| Photoperiod is one of the most important factors affecting fish reproduction.Fish in nature regulate reproductive activity such as sexual maturity and ovulation by sensing photoperiod changes to ensure higher survival rates of offspring.At present,photoperiod manipulation has become an effective tool to promote or delay the gonadal development of economic fishes,which greatly improves the economic benefits of fishes.A comprehensive understanding of the interaction between photoperiod and gonadal development of teleosts is of great significance for optimizing the artificial reproduction of teleosts.Studying the influence mechanism of photoperiod on fish ovarian development and grasping the dynamic response mechanism of ovarian autocrine factors in this process can supplement our understanding of fish adaptive physiology and reproductive physiology,and provide theoretical reference for fish actual production.In this study,zebrafish were used as experimental subjects.Fertility,plasma hormone content and expression of genes related to ovarian development of female zebrafish were detected under continuous dark(0L:24D,DD),natural photoperiod(14L:10D,LD)and continuous light(24L:0D,LL),focusing on the role of TGF-βsignaling pathway in this process,in order to provide a basis for further study of photoperiod regulation of zebrafish ovarian development.The specific research results are as follows:1.The effects of photoperiod on ovarian development of zebrafish were evaluated by analyzing the reproductive indicators of female zebrafish under different photoperiod conditions.The results showed that the gonadosomatic index(GSI),absolute fecundity(F),body length relative fecundity(F_L)of female zebrafish in DD group were apparently higher than those in LD group and LL group(P<0.05),but no significant difference was observed between LD and LL groups,and the body weight relative fecundity(F_W)was not significantly different under three photoperiods.The proportion of oocyte in previtellogenic(PV stage)was the highest in all three photoperiods,but the proportion of oocyte in PV stage in LL group was apparently higher than that in DD group(P<0.05).With the extension of light time,the number of oocytes in midvitellogenic stage(MV stage)and full-grown immature stage(FG stage)decreased successively,and the number of oocytes in DD group was significantly higher than that in LL group(P<0.05).The yolk diameter of fertilized eggs in DD group was significantly larger than that in LD and LL group(P<0.05),but no significant difference was observed between LD and LL groups.With the extension of light time,the absolute spawning and relative spawning index of zebrafish decreased gradually,and LL group was significantly lower than DD group(P<0.05);the fertilization rate and hatching rate of LL group were significantly lower than DD and LD group(P<0.05),but there was no significant difference between DD and LD groups.These results indicated that continuous darkness promoted ovarian development of zebrafish,and continuous light inhibited ovarian development of zebrafish.2.The concentrations of estradiol-17b(E2)and vitellogenin(VTG)in plasma and the relative expressions of esr and vtgs in zebrafish under different photoperiod conditions were detected by enzyme-linked immunosorbent assay(ELISA)and quantitative real-time PCR(q-PCR).The result showed that the levels of E2 and VTG in plasma of female zebrafish were relatively stable after 3 days of different photoperiod treatments.However,after 7 days of photoperiod treatment,the contents of E2 and VTG in plasma decreased with the prolongation of illumination time,and the DD group was significantly higher than the LL group(P<0.05).The expression patterns of three estrogen receptors(esr1,esr2a,esr2b)in the liver and ovary of zebrafish were similar under different photoperiod treatments.After 3 days of different photoperiod treatment,the relative expression of esr1,esr2a and esr2b in the liver and ovary of zebrafish remained stable;however,continuous dark treatment for 7 days significantly increased the expression of esr1 and esr2b in the liver(P<0.05).Continuous dark treatment for 7 days also significantly increased the expression of five vitellogenin genes(vtg1-vtg5)in the liver of zebrafish(P<0.05);however,no significant was observed in the expression patterns of vtg6 and vtg7 under three photoperiods.The above results indicate that continuous darkness promotes the synthesis of VTG by increasing E2 secretion and up-regulating the expression of esr,thereby promoting yolk development and ovarian development.3.The expression and localization of related genes and proteins in TGF-β-Smad/p38MAPK signaling pathway in zebrafish ovary were investigated by q-PCR,immunohistochemistry(IHC)and immunoblotting(WB).The results showed that the expression patterns of TGF-β-Smad/p38MAPK signaling pathway in zebrafish ovary were different under different photoperiod conditions.And the expression profile of TGF-βfamily ligand(tgfb3)and receptor(tgfbr2a,tgfbr2b,tgfbr1b)and downstream protein kinases(smad2,smad3a,p38β)were consisten.The relative expression levels of the above 6 genes were the highest in the DD group and the lowest in the LL group after 3days of treatment to different photoperiods,and the opposite tend were observed after 7days.The results of immunohistochemistry showed that phosphorylated smad2 protein signal was detected in the development of zebrafish oocytes from previtellogenic stage(PV)to full-grown immature stage(FG),but the expression position was different.In the oocytes of PV and EV stage,p-smad2 was mainly expressed in the nucleus.In MV and FG oocytes,p-smad2 was mainly expressed in the cytoplasm.Weak p-smad2 positive signals were detected in the follicular cells.However,photoperiod treatment was failure to affect the localization of p-samd2 in the zebrafish ovary.The results of western blot results showed that continuous light treatment significantly increased the level of phosphorylated p38MAPK protein in the ovaries of zebrafish after 3 days of different photoperiod treatment(P<0.05),but the difference disappeared after 7 days of photoperiod treatment.The above results suggest that photoperiod changes may affect the development of zebrafish ovaries by changing the expression pattern of tgfb3/receptor/protein kinase gene,but the deeper mechanism need to be further studied and explored. |
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