| Ⅰ . The role of kisspeptin/kisslr system expressed in the arcuate nucleus on the reproductive development and maintenance during the female rats in different periodsObjective: kisspeptin/kisslr system, expressed in the hypothalamic arcuate nucleus(ARC), has been declared as one of the most powerful factors in the reproductive axis according to recent research in the field of reproduction. Gonadotropin-releasing hormone (GnRH) is a neurohormone, secreted by the hypothalamus. It plays an important role in the regulating reproduction. The purpose of this study is to clear the kisspeptin, its receptor (kisslr), and gonadotropin releasing hormone expression, and to explore the possible influence on reproductive development and maintenance of developing female rats.Methods: The number of pregnant Wistar rats was ten and the rats were purchased.We divided their female pups into four groups (n=20) according to birth weight: 7 days and 21 days, 35 days, 63 days of the infancy, prepuberty, puberty and adult stage of postnatal development.Among the four groups, ARC of the brain and ovary were obtained,fixing in polyformaldehyde. Specific operations were as follows:(1) The vaginal smear daily was done for the pubertal and adult rats.(2) The serum levels of hormone were checked and analyzed by ELISA.(3) Hematoxylin-eosin (HE) staining was used to detect the ovary morphological changes of rats.(4) The levels of kiss 1-、kisslr- and gonadotropin-releasing hormone mRNA expression were detected by real time fluorescent quantitative PCR (qRT-PCR)method.(5) The expressions and location of kisspeptin/kisslr system and GnRH were examined by immunohistochemistry and western blot, resspectively.Results:(1) ELISA results demonstrated that the E2, LH and FSH secretion increased from infancy, prepuberty to puberty. However, Serum estrogen levels were reduced obviously among the adult rats.(2) Numerous primordial follicles gathered around and sporadic larger primary follicles located in the middle region of the ovary in the infancy-aged rat. Then,the secondary follicles were dominant in the ovary of the pre-pubertal rat. With further development, the growth follicles were observed and luteal tissues emerged because of estrous cycles in the pubertal rat. Finally, Corpora luteal cells were observed after ovulation in adult-aged rat. In addition, the number of atretic follicles and shrinking corpora luteal increased significantly.(3) The expression of kisspeptin/kisslr system increased along with aging, and the highest level was checked for adult rats. GnRH as well as kisspeptin/kisslr shows a similar expressional trend.Conclusion: The kisspeptin/kisslr system plays a role in the activation and maintenance of the reproductive function by regulating GnRH neuron, which indirectly regulates the serum FSH, LH, E2 levels.Ⅱ. The role of kisspeptin/kisslr system in the arcuate nucleus of female rats on puberty onset during negative energy balanceObjective: Reproductive endocrine disorder caused by hypoglycemia is very common in recent years. But potential mechanism of reproductive disorders even was puzzling. It is reported that leptin/leptinR- kisspeptin/kisslr system plays a critical role in reproductive field. Therefore, this study was to determine the role of leptin/leptinR played in the expression of kisspeptin/kisslr during negative energy balance and the role of leptin/leptinR-kisspeptin/kisslr on puberty onset.Methods: Wistar rats of the female aged 28 days were be split into four groups,namely as control group, fasting group, fasting+ saline/leptin group. Details were showed as bellow:(1) Body weight and serum leptin, LH, FSH and E2 hormone levels by ELISA were recorded;(2) The mRNA and protein levels of kisspeptin, kisslr, GnRH as well as the correlation were detected by qRT-PCR and western blot respectively;(3) The expressions of kisspeptin, kisslr and GnRH were tested by immunofluore-scence.Results:(1) There was a greater drop in yield with regard to body weight after 72h fasting and the leptin injection had no influence on body weight.(2) Enzyme linked immunosorbent assay showed that serum level of leptin and gonadotrophin concentration was lessened after 72h fasting versus control group.In contrast to fasting+ saline group, it was on the rise for the leptin,gonadotrophin levels in the leptin-treated rats. However, fasting and leptin made no difference on the E2 levels.(3) Results were showed by immunofluorescence that kisspeptin, kiss1r and GnRH expression was found in the cytoplasm but leptinR in the nucleus.(4) The expression of kissl-, kisslr-, GnRH- mRNA and reproduction endocrine peptides after 72h fasting were significantly decreased measured by qRT-PCR and western blot. Leptin administration could reverse this trend induced by fasting.(5) The leptinR expression of ARC in the hypothalamus increased after fasting and was restricted by leptin injection.Conclusion: leptin/leptinR makes the effects on the endocrine alteration during puberty onset by regulating the mRNA and protein expression of kiss1/kiss1r and it supports a certain experimental basis on exploring the concrete mechanism of puberty onset in the furture. |
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