| Gonadal somatic cells are the main component of fish gonad.Their important and even leading roles during gonadal differentiation and development of fish have attracted much attention.However,the study on them is limited to a few fish species.Their function in sex determination and differentiation is still unclear.Olive flounder(Paralichthys olivaceus)is an important maricultured fish and females grow much faster than males.This research mainly studied function of somatic cells during the flounder gonadal differentiation and development.Initially,characteristics and changing trends of proliferation of somatic cells during the gonadal differentiation and development processes were studied by using histological observation and expression patterns of the related genes including amh,oct4,cyp17a1,and 3β-hsd.And then,the order of action of somatic cells and germ cells was analyzed according to expression of the meiosis marker genes scp3 and dazl,and the genes related to sex steroid hormone synthesis cyp19a1 a and cyp11 b.At the same time,the effect of germ cells on somatic cells was evaluated with busulfan treatment.The results will provide a basis for clarifying roles of somatic cells in gonadal differentiation and development of the flounder and other fish.The main results are as follows:Before the gonadal differentiation,somatic cells were distributed around the filamentous primitive gonads.With development of the gonads,conical Sertoli cells and oval Leydig cells were gradually distributed around primordial germ cells(PGCs).After formation of ovarian cavity and vas deferens primordia as well as completion of the gonadal differentiation,the ovary was composed of oogonia and somatic cells including granulosa cells and theca cells,and the testis was composed of spermatogonia and somatic cells such as Sertoli and Leydig cells.Numbers of germ cells and somatic cells in the testis and ovary increased rapidly after the gonadal differentiation.Then seminal lobule in the testis and spawning plate in the ovary were formed,respectively.When the gonads developed to stage V,seminal lobule of the testis united,and the ovary formed a double-layer follicular cell structure.Ultrastructural observation under transmission electron microscope showed that granulosa cells of the ovary at stage II were oval or nearly round filled with chromatin,and theca cells were a long and narrow ellipse or cone shaped,mainly occupied by nucleus.Both often surrounded oocytes to form follicular cells.Leydig cells of the testis at stage II were oval in shape and filled between germ cells and seminal lobule,while Sertoli cells were cone-shaped or oblong in shape,participating in the formation of structure of seminal lobule and providing support for seminal lobule.During the gonadal differentiation,the results of real-time quantitative PCR(q PCR)and in situ hybridization(ISH)showed that amh was highly expressed in the gonads before the testicular differentiation,and mainly located in Sertoli cells,but didn’t express in the gonads before and during ovarian differentiation,suggesting that amh may initiate testicular differentiation.The results of q PCR,ISH,and immunohistochemical(IHC)showed that amh was mainly expressed in Sertoli cells around seminal lobules at stages I-V of the testis,and the highest expression of amh was detected in the testis at stage II.The 3β-hsd,oct4,and cyp17a1 were highly expressed at the early stage of the ovarian differentiation,and mainly expressed in the ovaries at stages I-V and located in oocytes at phases I and II,but only weakly expressed in Sertoli cells around seminiferous lobules at all stages of the testicular development,indicating that they play an important role in ovarian differentiation and oogenesis.So,amh can be used as a molecular marker of Sertoli cells in the testis,while 3β-hsd,oct4,and cyp17a1 are more suitable as molecular markers of oocytes in the ovary.Expression of meiosis marker genes scp3 and dazl was detected to learn the starting point of meiosis of the flounder germ cells.The results showed that the upregulated expression points of scp3 and dazl respectively appeared in the differentiated testis and ovary,suggesting that the flounder germ cells began meiosis only after the gonadal differentiation.Differential expression of genes related to sex steroid hormone synthesis cyp19a1 a and cyp11b2 was further analyzed.The former presented higher expression in the differentiated gonads,while the latter was slightly higher expression in the primitive gonads,but maintained at a low level in the differentiation gonads.However,levels of sex hormones testosterone(T)and estradiol(E2)all increased before and during the gonadal differentiation.T and E2 levels at 60 mm total length(TL)were the highest during the female differentiation,and their levels increased at 40 mm TL before the male differentiation.These results indicated that sex hormone levels seemed to have increased earlier than expression of cyp19a1 a and cyp11b2 in the gonads(including germ cells and somatic cells).RT-PCR results showed that scp3 and dazl were expressed in the flounder testes and ovaries at stages I-V,and expression of scp3 in the testes at stages II-V was significantly higher than that in the ovary(P < 0.05),and strong signals in primary spermatocytes of the testis were detected.While,expression of dazl in the ovary at stages I,II,and V was significantly higher than that in the testis(P < 0.05),and stronger signals were detected in oocytes of the ovary at stages I and II.Therefore,as molecular markers,scp3 is suitable for marking the meiosis process in the flounder testis,while dazl is more suitable for indicating the meiosis process of the ovary.Busulfan,a cytotoxic alkylating agent,is used to inhibit germ cell proliferation in some animal species.In fish species,studies with busulfan have been focused on the inhibition and transplantation of germ cells,and find that buulfan has effects on germ cells and resultes in transient or permanent sterility.However,effects of busulfan on gonadal somatic cells have not been fully studied.In this study,after intraperitoneal injection with 80(80B)or 120B(120B)mg/kg bufulfan in the adult flounder,both the bilateral gonads were atrophied,and the ovaries were discolored with adhesion to the visceral mass.Histological results indicated that germ cells in the gonads were detached,and there was a larger nucleus size and smaller cytoplasmic volume in spermatogonia.Numbers of spermatogonia and somatic cells in the testis were markedly less and greater,respectively(P < 0.05),while,in the ovary,numbers of oocytes and somatic cells were both less(P < 0.05).The q PCR analysis presented that,compared with the control groups,vasa expression in the ovary of the two treatment groups was not different,but its expression in the testis was markedly less(P < 0.05),indicating that the toxicity of busulfan may be sex-specific with there being greater effects in the males than the females.Furthermore,by using ISH,in the flounder ovaries of the two treatment groups,relative abundance of vasa and cyp19a1 a was very small in the cytoplasm of oocytes,while cyp19a1 a was still present in theca cells.In the testis,abundance of vasa was markedly less(P < 0.05)with there being very little vasa in spermatogonia and disrupted spermatogonia.In the 80 B treatment group,amh was in lesser abundance with there being very little amh in spermatogonia,however,with the 120 B treatment there was a large amh abundance in spermatogonia with there being disruption of structure of these germ cells and Sertoli cells.Busulfan,therefore,might inhibit the development of spermatogonia and lead to spermatogonia forming somatic-like cells in the flounder testis.After the flounder larvae in gonadal differentiation period was treated with 160 mg/kg busulfan,numbers of the PGC were markly less(P < 0.05),and few PGCs were observed in the gonads.These results implied that busulfan could affect the flounder gonadal differentiation and development,especially the development of spermatogonia in the testis. |
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