| It is well established that estrogens play key roles in female reproduction in vertebrates.The biological functions of estrogens are exerted via the estrogen receptors（ERs）,a series of ligand-dependent nuclear transcription factors.In mammals,accumulating evidence has suggested the significance of ERαin female reproduction.However,the precise physiological role and molecular mechanisms of estrogen/ERαsignaling in reproductive processes remains poorly defined in fish.In the present study,an ERα（esr1）mutant zebrafish line was generated via TALEN.The essential roles of ERαin ovarian maintenance and vitellogenesis in fish were demonstrated.The main points of this study were summarized as followed.1.Establishment of esr1 deficient zebrafish lineTo disrupt esr1 gene in zebrafish,TALEN target site for the zebrafish esr1 gene was designed.The TALEN target site was located on the third exon.The mutation rate was 50%（9/18）in the adult P0 fish.An 8-bp deletion mutation,inducing an open reading frame（ORF）shift and early termination of translation,was chosen to establish the esr1 mutant line.2.Morphology and ovarian histology of esr1 mutant female zebrafishAlthough the body length of wild-type and esr1 mutant zebrafish did not show significant difference during lifespan,the body weight of esr1 mutant female zebrafish was significantly higher at 90 days postfertilization（dpf）.As well,the GSI of esr1 mutant female zebrafish significantly increased at 60 and 90 dpf but dramatically declined at 180 dpf.Histological analyses showed similar ovarian developmental stage in both zebrafish lines in pubertal stage（60 dpf）and young adult stage（90 dpf）.However,in part of ovaries at 120 dpf and all ovaries at 180 dpf,MV,LV and FG follicles were absent and only early stage follicles（PG and PV stage follicles）and atretic follicles were left.3.esr1 mutant female zebrafish developed premature ovarian failureTracing assessment of fertility showed that esr1 mutant female zebrafish possessed normal fertility as wild-type females at 90 dpf.However,they showed signs of an age-dependent fertility loss from 105 dpf that the number of spawned esr1 mutant zebrafish reduced with age.By 180 dpf,all esr1 mutant females were infertile,while the age-matched wild-type females were still fertile.Moreover,esr1 deficient zebrafish produced more eggs compared to age-matched wild-type zebrafish from 90 dpf to 150 dpf.4.Steroidogenesis pathway in esr1 mutant female zebrafishWestern blot analyses showed that the pituitary protein levels of both FSHb and LHb remained unchanged in esr1 mutant zebrafish at 90 dpf,yet they both significantly declined by the age of 180 dpf.E2 level was significantly higher and 11-KT was unchanged in esr1 mutant females at 90 dpf.However,they both decreased dramatically by the age of 180 dpf.RT-PCR analysis showed that expression levels of steroidogenic genes,including star,hsd17b1,hsd17b3,cyp19a1a and fshr,increased significantly in esr1 mutant females at 90 dpf.However,the expression of all genes examined significantly decreased at 180 dpf.5.IGF system and mTOR signaling pathway in esr1 mutant female zebrafishRT-PCR analysis showed that at 90 dpf,the transcriptional levels of tsc genes were significantly down-regulated.Expression of several genes in mTOR signaling pathway increased significantly in esr1 mutant female zebrafish.Furthermore,the expression of IGFs was also significantly up-regulated.However,by the age of 180 dpf,the expression levels of both mTOR signaling pathway and the IGF system were comprehensively down-regulated,suggesting that it may be related to premature ovarian failure in esr1 mutant female zebrafish.6.Inhibition of mTOR signaling pathway led to ovarian failureIn order to clarify the role of mTOR signaling pathway in ovarian maintenance,in vivo exposure to rapamycin,an mTOR inhibitor,was conducted to adult wild-type female zebrafish.After treatment with 50 nM rapamycin for 14 days,these female zebrafish were infertile.GSI of them decreased significantly.Moreover,MV,LV and FG stage follicles were absent.Only PG and PV stage follicles and a few atretic follicles were found,suggesting that inhibition of mTOR signaling pathway led to ovarian failure.7.Vitellogenesis in esr1 mutant female zebrafishGenerally,LV stage follicles are filled with vitellogenic granules and the granules in FG follicles are well developed and large.However,vitellogenic granules were only found in the lateral part of esr1 mutant LV stage follicles.Moreover,only few large vitellogenic granules were found in esr1 mutant FG stage follicles and many of the vitellogenic granules in these follicles were small size ones.8.Expression profiles of estrogen receptors and Vtg genes in liverMales were defined as a good model for researches in vitellogenesis.In wild-type and esr1mutant male zebrafish,esr2a was found to be the most abundant mRNA of ER genes and significantly higher than the expression of other ER genes.While compared to wild-type zebrafish,the expression level of esr2a was unchanged and esr2b significantly decreased.Interestingly,the expression level of gper dramatically increased in esr1 mutant zebrafish.In wild-type zebrafish,vtg1 was the highest expressed Vtg gene.However,in esr1 mutant zebrafish,the baseline of vtg1 significantly decreased.While the expression level of vtg2 and vtg3 did not change markedly.9.Regulatory effect of nERs on gper expression in liver of zebrafishIn wild-type zebrafish,E2 significantly induced the expression of esr1 and gper in vitro and in vivo.But it had no effect on ERβs expression in vitro and decreased their expression in vivo.Upon ICI treatment,the nuclear ERs were inhibited.The induction of gper by E2 failed to be attenuated by ICI both in vitro and in vivo.Similarly,in esr1 mutant zebrafish,E2 induced expression of gper in vitro and in vivo.ICI did not attenuate the induction of gper by E2 in vitro and in vivo.PPT,selective activator of zfERβ,significantly increased the expression of esr1and gper in wild-type zebrafish in vivo.In esr1 mutant zebrafish,in vivo treatment with PPT significantly decreased the expression of gper.10.Regulatory effect of GPER on nER mRNA levels in liver of zebrafishIn wild-type zebrafish,G-1,a GPER agonist,did not significantly alter the expression levels of esr1,esr2a and esr2b in vitro.However,in vivo exposure to G-1 significantly up-regulated the expression of esr1 and esr2b,but decreased the expression of esr2a.In esr1mutant zebrafish,upon G-1 treatment,the expression of esr2a decreased in vitro and in vivo,but the expression of esr2b was induced in vivo.11.Functional significance of nERs in regulation of vitellogenesis in liver of zebrafishE2 increased the expression of vtg1,vtg2 and vtg3 significantly in wild-type zebrafish.However,such inductions were significantly attenuated by ICI.In esr1 mutant zebrafish,induction of Vtg expression by E2 was attenuated.Such inductions were also blocked by ICI.Treatment of PPT significantly induced the expression levels of vtg1,vtg2 and vtg3 in vivo in wild-type zebrafish.In esr1 mutant zebrafish,in vitro and in vivo treatment of PPT significantly up-regulated the vtg1 expression.12.Impact of GPER on vitellogenesis in liver of male zebrafishG-1 significantly induced the expression of Vtg genes in vitro and in vivo in wild-type zebrafish.While treated with E2 and G-15,the induction of Vtg genes by E2 was significantly attenuated by G-15 in vitro and in vivo.Similarly in esr1 mutant zebrafish,expression levels of Vtg genes increased significantly upon G-1 treatment in vitro and in vivo.In vivo treatment of G-15 significantly attenuated the induction of vtg1 and vtg3 by E2.Neither E2 alone nor co-treatment with G-15 altered the expression of vtg2 in vitro and in vivo.ICI decreased the expression of Vtg genes in wild-type zebrafish but not in esr1 mutant zebrafish.G-1significantly attenuated the inhibitory effect of ICI on Vtg genes in wild-type zebrafish and significantly increased expression of Vtg genes in esr1 mutant zebrafish. |
