Roles Of Amh/Amhy In Tilapia Reproduction And Breeding Of Fertile YY Pseudofemales

Anti-Müllerian hormone（Amh）,a member of the transforming growth factorβ（TGF-β）,was widely involved in the regulation of cell proliferation,differentiation,apoptosis and migration.In tetrapods,the original function of Amh was to promote the degeneration of the Müllerian duct in male embryos.Subsequent studies reported that Amh was also expressed in the Sertoli cells of the testis and granulosa cells of the ovaries.Amh could inhibit the proliferation of spermatogonia in the testis and the recruitment of primordial follicles in the ovaries during male and female gametogenesis.Teleosts lack Müllerian ducts,but they still have orthologues of mammalian amh and its type 2 receptor amhr2 gene.Similar to mammals,amh was mainly expressed in the Sertoli cells of zebrafish and medaka testis and in the granulosa cells of ovaries.Mutation of amh in mammals and teleosts resulted in similar defects.In recent years,duplicates of amh and amhr2 have been reported as the male sex determining genes in several teleost species.In Nile tilapia（Oreochromis niloticus）,amh（on the X chromosome）tandemly duplicated on the Y chromosome,and amh on Y chromosome degenerated as amh△-y.The duplicate amhy was proved to be the male sex determining gene of Nile tilapia.Tilapia have many advantages,such as short spawning cycles,and available of monosex populations,with males growing faster than females.Production of genetic all male tilapia（GMT）has been the first choice for aquaculture.However,fertile YY pseudofemales were required as the prerequisite for mass production of GMT.But until now,it was difficult to induce transdifferentiation of YY males into pseudofemales by estrogen treatment.Even if the YY male were successfully induced to pseudofemales they could not spawning normally,which seriously hindered the application of GMT in aquaculture.The purpose of this study was to analyze the function of amh/amh△-y/amhy in reproduction,and analyze the reason for lower sex reversal rate and infertility of YY pseudofemales.Present study will help to understand functional complementation or redundancy of duplicate genes especially tandem duplicates during evolution,and enrich the cognition of amh genes in male and female reproduction in teleosts,and even in vertebrates.Furthermore,it is helpful for breeding fertile YY pseudofemales,and lay a foundation for the application of GMT.The results were as follows:1.Expression of amh/amh△-y/amhy and amhr2 in Nile tilapia.In XX females,RT-q PCR（Quantitative real-time PCR）analysis showed that amh and amhr2 were highly expressed in primary growth follicles of ovaries at 30 and 60 dah（day after hatching）,then decreased at 90,150 and 180 dah.Immunohistochemistry showed that Amh was specifically expressed in granulosa cells of phase I and II follicles,but not in phase III and IV follicles.Amhr2 was expressed in oogonia,oocytes and granulosa cells of phase I follicle,but not in phase II to IV follicles.In addition,RT-q PCR and immunohistochemical results showed that Amh and Amhr2 were also expressed in the brain and pituitary.In male fish,immunofluorescence analysis showed that Amh/Amhy were expressed in Sertoli cells of XY and YY testis at 5 dah,with higher expression of Amhy in the YY fish compared with the XY fish and lower at 30,90 and 180 dah in YY fish than XY fish.Opposite expression profile of Amh was observed in XY fish during the same stage,very low at 5 dah and significantly up-regulated at 30,90 and 180 dah.2.Effects of amh/amh△-y/amhy and amhr2 mutation on folliculogenesis in XX females and XY pseudofemales.Using the existing F0 mutants as founder,the F2homozygous mutant fish of amh/amhr2 were successfully obtained.The gonadal phenotypes of the mutants were analyzed.Both amh^-/-and amhr2^-/-females displayed hypertrophic ovaries and infertility.Histological analysis showed that oogonia were significantly increased in the ovaries of amh^-/-and amhr2^-/-females at 90 dah,while there was no significant difference in the number of phase I and II follicles.PCNA（proliferating cell nuclear antigen）proliferation assay revealed excessive proliferation of oogonia in the amh^-/-and amhr2^-/-ovaries.At 180 dah,the wild type（WT）ovaries contained follicles from phase I to IV,while the amh^-/-and amhr2^-/-ovaries contained only phase I and II follicles,indicating that folliculogenesis was arrested at the primary growth stage.Number of oogonia,phase I and II follicles in amh^-/-and amhr2^-/-ovaries was significantly higher than that of WT ovaries.In addition,serum estrogen level was significantly down-regulated in the amh^-/-and amhr2^-/-fish,however,exogenous estrogen treatment could not rescue the arrested folliculogenesis.TUNEL（terminal deoxynucleotidyl transferase-mediated d UTP nick end-labeling）assay revealed significantly increased apoptosis of granulosa and theca cells in the ovaries of amh^-/-and amhr2^-/-,which may be one of the reasons for arrested folliculogenesis.Changes of gene expression on the brain-pituitary-gonadal axis caused by amh and amhr2 mutation were analyzed by RT-q PCR and in situ hybridization.gnrh3 in the brain,fsh and lh in the pituitary were significantly down-regulated,and follicle development related genes in the ovaries such as cyp17a1,cyp17a2,foxl2,inha,fshr and lhr were significantly down-regulated in the amh and amhr2 mutants compared with those in the wild type fish.These results indicated that both amh and amhr2 homozygous mutation resulted in excessive proliferation of oogonia,and folliculogenesis arrest at the primary growth stage in XX fish,which in turn,resulted in hypertrophic ovaries and infertility.Amh may regulate folliculogenesis not only at the gonad level,but also through the brain-pituitary-gonadal axis.In order to see if there is functional complementation or redundancy of the amh/amh△-y/amhy,single,double and triple mutations were established in XY fish.The XY fish with amh single mutation were male,and the induced（by estrogen treatment）amh^-/Y psedofemales were fertile.Both amhy single and amhy/amh△-y double mutation in XY fish resulted in male to female sex reversal,and the reversed pseudofemales were fertile.Although amh/amhy double mutation or amh/amh△-y/amhy triple mutation in XY fish were also resulted in male to female sex reversal,the mutants（XY pseudofemales）were infertile and displayed excessive proliferation of germ cells and arrested folliculogenesis at the primary growth stage,similar to the phenotypes observed in XX female with amh homozygous mutation.These results indicated that amhy could compensate the function of amh in the absence of amh,while amh△-y could not.3.Effects of amh/amh△-y/amhy mutation on spermatogenesis.In order to study the function of amh/amh△-y/amhy in spermatogenesis,mutation of amh(XY-amh^-/Y)and amh△-y(XY-amh△-y^X/-)in XY fish and mutation of amhy(YY-amhy^+/-)in YY fish were established.Comparison of cell proliferation using Brd U（5-Bromo-2′-deoxyuridine）incorporation assay showed that mutation of amh resulted in excessive proliferation of spermatogonia in XY male,which in turn,resulted in increased spermatozoa and GSI（Gonadosomatic index）.Normal spermatogenesis was observed in the XY-amh△-y^X/-male and YY male,the latter lacks the amh gene naturally.However,excessive proliferation of spermatogonia and increased GSI were also observed in the YY-amhy^+/-male fish,consistent with the phenotypes observed in the XY-amh^-/Y male fish.Considering amhy was still expressed at low levels during spermatogenesis,normal spermatogenesis observed in YY fish without the amh gene could be explained by compensation of amhy.Taking together,these results indicated that amh△-y has no function in spermatogenesis.4.Reasons for infertility of YY pseudofemales and breeding of fertile YY pseudofemales.In order to obtain fertile XY and YY pseudofemales,the male to female sex reversal of XY and YY male fish were induced by estrogen treatment（E2,200ug/g feed）for 3 months continuously from 5 dah.Results showed that the sex reversal rate of XY fish was nearly 100%,while the sex reversal rate was about 30%in YY fish under the same conditions.In the estrogen induced XY pseudofemales,normal folliculogenesis was observed,and these XY pseudofemales were fertile.Even though folliculogenesis occurred,decreased E2 and elevated 11-KT level were observed in the YY pseudofemales.These YY pseudofemales showed male genital papilla,and were infertile.Compared with only one copy of amhy in XY fish,two copies of amhy exist in YY fish.We speculated that this might be the reason of lower sex reversal rate of YY fish and infertility of YY pseudofemales.In order to test our hypothesis,we analyzed the expression of Amh/Amhy in XY and YY pseudofemales,constructed heterozygous(YY-amhy^+/-)and homozygous(YY-amhy^-/-)mutants of amhy in YY fish,and analyzed their phenotypes.In the ovaries of XY pseudofemales,Amh/Amhy was detected in the granulosa cells of phase I and II follicles,and was absent in the phase III and IV follicles.Cyp19a1a was expressed in the granulosa cells of phase III and IV follicles.These results indicated reciprocal expression pattern between Amhy and Cyp19a1a.In the ovaries of YY pseudofemales,Amhy was detected not only in the granulosa cells of phase I and II follicles,but also in the granulosa cells of phase III follicles.Therefore,expression of Cyp19a1a in granulosa cells of phase III and IV follicles was inhibited in YY pseudofemales.Similar sex reversal rate was observed between the YY-amhy^+/-and XY-WT male fish by E2 treatment.In addition,the sex reversed YY-amhy^+/-pseudofemales were fertile.The YY-amhy^-/-fish underwent male to female sex reversal with ovaries displaying the same phenotypes as those in XX-amh^-/-fish,with excessive proliferation of germ cells,increased GSI and arrested folliculogenesis.These results further support the notion that amhy could compensate for the function of amh in folliculogenesis in the absence of amh.Two copies of amhy in YY fish is the reason for lower sex reversal rate of YY fish and infertility of YY pseudofemales as they exert increased antagonism to exogenous estrogen.Elevated androgen level resulted in male sexual characteristics and infertility observed in YY pseudofemales.Based on these results,we combined estrogen treatment with androgen synthetase inhibiter to induce sex reversal in YY fish.As a result,the sex reversal rate was significantly increased from 30%to 90%.To inhibit the elevated androgen in the latter stage,low dose of estrogen and androgen synthetase inhibitor were used to treat YY pseudofemales at the vitellogenesis stage（150-180 dah）.These treatment resulted in female secondary sexual characteristics and fertile YY pseudofemales.In summary,our results demonstrated that amh inhibits the proliferation of germ cells during gametogenesis through amhr2.Both amh and amhr2 were found to be expressed in the brain and pituitary except the gonads,suggesting that amh may regulate the proliferation and development of germ cells through the brain-pituitary-gonadal axis.Mutation of amh in XX fish resulted in excessive proliferation of germ cells,arrested folliculogenesis at the primary growth stage and infertility in female.However,XY male fish with amh mutation were fertile.Mutation of amh/amh△-y/amhy further confirmed that amhy was the male sex determining gene in Nile tilapia.In the absence of amh,amhy could compensate for the function of amh in gametogenesis,while amh△-y has no function in gametogenesis due to defunctionalization.The present study provided a perfect case for analyzing the functional complementation or redundancy of duplicate genes.It not only enriched the cognition of amh gene in male and female reproduction in teleosts and even in vertebrates,but also laid a foundation for the breeding of fertile YY pseudofemales for the establishment of YY maintenance line and GMT production.