Effects Of Temperature On Gonadal Development And Endogenous Sex Hormones And Regulation Of Sex-related Factors Dmrt1 And Foxl2 In Hyriopsis Schlegeliis

Sex determination and sex differentiation are key factors that affect the formation of gender.Sex determination can be divided into genetic and environmental sex determination.The freshwater pearl mussel Hyriopsis schlegeliis belongs to the temperature-dependent sex determination in environmental sex determination,which is highly influenced by temperature-related factors.However,so far,the mechanism of the effect of temperature on sex differentiation of Hyriopsis schlegeliis is still unclear.Therefore,based on 20 years of previous laboratory work,this study further investigates the regulatory mechanism of temperature on sex development-related factors in Hyriopsis schlegeliis.The full-length c DNA sequence of Foxl2 in Hyriopsis schlegeliis was first cloned,and the expression and localization of Foxl2 and Dmrt1 in various tissues and different gonads were examined.Then,by inducing Hyriopsis schlegeliis in three temperature ranges（low temperature:16℃and 19℃;normal temperature:23℃;high temperature:27℃and 32℃）for eight weeks,the relationship between temperature and the expression of sex-regulating genes（Foxl2 and Dmrt1）,endogenous sex hormones（estradiol,progesterone,testosterone）,and Hyriopsis schlegeliis gonad development were explored.The main research results are as follows:（1）The Foxl2 gene of Hyriopsis schlegeliis was cloned by RACE-PCR.The full-length sequence of c DNA is 1598 bp,which contains 1275 bp ORF region and 63 bp5’-terminal UTR and 320 bp 3’-terminal UTR,ORF region encoding 404 amino acids,including forkhead frame domain,which is located between 167amino acids and257amino acids.After analysis,it was found that the relative molecular weight,hydrophilicity index and instability index of Foxl2 protein were 46.09 Kda,-0.884 and54.4,the protein was hydrophilic and unstable.Multiple sequence alignment and phylogenetic tree construction showed that Hyriopsis cumingii was closest to Hyriopsis schlegeliis,followed by Mercenaria mercenaria and Crassostrea virginica,and farthest from Mytilus galloprovincialis,indicating that Hyriopsis schlegeliis is highly conservative in evolutionary status.（2）The expression of Foxl2 and Dmrt1 genes in seven different tissues and in the gonads of Hyriopsis schlegeliis aged 7～43 months was analyzed using q RT-PCR technology.The results showed that Foxl2 and Dmrt1 genes were expressed at the highest levels in the gonadal tissues and at the lowest levels in the heart.In addition,there were differences in the expression levels of both genes in the other five tissues.The expression of Foxl2 in the ovary of female clam was significantly higher than that in the testis of male clam,while the expression of Dmrt1 in the testis of male clam was significantly higher than that in the ovary of female clam.In situ hybridization revealed that the positive signal of Foxl2 probe appeared in the oocytes of female clam and in the oocytes and mature eggs of hermaphroditic clam,while the positive signal of Dmrt1probe appeared in the spermatogonia of male clam and in the spermatogonia and sperm of hermaphroditic clam.（3）Through annual tracking observations,it was found that females dominated in Hyriopsis schlegeliis over 32 months of age,while males dominated in those under 18months of age.The expression of Foxl2 in female shrimp under 24 months of age showed a continuous upward trend,while it peaked in male clam at 29 months of age.The expression of Dmrt1 in male clam under 24 months of age showed an upward trend,while it peaks at 13 and 29 months of age in female mussels.（4）The results of the gonad section staining showed that during the induction process at 16℃in the low-temperature group,17-month-old female clam would undergo sex reversal and become male clam in the fourth and fifth week.In the low-temperature group at 19℃,29-month-old female clam would exhibit hermaphroditism in the sixth week and undergo sex reversal and become male clam in the seventh week.In the high-temperature group at 27℃,17-month-old male shrimp would undergo sex reversal and become female clam in the seventh and eighth weeks,and 29-month-old male clam would undergo sex reversal and become female clam in the seventh week.In the high-temperature group at 32℃,29-month-old male clam would undergo sex reversal and become female clam in the fifth and sixth weeks,and 40-month-old male clam would exhibit hermaphroditism in the seventh week.During the sex reversal process,mature eggs and sperm were observed to be released from the female follicles and male follicles of female and male clam,respectively,and many empty follicles appeared.The empty follicles subsequently contained a large number of spermatogonia and oocytes,leading to the occurrence of sex reversal.（5）The results of hormone measurements showed that there was no significant difference in progesterone levels between male and female mussels.Compared to the control group at normal temperatures,the estradiol levels in female Hyriopsis schlegeliis were lower and showed a decreasing trend when induced at 16°C and 19°C.On the other hand,when induced at 16°C and 32°C,the estradiol levels in male Hyriopsis schlegeliis were higher than the control group and showed an increasing trend.（6）The results of the Log₁₀（Dmrt1/Foxl2）expression level showed that under stimulation at 16℃and 19℃,the Log₁₀（Dmrt1/Foxl2）values in 17-month-old and 29-month-old female clam showed an increasing trend compared with the normal temperature group,while there was no significant change in 40-month-old female clam undergoing sex reversal into male clam,and the Log₁₀（Dmrt1/Foxl2）values were between[0,2].Under stimulation at 27℃and 32℃,the Log₁₀（Dmrt1/Foxl2）values in first-instar and second-instar male clam showed a decreasing trend compared with the normal temperature group,while there was no significant change in 40-month-old male clam and the Log₁₀（Dmrt1/Foxl2）values in the two genes during sex reversal into female clam were between[-2,-1].In summary,the Dmrt1 and Foxl2 genes are highly expressed in the gonads,with Dmrt1expressed at a higher level in the testis than in the ovary,and expressed in spermatogonia and sperm,while Foxl2 is expressed at a higher level in the ovary than in the testis,and expressed in oocytes and mature eggs.This indicates that the Dmrt1and Foxl2 genes are functionally conserved.Low temperatures of 19℃and below can induce sex reversal from female to male shrimp,with the Log₁₀（Dmrt1/Foxl2）values showing an increasing trend,accompanied by a decrease in estradiol content.High temperatures of 27℃and above can induce sex reversal from male to female clam,with the Log₁₀（Dmrt1/Foxl2）values showing a decreasing trend,accompanied by an increase in estradiol content.Therefore,temperature may regulate the expression of the Foxl2 and Dmrt1 genes,alter the endogenous sex hormone content in Hyriopsis schlegeliis,and regulate the differentiation of their reproductive follicles.