| Nuclear receptor liver receptor homolog-1(LRH-1/NR5A2)is an important transcription factor.In vertebrates,the structure of LRH-1 is highly consered.LRH-1acts as a key regulator of metabolism,development,cholesterol homeostasis and steroidogenesis.It is reported that,in rodent ovary,LRH-1 is localized specifically to granulosa cells of the follicle and to cells of the corpus luteum,but not in theca cells.In rat testis,LRH-1 mRNA and protein are expressed in Leydig and germ cells,but not in sertoli cells.Mice homozygous for a germline mutation of the LRH-1 gene die between embryonic day E6.5 and E7.5 in part because LRH-1 is necessary for formation of endoderm.In female rat,heterozygosity for a null mutation of LRH-1 leads to a decreased fertility and reduced reproductive ability of LRH-1+/-females.In male rat,LRH-1+/-mice showed lower plasma testosterone concentrations and lower seminal vesicles weights compared with LRH-1+/+ mice.In teleosts,the semiquantitative RT-PCR showed that LRH-1 expressed in the liver,ovary,intestines,fat,blood,head kidney,pituitary,kidney,swim bladder,and gill filament.It is reported that LRH-1could upregulate cyp19a1a/cyp19a1 b transcription in vitro in cyp19a1a/cyp19a1 b promoter.The genome of the Nile tilapia(Oreochromis niloticus)has been sequenced and opened,and eight gonadal transcriptomes have been sequenced and mapped to this reference genome by our group.To provide further insights into the function of LRH-1in teleost,here we examined the expression profile of LRH-1in different tissues in tilapia by RT-PCR.We documented the cellular localization of LRH-1 during variousstages of gonad development by immunohistochemistry.In addition,we used the CRISPR/Cas9 system to mutate LRH-1 in both sexes of the Nile tilapia to investigate its roles in steroidogenesis and oogenesis.The results are as follows:In the present study of tilapia,LRH-1 protein expression were found to be higher in the ovary than in the testis.The structure of LRH-1 in different species is highly conserved.We got polyclonal antibody of the orange-spotted grouper LRH-1 from Prof.Weimin Zhang' s lab(Sun Yat-Sen University).The specificity of the LRH-1 polyclonal antibody was confirmed by Western blot.Specific band(85kDa)was recognized in the ovary and testis in tilapia using grouper's LRH-1 antibody.LRH-1 immunostaining was observed by IHC in the XX and XY gonads.LRH-1 is predominantly expressed in steroidogenic cells.In the ovary,LRH-1 is expressed in the oogonia,oocyte,interstitial,and theca cells.In the testis,LRH-1 is expressed in the spermatogonia,spermatogoocyte and Leydig cells.IHC staining of adjacent sections in series from XX gonads confirmed that LRH-1 was co-expressed with Cyp19a1a/Cyp17a1/Cyp17a2 in the interstitial,and theca cells of the ovary.We mutate LRH-1 successfully by using CRISPR/Cas9.In 120 dah XX fish,mutation of LRH-1 resulted in reduced levels of Cyp19a1a/cyp19a1 a,scc,starI and 17?-estradiol(E2)compared with normal XX fish.In 210 dah XX fish,mutation of LRH-1 resulted in delayed ovary development and more interstitial cells were observed compared with normal XX fish.We use LRH-1 mutant F0 XY fish to cross with normal XX fish,and produced LRH-1 F1 generation.In 90 dah XX fish,LRH-1 heterozygous mutation resulted in reduced interstitial cells and the levels of Cyp19a1a/cyp19a1 a,cyp17a1?cyp17a2?star2?scc were also reduced.In 210 dah XX fish,LRH-1 heterozygous mutation resulted in delayed ovary and most eggs were not mature.In XY fish,LRH-1 heterozygous mutation resulted in delayed spermatogenesis,disorganized seminiferous lobules,decreased levels of Cyp19a1b/cyp19a1 b and17?-estradiol(E2).In summary,our results demonstrated that the expression pattern of LRH-1 in tilapia is similiar to mammals which is expressed in steroidogenic cells and germ cells.LRH-1 is an important regulator in ovary oogenesis and is a potential regulator of estradiol production,demonstrating that LRH-1 is conserved in expression and function in the vertebrate. |
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