The Roles Of Estrogen Receptors(esr1,esr2a And Esr2b) On Gametogenesis In Nile Tilapia

Estrogens play fundamental roles in regulating sex differentiation and reproductive activities and they act through estrogen receptors in all vertebrates.In tetrapods,there are two different estrogen receptor genes,Esr1 and Esr2,while in teleosts,due to unique genome replication,there are three estrogen receptor genes,esr1,esr2a and esr2b.To date,distinct roles of estrogen receptors have been characterized only in human and model organisms,including mouse,rat,zebrafish and medaka.Physiological role of estrogen/receptor signaling in reproduction remains poorly defined in non-model organisms,and which esr mediates the critical role of estrogen in sex determination and gametogenesis is still unknown in tilapia.In the present study,utilizing esr1 mutants which were generated by CRISPR/Cas9 and esr2a and esr2b mutants which were established in our lab,we analyzed the effects of esr deletion on gametogenesis and reproduction in female and male tilapia.The results are as follows:1.Generation and phenotype analysis of esr1 homozygous mutants.esr1 mutants were successfully generated by CRISPR/Cas9 and the target site was located on the first exon.The restriction enzyme HypAV located on the target site was selected to screen the F0 fish.Heterozygous mutants were obtained by mating esr1 mosaic F0 XY with WT XX fish.XX and XY heterozygous fish with an 8-bp deletion were selected by the polyacrylamide gel electrophoresis?PAGE?to breed homozygous mutants.Analysis of the gonadal phenotype of F2 male and female fish showed that,there was no differences in GSI?gonadal somatic index?,gonadal morphology,gametogenesis,serum E2 and 11-KT level and fertility between esr1^-/-and WT fish.The expressions of esr2a and esr2b in esr1^-/-XX and XY were examined by real-time PCR,and the results showed that there was no significant differences between esr1^-/-mutants and WT fish except up-regulated expression of esr2a in esr1-/-XX fish.2.The phenotypes analysis of esr2a mutants.The esr2a homozygous mutants had been established in our lab.Due to the limited time,only the ovarian phenotypes of esr2a-/-XX fish at 90 dah were analyzed.Previous study showed that at 90 dah?days after hatching?,WT XX ovaries contained phase I and phase II oocytes,while esr2a^-/-XX ovaries were filled with oogonia and phase I oocytes.The present study further analyzed the gonadal phenotypes of esr2a^-/-XX fish at 180 and 360 dah and esr2a^-/-XY fish.The results of present study showed that,at 180 dah,WT XX ovaries contained phase I to?oocytes,while the esr2a^-/-ovaries were filled with only phase I,phase II and a few phase III oocytes,indicating the oogenesis in the esr2a-/-ovaries was retard.The ovaries of esr2a-/-were smaller and the GSI of esr2a^-/-was significantly lower than those of the WT XX fish.But at 360 dah,esr2a^-/-ovaries were composed of all stages of oocytes?phase I to??,and the ovarian morphology,serum E2 level and fertility of esr2a^-/-XX showed no significant differences from those of the WT XX,indicating the retard oogenesis was restored.At 90 dah,WT XY testis contained spermatogonia and spermatocytes.At 180 dah,abundant spermatids and spermatozoa were observed in the WT XY testis.Compared with WT XY fish,at 90 and 180 dah,GSI,testis morphology,spermatogenesis,and serum 11-KT level of esr2a^-/-XY fish showed no significant differences from those of the WT XY fish,but less spermatogonia and smaller efferent duct area were observed in the esr2a-/-testes compared with the WT XY testes.esr2a^-/-males showed decreased fertility rate compared with that of the WT XY fish.Further analysis of the sperm revealed that some abnormal sperms were observed in the esr2a^-/-XY mutants.Consistently,the VCL?Curvilinear velocity?,VSL?Straight linear velocity?and BCF?Beat frequency of sperm flagella?of sperms from the esr2a^-/-XY mutants were significantly lower than those of the WT XY fish.Real-time PCR showed that the expression of esr1 and esr2b up-regulated in the esr2a-/-XX and XY mutants.3.The phenotypes analysis of esr2b mutants.The esr2b homozygous mutants had been established in our lab.Due to the limited time,only the ovarian phenotypes of esr2b^-/-XX fish at 90 dah were analyzed.Previous study showed that at 90 dah esr2b-/-XX ovaries contained phase I and phase II oocytes as WT XX ovaries.The present study further analyzed the gonadal phenotypes of esr2b^-/-XX fish at 180 and 360 dah and esr2b^-/-XY fish.The results of present study showed that,at 180 and 360 dah,the oogenesis of esr2b^-/-XX mutants proceeded normal as the WT XX fish.However,they were infertile due to abnormal development of the ovary,which failed to fuse the genital orifices.The esr2b^-/-XX mutants showed significantly higher level of serum E2 level than the WT XX.At 360 dah,most oocytes in the esr2b^-/-ovaries were degenerating.IHC?immunohistochemistry?showed that,positive signals from PCNA?cell proliferation marker?,Caspase3?cell apoptosis marker?and Cyp11b2?key enzyme responsible for the synthesis of the androgen 11-KT?were significantly higher in the esr2b^-/-XX mutants than WT XX.Compared with the WT XY fish,at 90 and 180 dah,GSI,spermatogenesis,efferent duct area and serum 11-KT level of esr2b^-/-XY showed no significant differences from those of the WT XY fish,but esr2b-/-XY mutants were infertile due to abnormal development of the testis which failed to fuse the genital orifices.Interestingly,sperms isolated from the dissected esr2b^-/-testes displayed fertilization rate similar to that of the sperms isolated from the WT XY fish.Further analysis of the sperm revealed that,the sperm from the esr2a^-/-XY mutants showed normal morphology.Consistently,no significant differences were observed in sperm VCL,VSL,BCF and the ratio of the abnormal sperm between the esr2b-/-and WT XY fish.Real-time PCR showed that the expression of esr1 and esr2a down-regulated in the esr2b-/-XX mutants and only the expression of esr2a significantly up--regulated in the esr2b^-/-XY mutants.4.The phenotypes analysis of esr2a^+/-esr2b^+/-XX fish and WT XX fish injected with PHTPP.Utilizing esr2a and esr2b mutants which were established in our lab,we established the esr2a^+/-esr2b^+/-mutants,and analyzed their gonadal phenotypes.The results showed that,compared with WT XX fish,at 180 dah,esr2a^+/-esr2b^+/-XX ovaries were filled with many oogonia and very few phase II and phase III oocytes.IHC showed that,positive signals from Cyp19a1a were significantly lower in the esr2a^+/-esr2b^-/-XX mutants than WT XX.Positive signals from Cyp11b2 were significantly higher in the esr2a^+/-esr2b^-/-XX mutants than WT XX.It was very difficult to establish the esr2a-/-esr2b-/-mutants due to infertile esr2a+/-esr2b-/-XX fish.PHTPP was a selective Esr2 antagonist of mammal.It had been proven in vitro that it could inhibit effectively esr2a and esr2b instead of esr1 in tilapia.After injected with PHTPP in vivo,female ovaries reversed to male testes at 180 dah histologically.In summary,in the present study,utilizing esr1 mutants which were generated by CRISPR/Cas9 and esr2a and esr2b mutants which were established in our lab,we analyzed their phenotypes.esr1 is dispensable for folliculogenesis and spermatogenesis;esr2a is required for folliculogenesis in female and normal sperm morphology in male;esr2b is required for ovarian duct and efferent duct development in female and male,respectively.Taken together,our study enriched the differential functions of esr1,esr2a and esr2b in fish reproduction and understanding the molecular mechanisms of esrs mediate estrogen regulating sexual differentiation and gametogenesis.