The Preliminary Study Of Blunt Snout Bream (Megalobrama Amblycephala) Kiss2/Kiss2R System On Reproductive Regulation

Blunt snout bream (Megalobrama amblycephala) belongs to Cypriniformes, cyprinid genus bream, also known as Wuchang fish, is an endemic species in China. In vertebrates sexual development and reproduction need a complex regulatory process, through the hypothalamus-pituitary-gonad (Hypothalamic-Pituitary-Gonad, HPG) axis of the reproductive endocrine regulation to regulate the gonadal development and maturation. Kiss/KissR been proposed to mediate neuroendocrine regulation of reproduction, the situation in M. amblycephala is unclear. By the end of rapid amplification (RACE), we cloned M. amblycephala Kiss2/Kiss2R gene cDNA sequence full-length, and applied quantitative real time PCR (qRT-PCR) to detect the expression pattern of Kiss2/Kiss2R in the adult male and female tissues; analyzed the Kiss2/Kiss2R expression during embryonic development and larval and juvenile period. Moreover, we used the tissue slice technology to identify the gonad development of M. amblycephala from stage Ⅰ to stage Ⅵ, and then through the qRT-PCR to detect the Kiss2/Kiss2R gene transcription in the brain, pituitary and gonad tissues of the samples collected during different stages. The results from the study could explain the regulatory role of Kiss2/Kiss2R in M. amblycephala, provide a reference to molecular mechanism of precocious puberty and basic information on screening functional gene of reproductive endocrine regulation. The main results were as follows:(1) According to the phylogenetic analysis results, we named the two genes we got, Kiss2and Kiss2R. Kiss2cDNA was623bp and contained an open reading frame (ORF) of378bp encoding a precursor protein of125amino acids (aa), with a untranslated5’ flank sequence of49bp and196bp of3region. When compared with Carassius auratus and Danio rerio, the amino acid sequence similarity was higher than80%. The putative functional motif is FNYNPFGLRF and the C-terminal cleavage site is GKR, has a poly A tail.(2) The complete Kiss2R cDNA has a length of1176bp, with442bp in3and161bp in5’UTR, consisting of an ORF of573bp that results in a predicted190aa, and only two transmembrane domains were founded. Two putative polyadenylation signal AAT (U) AAAs were founded and it also has a poly A tail.(3) Kiss2and Kiss2R are expressed in all tissues detected. In males, levels of Kiss2mRNAs were relatively higher in the muscle, followed by ovary and midbrain; midgut, heart, liver exhibited moderate levels of Kiss2mRNAs; while the females showed the highest Kiss2mRNA level in muscle, pituitary; testis is still higher than the other tissues. In another aspect, the expression of gonad Kiss2R was higher in females than that in males and it was vastly expressed in muscle in both sexes. Be similar to Kiss2, it shows a relatively higher expresstion in brain-pituitary-gonad axis.(4) The results showed that as soon as the fertilization was finished, the Kiss2and Kiss2R were in an activated state, then both mRNA level gradually declined. Kiss2mRNA level peaked at the blastocyst stage, while the Kiss2R was low. From midblastula stage to hatching, the Kiss2R had a fluctuation expression, the same as the Kiss2. Both Kiss2and Kiss2R dropped to the minimum at the hatching, then increases and keep a high level until7dph and15dpf, respectively, and then the drop appeares again. Significant differences are founded during different developmental stages for the embryonic and larval periods, between Kiss2and Kiss2R.(5) Gonadal stages are identified by histological observation. At the same gonadal stage, male M. amblycephala Kiss2has a similar expression pattern in brain, pituitary, both peak at the stage Ⅱ and Ⅳ, testis show the highest expression at the stage Ⅲ; female has the highest expresstion at stage Ⅱ; at the stage Ⅰ, male M. amblycephala Kiss2R at a higher expresstion level, then declines, but the expression in the testis is in fluctuation and get the highest level at stage Ⅲ; changes in the expression of the female brain and pituitary are obvious, the expresstion in brain and pituitary at stage Hand VI is higher, but the expression in the ovary is steady.