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Chromosoma Dec 2022In most eukaryotes, pairing of homologous chromosomes is an essential feature of meiosis that ensures homologous recombination and segregation. However, when the pairing...
In most eukaryotes, pairing of homologous chromosomes is an essential feature of meiosis that ensures homologous recombination and segregation. However, when the pairing process begins, it is still under investigation. Contrasting data exists in Mus musculus, since both leptotene DSB-dependent and preleptotene DSB-independent mechanisms have been described. To unravel this contention, we examined homologous pairing in pre-meiotic and meiotic Mus musculus cells using a three-dimensional fluorescence in situ hybridization-based protocol, which enables the analysis of the entire karyotype using DNA painting probes. Our data establishes in an unambiguously manner that 73.83% of homologous chromosomes are already paired at premeiotic stages (spermatogonia-early preleptotene spermatocytes). The percentage of paired homologous chromosomes increases to 84.60% at mid-preleptotene-zygotene stage, reaching 100% at pachytene stage. Importantly, our results demonstrate a high percentage of homologous pairing observed before the onset of meiosis; this pairing does not occur randomly, as the percentage was higher than that observed in somatic cells (19.47%) and between nonhomologous chromosomes (41.1%). Finally, we have also observed that premeiotic homologous pairing is asynchronous and independent of the chromosome size, GC content, or presence of NOR regions.
Topics: Animals; Mice; Male; In Situ Hybridization, Fluorescence; Meiosis; Meiotic Prophase I; Karyotype; Spermatocytes
PubMed: 35960388
DOI: 10.1007/s00412-022-00777-0 -
Toxics May 2023In recent years, male infertility has received global attention and seriously affected the quality of human fertility, and pyrethroids (type II pyrethroids), as...
In recent years, male infertility has received global attention and seriously affected the quality of human fertility, and pyrethroids (type II pyrethroids), as recognized environmental endocrine disruptors, may threaten male reproductive health. Therefore, in this study, we established an in vivo model for the development of testicular and germ cell toxicity induced by cyfluthrin and explored the role and mechanism of the gene-mediated P38 MAPK/JNK pathway in testicular and germ cell damage caused by cyfluthrin to find early and sensitive indicators and new therapeutic targets for the development of testicular damage. Firstly, 40 male Wistar rats (about 260 g) were divided into a control group (corn oil), low dose group (6.25 mg/kg), middle dose group (12.5 mg/kg) and high dose group (25 mg/kg). The rats were anesthetized and executed after 28 days of poisoning on alternate days. Then, HE staining, transmission electron microscopy, ELISA, q-PCR, Western blot, immunohistochemistry, double-immunofluorescence and TUNEL were used to observe the pathology, androgen levels, oxidative damage and altered expression of the key factors of the G3BP1 and MAPK pathways in rat testes. The results showed that, compared with the control group, the testicular tissue and spermatocytes were superficially damaged with an increasing dose of cyfluthrin; furthermore, it could interfere with the normal secretion of the hypothalamic-pituitary-gonadal axis (serum GnRH, FSH, T and LH levels) and cause hypergonadal dysfunction. A dose-dependent increase in MDA and a dose-dependent decrease in T-AOC indicated that the oxidative-antioxidative homeostatic balance was disrupted. The Western blot and qPCR analysis revealed that G3BP1, p-JNK1/2/3, P38 MAPK, p-ERK, COX1 and COX4 proteins and mRNA expression were decreased, and p-JNK1/2/3, p-P38MAPK, caspase 3/8/9 proteins and mRNA expression were significantly increased. The double-immunofluorescence and immunohistochemistry results showed that the protein expression of G3BP1 decreased with an increasing dose of staining, while the expression of JNK1/2/3 and P38 MAPK were increased significantly. The positive expressions of G3BP1 were mainly located in the testicular germinal epithelium and germ cell layer, and the positive expressions of JNK1/2/3 were mainly located in the testicular germinal epithelium and sperm cells, while the positive expressions of P38 MAPK were located in all levels of the germ cells and spermatozoa. Our results demonstrated that exposure to cyfluthrin caused testicular and spermatocyte damage in rats, which could cause pathomorphology, altered androgen levels and a decreased antioxidant capacity. When the intracellular antioxidant capacity was impaired, G3BP1 expression and activity were inhibited, causing activation of the P38 MAPK/JNK pathway and activation of the intracellular apoptotic pathway, which, in turn, led to germ cell apoptosis.
PubMed: 37235265
DOI: 10.3390/toxics11050451 -
Molecular Cell Dec 2021In animals, PIWI-interacting RNAs (piRNAs) silence transposons, fight viral infections, and regulate gene expression. piRNA biogenesis concludes with 3' terminal...
In animals, PIWI-interacting RNAs (piRNAs) silence transposons, fight viral infections, and regulate gene expression. piRNA biogenesis concludes with 3' terminal trimming and 2'-O-methylation. Both trimming and methylation influence piRNA stability. Our biochemical data show that multiple mechanisms destabilize unmethylated mouse piRNAs, depending on whether the piRNA 5' or 3' sequence is complementary to a trigger RNA. Unlike target-directed degradation of microRNAs, complementarity-dependent destabilization of piRNAs in mice and flies is blocked by 3' terminal 2'-O-methylation and does not require base pairing to both the piRNA seed and the 3' sequence. In flies, 2'-O-methylation also protects small interfering RNAs (siRNAs) from complementarity-dependent destruction. By contrast, pre-piRNA trimming protects mouse piRNAs from a degradation pathway unaffected by trigger complementarity. In testis lysate and in vivo, internal or 3' terminal uridine- or guanine-rich tracts accelerate pre-piRNA decay. Loss of both trimming and 2'-O-methylation causes the mouse piRNA pathway to collapse, demonstrating that these modifications collaborate to stabilize piRNAs.
Topics: Animals; Argonaute Proteins; Cell Separation; Drosophila melanogaster; Female; Flow Cytometry; Gene Expression; Gene Silencing; Genetic Techniques; Male; Methylation; Mice; Mice, Inbred C57BL; Mice, Transgenic; Protein Processing, Post-Translational; RNA, Double-Stranded; RNA, Small Interfering; Spermatocytes; Spermatogonia; Testis
PubMed: 34626567
DOI: 10.1016/j.molcel.2021.09.012 -
Andrology Jul 2023Precise spatiotemporal expression of cohorts of differentiation markers unique to spermatogonia, spermatocytes, and round spermatids punctuates spermatogenesis and... (Review)
Review
Precise spatiotemporal expression of cohorts of differentiation markers unique to spermatogonia, spermatocytes, and round spermatids punctuates spermatogenesis and ensures its completion. For example, genes coding for the synaptonemal complex or the acrosome or flagellum are expressed sequentially in a developmental stage- and germ cell-specific manner. But the transcriptional mechanisms governing the spatiotemporal order of gene expression within the seminiferous epithelium are poorly understood. Using the round spermatid-specific Acrv1 gene, which codes for the acrosomal protein SP-10 as a model, we learned that (1) the proximal promoter itself contains all the necessary cis-regulatory sequences, (2) an insulator prevents somatic cell expression of the testis-specific gene, (3) RNA II polymerase is loaded on the Acrv1 promoter but paused in spermatocytes, thus ensuring precise transcriptional elongation in round spermatids, and that (4) a transcriptional repressor binding protein of 43 kilodaltons (TDP-43) plays a role in maintaining the paused state in spermatocytes. Although the Acrv1 enhancer element has been narrowed down to 50 bp and its binding to a 47 kDa testis-abundant nuclear protein shown, the identity of the putative transcription factor responsible for activation of round spermatid-specific transcription remains elusive. Human male infertility is idiopathic with limited treatment options. Understanding transcriptional regulation of spermatogenesis has the potential to lead to future therapies for male infertility.
Topics: Mice; Male; Humans; Animals; Seminiferous Epithelium; Membrane Proteins; Spermatids; Gene Expression Regulation; Spermatogenesis; Testis; Infertility, Male; Gene Expression
PubMed: 36793255
DOI: 10.1111/andr.13410 -
Frontiers in Cell and Developmental... 2022Spermatogenesis holds considerable promise for human-assisted reproduction and livestock breeding based on stem cells. It occurs in seminiferous tubules within the...
Spermatogenesis holds considerable promise for human-assisted reproduction and livestock breeding based on stem cells. It occurs in seminiferous tubules within the testis, which mainly comprise male germ cells and Sertoli cells. While the developmental progression of male germ cells and Sertoli cells has been widely reported in mice, much less is known in other large animal species, including dairy goats. In this study, we present the data of single cell RNA sequencing (scRNA-seq) for 25,373 cells from 45 (pre-puberty), 90 (puberty), and 180-day-old (post-puberty) dairy goat testes. We aimed to identify genes that are associated with key developmental events in male germ cells and Sertoli cells. We examined the development of spermatogenic cells and seminiferous tubules from 15, 30, 45, 60, 75, 90, 180, and 240-day-old buck goat testes. scRNA-seq clustering analysis of testicular cells from pre-puberty, puberty, and post-puberty goat testes revealed several cell types, including cell populations with characteristics of spermatogonia, early spermatocytes, spermatocytes, spermatids, Sertoli cells, Leydig cells, macrophages, and endothelial cells. We mapped the timeline for male germ cells development from spermatogonia to spermatids and identified gene signatures that define spermatogenic cell populations, such as AMH, SOHLH1, INHA, and ACTA2. Importantly, using immunofluorescence staining for different marker proteins (UCHL1, C-KIT, VASA, SOX9, AMH, and PCNA), we explored the proliferative activity and development of male germ cells and Sertoli cells. Moreover, we identified the expression patterns of potential key genes associated with the niche-related key pathways in male germ cells of dairy goats, including testosterone, retinoic acid, PDGF, FGF, and WNT pathways. In summary, our study systematically investigated the elaborate male germ cells and Sertoli cells developmental patterns in dairy goats that have so far remained largely unknown. This information represents a valuable resource for the establishment of goat male reproductive stem cells lines, induction of germ cell differentiation , and the exploration of sequential cell fate transition for spermatogenesis and testicular development at single-cell resolution.
PubMed: 35938151
DOI: 10.3389/fcell.2022.944325 -
Animals : An Open Access Journal From... Oct 2022Male-derived sterility in cattle-yaks, a hybrid deriving from yak and cattle, is a challenging problem. This study compared and analyzed the histomorphological...
Male-derived sterility in cattle-yaks, a hybrid deriving from yak and cattle, is a challenging problem. This study compared and analyzed the histomorphological differences in testis between sexually mature yak and cattle-yak, and examined the transcriptome differences employing RNA-seq. The study found that yak seminiferous tubules contained spermatogenic cells at all levels, while cattle-yak seminiferous tubules had reduced spermatogonia (SPG) and primary spermatocyte (Pri-SPC), fewer secondary spermatocytes (Sec-SPC), an absence of round spermatids (R-ST) and sperms (S), and possessed large vacuoles. All of these conditions could have significantly reduced the volume and weight of cattle-yak testis compared to that of yak. RNA-seq analysis identified 8473 differentially expressed genes (DEGs; 3580 upregulated and 4893 downregulated). GO (Gene Ontology) and KEGG (Kyoto Encyclopedia of Genes and Genomes) enrichment evaluations for DEGs found their relation mostly to spermatogenesis and apoptosis. Among the DEGs, spermatogonia stem cell (SSCs) marker genes (, , , , , and ) and genes involved in apoptosis (, , , , , , and ) were significantly upregulated, while differentiation spermatogenic cell marker genes (, , , and ) and meiosis-related genes (, , , and ) were significantly downregulated in cattle-yak. Furthermore, the alternative splicing events in cattle-yak were substantially decreased than in yak, suggesting that the lack of protein subtypes could be another reason for spermatogenic arrest in cattle-yak testis.
PubMed: 36230452
DOI: 10.3390/ani12192711 -
International Journal of Molecular... Dec 2021MFN1 (Mitofusin 1) and MFN2 (Mitofusin 2) are GTPases essential for mitochondrial fusion. Published studies revealed crucial roles of both Mitofusins during embryonic...
MFN1 (Mitofusin 1) and MFN2 (Mitofusin 2) are GTPases essential for mitochondrial fusion. Published studies revealed crucial roles of both Mitofusins during embryonic development. Despite the unique mitochondrial organization in sperm flagella, the biological requirement in sperm development and functions remain undefined. Here, using sperm-specific Cre drivers, we show that either or knockout in haploid germ cells does not affect male fertility. The and double knockout mice were further analyzed. We found no differences in testis morphology and weight between -deficient mice and their wild-type littermate controls. Spermatogenesis was normal in double knockout mice, in which properly developed TRA98+ germ cells, SYCP3+ spermatocytes, and TNP1+ spermatids/spermatozoa were detected in seminiferous tubules, indicating that sperm formation was not disrupted upon MFN deficiency. Collectively, our findings reveal that both MFN1 and MFN2 are dispensable for sperm development and functions in mice.
Topics: Animals; Female; GTP Phosphohydrolases; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Mitochondria; Mitochondrial Dynamics; Sperm Maturation; Spermatocytes; Spermatogenesis; Spermatozoa; Testis
PubMed: 34948301
DOI: 10.3390/ijms222413507 -
PNAS Nexus Jul 20222,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a potent and environmentally persistent endocrine disrupting chemical. Our previous work demonstrated the latent...
2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a potent and environmentally persistent endocrine disrupting chemical. Our previous work demonstrated the latent reproductive maladies of early-life TCDD exposure in zebrafish. Zebrafish acutely exposed to low, environmentally relevant levels of TCDD (50 pg/mL) during two windows of sexual differentiation in development (1 hour of exposure at 3 and 7 weeks postfertilization) were later infertile, showed a reduction in sperm, and exhibited gene expression consistent with an altered microenvironment, even months after exposure. Due to the highly heterogeneous cell- type and -stage landscape of the testes, we hypothesized various cell types contribute markedly different profiles toward the pathology of TCDD exposure. To investigate the contributions of the diverse cell types in the adult zebrafish testes to TCDD-induced pathology, we utilized single-cell RNA-seq and the 10x Genomics platform. The method successfully captured every stage of testicular germ cell development. Testes of adult fish exposed during sexual differentiation to TCDD contained sharply decreased populations of late spermatocytes, spermatids, and spermatozoa. Spermatogonia and early spermatocyte populations were, in contrast, enriched following exposure. Pathway analysis of differentially expressed genes supported previous findings that TCDD exposure resulted in male infertility, and suggested this outcome is due to apoptosis of spermatids and spermatozoa, even years after exposure cessation. Increased germ cell apoptosis was confirmed histologically. These results provide support for an environmental exposure explanation of idiopathic male infertility.
PubMed: 35799832
DOI: 10.1093/pnasnexus/pgac060 -
Nan Fang Yi Ke Da Xue Xue Bao = Journal... Jun 2023To explore the interaction between Tubulin beta 4B class IVb (TUBB4B) and Agtpbp1/cytosolic carboxypeptidase- like1 (CCP1) in mouse primary spermatocytes (GC-2 cells)...
OBJECTIVE
To explore the interaction between Tubulin beta 4B class IVb (TUBB4B) and Agtpbp1/cytosolic carboxypeptidase- like1 (CCP1) in mouse primary spermatocytes (GC-2 cells) and the role of TUBB4B in regulating the development of GC-2 cells.
METHODS
Lentiviral vectors were used to infect GC-2 cells to construct TUBB4B knockdown and negative control (NC-KD) cells. The stable cell lines with TUBB4B overexpression (Tubb4b-OE) and the negative control (NC-OE) cells were screened using purinomycin. RT-qPCR and Western blotting were used to verify successful cell modeling and explore the relationship between TUBB4B and CCP1 expressions in GC-2 cells. The effects of TUBB4B silencing and overexpression on the proliferation and cell cycle of GC-2 cells were evaluated using CCK8 assay and flow cytometry. The signaling pathway proteins showing significant changes in response to TUBB4B silencing or overexpression were identified using Western blotting and immunofluorescence assay and then labeled for verification at the cellular level.
RESULTS
Both TUBB4B silencing and overexpression in GC-2 cells caused consistent changes in the mRNA and protein expressions of CCP1 ( < 0.05). Similarly, TUBB4B expression also showed consistent changes at the mRNA and protein after CCP1 knockdown and restoration ( < 0.05). TUBB4B knockdown and overexpression had no significant effect on proliferation rate or cell cycle of GC-2 cells, but caused significant changes in the key proteins of the nuclear factor kappa-B (NF-κB) signaling pathway (p65 and p-p65) and the mitogen-activated protein kinase (MAPK) signaling pathway (ErK1/2 and p-Erk1/2) ( < 0.05); CCP1 knockdown induced significant changes in PolyE expression in GC-2 cells ( < 0.05).
CONCLUSIONS
TUBB4B and CCP1 interact via a mutual positive regulation mechanism in GC-2 cells. CCP-1 can deglutamize TUBB4B, and the latter is involved in the regulation of NF-κB and MAPK signaling pathways in primary spermatocytes.
Topics: Animals; Male; Mice; GTP-Binding Proteins; Mitogen-Activated Protein Kinases; NF-kappa B; RNA, Messenger; Serine-Type D-Ala-D-Ala Carboxypeptidase; Signal Transduction; Spermatocytes; Tubulin
PubMed: 37439173
DOI: 10.12122/j.issn.1673-4254.2023.06.16 -
PeerJ 2022Leptin is a 16 kDa hormone encoded by obese () gene in adipocytes. This molecule not only regulates energy metabolism but also plays a role in the reproduction of...
Leptin is a 16 kDa hormone encoded by obese () gene in adipocytes. This molecule not only regulates energy metabolism but also plays a role in the reproduction of mammals. Leptin and its receptor () have been found in male reproductive systems of human, bovine, equine and pig. The effects of leptin on sperm quality vary widely from different research findings. However, the presence of leptin and its receptor in the ram reproductive system and the effect of leptin on sperm quality have not reported yet. In the present study, we found that the was highly expressed in primary and secondary spermatocytes of the testes, was highly expressed in secondary spermatocytes of the testes. The expressions of were in stereocilia of epididymis and in columnar cells of epididymal caput and cauda, the expressions of were in columnar cells of epididymis and in stereocilia of epididymal and cauda. The presence of both and in testes, epididymis and sperm were confirmed through RT-PCR, immunolocalization and Western blot analyses. The RT-qPCR results indicated and had higher expression levels in epididymal sperm than that of the ejaculated sperm in rams. When sperm were treated with 5 ng/mL leptin, the progressive motility ( < 0.01), straight-line velocity (VSL) ( < 0.05), average path velocity (VAP) ( < 0.05), membrane mitochondrial potential (MMP) ( < 0.01) and viability ( < 0.05) significantly increased, while DNA fragmentation index (DFI) and reactive oxygen species (ROS) significantly decreased compared to the control ( < 0.01), and the other semen parameters such as acrosome integrity and acrosome reaction rate had no significant changes between groups ( > 0.05). In conclusion, this is probably the first report describing localization of leptin and its receptors in the reproductive system of rams and their effects on sperm quality parameters. Our findings suggest that 5 ng/mL leptin treatment enhanced sperm motility, viability and MMP, and decrease DFI and ROS without obvious influence on the acrosome reaction in ram sperm. The potential mechanisms may be related to leptin's ability to reduce the oxidative stress and apoptosis of sperms and improve their mitochondrial function and energy supply, therefore, to maintain the physiological homeostasis of the sperm.
Topics: Male; Animals; Sheep; Cattle; Horses; Humans; Swine; Semen; Leptin; Reactive Oxygen Species; Sperm Motility; Spermatozoa; Sheep, Domestic; Genitalia
PubMed: 36187750
DOI: 10.7717/peerj.13982