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Chromosomal distribution of miRNA genes and miRNA gene clusters identified in this study. (A) Chromosomal distribution of the 141 miRNA genes in the mouse genome. (B) Chromosomal distribution of 19 miRNA gene clusters and 28 miRNA genes exclusively or preferentially expressed in the testis. The mouse chromosomes were drawn to scale and aligned by their centromere position. Nineteen miRNA clusters (miRcs1 – 19) are indicated on the left and the 28 testis-specific or testis-preferential miRNAs are marked on the right. The miRc-19 genomic fragment containing the 11 X-linked testis-specific or testis-preferential miRNAs is enlarged and shown to the right of the X chromosome. The solid bars represent repetitive sequences and the open bars stand for the unique miRNA-coding sequences.
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Using a new small RNA cloning method, we identified 141 miRNAs from the mouse testis, of which 29 were novel. The 141 miRNAs were mapped onto all chromosomes except the Y chromosome and 2/3 of these miRNA genes exist as clusters. approximately 70% of these miRNA genes were located in intronic or intergenic regions, whereas the remaining miRNAs were...
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... encoding these miRNAs were found on all chromosomes but the Y chromosome ( Fig. 2A and Supplementary Table 4). Interestingly, 27 out of the 141 miRNAs (19%) were located on the X chromosome ( Fig. 2A). A total of 19 miRNA gene clusters (miRcs) containing 94 miRNA genes (67%) were identified, and these clusters were randomly distributed across the mouse genome (Fig. 2B). The average size of a miRNA gene cluster is ∼ ...
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... encoding these miRNAs were found on all chromosomes but the Y chromosome ( Fig. 2A and Supplementary Table 4). Interestingly, 27 out of the 141 miRNAs (19%) were located on the X chromosome ( Fig. 2A). A total of 19 miRNA gene clusters (miRcs) containing 94 miRNA genes (67%) were identified, and these clusters were randomly distributed across the mouse genome (Fig. 2B). The average size of a miRNA gene cluster is ∼ 6.0 kb, containing an average of ∼ 5 miRNA genes (Supplementary Table 5). The longest cluster, miRc-19 (62,361 bp) on ...
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... miRNAs were found on all chromosomes but the Y chromosome ( Fig. 2A and Supplementary Table 4). Interestingly, 27 out of the 141 miRNAs (19%) were located on the X chromosome ( Fig. 2A). A total of 19 miRNA gene clusters (miRcs) containing 94 miRNA genes (67%) were identified, and these clusters were randomly distributed across the mouse genome (Fig. 2B). The average size of a miRNA gene cluster is ∼ 6.0 kb, containing an average of ∼ 5 miRNA genes (Supplementary Table 5). The longest cluster, miRc-19 (62,361 bp) on chromosome X, contains 20 miRNAs. miRNA clusters are found on both the plus (58%) and minus (42%) strands. The majority of miRNA genes (∼ 70%) were located in intergenic ...
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... genomic locations of the 6 testis-specific and 22 testispreferential miRNAs were further analyzed (Supplementary Table 7 and Fig. 2B). Twenty-one of the 28 miRNA genes were located in intergenic regions (75%), 3 were mapped to introns, and 4 were from exons or both exons and introns. Interestingly, 11 of the 28 miRNA genes were on the X chromosome, suggesting that the X chromosome encodes more miRNAs preferentially or exclusively expressed in spermatogenic cells. In ...
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... 11 of the 28 miRNA genes were on the X chromosome, suggesting that the X chromosome encodes more miRNAs preferentially or exclusively expressed in spermatogenic cells. In addition, three testis-specific miRNAs (mir-t19, mir-t25, and mir-t27) were located on the miRNA clusters miRc-16 and miRc-19 ( Fig 2B). The miRc-16 located on chromosome 17 encoded two miRNAs (mir-t19 and mirt20), while all of the 11 X-linked testis-specific or testispreferential miRNA genes were located within the miRc-19 (Fig. 2B). ...
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... cells. In addition, three testis-specific miRNAs (mir-t19, mir-t25, and mir-t27) were located on the miRNA clusters miRc-16 and miRc-19 ( Fig 2B). The miRc-16 located on chromosome 17 encoded two miRNAs (mir-t19 and mirt20), while all of the 11 X-linked testis-specific or testispreferential miRNA genes were located within the miRc-19 (Fig. 2B). More interestingly, each of these 11 X-linked miRNA genes within the miRc-19 was flanked by repetitive sequences at both the 5′ and the 3′ sides and all of them are clustered within a region of ∼62 kb, where no mRNA-coding genes are found. The significance of this unique genomic structure of X-linked miRNAs deserves further ...
Citations
... Several studies have highlighted the role of small RNAs (siRNAs, piRNAs, miRNAs) in spermatogenesis and, therefore, in male fertility (reviewed in [6,17,29,[32][33][34][35][36][37][38]). In particular, studies have shown that miRNAs can be expressed exclusively or preferentially in the testis and in specific testicular cell types, as shown by Gan et al., who found miRNAs to be more abundant in spermatogonia than in other testis cell types [34,[39][40][41]. In addition to specific expression in male reproductive tissues, the role of miRNA has also been highlighted in steroidogenesis and in zygotic and early embryo reprogramming [35]. ...
Oncological treatments have dramatically improved over the last decade, and as a result, survival rates for cancer patients have also improved. Quality of life, including concerns about fertility, has become a major focus for both oncologists and patients. While oncologic treatments are often highly effective at suppressing neoplastic growth, they are frequently associated with severe gonadotoxicity, leading to infertility. For male patients, the therapeutic option to preserve fertility is semen cryopreservation. In prepubertal patients, immature testicular tissue can be sampled and stored to allow post-cure transplantation of the tissue, immature germ cells, or in vitro spermatogenesis. However, experimental techniques have not yet been proven effective for restoring sperm production for these patients. MicroRNAs (miRNAs) have emerged as promising molecular markers and therapeutic tools in various diseases. These small regulatory RNAs possess the unique characteristic of having multiple gene targets. MiRNA-based therapeutics can, therefore, be used to modulate the expression of different genes involved in signaling pathways dysregulated by changes in the physiological environment (disease, temperature, ex vivo culture, pharmacological agents). This review discusses the possible role of miRNA as an innovative treatment option in male fertility preservation–restoration strategies and describes the diverse applications where these new therapeutic tools could serve as fertility protection agents.
... It has been demonstrated that miRNA expressions pattern differs between immature and mature testes in human [24], rhesus monkey [25] and pig [26]. In contrast, the microarray analyses showed that the most testicular miRNAs are preferentially expressed in the meiotic germ cells [27,28]. The expression of Mir214 is abundant in pachytene spermatocytes and this miRNA plays a key role in meiosis by targeting heat shock proteins [29]. ...
Mouse Pxt1 gene is expressed exclusively in male germ cells and encodes for a small, cell death inducing protein. However, upon PXT1 interaction with BAG6, cell death is prevented. In transiently transfected cell lines the PXT1 expression triggered massive cell death, thus we ask the question whether the interaction of PXT1 and BAG6 is the only mechanism preventing normal, developing male germ cells from being killed by PXT1. The Pxt1 gene contains a long 3'UTR thus we have hypothesized that Pxt1 can be regulated by miRNA. We have applied Pxt1 knockout and used Pxt1 transgenic mice that overexpressed this gene to shed more light on Pxt1 regulation. Using the ELISA assay we have demonstrated that PXT1 protein is expressed in adult mouse testis, though at low abundance. The application of dual-Glo luciferase assay and the 3'UTR cloned into p-MIR-Glo plasmid showed that Pxt1 is regulated by miRNA. Combining the use of mirDB and the site-directed mutagenesis further demonstrated that Pxt1 translation is suppressed by Mir6996-3p. Considering previous reports and our current results we propose a model for Pxt1 regulation in the mouse male germ cells.
... It is conceivable that miRNAs can regulate meiosis and thus spermatogenesis [17] by regulating mRNA degradation and disrupting mRNA translation [18,19]. While recent studies focused on miRNA tissue expression in rodents and humans, miRNA data for dogs are lacking [20,21]. ...
High-throughput in-silico techniques help us understand the role of individual proteins, protein–protein interaction, and their biological functions by corroborating experimental data as epitomized biological networks. The objective of this investigation was to elucidate the association of miRNA-mediated genes in the regulation of dog testes development from immature to adult form by in-silico analysis. Differentially expressed (DE) canine testis miRNAs between healthy immature (2.2 ± 0.13 months; n = 4) and mature (11 ± 1.0 months; n = 4) dogs were utilized in this investigation. In silico analysis was performed using miRNet, STRING, and ClueGo programs. The determination of mRNA and protein expressions of predicted pivotal genes and their association with miRNA were studied. The results showed protein–protein interaction for the upregulated miRNAs, which revealed 978 enriched biological processes GO terms and 127 KEGG enrichment pathways, and for the down-regulated miRNAs revealed 405 significantly enriched biological processes GO terms and 72 significant KEGG enrichment pathways (False Recovery Rate, p < 0.05). The in-silico analysis of DE-miRNA’s associated genes revealed their involvement in the governing of several key biological functions (cell cycle, cell proliferation, growth, maturation, survival, and apoptosis) in the testis as they evolve from immature to adult forms, mediated by several key signaling pathways (ErbB, p53, PI3K-Akt, VEGF and JAK-STAT), cytokines and hormones (estrogen, GnRH, relaxin, thyroid hormone, and prolactin). Elucidation of DE-miRNA predicted genes’ specific roles, signal transduction pathways, and mechanisms, by mimics and inhibitors, which could perhaps offer diagnostic and therapeutic targets for infertility, cancer, and birth control.
... Additionally, Yan et al found 19 miRNAs differentially expressed between immature and mature mice testes (14 up regulated and five down regulated) [94], while Lian et al used RT-qPCR assay and found 122 miRNAs differentially expressed in the immature and mature porcine testes [95]. Furthermore, Ro et al reported cloning and expression profiling of 141 miRNAs expressed in mice testes of which 28 are preferentially or exclusively expressed in testes [96]. It is noteworthy that Torley et al used sheep as a model with the aim to identify the expression of miRNAs in mammalian fetal gonad and revealed significant differences between testes among 24 miRNAs at gestational day (GD) 42 and 43 miRNAs at GD75. ...
Infertility is a common problem affecting one in six couples and in 30% of infertile couples, the male factor is a major cause.
A large number of genes are involved in spermatogenesis and a significant proportion of male infertility phenotypes are of
genetic origin. Studies on infertility have so far primarily focused on chromosomal abnormalities and sequence variants in
protein-coding genes and have identified a large number of disease-associated genes. However, it has been shown that a
multitude of factors across various omics levels also contribute to infertility phenotypes. The complexity of male infertility has
led to the understanding that an integrated, multi-omics analysis may be optimal for unravelling this disease. While there is a
vast array of different factors across omics levels associated with infertility, the present review focuses on known factors from
the genomics, epigenomics, transcriptomics, proteomics, metabolomics, glycomics, lipidomics, miRNomics, and integrated
omics levels. These include: repeat expansions in AR, POLG, ATXN1, DMPK, and SHBG, multiple SNPs, copy number variants
in the AZF region, disregulated miRNAs, altered H3K9 methylation, differential MTHFR, MEG3, PEG1, and LIT1 methylation,
altered protamine ratios and protein hypo/hyperphosphorylation. This integrative review presents a step towards a
multi-omics approach to understanding the complex etiology of male infertility. Currently only a few genetic factors, namely
chromosomal abnormalities and Y chromosome microdeletions, are routinely tested in infertile men undergoing intracytoplasmic
sperm injection. A multi-omics approach to understanding infertility phenotypes may yield a more holistic view of
the disease and contribute to the development of improved screening methods and treatment options. Therefore, beside discovering
as of yet unknown genetic causes of infertility, integrating multiple fields of study could yield valuable contributions
to the understanding of disease development. Future multi-omics studies will enable to synthesise fragmented information
and facilitate biomarker discovery and treatments in male infertility.
... These genes belong to protein-coding, non-coding RNAs as well as pseudogenes and to be experimentally confirmed annotation classification (Figure 4d, Additional file 1: Figures S13 b-d). Among these genes include previously characterized human testis-specific lncRNA SPATA42 (Xie et al., 2020) and miRNA miR-202 (Ro et al., 2007). In mouse testis, genes with no ortholog in other seven species include protein-coding gene Cypt4 (Kato & Nozaki, 2012), lncRNAs Rbakdn (Liu et al., 2021), and pseudogene 1700019M22Rik (Kato & Nozaki, 2012). ...
One of the main challenges in analyzing gene expression profiles across species is the dependence on determining corresponding genes between species. Homology-based approaches fail to account for the contribution of non-homologous genes to the phenotype, genes' functional divergence, and rewiring of pathways. Homology-independent methods based on joint matrix factorization provide a potential solution, but biological interpretations with existing approaches are difficult. We developed a novel joint matrix factorization method that we call the orthogonal shared basis factorization (OSBF) to compare functionally similar phenotypes across species. OSBF utilizes a similar correlation structure within individual datasets to estimate interpretable matrix factors. This homology-independent approach places cellular phenotypes in a common coordinate system that can summarize gene expression patterns shared by different organisms and quantifies the role of all genes in the phenotype independent of their homology relationships and annotation. OSBF is available on https://github.com/amalthomas111/SBF.
... miRNAs and siRNAs (small interfering RNAs), both of which are Argonaute-bound small RNAs, are essential for mammalian spermatogenesis (Hayashi et al., 2008). During the active transcription of meiotic genes in pachytene spermatocytes and round spermatocytes, miRNAs were significantly enriched (Ro et al., 2007;Kotaja, 2014). In addition, knocking out two miRNA clusters (mir-34B/C and-449A/B/C) will lead to the imbalance of expression of many key genes and the failure of multiciliogenesis in spermatogenic output tubules of mouse testis Figure 5). ...
Emerging evidence shows that m6A is the most abundant modification in eukaryotic RNA molecules. It has only recently been found that this epigenetic modification plays an important role in many physiological and pathological processes, such as cell fate commitment, immune response, obesity, tumorigenesis, and relevant for the present review, gametogenesis. Notably the RNA metabolism process mediated by m6A is controlled and regulated by a series of proteins termed writers, readers and erasers that are highly expressed in germ cells and somatic cells of gonads. Here, we review and discuss the expression and the functional emerging roles of m6A in gametogenesis and early embryogenesis of mammals. Besides updated references about such new topics, readers might find in the present work inspiration and clues to elucidate epigenetic molecular mechanisms of reproductive dysfunction and perspectives for future research.
... Many expression profiling studies indicate that miRNAs play essential roles in gonadal development. These include specific testis and ovary miRNA signatures and differential expression analyses at key stages of gonad development performed in a variety of mammalian species, including mouse [Ro et al., 2007;Yan et al., 2007;Mishima et al., 2008;Song et al., 2009;Buchold et al., 2010], sheep [Torley et al., 2011;Bai et al., 2019], pig [Lian et al., 2012;Ran et al., 2015;Ding et al., 2020], human [Yang et al., 2013], and dog [Kasimanickam and Kasimanickam, 2015]. Here, we will focus exclusively on mammalian gonadal sex determination and differentiation, and no discussion on the roles of miRNAs in either the function of the adult mammalian testis or the mechanisms of sex determination in non-mammalian species will be included. ...
Non-coding RNAs (ncRNAs) are a group of RNAs that do not encode functional proteins, including long non-coding RNAs (lncRNAs), microRNAs (miRNAs), PIWI-interacting RNAs (piRNAs), and short interfering RNAs (siRNAs). In the last 2 decades an effort has been made to uncover the role of ncRNAs during development and disease, and nowadays it is clear that these molecules have a regulatory function in many of the developmental and physiological processes where they have been studied. In this review, we provide an overview of the role of ncRNAs during gonad determination and development, focusing mainly on mammals, although we also provide information from other species, in particular when there is not much information on the function of particular types of ncRNAs during mammalian sexual development.
... More recently, both miRNA and miRNA* were shown to coexist and function [139,216]. Additionally, the ratio of 5p and 3p strands was found to be tissue-specific in mice [157]. Hence, the nomenclature of miR-#-5p or miR-#-3p is recommended. ...
The discovery of various noncoding RNAs (ncRNAs) and their biological implications is a growing area in cell biology. Increasing evidence has revealed canonical and noncanonical functions of long and small ncRNAs, including microRNAs, long ncRNAs (lncRNAs), circular RNAs, PIWI-interacting RNAs, and tRNA-derived fragments. These ncRNAs have the ability to regulate gene expression and modify metabolic pathways. Thus, they may have important roles as diagnostic biomarkers or therapeutic targets in various diseases, including neurodegenerative disorders, especially Parkinson’s disease. Recently, through diverse sequencing technologies and a wide variety of bioinformatic analytical tools, such as reverse transcriptase quantitative PCR, microarrays, next-generation sequencing and long-read sequencing, numerous ncRNAs have been shown to be associated with neurodegenerative disorders, including Parkinson’s disease. In this review article, we will first introduce the biogenesis of different ncRNAs, including microRNAs, PIWI-interacting RNAs, circular RNAs, long noncoding RNAs, and tRNA-derived fragments. The pros and cons of the detection platforms of ncRNAs and the reproducibility of bioinformatic analytical tools will be discussed in the second part. Finally, the recent discovery of numerous PD-associated ncRNAs and their association with the diagnosis and pathophysiology of PD are reviewed, and microRNAs and long ncRNAs that are transported by exosomes in biofluids are particularly emphasized.
... The silencing of genes is an essential biological phenomenon by which numerous cellular processes including self-renewal, proliferation, differentiation, and apoptosis could be fine-tuned (10). Moreover, studies have reported that miRNAs are highly expressed and they are involved in the process of spermatogenesis (11)(12)(13)(14)(15)(16)(17). In line with this study, the loss of DICER (a protein which facilitates the activation of the RISC activation) could be resulted in a defect in germ cell development (18,19). ...
Objective:
Numerous evidence indicates that microRNAs (miRNAs) are critical regulators in the spermatogenesis process. The aim of this study was to investigate miR-106b cluster regulates primordial germ cells (PGCs) differentiation from human mesenchymal stem cells (MSCs).
Materials and methods:
In this experimental study, samples containing male adipose (n: 9 samples- age: 25-40 years) were obtained from cosmetic surgeries performed for the liposuction in Imam Khomeini Hospital. The differentiation of MSCs into PGCs was accomplished by transfection of a lentivector expressing miR-106b. The transfection of miR-106b was also confirmed by the detection of a clear green fluorescent protein (GFP) signal in MSCs. MSCs were treated with bone morphogenic factor 4 (BMP4) protein, as a putative inducer of PGCs differentiation, to induce the differentiation of MSCs into PGCs (positive control). After 4 days of transfection, the expression of miR-106b, STELLA, and FRAGILIS genes was evaluated by real-time polymerase chain reaction (PCR). Also, the levels of thymocyte differentiation antigen 1 (Thy1) protein was assessed by the western blot analysis. The cell surface expression of CD90 was also determined by immunocytochemistry method. The cytotoxicity of miR-106b was examined in MSCs after 24, 48, and 72 hours using the MTT assay.
Results:
MSCs treated with BMP4 or transfected by miR-106b were successfully differentiated into PGCs. The results of this study also showed that the expression of miR-106b was significantly increased after 48 hours from transfection. Also, we showed STELLA, FARGILIS, as well as the protein expression of Thy1, was significantly higher in MSCs transfected by lentivector expressing miR-106b in comparison with MSCs treated with BMP4 (P≤0.05). MTT assay showed miR-106b was no toxic during 72 hours in 1 μg/ml dose, that this amount could elevated germ cells marker significantly higher than other experimental groups (P≤0.05).
Conclusion:
According to this findings, it appears that miR-106b plays an essential role in the differentiation of MSCs into PGCs.
... Notably, as an important physiological process, spermatogenesis is also regulated by miRNAs to some extent [61]. miRNAs are abundantly enriched during the active transcription of meiotic genes in male germ cells, especially in pachytene spermatocytes and round spermatids [62]. Many genes, such as Rsbn1, participate in spermatogenesis and are involved in the regulation of translation by miRNAs [62]. ...
... miRNAs are abundantly enriched during the active transcription of meiotic genes in male germ cells, especially in pachytene spermatocytes and round spermatids [62]. Many genes, such as Rsbn1, participate in spermatogenesis and are involved in the regulation of translation by miRNAs [62]. In addition, miRNA clusters (miR-34b/c and miR-449a/b/c) are indispensable for spermatogenesis and male efferent ductule ciliogenesis [63,64]. ...
Emerging evidence shows that m6A, one of the most abundant RNA modifications in mammals, is involved in the entire process of spermatogenesis, including mitosis, meiosis, and spermiogenesis. “Writers” catalyze m6A formation on stage-specific transcripts during male germline development, while “erasers” remove m6A modification to maintain a balance between methylation and demethylation. The different functions of RNA-m6A transcripts depend on their recognition by “readers”. m6A modification mediates RNA metabolism, including mRNA splicing, translation, and degradation, as well as the maturity and biosynthesis of non-coding RNAs. Sperm RNA profiles are easily affected by environmental exposure and can even be inherited for several generations, similar to epigenetic inheritance. Here, we review and summarize the critical role of m6A in different developmental stages of male germ cells, to understand of the mechanisms and epigenetic regulation of m6A modifications. In addition, we also outline and discuss the important role of non-coding RNAs in spermatogenesis and RNA modifications in epigenetic inheritance.
