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Comparison of differentially expressed gene (DEG) and differential trascript usage (DTU) gene numbers in all four group comparisons. (A, B, C, D) Venn diagrams display number and proportion of genes that are differentially expressed, have a DTU event, or both in the (A) Sertoli cell-only versus SPG, (B) SPG versus SPC, (C) SPC versus SPD, and (D) SPD versus CTR group comparisons. Yellow = differential gene expressions, blue = DTU genes.
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The process of spermatogenesis—when germ cells differentiate into sperm—is tightly regulated, and misregulation in gene expression is likely to be involved in the physiopathology of male infertility. The testis is one of the most transcriptionally rich tissues; nevertheless, the specific gene expression changes occurring during spermatogenesis are...
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... found between 1,062 and 2,153 DTU genes in each of the four comparisons (Tables S8-S11). By comparing DTU genes to DEGs, we found an overlap of less than 8% in all four comparisons, indicating that the expression of most genes is regulated either at the pre-or the post-transcriptional level (Fig 4) and that only few genes are regulated at both levels. Furthermore, we found that the proportion of DEGs to DTU genes in all group comparisons was 2:1 ( Fig. 4A-C), except for SPD versus CTR, where this ratio was inversed with more DTU genes than DEGs (Fig 4D). ...
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... genes to DEGs, we found an overlap of less than 8% in all four comparisons, indicating that the expression of most genes is regulated either at the pre-or the post-transcriptional level (Fig 4) and that only few genes are regulated at both levels. Furthermore, we found that the proportion of DEGs to DTU genes in all group comparisons was 2:1 ( Fig. 4A-C), except for SPD versus CTR, where this ratio was inversed with more DTU genes than DEGs (Fig ...
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... comparing DTU genes to DEGs, we found an overlap of less than 8% in all four comparisons, indicating that the expression of most genes is regulated either at the pre-or the post-transcriptional level (Fig 4) and that only few genes are regulated at both levels. Furthermore, we found that the proportion of DEGs to DTU genes in all group comparisons was 2:1 ( Fig. 4A-C), except for SPD versus CTR, where this ratio was inversed with more DTU genes than DEGs (Fig 4D). ...
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... general biological functions (e.g., RNA metabolism, cell survival) were enriched among the DTU genes in each group comparison. To further classify the biological pathways enriched among DEGs and DTU genes, we performed pathway analysis via the Reactome Knowledgebase ( Gillespie et al, 2022), which confirmed that germ cell-specific and general pathways are enriched among DEGs (Fig.S4) and DTU genes (Fig.S5), respectively. ...
Citations
... All experiments followed the Portuguese (Decreto-Lei n° 113/2013) and European (Directive 2010/63/EU) legislations, concerning housing, husbandry, and animal welfare. Kuss et al., 2023), were extracted using the Direct-zol RNA Microprep kit (Zymo Research), following the manufacturer's instructions. RNA quality was estimated by electrophoresis (Agilent Technologies), with all samples having a RNA integrity number (RIN) >4.5 (range: 4.5-5.6), ...
Male germ cells share a common origin across animal species, therefore they likely retain a conserved genetic program that defines their cellular identity. However, the unique evolutionary dynamics of male germ cells coupled with their widespread leaky transcription pose significant obstacles to the identification of the core spermatogenic program. Through network analysis of the spermatocyte transcriptome of vertebrate and invertebrate species, we describe the conserved evolutionary origin of metazoan male germ cells at the molecular level. We estimate the average functional requirement of a metazoan male germ cell to correspond to the expression of approximately 10,000 protein-coding genes, a third of which defines a genetic scaffold of deeply conserved genes that has been retained throughout evolution. Such scaffold contains a set of 79 functional associations between 104 gene expression regulators that represent a core component of the conserved genetic program of metazoan spermatogenesis. By genetically interfering with the acquisition and maintenance of male germ cell identity, we uncover 161 previously unknown spermatogenesis genes and three new potential genetic causes of human infertility. These findings emphasize the importance of evolutionary history on human reproductive disease and establish a cross-species analytical pipeline that can be repurposed to other cell types and pathologies.
... For histological evaluation, two independent testicular sections from each testis were stained with periodic acid-Schiff/hema-toxylin and were evaluated based on the Bergmann and Kliesch scoring method 33 as previously described. 34 Preparation of single-cell suspensions from testicular biopsies For the extraction of pure germ cell subtypes, testicular biopsies were digested into a single-cell suspension as previously published. 16 The digestion was based on mechanically chopping up the testicular tissue with a sterile blade into 1 mm 3 pieces and a two-step enzymatic incubation: first, with MEMa (ThermoFisher scientific, Gibco, Cat# 22561021) with 1 mg/mL collagenase IA (Merck/Sigma Aldrich, Cat# C9891) at 37 C for 10 min and, second, with Hank's balanced salt solution (HBSS) containing 4 mg/mL trypsin (Thermo Fisher Scientific, Gibco, Cat# 27250018) and 2.2 mg/mL of DNase I (Merck/Sigma-Aldrich, Cat# DN25) at 37 C for 8-10 min and strong pipetting in between. ...
Sperm production and function require the correct establishment of DNA methylation patterns in the germline. Here, we examined the genome-wide DNA methylation changes during human spermatogenesis and its alterations in disturbed spermatogenesis. We found that spermatogenesis is associated with remodeling of the methylome, comprising a global decline in DNA methylation in primary spermatocytes followed by selective remethylation, resulting in a spermatids/sperm-specific methylome. Hypomethylated regions in spermatids/sperm were enriched in specific transcription factor binding sites for DMRT and SOX family members and spermatid-specific genes. Intriguingly, while SINEs displayed differential methylation throughout spermatogenesis, LINEs appeared to be protected from changes in DNA methylation. In disturbed spermatogenesis, germ cells exhibited considerable DNA methylation changes, which were significantly enriched at transposable elements and genes involved in spermatogenesis. We detected hypomethylation in SVA and L1HS in disturbed spermatogenesis, suggesting an association between the abnormal programming of these regions and failure of germ cells progressing beyond meiosis.
... At maturity (10 years), genes such as PRM1, HMGB4, SPA17, and TSACC were significantly expressed. All of these genes are closely related to the reproductive system and play key roles in testicular development, spermatogenesis, the packaging of sperm DNA, and sperm maturation [70][71][72]. In summary, for sexually immature versus sexually mature Mongolian horses, the difference in function of differential genes is huge. ...
This study aimed to investigate differences in testicular tissue morphology, gene expression, and marker genes between sexually immature (1-year-old) and sexually mature (10-year-old) Mongolian horses. The purposes of our research were to provide insights into the reproductive physiology of male Mongolian horses and to identify potential markers for sexual maturity. The methods we applied included the transcriptomic profiling of testicular cells using single-cell sequencing techniques. Our results revealed significant differences in tissue morphology and gene expression patterns between the two age groups. Specifically, 25 cell clusters and 10 cell types were identified, including spermatogonial and somatic cells. Differential gene expression analysis highlighted distinct patterns related to cellular infrastructure in sexually immature horses and spermatogenesis in sexually mature horses. Marker genes specific to each stage were also identified, including APOA1, AMH, TAC3, INHA, SPARC, and SOX9 for the sexually immature stage, and PRM1, PRM2, LOC100051500, PRSS37, HMGB4, and H1-9 for the sexually mature stage. These findings contribute to a deeper understanding of testicular development and spermatogenesis in Mongolian horses and have potential applications in equine reproductive biology and breeding programs. In conclusion, this study provides valuable insights into the molecular mechanisms underlying sexual maturity in Mongolian horses.
... FRAGILIS, another germline specifier differently involved in the developmental stages (Lange et al., 2003), was only barely affected by the EDs exposure, as its expression resulted altered only in PFOS and BPA + PFOA samples, in which a dramatic reduction of the transcript was observed. OVOL1, a marker linked to the activation of spermatocyte-specific genes (Siebert-Kuss et al., 2023), appeared diversely regulated by the pollutants, as it was highly upregulated by PFOS, whereas BPA, PFOA, BPA + PFOS and BPS + PFOA reduced its expression. We finally checked for PIWIL2, a germline specifier essential for spermatogenesis: this marker was downregulated from BPs, alone and in combination with PFs, with the only exception of BPS + PFOS; the same effect was observed when hiPSCs were exposed to the cocktail containing all the EDs. ...
Bisphenols and Perfluoroalkyls are chemical compounds widely used in industry known to be endocrine disruptors (EDs). Once ingested through contaminated aliments, they mimic the activity of endogenous hormones leading to a broad spectrum of diseases. Due to the extensive use of plastic in human life, particular attention should be paid to antenatal exposure to Bisphenols and Perfluoroalkyls since they cross the placental barrier and accumulates in developing embryo. Here we investigated the effects of Bisphenol-A (BPA), Bisphenol-S (BPS), perfluorooctane-sulfonate (PFOS) and perfluorooctanoic-acid (PFOA), alone or combined, on human-induced pluripotent stem cells (hiPSCs) that share several biological features with the stem cells of blastocysts. Our data show that these EDs affect hiPSC inducing a great mitotoxicity and dramatic changes in genes involved in the maintenance of pluripotency, germline specification, and epigenetic regulation. We also evidenced that these chemicals, when combined, may have additive, synergistic but also negative effects. All these data suggest that antenatal exposure to these EDs may affect the integrity of stem cells in the developing embryos, interfering with critical stages of early human development that might be determinant for fertility. The observation that the effects of exposure to a combination of these chemicals are not easily foreseeable further highlights the need for wider awareness of the complexity of the EDs effects on human health and of the social and economic burden attributable to these compounds.
Male germ cells share a common origin across animal species, therefore they likely retain a conserved genetic program that defines their cellular identity. However, the unique evolutionary dynamics of male germ cells coupled with their widespread leaky transcription pose significant obstacles to the identification of the core spermatogenic program. Through network analysis of the spermatocyte transcriptome of vertebrate and invertebrate species, we describe the conserved evolutionary origin of metazoan male germ cells at the molecular level. We estimate the average functional requirement of a metazoan male germ cell to correspond to the expression of approximately 10,000 protein-coding genes, a third of which defines a genetic scaffold of deeply conserved genes that has been retained throughout evolution. Such scaffold contains a set of 79 functional associations between 104 gene expression regulators that represent a core component of the conserved genetic program of metazoan spermatogenesis. By genetically interfering with the acquisition and maintenance of male germ cell identity, we uncover 161 previously unknown spermatogenesis genes and three new potential genetic causes of human infertility. These findings emphasize the importance of evolutionary history on human reproductive disease and establish a cross-species analytical pipeline that can be repurposed to other cell types and pathologies.
Sperm production and function require the correct establishment of DNA methylation patterns in the germline. Here, we examined the genome-wide DNA methylation changes during human spermatogenesis and its alterations in disturbed spermatogenesis. We found that spermatogenesis is associated with remodeling of the methylome, comprising a global-decline in DNA methylation in primary spermatocytes followed by selective remethylation, resulting in a spermatid-specific methylome. Hypomethylated regions in spermatids were enriched in specific transcription factor binding sites for DMRT and SOX family members and spermatid-specific genes. Intriguingly, while SINEs displayed differential methylation throughout spermatogenesis, LINEs appeared to be protected from changes in DNA methylation. In disturbed spermatogenesis, germ cells exhibited considerable DNA methylation changes, which were significantly enriched at transposable elements and genes involved in spermatogenesis. We detected hypomethylation in SVA and L1HS in disturbed spermatogenesis, suggesting an association between the abnormal programming of these regions and failure of germ cells progressing beyond meiosis.
Sperm flagellum plays a critical role in male fertility. Here, we generated Ccdc183 knockout (KO) mice using the CRISPR/Cas9 system to reveal the protein function of CCDC183 in spermiogenesis. We demonstrated that the absence of CCDC183 causes male infertility with morphological and motility defects in spermatozoa. Due to the lack of CCDC183, centrioles after elongation of axonemal microtubules do not connect the cell surface and nucleus during spermiogenesis, which causes subsequent loss of cytoplasmic invagination around the flagellum. As a result, the flagellar compartment does not form properly and cytosol-exposed axonemal microtubules collapse during spermiogenesis. In addition, ectopic localization of accessory structures such as the fibrous sheath and outer dense fibers, and abnormal head shape due to abnormal sculpting by the manchette are observed in Ccdc183 KO spermatids. Our results indicate that CCDC183 plays an essential role in cytoplasmic invagination around the flagellum to form functional spermatozoa during spermiogenesis.
Cardiovascular disease (CVD) is the most fatal disease that causes sudden death, and inflammation contributes substantially to its occurrence and progression. The prevalence of CVD increases as the population ages, and the pathophysiology is complex. Anti-inflammatory and immunological modulation are the potential methods for CVD prevention and treatment. High-Mobility Group (HMG) chromosomal proteins are one of the most abundant nuclear nonhistone proteins which act as inflammatory mediators in DNA replication, transcription, and repair by producing cytokines and serving as damage-associated molecular patterns in inflammatory responses. The most common and well-studied HMG proteins are those with an HMGB domain, which participate in a variety of biological processes. HMGB1 and HMGB2 were the first members of the HMGB family to be identified and are present in all investigated eukaryotes. Our review is primarily concerned with the involvement of HMGB1 and HMGB2 in CVD. The purpose of this review is to provide a theoretical framework for diagnosing and treating CVD by discussing the structure and function of HMGB1 and HMGB2.
