Research Of Molecular Characteristics And Expression And Localization Of UCHL1 And ESR1 In The Male Gonads Of Chinese Giant Salamander (Andriasdavidianus)

Because of overexploitation,habitat loss and pollution,the number of the wild Chinese giant salamander(CGS)is undergoing a sharp decline.Study of reproductive physiology and artificial reproduction has a great significance for the recovery of the population of CGSs.We firstly studied anatomically and histologically the gonad development of CGSs.Secondly,we cloned and analyzed the molecular properties of UCHL1 and ESR1 of the CGSs.Thirdly,the localization and expression of UCHL1 and ESR1 in the gonad were detected by RT-PCR,RT-qPCR and immunohistochemistry.The results are as follows:1.In6-month-old CGSs,it is difficult to distinguish the male and female gonad anatomically.The male and femalegonads of CGSsare long-strip shape.From 12-month-old to the sexual matural CGSs,the shapeof testis isprolate ellipsewhile the shapeof ovary is a long-strip.The time of the gender differentiation of the CGSs is between 6-month-old and 12-month-old.H.E.staining resulrs showed that some large nuclear cells formed outer wall,and the centralis was latticed cavity with fibrocyte single cells in the gonads of 6-month-old CGSs.In 12-month-old CGSs,the primary seminiferous lobules appeared and interstitial tissues are rich.In 2-year-old CGSs,the germ cells and somatic cells formed epithelium of seminiferous lobules and there was lobular lumen.In 3-year-old CGSs,seminiferous lobules increased and the lobular lumen enlarged.In adult CGSs,the germ cells distributed regularly from base to top of seminiferous lobules.A large number of maturalspermlocaled at the top of seminiferous lobules in nonreproductive period,while the seminiferous lobules were filled by the maturalsperm in reproduction period.2.The CGSs UCHL1 gene includes 1087 bp,ORF contains 669 bp and encods 222 amino acids.The CGSs UCHL1 protein has one O-glycosylation site,fourteen potential phosphorylation sites.The homology of the CGSs UCHL1 protein with H.sapiensis is 73.99%.RT-qPCR results showed thatthe peak level of UCHL1 mRNA appeared in testis of 2-year-old CGSs.Further revealed UCHL1 mRNA expression was significantly lower inreproduction period thanno-reproductive phase.In the gonads of the6-month-old CGSs UCHL1 localized in cell nucleus.Intestis of the 12-month-old to 3-year-old CGSs,localization of UCHL1 was in the cytoplasm and the cells formed the seminiferous lobules.In testes of the adult CGSs,the UCHL1 distributed intercellular space and the UCHL1 was an ingredient forming niches of differentiation germ cells in the seminiferous lobules.3.The CGSs ESR1 gene includes 2585 bp,ORF contains 1755 bp and encods 584 amino acids.The CGSs ESR1 protein contains ten O-glycosylation sites,three N-glycosylation sites,fifty-nine potential phosphorylation sites.The homology of the CGSs ESR1 protein with Andrias japonicus is 99% and about 70% with mammals.RT-qPCR results showed that a trend of rising of ESR1 in testis from 12-month-oldto 3-year-old CGSs.Immunohistochemistry results showed that in testis at different development stages,ESR1 proteins are located in testicular germ cells and leydig cells,but the mature sperm do not expresses ESR1.The above results showed that the time of gender differentiation of theCGSs is between 6-month-old and 12-month-old.In the male gonads,the CGSs UCHL1 has constitutive expression(the nucleus,cytoplasm and cell membrane positioning)and secretory type(dividing germ cells in different niches).UCHL1 may be associated with the development of the testes and guaranteed the synchronous development of germ cells of CGSs.ESR1 gene is highly conserved in vertebrates.ESR1 proteins are located in reproductive cells and leydig cells in the CGSs testis.The ESR1 participate in maintaining the survival of germ cells and normal spermatogenesis.