Establishment and comparative analysis of three DNA damage models of GC-1 cells / 中国药理学通报

Aim To establish three kinds of DNA damage models of mouse testicular spermatogonia cell line GC-1 cells and analyze their similarities and differences. Methods GC-1 cells were treated with UVB irradiation, D-galactose(D-Gal) or bleomycin (BLM), respectively. Then the expression and localization of 'Y-H2AX were detected by Western blot and immunofluorescence, the expression and localization of 8-OHdG were measured by immunofluorescence, and the expression levels of p-p53 and p21 were measured by Western blot. Results The expression of -y-H2AX in GC-1 cell reached to the peak 4 h after UVB irradiation and 6 h after D-Gal stimulation, whereas -y-H2AX expression gradually increased after BLM stimulation, and the higher the concentration of BLM,the shorter the time to reach the peak. The results of immunofluorescence showed that 8-OHdG expression was observed in the nucleus and cytoplasm of GC-1 cells after UVB irradiation and BLM stimulation, and the longer the culture time, the more the expression in the nucleus. In contrast, the expression of 8-OHdG was observed in the cytoplasm and reached the peak at 6 h in the D-Gal stimulated GC-1 cells. After UVB irradiation, the protein expression levels of p-p53 gradually increased, while p21 protein expression appeared later than that of p-p53; in the D-Gal stimulated GC-1 cells, the protein expression levels of p-p53 reached the peak at 6 h, and p21 protein expression reached the peak at 12 h; after low concentration BLM stimulation, the protein expression levels of p-p53 and p21 continuously increased, and after high concentration BLM stimulation, the protein expression levels of p-p53 and p21 reached its peak at 2 h, then decreased at 4 h. Conclusions Three kinds of DNA damage models of GC-1 cells are successfully established, and the DNA damage in GC-1 cells treated with D-Gal is mild, while the DNA damage in GC-1 cells treated by UVB irradiation and BLM stimulation is more severe.

Ionizing Radiation-Induced RPL23a Reduction Regulates Apoptosis / 生物医学与环境科学（英文）

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.

Mechanistic target of rapamycin kinase (Mtor) is required for spermatogonial proliferation and differentiation in mice / 亚洲男科学杂志(英文版)

Spermatogonial development is a vital prerequisite for spermatogenesis and male fertility. However, the exact mechanisms underlying the behavior of spermatogonia, including spermatogonial stem cell (SSC) self-renewal and spermatogonial proliferation and differentiation, are not fully understood. Recent studies demonstrated that the mTOR complex 1 (mTORC1) signaling pathway plays a crucial role in spermatogonial development, but whether MTOR itself was also involved in any specific process of spermatogonial development remained undetermined. In this study, we specifically deleted Mtor in male germ cells of mice using Stra8-Cre and assessed its effect on the function of spermatogonia. The Mtor knockout (KO) mice exhibited an age-dependent perturbation of testicular development and progressively lost germ cells and fertility with age. These age-related phenotypes were likely caused by a delayed initiation of Mtor deletion driven by Stra8-Cre. Further examination revealed a reduction of differentiating spermatogonia in Mtor KO mice, suggesting that spermatogonial differentiation was inhibited. Spermatogonial proliferation was also impaired in Mtor KO mice, leading to a diminished spermatogonial pool and total germ cell population. Our results show that MTOR plays a pivotal role in male fertility and is required for spermatogonial proliferation and differentiation.

Identification of sumoylated targets in proliferating mouse spermatogonia and human testicular seminomas / 亚洲男科学杂志(英文版)

Spermatogenesis is regulated by a complex network of posttranslation modifications. Sumoylation (a modification by small ubiquitin-like modifiers, or SUMO proteins) was identified as an important cellular event in different cell types. SUMO proteins are highly expressed in the testis, and their role during spermatogenesis has begun to be elucidated. Given the important role of sumoylation in the regulation of mitosis and cancer progression in other tissues, the aim of the current study was to identify the targets of SUMO in proliferating mouse spermatogonia and human seminoma tissues and to initially examine the level of sumoylation in relation to the proliferative activity of the tissues. Using freshly purified spermatogonia and C18-4 spermatogonia cell line, mass spectrometry analysis identified several SUMO targets implicated into the proliferation of spermatogonia (such as heat shock protein 60 [HSP60] and prohibitin). Tissue array and western blot approaches showed that SUMO expression is a prominent feature of human seminomas and that the proliferative activity of the tumor tissues was positively correlated with the level of SUMO expression. Downregulation of sumoylation with si-RNA was not sufficient to significantly affect the proliferation of C18-4 spermatogonia; however, SUMO overexpression increased the proliferation rate of the cells. These data suggest that cells are more sensitive to an elevated level of SUMO, and that this situation may lead to an upregulated cellular proliferation and, possibly, cancer. Mass spectrometry analysis identified around a hundred SUMO targets in seminoma samples. Notably, many of the identified proteins (such as proliferating cell nuclear antigen [PCNA], DNA topoisomerase 2-alpha [Top2A], prohibitin, 14-3-3 protein, and others) were implicated in oncogenic transformation and cancer progression.

Mechanistic target of rapamycin kinase (Mtor) is required for spermatogonial proliferation and differentiation in mice / 亚洲男科学杂志(英文版)

Spermatogonial development is a vital prerequisite for spermatogenesis and male fertility. However, the exact mechanisms underlying the behavior of spermatogonia, including spermatogonial stem cell (SSC) self-renewal and spermatogonial proliferation and differentiation, are not fully understood. Recent studies demonstrated that the mTOR complex 1 (mTORC1) signaling pathway plays a crucial role in spermatogonial development, but whether MTOR itself was also involved in any specific process of spermatogonial development remained undetermined. In this study, we specifically deleted Mtor in male germ cells of mice using Stra8-Cre and assessed its effect on the function of spermatogonia. The Mtor knockout (KO) mice exhibited an age-dependent perturbation of testicular development and progressively lost germ cells and fertility with age. These age-related phenotypes were likely caused by a delayed initiation of Mtor deletion driven by Stra8-Cre. Further examination revealed a reduction of differentiating spermatogonia in Mtor KO mice, suggesting that spermatogonial differentiation was inhibited. Spermatogonial proliferation was also impaired in Mtor KO mice, leading to a diminished spermatogonial pool and total germ cell population. Our results show that MTOR plays a pivotal role in male fertility and is required for spermatogonial proliferation and differentiation.

PLZFposc-KITpos-delineated A1-A4-differentiating spermatogonia by subset and stage detection upon Bouin fixation / 亚洲男科学杂志(英文版)

While hallmarks of rodent spermatogonia stem cell biomarkers' heterogeneity have recently been identified, their stage and subset distributions remain unclear. Furthermore, it is currently difficult to accurately identify subset-specific SSC marker distributions due to the poor nuclear morphological characteristics associated with fixation in 4% paraformaldehyde. In the present study, testicular cross-sections and whole-mount samples were Bouin fixed to optimize nuclear resolution and visualized by immunohistochemistry (IHC) and immunofluorescence (IF). The results identified an expression pattern of PLZFhighc-KITpos in A1 spermatogonia, while A2-A4-differentiating spermatogonia were PLZFlowc-KITpos. Additionally, this procedure was used to examine asymmetrically expressing GFRA1 and PLZF clones, asymmetric Apr and false clones were distinguished based on the presence or absence of TEX14, a molecular maker of intercellular bridges, despite having identical nuclear morphology and intercellular distances that were <25 μm. In conclusion, this optimized Bouin fixation procedure facilitates the accurate identification of spermatogonium subsets based on their molecular profiles and is capable of distinguishing asymmetric and false clones. Therefore, the findings presented herein will facilitate further morphological and functional analysis studies and provide further insight into spermatogonium subtypes.

PLZFposc-KITpos-delineated A1-A4-differentiating spermatogonia by subset and stage detection upon Bouin fixation / 亚洲男科学杂志(英文版)

While hallmarks of rodent spermatogonia stem cell biomarkers' heterogeneity have recently been identified, their stage and subset distributions remain unclear. Furthermore, it is currently difficult to accurately identify subset-specific SSC marker distributions due to the poor nuclear morphological characteristics associated with fixation in 4% paraformaldehyde. In the present study, testicular cross-sections and whole-mount samples were Bouin fixed to optimize nuclear resolution and visualized by immunohistochemistry (IHC) and immunofluorescence (IF). The results identified an expression pattern of PLZFhighc-KITpos in A1 spermatogonia, while A2-A4-differentiating spermatogonia were PLZFlowc-KITpos. Additionally, this procedure was used to examine asymmetrically expressing GFRA1 and PLZF clones, asymmetric Apr and false clones were distinguished based on the presence or absence of TEX14, a molecular maker of intercellular bridges, despite having identical nuclear morphology and intercellular distances that were <25 μm. In conclusion, this optimized Bouin fixation procedure facilitates the accurate identification of spermatogonium subsets based on their molecular profiles and is capable of distinguishing asymmetric and false clones. Therefore, the findings presented herein will facilitate further morphological and functional analysis studies and provide further insight into spermatogonium subtypes.

CEP55 may be a potential therapeutic target for non-obstructive azoospermia with maturation arrest / 南方医科大学学报

OBJECTIVE@#To explore the effect of small interfering RNA (siRNA)-mediated CEP55 gene silencing on the proliferation of mouse spermatogonia.@*METHODS@#Six patients with azoospermia diagnosed to have maturation arrest (3 cases) or normal spermatogenesis (3 cases) based on testicular biopsy between January 1 and December 31, 2017 in our center were examined for differential proteins in the testicular tissue using isobaric tags for relative and absolute quantitation (iTRAQ), and CEP55 was found to differentially expressed between the two groups of patients. We constructed a CEP55 siRNA for transfection in mouse spermatogonia and examined the inhibitory effects on CEP55 expressions using Western blotting and qPCR. The effect of CEP55 gene silencing on the proliferation of mouse spermatogonia was evaluated with CCK8 assay.@*RESULTS@#In the testicular tissues from the 6 patients with azoospermia, iTRAQ combined with LC/MS/MS analysis identified over two hundred differentially expressed proteins, among which CEP55 showed the most significant differential expression between the patients with maturation arrest and those with normal spermatogenesis. The cell transfection experiment showed that compared with the cells transfected with the vehicle or the negative control sequence, the mouse spermatogonia transfected with CEP55 siRNA showed significantly lowered expressions of CEP55 mRNA and protein ( < 0.05) and significantly decreased proliferation rate as shown by CCK8 assay ( < 0.05).@*CONCLUSIONS@#CEP55 may play a key role in spermatogenesis and may serve as a potential therapeutic target for non-obstructive azoospermia with maturation arrest.

Stimulated by retinoic acid gene 8 (Stra8) plays important roles in many stages of spermatogenesis / 亚洲男科学杂志(英文版)

To clarify the functions and mechanism of stimulated by retinoic acid gene 8 (Stra8) in spermatogenesis, we analyzed the testes from Stra8 knockout and wild-type mice during the first wave of spermatogenesis. Comparisons showed no significant differences in morphology and number of germ cells at 11 days postpartum, while 21 differentially expressed genes (DEGs) associated with spermatogenesis were identified. We speculate that Stra8 performs many functions in different phases of spermatogenesis, such as establishment of spermatogonial stem cells, spermatogonial proliferation and self-renewal, spermatogonial differentiation and meiosis, through direct or indirect regulation of these DEGs. We therefore established a preliminary regulatory network of Stra8 during spermatogenesis. These results will provide a theoretical basis for further research on the mechanism underlying the role of Stra8 in spermatogenesis.

Stimulated by retinoic acid gene 8 (Stra8) plays important roles in many stages of spermatogenesis / 亚洲男科学杂志(英文版)

To clarify the functions and mechanism of stimulated by retinoic acid gene 8 (Stra8) in spermatogenesis, we analyzed the testes from Stra8 knockout and wild-type mice during the first wave of spermatogenesis. Comparisons showed no significant differences in morphology and number of germ cells at 11 days postpartum, while 21 differentially expressed genes (DEGs) associated with spermatogenesis were identified. We speculate that Stra8 performs many functions in different phases of spermatogenesis, such as establishment of spermatogonial stem cells, spermatogonial proliferation and self-renewal, spermatogonial differentiation and meiosis, through direct or indirect regulation of these DEGs. We therefore established a preliminary regulatory network of Stra8 during spermatogenesis. These results will provide a theoretical basis for further research on the mechanism underlying the role of Stra8 in spermatogenesis.

Hydrogen alleviates high-level low-dose irradiation-induced injury of male reproductive system in mice / 第二军医大学学报

Objective To explore the protective effects of molecular hydrogen on high-level low-dose irradiation-induced male reproductive system injury in mice and the underlying mechanism. Methods Cultured spermatogonia-derived cell line GC-1 spg was randomized into control group, hydrogen group, 4 Gy radiation group and 4 Gy radiation+hydrogen group. The apoptotic rate of the cells was detected by flow cytometry assay at 24 h after treatment in each group. Seventy-two male BALB/c mice were randomized into control group, hydrogen group, 0.25 Gy radiation group, 0.25 Gy radiation+ hydrogen group, 0.5 Gy radiation group and 0.5 Gy radiation+hydrogen group, with 12 mice in each group. The hydrogen treatment was conducted by hydrogen-rich water administration and high-concentration hydrogen gas inhalation. At 24 h after treatment, the testes were dissected and sectioned for H-E staining, and blood samples from the internal canthus vein were collected to determine the levels of gonadotropin-releasing hormone (GnRH), follicle-stimulating hormone (FSH), luteinizing hormone (LH), and testosterone using ELISA. At 4 weeks after radiation, the bilateral epididymides were isolated to prepare sperm suspensions, and the DNA damage of the spermatozoa was examined using the neutral single cell gel electrophoresis. Results The 24 h apoptosis rate of GC-1 spg cells was significantly decreased in the 4 Gy radiation+hydrogen group compared with the 4 Gy radiation group (t=7.186, P<0.01). Hydrogen obviously reverted the histological damage of the testes induced by 0.5 Gy irradiation, significantly inhibited 0.25 Gy and 0.5 Gy radiation-caused surge of FSH (t=3.195 8, P=0.019; t=10.723 4, P<0.05), and significantly ameliorated comet tailing and damage of the sperm DNA at 4 weeks after radiation (tail area t0.25 Gy=16.592 3, t0.5 Gy=15.891 5; tail DNA t0.25 Gy=11.296 5, t0.5 Gy=13.785 0; tail DNA% t0.25 Gy=26.834 0, t0.5 Gy=10.325 7; tail length t0.25 Gy=16.865 4, t0.5 Gy=15.441 2; tail moment t0.25 Gy=26.979 4, t0.5 Gy=13.174 2; Olive tail moment t0.25 Gy=24.752 4,t0.5 Gy=6.896 1; all P<0.05). Conclusion Molecular hydrogen protects male mouse reproductive system from high-level low-dose radiation through reducing spermatogonium apoptosis, adjusting hormone disturbance and ameliorating sperm DNA damage.

Rhodiola polysaccharide effect on spermatogonial stem cell proliferation in vitro / 中国组织工程研究

BACKGROUND:To establish a rapid and effective method to obtain sufficient spermatogonial stem cels that can meet the clinical need is urgent to be solved in the spermatogonial stem cel transplantation. OBJECTIVE:To study the effect of rhodiola polysaccharide on the proliferation of spermatogonial stem celsin vitro. METHODS:Under sterile conditions, spermatogonial stem cels and Sertoli cels were isolated from the testis of mice, and spermatogonial stem cels were seeded onto the feed layer of Sertoli cels. Then, the co-cultured cels were assigned into experimental group 1 (simple cel culture medium), experimental group 2 (cel culture medium containing 150 mg/L rhodiola polysaccharide) and experimental group 3 (cel culture medium containing 150 mg/L rhodiola polysaccharide, 1 U/L leukemia inhibitory factor and 10 μg/L glial cel line-derived neurotrophic factor). After 7 days of co-culture, flow cytometry was used to detect cel proliferation in vitro, and cel viability and positive expression of GFRa-1, Thy-1 and C-kit were calculated. RESULTS AND CONCLUSION:After 7 days of co-culture, the cels grew rapidly and presented with colony and thyrsiform growth, and the number of cel masses increased significantly, al of which were in line with the proliferative features of spermatogonial stem cels. The GFRa-1, Thy-1 and C-kit proteins were expressed in the cel membrane and cytoplasm, mainly in the cel membrane. The viability of spermatogonial stem cels and positive expression of GFRa-1 and Thy-1 were ranked as folows: experimental group 3 > experimental group 2 > experimental group 1, and there were significant differences between groups (P < 0.05). The positive expression of C-kit had no difference between experimental groups 1 and 2, but it was significantly higher in the experimental group 3 than the other two groups (P < 0.05). These findings indicate that rhodiola polysaccharide used alone or combined with leukemia inhibitory factor and glial cel line-derived neurotrophic factor can enhance the proliferative ability of spermatogonial stem celsin vitro.

Spermatogonial stem cells as a therapeutic alternative for fertility preservation of prepubertal boys / Espermatogônias-tronco como uma alternativa para a preservação da fertilidade de meninos pré-púberes

ABSTRACT Spermatogonial stem cells, which exist in the testicles since birth, are progenitors cells of male gametes. These cells are critical for the process of spermatogenesis, and not able to produce mature sperm cells before puberty due to their dependency of hormonal stimuli. This characteristic of the reproductive system limits the preservation of fertility only to males who are able to produce an ejaculate. This fact puts some light on the increase in survival rates of childhood cancer over the past decades because of improvements in the diagnosis and effective treatment in pediatric cancer patients. Therefore, we highlight one of the most important challenges concerning male fertility preservation that is the toxic effect of cancer therapy on reproductive function, especially the spermatogenesis. Currently, the experimental alternative for fertility preservation of prepubertal boys is the testicular tissue cryopreservationfor, for future isolation and spermatogonial stem cells transplantation, in order to restore the spermatogenesis. We present a brief review on isolation, characterization and culture conditions for the in vitro proliferation of spermatogonial stem cells, as well as the future perspectives as an alternative for fertility preservation in prepubertal boys. The possibility of restoring male fertility constitutes a research tool with an huge potential in basic and applied science. The development of these techniques may be a hope for the future of fertility preservation in cases that no other options exist, e.g, pediatric cancer patients.

RESUMO As espermatogônias-tronco, presentes nos testículos desde o nascimento, são as células progenitoras dos gametas masculinos, e, desse modo, críticas para o processo de espermatogênese. Antes da puberdade, essas células não são capazes de produzir espermatozoides maduros, o que só ocorrerá após o estímulo hormonal. Essa característica do sistema reprodutivo limita a possibilidade de preservação da fertilidade apenas para homens capazes de produzir um ejaculado. Tal fato coloca em evidência o aumento nas taxas de sobrevivência de crianças com câncer nas últimas décadas, devido principalmente à melhora no diagnóstico e ao tratamento dos pacientes pediátricos. Dessa forma, destaca-se um dos mais importantes desafios relativos à preservação da fertilidade masculina, que é o efeito tóxico das terapias anticâncer para o sistema reprodutivo, especialmente a espermatogênese. Tendo isso em vista, a alternativa experimental atualmente estudada para a preservação da fertilidade de pacientes pré-púberes é a criopreservação de tecido testicular para futuro isolamento e transplante de espermatogônias-tronco, a fim de restabelecer a espermatogênese. Apresentamos aqui uma breve revisão sobre isolamento, caracterização e condições de cultivo para a proliferação de espermatogônias-tronco, bem como as futuras perspectivas, como alternativa para preservação da fertilidade de meninos pré-púberes. A possibilidade de restabelecer a fertilidade masculina é uma ferramenta de pesquisa com potencial enorme de uso na pesquisa básica e aplicada. O desenvolvimento dessas técnicas pode fornecer uma esperança futura de preservação de fertilidade nos casos em que não há nenhuma outra opção, como para os pacientes pediátricos de câncer.

Conserved expression of ubiquitin carboxyl-terminal esterase L1 (UCHL1) in mammalian testes.

Spermatogonia, the adult germ cells that initiate spermatogenesis in mammalian testis, are capable of dividing both mitotically and meiotically. Isolation and preservation of spermatogonia helps in preserving genetic pool of endangered animals. In this context, identification of marker(s) that can distinguish spermatogonia from other cells in testis gains significance. Here, we examined the expression of ubiquitin carboxyl-terminal esterase L1 (UCHL1) gene and protein in the testes of several mammals, including highly endangered species. Semi-quantitative-reverse transcriptase-polymerase chain reaction (RT-PCR) analysis showed presence of UCHL1 amplicon of 442 bp in all the 18 mammals studied. Nucleotide sequence analysis of these amplicons and their predicted protein sequences revealed 88-99% and 95-100% homology with available human UCHL1 and UCHL1 sequences of other available species in the GenBank, respectively. Western blot analysis showed that UCHL1 protein size was unique in all wild mammals. Immunohistology results confirmed UCHL1 expression in the spermatogonia/gonocytes in testes of several mammals belonging to eight distinct families including highly endangered Felidae, Canidae and Cercopithecoidae. These findings suggest that UCHL1 expression is conserved in the mammalian testis, and could be used as a specific marker for gonocytes/spermatogonia for developing male germ-cell based conservation techniques.

Spermatogonial stem cells and their biological characteristics:current situation, advances and applications / 中国组织工程研究

BACKGROUND:Spermatogonial stem cel s with abilities of differentiation, self renewal and proliferation are a kind of adult stem cel s that can transfer genetic information into offspring, which have great application prospects in medicine, genetics and zoology. OBJECTIVE:To review the source, biological characteristics, and application of spermatogonial stem cel s as wel as self-renewal and molecular regulation underlining these differentiations. METHODS:PubMed and CNKI databases were searched by the first author using key words of“spermatogonial stem cel , biological characteristics, self-renewal, differentiation”in English and in Chinese, respectively, to retrieve relevant articles published from 1990 to 2015. Literatures addressing spermatogonial stem cel s were included, and 46 articles were chosen for further analysis eventual y. RESULTS AND CONCLUSION:Spermatogonial stem cel s can be cultured in vitro, cryopreserved, and genetical y modified as wel as used for al ogeneic or xenogeneic transplantation, al of which contribute to understanding the mechanisms of spermatogenesis, thereby providing new means for treatment of male sterile disease and genetic diseases and providing new hopes for chemotherapy-induced germ cel damage in young cancer patients. Microenvironment and Plzf, GDNF, SCF/c-Kit signaling pathways can play an important role in the regulation of spermatogonial stem cel self-renewal and differentiation. As a cel model, spermatogonial stem cel s become an important tool for the researches on spermatogenesis mechanism, regeneration of spermatogenesis in sterile individuals and reproduction of transgenic animals.

Vascular differentiation of multipotent spermatogonial stem cells derived from neonatal mouse testis

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.

Mouse testicular gene expression pattern differences between spermatogonial stem cell proliferative and differential stages / 中华泌尿外科杂志

Objective To detect the mouse testicular gene expression pattern differences be-tween spermatogonial stem cell (SSC) proliferative and differential stages and study the molecular reg-ulation mechanism in SSC proliferation and differentiation. Methods With the interval of 24 days, male Kunming mice were injected intraperitoneally with two doses of busulfan (10 mg/kg) to establish spermatogenesis regeneration models. 36 k Mouse Genome Array was used to detect the differential gene expression profiles between the stages of SSC proliferation and differentiation. Bioinforrnsties analysis was conducted in GO (gene ontology) and KEGG (Kyoto Encyclopedia of Genes and Ge-nomes) pathway to describe the potential roles that may play in spermatogonial stern cells behavior regulation. Results Nine hundred and eleven differential expression genes were identified by gene arrays in mice testes, consisting of 608 up-regulated and 303 down-regulated in SSC proliferation stage and SSC differentiation stage. The differential expression genes were classified by their biological process, molecular function and cellular component, respectively. Alterations with statistical signifi-cance (P<0.05)appeared in 84 KEG(;signal pathways, including Notch and Wnt signaling pathways which had been proved to be important for stem cell maintenance. Fifty-six differential expression genes were selected as genes related to stem cells, among which 40 genes were up-regulated, including some stem cell biomarkers(such as Cd9, StraS, hgbl-, Oct4 and Thyl)and some growth factors(such as Fgf2, Pdgfa and Csfl). Conclustion The regulation of SSC proliferation and differentiation involves inmany differentially expressed genes in various signal pathways. This study provides a molecular basis for the elucidation of the molecular mechanism behind self-renewal and differentiation of spermatogonial stem cells.

Long-term culture and transplantation of spermatogonial stem cells from BALB/c mice / 中华泌尿外科杂志

Objective To establish a long-term culture system for mouse spermatogonial stem cells(SSCs). Methods Testis cells from 4-6 days postpartum male transgenic BALB/C mice were collected by a modified two-step enzymatic digestion method.After three differential adherence selections,the enriched germ cells were finally suspended in StemPro-34 SFM medium supplemented with other nutrients factors and plated on mouse embryonic fibroblast(MEF)feeder layer.20 ng/ml Glial cell line-derived neurotrophic factor,10 ng/ml basic fibroblast growth factor and 200 ng/ml GDNF-family receptor al were added to the serum-free medium to promote SSCs proliferation.Aduh male BALB/C mice,4-5 weeks old,underwent intraperitoneal injection of 40 mg/kg busulfan as recipient mice.Cultured SSCs were also injected into the seminiferous tubules of the left recipient testis through micromanipulator and right testis as self-control.Testes of recipient mice were observed by a fluorescence stereomicroscope and HE stains at 2 months after transplantation. Results By improved digestion method,the vitality of isolated testis cells was more than 98%and the stem cells was enriched about 18.5 fold. 1-2 days after transferred to MEF feeder, the round germ cells started to proliferate and had the shape of paired or aligned undifferentiated spermatogonia connected by cytoplasmic bridges. After 3-4 days, SSCs proliferated continuously and formed typical colonies. SSCs from BALB/c mice could be cultured and passaged in a steady state for 3 months. Cryostat section through the transplanted testis showed that most of seminiferous tubules were filled with germ cells expressing EGFP.HE staining further showed clearly that seminiferous tubules contained complete spermatogenesis.Conclusions SSCs from BALB/c mice could be cultured in an improved culture system for 3 months.The culture system could facilitate understanding the regulatory mechanism that governs SSCs and might provide an opportunity for the cure of infertility.

The extract of "shoe flower" (Hibiscus rosea sinensis L) leaves inhibit the spermatogenesis of ddy strain mice.

This study was conducted in order to develop male contraception from plants, namely the "shoe flower" (Hibiscus rosea sinensis L) leaves. The objective of this study was to find out whether the extract of "shoe flower" leaves could inhibit the process of spermatogenesis on ddy strain mice. This research was performed in 3 groups and each group consisted of 8 mice. The control group was given 1% carboxy methyl cellulose (CMC) in 0.5 ml aquabides. The treatment group I was given the extract of "shoe flower" leaves 700 mg/kg BW and 1% CMC in 0.5 ml aquabides, and the second treatment group was given the extract of "shoe flower" leaves, 800 mg/kgBW and 1% CMC in 0.5 ml aquabides. The treatment were given for 40 days in accordance with the spermatogenesis cycle. Then, the production of histological slides of the mice testis and the observation of the slides using light microscope with magnification of 100x and 400x were done. Further, counting of the spermatogenic cells was done. At last the pictures of seminiferous tubulus cross-section of the three groups which consisted of spermatogenic cells were taken through light microscope with magnification of 100x and 400x using Fuji camera and Fuji film, 200 ASA. The results showed significant differences between the control, treatment I, and treament II group. There were decreased numbers of spermatogonia, pachyten primary spermatocytes and spermatids in treatment groups (P<0.01). The result of this study showed that the extract of "shoe flower" (Hibiscus rosea sinensis L) leaves, inhibited the process of spermatogenesis of ddy strain mice. It is hoped that the result of this study can be developed into a male contraception.

Successful treatment of unilateral cryptorchid boys risking infertility with LH-RH analogue

INTRODUCTION: Infertility is the primary concern for boys with uni- or bilateral undescended testes. An early and seemingly successful orchiopexy does not improve fertility in a substantial number of cryptorchid males. We confirmed that LH-RH analogue (LH-RHa) treatment induces an increase in and maturation of the germ cells; however, it was uncertain if treatment would improve the chance of fertility later in life. MATERIALS AND METHODS: Thirty unilateral cryptorchid boys, with an average age of 3 years at the time of surgery, were included in the study. Testicular biopsy showed that they had impaired testicular maturation and were therefore at high risk for infertility. Fifteen of the 30 unilateral cryptorchid boys were treated with 10 µg LH-RHa (Buserelin) nasal spray, administered on alternate days for a period of 6 months, following orchiopexy. The control group consisted of 15 cryptorchid boys who had been treated by Schoemakers type of orchiopexy, alone. After puberty, the ejaculates of both groups were analyzed. RESULTS: All males in the untreated group were severely oligospermic, with 20 percent being azoospermic. In contrast, 86 percent of the treated ex-cryptorchid males had a sperm concentration within the normal range; this was significantly different from the sperm concentration found in the untreated group (p = 0.000008). CONCLUSION: For the first time, we demonstrate that infertility in cryptorchidism can be successfully corrected when suitably treated with a LH-RHa. Sperm parameters normalized following therapy in the majority of cryptorchid males who, untreated, would have remained infertile. This innovative hormonal treatment will have a profound effect on the current recommended surgical treatment of boys with undescended testes.