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Cellular & Molecular Biology Letters Sep 2023In recent years, N6-methyladenosine (mA) methylation modification of mRNA has been studied extensively. It has been reported that mA determines mRNA fate and...
BACKGROUND
In recent years, N6-methyladenosine (mA) methylation modification of mRNA has been studied extensively. It has been reported that mA determines mRNA fate and participates in many cellular functions and reactions, including oxidative stress. The PLOD2 gene encodes a protein that plays a key role in tissue remodeling and fibrotic processes.
METHODS
The mA methylation and expression levels of PLOD2 were determined by mA methylated RNA immunoprecipitation sequencing (MeRIP-seq) and MeRIP-quantitative polymerase chain reaction (qPCR) in the testes of varicocele rats compared with control. To determine whether IGF2BP2 had a targeted effect on the PLOD2 mRNA, RNA immunoprecipitation-qPCR (RIP-qPCR) and luciferase assays were performed. CRISPR/dCas13b-ALKBH5 could downregulate mA methylation level of PLOD2, which plays an important role in PLOD2-mediated cell proliferation and apoptosis in GC-2 cells.
RESULTS
PLOD2 was frequently exhibited with high mA methylation and expression level in the testes of varicocele rats compared with control. In addition, we found that IGF2BP2 binds to the mA-modified 3' untranslated region (3'-UTR) of PLOD2 mRNA, thereby positively regulating its mRNA stability. Targeted specific demethylation of PLOD2 mA by CRISPR/dCas13b-ALKBH5 system can significantly decrease the mA and expression level of PLOD2. Furthermore, demethylation of PLOD2 mRNA dramatically promote GC-2 cell proliferation and inhibit cell apoptosis under oxidative stress.
CONCLUSION
As a result, we found that varicocele-induced oxidative stress promoted PLOD2 expression level via mA methylation modification. In addition, targeting mA demethylation of PLOD2 by CRISPR/dCas13b-ALKBH5 system can regulate GC-2 cell proliferation and apoptosis under oxidative stress. Taken together, our study has acquired a better understanding of the mechanisms underlying male infertility associated with oxidative stress, as well as a novel therapeutic target for male infertility.
Topics: Male; Animals; Rats; Humans; Spermatocytes; Varicocele; 3' Untranslated Regions; Adenosine; Infertility, Male; Procollagen-Lysine, 2-Oxoglutarate 5-Dioxygenase; RNA-Binding Proteins
PubMed: 37670228
DOI: 10.1186/s11658-023-00475-4 -
Journal of Advanced Research Sep 2023The R-loop is a naturally formed three-strand nucleic acid structure that recently has been reported to participate in multiple biological processes and helped answer...
INTRODUCTION
The R-loop is a naturally formed three-strand nucleic acid structure that recently has been reported to participate in multiple biological processes and helped answer some previously unexplained scientific questions. Meiosis process involves multiple chromatin-related events such as DNA double-stranded breaks (DSB) formation, repairing and transcriptional dynamics.
OBJECTIVES
Explore the regulatory roles and physiological functions of R-loops in the mammalian meiosis process.
METHODS
In our study, using genome-wide S9.6 CUT & Tag seq, we first mapped the genomic distribution and dynamic changes of R-loop during the meiotic process in mice, from spermatogonia to secondary spermatocytes. And we further explore the role of R-loop in physiological conditions by constructing conditional knockout mice of Rnaseh1, which deleted the R-loop endonuclease before meiosis entry.
RESULTS
R-loop predominantly distributes at promoter-related regions and varies across different meiotic stages. By joint analysis with the corresponding transcriptome, we found that the R-loop was closely related to transcription during the meiotic process. The high frequency of promoter-related R-loop in meiotic cells is usually accompanied by high transcription activity, and we further verified this in the leptotene/zygotene to the pachytene transition process. Moreover, the lack of RNase H1 caused sterility in male mice with R-loop accumulation and abnormal DSB repair in spermatocytes. Further analysis showed that abnormal R-loop accumulation in the leptotene/zygotene stages influenced transcriptional regulation in the pachytene stage.
CONCLUSION
The mutual regulation of the R-loop and transcription plays an essential role in spermatogenesis. And R-loop is also important for the normal repair process of DSB during meiosis.
Topics: Male; Mice; Animals; R-Loop Structures; DNA Breaks, Double-Stranded; Meiosis; Spermatogenesis; Spermatocytes; Mice, Knockout; Mammals
PubMed: 36396044
DOI: 10.1016/j.jare.2022.10.016 -
Frontiers in Endocrinology 2024Infertility affects approximately 10-15% of couples worldwide who are attempting to conceive, with male infertility accounting for 50% of infertility cases. Male... (Review)
Review
Infertility affects approximately 10-15% of couples worldwide who are attempting to conceive, with male infertility accounting for 50% of infertility cases. Male infertility is related to various factors such as hormone imbalance, urogenital diseases, environmental factors, and genetic factors. Owing to its relationship with genetic factors, male infertility cannot be diagnosed through routine examination in most cases, and is clinically called 'idiopathic male infertility.' Recent studies have provided evidence that microRNAs (miRNAs) are expressed in a cell-or stage-specific manner during spermatogenesis. This review focuses on the role of miRNAs in male infertility and spermatogenesis. Data were collected from published studies that investigated the effects of miRNAs on spermatogenesis, sperm quality and quantity, fertilization, embryo development, and assisted reproductive technology (ART) outcomes. Based on the findings of these studies, we summarize the targets of miRNAs and the resulting functional effects that occur due to changes in miRNA expression at various stages of spermatogenesis, including undifferentiated and differentiating spermatogonia, spermatocytes, spermatids, and Sertoli cells (SCs). In addition, we discuss potential markers for diagnosing male infertility and predicting the varicocele grade, surgical outcomes, ART outcomes, and sperm retrieval rates in patients with non-obstructive azoospermia (NOA).
Topics: Humans; Male; MicroRNAs; Semen; Infertility, Male; Spermatogenesis; Phenotype; Biomarkers
PubMed: 38449855
DOI: 10.3389/fendo.2024.1293368 -
Development (Cambridge, England) Jul 2023Retinoic acid (RA) is the proposed mammalian 'meiosis inducing substance'. However, evidence for this role comes from studies in the fetal ovary, where germ cell...
Retinoic acid (RA) is the proposed mammalian 'meiosis inducing substance'. However, evidence for this role comes from studies in the fetal ovary, where germ cell differentiation and meiotic initiation are temporally inseparable. In the postnatal testis, these events are separated by more than 1 week. Exploiting this difference, we discovered that, although RA is required for spermatogonial differentiation, it is dispensable for the subsequent initiation, progression and completion of meiosis. Indeed, in the absence of RA, the meiotic transcriptome program in both differentiating spermatogonia and spermatocytes entering meiosis was largely unaffected. Instead, transcripts encoding factors required during spermiogenesis were aberrant during preleptonema, and the subsequent spermatid morphogenesis program was disrupted such that no sperm were produced. Taken together, these data reveal a RA-independent model for male meiotic initiation.
Topics: Animals; Female; Male; Testis; Tretinoin; Spermatogenesis; Spermatogonia; Spermatozoa; Meiosis; Mammals
PubMed: 37350382
DOI: 10.1242/dev.201638 -
BioRxiv : the Preprint Server For... Dec 2023Meiotic progression requires coordinated assembly and disassembly of protein complexes involved in chromosome synapsis and meiotic recombination. The AAA+ ATPase TRIP13...
Meiotic progression requires coordinated assembly and disassembly of protein complexes involved in chromosome synapsis and meiotic recombination. The AAA+ ATPase TRIP13 and its orthologue Pch2 are instrumental in remodeling HORMA domain proteins. Meiosis-specific HORMAD proteins are associated with unsynapsed chromosome axes but depleted from the synaptonemal complex (SC) of synapsed chromosome homologues. Here we report that TRIP13 localizes to the synapsed SC in early pachytene spermatocytes and to telomeres throughout meiotic prophase I. Loss of TRIP13 leads to meiotic arrest and thus sterility in both sexes. -null meiocytes exhibit abnormal persistence of HORMAD1 and HOMRAD2 on synapsed SC and chromosome asynapsis that preferentially affects XY and centromeric ends. These findings confirm the previously reported phenotypes of the hypomorph alleles. heterozygous () mice also exhibit meiotic defects that are less severe than the -null mice, showing that TRIP13 is a dosage-sensitive regulator of meiosis. Localization of TRIP13 to the synapsed SC is independent of SC axial element proteins such as REC8 and SYCP2/SYCP3. The N- or C-terminal FLAG-tagged TRIP13 proteins are functional and recapitulate the localization of native TRIP13 to SC and telomeres in knockin mice. Therefore, the evolutionarily conserved localization of TRIP13/Pch2 to the synapsed chromosomes provides an explanation for dissociation of HORMA domain proteins upon chromosome synapsis in diverse organisms.
PubMed: 37808842
DOI: 10.1101/2023.09.25.559355 -
International Journal of Biological... Dec 2023The variety of species can be efficiently increased by interspecific hybridization. However, because the males in the hybrid progeny are usually sterile, this heterosis...
The variety of species can be efficiently increased by interspecific hybridization. However, because the males in the hybrid progeny are usually sterile, this heterosis cannot be employed when other cattle and yaks are hybridized. While some system-level studies have sought to explore the etiological basis for male cattle-yak sterility, no systematic cellular analyses of this phenomenon have yet been performed. Here, single-cell RNA sequencing and UPHLC-MS/MS targeted metabolomics methods were used to study the differences in testicular tissue between 4-year-old male yak and 4-year-old male cattle-yak, providing new and comprehensive insights into the causes of male cattle-yak sterility. Cattle-yak testes samples detected 6 somatic cell types and one mixed germ cell type. Comparisons of these cell types revealed the more significant differences in Sertoli cells (SCs) and [Leydig cells and myoid cells (LCs_MCs)] between yak and cattle-yak samples compared to other somatic cell clusters. Even though the LCs and MCs from yaks and cattle-yaks were derived from the differentiation of the same progenitor cells, a high degree of overlap between LCs and MCs was observed in yak samples. Still, only a small overlap between LCs and MCs was observed in cattle-yak samples. Functional enrichment analyses revealed that genes down-regulated in cattle-yak SCs were primarily enriched in biological activity, whereas up-regulated genes in these cells were enriched for apoptotic activity. Furthermore, the genes of up-regulated in LCs_MCs of cattle-yak were significantly enriched in enzyme inhibitor and molecular function inhibitor activity. On the other hand, the genes of down-regulated in these cells were enriched for signal receptor binding, molecular function regulation, positive regulation of biological processes, and regulation of cell communication activity. The most significant annotated differences between yak and cattle-yak LCs_MCs were associated with cell-to-cell communication. While yak LCs_MCs regulated spermatogenic cells at spermatogonia, spermatocyte, and spermatid levels, no such relationships were found between cattle-yak LCs_MCs and germ cells. This may suggest that the somatic niche in male cattle-yak testes is a microenvironment that is ultimately not favorable for spermatogenesis.
Topics: Humans; Animals; Cattle; Male; Tandem Mass Spectrometry; Testis; Infertility, Male; Spermatogenesis; Sequence Analysis, RNA
PubMed: 37716658
DOI: 10.1016/j.ijbiomac.2023.126831 -
Frontiers in Endocrinology 2023Cryopreservation of immature testicular tissue (ITT) is currently the only option to preserve fertility of prepubertal patients. Autologous transplantation of ITT may...
BACKGROUND
Cryopreservation of immature testicular tissue (ITT) is currently the only option to preserve fertility of prepubertal patients. Autologous transplantation of ITT may not be safe or appropriate for all patients. Therefore, methods to mature ITT are needed.
OBJECTIVES
Aim to investigate the feasibility of inducing spermatogenesis from ITT cryopreserved for pediatric patients prior to initiation of gonadotoxic therapy.
MATERIALS AND METHODS
Cryopreserved-thawed ITT from prepubertal and peripubertal patients were cultured for 7, 16, and 32 days in medium with no hormones or supplemented with 5 IU/L FSH, 1 IU/L hCG, or 5IU/L FSH+1 IU/L hCG. Samples were evaluated histologically to assess tissue integrity, and immunofluorescence staining was performed to identify VASA (DDX4)+ germ cells, UCHL1+ spermatogonia, SYCP3+ spermatocytes, CREM+ spermatids, SOX9+ Sertoli cells. Proliferation (KI67) and apoptosis (CASPASE3) of germ cells and Sertoli cells were also analyzed. Sertoli and Leydig cell maturation was evaluated by AR and INSL3 expression as well as expression of the blood testis barrier protein, CLAUDIN11, and testosterone secretion in the culture medium.
RESULTS
Integrity of seminiferous tubules, VASA+ germ cells and SOX9+ Sertoli cells were maintained up to 32 days. The number of VASA+ germ cells was consistently higher in the peripubertal groups. UCHL1+ undifferentiated spermatogonia and SOX9+ Sertoli cell proliferation was confirmed in most samples. SYCP3+ primary spermatocytes began to appear by day 16 in both age groups. Sertoli cell maturation was demonstrated by AR expression but the expression of CLAUDIN11 was disorganized. Presence of mature and functional Leydig cells was verified by INSL3 expression and secretion of testosterone. Gonadotropin treatments did not consistently impact the number or proliferation of germ cells or somatic cells, but FSH was necessary to increase testosterone secretion over time in prepubertal samples.
CONCLUSION
ITT were maintained in organotypic culture for up to 32 days and spermatogonia differentiated to produce primary spermatocytes in both pre- and peripubertal age groups. However, complete spermatogenesis was not observed in either group.
Topics: Male; Humans; Child; Fertility Preservation; Organ Culture Techniques; Cryopreservation; Testosterone; Follicle Stimulating Hormone
PubMed: 37701899
DOI: 10.3389/fendo.2023.1242263 -
IScience Sep 2023WNK1 is an important regulator in many physiological functions, yet its role in male reproduction is unexplored. In the male germline, WNK1 is upregulated in...
WNK1 is an important regulator in many physiological functions, yet its role in male reproduction is unexplored. In the male germline, WNK1 is upregulated in preleptotene spermatocytes indicating possible function(s) in spermatogenic meiosis. Indeed, deletion of in mid-pachytene spermatocytes using the mouse led to male sterility which resembled non-obstructive azoospermia in humans, where germ cells failed to complete spermatogenesis and produced no sperm. Mechanistically, we found elevated MTOR expression and signaling in the -depleted spermatocytes. As MTOR is a central mediator of translation, we speculated that translation may be accelerated in these spermatocytes. Supporting this, we found the acrosome protein, ACRBP to be prematurely expressed in the spermatocytes with deletion. Our study uncovered an MTOR-regulating factor in the male germline with potential implications in translation, and future studies will aim to understand how WNK1 regulates MTOR activity and impact translation on a broader spectrum.
PubMed: 37694147
DOI: 10.1016/j.isci.2023.107616 -
Science (New York, N.Y.) Mar 2024The extent to which prophage proteins interact with eukaryotic macromolecules is largely unknown. In this work, we show that cytoplasmic incompatibility factor A (CifA)...
The extent to which prophage proteins interact with eukaryotic macromolecules is largely unknown. In this work, we show that cytoplasmic incompatibility factor A (CifA) and B (CifB) proteins, encoded by prophage WO of the endosymbiont alter long noncoding RNA (lncRNA) and DNA during sperm development to establish a paternal-effect embryonic lethality known as cytoplasmic incompatibility (CI). CifA is a ribonuclease (RNase) that depletes a spermatocyte lncRNA important for the histone-to-protamine transition of spermiogenesis. Both CifA and CifB are deoxyribonucleases (DNases) that elevate DNA damage in late spermiogenesis. lncRNA knockdown enhances CI, and mutagenesis links lncRNA depletion and subsequent sperm chromatin integrity changes to embryonic DNA damage and CI. Hence, prophage proteins interact with eukaryotic macromolecules during gametogenesis to create a symbiosis that is fundamental to insect evolution and vector control.
Topics: Animals; Male; Cytoplasm; DNA; Prophages; RNA, Long Noncoding; Spermatozoa; Wolbachia; Paternal Inheritance; Viral Proteins; Drosophila melanogaster; Bacterial Proteins; Deoxyribonucleases
PubMed: 38452081
DOI: 10.1126/science.adk9469 -
BioRxiv : the Preprint Server For... Dec 2023Spermatogenesis is a unidirectional differentiation process that generates haploid sperm, but how the gene expression program that directs this process is established is...
Spermatogenesis is a unidirectional differentiation process that generates haploid sperm, but how the gene expression program that directs this process is established is largely unknown. Here we determine the high-resolution 3D chromatin architecture of male germ cells during spermatogenesis and show that CTCF-mediated 3D chromatin predetermines the gene expression program required for spermatogenesis. In undifferentiated spermatogonia, CTCF-mediated chromatin contacts on autosomes pre-establish meiosis-specific super-enhancers (SE). These meiotic SE recruit the master transcription factor A-MYB in meiotic spermatocytes, which strengthens their 3D contacts and instructs a burst of meiotic gene expression. We also find that at the mitosis-to-meiosis transition, the germline-specific Polycomb protein SCML2 resolves chromatin loops that are specific to mitotic spermatogonia. Moreover, SCML2 and A-MYB establish the unique 3D chromatin organization of sex chromosomes during meiotic sex chromosome inactivation. We propose that CTCF-mediated 3D chromatin organization enforces epigenetic priming that directs unidirectional differentiation, thereby determining the cellular identity of the male germline.
PubMed: 38076840
DOI: 10.1101/2023.11.30.569508