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*

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 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*

Male ; Meiosis ; Protein Tyrosine Phosphatase, Non-Receptor Type 11 ; Spermatocytes ; Spermatogenesis ; Spermatogonia ; Tyrosine

Cimetidine is an H2 receptor antagonist that has an antiandrogenic effect. It intervenes with the conversion of testosterone into estrogen in the Sertoli cells with accompanying testicular structural changes. In the present study, the microscopic and the ultrastructural changes induced by cimetidine and the effect of vitamin B12 as a protective agent on rat testes were studied. Immunoexpression of estrogen receptor β (ERβ) in testes was evaluated. Twenty-four adult male rats were divided into four groups: control, cimetidine-treated, vitamin B12 treated, and combined cimetidine and vitamin B12 treated. The experimental rats were administered with cimetidine and/or vitamin B12 for 52 days. Group II rats showed marked atrophy of the seminiferous tubules with a significant increase in tubular diameter and decrease in the tubular luminal and epithelial areas. Ultrastructure of this group showed irregular Sertoli cells with basal cytoplasmic vacuolation and significantly thickened basement membrane. ERβ immunoexpression was similar to controls. Group III rats showed near normal seminiferous tubular structures with minimal cellular alterations and the immunoreactivity of the testicular sections was very close to normal. However, group IV rats showed markedly immunopositive detached cells, spermatids, and primary spermatocytes. Cimetidine interferes with the control of spermatogenesis as evidenced by microscopic and ultrastructural studies and affection of ERβ receptors and vitamin B12 has a protective action against this harmful effect.
Adult ; Animals ; Basement Membrane ; Cimetidine ; Cytoplasm ; Estrogens ; Humans ; Male ; Phenobarbital ; Rats* ; Seminiferous Epithelium* ; Seminiferous Tubules ; Sertoli Cells ; Spermatids ; Spermatocytes ; Spermatogenesis ; Testis ; Testosterone ; Vitamin B 12* ; Vitamins*

OBJECTIVE: Recapitulation of the spermatogenesis process in vitro is a tool for studying the biology of germ cells, and may lead to promising therapeutic strategies in the future. In this study, we attempted to transdifferentiate Wharton's jelly-derived mesenchymal stem cells (WJ-MSCs) into male germ cells using all-trans retinoic acid and Sertoli cell-conditioned medium. METHODS: Human WJ-MSCs were propagated by the explant culture method, and cells at the second passage were induced with differentiation medium containing all-trans retinoic acid for 2 weeks. Putative germ cells were cultured with Sertoli cell-conditioned medium at 36℃ for 3 more weeks. RESULTS: The gene expression profile was consistent with the stage-specific development of germ cells. The expression of Oct4 and Plzf (early germ cell markers) was diminished, while Stra8 (a premeiotic marker), Scp3 (a meiotic marker), and Acr and Prm1 (postmeiotic markers) were upregulated during the induction period. In morphological studies, approximately 5% of the cells were secondary spermatocytes that had completed two stages of acrosome formation (the Golgi phase and the cap phase). A few spermatid-like cells that had undergone the initial stage of tail formation were also noted. CONCLUSION: Human WJ-MSCs can be transdifferentiated into more advanced stages of germ cells by a simple two-step induction protocol using retinoic acid and Sertoli cell-conditioned medium.
Acrosome ; Biology ; Germ Cells* ; Humans* ; In Vitro Techniques ; Male* ; Mesenchymal Stromal Cells* ; Methods ; Spermatocytes ; Spermatogenesis ; Tail ; Transcriptome ; Tretinoin

The reproductive system and gonad development and germ cell occurrence of Whitmania pigra have been studied by using tissue section electron microscope techniques. W. pigra has completely independent male and female reproduction system, which lasts 11 months. The development of spermary started before the development of ovary. When egg cell is only a primordial germ cell, sperm has an initially complete form. Meanwhile, sperm cells and egg cells orderly development and synchronously mature. According to the development of sperm cells and egg cells, the development of cycle of the spermary could be divided into 6 stages: proliferating stage (1-3 months of age), growing stage (4-5 months of age), resting stage (6-8 months of age), maturing stage (9 months of age), spawning stage (10 months of age) and degradation stage (11 months of age). The development of cycle of the ovary could be divided into 6 stages: forming stage (1-2 months of age), proliferating stage (3-4 months of age), growing stage (5-8 months of age), maturing stage (9 months of age), spawning stage (10 months of age) and resting stage (11 months of age). W. pigra is a synchronous hermaphrodite animal, the development of cycle of the spermary and ovary each has six stages, sperm cells and egg cells orderly development and synchronously mature.
Animals ; Female ; Gonads ; cytology ; Leeches ; growth & development ; Male ; Ovary ; cytology ; Ovum ; cytology ; Reproduction ; Spermatocytes ; cytology

Adult ; Chromatids/genetics* ; Chromosome Aberrations ; Humans ; Infertility, Male/genetics* ; Male ; Meiosis ; Metaphase/genetics* ; Spermatocytes/physiology*

Humans ; Male ; Metaphase/physiology* ; Sex Chromosomes/ultrastructure* ; Spermatocytes/ultrastructure* ; Spermatogenesis ; Testis/ultrastructure*