| Aromatase,encoded by CYP19A1 gene,is the key rate-limiting enzyme to promote the conversion of androgen to estrogen,which was firstly considered as a female sex hormone.In recent years,it has been proved that aromatase/estrogen plays an important role in male physiology and reproduction.At present,the research on the effects of aromatase and estrogen on chicken semen quality and reproductive performance has not been carried out in depth.The expression regulation mechanism of chicken aromatase is different from that of mammals.This study firstly focused on the transcriptional regulation of aromatase,intending to solve which transcription factors regulating the expression of CYP19A1,then laid the ground for the subsequent site-specific regulation of CYP19A1 expression theoretically.After that,aromatase was overexpressed and inhibited in roosters testis to find auxiliary markers for improving semen quality and prolonging breeding of roosters,via revealing the effects on roosters reproductive traits at the level of transcription,protein,metabolism and extracellular vesicles.The main results are as follow:1.The conservation of aromatase among species was analyzed by sequence alignment method,and the tertiary structure of chicken aromatase protein was simulated by homology modeling method.It was found that the three-dimensional structure of chicken aromatase was highly similar to that of human aromatase,and the evolution of aromatase was extremely conservative.Immunofluorescence and immunohistochemistry results showed that CYP19A1,ESR1,ESR2 and NR5A2 were expressed in chicken follicle theca cells layer.Chicken theca cells and passage cells were cultured to provide a suitable cell model for exploring the transcriptional regulation mechanism of chicken aromatase.The q PCR was used to detect the m RNA expression levels of CYP19A1,ESR1,ESR2 and NR5A2 in theca cells.It was found that CYP19A1 and NR5A2 expression levels decreased sharply with the passage of time,while the expression level of ESR2 increased,and the expression level of ESR1 did not change significantly.2.CYP19A1 promoter activity was analyzed by the luciferase reporter assay.It was suggested that p GL3-P3(-368 bp to-9 bp)was required for CYP19A1 transcriptional activity and showed fully intact promoter activity.ESR1 enhanced the activity of fragment P3(from-368 bp to-9 bp)and repressed the activity of fragment P2(from-511 bp to-9 bp)of the CYP19A1 promoter.NR5A2 enhanced the activity of fragment P3(from-368 bp to-9 bp)and inhibited the activity of fragment P4(from-197 bp to-9bp)of the CYP19A1 promoter.Western blotting results showed that the overexpression of ESR1,ESR2 and NR5A2 could upregulate CYP19A1 protein expression,and ESR1,ESR2 and NR5A2 formed a network module,in which each node regulated CYP19A1 expression by mutual constraints.3.It was proved that CYP19A1 was highly expressed in Leydig cells of testis in control group and low concentration lentivirus injection group,and increased in Sertoli Cells of testis in high concentration lentivirus injection group via using different concentrations of lentivirus overexpression CYP19A1 vector to inject rooster testis.Overexpression of CYP19A1 at low concentration in testis decreased the testosterone content of seminal plasma and increased the ratio of testosterone to estrogen,while overexpression of CYP19A1 at high concentration in testis increased the estrogen content,decreased the testosterone content and the ratio of testosterone to estrogen.4.Transcriptome and proteome sequencing analysis of rooster testis with highly overexpressed CYP19A1 showed that CYP19A1 altered the expression levels of genes and proteins such as HSD3B1,PLTP,WNT5 A and SLC9A3R1 through regulating multiple biological processes,such as sex hormone synthesis pathway,WNT signaling pathway and immune response,resulting in abnormal morphology of roosters testicular organs and damaged cytoskeleton of Sertoli Cells,also decreasing of the frequency of sperm flagella and sperm motility.The results showed that there was a complex and coordinated biological process between sperm motility and fertility,and it confirmed that CYP19A1 played an indispensable role in male reproductive organ development,semen quality,fertility and sex hormone synthesis.5.Extracellular vesicles(EVs)of chicken seminal plasma in each treatment group were successfully isolated.EVs physical morphology and particle size in the seminal plasma of the control group,low concentration lentivirus injection group and high concentration lentivirus injection group did not change significantly,but the expression of some proteins in the EVs changed significantly.EVs proteome sequencing results showed most of the differential proteins in the low concentration lentivirus CYP19A1 injection group were enriched into the energy metabolism pathways,and most of the differential proteins in the high concentration lentivirus CYP19A1 injection group were enriched into the immune disease pathways.Western blotting results suggested that SOD3 and CAV1 may be assistant markers to judge the fertility of roosters.6.Metabolomic profiling of semen showed that aromatase activity could affect the metabolic pathways such as taurine,steroid hormone synthesis,glycerophospholipids and cholesterol.The contents of threonine and other essential amino acids increased,so did the metabolites of taurine,taurocholate and glycine goose deoxycholic acid,while the precursors of synthetic estrogen and testosterone decreased,thus affecting the reproductive traits of roosters.Based on the above results,it is revealed that the transcriptional regulation of chicken CYP19A1 in gonads is different from that of mammals.ESR1,ESR2 and NR5A2 regulated the expression of CYP19A1 through mutual constraints.Aromatase changed hormone secretion,testicular phenotype and sperm motility by affecting the protein composition of chicken steroid hormone synthesis pathway,taurine metabolism pathway,WNT signal pathway and EVs of seminal plasma,which played an important role in chicken semen quality and fertility. |
PDF Full Text Request