Recent studies have confirmed that diverse adult tissue cells can be reprogrammed and induced to pluripotency, that is so-called induced pluripotent stem cells (iPS cells). But most of these dedifferentiated processes are induced by gene delivery with retroviral vectors. Some of the delivered genes are cancer causing. So, in current situation, these adult-derived embryonic stem-like cells cannot be used in clinical therapy to cure human diseases. Recently some articles that were published in the authoritative journals are receiving attentions. They show that, in mice and human, spermatogonial stem cells (SSCs) can be used for generating pluripotent stem cells without the exogenous genes and retroviruses, and they can also be used for autologous transplantation without ethical problems. These findings suggest that human SSCs may have considerable potential for cell-based, autologous organ regeneration therapy for various diseases. In this review, we describe and compare the methods that have been used to isolate, purificate and culture SSCs. We also describe the recent results in which SSCs can be transformed into pluripotent stem cells, and the pluripotent stem cells have potential applications in regenerative medicine and genetic medicine.
Cell Culture Techniques ; methods ; Cell Dedifferentiation ; physiology ; Cells, Cultured ; Humans ; Male ; Pluripotent Stem Cells ; cytology ; Spermatogonia ; cytology ; Stem Cells ; cytology

The isolation and culturation of SSCs of different stage of Wuzhishan Mini Porcine (WZSP) with different way of enzymatic digestion and culturation were deaded in this study. The results of the experiment described are as the following: The proper time of isolation and culturation of SSCs of WZSP is 1-20 old days. Different old of piglets with different method. Using DMEM medium as a fundmental culture medium add different gradient at 34 degrees C in a water-saturated atmosphere of 95% air, 5% CO2. The mulberry-shaped SSCs clusters appeared as original generation in 7-8 days culture. The SSCs clusters developed half-suspendedly in the culture medium. SSCs alkaline phosphatase (AKP) staining expressed positively. Mouse embryonic fibroblast was used as feeder layer for the SSCs passage cultured, The SSCs show good attached attributes, but the number of SSCs decreased quickly after 4 days culture. By seminiferous cord fragment culturation can also appear SSCs clusters in 5 days, The SSCs clusters developed half-suspendedly in the culture medium. In addition, the testes placed in cold (4 degrees C) PBS banlanced salt solution for 24 h also can be used as a good matierials for preparation of SSCs. These results indicate that the method of solation and culturation of SSCs are very correct and efficient, all these can be utilized as a good reference for future studies.
Animals ; Cells, Cultured ; Male ; Mice ; Spermatogonia ; cytology ; Stem Cells ; physiology ; Swine

Spermatogenesis is a process consisting of spermatogonial proliferation, spermatocytic meiosis, and spermiogenesis, and is also considered to be a process in which heterochromatins gradually aggregate and finally reach a highly condensed formation in the sperm head. Recent studies show that epigenetic regulation plays a key role in spermatogenesis. This review discusses the mechanisms of epigenetic regulation in spermatogenesis in three aspects, DNA methylation, histone modification, and noncoding RNAs. These factors are essential for spermatogenesis, fertilization, and embryogenesis by mutual regulation as well as by gene expression regulation, transposon activation, sex chromosome inactivation, and genome imprinting.
DNA Methylation ; Embryonic Development ; Epigenesis, Genetic ; physiology ; Genomic Imprinting ; Humans ; Male ; Meiosis ; Spermatogenesis ; genetics ; Spermatogonia ; cytology ; physiology

AIMTo isolate and transplant germ cells from adult mouse testes for transplantation.

METHODSIn order to distinguish transplanted cells from endogenous cells of recipients, donor transgenic mice expressing green fluorescent protein (GFP) were used. Germ cells were collected from the donors at 10-12 weeks of age and spermatogonia were concentrated by percoll fractionation and transplanted into recipient seminiferous tubules that had been previously treated with busulfan at 5 weeks of age to remove the endogenous spermatogenic cells.

RESULTSTwenty weeks after the transplantation, a wide spread GFP signal was observed in the recipient seminiferous tubules. The presence of spermatogenesis and spermatozoa was confirmed in sections of 12 out of 14 testes transplanted (86 %). However, when germ cells were transplanted without concentration the success rate was zero (0/9).

CONCLUSIONGerm cells from adult mouse testes can be successfully transplanted into recipient seminiferous tubules if the cell population is rich in spermatogonia and the percoll fractionation is useful in obtaining such a cell population.

Animals ; Cell Fractionation ; Green Fluorescent Proteins ; Luminescent Proteins ; genetics ; Male ; Mice ; Mice, Inbred C57BL ; Seminiferous Tubules ; cytology ; physiology ; Spermatogenesis ; physiology ; Spermatogonia ; physiology ; transplantation ; Testis ; cytology

OBJECTIVEThe culture of human spermatogonial stem cells (SSC) has not been studied in detail yet. Here we tried to explore the optimized culture method of human SSC by using several different co-culture systems.

METHODSThe alpha6 +Thy-1 +c-kit- cells acquired by the immunomagnetic beads sorting technique were cultured in different co-culture systems. Their morphological, biological characteristics and survival rates were intensively observed by microscopic or immunocytochemical assay. The long-term survival rate of human SSC during culture period was evaluated by germ cell transplantation technique.

RESULTSThe alpha6 +Thy-1 +c-kit- cells could stably survive in the DMEM and DMEM/F12 mediums with fetal bovine serum (FBS) on feeder layer. The survival rates within 1 week were more than 90%. The long-time culture showed the cells were gradually attached on the surface of Sertoli cells by the manner of scattered single cell or accumulated masses. Part of the SSC became more tightly attachment with Sertoli cells or mounted among the Sertoli cells. They could survive or even proliferate for more than 3 months in vitro. Germ cells transplantation study showed that some alpha6 +Thy-1 +c-kit- cells labeled by PKH26 could resided on the basal membrane of seminiferous tubule of nude mice, appearing as single or coupled cells 2 months later after transplantation. The function evaluation of the cultured cells by counting the fluorescent cells in the seminiferous tubule showed 54.9% and 9.2% of SSC in the alpha6 +Thy-1 +c-kit- cells were still remained after cultured for 2 and 4 weeks, respectively.

CONCLUSIONHuman SSC could maintain survival in vitro for more than 3 months, but it was still need to seek for a more optimized and successful culture system for its efficient expansion and proliferation. Thus it will open up a wide prospect for the understanding of the biology of human SSC and the treatment of male sterility.

Adult ; Cell Culture Techniques ; Cell Survival ; Cells, Cultured ; Coculture Techniques ; Humans ; Male ; Sertoli Cells ; cytology ; Spermatogonia ; cytology ; physiology ; Stem Cell Transplantation ; Stem Cells ; cytology

We previously reported the successful establishment of embryonic stem cell (ESC)-like multipotent spermatogonial stem cells (mSSCs) from neonatal mouse testis. Here, we examined the ability of mSSCs to differentiate into vascular endothelial cells and smooth muscle cells, and compared to that of mouse ESCs. We used real-time reverse transcriptase polymerase chain reaction and immunohistochemistry to examine gene expression profiles of mSSCs and ESCs during in vitro vascular differentiation. Both mSSCs and ESCs exhibited substantial increase in the expression of mesodermal markers, such as Brachyury, Flk1, Mesp1, Nkx2.5, and Islet1, and a decrease in the expression of pluripotency markers, such as Oct3/4 and Nanog during the early stage of differentiation. The mRNA levels of vascular endothelial (VE)-cadherin and CD31 gradually increased in both differentiated mSSCs and ESCs. VE-cadherin- or CD31-positive cells formed sprouting branch-like structures, as observed during embryonic vascular development. At the same time, vascular smooth muscle cell-specific markers, such as myocardin and alpha-smooth muscle actin (SMA), were also highly expressed in differentiated mSSCs and ESCs. Immunocytochemical analysis revealed that the differentiated cells expressed both alpha-SMA and SM22-alpha proteins, and exhibited the intracellular fibril structure typical of smooth muscle cells. Overall, our findings showed that mSSCs have similar vascular differentiation abilities to those of ESCs, suggesting that mSSCs may be an alternative source of autologous pluripotent stem cells for vascular regeneration.
Animals ; Animals, Newborn ; Biological Markers/metabolism ; Cell Differentiation/physiology ; Embryonic Stem Cells/cytology/physiology ; Endothelial Cells/*cytology/physiology ; Gene Expression ; Gene Expression Profiling ; Humans ; Immunohistochemistry ; Male ; Mice ; Muscle, Smooth, Vascular/*cytology/physiology ; Myocytes, Smooth Muscle/*cytology/physiology ; Pluripotent Stem Cells/*cytology/physiology ; Real-Time Polymerase Chain Reaction ; Spermatogonia/*cytology/physiology ; Testis/*cytology/physiology

The effect of ginsenosides on proliferation of type A spermatogonia was investigated in 7-day-old mice. Spermatogonia were characterized by c-kit expression and cell proliferation was assessed by immunocytochemical demonstration of proliferating cell nuclear antigen (PCNA). After 72-h culture, Sertoli cells formed a confluent monolayer to which numerous spermatogonial colonies attached. Spermatogonia were positive for c-kit staining and showed high proliferating activity by PCNA expression. Ginsenosides (1.0 approximately10 microg/ml) significantly stimulated proliferation of spermatogonia. Activation of protein kinase C (PKC) elicited proliferation of spermatogonia at 10(-8) to 10(-7) mol/L and the PKC inhibitor H(7) inhibited this effect. Likewise, ginsenosides-stimulated spermatogonial proliferation was suppressed by combined treatment of H(7). These results indicate that the proliferating effect of ginsenosides on mouse type A spermatogonia might be mediated by a mechanism involving the PKC signal transduction pathway.
Animals ; Cell Proliferation ; drug effects ; Enzyme Activation ; Ginsenosides ; pharmacology ; Male ; Mice ; Mice, Inbred ICR ; Proliferating Cell Nuclear Antigen ; analysis ; Protein Kinase C ; physiology ; Spermatogonia ; cytology ; drug effects

Nanos2, a member of the Nanos2 gene family, is a specific gene in male germ cells and encodes an evolutionarily conserved RNA binding protein expressed in male primordial germ cells (PGCs) during the embryonic period as well as in the spermatogonial stem cells (SSCs) of the testis. In the embryonic period, Nanos2 promotes the development of male PGCs and inhibits them from meiosis. In the process of spermatogenesis, Nanos2 suppresses the differentiation of SSCs in the testis and maintains the stability of the SSC pool. The knockout of Nanos2 may cause the disappearance of germ cells and sterility in male mice while its overexpression in the testis may lead to accumulation of SSCs in seminiferous tubules. Besides, Nanos2 is involved in the degradation of specific RNAs and possibly associated with some diseases of the male reproductive system. This review focuses on the recent progress in the studies of Nanos2 in the male reproductive system.
Animals ; Cell Differentiation ; Gene Knockout Techniques ; Male ; Meiosis ; Mice ; RNA ; metabolism ; RNA-Binding Proteins ; genetics ; metabolism ; Spermatogenesis ; physiology ; Spermatogonia ; Spermatozoa ; Testis ; cytology

The effects of over-expression of testis-specific expressed gene 1 (TSEG-1) on the viability and apoptosis of cultured spermatogonial GC-1spg cells were investigated, and the immortal spermatogonial cell line GC-1spg (CRL-2053™) was obtained as the cell model in order to explore the function of TSEG-1. We transfected the eukaryotic vector of TSEG-1, named as pEGFP-TSEG-1 into cultured spermatogonial GC-1spg cells. Over-expression of TSEG-1 inhibited the proliferation of GC-1spg cells, and arrested cell cycle slightly at G0/G1 phase. Transfection of TSEG-1 attenuated the transcript levels of Ki-67, PCNA and cyclin D1. In addition, over-expression of TSEG-1 induced early and late apoptosis, and reduced the mitochondrial membrane potential of GC-1spg cells. Moreover, transfection of TSEG-1 significantly enhanced the ratio of Bax/Bcl-2 and transcript levels of caspase 9, and decreased the expression of Fas and caspase 8 in GC-1spg cells. These results indicated over-expression of TSEG-1 suppresses the proliferation and induces the apoptosis of GC-1spg cells, which establishes a basis for further study on the function of TSEG-1.
Animals ; Caspase 8 ; biosynthesis ; genetics ; Cell Line ; Cyclin D1 ; biosynthesis ; genetics ; G1 Phase ; physiology ; Histones ; genetics ; metabolism ; Ki-67 Antigen ; biosynthesis ; genetics ; Male ; Mice ; Proliferating Cell Nuclear Antigen ; biosynthesis ; genetics ; Resting Phase, Cell Cycle ; physiology ; Spermatogonia ; cytology ; metabolism ; bcl-2-Associated X Protein ; biosynthesis ; genetics