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Cells Feb 2020The molecular basis of residual histone retention after the nearly genome-wide histone-to-protamine replacement during late spermatogenesis is a critical and open...
The molecular basis of residual histone retention after the nearly genome-wide histone-to-protamine replacement during late spermatogenesis is a critical and open question. Our previous investigations showed that in postmeiotic male germ cells, the genome-scale incorporation of histone variants TH2B-H2A.L.2 allows a controlled replacement of histones by protamines to occur. Here, we highlight the intrinsic ability of H2A.L.2 to specifically target the pericentric regions of the genome and discuss why pericentric heterochromatin is a privileged site of histone retention in mature spermatozoa. We observed that the intranuclear localization of H2A.L.2 is controlled by its ability to bind RNA, as well as by an interplay between its RNA-binding activity and its tropism for pericentric heterochromatin. We identify the H2A.L.2 RNA-binding domain and demonstrate that in somatic cells, the replacement of H2A.L.2 RNA-binding motif enhances and stabilizes its pericentric localization, while the forced expression of RNA increases its homogenous nuclear distribution. Based on these data, we propose that the specific accumulation of RNA on pericentric regions combined with H2A.L.2 tropism for these regions are responsible for stabilizing H2A.L.2 on these regions in mature spermatozoa. This situation would favor histone retention on pericentric heterochromatin.
Topics: Animals; Cell Nucleus; Genome, Human; Heterochromatin; Histones; Humans; Male; Mice; Mice, Knockout; NIH 3T3 Cells; RNA Recognition Motif Proteins; RNA, Nuclear; RNA-Binding Motifs; Spermatocytes; Spermatogenesis; Transfection
PubMed: 32085641
DOI: 10.3390/cells9020474 -
Frontiers in Cell and Developmental... 2023Sevoflurane (Sev) is one of the commonly used inhalation anesthetic chemicals in clinics. It has great impact on spermatogenesis and fertilization in male animals. The...
Sevoflurane (Sev) is one of the commonly used inhalation anesthetic chemicals in clinics. It has great impact on spermatogenesis and fertilization in male animals. The underlying mechanism remains largely unexplored. Based on our previous research, we hypothesized that Sev induced iron metabolism disturbance in the testis and epididymis and inhibited the spermatogenesis. In this study, two-month-old C57BL/6 male mice were treated with 3% Sev for 6 h, and their fertility (including sperm concentration, sperm mobility, and the number of offspring) was evaluated. Mice testis, epididymis, and sperm were harvested and subjected to Western blot analysis and immunofluorescence analysis. Iron levels were reflected by the gene expression of iron metabolism-related proteins (including ferritin, TfR1, and FpN1) and ICP-MS and Perl's iron staining. Electron transport and oxidative phosphorylation levels were measured by Oxygraph-2k and ATP contents. The activity of ribonucleotide reductase was evaluated by assay kit. DNA synthesis status in testis and/or epididymis was marked with BrdU. Cell proliferation was evaluated by double immunofluorescence staining of specific protein marker expression. Our results revealed that the mice exposed to Sev showed damaged testicular and epididymis structure and significantly reduced the sperm concentration, sperm motility, and fertility. Sev decreases the iron levels through down-regulating the expression of H-ferritin, L-ferritin, and FpN1, and up-regulating the expression of TfR1 in the testis and epididymis. Iron levels also significantly reduced in germ cells which decrease the number of germ cells, including sperm, Sertoli cells, and primary spermatocyte. Iron deficiency not only decreases electron transport, oxidative phosphorylation level, and ATP production but also suppresses the activity of ribonucleotide reductase and the expression of Ki67, DDX4, GATA1, and SCP3, indicating that Sev affects the spermatogenesis and development. Meanwhile, Sev impaired the blood-testis barrier by decreasing the ZO1 expression in the testis and epididymis. The damage effect induced by Sev can be significantly ameliorated by iron supplementation. In conclusion, our study illustrates a new mechanism by which Sev inhibits spermatogenesis and fertility through an oxidative phosphorylation pathway due to iron deficiency of epididymis and testis or sperm. Furthermore, the damaging effects could be ameliorated by iron supplementation.
PubMed: 37346174
DOI: 10.3389/fcell.2023.1184632 -
PeerJ 2023The sperm of Chinese mitten crab () have special noncondensed nuclei. The formation and stability of the special nuclei are closely related to the correct folding of...
BACKGROUND
The sperm of Chinese mitten crab () have special noncondensed nuclei. The formation and stability of the special nuclei are closely related to the correct folding of proteins during spermatogenesis. P4HB plays a key role in protein folding, but its expression and role in the spermatogenesis of are unclear.
OBJECTIVE
To investigate the expression and distribution characteristics of P4HB in the spermatogenesis of as well as its possible role.
METHODS
The testis tissues of adult and juvenile were used as materials. We utilized a variety of techniques, including homology modeling, phylogenetic analysis, RT-qPCR, western blotting, and immunofluorescence staining to predict the protein structure and sequence homology of P4HB, analyze its expression in the testis tissues, and localize and semi-quantitatively assess its expression in different male germ cells.
RESULTS
The sequence of P4HB protein in shared a high similarity of 58.09% with the human protein disulfide isomerase, and the phylogenetic tree analysis indicated that the protein sequence was highly conserved among crustaceans, arthropods, and other animals species. P4HB was found to be expressed in both juvenile and adult testis tissues, with different localization patterns observed all over the developmental stages of male germ cells. It was higher expressed in the spermatogonia, spermatocytes, and stage I spermatids, followed by the mature sperm than in the stage II and III spermatids. The subcellular localization analysis revealed that P4HB was predominantly expressed in the cytoplasm, cell membrane, and extracellular matrix in the spermatogonia, spermatocytes, stage I and stage II spermatids, with some present in specific regions of the nuclei in the spermatogonia. In contrast, P4HB was mainly localized in the nuclei of stage III spermatids and sperm, with little expression observed in the cytoplasm.
CONCLUSION
P4HB was expressed in the testis tissues of both adult and juvenile , but the expression and localization were different in male germ cells at various developmental stages. The observed differences in the expression and localization of P4HB may be an essential factor in maintaining the cell morphology and structure of diverse male germ cells in . Additionally, P4HB expressed in the nuclei of spermatogonia, late spermatids, and sperm may play an indispensable role in maintaining the stability of the noncondensed spermatozoal nuclei in .
Topics: Animals; Male; Phylogeny; Procollagen-Proline Dioxygenase; Protein Disulfide-Isomerases; Semen; Spermatogenesis; Spermatogonia; Testis; Brachyura
PubMed: 37334119
DOI: 10.7717/peerj.15547 -
Genes Dec 2023is the most widely distributed freshwater shrimp in China, with important economic value and great potential for development. The forkheadboxL2 () gene has been found...
is the most widely distributed freshwater shrimp in China, with important economic value and great potential for development. The forkheadboxL2 () gene has been found to be involved in the reproductive development of many crustaceans. To understand the role of the gene in the gonad development of , we designed CDS-specific primers for the () gene and cloned its CDS sequence using RT-PCR. The nucleotide and protein sequence information was then analyzed through bioinformatics analysis. The expression and subcellular localization of in various tissues were detected using qRT-PCR and in situ hybridization. The effects of knockdown on gonad development were investigated using RNA interference. The results showed that the CDS length of the gene was 1614 bp and encoded 537 amino acids. Protein sequence comparison and phylogenetic analysis showed that was the closest relative to Crayfish. qRT-PCR analysis indicated that the expression level of in the testis was significantly higher (>40 fold) than that in the ovary ( < 0.01). The in situ hybridization results showed that was expressed in both the cytoplasm and the nucleus of egg cells, and that the expression was strongest in egg cells at the early stage of yolk synthesis, while weak in the secondary oocytes. The positive signal was strongest in the spermatocyte nucleolus, while only a trace signal was observed in the cytoplasm. After interfering with the gene using dsRNA, the expression of in the RNA interference group was significantly lower than that in the control group, and this interference effect lasted for one week. Moreover, the gonad index of the experimental group was significantly lower than that of the control group ( < 0.05) after 10 days of cultivation following knockdown. The expression levels of the and genes, which are related to gonad development, decreased significantly after gene interference. The results suggest that the gene is involved in the growth and development of gonads, particularly in the development of testis, and is related to the early development of oocytes. This study provides a theoretical basis for the artificial breeding of .
Topics: Male; Animals; Female; Astacoidea; Phylogeny; Amino Acid Sequence; Polymerase Chain Reaction; Cloning, Molecular
PubMed: 38137012
DOI: 10.3390/genes14122190 -
Frontiers in Endocrinology 2021To achieve spermatogenesis , one of the most challenging processes to mimic is meiosis. Meiotic problems, like incomplete synapsis of the homologous chromosomes, or...
To achieve spermatogenesis , one of the most challenging processes to mimic is meiosis. Meiotic problems, like incomplete synapsis of the homologous chromosomes, or impaired homologous recombination, can cause failure of crossover formation and subsequent chromosome nondisjunction, eventually leading to aneuploid sperm. These meiotic events are therefore strictly monitored by meiotic checkpoints that initiate apoptosis of aberrant spermatocytes and lead to spermatogenic arrest. However, we recently found that, derived meiotic cells proceeded to the first meiotic division (MI) stage, despite displaying incomplete chromosome synapsis, no discernible XY-body and lack of crossover formation. We therefore optimized our culture system of meiosis from male germline stem cells (mGSCs) in order to achieve full chromosome synapsis, XY-body formation and meiotic crossovers. In comparison to previous culture system, the -generated spermatocytes were transferred after meiotic initiation to a second culture dish. This dish already contained a freshly plated monolayer of proliferatively inactivated immortalized Sertoli cells supporting undifferentiated mGSCs. In this way we aimed to simulate the multiple layers of germ cell types that support spermatogenesis in the testis. We found that in this optimized culture system, although independent of the undifferentiated mGSCs, meiotic chromosome synapsis was complete and XY body appeared normal. However, meiotic recombination still occurred insufficiently and only few meiotic crossovers were formed, leading to MI-spermatocytes displaying univalent chromosomes (paired sister chromatids). Therefore, considering that meiotic checkpoints are not necessarily fully functional , meiotic crossover formation should be closely monitored when mimicking gametogenesis to prevent generation of aneuploid gametes.
Topics: Aneuploidy; Animals; Azoospermia; Cell Differentiation; Cell Line; Cell Proliferation; Chromosome Pairing; Chromosomes; Male; Meiosis; Mice; Mice, Inbred DBA; Sertoli Cells; Spermatocytes; Spermatogenesis; Spermatozoa; Testis
PubMed: 34721307
DOI: 10.3389/fendo.2021.761249 -
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 -
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 -
Journal of Biomedical Physics &... Apr 2023Substantial evidence indicates that exposure to extremely low frequency-electromagnetic fields (ELF-EMFs) affects male reproductive system.
BACKGROUND
Substantial evidence indicates that exposure to extremely low frequency-electromagnetic fields (ELF-EMFs) affects male reproductive system.
OBJECTIVE
The goal of this study was to evaluate the effects of long-term irradiation with ELF-EMF on sperm quality and quantity and testicular structure.
MATERIAL AND METHODS
In this case-control study, sixty male Sprague-Dawley rats were randomly divided into six groups. Experimental groups were exposed to ELF-EMF (50 Hz EMF, 100 µT) for either 1 h/day for 52 days (Group 1), 4 h/day for 52 days (Group 3), 1 h/day for 5 days (Group 5), 4 h/day for 52 days (Group 7). Groups 2, 4, 6 and 8 were only sham exposed at durations equal to Groups 1, 3, 5 and 7, respectively.
RESULTS
Both count and motility of sperms were significantly decreased in animals exposed to ELF-EMF (1 h/day for 52 days, 4 h/day for 52 day, and 4 h/day for 5 days) compared to the sham-exposed groups (<0.05). Serum testosterone levels showed a significant decrease in the animals exposed to ELF-EMF (4 h/day for 5 days) compared to the control groups (<0.05). A significant decrease was observed in the volume of the seminiferous tubules, seminiferous tubules epithelium and interstitial tissue in the animals exposed to ELF-EMF for 4 h/day for 5 days. Tubules length was also reduced by 18% in animals exposed to ELF-EMF (4 h/day for 5 days).
CONCLUSION
Our results show that ELF-EMF can reduce spermatocyte count and motility and is able to induce structural changes in testicular tissue.
PubMed: 37082547
DOI: 10.31661/jbpe.v0i0.2011-1234