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Reproductive Biology and Endocrinology... Jun 2018Spermatogenesis in most mammals (including human and rat) occurs at ~ 3 °C lower than body temperature in a scrotum and fails rapidly at 37 °C inside the abdomen....
BACKGROUND
Spermatogenesis in most mammals (including human and rat) occurs at ~ 3 °C lower than body temperature in a scrotum and fails rapidly at 37 °C inside the abdomen. The present study investigates the heat-sensitive transcriptome and miRNAs in the most vulnerable germ cells (spermatocytes and round spermatids) that are primarily targeted at elevated temperature in a bid to identify novel targets for contraception and/or infertility treatment.
METHODS
Testes of adult male rats subjected to surgical cryptorchidism were obtained at 0, 24, 72 and 120 h post-surgery, followed by isolation of primary spermatocytes and round spermatids and purification to > 90% purity using a combination of trypsin digestion, centrifugal elutriation and density gradient centrifugation techniques. RNA isolated from these cells was sequenced by massive parallel sequencing technique to identify the most-heat sensitive mRNAs and miRNAs.
RESULTS
Heat stress altered the expression of a large number of genes by ≥2.0 fold, out of which 594 genes (286↑; 308↓) showed alterations in spermatocytes and 154 genes (105↑; 49↓) showed alterations in spermatids throughout the duration of experiment. 62 heat-sensitive genes were common to both cell types. Similarly, 66 and 60 heat-sensitive miRNAs in spermatocytes and spermatids, respectively, were affected by ≥1.5 fold, out of which 6 were common to both the cell types.
CONCLUSION
The study has identified Acly, selV, SLC16A7(MCT-2), Txnrd1 and Prkar2B as potential heat sensitive targets in germ cells, which may be tightly regulated by heat sensitive miRNAs rno-miR-22-3P, rno-miR-22-5P, rno-miR-129-5P, rno-miR-3560, rno-miR-3560 and rno-miR-466c-5P.
Topics: Animals; Gene Expression; Gene Expression Profiling; Hot Temperature; Male; Rats; Rats, Sprague-Dawley; Spermatocytes; Spermatogenesis
PubMed: 29859541
DOI: 10.1186/s12958-018-0372-8 -
ELife May 2021The male germ cells must adopt the correct morphology at each differentiation stage for proper spermatogenesis. The spermatogonia regulates its differentiation state by...
The male germ cells must adopt the correct morphology at each differentiation stage for proper spermatogenesis. The spermatogonia regulates its differentiation state by its own migration. The male germ cells differentiate and mature with the formation of syncytia, failure of forming the appropriate syncytia results in the arrest at the spermatocyte stage. However, the detailed molecular mechanisms of male germ cell morphological regulation are unknown. Here, we found that EXOC1, a member of the Exocyst complex, is important for the pseudopod formation of spermatogonia and spermatocyte syncytia in mice. EXOC1 contributes to the pseudopod formation of spermatogonia by inactivating the Rho family small GTPase Rac1 and also functions in the spermatocyte syncytia with the SNARE proteins STX2 and SNAP23. Since EXOC1 is known to bind to several cell morphogenesis factors, this study is expected to be the starting point for the discovery of many morphological regulators of male germ cells.
Topics: Animals; Cell Differentiation; Gene Expression Regulation, Developmental; Giant Cells; Male; Mice; Mice, Inbred C57BL; Spermatocytes; Spermatogenesis; Spermatogonia; Vesicular Transport Proteins
PubMed: 33973520
DOI: 10.7554/eLife.59759 -
Oncotarget Jul 2017Bisphenol A (BPA) acts as xenoestrogen and has a great impact on disorders of human reproductive system. However, the mechanism through which BPA can affect human...
Bisphenol A (BPA) acts as xenoestrogen and has a great impact on disorders of human reproductive system. However, the mechanism through which BPA can affect human testicular function remains to be identified. GPR30 is a novel membrane estrogen receptor with high-affinity and low-capacity binding to estrogens. We demonstrated that estrogen receptor α (ERα), estrogen receptor β (ERβ) as well as GPR30 are expressed in mouse spermatocyte-derived GC-2 cells using Real-time PCR. We treated the cells with different doses of BPA and found that even low doses of BPA can inhibit GC-2 cell growth using MTT assay. To make sure which receptor is responsible for the biological function of BPA, we used ER down-regulator ICI and indicated that BPA could bind to GPR30. We also observed that BPA was able to induce Erk1/2 phosphorylation in GC-2 cells and proved that this process was mediated by GPR30-related EGFR-MAPK pathway using western blot. By Real-time PCR, we found that the expression of c-Fos was up-regulated and Cyclin D1 gene was down-regulated, in the presence of BPA and ICI. The results of MTT assay, comet assay and flow cytometry indicated that the activation of GPR30 induced by BPA inhibited the cell growth and induced cell apoptosis and ICI, GPR30 siRNA, EGFR inhibitor (AG), and MAPK (PD) inhibitor could partially reverse this effect. Immunohistochemistry on the testis of BPA -damaged mice showed that BPA induced spermatocyte apoptosis without affecting the seminiferous tubules and spermatocyte. In conclusion, BPA triggered spermatocyte apoptosis via GPR30.
Topics: Animals; Apoptosis; Benzhydryl Compounds; Biomarkers; Cell Line; Cell Proliferation; Dose-Response Relationship, Drug; ErbB Receptors; Gene Expression; Gene Expression Regulation; Genes, fos; Male; Mice; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Phenols; Phosphorylation; Receptors, Estrogen; Signal Transduction; Spermatocytes
PubMed: 28446726
DOI: 10.18632/oncotarget.16923 -
Protocol for isolation and proteostatic analysis of sub-populations of spermatogenic cells in mouse.STAR Protocols Jun 2022Spermatogenesis generates heterologous cell populations which, if not distinguished clearly, often hinder mechanistic and etiological studies. Here, we present a...
Spermatogenesis generates heterologous cell populations which, if not distinguished clearly, often hinder mechanistic and etiological studies. Here, we present a protocol to identify and isolate populations of mouse spermatogenic cells, including spermatogonial stem cells (SSCs), spermatocytes, and haploid spermatids. We also describe absolute quantification of mRNA copy numbers in SSCs. The isolated cells can be used for analyzing nascent protein synthesis and protein degradation, two main events that maintain cellular proteostasis important for healthy and long-term production of male gametes. For complete details on the use and execution of this protocol, please refer to Zou et al. (2021).
Topics: Adult Germline Stem Cells; Animals; Haploidy; Male; Mice; Spermatids; Spermatocytes; Spermatogenesis
PubMed: 35600921
DOI: 10.1016/j.xpro.2022.101398 -
Zoological Research Jul 2021Normal spermatogenic processes require the scrotal temperature to be lower than that of the body as excessive heat affects spermatogenesis in the testes, reduces sperm...
Normal spermatogenic processes require the scrotal temperature to be lower than that of the body as excessive heat affects spermatogenesis in the testes, reduces sperm quality and quantity, and even causes infertility. Endoplasmic reticulum stress (ERS) is a crucial factor in many pathologies. Although several studies have linked ERS to heat stress, researchers have not yet determined which ERS signaling pathways contribute to heat-induced testicular damage. Melatonin activates antioxidant enzymes, scavenges free radicals, and protects the testes from inflammation; however, few studies have reported on the influence of melatonin on heat-induced testicular damage. Using a murine model of testicular hyperthermia, we observed that heat stress causes both ERS and apoptosis in the testes, especially in the spermatocytes. These observations were confirmed using the mouse spermatocyte cell line GC2, where the and signaling pathways were activated during heat stress. Knockout of the above genes effectively reduced spermatocyte damage caused by heat stress. Pretreatment with melatonin alleviated heat-induced apoptosis by inhibiting the and signaling pathways. This mitigation was dependent on the melatonin receptors. experiments verified that melatonin treatment relieved heat-induced testicular damage. In conclusion, our results demonstrated that ATF6 and PERK are important mediators for heat-induced apoptosis, which can be prevented by melatonin treatment. Thus, our study highlights melatonin as a potential therapeutic agent in mammals for subfertility/infertility induced by testicular hyperthermia.
Topics: Activating Transcription Factor 6; Animals; Apoptosis; Gene Expression Regulation; Hot Temperature; Male; Melatonin; Mice; Mice, Inbred ICR; Signal Transduction; Spermatocytes; Testis; eIF-2 Kinase
PubMed: 34254745
DOI: 10.24272/j.issn.2095-8137.2021.041 -
Reproduction (Cambridge, England) Feb 2016Recent findings suggest diverse and potentially multiple roles of small ubiquitin-like modifier (SUMO) in testicular function and spermatogenesis. However, SUMO targets...
Recent findings suggest diverse and potentially multiple roles of small ubiquitin-like modifier (SUMO) in testicular function and spermatogenesis. However, SUMO targets remain uncharacterized in the testis due to the complex multicellular nature of testicular tissue, the inability to maintain and manipulate spermatogenesis in vitro, and the technical challenges involved in identifying low-abundance endogenous SUMO targets. In this study, we performed cell-specific identification of sumoylated proteins using concentrated cell lysates prepared with de-sumoylation inhibitors from freshly purified spermatocytes and spermatids. One-hundred and twenty proteins were uniquely identified in the spermatocyte and/or spermatid fractions. The identified proteins are involved in the regulation of transcription, stress response, microRNA biogenesis, regulation of major enzymatic pathways, nuclear-cytoplasmic transport, cell-cycle control, acrosome biogenesis, and other processes. Several proteins with important roles during spermatogenesis were chosen for further characterization by co-immunoprecipitation, co-localization, and in vitro sumoylation studies. GPS-SUMO Software was used to identify consensus and non-consensus sumoylation sites within the amino acid sequences of the proteins. The analyses confirmed the cell-specific sumoylation and/or SUMO interaction of several novel, previously uncharacterized SUMO targets such as CDK1, RNAP II, CDC5, MILI, DDX4, TDP-43, and STK31. Furthermore, several proteins that were previously identified as SUMO targets in somatic cells (KAP1 and MDC1) were identified as SUMO targets in germ cells. Many of these proteins have a unique role in spermatogenesis and during meiotic progression. This research opens a novel avenue for further studies of SUMO at the level of individual targets.
Topics: Animals; Cells, Cultured; HEK293 Cells; Humans; Male; Mice; Mice, Inbred C57BL; Organ Specificity; Spermatids; Spermatocytes; Spermatogenesis; Sumoylation; Testis
PubMed: 26701181
DOI: 10.1530/REP-15-0239 -
Asian Journal of Andrology 2020The transition from spermatogonia to spermatocytes and the initiation of meiosis are key steps in spermatogenesis and are precisely regulated by a plethora of proteins....
The transition from spermatogonia to spermatocytes and the initiation of meiosis are key steps in spermatogenesis and are precisely regulated by a plethora of proteins. However, the underlying molecular mechanism remains largely unknown. Here, we report that Src homology domain tyrosine phosphatase 2 (Shp2; encoded by the protein tyrosine phosphatase, nonreceptor type 11 [Ptpn11] gene) is abundant in spermatogonia but markedly decreases in meiotic spermatocytes. Conditional knockout of Shp2 in spermatogonia in mice using stimulated by retinoic acid gene 8 (Stra8)-cre enhanced spermatogonial differentiation and disturbed the meiotic process. Depletion of Shp2 in spermatogonia caused many meiotic spermatocytes to die; moreover, the surviving spermatocytes reached the leptotene stage early at postnatal day 9 (PN9) and the pachytene stage at PN11-13. In preleptotene spermatocytes, Shp2 deletion disrupted the expression of meiotic genes, such as disrupted meiotic cDNA 1 (Dmc1), DNA repair recombinase rad51 (Rad51), and structural maintenance of chromosome 3 (Smc3), and these deficiencies interrupted spermatocyte meiosis. In GC-1 cells cultured in vitro, Shp2 knockdown suppressed the retinoic acid (RA)-induced phosphorylation of extracellular-regulated protein kinase (Erk) and protein kinase B (Akt/PKB) and the expression of target genes such as synaptonemal complex protein 3 (Sycp3) and Dmc1. Together, these data suggest that Shp2 plays a crucial role in spermatogenesis by governing the transition from spermatogonia to spermatocytes and by mediating meiotic progression through regulating gene transcription, thus providing a potential treatment target for male infertility.
Topics: Animals; Cell Cycle Proteins; Cell Line; Cell Survival; Chondroitin Sulfate Proteoglycans; Chromosomal Proteins, Non-Histone; Gene Expression Regulation; Gene Knockdown Techniques; Infertility, Male; Male; Meiosis; Mice; Mice, Knockout; Mice, Transgenic; Phosphate-Binding Proteins; Protein Tyrosine Phosphatase, Non-Receptor Type 11; Rad51 Recombinase; Real-Time Polymerase Chain Reaction; Spermatocytes; Spermatogenesis; Spermatogonia
PubMed: 31210146
DOI: 10.4103/aja.aja_49_19 -
Cell Reports Jan 2024Dynamic chromosome remodeling and nuclear compartmentalization take place during mammalian meiotic prophase I. We report here that the crucial roles of male...
Dynamic chromosome remodeling and nuclear compartmentalization take place during mammalian meiotic prophase I. We report here that the crucial roles of male pachynema-specific protein (MAPS) in pachynema progression might be mediated by its liquid-liquid phase separation in vitro and in cellulo. MAPS forms distinguishable liquid phases, and deletion or mutations of its N-terminal amino acids (aa) 2-9 disrupt its secondary structure and charge properties, impeding phase separation. Maps pachytene spermatocytes exhibit defects in nucleus compartmentalization, including defects in forming sex bodies, altered nucleosome composition, and disordered chromatin accessibility. Maps male mice expressing MAPS protein lacking aa 2-9 phenocopy Maps mice. Moreover, a frameshift mutation in C3orf62, the human counterpart of Maps, is correlated with nonobstructive azoospermia in a patient exhibiting pachynema arrest in spermatocyte development. Hence, the phase separation property of MAPS seems essential for pachynema progression in mouse and human spermatocytes.
Topics: Humans; Male; Mice; Animals; Chromatin; Meiosis; Pachytene Stage; Phase Separation; Meiotic Prophase I; Spermatocytes; Mammals
PubMed: 38175751
DOI: 10.1016/j.celrep.2023.113651 -
Biochimica Et Biophysica Acta.... Sep 2022Genome stability depends on chromosome congression and alignment during cell division. Kinesin-7 CENP-E is critical for kinetochore-microtubule attachment and chromosome...
Genome stability depends on chromosome congression and alignment during cell division. Kinesin-7 CENP-E is critical for kinetochore-microtubule attachment and chromosome alignment, which contribute to genome stability in mitosis. However, the functions and mechanisms of CENP-E in the meiotic division of male spermatocytes remain largely unknown. In this study, by combining the use of chemical inhibitors, siRNA-mediated gene knockdown, immunohistochemistry, and high-resolution microscopy, we have found that CENP-E inhibition results in chromosome misalignment and metaphase arrest in dividing spermatocyte during meiosis. Strikingly, we have revealed that CENP-E regulates spindle organization in metaphase I spermatocytes and cultured GC-2 spd cells. CENP-E depletion leads to spindle elongation, chromosome misalignment, and chromosome instability in spermatocytes. Together, these findings indicate that CENP-E mediates the kinetochore recruitment of BubR1, spindle assembly checkpoint and chromosome alignment in dividing spermatocytes, which finally contribute to faithful chromosome segregation and chromosome stability in the male meiotic division.
Topics: Animals; Chromosomal Proteins, Non-Histone; Chromosome Segregation; Genomic Instability; Kinesins; Kinetochores; Male; Mice; Mitosis; Spermatocytes
PubMed: 35680098
DOI: 10.1016/j.bbamcr.2022.119306 -
Epigenetics Apr 2016H1T is a linker histone H1 variant that is highly expressed at the primary spermatocyte stage through to the early spermatid stage of spermatogenesis. While the...
H1T is a linker histone H1 variant that is highly expressed at the primary spermatocyte stage through to the early spermatid stage of spermatogenesis. While the functions of the somatic types of H1 have been extensively investigated, the intracellular role of H1T is unclear. H1 variants specifically expressed in germ cells show low amino acid sequence homology to somatic H1s, which suggests that the functions or target loci of germ cell-specific H1T differ from those of somatic H1s. Here, we describe the target loci and function of H1T. H1T was expressed not only in the testis but also in tumor cell lines, mouse embryonic stem cells (mESCs), and some normal somatic cells. To elucidate the intracellular localization and target loci of H1T, fluorescent immunostaining and ChIP-seq were performed in tumor cells and mESCs. We found that H1T accumulated in nucleoli and predominantly targeted rDNA repeats, which differ from somatic H1 targets. Furthermore, by nuclease sensitivity assay and RT-qPCR, we showed that H1T repressed rDNA transcription by condensing chromatin structure. Imaging analysis indicated that H1T expression affected nucleolar formation. We concluded that H1T plays a role in rDNA transcription, by distinctively targeting rDNA repeats.
Topics: Amino Acid Sequence; Animals; Cell Nucleolus; Chromatin; DNA, Ribosomal; Gene Expression Regulation, Developmental; Germ Cells; Histones; Male; Mice; Mouse Embryonic Stem Cells; Sequence Homology, Amino Acid; Spermatids; Spermatocytes; Spermatogenesis; Testis; Transcription, Genetic
PubMed: 27018843
DOI: 10.1080/15592294.2016.1159369