Establishment Of Two ERβ Knockout Lines And Their Functional Study In Nile Tilapia

Estrogens are critical for sex determination and differentiation in fish,but the molecular mechanism behind them remains largely unknown.Estrogens regulate the expression of their target genes mainly through estrogen receptors(ER).As the same as most of teleosts,there are three estrogen receptor subtypes in Nile tilapia(Oreochromis niloticus): ERα,ERβ1 and ERβ2.In tilapia,which ER mediates the critical role of estrogen in sex determination,as well as the target downstream genes and its mechanism,is still unknown.Recently,the development of screening of ER specific agonists and antagonists,as well as the establishment of the CRISPR / Cas9 system in farmed fish in our lab provide valuable tools to elucidate the mechanism of ER mediated sex determination.Therefore,in this study,we aim to study the roles of two ER?(7)ER?(16)and ER?(17)(8)in sex determination using transcriptional activation in vitro and gene knockout by CRISPR/Cas9 in vivo.The results are in the following:1)Screening agonists and antagonists of ER specific to tilapia.In vitro analyses,estrogen E2(17β-Estradiol,E2)can induce the luciferase activity in a dose-dependent manner by ERα,ERβ1 and ERβ2.The EC50 of ERα,ERβ1 and ERβ2 was 1.9×10-9M,16×10-12 M and 1.3×10-10 M,respectively.The binding ability of estrogens to ERβ subtypes was 100 times higher than that of ERα in tilapia.Furthermore,to detect tilapia specific agonists and antagonists of ERs,we test whether agonists and antagonists of ERs specific to mammalian also have effects on tilapia using GAL4 system.The results showed that the mammalian ERβ specific agonist WAY can specifically activate the activity of ERβ2,but not ERα and ERβ1;the mammalian ERβ specific antagonist PHTPP can specifically inhibit the activity of the ERβ1 and ERβ2,but not ERα;the mammalian ERα-specific agonist PPT and antagonist MPP had no effect on any of the three ERs in tilapia.This indicated that the agonist and antagonist of mammalian ER are not entirely applicable to tilapia.2)The establishment of ERβ mutant lines in tilapia.The two ERβ subtypes(ERβ1 and ERβ2)were knockout using CRISPR/Cas9 in tilapia.The target mutant sites adjacent to protospacer motif(PAM)were designed on the 5th exon of ERβ1(BtsIMut I)and the 4th exon of ERβ2(Hpy188I),respectively.Two particular restriction enzymes mentioned above were used to screen the ERβ1 and ERβ2 deficent F0 fish.The F1 offspring were obtained by outcrossing the ERβ1(mutant rate 46%)and ERβ2(mutant rate 90%)chimeric XY fish with the wild-type XX fish,respectively.The heterozygous ERβ1+/-and ERβ2+/-fish were screened by the polyacrylamide gel electrophoresis(PAGE)from F1 offspring.The 17 heterozygous ERβ1+/-and 7 wild-type ERβ1+/+ fish were detected from the 24 F1 fish.In-frame(-13 bp,-5bp,-2bp,+11bp and-11bp)and frame-shift(-39bp)mutations induced at the target site were confirmed by Sanger sequencing in the heterozygous ERβ1+/-fish.Similarly,the 12 heterozygous ERβ2+/-and 9 wild-type ERβ2+/+ fish were detected from the 21 F1 fish.In-frame(-11 bp and +7bp)and frame-shift(-6bp)mutations induced at the target site were confirmed by Sanger sequencing in the heterozygous ERβ2+/-fish.The homozygous ERβ1-/-(-13bp)and ERβ2-/-(-11bp)F2 fish were obtained by outcrossing the heterozygous XY fish with the heterozygous XX fish,respectively.3)Histological studies and immunohistochemistry detection in F1 fish.After 120 and 180 dah(days after hatching),phase II oocytes were observed in the ovary of the wild-type ERβ1+/+XX fish,wheras only oogonia and a few phase I oocytes were observed in the heterozygous ERβ1+/-XX fish.Compared to the wild-type fish,both the key enzyme for the synthesis of estrogen,Cyp19a1 a,and the marker of germ cells,Vasa,were expressed normally in the gonad of ERβ1+/-as the wild-type fish.However,the numbers of oocytes in phase I and phase II,which were labeled using 42SP50,were significantly reduced.These results suggested that the retardation of oogenesis in the ERβ1+/-XX fish may be due to haploinsufficiency.In contrast,no obvious phenotypes in gonad were detected in the ERβ2+/-XX fish at 120 and 180 dah,and the corresponding ovaries were also filled with phase II oocytes,just as same as wild-type ovaries.After sexual maturation,both ERβ1+/-and ERβ2+/-heterozygous mutations were fertile.Homozygous knockout fish were obtained by sister crossing.4)Histological studies and immunohistochemistry detection in F2 fish.At 30 and 90 dah,all homozygous ERβ1-/-and ERβ2-/-XX fish expressed Cyp19a1 a but not Dmrt1,as the same as the wild-type XX fish;And all homozygous ERβ1-/-and ERβ2-/-XY fish expressed Dmrt1 but not Cyp19a1 a,which is exactly same pattern as the wild-type XY fish.These results indicated that mutant of ERβ1 and ERβ2 did not cause sex reversal in XX fish.The reason could be that the knockout of one of the ERβ will be compensated by another ERβ or ERα.Establishment of and study on single ER knockout of ERα,double knockout of ER and even triple knockout of ER model are needed.In summary,the agonists and antagonists of ERs specific to mammalian were not applicable for the ERs in tilapia,and therefore we need to find tilapia specific agonists and antagonists to elucidate the mechanism of ER-mediated sex determination.The sex was not reversed by mutation of either ERβ1 or ERβ2 alone.However,the establishment of ERβ knockout lines made a foundation for the subsequent double knockout and triple knockout model establishment,as well as for further in-depth analysis on which ER/ERs involved in sex determination,differentiation and maintenance.