OBJECTIVETo study the expression of Annexin A7 in the mouse testis, especially in different types of spermatogonia.

METHODSWe prepared Annexin A7 recombinant protein using prokaryotic expression, adsorbed the Annexin A7 antibody with it after identified by mass spectrometry, and detected the expression of Annexin A7 by Western-blot and immunohistochemistry.

RESULTSAnnexin A7 was expressed in a development-dependent manner in the spermatogonia of the prepubertal mice and in the type-A single (As) and type-A paired (Apr) spermatogonia of adult mice. These results were confirmed by the co-localization of Annexin A7 and Stra8, a known determinant of differentiated spermatogonial stem cells (SSCs).

CONCLUSIONAnnexin A7 is the internal factor of As and Apr spermatogonia, which might be involved in the biological functions of SSCs.

Animals ; Annexin A7 ; metabolism ; Male ; Mice ; Spermatogonia ; cytology ; metabolism ; Stem Cells ; cytology ; metabolism

OBJECTIVESTo detect the changes of DNA ploidy of spermatogenic cells in testis and epididymis.

METHODSRight epididymides and testes from 15 fertile youth donors who died of accident were collected. Samples of spermatogenic cells in different regions of epididymis (caput, corpus and cauda) and tests were collected. DNA of spermatogenic cells were detected by flow cyctometry (FCM).

RESULTSThe haploid(1n), diploid(2n) and tetraploid(4n) spermatogenic cells were existed in different regions of epididymis and testis. The 1n cells increased from (24.87 +/- 7.28)% in testis to (96.33 +/- 1.58)% in epididymis cauda, there were significant differences among regions of testis and epididymis caput, corpus(P < 0.01), and the difference among regions of epididymis corpus and epididymis cauda were also significant(P < 0.05). While the percentages of 2n and 4n cells decreased from (63.07 +/- 8.96)% and (9.43 +/- 3.83)% in tesits to (2.47 +/- 0.93)% and (1.17 +/- 0.95)% in epididymis respectively. There was significant difference of 2n cells between testis and epididymis caput, corpus(P < 0.01), and was also remarkable difference between epididymis corpus and cauda (P < 0.05). There was no difference of 4n cells between testis and epididymis caput(P > 0.05). There were significant difference among regions of epididymis caput, corpus and cauda(P < 0.05).

CONCLUSIONSThe percentage of immature spermatogenic cells decreased along with passing through the epididymis.

Adult ; DNA ; analysis ; Epididymis ; metabolism ; Flow Cytometry ; Humans ; Male ; Spermatogonia ; metabolism ; Testis ; metabolism

Meiotic initiation is a critical step in gametogenesis. Recently, some genes required for meiotic initiation have been identified. However, meiosis-initiating factors and the underlying mechanisms are far from being fully understood. We have established a long-term culture system of spermatogonial stem cells (SSCs) and an in vitro model of meiotic initiation using mouse SSCs. Our previous study revealed that the RNA-binding protein RBFOX2 may regulate meiotic initiation, but the role and the mechanism need to be further elucidated. In this study, we constructed RBFOX2 knockdown SSC lines by using lentivirus-mediated gene delivery method, and found that the knockdown SSCs underwent normal self-renewal, mitosis and differentiation. However, they were unable to initiate meiosis when treated with retinoic acid, and they underwent apoptosis. These results indicate that RBFOX2 plays an essential role in meiotic initiation of spermatogonia. This work provides new clues for understanding the functions of RNA-binding proteins in meiotic initiation.
Mice ; Male ; Animals ; Spermatogonia/metabolism* ; Meiosis/genetics* ; Cell Differentiation ; Tretinoin/pharmacology* ; Mitosis ; Testis/metabolism*

With the rapid development of gene editing technology, the study of spermatogonial stem cells (SSCs) holds great significance in understanding spermatogenesis and its regulatory mechanism, developing transgenic animals, gene therapy, infertility treatment and protecting rare species. Biogenesis of lysosome-related organelles complex 1 subunit 1 (BLOC1S1) is believed to have anti-brucella potential. Exploring the impack of BLOC1S1 on goat SSCs not only helps investigate the ability of BLOC1S1 to promote SSCs proliferation, but also provides a cytological basis for disease-resistant breeding research. In this study, a BLOC1S1 overexpression vector was constructed by homologous recombination. The BLOC1S1 overexpression cell line of goat spermatogonial stem cells was successfully constructed by lentivirus packaging, transfection and puromycin screening. The overexpression efficiency of BLOC1S1 was found to be 18 times higher using real time quantitative PCR (RT-qPCR). Furthermore, the results from cell growth curve analysis, flow cytometry for cell cycle detection, and 5-ethynyl-2'-deoxyuridine (EdU) staining showed that BLOC1S1 significantly increased the proliferation activity of goat SSCs. The results of RT-qPCR, immunofluorescence staining and Western blotting analyses revealed up-regulation of proliferation-related genes (PCNA, CDK2, CCND1), and EIF2S3Y, a key gene regulating the proliferation of spermatogonial stem cells. These findings strongly suggest that the proliferative ability of goat SSCs can be enhanced through the EIF2S3Y/ERK pathway. In summary, this study successfully created a goat spermatogonial stem cell BLOC1S1 overexpression cell line, which exhibited improved proliferation ability. This research laid the groundwork for exploring the regulatory role of BLOC1S1 in goat spermatogonia and provided a cell platform for further study into the biological function of BLOC1S1. These findings also establish a foundation for breeding BLOC1S1 overexpressing goats.
Animals ; Male ; Goats ; Stem Cells ; Spermatogonia/metabolism* ; Cell Proliferation ; Flow Cytometry ; Testis/metabolism*

Continuous self-renewal and differentiation of spermatogonial stem cells (SSCs) is vital for maintenance of adult spermatogenesis. Although several spermatogonial stem cell regulators have been extensively investigated in rodents, regulatory mechanisms of human SSC self-renewal and differentiation have not been fully established. We analyzed single-cell sequencing data from the human testis and found that forkhead box P4 (FOXP4) expression gradually increased with development of SSCs. Further analysis of its expression patterns in human testicular tissues revealed that FOXP4 specifically marks a subset of spermatogonia with stem cell potential. Conditional inactivation of FOXP4 in human SSC lines suppressed SSC proliferation and significantly activated apoptosis. FOXP4 expressions were markedly suppressed in tissues with dysregulated spermatogenesis. These findings imply that FOXP4 is involved in human SSC proliferation, which will help elucidate on the mechanisms controlling the fate decisions in human SSCs.
Adult ; Humans ; Male ; Cell Differentiation ; Cell Proliferation ; Forkhead Transcription Factors/metabolism* ; Spermatogenesis/genetics* ; Spermatogonia/metabolism* ; Stem Cells/metabolism* ; Testis/metabolism*

OBJECTIVESTo study the expression and the significance of telomerase gene hTERT in testes of infertile male.

METHODSBy using in situ hybridization(ISH) techniques, the expression of telomerase gene hTERT mRNA in testes of 47 infertile male and 10 normal testicular tissues were observed.

RESULTSIn male testes, there was a positive correlation between the expression of hTERT and the quantity and density of germ cells(spermatogonia, spermatocyte, spermatid). The expression of hTERT in some germinal cell of maturation arrest patients were not significantly different with those of normal.

CONCLUSIONSOur results suggest that the deficiency of telomerase might be a factor for germinal cell maturation arrest and there might be some other etiological factors in these patients. Our study provides experimental groundwork for the gene therapy of male infertility.

Humans ; Infertility, Male ; enzymology ; Male ; Spermatids ; Spermatocytes ; Spermatogenesis ; Spermatogonia ; Telomerase ; deficiency ; genetics ; metabolism ; Testis ; enzymology ; physiology

Objective: The expression patterns of ribosomal large subunit protein 23a (RPL23a) in mouse testes and GC-1 cells were analyzed to investigate the potential relationship between RPL23a expression and spermatogonia apoptosis upon exposure to X-ray. Methods: Male mice and GC-1 cells were irradiated with X-ray, terminal dUTP nick end-labelling (TUNEL) was performed to detect apoptotic spermatogonia Results: Ionizing radiation (IR) increased spermatogonia apoptosis, the expression of RPL11, MDM2 and p53, and decreased RPL23a expression in mice spermatogonia Conclusion These results suggested that IR reduced RPL23a expression, leading to weakened the RPL23a-RPL11 interactions, which may have activated p53, resulting in spermatogonia apoptosis. These results provide insights into environmental and clinical risks of radiotherapy following exposure to IR in male fertility. The graphical abstract was available in the web of www.besjournal.com.
Animals ; Apoptosis/genetics* ; Gene Expression Regulation ; Male ; Mice ; Ribosomal Proteins/metabolism* ; Signal Transduction ; Spermatogonia/radiation effects*

Spermatogonial stem cells (SSCs) transmit genetic information to the next progeny in males. Thus, SSCs are a potential target for germline modifications to generate transgenic animals. In this study, we report a technique for the generation of transgenic rats by in vivo manipulation of SSCs with a high success rate. SSCs in juvenile rats were transduced in vivo with high titers of lentivirus harboring enhanced green fluorescent protein and mated with wild-type females to create founder rats. These founder rats expressed the transgene and passed on the transgene with an overall success rate of 50.0%. Subsequent generations of progeny from the founder rats both expressed and passed on the transgene. Thus, direct modification of SSCs in juvenile rats is an effective means of generating transgenic rats through the male germline. This technology could be adapted to larger animals, in which existing methods for gene modification are inadequate or inapplicable, resulting in the generation of transgenic animals in a variety of species.
Animals ; Green Fluorescent Proteins ; Lentivirus ; Male ; Rats ; Rats, Transgenic ; Spermatogonia/metabolism*

Spermatogonial stem cells are a population of immortal cells, capable of self-renewal and multi-directional differentiation. The theory of the "stem cell niche" was originally proposed for the hematopoietic system, and niches also exist in testicular tissues. The niche for spermatogonial stem cells is a semi-isolation system, characteristic of specific number regulation and changing with the age. The self-renewal of spermatogonial stem cells is regulated by two endogenous factors, nanos2 and Plzf, as well as by the niche cell-excreted factor, glial-derived neurotrophic factor (GDNF).
Animals ; Carrier Proteins ; metabolism ; Glial Cell Line-Derived Neurotrophic Factor ; metabolism ; Male ; Mice ; RNA-Binding Proteins ; Spermatogonia ; cytology ; metabolism ; Stem Cells ; cytology ; metabolism ; Testis ; cytology ; metabolism

OBJECTIVETo investigate the expression of transient receptor potential melastatin (TRPM) and transient receptor potential vanilloid (TRPV) channel family genes in rat spermatogenic cells.

METHODSRat spermatogenic cells were isolated by a mechanical procedure and the total RNA was extracted using TRIzol reagent. TRPM and TRPV channel family genes were amplified by RT-PCR and the presence of the target genes was detected by agarose gel electrophoresis. The relative gene expression levels were measured by real-time quantitative RT-PCR.

RESULTSTRPV5, TRPM3, TRPM4 and TRPM7 mRNAs were expressed in rat spermatogenic cells, but TRPV1, TRPV2, TRPV3, TRPV4, TRPV6, TRPM1, TRPM2, TRPM5, TRPM6, TRPM7 and TRPM8 mRNAs were not detected. The relative expressions of TRPM and TRPV mRNA were determined by quantitative real-time RT-PCR. TRPM7 expression was the highest among all the TRPM subtypes in rat spermatogenic cells, at a level equivalent to (0.0430-/+0.0034)% of beta-actin expression. TRPM3 and TRPM4 were also highly expressed, but their expression levels were only approximately 56% and 63% of that of TRPM7, respectively. For the TRPV subfamily, only TRPV5 mRNA was abundantly expressed at the level of (0.0157-/+0.0029)% relative to that of beta-actin.

CONCLUSIONTRPV5, TRPM3, TRPM4 and TRPM7 mRNAs were coexpressed in spermatogenic cells in rats, among which TRPM4 and TRPM7 mRNA were expressed at high levels. TRPM4 and TRPM7 channels may be involved in the regulation of growth, differentiation and maturation of rat spermatogenic cells and are associated with the generation of the sperms.

Animals ; Male ; RNA, Messenger ; genetics ; metabolism ; Rats ; Rats, Sprague-Dawley ; Spermatocytes ; cytology ; metabolism ; Spermatogonia ; cytology ; metabolism ; TRPM Cation Channels ; genetics ; metabolism ; TRPV Cation Channels ; genetics ; metabolism