| Most of the gonadal development of Hyriopsis schlegeliis are unisexual,and a few individuals show hermaphroditism at 26-34 months of age.However,the factors affecting gonadal development and hermaphroditism are unknown.In this study,the hermaphroditism of the gonads of H.schlegeliis was screened and observed for two consecutive years,and differentiation of reproductive follicles was induced by five temperatures.The gonads of H.schlegeliis in different periods were detected by qRT-PCR.At the same time,in situ hybridization and Western blot were used to determine the cell location and protein quantification of Dmrt1,a key gene for sex determination,to explore the effect of temperature on the gonadal development of H.schlegeliis.1.Investigation on sex differentiation of H.schlegeliis in natural cultureUnder natural conditions,the hermaphroditism of female follicles also appeared in 6-month-old H.schlegeliis,and the hermaphroditism of the selected hermaphroditic H.schlegeliis at 28 and 29 months of age showed different sex differentiation at about30 months of age: 77.8% became males,16.7% remained hermaphroditism at 38 months of age,and 5.5% became females.Detection hermaphrodite clam gonad growth to 31 months,found that the existence of three types of reproductive follicles:female follicles,male follicles and female and male germ cells coexist in follicles,and along with the development of the same hermaphrodite clam development in a different state: some male reproductive follicles in hermaphrodite clam accounted for99%,basic no cavity;Some female and male reproductive follicles are 50% each,with small or no cavities.Some female and male reproductive follicles are 50% each,but have large cavities.2.Spatial and temporal mRNA expressions of sex-related genes Tra2 a,Fem1b,Fem1 c,Sox9,Dmrt1 and Foxl2 in gonadal tissues of H.schlegeliisAnalysis of spatiotemporal expression of sex-related genes in the gonads of H.schlegeliis embryos,visceral mass at 1-7 months of age,female and male at 27-32 months of age,and hermaphroditic mussel at 29 and 31 months of age.The results showed: The mRNA expression levels of Tra2 a,Fem1b,Fem1 c and Sox9 at 1-7months of age showed no significant change compared with those at embryonic stage except for 5 months of age.The mRNA levels of Fem1 b and Fem1 c showed the same trend,which was roughly consistent with that of Tra2 a.Sox9 had the same change trend as Fem1 B and Fem1 c except at 5 months of age.The mRNA level of Dmrt1 gene at 1-7 months of age was almost not expressed at 1-5 months of age,but increased significantly at 6 and 7 months of age compared with that at embryonic stage.The four genes Tra2 a,Fem1b,Fem1 c and Sox9 were expressed in the gonadal tissues of both female and male clams at 27-32 months of age,and the expression trend of Tra2 a,Fem1b,Fem1 c and Sox9 in the gonadal tissues of both female and male clams at 27-30 months of age was consistent.At 31-32 months of age,the expression trend of Tra2 a,Fem1b and Fem1 c was consistent,while the expression trend of Sox9 was opposite.The mRNA expression levels of Tra2 a and Sox9 in male and female clams were higher than those in female clams.The expression of Dmrt1 in27-month-old males was higher than that in females,and Dmrt1 was specifically expressed in the gonadal tissue of 28-32 month-old males.The expression levels of Tra2 a,Fem1b and Dmrt1 at 29 and 31 months of age were firstly increased,then decreased,and showed an opposite trend to that of Sox9 at 31 months of age.The expression of Foxl2 in female and hermaphrodite mussels was significantly higher than that in male mussels.The results showed that hermaphrodite or sex reversal mussels.3.Effects of temperature on differentiation of reproductive follicles and mRNA expression of genes related to sex regulation in H.schlegeliisUnder the temperature stimulation of 15℃,19℃,23℃,27℃ and 32℃,the13-month-old and 26-month-old mussels were cultured for one month,respectively.At the end of the experiment,the sex of the mussels was detected by paraffin section and the mRNA expression of the sex-related gene Tra2 a,Fem1b,Sox9,Dmrt1 and Foxl2 in the gonads was detected by qRT-PCR.The results showed that 12.5% of the males in the 14-month-old group became hermaphrodite under 32℃ conditions.Under condition 27℃,sex reversal occurred in 37.5% of males and 14.29% of females.The qRT-PCR results showed that the expression trend of Tra2 a,Fem1b and Sox9 in 14-month-old female clams was similar at five temperatures,and the expression trend of Tra2 a,Fem1b and Sox9 in 14-month-old female clams was not significantly changed.The expression of Dmrt1 was significantly higher at 19℃ and23℃ than at other temperatures.The expression of Foxl2 at 19℃ and 23℃ was significantly lower than that at other temperatures.The expression trend of Tra2 a,Fem1b and Sox9 was similar at five temperatures,and the expression of Dmrt1 at19℃,23℃,27℃ and 32℃ was significantly higher than that at other temperatures.There was no significant change in the expression of below,but it was significantly higher than that of 15℃.Foxl2 expression at 27℃ was higher than that at other temperatures.The expression trend of Tra2 a,Fem1b and Sox9 was similar at five temperatures,and there was no significant change in the expression.The expression of Dmrt1 at 19℃ was significantly higher than that at other temperatures.Foxl2 expression at 27℃ was significantly higher than that at 19℃,23℃ and 32℃,27 months of age.Tra2 a and Fem1 b expression trends were similar at five temperatures,and the expression of Tra2 a and Fem1 b did not change significantly.The expression of Sox9 was significantly higher at 23℃ than at other temperatures.The expression of Dmrt1 at 32℃ was significantly higher than that at other temperatures.The results showed that temperature affected the differentiation of reproductive follicles.High temperature(27℃ and above)could easily induce the reversal of male to female,and low temperature(23℃ and below)could easily induce the reversal of female to male,among which 19℃ and 27℃ had a greater effect,and the early stage of gonad development was more affected.The five genes Tra2 a,Fem1b,Sox9,Dmrt1 and Foxl2 were all involved in the differentiation of reproductive follicles induced by temperature,and the effect of Dmrt1 was obvious.4.Localization and expression of Dmrt1 gene in gonadal tissue of H.schlegeliis tissue localization and quantification of sex-determining key gene Dmrt1In this study,in situ hybridization was used to localize Dmrt1 mRNA,and Western blot was used to quantitate Dmrt1 protein in the gonadal tissues of female,male and hermaphrodites aged 31 months.The results showed that Dmrt1 mRNA positive signal was mainly localized in spermatogonia,morula and mature oocytes.The amount of protein expressed by Dmrt1 gene in monoecious mussels showed two trends: higher than that of contemporaneous males and lower than that of contemporaneous males.In conclusion,the reproductive follicles of H.schlegeliis are pluripotent,and the differentiation is affected by temperature.High temperatures of 27℃ and above are easy to induce the reversal or hermaphroditism of male to female,while low temperatures of 23℃ and below are easy to induce the reversal of female to male.It is possible that temperature regulates the differentiation of reproductive follicles by regulating the expression of sex-related genes such asDmrt1. |
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