Gonadal Development,Sperm Biology,Expression And Methylation Of Cyp19a1 Gene Of Oreochromis Niloticus?×O.aureus?

Because of the males tilapia grow significantly faster the females,interspecific hybridization of Oreochromis niloticus ? × O.aureus ? was widely used in order to obtain high male rates above 95% in production.In order to further explore the mechanism of male percentage deviation in the offspring,this paper observes the sex differentiation,gonadal development,maturation and viability of the spermatozoa,and the m RNA expression and methylation level of the promoter region of the cyp19a1 a gene in the gonadal developmental process,to provide basic data for sex determination biology of the hybrid tilapia.The gender type and gonad development of O.niloticus?×O.aureus? at 0.5-4.5 month were observed by microscopic histological observation.The results showed that the primordial gonad of O.niloticus?)×O.aureus?was undifferentiated at 0.5-month-old.The primordial germ cells in the testis differentiated into spermatogonia around 1 month,primary spermatocytes occurred at the age of 2.5 month,while secondary spermatocytes presented around 3-month-old.The germ cells presence different developmental stages in the testis and sparse sperm dispersed in ampulla cavity at the age of 3.5-4.5 months,while intensive sperm dispersed in ampulla cavity around 4.5 months.Among the samples,female individuals were also detected at the 2.5,3.5,4,4.5 months,respectively,the oocytes were in phase ? at 2.5-3.5 months ovarian,and phase ? at 4,4.5 month-old.This study suggested that there were two kinds of gender existed in hybrids with the male dominated,the testis and ovarian in the hybrids could develop normally.The sperm morphology of O.niloticus?× O.aureus? and O.niloticus were observed by scanning electron microscopy technique,which was composed of head,middle and tail piece,there existed "normal type" and "short-tailed type" sperms in two species.Sperm density was measured with hemocytometer,it was?2.39±0.43?×109 spermatozoa m L-1 and?4.38±0.57?×109 spermatozoam L-1 for O.niloticus?× O.aureus? and O.niloticus,respectively.The fast moving time,lifespan andactivation rate were compared under different gradients p H,K⁺ and Ca²⁺ solutions.Optimum sperm activation was found when p H=7.5,the activation rate reached to 90%,the fast moving time and lifespan of O.niloticus?× O.aureus? sperm were lower than O.niloticus.Sperm fast moving time reached the highest at K⁺=100mmol/L,it was 57.76±10.88 s and 64.69±9.10 s for O.niloticus?× O.aureus? and O.niloticus,respectively,while sperm lifespan and activation rate reached the highest?90%?at K⁺=75mmol/L with 470.27±4.63 s and 513.97±5.33 s.Sperm motility reached the maximum under Ca²⁺=37.6mmol/L,the fast moving time and lifespan of O.niloticus?× O aureus? and O.niloticus sperm were 31.56±4.28 s,40.56±4.28 s,and 112.78±4.58 s,116.11 ±7.43 s,respectively.In comparison to O.niloticus,the sperm morphology of O.niloticus?× O.aureus? was normal with lower sperm density,and lower sperm motility at the optimum p H,K⁺ and Ca²⁺ solutions.Through transcriptome sequencing and we found that they have same cyp19a1 a CDS region between?Oreochromis niloticus ??×?O.aureus ??and O.niloticus,and they have the same promoter and noncoding region by PCR cloning.Taking O.niloticus testis and ovary cyp19a1 a m RNA expression for the reference,it is showed that m RNA expression level of cyp19a1 a gene in testis and ovary of?Oreochromis niloticus ??×?O.aureus ??decreasing trend,which was lower than O.niloticus and the ovaries were higher than testis at 40,60,80 and 120 days.The promoter?SP1,SP2,SP3?of cyp19a1 a BSP methylation sequencing analysis at the same time point,and methylation level in the ovary was higher than that of the testis,it is different that the methylation level of SP1 and SP2 during the development,SP1 and SP2 showed an increasing trend in the testis,SP1 appears to increasing trend,however the SP2 showed a trend of decreasing after increasing.It was not obvious that methylation level of SP3 between?Oreochromis niloticus ??×?O.aureus ??and O.niloticus.on the whole,the methylation level testis and ovarian higher than the O.niloticus.