| The relaxin family is a class of neuropeptide hormones that includes human relaxin 1（H1 relaxin）,human relaxin 2（H2 relaxin）,human relaxin 3（H3 relaxin）,insulin-like peptide 3（INSL3）,insulin-like peptide 4（INSL4）,insulin-like peptide 5（INSL5）,and insulin-like peptide 6（INSL6）.Rln3 in mice is expressed in the brain and testis,and is mainly involved in reproduction,stress response,feeding activity,memory activity,osmotic pressure regulation,anxiety response and other processes.Although rln3 gene is highly expressed in testis of mammals and fish,there are few studies on its effects on testis development and spermatogenesis in males,which need further study.Nile tilapia（Oreochromis niloticus）is a worldwide farmed fish.The spawning cycle of female fish is about 14 days and sexual maturity is about one year.The sexual maturity of male fish is about 6 months.In addition,our laboratory successfully obtained unisexual fish fry with large individual is convenient for microscopic injection and sampling,which is a good model for studying fish spermatogenesis in fish.Previous studies in our group have shown that there are two relaxin 3（rln3）genes in Nile tilapia:rln3a and rln3b.Both are expressed in the Leydig cell of the testis and in the brain,and the expression of rln3a gene in testis was significantly higher than that of rln3b gene,and the expression of rln3b gene in brain was significantly higher than that of rln3a gene.In the present study,we investigated the molecular mechanism of Rln3a/3b on regulating spermatogenesis,maturation and fertility by designing a series of experiments based on the construction of rln3a/3b mutant lines in Nile tilapia（Oreochromis niloticus）.In the present study,the effects of homozygous knockout lines of rln3a on testis development,spermatogenesis,maturation,fertility and androgen synthesis were explored through H.E.,immunohistochemistry and sperm motility tests.The molecular mechanism of Rln3a regulating androgen synthesis was studied by in vitro treatment of rln3a+/+XY and rln3a-/-XY testis.The rln3b heterozygous mutant line was established by CRISPR/Cas9 technology,and the effect of Rln3b on the development of Nile tilapia testis and steroidgenesis was studied.The specific research results are as follows:1.Effects of homozygous knockout of rln3a on testis development,spermatogenesis,sperm morphology,fertility and androgen synthesis of Nile tilapia.The results of H.E.,immunohistochemistry and Real-time PCR showed that compared with the testis of rln3a+/+XY,rln3a-/-XY GSI was significantly increased,semen volume was increased,but the number of mature sperm and various germ cells was significantly decreased.Immunofluorescence staining of sperm and sperm motility analysis showed that the sperm of rln3a-/-XY were malformed,with double tail phenomenon,and the average linear movement of sperm,sperm motility were significantly reduced.Rln3a-/-XY offspring all died around the 6th day after artificial fertilization and the survival rate is significantly reduced.Moreover,the knockout of rln3a gene significantly reduced the level of androgen and the protein levels of steroid hormone synthesis genes（St AR1,Cyp17a1,Cyp11b2）,which may be the reason why Rln3a affects spermatogenesis,maturation and fertility.2.Androgen 11-KT successfully rescued the phenotype of decreased sperm motility and decreased fertility due to caused by the deletion of rln3a in Nile tilapia.Male rln3a homozygous fish were treated with 11-KT（200ug/g（feed weight））for 2 months from 4 months of age.The results showed that the rln3a-/-+11-KT group had significantly higher sperm forward motility ratio and total sperm viability compared with the rln3a-/-group,but there was no significant difference from the rln3a+/+group.The proportion of motility and the proportion of inactive sperm was significantly lower than that of the rln3a-/-group,and there was no significant difference from the rln3a+/+group.In terms of the survival rate of offspring,after11-KT rescue,the fertility of the rescue group was significantly higher than that of the rln3a-/-XY group,and the survival rate was significantly increased.3.Rln3a affects androgen synthesis through the c AMP/PKA signaling pathway.Real-time PCR results showed that the expression level of Gn RH1,kisspeptin2,fshβand lhβin the brain and pituitary of rln3a homozygous knockout fish.The expression of rln3b gene was also significantly decreased in the brain and testis of homozygous rln3a knockout fish,suggesting that both Rln3a/3b may be involved in the regulation of the HPG axis（Hypothalamus-pituitary-gonad）,and there may be no compensatory relationship between rln3a and rln3b genes.Western blotting and Real-time PCR detection showed that the expressions of genes prkaca and prkacb in the c AMP/PKA signaling pathway of rln3a-/-XY testis were significantly decreased,and the protein expressions of CREB and P-CREB were significantly decreased compared with those of control fish rln3a+/+XY.According to the above results,it was suggested that Rln3a might affect androgen synthesis through the c AMP/PKA signaling pathway.Through in vitro treatment experiments of rln3a+/+XY testis,it was found that the h CG treated group(rln3a+/+-h CG)significantly increased the expression of rln3a gene,indicating that the expression of Rln3a in the testis was regulated by gonadotropin（GTH）.In addition,Real-time PCR results showed that the c AMP/PKA signaling pathway inhibitor H89 could inhibit the expression of androgen synthesis pathway genes St AR1,cyp17a1,cyp11a1 and hsd11b2 in both the control group(rln3a+/+-vehicle)and the h CG treated group(rln3a+/+-h CG).Western blotting analysis revealed that H89 treatment significantly reduced the protein levels of St AR1 and Cyp17a1,as well as the levels of CREB and P-CREB in the c AMP/PKA signaling pathway.The response mechanism of testis to h CG treatment and Rln3a affects the molecular mechanism of androgen synthesis were further studied by in vitro treatment experiments with rln3a-/-XY testis.The results showed that the expression levels of St AR1,cyp17a1,cyp11a1 and hsd11b2 genes were significantly decreased in the rln3a-/--vehicle group compared with the rln3a+/+-vehicle group.However,after h CG treatment(rln3a-/--h CG),the expression levels of St AR1,cyp17a1 and hsd11b2 genes in the testis of rlm3a-/-were significantly up-regulated,but the expression levels of St AR1 and cyp17a1 genes were significantly lower than those in the rln3a+/+-vehicle group and the rln3a+/+-h CG group,further demonstrating that Rln3a plays an irreplaceable role in the androgen synthesis process of the HPG axis and testis.In addition,in vitro treatment of the testis of rln3a-/-male by Forskolin(rln3a-/--Forskolin),an agonist in the c AMP/PKA signaling pathway,showed that the genes St AR1,cyp11a1 and hsd11b2 in the androgen synthesis pathway were significantly up-regulated compared with those in the rln3a-/--vehicle group.The expression levels of cyp11a1 and hsd11b2 genes were not different from those of rln3a+/+testis.These experiments further demonstrated that Rln3a affects androgen synthesis through the c AMP/PKA signaling pathway.4.Expression pattern of rln3 gene and its receptor in brain divisions of Nile tilapia.The brain of Nile tilapia can be divided into 7 parts,which are Olfactory bulb,Telencephalon,Hypothalamus,Optic tectum,Diencephalon,Cerebellum and Myelencephalon.rln3 receptors genes include rxfp3,rxfp3.2a,rxfp3.3b,and rxfp3.3a3.Real-time PCR results showed that rln3a/3b genes and its receptor were expressed in all 7 brain divisions of adult tilapia,and the highest expression of rln3a/3b genes was found in the Diencephalon,and the expression of rln3a gene in the Olfactory bulb of female fish was significantly higher than that of male fish,while the expression of rln3b gene in the Myelencephalon of female fish was significantly higher than that of male fish.Further analysis of the expression level of rln3a/3b genes in the brain divisions,the results are as follows:rln3b gene expression in the Olfactory bulb and Telencephalon of male fish is significantly higher than rln3a gene,there is no significant difference in female fish.The expression level of rln3b gene in the Hypothalamus of both female and male fish was significantly higher than that of rln3a gene,suggesting that Rln3b has a profound effect on the hypothalamic-pituitary-gonad axis（HPG axis）of Nile tilapia.The expression of rln3 receptor genes in brain divisions is as follows:the expression of rxfp3 gene is the highest in the Hypothalamus of male fish and the highest in the Optic tectum of female fish.The expression level of rxfp3.2a gene was the highest in the Olfactory bulb of male fish and in the Telencephalon of female fish.The highest expression of rxfp3.3a3 gene was found in the Cerebellum of males,and in the Olfactory bulb and Myelencephalon of females.rxfp3.3b gene was highest expressed in the Optic tectum of both female and male fish.The expression of rxfp3.2a gene in Hypothalamus and Cerebellum of male fish was significantly higher than that of female fish.The expression of rxfp3.3a3 gene in Hypothalamus and Optic tectum of male fish was significantly higher than that of female fish.The expression of rxfp3 and rxfp3.3b genes in brain divisions did not differ between male and female fish.Since the expression levels of receptors rxfp3.2a and rxfp3.3a3 in the hypothalamus of male tilapia are all higher than those of female tilapia,it is speculated that rln3 gene may bind to receptors rxfp3.2a and rxfp3.3a3 in male tilapia,thus affecting the fertility of male fish.5.Effects of Rln3b on steroidgenesis synthesis in Nile tilapia.The rln3b gene was successfully mutated through CRISPR/Cas9 gene editing technology and established to the F1 generation.The analysis found that the rln3b mutant line had no significant changes in the testis of male fish at the age of 3 months.The morphology of the testis at 6 months of age changed significantly,with transparent vesicles,the gonadal index GSI was significantly reduced,and the morphology of the seminiferous lobules changed from elongated to irregular round.Moreover,the expression levels of steroidgenesis related enzymes genes St AR1,cyp11a1 and cyp17a1 were significantly decreased.In conclusion,Rln3a affects androgen synthesis through c AMP/PKA signaling pathway,and then plays an important role in spermatogenesis,maturation and fertility of Nile tilapia.Through CRISPR/Cas9 mutation of rln3b,we found that Rln3b also plays an indispensable role in regulating testis development and steroidgenesis of Nile tilapia.This study is the first to reveal an regulatory role of Rln3 in male reproduction in teleosts through gene editing,laying a foundation for the study of reproductive regulation in vertebrate. |
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