Study On The Role Of Non-Coding RNA And Rik08 In Male Infertility

In recent years,infertility is becoming a major problem affecting human health,with data suggesting that about 10%-15%of couples with childbearing age are unable to reproduce normally,half of which is male.Therefore,it is more important to study the mechanism of the related diseases that cause male infertility,and we study the testicular development stagnation caused by cryptorchidism,testicular cancer and gene deletion.Idiopathic diseases of the reproductive system are important factors leading to male infertility.Many studies have shown that microRNAs(miRNAs)regulate the expression of multiple genes that play a significant role in spermatogenesis and development.We previously showed that microRNA-210(miR210)is one of the markedly upregulated microRNAs in the testes of sterile males with maturation arrest(MA).However,the role of miR-210 in spermatogenesis remains unknown.In this study,we found that miR-210 is highly expressed not only in patients with MA but also in patients with cryptorchidism.In addition,miR-210 inhibits the expression of Nuclear Receptor Subfamily 1,Group D,Member 2(NR1D2)both in vitro and in vivo,particularly in cryptorchidic tissues.To facilitate further research,we established a mouse model of cryptorchidism and were surprised to discover that the miR-210 expression pattern was in accordance with that of patients with cryptorchidism.Thus,we propose that miR-210 may serve as a biomarker of cryptorchidism in clinical tests.Intermediate-size noncoding RNAs(imsncRNAs)have been shown to play important regulatory roles in the development of several eukaryotic organisms.In this research,we selected imsnc761 as a research target.Expression analyses in a previous study showed that imsnc761 was downregulated in maturation-arrested testis tissue compared with the level in normal controls.In this study,we found that imsnc761 could interact with DEAD-box helicase 6(DDX6)to induce NT2 cell apoptosis and proliferation inhibition via the p53 pathway.This interaction between imsnc761 and DDX6 also inhibited mitochondrial function and specific gene transcription and translation.To facilitate further research,we used label-free quantification method to analyse the associated differences in KEGG pathways and biological processes.This confirmed the changes of several specific pathways,which matched our molecular experimental results.The imsnc761 sequence was also identified as snoRNA,and for the first time,our study pointed out that DDX6 can modulate the proliferation and apoptosis of testicular cancer cells by interacting with small fragments of RNA.Many specific genes expressed in testicular tissue are crucial for testicular development and spermatogenesis.In the third part of the study,we selected Rik08 as the object of study,which only expressed in testes,using CRISPR technology to construct the gene knockout mice.Then we found that the male Rik08 deficient mice testes had developmental disorders and the spermatogenic arrest.Rik08-/-mice were unable to produce mature sperm and infertile.After a series of experiments,it was confirmed that the Rik08-/-mice were pachytene spermatocytes arrest while the hormones’expression were normal.At the same time,we used quantitative proteomic method to analyse the testes from wild type and knockout mice,and then used bioinformatics sites to cluster the differential proteins to find out five genes which directly related to meiosis as a starting point for deeper research.