluctuations in melatonin caused by external light stimuli, and that inhibitory effects on gonadal development are partially controlled by interactions within the melatonin–DA system.

Gonadotropin-inhibitory hormone (GnIH) and its receptor in the female pig: cDNA cloning, expression in tissues and expression pattern in the reproductive axis during the estrous cycle

Since its discovery, gonadotropin-inhibitory hormone (GnIH) has appeared to act as a key neuropeptide in the control of vertebrate reproduction. GnIH acts via the novel G protein-coupled receptor 147 (GPR147) to inhibit gonadotropin release and synthesis. To determine the physiological functions of GnIH in the pig, a study was conducted to clone and sequence the cDNA of the GnIH precursor and GPR147. Our results demonstrated that the cloned pig GnIH precursor cDNA encoded three LPXRF and that its receptor possessed typical transmembrane features. Subsequently, tissue expression studies revealed that GnIH was mainly expressed in the brain, corresponding largely with the tissue expression patterns of GPR147 in the pig. The expression patterns in the reproductive axis of the female pig across the estrous cycle were also systemically investigated. The hypothalamic levels of both GnIH and its receptor mRNA were lowest in estrus and peaked in the proestrus and diestrus phases. The highest pituitary GnIH mRNA level was detected in the metestrus, and its receptor displayed a somewhat similar pattern of expression to that of the ligand. However, the expression patterns of GnIH and GPR147 were negatively correlated in the ovary. Immunolocalization in the ovary during the estrous cycle revealed that the immunoreactivities of GnIH and GPR147 were mainly localized in the granulosa and theca cells of the antral follicles during proestrus and estrus and in the luteal cells during metestrus and diestrus. Taken together, this research provided molecular and morphological data for further study of GnIH in the pig.