The molecular characteristics in different procedures of spermatogenesis
Introduction
Infertility is an inevitable challenge in reproductive medicine. Male infertility accounts for 20–30% of cases(Jungwirth et al., 2012, Vander Borght and Wyns, 2018). Most infertile men have abnormal semen quality. The development of assisted reproductive technology has effectively improved the fertility of men with abnormal semen, enabling them to obtain more fertility opportunities. Azoospermia is one of the most serious male infertility diseases and includes obstructive azoospermia and nonobstructive azoospermia (NOA)(Wosnitzer et al. 2014). The major indication for adopting ART in infertile men is the presence of viable sperm in the testicles(Gross et al. 2020). For patients with obstructive azoospermia(OA), the testis usually has normal spermatogenesis. Therefore, removing the obstruction through surgery or obtaining sperm through sperm retrieval surgery can solve the fertility problem(Akerman et al. 2020). However, for patients with NOA, spermatogenic function in the testis has failed. Although the development of microsurgery has improved the chances of obtaining sperm, the outcome is often unsatisfactory because no sperm is found or the sperm is inactive(Halpern et al., 2019, Gross et al., 2020). To date, there is no effective treatment to improve spermatogenesis failure in NOA patients. Increasing the fertility opportunities of patients with NOA remains a major challenge. To overcome this challenge, the molecular regulatory network in different spermatogenesis processes should first be understood in detail.
RNA m6A modification has recently been explored as a posttranscriptional modification that occurs in almost all kinds of RNA, including tRNA, rRNA, mRNA and noncoding RNA (ncRNA) (Meyer et al., 2012, Linder et al., 2015, Brown et al., 2016, Pendleton et al., 2017, Warda et al., 2017, Du et al., 2018, Ji et al., 2018, Chen et al., 2019). Previous studies have found that the RNA m6A regulators METTL3/14 participate in the self-renewal and differentiation of spermatogonial stem cells and that AKBH5 and YTHDC1 are involved in spermatocyte mitosis by regulating the RNA m6A modification of spermatogenic cells(Zheng et al., 2013, Hsu et al., 2017, Lin et al., 2017). Knockout of these RNA m6A regulators caused spermatogenesis dysfunction and induced infertility. Based on analytic results of single-cell sequencing data in NOA (Wang et al. 2018), we unexpectedly found that almost all spermatogenic cells and somatic cells have dysregulated RNA m6A regulators (Cai et al. 2021). This suggests that RNA m6A regulators have important roles in maintaining normal spermatogenesis.
During spermatogenesis, microRNAs (miRNAs) are expressed in different testicular cells in a cell- and step-specific manner(Chen et al., 2017, Zhou et al., 2019, Cai et al., 2020). It is an important part of the coordinated molecular regulatory network during spermatogenesis. Additionally, studies have shown that the formation of miRNAs is regulated by RNA m6A regulators such as METTL3(Alarcon et al., 2015b, Di Timoteo et al., 2020, Lin et al., 2020). Meanwhile, miRNA can also reversely affect the binding of METTL3 through the miRNA-mRNA axis, thereby changing the RNA m6A level(Chen et al. 2015). Other RNA m6A regulators, including HNRNPA2B1, YTHDF1, YTHDF2, and METTL14, have also been found to interact with miRNAs to exert molecular regulation(Alarcon et al., 2015a, Zhou et al., 2017, Chen et al., 2019, Lin et al., 2020, Xu et al., 2020). Therefore, miRNAs are closely related to RNA m6A regulators, and miRNAs may play an important role during spermatogenesis.
It is known that m6A regulators and miRNAs play an important role in the regulation of spermatogenesis. We asked which 6A regulators and miRNAs are involved in regulating spermatogenesis in different spermatogenic cells.Data-based bioinformatic analysis can provide important reference value for basic and clinical research. Based on this, we reanalyzed the open-access microarray dataset GSE45887 of NOA published in the GEO database (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE45887). Based on testicular histopathology, NOA testis samples were divided into three types, including Sertoli-cell-only syndrome (SCOS), meiotic arrest (MA), and postmeiotic arrest (PA), which represented the loss of three germ cell populations, including whole spermatogenic cells, postmeiotic spermatogenic cells, and a mixture of late spermatids and spermatozoa, respectively. After comparison with normal testes, their molecular characteristics were obtained.
Section snippets
Microarray data
We searched the GEO database and found four NOA datasets from human testicular biopsy samples (GSE45885, GSE45887, GSE51111 and GSE9210). GSE9210 were excluded due to containing animal data. The human samples in GSE45885 and GSE51111 are part of the human samples in GSE45887; therefore, GSE45885 and GSE51111 were excluded. Finally, GSE45887 was selected for further bioinformatic analysis. GSE45887 contained testicular biopsy specimens from 16 patients with NOA and 4 patients with normal
Identification of DEGs in three germ cell populations
Differential expression analysis of data in three types of NOA was performed with that in NS. According to the limma package (P < 0.01), there were 624 DEGs in the PA group, of which 82.29% were downregulated and 17.71% were upregulated (Fig. 1A). In the MA group, there were a total of 1177 DEGs, of which 72.23% were downregulated and 27.27% were upregulated (Fig. 1B). In the SCOS group, there were a total of 6022 DEGs, of which 57.01% were downregulated and 42.99% were upregulated (Fig. 1C).
Functional enrichment analysis of DEGs and GSEA of all genes in three germ cell populations
To
Discussion
Spermatogenesis refers to the process of cell differentiation and development from spermatogonial stem cells to spermatozoa, which includes more than a dozen kinds of germ cells. During spermatogenesis, different types of germ cells have different regulatory mechanisms. Therefore, with the increase in the types of spermatogenic cells, the regulatory mechanisms are more complex. In our study, PA, MA and SCOs were characterized by a lack of a mixture of late spermatids and spermatozoa,
Funding xxx
Shandong Province Medical and Health Technology Development Plan, NO: 202004050054.
Ethical approval xxx
This article does not contain any studies with human participants or animals performed by any of the authors.
Authors’ roles xxx
GB, XXZ and LLL collected the data and wrote the manuscript. ZLC and JX designed the study, analyzed data and wrote manuscript. HJL and BY designed and supervised the study. All authors read and approved the final version of the manuscript.
Data Availability Statement xxx
The data underlying this article are available in the article and in its online supplementary material.
Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
References (49)
- et al.
Sperm Extraction in Obstructive Azoospermia: What's Next?
Urol. Clin. North Am.
(2020) - et al.
HNRNPA2B1 is a mediator of m(6)A-dependent nuclear RNA processing events
Cell
(2015) - et al.
The roles of cirRNA in the development of germ cells
Acta Histochem.
(2020) - et al.
m(6)A RNA methylation is regulated by microRNAs and promotes reprogramming to pluripotency
Cell Stem Cell
(2015) - et al.
The roles of microRNAs in regulation of mammalian spermatogenesis
J. Anim. Sci. Biotechnol.
(2017) - et al.
Modulation of circRNA metabolism by m(6)A modification
Cell Rep.
(2020) - et al.
Round spermatid injection
Urol. Clin. North Am.
(2020) - et al.
Fertility-enhancing male reproductive surgery: glimpses into the past and thoughts for the future
Fertil. Steril.
(2019) - et al.
European Association of Urology guidelines on Male Infertility: the 2012 update
Eur. Urol.
(2012) - et al.
Comprehensive analysis of mRNA methylation reveals enrichment in 3' UTRs and near stop codons
Cell
(2012)
Distinct m(6)A methylome profiles in poly(A) RNA from Xenopus laevis testis and that treated with atrazine
Chemosphere
Fertility and infertility: Definition and epidemiology
Clin. Biochem.
ALKBH5 is a mammalian RNA demethylase that impacts RNA metabolism and mouse fertility
Mol. Cell
Genome-wide maps of m6A circRNAs identify widespread and cell-type-specific methylation patterns that are distinct from mRNAs
Cell Rep
Altered of microRNA expression level in oligospermic patients
Iran. J. Reprod. Med.
N6-methyladenosine marks primary microRNAs for processing
Nature
An automated method for finding molecular complexes in large protein interaction networks
BMC Bioinf.
MicroRNA expression profiles of seminoma from paraffin-embedded formalin-fixed tissue
Virchows Arch.
Long noncoding RNAs expression profile and long noncoding RNA-mediated competing endogenous RNA network in nonobstructive azoospermia patients
Epigenomics
Methyltransferase-like protein 16 binds the 3'-terminal triple helix of MALAT1 long noncoding RNA
Proc. Natl. Acad. Sci. USA
RNA N6-methyladenosine modification, spermatogenesis, and human male infertility
Mol. Hum. Reprod.
N6-methyladenosine modification controls circular RNA immunity
Mol. Cell
SUMOylation of the m6A-RNA methyltransferase METTL3 modulates its function
Nucleic Acids Res.
Ythdc2 is an N(6)-methyladenosine binding protein that regulates mammalian spermatogenesis
Cell Res.
Cited by (5)
Joint analysis of m<sup>6</sup>A and mRNA expression profiles in the testes of idiopathic nonobstructive azoospermia patients
2022, Frontiers in Endocrinology
- 1
Contributed equally.