Sex determination is a way to determine the direction of differentiation,while sex differentiation refers to the process of undifferentiated gonads with two-way potential developing into spermary or ovary and secondary sexual characteristics after a series of events that occur in a programmed way.Sex reversal refers to the process that an organism changes from its original sex to another sex through a series of biological pathways on the basis of its original sex.The organism that can naturally undergo sex reversal is called hermaphrodite.In addition to bisexual animals,hermaphrodites can be divided into two types,including male precocious and female precocious,and the common species in fish are the Lates calcarifer and Monopterus albus.In the sex reversal of fish,estrogen is closely related to sexual reversal in fish,Hsd17b1 gene as a highly related gene for estrogen,The lack of expression of the Hsd17b1 gene will lead to the decline of the reproductive performance of animals,and when it is overexpressed,it will lead to the maleness of animals.However,it is not clear whether the Hsd17b1 gene plays a certain role in sex reversal animals.Therefore,we studied the Hsd17b1 gene of two sex reversal animals(Lates calcarifer and Monopterus albus)in order to explore the role of this gene in sex reversal animals.The specific work is as follows:1.We cloned and analyzed the Hsd17b1 gene in Lates calcarifer.The cloned c DNA fragment of the Hsd17b1 gene in Lates calcarifer was 1287 bp in length,including a 3’untranslated region(UTR)of 385bp and an open reading frame(ORF)of 873 bp,encoding a total of 290 amino acids,including the NADB Rossmann super family conserved domain.The gene was relatively conserved in evolution,the similarity of over 50%with that in other species.Previous studies found that the Hsd17b1 gene was expressed in granulosa cells,while the Zar1 gene was specifically expressed in germ cell,thus,we also isolated and analyzed the Zar1 gene in the sea bass,and the cloned Zar1 c DNA fragment of Lates calcarifer was 1192bp,including 16 bp in the 5’UTR,165 bp in the 3’UTR,and 1011 bp in the ORF.This sequence encoded336 amino acids in total,it contains special zinc finger domain.2.The expression of Hsd17b1 and Zar1 in different tissues of Lates calcarifer was analyzed.The results showed that the Hsd17b1 and Zar1 genes were specifically expressed only in the ovaries of the eight individual tissues of Lates calcarifer(intestine,hypothalamus,heart,liver,spleen,kidney,testes,and ovaries).The results of chemical in situ hybridization showed that neither the Hsd17b1 antisense probe nor the Zar1 antisense probe detected a positive signal in the testis.The positive signal of Hsd17b1 in the ovary was detected in oogonium,primary growth stage oocytes,and granulosa cells of mature oocytes.However,compared to growing oocytes,the signal detected in oocytes was stronger,while almost no signal was detected in mature oocytes.The positive signal of Zar1 in the ovary was detected in both oogonium and primary growth stage oocytes,with the strongest positive signal of Zar1 detected in growing oocytes.The signal in oocytes was weak,while there was almost no signal in mature oocytes.3.We first studied the expression patterns of Hsd17b1 of Lates calcarifer which is a male precocious hermaphrodite fish,and then we also investigated the mechanism of Hsd17b1 gene in another female precocious hermaphrodite fish which is Monopterus albus.Thus,we cloned and analyzed the Hsd17b1c DNA fragment of Monopterus albus,which has a length of 1101 bp,including a 5’UTR of 81 bp,a 3’UTR of 147 bp,and a 873 bp ORF,encoding 290 amino acids.Like the pointed snouted bass,the Hsd17b1gene of the yellow eel also contains a NADB Rossmann super family conserved domain.In addition,the similarity between the proteins encoded by the yellow eel and pointed snouted bass is as high as 90.7%.4.We analyzed the tissue expression of the Hsd17b1 gene in Monopterus albus,and the results showed that the Hsd17b1 gene was specifically expressed in eight individual tissues of the eel(intestine,hypothalamus,heart,liver,spleen,kidney,testes,and ovaries),only in the ovaries.In the gonads of Monopterus albus at different developmental stages(ovaries,early intersex,mid intersex,late intersex,and testes),the results showed that the expression of Hsd17b1 showed a trend of first increasing and then decreasing,peaking at mid intersex.From female to early intersex,and then to mid intersex,the expression of Hsd17b1 gradually increased.The expression was relatively highest in mid intersex,and then began to gradually decrease.The expression was relatively low in late intersex,in males,Hsd17b1 was almost not expressed in testis.Meanwhile,in situ hybridization results showed that no Hsd17b1 positive signal was detected in the late-stage gonads and testes of the eel;The positive signal of Hsd17b1 in the ovary was detected in oogonium,primary growth stage oocytes,and granulosa cells of mature oocytes.Compared with growing oocytes,the signal detected in oocytes was stronger,while in mature oocytes,almost no positive signal was detected.In the early intersex gonads,the results were consistent with those in the ovaries.Hsd17b1 positive signals were detected in oogonium,primary growth stage oocytes,and granulosa cells of mature oocytes.Compared with growing oocytes,stronger signals were detected in oocytes,while almost no positive signals were detected in mature oocytes.In the intermediate gonads,the results are similar to those in the ovaries.Hsd17b1 positive signals are detected in oogonium,primary growth stage oocytes,and granulosa cells of mature oocytes.Compared with growing oocytes,the signals detected in oocytes are stronger,and all signals are stronger than those in the ovaries.Positive signals are almost undetectable in mature oocytes,no positive signals were detected in the degenerated oocytes.5.Two experimental groups and a control group were set up to treat the gonads of Monopterus albus,with three replicates in each group.The control group consisted of 8×10-9mol/L estrogen and 8×10-9mol/L androgen,while the control group consisted of 0.1%DMSO.After 72 hours of treatment,the results showed that the relative expression of the Hsd17b1 gene in the early intersexual gonads of Monopterus albus treated with estrogen and androgen was significantly increased(P<0.05).In the intermediate gonads,estrogen and androgen have an upregulation effect on the expression of the Hsd17b1 gene in Monopterus albus,but not significantly.In the ovaries,there was no change in the expression of the Hsd17b1 gene after estrogen treatment,while there was a slight upregulation after androgen treatment.These results indicated that the Hsd17b1 gene plays an important role in the gonadal development of sex reversal fish,and mainly plays a role in the early oogenesis and ovarian development,which may be involved in the process of sex reversal.The findings of this study will provide a theoretical basis for elucidating the role of the Hsd17b1 gene in gonadal development and regulation of sex reversal in fish. |