Relationship Between Rspo1 And Zebrafish Danio Rerio Ovary Development

R-Spondinl (Rspol) is a potential female-determining gene in mammals (i.e. human and mouse), whoes differential expression associates with sex determination. However, no attempts have been made to characterize Rspol in fish. In this study, we first cloned and analyzed zebrafish Danio rerio Rspo1(zRspol) and confirmed a single Rspol transcript in both developing and adult zebrafish. By fusing zebrafish Rspol with a green fluorescence gene, we found that zRspol disperses in cytosol, locates on Golgi apparatus and, cell membrane but not nucleus in fish epithelioma papulosum cyprinid (EPC) cells, zebrafish liver cells (ZFL) and human kidney HEK293T cells. Subcellular analysis shows that zRspol locates on cell membrane, suggesting that zRspol could function onβ-catenin signaling pathway. In addition zRspol does not locate on nucleus suggests that zRspol differs from other mammalian R-spondins in some aspects.Rspol was suspected to control PGCs (Primordial germ cells) division, proliferation and somatic cell differentiation in male and female mammalians. The change of Rspol expression potentially affects other molecular events during gonads differentiation. Through real-time PCR method, we observed zRspol expression throughout the development of zebrafish. We also observed a consistently higher zRspol expression in adult ovary than that in testis, suggesting roles of zRspol in PGCs, and gonad differentiation. A high expression of zRspol was detected in some non-gonadal organs(e.g. muscles, kidneys) as well. Our in situ hybridization results demonstrated that zRspol expresses in both premature germ cells (ogania, primary oocytes in ovaries, spermatogonias and spermatocytes in testes) and somatic cells (granulosa cells and theca cells of early and late cortical-alveolar stage follicles in ovaries, leydig cells in testes) of gonads during gonadal development, suggesting role(s) of Rspol in zebrafish gonads development and differentiation.Zebrafish ovary contains follicles of all differentiating stages. Primary follicles develop to mature eggs in adult female ovary. Cox-2, which normally expresses in granulosa cells in mammalian ovaries and whose expression is regulated byβ-catenin signaling, controls follicular maturation and predicts ovulation time. Rspol, a secretory protein, determines mammalian gonad development direction through regulatingβ-catenin. However, neither mechanism(s) controlling vertebrate cox-2 expression nor the function of Rspol has been investigated in ovaries. We have observed zRspol transcripts in granulosa cells from zebrafish ovaries. In this study, we firstly documented zRspol and cox-2 temporal expression during follicular development in zebrafish ovary. Both of the two genes share a similar expression pattern—reaching a peak at late stageⅢof follicle growth and decreasing during the transition of late stageⅢto stageⅣof follicle growth. Then we therefore suspected that cox-2 might be regulated by zRspol in ovaries. We provide direct evidence supporting that cox-2 expression is controlled by zRspol through quantifying cox-2 transcripts in zRspol up-and down-expressed in vitro cultured ovarian follicle cells. Together, we conclude that zRspol product could participate in the regulation of cox-2 expression during ovarian follicle development. MT (17α-methyl-testosterone) treatment resulted in expression reduction of both zRspol and cox-2 suggesting that zRspol expression and its down signaling could be controlled by hormone. Our data support that Rspol could controlβ-catenin signaling.Except for cox-2, the expression of cyp19ala, able to transform androgen to estrogen, was controlled by zRspol in in vitro cultured zebrafish follicle cells. Our study show that the presence of hCG is able to turn over the regulation of cypl9al a expression by zRspo1:Cyp19a1a expression is up-regulated with hCG treatment and down-regulated without hCG when zRspol was over-expressed. Accompanied by the decrease of cypl9ala expression resulted from zRspol over-expression and hCG absence, Estrogen product level declined correspondingly.In conclusion, my study established that 1) zRspol locates in cytosol, Golgi apparatus and membrane,2) zRspol differentially expresses in zebrafish gonalds,3) zRspol controls the expression of gene cox2 and 4) zRspol regulates estrogen synthesase cypl9ala expression depending upon the presence of hCG.