Mammalian testis exhibits remarkably high transcriptome complexity, and spermatogenesis undergoes two periods of transcriptional cessation. These make the RNA-binding proteins (RBPs) the utmost importance during male germ cell development. Heterogeneous nuclear ribonucleoproteins (hnRNPs) are a large family of RBPs implicated in many steps of RNA processing; however, their roles in spermatogenesis are largely unknown. Here, we investigated the expression pattern of 12 hnRNP family members in mouse testes and found that most detected members are highly expressed in the testis. Furthermore, we found that most of the detected hnRNP proteins (hnRNPD, hnRNPK, hnRNPQ, hnRNPU, and hnRNPUL1) display the highest signals in the nuclei of pachytene spermatocytes, round spermatids, and Sertoli cells, whereas hnRNPE1 exclusively concentrates in the manchette of elongating spermatids. The expression of these hnRNP proteins showed both similarities and specificity, suggesting their diverse roles in spermatogenesis.
Mice ; Male ; Animals ; Heterogeneous-Nuclear Ribonucleoproteins/metabolism* ; Spermatogenesis/genetics* ; Testis/metabolism* ; Spermatids/metabolism* ; Sertoli Cells ; Spermatocytes/metabolism* ; RNA-Binding Proteins/metabolism* ; Mammals

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 (P < 0.05). Similarly, TUBB4B expression also showed consistent changes at the mRNA and protein after CCP1 knockdown and restoration (P < 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) (P < 0.05); CCP1 knockdown induced significant changes in PolyE expression in GC-2 cells (P < 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.
Animals ; Male ; Mice ; GTP-Binding Proteins/metabolism* ; Mitogen-Activated Protein Kinases/metabolism* ; NF-kappa B/metabolism* ; RNA, Messenger ; Serine-Type D-Ala-D-Ala Carboxypeptidase/metabolism* ; Signal Transduction ; Spermatocytes ; Tubulin/genetics*

OBJECTIVE: To study the protective effect of hyperoside (Hyp) against ydrogen peroxide (H₂O₂)- induced oxidative damage in mouse spermatocytes GC-2 cells and explore the role of the Keap1/Nrf2/HO-1 pathway in this protective mechanism. METHODS: GC-2 cells were treated with 2.5 mmol/L azaacetylcysteine (NAC), 50, 100, and 200 μmol/L hyperoside, or the culture medium for 48 h before exposure to H₂O₂ (150 μmol/L) for 2 h. CCK-8 assay was used to detect the changes in cell viability, and cell apoptosis was analyzed using flow cytometry. Enzyme-linked immunosorbent assay (ELISA) was used to detect the levels of superoxide dismutase (SOD), glutathione peroxidase (GSH-PX), catalase (CAT) activity and malondialdehyde (MDA) in the culture medium. Western blotting and RT-qPCR were used to detect the protein and mRNA expression levels of nuclear factor erythroid 2-related factor2 (Nrf2), Kelch-like ECH-associated protein 1 (Keap1), and heme oxygenase-1 (HO-1); the nuclear translocation of Nrf2 was detected using immunofluorescence assay. RESULTS: Exposure to H₂O₂ significantly lowered the proliferation rate, reduced the activities of SOD, GSH and CAT, and obviously increased MDA content, cell apoptosis rate, and the expressions of Keap1 and Nrf2 mRNA and Keap1 protein in GC-2 cells (P < 0.05 or 0.01). Treatment of the cells prior to H₂O₂ exposure with either NAC or 200 μmol/L hyperoside significantly increased the cell proliferation rate, enhanced the activities of SOD, GSH-PX and CAT, and lowered MDA content and cell apoptosis rate (P < 0.05). Treatment with 200 μmol/L hyperoside significantly decreased the mRNA and protein expressions of Keap1 and increased the expressions of HO-1 mRNA and the protein expressions of Nrf2 and HO-1 (P < 0.05 or 0.01). Hyperoside also caused obvious nuclear translocation of Nrf2 in the cells (P < 0.05). CONCLUSION Hyperoside protects GC-2 cells against H₂O₂- induced oxidative damage possibly by activation of the Keap1/Nrf2/HO-1 signaling pathway.
Animals ; Antioxidants/metabolism* ; Heme Oxygenase-1/metabolism* ; Hydrogen Peroxide/pharmacology* ; Kelch-Like ECH-Associated Protein 1/metabolism* ; Male ; Mice ; NF-E2-Related Factor 2/metabolism* ; Oxidative Stress ; Quercetin/analogs & derivatives* ; RNA, Messenger/metabolism* ; Spermatocytes/metabolism* ; Superoxide Dismutase/metabolism*

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.
Animals ; Cell Cycle Proteins/genetics* ; Cell Line ; Cell Survival ; Chondroitin Sulfate Proteoglycans/genetics* ; Chromosomal Proteins, Non-Histone/genetics* ; Gene Expression Regulation ; Gene Knockdown Techniques ; Infertility, Male ; Male ; Meiosis/genetics* ; Mice ; Mice, Knockout ; Mice, Transgenic ; Phosphate-Binding Proteins/genetics* ; Protein Tyrosine Phosphatase, Non-Receptor Type 11/genetics* ; Rad51 Recombinase/genetics* ; Real-Time Polymerase Chain Reaction ; Spermatocytes/metabolism* ; Spermatogenesis/genetics* ; Spermatogonia/metabolism*

OBJECTIVETo explore the effects of long-term exposure to particulate matter 2.5 (PM2.5) from automobile exhaust on the reproductive function of Sprague Dawley (SD) rats.

METHODSForty-five male SD rats, weighing 80 - 94 g and aged 28 days, were randomly assigned to receive intra-tracheal administration of 0.9% normal saline (control group, n = 15), PM2. 5 at 2 μg per 100 g body weight per day (low-dose PM2.5 group, n = 15), and PM2.5 at 16 μg per 100 g body weight per day (high-dose PM2.5 group, n = 15), qd, for 60 successive days. After the last 24-hour exposure, 10 rats were taken from each group for copulation with normal female ones, while the others were sacrificed, their testes removed for sperm count and deformity, pathological examination, and determination of the Connexin43 expression.

RESULTSThe conception rate was significantly decreased in the low- and high-dose PM2.5 groups as compared with that of the control (70% and 50% vs 100%), and so were the sperm count and quality. The rats in the PM2.5-exposed groups showed significantly disordered histological structure of the seminiferous tubules, reduced sperm count in the testicular lumen, some exfoliated secondary spermatocytes, downregulated Connexin43 expression in the testis, and damaged blood-testis barrier.

CONCLUSIONLong-term exposure to PM2.5 from automobile exhaust damages the reproductive function of male SD rats.

Animals ; Blood-Testis Barrier ; Body Weight ; Connexin 43 ; metabolism ; Down-Regulation ; Fertilization ; Male ; Particulate Matter ; toxicity ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Reproduction ; Seminiferous Tubules ; Sperm Count ; Spermatocytes ; Testis ; metabolism ; pathology ; Vehicle Emissions ; toxicity

OBJECTIVETo investigate the effects of single heat stress treatment on spermatogenic cells in mice.

METHODSWe randomly divided 36 C57 male mice into a control and a heat stress treatment group and submerged the lower part of the torso in water at 25 °C and 43 °C, respectively, both for 15 minutes. At 1, 7, and 14 days after treatment, we obtained the testicular organ indexes, observed the changes in testicular morphology by HE staining, and determined the location and expression levels of the promyelocytic leukemia zinc finger (PLZF) and synaptonemal comlex protein-3 (SCP-3) in the testis tissue by immunohistochemistry and Western blot.

RESULTSThe testicular organ index was significantly lower in the heat stress treatment than in the control group (P < 0.05). Compared with the controls, the heat shock-treated mice showed loosely arranged spermatogenic cells scattered in the seminiferous tubules at 1 day after heat stress treatment, atrophied, loosely arranged and obviously reduced number of spermatogenic cells at 7 days, and relatively closely arranged seminiferous tubules and increased number and layers of spermatogenic cells at 14 days. The number of SCP-3 labelled spermatocytes obviously decreased in the heat stress-treated animals at 1 and 7 days and began to increase at 14 days. The PLZF protein expression was significantly reduced in the heat stress treatment group at 1 day as compared with that in the control (0.19 ± 0.12 vs 0.64 ± 0.03, P < 0.01), but elevated to 0.77 ± 0.02 at 7 and 14 days, even remarkably higher than in the control animals (P < 0.01).

CONCLUSIONHeat stress treatment can induce short-term dyszoospermia in mice, which can be recovered with the prolonged time after treatment.

Animals ; Blotting, Western ; Hot Temperature ; Immunohistochemistry ; Male ; Mice ; Nuclear Proteins ; metabolism ; Promyelocytic Leukemia Protein ; Seminiferous Tubules ; cytology ; Spermatocytes ; cytology ; pathology ; Testis ; metabolism ; Transcription Factors ; metabolism ; Tumor Suppressor Proteins ; metabolism

OBJECTIVETo prepare a specific polyclonal antibody against full-length SUN5 for detecting the expression of SUN5 in human germ cells.

METHODSBioinformatic methods were used to compare the full-length SUN5 and its variant SUN5β, and a short peptide was designed based on the differential region to prepare SUN5 antibody. The prepared antibody was used to detect the expression of SUN5 in Ntera-2 cells and in human germ cells by Western blotting and immunofluorescence assay.

RESULTSThe short peptide was correctly synthesized and SUN5 antibody was obtained and purified. Western blotting showed that the prepared antibody was capable of recognizing full-length SUN5 in Ntera-2 cells, and SUN5 expression was localized on the nuclear membrane and in the cytoplasm as shown by immunofluorescence assay. Using this antibody, we detected SUN5 expression in the spermatocytes, round spermatids and sperms in human germ cells.

CONCLUSIONWe successfully prepared SUN5-specific antibody. SUN5 is expressed in the spermatocytes, round spermatids and sperms in human germ cells, suggesting its important role in spermatogenesis.

Antibodies ; chemistry ; Blotting, Western ; Cytoplasm ; metabolism ; Fluorescent Antibody Technique ; Humans ; Male ; Nuclear Envelope ; metabolism ; Proteins ; immunology ; metabolism ; Spermatids ; metabolism ; Spermatocytes ; metabolism ; Spermatogenesis ; Spermatozoa ; metabolism

RNA binding proteins (RBPs) regulate the function of cells by interacting with nascent transcripts and therefore are receiving increasing attention from researchers for their roles in tissue development and homeostasis. The polypyrimidine tract binding (PTB) protein family of RBPs are important posttranscriptional regulators of gene expression. Further investigations on the post-transcriptional regulation mechanisms and isoforms of PTB proteins in the spermatogenesis show that PTB protein 1 (Ptbp1) is a predominant isoform in mitotic cells (spermatogonia), while Ptbp2 predominates in meiotic spermatocytes and postmeiotic spermatids and binds to the specific 3' untranslated region (3' UTR) of the phosphoglycerate kinase 2 (Pgk-2) mRNA, which helps to stabilize Pgk-2 mRNA in male mouse germ cells. In case of Ptbp2 inactivation in the testis, the differentiation of germ cells arrests in the stage of round spermatids, with proliferation of multinucleated cells in the seminiferous tubule, increased apoptosis of spermatocytes, atrophy of seminiferous tubules, and lack of elongating spermatids, which consequently affects male fertility. This article presents an overview on the structure of the PTB protein and its role in regulating mammalian spermatogenesis.
Animals ; Atrophy ; Gene Expression Regulation ; physiology ; Heterogeneous-Nuclear Ribonucleoproteins ; metabolism ; physiology ; Homeostasis ; Isoenzymes ; metabolism ; Male ; Mice ; Nerve Tissue Proteins ; metabolism ; physiology ; Phosphoglycerate Kinase ; metabolism ; Polypyrimidine Tract-Binding Protein ; metabolism ; physiology ; RNA, Messenger ; metabolism ; RNA-Binding Proteins ; Seminiferous Tubules ; pathology ; Spermatids ; metabolism ; Spermatocytes ; metabolism ; Spermatogenesis ; physiology ; Spermatogonia ; metabolism ; Testis ; metabolism

OBJECTIVETo observe the effect of Liuweidihuang Pills in relieving cellphone electromagnetic radiation-induced histomorphological abnormality, oxidative injury, and cell apoptosis in the rat testis.

METHODSThirty adult male SD rats were equally randomized into a normal, a radiated, and a Liuweidihuang group, the animals in the latter two groups exposed to electromagnetic radiation of 900 MHz cellphone frequency 4 hours a day for 18 days. Meanwhile, the rats in the Liuweidihuang group were treated with the suspension of Liuweidihuang Pills at 1 ml/100 g body weight and the other rats intragastrically with the equal volume of purified water. Then all the rats were killed for observation of testicular histomorphology by routine HE staining, measurement of testicular malondialdehyde (MDA) and glutathione (GSH) levels by colorimetry, and determination of the expressions of bax and bcl-2 proteins in the testis tissue by immunohistochemistry.

RESULTSCompared with the normal controls, the radiated rats showed obviously loose structure, reduced layers of spermatocytes, and cavitation in the seminiferous tubules. Significant increases were observed in the MDA level (P < 0.01) and bax expression (P < 0.01) but decreases in the GSH level (P < 0.01) and bcl-2 expression (P < 0.01) in the testis issue of the radiated rats. In comparison with the radiated rats, those of the Liuweidihuang group exhibited nearly normal testicular structure, significantly lower MDA level (P < 0.05), bax expression (P < 0.01), and bcl-2 expression (P < 0.01).

CONCLUSIONLiuweidihuang Pills can improve cellphone electromagnetic radiation-induced histomorphological abnormality of the testis tissue and reduce its oxidative damage and cell apoptosis.

Animals ; Apoptosis ; drug effects ; radiation effects ; Body Weight ; drug effects ; radiation effects ; Cell Phone ; Drugs, Chinese Herbal ; pharmacology ; Electromagnetic Radiation ; Glutathione ; metabolism ; Male ; Malondialdehyde ; metabolism ; Oxidative Stress ; Radiation-Protective Agents ; pharmacology ; Rats ; Rats, Sprague-Dawley ; Seminiferous Tubules ; drug effects ; radiation effects ; Spermatocytes ; drug effects ; metabolism ; radiation effects ; Staining and Labeling ; Testis ; drug effects ; metabolism ; pathology ; radiation effects

Uchl1 was found to be involved in spermatocyte apoptosis. The aim of the present study was to test whether Uchl1 and its associated proteins Jab1 and p27(kip1) were involved in spermatogenic damages in response to heat-stress in cryptorchidism. Hematoxylin and eosin (HE) staining and DNA end labeling (TUNEL) were used to observe morphological and apoptotic characteristics of spermatogenic cells; Immunohistochemical analysis was used to detect changes of Uchl1 and its associated proteins Jab1 and p27(kip1) in response to heat-stress from cryptorchidism leading to spermatocyte losses; And protein affinity analysis (pull-down) and immunofluorescence co-localization were used to verify the relevance among the three proteins in spermatocytes. The results showed that, Jab1 and p27(kip1), in parallel to Uchl1, increased in spermatocytes of apoptotic appearances in response to heat-stress, but not in multinucleated giant cells; Jab1 bound to Uchl1 in testis protein extracts, and co-localized with Uchl1 and p27(kip1) specifically in spermatocytes with apoptotic appearances. These results suggest that the accumulation of Uchl1 protein is involved in the heat-stress-induced spermatocyte apoptosis through a new pathway related with Jab1 and p27(kip1), but not the formation of multinucleated giant cells.
Animals ; Apoptosis ; COP9 Signalosome Complex ; Cryptorchidism ; pathology ; Cyclin-Dependent Kinase Inhibitor p27 ; metabolism ; Hot Temperature ; Intracellular Signaling Peptides and Proteins ; metabolism ; Male ; Mice ; Peptide Hydrolases ; metabolism ; Spermatocytes ; cytology ; metabolism ; Stress, Physiological ; Ubiquitin Thiolesterase ; metabolism