The Effects Of Kisspeption-10 On The Initiation Of Egg Laying In Quail

Kisspeptins consist of several peptides encoded by Kiss-1 gene, which bind to the specific receptor of G protein-coupled receptor 54 (GPR54) to exert the biological function. Mice and humans lacking a functional GPR54 fail to undergo puberty and exhibit hypogonadotropic hypogonadism, indicating that Kisspeptin signaling pathway plays an essential role in the onset of reproduction. Studies in several mammalian species have shown that Kisspeptins stimulate the secretion of gonadotropins from the pituitary by stimulating the release of GnRH from the forebrain after the activation of GPR54, which expressing in GnRH neurons. In this study, hens during high laying period and quail during early laying period were used as the model, and the methods of immunohistochemical (IHC) and intraperitoneal injection (i.p.) were employed in the present study. The objective of this study was to:(1) whether Kisspeptin-like immunoreactive substances exits in the brain of female birds; (2) The effect of Kisspeptin-10 on the onset of egg laying and possible mechanisms involved in the process.1 Kisspeptin-like immunoreactive substances in female avian brainIn this study, IHC was used to locate Kisspeptin-10 expression in hypothalamus and thalamus of hens during high laying period. Kisspeptin expression in the hypothalamus of chickens during high laying stages was detected and quantitated by western blot. The results showed that:Kisspeptin immunoreactive neurons were observed in the paraventricular nucleus (PVN), nucleus dorsolateralis anterior (DLA), superficial gray matter fiber layer (SGFS), nucleus intercalates thalami (ICT). The same antibody, with WB anchoraged the target protein (MW=15-17 KD) on hens and quail suggested that there may be Kisspeptin-like immunoreactive substances in female avian hypothalamus.2 The effect of Kisspeptin-10 on egg laying of quailsTwenty-four hours after hatching was defined as 1 day old, at 20 days old,75 quails were randomly divided into three groups (25 quails per group,5 replicates per group):the control group (Con,0.3 mL sterilized physiological saline), low dose treatment group (L, 10 nmol per quail), high dose treatment group (H,100 nmol per quail). Quails were subjected daily intraperitoneal injection (i.p.) with Kisspeptin-10 or saline from 20 days of age. Injection has been lasted for 22 days and animals were allowed ad lib access to food and water during the whole experiment. Body weight and the information of egg laying has been recorded for 8 weeks. Quails were killed by decapitation to collect blood, reproductive organs, liver and record the amount of large follicles. Hypothalamus, pineal gland, pituitary, ovary (small follicles) and granulosa layer of F1 follicles were sampled and put into the liquids nitrogen for further analysis. Radioactive immunoassay (RIA) was used to measure the concentration of LH, FSH, E2, Leptin as well as estradiol (E2) in blood. Real-time fluorescence quantitative PCR was used to detect the relevant genes expression in hypothalamus-pituitary-gonadal axis (HPG axis), and western blot was applied to measure protein expression of FTO and the Leptin receptor (LepR) in hypothalamus. The results showed that:(1) the repeated Kisspeptin-10 i.p. injection had notably promoted laying performance (P<0.01), while there was no significant difference among date (P=0.981) and none interaction between treatment and date (P=0.471); (2) the amount of large yellow follicles and reproductive oviduct weight in L group were significantly increased compared with control group (P<0.05), and showing the trend to increase in H group(P= 0.07); (3) The expression of GnRH-1 mRNA and POMC mRNA in hypothalamus was markedly up-regulated by high dosage of Kisspeptin-10 injection compared with the control group (P<0.05). Comtrasted to the control group, LH and GnRHR-â…¡genes expression in pituitary were significantly increased, while FSH mRNA expression was decreased by Kisspeptin-10 administration(P<0.05). LHR and FSHR mRNA expression in small yellow follicles showed a significant decrease (P<0.05), and the expression of LHR, FSHR and GnRH-â… mRNA in the granulosa layer of F1 follicles were also markedly decreased compared with the control group (P<0.05). However, the level of FTO and LepR gene and protein did not changed by Kisspeptin-10 injection (P>0.05). The results suggested that repeated injection of Kisspeptin-10 promoted early egg laying rate and accelerated entry into reproductive period, which may be induced by a faster maturation pace, or possibly directly stimulate central reproductive system to express GnRH, or directly affect on peripheral reproductive system.