Research On DNA Methylation And Imprinting Gene Expression In Porcine Testis Before Sexual Maturity

Since the term’epigenetic’was proposed, epigenetics came into view.After research, it was discovered that epigenetic modification contained DNA methylation, histone modification, genomic imprinting and other means. This experiment used4,14,30,60,210-day testis of tri-crossbreeding pig as experimental material, observed morphology by H.E staining and analysed developmental state of testis. Simultaneously, explored the change of methylation degree between different age testicle by methylation-specific immunofluorescence. Then using musle,uterus and ovary of adult sows as control,detected and analysed the expression of imprinting gene in different tissues of pig. The results were as follows:1. The morphological observation and degree of methylation detection in porcine testis(1) The diameter of4-day seminiferous tubules were the smallest, and there was no significant difference between14and30-day seminiferous tubules diameter but had obviously increased over the4-days;60-day seminiferous tubules diameter was significantly greater than30-days;210-day seminiferous tubules diameter was the greatest.(2) The interstitial area of4-day was larger; the volume of interstitial cell and nucleus in4-day were larger, and the number of interstitial cell in4-day was the largest. The interstitial area of14-day was the largest, and the same to the volume of interstitial cell and nucleus in14-day, while the number of interstitial cell in14-day was less than4-days. The interstitial area of30-day was less than4-days; the volume of interstitial cell and nucleus in30-day were obviously smaller than14-day,but larger than4-day,and the number of interstitial cell in30-day was less than14-days.The interstitial area of60-day had no much difference with30-days,while the volume of interstitial cell and nucleus in60-day was significantly smaller than30-days;the number of interstitial cell in60-day was more than14-days.The interstitial area of 210-day was the smallest; the volume of interstitial cell and nucleus in210-day had no significant difference with60-day,and the number of interstitial cell in210-day was the least.(3) In seminiferous tubule of4-day, supporting cells arranged close as fence inside the inophragma, few single spermatogeniums(small and round, nucleus was large and round, occupied more than90%volume of cell, deep stained in nuclear) could be observed between supporting cells,1-3primordial germ cells(large and round, light stained in endochylema, round nucleus, caryotheca was distinct) were observed near supporting cells dispersedly in the tubules.In14-day seminiferous tubule, there was no much difference in cellular kind and location, but the number of supporting cell was more than4-days,while the number of primordial germ cell and spermatogenium didn’t change obviously to4-days. In30-day seminiferous tubule, the number of supporting cell arranging loose inside inophragma was less than4,14-days obviously. The number of primordial germ cell and spermatogenium in30-day seminiferous tubule increased, and primordial germ cell was closer inophragma.In60-day seminiferous tubule,the number of supporting cell inside inophragma and spermatogenium was more than4,14,30-day remarkably, while the number of primordial germ cell was less than4,14-day obviously.1-3primary spermatocyte and spermatid could be observed in60-day seminiferous tubule.In210-day seminiferous tubule,the number of supporting cell arranging more loosely was significant less than30-days. Spermatogenium was near and inside inophragma, spermatid was near lumen, primary spermatocyte located between spermatogenium and spermatid. The cellular distribution of spermatogenium, primary spermatocyte and spermatid was multi-layer. Mature supporting cell—sertoli cell was observed in multi-cell layer, and mature spermatozoa was observed in the center of lumen, but no any primordial germ cell and secondary spermatocyte were found.The number of germ cells observed in210-day was the largest.(4) The result of methylation degree detection showed that the interstitial average fluorescence intensity was higher than tubule’s in the same age testis. In seminiferous tubule,60-day average fluorescence intensity was the highest while 14-day was the lowest.30and210-day average fluorescence intensity had no significant difference(P＞0.05),and both were remarkably higher than4-days(P＜0.05). In interstitial substance,60-day average fluorescence intensity was the highest and14-day was the lowest.4and30-day average fluorescence intensity had no significant difference(P＞0.05),and both were remarkably lower than210-days(P＜0.05).2. The research of impring gene expressionDetected the expression of paternal imprinting genes(H19,MEG3),maternal imprinting genes(PEG10、PEG5、PLAGL1、DIRAS3),and potential imprinting gene(IGF2r) in different age testis of tri-crossbreeding pig(musle, uterus and ovary of swine as control). The results were as follows:(1) In testis, all detected genes expressed, but H19and DIRAS3didn’t express in individual samples.In control,DIRAS3and MEG3expressed in all tissues.H19, PEG10,PEG5expressed in uterus and ovary,but not in musle.PLAGL1did not express in all control tissues.H19,DIRAS3,PEG5and PEG10expressed or not in some control samples.(2) The result of gene relative expression in different age testis showed that the expression of H19had no significant difference among4,30,60and210day(P＞0.05). The same to4,14,60,210day,but30-day expression was significant higher than that in14-day(P＜0.05).To MEG3,the expression in30-day was the highest while60-day expression was remarkably higher than14-day(P＜0.05).4and210-day had no significant difference(P＞0.05),and both were remarkably lower than others(P＞0.05). To PEG10,the expression in4and30-day had no significant difference(P＞0.05) and both were remarkably higher than14-day(P＞0.05),while60and210-day had no significant difference(P＞0.05) and both were remarkably lower than others(P＞0.05). To PLAGL1,30and60-day had no significant difference in expression(P＞0.05) and both were remarkably lower than4-day(P＞0.05), while14and210-day had no significant difference(P＞0.05) and both were remarkably lower than others(P＞0.05). To PEG5,the expression in210-day was the lowest, while60-day expression was significant higher than that in14-day(P＜0.05).4and30-day expression had no significant difference(P＞0.05) and both were remarkably higher than others(P＞0.05). To IGF2r,14and210-day had no significant difference in expression(P>0.05) and both were remarkably lower others(P>0.05),while4and30-day had no significant difference(P>0.05) and both were remarkably lower than60-day (P>0.05).The result indicated that:during4-to30-day testis,interstitial cell developed mainly and seminiferous tube developed slowly. In the same age testis,the degree of methylation in interstitial region was higher than that in tube; the methylation degree of testis flucuated with age changed; the expression of all detected genes in testis varied with age-change.