GnRH gene products downregulate their neighboring genes encoding protein tyrosine phosphatases

The decapeptide gonadotropin-releasing hormone (GnRH) has multiple functions, including the regulation of gonadotropin secretion, reproductive behavior, and cell proliferation. In this paper, we have found that in the medaka (Oryzias latipes) genome, gonadotropin-releasing hormone (GnRH) genes are adjacent to type IV protein tyrosine phosphatase (PTP) genes encoding PTPα and PTPε, which are known to regulate cellular activity via interacting with voltage-gated potassium channel. We have subsequently demonstrated using an in vitro medaka whole-brain culture system that GnRH downregulates the PTPα and PTPε gene expression. Inhibition of intracellular type IV PTP signaling, which probably results in the modulation of cellular activity, may account for multiplicity of GnRH function.     

GnRH systems in masu salmon and barfin flounder

Multiple forms of the gonadotropin-releasing hormone (GnRH) exist in teleost fish. A salmonid fish, masu salmon Oncorhynchus masou has salmon GnRH (sGnRH) and chicken GnRH-II (cGnRH-II). sGnRH neurons were scattered from the olfactory nerve through the ventral telencephalon (VT) and the preoptic area (POA). sGnRH but not cGnRH-II was detected in the pituitary. sGnRH mRNA levels in the VT and the POA increased during gonadal maturation, suggesting that sGnRH neurons in these areas are involved in gonadal maturation. sGnRH neurons were first detected in a cluster near the olfactory epithelium 40 days after fertilization. sGnRH neurons were not detected in the brain by the olfactory epithelia lesion, suggesting that sGnRH neurons are derived from the olfactory epithelium. A pleuronectiform fish, barfin flounder Verasper moseri has sGnRH, cGnRH-II and seabream GnRH (sbGnRH). sGnRH and cGnRH-II-immunoreactive fibers were observed throughout the brain, but not in the pituitary. sbGnRH neurons were located in the POA and sent fibers to the pituitary, indicating that sbGnRH is involved in GTH secretion. Judging from the location of neuronal somata and their projections, it is indicated that three GnRH systems exist in the barfin flounder; the TN-, the MT- and the POA-GnRH system. However, in masu salmon, clear anatomical identification of the TN- and the POA-GnRH system is difficult, because the GnRH neurons located in the ventral forebrain are consecutive and the GnRH form produced in these neurons is the same (sGnRH). Thus, it is suggested in masu salmon that sGnRH neurons are derived from the olfactory epithelium, migrate into the brain and play different roles according to the location in the brain.     

Involvement of Gonadotropin-Releasing Hormone in Thyroxine Release in Three different forms of Teleost Fish: Barfin Founder, Masu Salmon and Goldfish

Effects of gonadotropin-releasing hormone (GnRH) on thyroxine (T₄) release in vivo and in vitro were studied in barfin flounder Verasper moseri, masu salmon Oncorhynchus masou and goldfish Carassius auratus. Seabream GnRH (sbGnRH) at a dose of 200 ng/50 g body weight (BW) significantly increased plasma T₄ levels 1 h after the in vivo injection in the barfin flounder, but thereafter the levels normalized. Salmon GnRH (sGnRH) significantly increased plasma T₄ levels l h after the injection with a significant return to initial levels in male masu salmon and male goldfish. In contrast, sGnRH and cGnRH-II in barfin flounder, and cGnRH-II in male masu salmon and male goldfish were not effective in stimulating T₄ release. To clarify direct involvement of GnRH in T₄ release, dissected lower jaw including scattered thyroid follicles was incubated with sbGnRH (1 μg/well) in barfin flounder, and with two doses (0.1 and 1 μg/well) of sGnRH in masu salmon and goldfish in vitro. T₄ concentrations of control were stable during 24 h. Incubation of lower jaw with high dose (1 μg/well) of GnRH significantly (P<0.05) increased T₄ concentrations of incubation medium at 1 h in all experimental fishes. These results indicate that direct stimulation of T₄ secretion by GnRH occurs widely in teleost fish.     

GnRH isoforms expression in relation to the gonadal cycle and to dominance rank in the Gilthead seabream, Sparus aurata

The manner in which behavior influences the gonadotropin-releasing hormone (GnRH) axis in hermaphroditic fishes is not understood. The Gilthead seabream, Sparus aurata, is a protandrous hermaphrodite with a complex gonadal cycle consisting of a quiescent, pre-spawning, spawning, and post-spawning stage. On two separate experiments, I used real-time quantitative PCR to measure the mRNA expression of three GnRH isoforms in homogenized seabream whole-brain extracts. In the first experiment, I measured the levels of GnRH-1, GnRH-2, and GnRH-3 mRNA throughout the gonad cycle. All three GnRH mRNAs increase around the peak of the spawning season (December). GnRH-3 mRNA expression is also elevated in August, which coincides with the beginning of gonad differentiation. All three GnRH mRNAs have the lowest expression levels in the month of September. There was no difference between males and females in the expression levels of any of the three GnRH mRNA. In the second experiment, I measured individual dominance ranks in six groups of fish, three during quiescence and three during spawning. GnRH-1 mRNA expression was positively correlated with dominance rank only during the quiescent period. The more dominant fish tended to have higher GnRH-1 mRNA expression. The existence of a quiescent-only correlation between GnRH-1 mRNA and dominance rank suggests a mechanism by which activation of gonad maturation could occur first in the most dominant ambisexual fish.     

GnRH and gpcr: laser-captured single cell gene profiling

We have developed a novel single cell real-time quantitative PCR technique, which incorporates harvesting marker-identified single cells using laser-capture. Here, for the first time in a vertebrate species, using this innovative single cell gene profiling technique, we report the presence of G-protein coupled receptors in individual gonadotropin-releasing hormone (GnRH) neurons and endocrine cells of the pituitary of the tilapia Oreochromis niloticus. The differential expression of multiple combinations of three GnRH receptor types (R1, R2 and R3) in individual gonadotropic and nongonadotropic cells demonstrates cellular and functional heterogeneity. The differential use of GnRH receptors in corticotropes, melanotropes and thyrotropes during gonadal maturation and reproductive behaviors suggests new roles for these hormones. Further, we provide evidence of the structure of a novel nonmammalian G-protein coupled receptor (GPR54) for kisspeptins, encoded by Kiss-1 gene, which is highly conserved during evolution and expressed in GnRH1, GnRH2 and GnRH3 neurons. We hypothesize GPR54 stimulates GnRH secretion and is crucial for pubertal maturation. We speculate, the use of this method will allow the identification and quantification of known and unknown genes in single cells, which would greatly facilitate our understanding of the complex interactions that govern the physiology of individual cells in vertebrates species.     

Changes in GnRH levels in the brain and pituitary gland during migrations of sockeye salmon and chum salmon

Levels of two moleculer types of gonadotropin-releasing hormone (GnRH), salmon GnRH (sGnRH) and chicken GnRH–II (cGnRH–II) in the various brain regions and pituitary gland of sockeye salmon (Oncorhynchus nerka) and chum salmon (O. keta) during smoltification and spawning migration, respectively, were measured using specific time-resolved fluoroimmunoassay (TR-FIA) systems. Changes in sGnRH levels in different brain regions tended to be specifically synchronized with serum thyroid hormone or pituitary gonadotropin (GTH) levels during smoltification and spawning migration, respectively. In contrast, cGnRH–II levels did not show such synchronized changes. SGnRH and cGnRH–II in various brain regions might have different roles during smoltification and spawning migration of salmonid fishes.     

Molecular cloning and expression of caspase-3 in the protandrous cinnamon clownfish, Amphiprion melanopus, during sex change

The caspase-3 appears to be a key protease in the apoptotic pathway. We identified caspase-3 complementary DNAs from the ovaries of the protandrous cinnamon clownfish (Amphiprion melanopus), and investigated its mRNA and proteins, and activity levels during the sex change (I, mature male; II, male at 90 days after removing of the female; and III, mature female). The nucleotide sequence of the caspase-3 cDNA was 969 base pairs in length with open reading frames encoding peptides of 282 amino acids. The caspase-3 mRNA and protein, and activity levels in stages of the mature gonad are higher than those of the development gonad stage. To understand the effect of gonadotropin-releasing hormone (GnRH) on gonad apoptosis, we examined expression of genes caspase-3 mRNA and activity level in immature cinnamon clownfish gonads after GnRH analogue (GnRHa). The findings support the hypothesis that caspase-3 expression is associated with both testicular and ovarian development, and suggests that it may play a role in the control of ovarian development in cinnamon clownfish. Also, we demonstrate that GnRH agonists stimulate caspase-3 production which can in turn stimulate apoptosis. The present study provides a framework for better understanding of the role of caspase-3 during sex change processes in fish.     

Gonadotropin-releasing hormone (GnRH) neuronal systems in the pejerrey, Odontesthes bonariensis (Atheriniformes)

The brain of the pejerrey (Odontesthes bonariensis) has recently been shown to contain three forms of gonadotropin-releasing hormone (GnRH): salmon GnRH (sGnRH), chicken GnRH-II (cGnRH-II) and pejerrey GnRH (pjGnRH), nevertheless neuroanatomical studies on the distribution of these peptides are lacking. In this study we investigated the distribution of immunoreactive GnRH in the brain of adult pejerrey. Four different policlonal antisera and a monoclonal antibody against different GnRH variants were applied on cryosections and visualized using the ABC method. Three antisera (PBL#49, sGnRH#2 and cII741) revealed three different immunoreactive areas: the terminal nerve ganglion (at the junction between the olfactory bulbs and the anterior telencephalon), the preoptic area just anterior to the hypothalamus and the midbrain tegmentum. Fibers immunoreactive to GnRH were detected in different brain areas: the olfactory bulbs, the ventral thelencephalon, the hypothalamus, the mesencephalic area and an important innervation entering into the pituitary gland. Two other antibodies (LRH13 and s1668) labeled the two nuclei corresponding to the forebrain but not the midbrain tegmentum. As both antibodies have low crossreactivity to cGnRH-II, the data suggest that this group of cells express cGnRH-II. In summary, three different areas with immunoreactivity to GnRH were detected in the pejerrey brain. The distribution of sGnRH, pjGnRH and cGnRH-II expressing neurons, is discussed.     

The existence of gonadotropin-releasing hormone (GnRH) immunoreactivity in the ovary and the effects of GnRHs on the ovarian maturation in the black tiger shrimp Penaeus monodon

Immunocytochemical localization using antibodies against five isoforms of gonadotropin-releasing hormone (GnRH), namely, luteinizing hormone-releasing hormone (LHRH), salmon (s)GnRH, octopus (oct)GnRH, lamprey (l)GnRH-I, and lGnRH-III, showed that only lGnRH-I immunoreactivity (ir-lGnRH-I) was localized in follicular cells of proliferative, vitellogenic, and mature ovaries. The effects of exogenous GnRHs on the ovarian maturation cycle of Penaeus monodon were compared by treating female broodstocks with LHRH, sGnRH, and lGnRH-I. The cycle of ovarian maturation in both eyestalk-ablated and eyestalk-intact shrimp administered with the three isoforms of GnRH was significantly shorter than that of the control animals. Moreover, administrations of all GnRH isoforms showed similar numbers of spawned eggs and the percentage of successful fertilization as in the control animals. These findings suggest that GnRHs may be highly conserved peptides that play an important role in inducing the ovarian maturation in the shrimp.     

Effects of photoperiod on gonadotropin-releasing hormone levels in the brain and pituitary of underyearling male barfin flounder

ABSTRACT:   A pleuronectiform fish, the barfin flounder Verasper moseri , expresses three gonadotropin-releasing hormone (GnRH) forms in the brain: salmon GnRH (sGnRH), chicken GnRH-II (cGnRH-II) and seabream GnRH (sbGnRH). To clarify the effects of photoperiod on GnRH systems, changes in brain and pituitary GnRH peptide levels were examined using time-resolved fluoroimmunoassays. In experiment 1, 5-month-old male barfin flounder (mean total length 9.0 cm, body weight 11.0 g) were divided into short (8:16 h light : dark [L:D] cycle; lights on 08.00–16.00 hours) and long photoperiod (16:8 h L:D cycle; lights on 04.00–20.00 hours) groups in mid September and maintained until November under natural water temperature (19.3–15.2°C). Brain sGnRH concentrations were significantly higher in the 16:8 h L:D group than in the 8:16 h L:D group, whereas no significant differences were observed in total length, body weight, plasma testosterone concentration, brain cGnRH-II concentration and pituitary sbGnRH content. In experiment 2, 7-month-old male barfin flounder (mean total length 16.5 cm, body weight 76.8 g) were divided into short and long photoperiod groups in mid December and maintained until February under natural water temperature (12.5–6.6°C). Total length, body weight and condition factor were significantly greater in the 16:8 h L:D group than in the 8:16 h L:D group, whereas no significant differences were observed in plasma testosterone concentration and GnRH levels in the brain and pituitary. These results indicate that levels of sGnRH in barfin flounder are influenced by photoperiodic treatment dependent on water temperature and/or body size.     

The origin of the mammalian form of GnRH in primitive fishes

The presence of neuroendocrine hormones in extant agnathan fishes suggests that a method of control involving these hormones was operating 500–600 million years ago in emerging vertebrates. Data on a limited number of species show that several members of the GnRH family of peptides may have arisen in non-teleost fishes. Lamprey (Petromyzon marinus) GnRH has a unique composition and has not been detected in other vertebrates. It is not yet clear whether the chicken II GnRH-like molecule arose in cartilaginous fishes, but a chromatographically and immunologically similar molecule is found in dogfish (Squalus acanthias) and ratfish (Hydrolagus colliei). Finally, a mammalian GnRH-like molecule is detected in three primitive bony fish: sturgeon (Acipenser transmontanus), reed fish (Calamoichthys calabaricus), and alligator gar (Lepidosteus spatula). Minor forms are also present, but are not yet characterized. Clearly, the basic structure of GnRH peptides was established in primitive fish. In contrast, at least three other identified forms of GnRH have been detected in teleosts or tetrapods: Salmon I, catfish I, and chicken I GnRH. Evidence for the presence of members of the GnRH family and the neurohypophysial hormone family in primitive fishes argues for the importance of neuroendocrine control throughout the history of vertebrates.     

Gonadotropin-releasing hormone receptors: neuroendocrine regulators and neuromodulators

Several lines of evidence support the idea that more than one gonadotropin-releasing hormone receptor (GnRH-R) subtype exists to accommodate the presence of multiple GnRH forms seen in vertebrate species. The development of pituitary endocrine cells, GnRH-Rs type IA (GfA), IB (GfB) and type III in the pituitary and their synchronization with the establishment of GnRH1-3 regulatory pathways reflect a neuroendocrine function for the GnRH-Rs. Based on developmental, morphological and biochemical evidence it can be hypothesized that GnRH1 regulates gonadotrophs through GnRH-R type IA, GnRH2 regulates prolactin secretion through GnRH-R type IB and GnRH3 regulates growth hormone secretion through GnRH-R type III. Therefore, the three native GnRH variants in advance teleost like tilapia might have their respective cognate receptors, each being capable of regulating different and, to some degree the same pituitary endocrine cells. In the brain, the expression pattern of GnRH-R type IB and type III parallels the distribution of GnRH2 and GnRH3 fiber network, which suggests their role in neuromodulation and reproductive behavior.     

Immunohistochemical localization of three GnRH systems in brain and pituitary of Japanese flounder

ABSTRACT:   To clarify the possible roles of gonadotropin-releasing hormone (GnRH) in the reproduction of Japanese flounder Paralichthys olivaceus , localization of salmon GnRH (sGnRH), chicken GnRH-II (cGnRH-II), and sea bream GnRH (sbGnRH) immunoreactive (ir) cell bodies and fibers in the brain and pituitary were examined together with follicle stimulating hormone (FSH) and luteinizing hormone (LH)-ir cells in the pituitary by immunohistochemistry. sGnRH-ir cell bodies were localized in the ventromedial part of the rostral olfactory bulb and cGnRH-II-ir cell bodies were restricted to the midbrain tegmentum, while sbGnRH-ir cell bodies were evident in the preoptic area. sGnRH-ir fibers were distributed throughout the brain, especially abundant in the forebrain. cGnRH-II-ir fibers were also scattered in many areas of the brain with abundance in the midbrain, but sbGnRH-ir fibers were observed in the preoptic–hypothalamic area and innervated the pituitary. In the pituitary, neither sGnRH-ir fibers nor cGnRH-II-ir fibers were found, but sbGnRH-ir fibers were profuse in the neurohypophysis and invaded the proximal pars distalis, targeting FSH and LH cells. These results suggest that three GnRH systems can play different physiological roles in the brain of Japanese flounder. Among them, sbGnRH is considered to be involved in reproduction by stimulating gonadotropin secretion, while sGnRH and cGnRH-II can function as a neurotransmitter and/or neuromodulator within the brain in this species.     

Development of a time-resolved fluoroimmunoassay for octopus gonadotropin-releasing hormone

We have developed a time-resolved fluoroimmunoassay (TR-FIA) for octopus gonadotropin-releasing hormone (oct-GnRH) to determine the profiles of oct-GnRH peptide levels in cephalopods. The sensitivity and the intra-assay and inter-assay coefficients of variation were 4.9 pg/well and 6.8 (n = 10) and 2.7% (n = 5), respectively. Anti-oct-GnRH antibody was tested on all known forms of GnRH and found to cross-react with lamprey GnRH-II (27.1%), annelid GnRH (3.36%), tunicate GnRH-I (0.92%), dogfish GnRH (0.51%), and scallop Patinopecten yessoensis GnRH (0.05%). The displacement curve obtained for serially diluted brain extracts of three cephalopods, the spear squid Loligo bleekeri, swordtip squid Loligo edulis, and North Pacific giant octopus Octopus dofleini, paralleled the oct-GnRH standard curve. The presence of oct-GnRH in the central nervous system (CNS) of these cephalopods was further examined by a combination of reverse-phase high performance liquid chromatography and oct-GnRH TR-FIA. CNS extracts from these cephalopods showed peaks with retention times similar to that of synthetic oct-GnRH. These results indicate that this novel oct-GnRH TR-FIA is widely applicable for oct-GnRH measurement in cephalopods.     

尼罗罗非鱼雌雄鱼肌肉组织差异表达基因的筛选

应用引物退火控制技术(ACP)筛选尼罗罗非鱼雌雄鱼肌肉组织差异表达基因,寻找与雌雄鱼肌肉生长发育相关的候选基因。本实验从同等条件下养殖的尼罗罗非鱼群体中随机选取雌、雄鱼各5尾组成RNA池,采用引物退火控制技术,分析了两组个体肌肉组织差异表达基因。利用20对随机引物差异显示扩增,共获得8条ESTs,其中5个已知的ESTs分别为转录变体3(LOC100691543)、60S核糖体蛋白(RL3)、小白蛋白β样蛋白、肌型肌酸激酶M2-CK和转录因子Sox4,其余3个为未知的ESTs。实时定量PCR分析各差异表达基因在尼罗罗非鱼雌雄肌肉组织中的表达发现,8个差异表达基因中转录变体3与ACP6-Y在尼罗罗非鱼雄鱼肌肉组织中的表达均极显著高于雌鱼(P0.01),ACP3-X、60S核糖体蛋白(RL3)、小白蛋白β样蛋白、ACP15-X、肌型肌酸激酶M2-CK与转录因子Sox4在尼罗罗非鱼雌鱼肌肉组织中的表达均极显著高于雄鱼(P0.01)。结果表明,应用引物退火控制技术筛选获得了8个可能参与了雌雄鱼肌肉生长发育调控的ESTs,为进一步筛选雌雄鱼肌肉生长发育相关候选基因奠定了基础。     

中华鳖GnRH 1基因cDNA克隆及表达特征

为了解GnRH基因在中华鳖(Pelodiscus sinensis)性腺和胚胎发育过程中的表达特征,采用cDNA末端快速扩增(RACE)技术从中华鳖全脑中获得与生长生殖调控密切相关的GnRH1基因全长cDNA,并运用实时荧光定量PCR(qRT-PCR)技术检测GnRH1在成鳖不同组织和胚胎发育时期的表达水平。结果显示:中华鳖GnRH1基因cDNA全长546 bp,其中5′非编码区(5′UTR)99 bp,3′非编码区(3′UTR)168 bp,开放阅读框(ORF)279 bp,编码92个氨基酸,分子质量为10.23 ku,理论等电点pI为5.65,具有N端信号肽(1~23 aa)、核心十肽区域(24~33 aa)、断裂位点GKR(34~36 aa)及相关肽区域(37~92 aa),符合GnRH蛋白典型结构特征。系统进化树结果显示,中华鳖GnRH1基因和绿海龟(Chelonia mydas)、墨西哥箱龟(Terrapene carolina mexicana)及西部锦龟(Chrysemys picta bellii)GnRH1基因聚为一支。qRT-PCR结果表明,GnRH1基因在中华鳖雌雄个体的8个组织中均有表达,在脑和性腺组织中高表达,且具有性别差异,雄性中华鳖中的表达显著高于雌性(P<0.05);在10个胚胎发育时期均表达,且随发育时间的后移,表达量显著增加,在第16期达到峰值。GnRH1基因可能在中华鳖生长及性腺分化中具有重要作用。     

Reproduction phase-related variations in the GnRH immunoreactive fibers in the pineal of the Indian major carp Cirrhinus mrigala (Ham.)

We studied GnRH immunoreactivity in the pineal gland of the Indian major carp Cirrhinus mrigala during different phases of reproductive cycle. In the resting phase (December–January), GnRH immunoreactive (-ir) fibers were organized as paired fascicles above the posterior commissure that ascend in the stalk and distribute widely in the pineal gland. The GnRH-ir fiber density significantly declined (P<0.001) during the preparatory phase (February–April) and the fibers disappeared thereafter. While no GnRH fibers were seen during the prespawning (May–June) and spawning (July–August), isolated GnRH-ir fibers reappeared in the postspawning phase. Since no GnRH cell bodies were detected in the pineal, these GnRH-ir fibers seem to be of central origin. The results reveal a distinct reciprocal relationship between the GnRH immunoreactivity in the pineal and the status of the ovarian maturity; the fibers appeared in the pineal only during the period of ovarian quiescence. While the functional significance of these cyclic changes in GnRH is yet to be determined, we suggest that the decapeptide may serve as a transmitter of central origin that modulates the activity of the pineal gland.     

The first assessment of cyanobacterial and diazotrophic diversities in the Japan Sea

The diversity of cyanobacteria and diazotrophs in the Japan Sea was investigated by analyzing sequences of cyanobacterial 16S rRNA genes and nitrogen fixation genes (nifH) from seawater sampled at depths ranging from the surface to 100?m at two stations. Of the 107 cyanobacterial 16S rRNA gene sequences obtained, 97 and three sequences were assigned to Synechococcus sub-cluster 5.1 and Prochlorococcus HL (II), respectively. Unlike other oceanic regions, at our two sampling stations the composition ratio of the sequences assignable to Synechococcus sub-cluster 5.3 was relatively high (8?%). No sequences of diazotrophic cyanobacteria were found in the cyanobacterial 16S rRNA genes. In the nifH clone library (36 sequences), ten sequences were identified as a UCYN-A group of diazotrophic cyanobacteria; the other 26 sequences (72?%) were assigned to proteobacteria. These results suggest that heterotrophic bacteria, including UCYN-A, dominate the diazotrophic community in the Japan Sea. Our study reveals the dominance of Synechococcus in cyanobacterial community and (photo)heterotrophic diazotrophs in the diazotrophic community in the Japan Sea, suggesting its unique characteristics.     

In vivo activity of native GnRHs and their analogues on ovulation in the African catfish, Clarias gariepinus (Burchell)

Four distinct forms of native gonadotropin‐releasing hormone (GnRH) and two newly designed analogues were tested for their in vivo activity to induce ovulation in African catfish. The effects of these peptides on ovulatory parameters were compared with those of carp pituitary and [d ‐Ala⁶, Pro⁹‐NEt]‐mammalian GnRH analogue (mGnRHa), two tested ovulation‐inducing agents in African catfish. Assessment of ovulation was carried out by determining the ovulation ratio and the relative quantity of egg produced. From the results of the experiments, the order of potency of the native GnRH peptides is summarized as chicken GnRH‐II (cGnRH‐II) >salmon GnRH (sGnRH) >mammalian GnRH >chicken GnRH‐I (cGnRH‐I). Chicken GnRH‐II was as potent as mGnRHa while cGnRH‐I was totally ineffective. The new d ‐Orn⁶‐cGnRH‐II and d ‐Orn⁶‐sGnRH with a substitution at position 6 with d ‐isomer residue were as potent as the most extensively used mGnRHa, indicating that the position 6 modification might be more crucial than the substitution at the C‐terminal. On the basis of our results, the potential use and incorporation of cGnRH‐II and sGnRH for the development of more generic spawning induction therapies are suggested.