Regulation Of Tac3 / Tacr3 In The Propagation Of Black - Line Hamsters

Reproductive activity is an extremely complex process in animals especially in mammal, many factors in this process regulate the activity of the hypothalamic pituitary gonadal axis through complex neuroendocrine system. The animals occur reproductive behavior only in particular season to get maximum reproductive success rate which called seasonal reproductive. Seasonal reproduction is one of the major influential factors of animal population fluctuations, Tac3/Tacr3 in regulating animal seasonal reproduction plays an important role.Tac3 ecoded Neural excitation peptide B(NKB), NKB is a kind of peptide containing a certain ten amino acid;Tacr3 ecoded NK3 R, NK3 R is the natural recepters of NKB, belongs to the typical rhodopsin seven transmembrane G protein coupled receptor family.Cricetulus barabensis is typical seasonal breeding animals which is the ideal model to study mechanism of seasonal breeding. Its breeding peak are spring and Autumn. This experiment used Cricetulus barabensis as research materials explore differentially expressed of Tac3/Tacr3 in different organizations, different development phase and different seasons by biological methods and technologies, which indicated the mechanism of Tac3/Tacr3 participate in seasonal reproductive regulation and learned the seasonal outbreak mechanism of Cricetulus barabansis populations from molecular level.Firstly, this study obtained Tac3 cDNA 396bp(Genbank loading number: KU519331) in Cricetulus barabansis by PCR amplfied, which including the complete encoding sequence 360 bp, Tacr3 cDNA 1256bp(Genbank loading number: KU519332), including encoding sequence 1244 bp and bioinformatics analysis was carried out to construct phylogenetic trees.Tac3 ecoded 119 amino acid residues, the first 21 for the signal peptide region, C end contained a highly conserved structural functional domain characteristic ten peptid-Asp-Met-His-Asp-Phe-Phe-Val-Gly-Leu-Met, which accorded with characteristics of tachykinin family. The tachykinin conservative carboxyl terminus was the basic structure of the receptor. The high conservation of evolution showed that this kind of peptide played an important role in life activities. The encoding area of Tacr3 was 1244 bp, it encoded 414 amino acid residues, the two level structure analysis showed strong overall hydrophobicity. Correlation structure analysis of the obtained amino acid sequences by SMART found that there are 7 distinct highhydrophobic regions in the sequence. The seven hydrophobic regions composed of seven complete alpha helical domains, it belonged to the typical rhodopsin seven transmembrane G protein coupled receptor family. The phylogenetic tree was constructed by using MEGA 5.0 software to construct the Tac3/Tacr3 nucleotide sequences with Cricetulus griseus、Mesocricetus auratus、Bos taurus、Homo sapiens and many other species.Secondly, the real-time fluorescence quantitative PCR was used to detect the Tac3 and Tacr3 relative expression in Cricetulus barabensis in different organs and different developmental stages and different seasons. The enzyme-linked immunosorbent assay was used to detect the serum concentrations of NKB/NK3R( proteins encoded encoded by Tac3/Tacr3) in different developmental stages and in different seasons to explore the regulation and control of reproductive activities by the system.The expression of different organs found that Tac3 and Tacr3 expression levels was significantly higher in the Cricetulus barabensis hypothalamus than in the livers,kidneys,spleen and so on(P<0.01), serum protein levels had no significant differences in different developmental stages(P>0.05), all of these indicated that the Tac3/Tacr3 system played an important role in the central nervous system,hypothalamus is the key part of Tac3/Tacr3 system.Exploration of developmental expression pattern found that Tac3 and Tacr3 had the highest expression( P<0.05) in female Cricetulus barabensis larvae,had the highest expression level(P<0.05) in adult male Cricetulus barabensis,infered that the system involved in the regulation of Cricetulus barabensis reproductive start, and there are male and female shaped in time, it mainly reflected in the changes in the development period.In the seasonal expression research, the expression of Tac3 in the hypothalamus of Cricetulus barabensis was much higher in the spring and autumn than summer and winter(P<0.05), which consistent with the seasonal expression pattern of GnRH and seasonal breeding of Cricetulus barabensis. It was inferred that the expression of Tac3 could promote the secretion of GnRH, it played a role in promoting reproductive regulation of Cricetulus barabensis. Tacr3 receptor gene in female Cricetulus barabensis hypothalamus was higher expressed in summer and autumn than spring and winter(P<0.05), the male individuals were higher in spring and autumn(P<0.05),that was roughly the same as the expression of GPR54(KiSS1 receptor) in thehypothalamus. We speculated that Tac3/Tacr3 system and KiSS1/GPR54 system in striped hamsters were inextricably linked. Tacr3 had the highest expression level(P<0.05) in summer in hypothalamus of female Cricetulus barabensis. it was concluded that this phenomenon might be related to the length of illumination. The expression of Tacr3 was significantly affected by the positive regulation of the length of light. But the expression level was not the highest in the ovary, central and peripheral manifestations were not consistent, it might due to the complexity of the physiological regulation mechanism.Generally speaking, this study exploreed the gene differential expression of Tac3 and Tacr3 in different organs, different developmental stages and different seasons of Cricetulus barabensis, and the endocrine system involved in the regulation of neuropeptide Cricetulus barabensis mechanism of seasonal breeding from the molecular level, and the genetic mechanism of population fluctuation and outbreak of animal population to provide a theoretical basis for rodent control.