| Background:During the course of development in mammalian, hypothalamus-pituitary-gonad (HPG) axis play a key role. HGP axis is the fundament of normal reproductive function in both man and woman. GnRH release impulse in hypothalamus is the signal of pubertal onset. A number of neuropeptides and neurotransmitters participate in the regulation of GnRH. NMDA, NO, leptin et al have positive regulating roles, but NPY and GABA et al have negative roles. The transcription of GnRH is also under the negative feedback regulation of gonad steroid. KiSS-1 is a new gene found in mammalian, which regulate the release of GnRH. GPR54 is the receptor of kisspeptin. The individuals who suffered the mutations of GPR54 gene happened isolated hypogonadotropic hypogonadism. KISS-1 and GPR54 may have close relation with the onset of puberty. Exploring of function and mechanism of KiSS-1 and GPR54 in reproductive regulation is relatively few. This research will improve our knowledge about puberty onset and reproductive regulation, and provide new target for reproductive regulation and reproductive disease therapy.Objective:1. Hypothalamus is the center of reproductive endocrinal regulation. By the technique of molecular biology, to evaluate the stimulating role of KiSS-1 to GnRH, explore the expression level of KiSS-1 in rat hypothalamus.2. To explain the function of KiSS-1 and GPR54 in development, we further approached the mechanism of KiSS-1 regulating GnRH.3. To investigate the probably direct role of KiSS-1 and GPR54 in gonad, explored the expression of KiSS-1 and GPR54 in rat testis. Evaluate the function of KiSS-1 and GPR54 in the whole hypothalamus-pituitary-gonad axis.Methods:1. Explored the expression of KiSS-1, GPR54 and GnRH in hypothala- mus from different developmental stage of male rats. Approached the role of KiSS-1 at sex development onset and normal reproductive regulation. Detected the level of testosterone by chemoluminescence in the serum of rats' icved with kisspeptin.2. Ater icved with kisspeptin, adult male rats were killed. The expression of GnRH mRNA in hypothalamus and FSH-βand LH-βmRNA in pituitary were detected by RT-PCR and real-time RT-PCR.3. Male castrated pubertal rats were ip injected with NMDA at a dose of 40mg/BW or MK-801 at a dose of 1mg/BW. Rats injected with NS served as negative control. The expression of KiSS-1 mRNA in hypothalamus was compared.4. Detection of KiSS-1 and GPR54 at the testis from different developmental stage rats was conducted by immunohistochemistry technique.5. Statistics methods: all the data was compared and statisticed with the soft of SPSS Version 11.5.Results:1. The result from RT-PCR manifested that KiSS-1 expression in rat hypothalamus rose gradually during development. It was highest at puberty stage, and dropped a little at adult rats at a relative high level. KiSS-1 mRNA of prepubertal, early pubertal, pubertal and adult rats was 1.7,2.1,3.5,2.0 times higher than that of infantile rats, respectively. Correlation analysis revealed that GnRH expression correlate positive with KiSS-1 expression. The activation of GnRH neurons were later than KiSS-1. Expression of GnRH was highest in puberty rats. Prepubertal, early pubertal, pubertal and adult rats GnRH mRNA was 1.1, 1.9, 2.4, 1.9 times higher than that of infantile rats, respectively. GPR54 expressed at different development stages. The transcription level of this gene was significantly lower in infantile and prepubertal rats than that of pubertal and adult rats.2. No rising of GnRH mRNA was detected after kisspeptin was icv injected. But FSH-βand LH-βmRNA levels in pituitaries rose significantly. After kisspeptin injected for 2 hours, rat serum testosterone also rose greatly.3. Compared with the control, NMDA treatment lowered the expression of KiSS-1 mRNA, but MK-801 had an opposite result.4. Both KiSS-1 and GPR54 expressed in rat testis. Immunohistochemistry technique manifested all the immunoreaction positive cells were germ cells. There was no expression in interstitial cells. KiSS-1 and GPR54 express highest in puberty rat testis and lowest in prepubertal rats.Conclusion:1. KiSS-1 and its receptor GPR54 is one of the factors that regulate gonad axis. Though no regulating role of KiSS-1 to GnRH gene expression was found in this study, kisspeptin treatment may stimulate the expression and release of GnRH peptide. It was manifestied by that LH and FSH expression increased and serum testosterone level rose greatly.2. Expression of KiSS-1 and GnRH genes correlate positively. KiSS-1 may participate in GnRH regulation. Its physiology functions relevant with sex development onset and reproductive regulation. For KiSS-1 and GPR54 express highest at pubertal rats, the expression of the genes is related with sex development of mammalian.3. Exciting amino acids can regulate expression of KiSS-1 directly or indirecly. NMDA has an upregulate function, and its non-completion receptor antagonist can down regulate the expression of KiSS-1. It seems that KiSS-1 was at the downstream of NMDA and KiSS-1 may mediate the regulating role of other regulate agents to GnRH.4. Both KiSS-1 and GPR54 expressed in rat testis germ cells. They may take part in the course of germ cells maturation. It seems that KiSS-1 and GPR54 in mammalian gonad has a direct physiological functions. |
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