OBJECTIVETo perform a preliminary study on the dysfunction of spermatogonial stem cells (SSCs) induced by cyclophosphamide.

METHODSAccording to development stage of spermatogenesis in Wistar rat, 72 rats were divided into three groups, 1, 3 and 9 weeks groups, respectively. Then 24 rats per group were randomly divided into experimental and control groups, and 100 mg/kg cyclophosphamide was injected by intraperitoneal injection in experimental groups, with the same volume of normal saline in the control. Apoptosis of spermatogenic cells were detected after 24 hours by TUNEL. Then 60 one-week rats, whose germ cells were only SSCs, were randomly divided into experimental and control group. We detected c-Kit by immunohistochemistry and cell cycle by flow cytometry at 24 hours, 3 and 9 weeks.

RESULTSThere was no significant difference in apoptosis of germ cells in 1 week between experimental group and control group ( P > 0.05); however, there were significant differences among other groups. The ratio of S stage and c-Kit expression of spermatogonial cells were decreased in one-week rats at 24 hours, 3 and 9 weeks in experimental group compared with the control, respectively (P < 0.01).

CONCLUSIONCyclophosphamide does not significantly induce SSCs apoptosis. It may be more important to interfere the proliferation and differentiation of SSCs.

Animals ; Apoptosis ; drug effects ; Cell Differentiation ; drug effects ; Cell Proliferation ; drug effects ; Cells, Cultured ; Cyclophosphamide ; pharmacology ; Male ; Proto-Oncogene Proteins c-kit ; metabolism ; Random Allocation ; Rats ; Rats, Wistar ; Spermatogonia ; cytology ; drug effects ; metabolism ; Stem Cells ; cytology ; drug effects

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

OBJECTIVETo study the effects of growth factors on the survival and proliferation of human spermatogonial stem cells (SSCs) in vitro.

METHODSSSCs were treated with the growth factors SCF, LIF and bFGF added to the culture, each at the concentrations of 0, 5, 10 and 20 microg/L and repeated three times. The survival time and proliferation rate of the cells were determined every 8-12 hours and their morphological features observed with the light microscope and electron microscope.

RESULTSThe survival time and proliferation rate of the SSCs were significantly increased in the treated groups as compared with the control (P < 0.05).

CONCLUSIONThe growth factors SCF, LIF and bFGF can promote the survival and proliferation of SSCs in vitro.

Cell Proliferation ; drug effects ; Cell Survival ; drug effects ; Cells, Cultured ; Fibroblast Growth Factor 2 ; pharmacology ; Humans ; Intercellular Signaling Peptides and Proteins ; pharmacology ; Leukemia Inhibitory Factor ; pharmacology ; Male ; Spermatogonia ; cytology ; drug effects ; Stem Cell Factor ; pharmacology ; Stem Cells ; cytology ; drug effects

OBJECTIVETo explore the role of neuregulin (neural regulation of protein, NRG) in the process of mouse spermatogonia proliferation.

METHODSMouse testis fragments were cultured in the medium DMEM containing purified NRG1beta or NRG3 at the concentration of 50, 100 and 200 ng/ml, respectively, followed by BrdU immunohistochemical staining and determination of the proliferation rate of spermatogonia.

RESULTSCompared with the control group, neuregulin significantly promoted the proliferation of spermatogonia (P < 0.05). The proliferation rates of spermatogonia cultured in the medium with 50, 100 and 200 ng/ml of NRG13 were 1.69, 1.55 and 1.86 times, and those with 50, 100 and 200 ng/ml of NRG3 were 1.35, 1.54 and 2.11 times that of the control.

CONCLUSIONNRG1beta and NRG3 can promote the proliferation of mouse spermatogonia, and NRG is expected to be applied in the treatment of male infertility.

Animals ; Cell Proliferation ; drug effects ; Cells, Cultured ; Intracellular Signaling Peptides and Proteins ; pharmacology ; Male ; Mice ; Mice, Inbred C57BL ; Neuregulin-1 ; pharmacology ; Signal Transduction ; Spermatogonia ; cytology ; metabolism

OBJECTIVE: To determine the molecular mechanism of germ cell apoptosis via investigating the effect of PFT-α on the expression of p53 and bcl-2/bax during experimental cryptorchid cell apoptosis. METHODS: Male Sprague-Dawley rats were assigned into 4 groups: a sham-operated group, a cryptorchid group, a cryptorchid+p53 inhibitor (p53 inhibitor-alpha, PFT-α) group, and a cryptorchid+dissolvent of PFT-α [dimethyl sulphoxide (DMSO)] group. Unilateral cryptorchidism was surgically induced in the rats of the cryptorchid group, PFT-α group, and cryptorchid+dissolvent of PFT-α group. The rats in the PFT-α group and cryptorchid+dissolvent of PFT-α group were intra-peritoneally injected PFT-α and dissolvent of PFT-α, respectively, once a day. The rats were killed on the 7th day after the surgery. The morphology of spermatogenic epithelium at the side of surgery in the rats was observed under light microscope. The apoptosis of spermatogenic cells in the unilateral cryptorchidism was evaluated by TUNEL and flow cytometry (FCM). The protein expression levels of p53, bcl-2, and Bax were detected by Western blot and immunohistochemical assay in turn. RESULTS: Compared with the cryptorchid groups and the cryptorchid+dissolvent of PFT-α group, the seminiferous epithelium of the cryptorchid+p53 inhibitor group appeared orderly, with thicker cell layers and lower apoptosis index, weak protein expression level of p53/Bax and strong protein expression level of bcl-2. CONCLUSION PFT-α inhibits the germ cell apoptosis caused by the experimental cryptorchidism via increasing the expression of bcl-2 and decreasing the expression of p53 and bax.
Animals ; Apoptosis ; Benzothiazoles ; pharmacology ; Cryptorchidism ; pathology ; Disease Models, Animal ; Humans ; Male ; Rats ; Rats, Sprague-Dawley ; Spermatogonia ; cytology ; drug effects ; Toluene ; analogs & derivatives ; pharmacology

OBJECTIVETo investigate the occurrence of germ cell apoptosis and the expression of Bcl-2/Bax after aniso-doses arsenic (As2O3) administration in the testes of the adult male rats.

METHODForty healthy male Sprague-Dawley rats were divided randomly into four groups and were administered respectively with 0 (control group), 0.375, 0.75, 1.5 mg x kg(-1) of As2O3 by intragastric administration consecutively for 16 weeks. The numbers of testicular sperm head were counted and the coefficient of testicular viscera and daily sperm production (DSP) were calculated in every group. The apoptosis of germ cell were assessed by in situ terminal deoxynucleotityl transferase mediated dTUP nick end labeling (TUNEL) technique. The expression of Bel-2/Bax in spermatogenic cells of different grades were located and quantitated by the method of immunohistochemistry.

RESULT(1) In the testes of As2O3-treated rats, the coefficient of testicular viscera, the number of testicular sperm head and DSP in the middle and high dose groups decreased significantly, compared with those in the control group (P < 0.01); however the apoptosis index of germ cell (AI) significantly increased compared with that in the control group (P < 0.01). Bcl-2 expression of middle dose group and high dose group decreased significantly compared with that in the control group (P < 0.01). Bax expression of middle dose group and high dose group increased significantly compared with that in the control group (P < 0.01); (2)The dependability analysis between DSP and AI showed a negative correlation (r = -0. 563, P <0. 01). (3) The negative correlation was significant between AI and Bcl-2 expression of spermatogenic cells (r = -0.825, P < 0.01); The positive correlation was significant between AI and Bax expression of spermatogenic cells (r = 0.710, P < 0.01).

CONCLUSIONOne of the mechanisms of male reproduction toxicity of As2O3 might be that Bcl-2 expression is inhibited and Bax expression is encouraged which induces the decrease of quantity of sperm cells.

Animals ; Apoptosis ; drug effects ; Arsenicals ; pharmacology ; Gene Expression ; drug effects ; Male ; Oxides ; pharmacology ; Proto-Oncogene Proteins c-bcl-2 ; genetics ; metabolism ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Spermatogonia ; cytology ; drug effects ; metabolism ; bcl-2-Associated X Protein ; genetics ; metabolism

AIMTo investigate whether estrogen stimulates the proliferation of spermatogonia or induces spermatogenesis in cryptorchid mice.

METHODSMice were surgically rendered cryptorchid, then treated with different doses of 17beta-estradiol (E2) s.c. once a day. Mice were killed at sexual maturity (45 days of age), and histological analysis and immunofluorescence were performed. Serum follicle stimulating hormone (FSH), estradiol, testosterone and luteinizing hormone (LH) were measured.

RESULTSLow doses of E2 had no notable effect on spermatogonia, but at higher doses, E2 stimulated the proliferation of spermatogonia.

CONCLUSIONE2 has a dose-related mitogenic effect on spermatogonia.

Animals ; Cell Division ; drug effects ; Cryptorchidism ; physiopathology ; Disease Models, Animal ; Estradiol ; blood ; pharmacology ; Follicle Stimulating Hormone ; blood ; Luteinizing Hormone ; blood ; Male ; Mice ; Spermatogonia ; cytology ; drug effects ; pathology ; Testosterone ; blood

An ideal animal model of azoospermia would be a powerful tool for the evaluation of spermatogonial stem cell (SSC) transplantation. Busulfan has been commonly used to develop such a model, but 30%-87% of mice die when administered an intraperitoneal injection of 40 mg kg^-1. In the present study, hematoxylin and eosin staining, Western blot, immunofluorescence, and quantitative real-time polymerase chain reaction were used to test the effects of busulfan exposure in a mouse model that received two intraperitoneal injections of busulfan at a 3-h interval at different doses (20, 30, and 40 mg kg^-1) on day 36 or a dose of 40 mg kg^-1 at different time points (0, 9, 18, 27, 36, and 63 days). The survival rate of the mice was 100%. When the mice were treated with 40 mg kg^-1 busulfan, dramatic SSC depletion occurred 18 days later and all of the germ cells were cleared by day 36. In addition, the gene expressions of glial cell line-derived neurotrophic factor (GDNF), fibroblast growth factor 2 (FGF2), chemokine (C-X-C Motif) ligand 12 (CXCL12), and colony-stimulating factor 1 (CSF1) were moderately increased by day 36. A 63-day, long-term observation showed the rare restoration of endogenous germ cells in the testes, suggesting that the potential period for SSC transplantation was between day 36 and day 63. Our results demonstrate that the administration of two intraperitoneal injections of busulfan (40 mg kg^-1 in total) at a 3-h interval to mice provided a nonlethal and efficient method for recipient preparation in SSC transplantation and could improve treatments for infertility and the understanding of chemotherapy-induced gonadotoxicity.
Adult Germline Stem Cells/transplantation* ; Animals ; Azoospermia/chemically induced* ; Busulfan/toxicity* ; Disease Models, Animal ; Infertility, Male/chemically induced* ; Injections, Intraperitoneal ; Male ; Mice ; Spermatogenesis/drug effects* ; Spermatogonia/drug effects* ; Stem Cell Transplantation/methods*

Zearalenone (ZEA), a nonsteroidal estrogenic mycotoxin, is known to cause testicular toxicity in animals. In the present study, the effects of ZEA on spermatogenesis and possible mechanisms involved in germ cell injury were examined in rats. Ten-week-old Sprague-Dawley rats were treated with 5 mg/kg i.p. of ZEA and euthanized 3, 6, 12, 24 or 48 h after treatment. Histopathologically, spermatogonia and spermatocytes were found to be affected selectively. They were TUNEL-positive and found to be primarily in spermatogenic stages I-VI tubules from 6 h after dosing, increasing gradually until 12 h and then gradually decreasing. Western blot analysis revealed an increase in Fas and Fas ligand (Fas-L) protein levels in the ZEA-treated rats. However, the estrogen receptor (ER)alpha expression was not changed during the study. Collectively, our data suggest that acute exposure of ZEA induces apoptosis in germ cells of male rats and that this toxicity of ZEA is partially mediated through modulation of Fas and Fas-L systems, though ERalpha may not play a significant role.
Animals ; Antigens, CD95/*immunology ; Apoptosis/*drug effects/immunology ; Estrogens, Non-Steroidal/*toxicity ; Fas Ligand Protein/*immunology ; Histocytochemistry ; Immunoblotting ; In Situ Nick-End Labeling ; Male ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Spermatocytes/cytology/*drug effects/immunology ; Spermatogenesis/drug effects/immunology ; Spermatogonia/drug effects/immunology ; Testis/cytology/*drug effects/immunology ; Zearalenone/*toxicity

The present study identified the favorable environment conditions for Spermatogomial stem cells in vitro according to their unique biological properties. Three growth factors, stem cell factor (SCF), leukemia inhibitory factor (LIF) and basic fibroblast growth factor (bFGF) were all found to independently contribute to the proliferation of mouse spermatogonial stem cell. The percentage of cell proliferation significantly enhanced by SCF at 30 ng/mL but decreased with heightening its combination after cultured 120 hours. The mice spermatogonial stem cells were significantly proliferated after 120 hours' culture with 10 ng/mL and 20 ng/mL (P < 0.01) of LIF, between 20 ng/mL and 50 ng/mL (P < 0.01) for bFGF. SCF and bFGF were significantly enhanced mice spermatogonial stem cells proliferation after these three factors combination. For LIF, no obvious effect was observed.
Animals ; Cell Division ; drug effects ; Cells, Cultured ; Dose-Response Relationship, Drug ; Fibroblast Growth Factor 2 ; pharmacology ; Growth Inhibitors ; pharmacology ; Interleukin-6 ; Leukemia Inhibitory Factor ; Lymphokines ; pharmacology ; Male ; Mice ; Spermatogonia ; drug effects ; physiology ; Stem Cell Factor ; pharmacology ; Stem Cells ; drug effects ; physiology