| Relaxins are a family of peptides that have practical roles in mammalian pregnancy preparing the birth canal for delivery, resetting cardiovascular and osmoregulatory control and promoting growth and development of reproductive structures. And recent s tudies have revealed the presence of six orthologous relaxin family genes:RLN1/2, and-3, and insulin-like peptide (INSL)-3,-4,-5,and -6 in vertebrates. Relaxin3 (rln3), a new novel member of relaxin family, is involved in the regulation of arousal, feeding, stress responses, cognition, osmoregulation and anxiety-related behavior in mammals. Large scale synteny and phylogentic analysis revealed that two rln3 paralogues emerged during fish specific genome duplication in tilapia. But we are know few fuctions of the two rln3 in teleost. The Nile tilapia (Oreochromis niloticus), a XX/XY sex determination system with a gonochoristic teleost, has become one of a most important species in the word aquaculture. Besides, it’s a very good experimental model to understanding the developmental sex determination with genetic basis because of the feasibility of monosex populations and the whole genome sequence was opened. In the present study, real-time PCR and RT-PCR were experimented to study the rln3a expression in gonads and rln3b expression in brain, respectively. In addition, the function of rln3a gene in tilaipa sex determination and differentiation was elucidated by knockout, and the function of rln3b was experimented by salinity. The main results are as follows:1) The tilapia rln3a and rln3b genes, identified from the testis of adult tilapia, consists of 444 and 456 nucleotides and the ORF encodes a protein of 147 and 151 amino acids, respectively. Phylogenetic analysis showed that rln3a together with 3b from, teleosts clustered into one clade while rln3 from tetrapods and Coelacanth formed a distinct clade. Surprisingly, synteny analysis indicated that RNL3b, but not 3a, is the orthologous gene due to the conserved synteny of RFX1, CC2DL1A and NANOS3 genes at the upstream rln3 gene in tetrapods, coelacanth and rln3b in teleosts.2) Tilapia rln3a is most highly expressed in the testis, meanwhile, it was also expressed in the brain, pituitary and kidney at less transcript level than that of testis. In contrast, rln3b was found to be most abundantly expressed in the brain. In addition, it is also expressed in the pituitary and testis at much lower efficiency than that of brain. rln3a expression increased sharply from 30 dah in XY gonads, further elevated significantly at 90 dah and peaked at 150 dah, while in the XX gonads, it remains the basal level at all the stages. The expression of rln3b was abundantly expressed from 150 dah in XY gonads, while its expression had remained at a very low level during all stages in XX gonads.3) Histological observation demonstrated that rln3a knock-out through CRISPR/Cas9 system lead to the depletion of both spermatocytes and spermatids in the testes XY fish at 90 and 150 dah, whereas the meiotic germ cells at different phase were observed in the control testis. Meanwhile, the proliferation of spermatogonial cells was also delayed at 90 dah, it was partially restored from 150 dah in the testis of rln3a-deficient XY fish. IHC analysis also demonstrated absence of Vasa and Ef1a1b (germ cell marker) expression confirming the loss of germ line cells in the testis of 90 dah in rln3a-deficient tilapia in comparison with control testis. In the 150 dah, Vasa expression was also restricted in the proliferated spermatogonial cell, while it could be detected in both spermaogonia and spermatocytes.In the 240 dah, the indel fish recovered normal compared with the control.4) To investigate the osmoregulatory functions of both rln3a and rln3b, real-time PCR was used to investigate the expression profiles of both rln3a and rln3b in the brain under the acute salt tress with different concentration and different treatment time span. Total RNA were isolated from whole brains of both control fish and salt tress treaded fish. Notably, transcript levels of rln3b was significantly up-regulated under the salt tress by 8 day, while no evident change was found by 4 day at 8ppt. Furthermore, significant increase of rln3b transcript level was detected by both 4 and 8 day at 20ppt, but the rln3a was not change at any conditions.In summary, we identified two paralogous rln3 genes, which displays distinct expression profiles and functions, from tilapia. Our data highlights that rln3a, with highest transcript in the testis, plays essential roles in spermatogenesis as revealed by knockout experiments. On the other hand, rln3b, with most abundant expression in brain, is involved in osmoregulations. |
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