Circular RNA regulates male spermatogenesis: a narrative review

Spermatogenesis was crucial for adult male animals to achieve reproductive function, and this complex physiological process required timely and moderate expression of related genes. A large number of epigenetic regulatory factors were involved, including cyclic RNA. Circular RNA had various characteristics such as rich expression, evolutionary conservation, cell or tissue specificity, and higher resistance to exonuclease or ribonuclease degradation. It can regu‑ late the expression of parental genes and function as mRNA traps, miRNAs, or proteins in the corpus cavernous; it can also participate in the process of spermatogenesis through RNA‑binding proteins, including the formation of repro‑ ductive stem cells, sperm formation, seminal plasma composition, and testicular tissue formation.


Background
Spermatogenesis is the process by which male reproductive cells proliferate and differentiate into mature sperm.Epigenetic regulation played an important role in spermatogenesis.It had confirmed that RNA exists in sperm with the rapid development of molecular biology, gene chip technology, and high-throughput sequencing technology.RNA played an important role in spermatogenesis.With in-depth research on non-coding RNA, a new class of star molecules had been reported: circular RNA [1].Circular RNA was a type of non-coding RNA.As a novel transcript form, the role of circular RNA in human germ cell development and its relationship with early embryonic development had been less studied.Circular RNA was not composed of exons arranged in the normal order of the genome but rather was connected endto-end at the 5′ and 3′ ends, forming a covalently closed single-stranded circular molecule.Thousands of endogenous circular RNA had been detected in cells of many different species.Circular RNA had various characteristics such as rich expression, evolutionary conservation, cell or tissue specificity, and higher resistance to exonuclease or ribonuclease degradation.Circular RNA was a special type of endogenous non-coding RNA (ncRNA) that was selectively cleaved and widely expressed in eukaryotic cells.It was a new research hotspot in the RNA family, following microRNA (miRNA) and long non-coding RNA (lncRNA).Circular RNA included endogenous RNA molecules composed of exons and RNA molecules derived from introns.Circular RNA had been proven widely present in various eukaryotic organisms.Circular RNA was a single-stranded, covalently closed RNA molecule that was ubiquitous in species from viruses to mammals.Significant progress had been made in the biogenesis, regulation, localization, degradation, and modification of circular RNA.Circular RNA exerts its biological functions by acting as transcriptional regulators, miR sponges, and protein templates.Extracellular vesicles (EVs) can serve as signal carriers to transmit the proteins, lipids, and nucleic acid (mRNAs, miRNAs, lncRNAs, and circular RNAs) molecules they contain, playing various roles in the interactions between sperm cells.Circular RNA had potential biological significance [2].

Spermatogenesis
Spermatogenesis refers to the process in which the sperm was in the seminiferous tubules of adult male animals, and different spermatogenic cells develop and differentiate according to different steps in the seminiferous tubules.Multidimensional pathways regulated spermatogenesis, such as follicle-stimulating hormone (FSH), luteinizing hormone (LH), and inhibin B (INH B) as well as local regulation of paracrine, autocrine, and cellular hormones in the testes.This complex physiological process requires timely and moderate expression of relevant genes, with a large number of epigenetic regulatory factors involved, such as histone modification, DNA methylation, genomic imprinting, X chromosome inactivation (it refers to the phenomenon in which one of the two X chromosomes in a woman lose activity; it was packaged as heterochromatin, which suppresses gene function and silences it), and non-coding RNA [3].Circular RNA was an important executor of epigenetic regulation in spermatogenesis.

Types and potential functions of circular RNA
The known circular RNA can be divided into five categories based on their formation mode.(1) Exon circular RNA (ecRNA) is entirely composed of single or multiple exons and does not contain introns.There may be three possible mechanisms for its formation: cyclization dependent on spliceosomes, cyclization driven by cis-elements, and cyclization mediated by RNA-binding protein (RBP).The three are not isolated and can work synergistically to exert their functions.(2) Intergenic circular RNA is a region located between two genes, with an unclear mechanism of formation.(3) Circular intronic RNA (ciRNA) is composed of a single intron and originates from the intron lasso structure generated by conventional pre-mRNA splicing.It was not degraded and accumulated in cells [4].(4) EIciRNA contained both introns and exons.( 5) Other circRNAs do not belong to the above four categories.Circular RNA was an important epigenetic regulatory factor.It can participate in the transcription and regulation of posttranscriptional gene expression.Circular RNA had the following functions: (1) it can regulate the expression of parental genes.Some intron-derived circular RNAs were abundant in the nucleus and regulated the transcriptional activity of their own coding genes through cis-or trans-interactions with RNA polymerase II.Cai Y et al. detected that circEIF3J and circPIAP2 can interact with small ribonucleoproteins in human cells, regulating host gene transcription in a cis-reaction manner [5].(2) Circular RNA was mainly composed of exons; it may be involved in the generation of mRNA, and the formation of circular RNA can compete with linear mRNA [6].However, this evidence did not support this hypothesis, such as a receptor site mutation in exon 4 or exon 5 of the flavin mononucleotide (fmn) gene, which can lead to corresponding circular RNA deletion but does not affect the splicing efficiency of linear RNA [7].Therefore, it was not known whether mRNA traps are byproducts of circular RNA biogenesis or definite biological significance.(3) There were binding sites for miRNA on circular RNA, which can act as the sponge of miRNA [8], bind with corresponding miRNA, relieve the inhibitory effect of miRNA on mRNA, increase the expression level of target gene mRNA, and regulate target genes at the post transcriptional level, such as circular-RNAcTFRC, circular-PSMC3, and circular-SETD3.CircRNA-cTFRC acts as the sponge of miRNA-107, promoting the expression of host gene TFRC [9].(4) Some circular RNAs have one or more binding sites for RBP, which can serve as the sponge of protein molecules.Circular RNA can bind to various RBPs, such as Argonautes, RNA Pol II, and MBL, and may regulate the function of RBPs through the competitive mode of miRNA sponge [10].(5) Circular RNA was considered to have no translation function, but research suggests that it can initiate translation function within cells by having an internal ribosome entry site.Testing of some artificially synthesized circular RNAs revealed that they contain an internal ribosome entry site.These circular RNAs may contain functions that encode proteins [11].

Circular RNA regulated male spermatogenesis
Human spermatogenesis refers to the physiological process of proliferation and differentiation of the spermatogonia into mature sperm.It was the result of programmed expression and mutual regulation of specific genes in a specific time and space under the fine regulation of the hypothalamic pituitary testicular endocrine axis and local microenvironment of the testis in the body.The comprehensive and complete gene expression profile of spermatogenic cells during testicular development strongly confirms the existence of specific types and differential levels of gene expression in each stage of spermatogenesis [12].Epigenetics regulates gene dynamic expression during spermatogenesis and maintains normal spermatogenesis through various methods such as histone modification, DNA methylation, chromatin recombination, and non-coding RNA regulation [13].

Expression and function of circular RNA in human testicular tissue
At present, thousands of circular RNA had been identified in human normal tissues (brain, heart, liver, lung, stomach, colon, etc.) and disease tissues (esophageal squamous cell carcinoma, gastric cancer, colorectal cancer, breast cancer, renal clear cell carcinoma, etc.) [14].Zhu F et al. predicted a total of 15,996 circular RNAs with read count values ≥ 1 in human testicular tissues using circular Seq technology [15].Among the host genes that make up these 15,996 circular RNAs, 1017 host genes can form loops. GO enrichment analysis showed that these genes were mainly related to germ cell development, meiotic cell cycle, and other factors.Most genes can produce up to 6 circular RNAs, with a minority producing more than 6 circular RNAs.The human testicular tissue, like other tissues, contains abundant circular RNAs, and most of these circular RNAs may be related to the expression of testis specific genes and dynamic changes in gene expression during spermatogenesis.HIST1H2BA [16], which played an important role in the transformation of histones into protamine in the sperm nucleus during spermatogenesis, and LRWD1 [17], which was closely related to Sertoli cell-only syndrome, were host genes corresponding to circular RNAs.I supposed the circular RNAs derived from the testes and sperm mainly come from the exon regions of genes.According to the location of host genes, they were widely distributed on all chromosomes, including the mitochondrial genome.These circular RNAs mainly participated in germ cell processes, meiotic events, epigenetic modifications, sperm motility, and fertilization, thus playing a crucial role in male reproduction.

Expression and function of circular RNA in human seminal plasma
Hundreds of circular RNAs had been detected in normal human saliva, and thousands of circular RNAs had been detected in the peripheral blood, indicating the widespread presence of circular RNAs in body fluids [18].Semen, which was as an important bodily fluid in humans, also contained abundant circular RNAs.Zhang et al. collected normal male semen and separated the seminal plasma.After storage at room temperature for 0 h, 12 h, and 24 h, there was no significant change in the expression level of circular RNA, indicating that the stability of circular RNA in seminal plasma remained good after 24 h in vitro [19].The high stability of circular RNA was inevitably related to its circular closed structure and the digestion characteristics of ribonuclease R (RNase R).It also speculated that circular RNA in human seminal plasma might exist in the form of protein binding.

Expression and function of circular RNA in human sperm
Research had confirmed that circular RNA in the sperm played a significant role in spermatogenesis, motility, fertilization, and early embryonic development events [20].Manfrevola et al. screened for differentially expressed sperm circular RNAs between asthenozoospermia patients and normal males, and the results showed that sperm circular RNAs come from a wide range of sources.Eighty-three percent of sperm circular RNAs were derived from exons, 5% from introns, and 3% from intergenic regions.The content of other sources of circular RNAs was relatively low [21].The GO enrichment analysis results indicate that differentially expressed circular RNAs were mainly enriched in cellular components such as sperm flagella, primary flagella, flagella axons, cilia morphogenesis, regulation of cilia oscillation frequency, determination of epithelial cilia movement with left/right asymmetry of the body axis, assembly of axon motor protein arm complexes, and molecular functions such as methylated histone binding, histone acetyltransferase activity, and histone methyltransferase activity.I supposed sperm circular RNAs participated in the formation of the biological structure of sperm tail flagella axons and played an important role in sperm forward movement.Song Y et al. focused on studying circNAPE-PLDio1, which is one of the circular RNA subtypes of the N-acyl photoshatitylethanolamine photosphosphopase D (NAPEPLD) transcript and was expressed in both human and mouse sperm [22].In unfertilized mouse oocytes, expression analysis showed that circNAPEPDio1 and circNAPEPDio2 were at low and high levels, respectively.After fertilization, the expression of circNAPEPDio1 significantly increased, while the expression of circ-NAPEPDio2 remained unchanged.This suggested that circNAPEPLDio1 may represent the cytoplasmic contribution of the father to the fertilized oocyte and served as a miRNA bait to regulate the first stage of embryonic development in the fertilized oocyte.

Circular RNA participated in male infertility
Tang W used RT qPCR to screen for sperm circular RNAs in normal males and males with asthenozoospermia and detected differential expression of 5 circular RNAs between the two groups [23].Compared with healthy males, the expression of hsa circle MY09B was significantly downregulated in patients with asthenozoospermia, while the differences in hsa circle WHSCI (8), hsa circle 1, and hsa circle CAMSAPl were statistically significant.According to the gene database in the National Center for Biotechnology Information (NCBI), the MY09B (myosin IXB) gene was located at 19p13.11 and contains 40 exons.It was widely expressed in tissues and organs of the human reproductive system, and its function has predicted the development and functional diseases of the human reproductive system [24].The WHSCl gene, also known as the nuclear receptor-binding SET domain protein 2 (NSD2) gene, was located at 4p16.3 and contains a total of 29 exons.It was widely expressed in tissues and organs of the human reproductive system, with its expression value in the testes being the highest among currently studied tissues, indicating its association with the function of the human reproductive system, especially the male reproductive system.The CAMSAP1 gene, located at 9q34.3, contains a total of 19 exons and was widely expressed in tissues and organs of the human reproductive system.Its expression levels in reproductive system tissues, from high to low, are as follows: testicles, endometrium, ovaries, and prostate, indicating that it was related to the function of the human reproductive system, especially the male reproductive system [25].The biological function of circular RNA can provide potential targets for the clinical diagnosis and treatment of male infertility.Its correlation with the human reproductive system, especially the male reproductive system, still needed further exploration.

Conclusion
The sperm is essential for the fertilization process as a male gamete.It was believed that biological events such as transcription and translation do not occur within the sperm as a highly differentiated terminal cell.However, people had gained a more microscopic and comprehensive understanding of the sperm with the rapid development of molecular biology, gene chip technology, high-throughput sequencing technology, and other methods.The circular RNAs derived from the testes and sperm mainly come from the exon regions of genes, and they were widely distributed on all chromosomes, including the mitochondrial genome, based on the location of host genes.These circular RNAs mainly participated in germ cell processes, meiotic events, epigenetic modifications, sperm motility, and fertilization, thus playing a crucial role in male reproduction [26].With the gradual development of bioinformatics technology and highthroughput sequencing methods, more and more circular RNA had been identified in the field of mammalian reproduction.The discovery of circular RNA provided direction for screening biomarkers for diagnosis and treatment of reproductive diseases, added new regulatory network for in-depth analysis of the regulatory mechanisms in reproductive cells and embryonic development, and opened up new perspectives for studying mammalian reproductive development.It was not yet clear whether the complex regulatory network between newly emerging circular RNA and RNA/proteins would break the previously recognized regulatory mechanisms.At present, the research on circular RNA in the field of mammalian reproduction was still in its early stages, and its important biological functions in the reproductive field still needed further exploring.I think further progress would be made in the research of circular RNA in the reproductive field with the efforts of researchers to explore circular RNA.