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Objective: To explore the change in the proliferation of spermatogenic cells in the male mouse with infertility induced by exogenous estradiol benzoate (EB)., Methods: Sixty male mice aged 4 weeks were randomly divided into a control, a low-dose EB, and a high-dose EB group to be injected intramuscularly with corn oil at 150 μl or EB at 5 or 10 mg/kg, respectively, every other day for 4 weeks. Then, we obtained the weight and indexes of the testis, performed HE staining of the paraffin sections of the testis tissue and epididymal cauda, counted the spermatozoa in the epididymal sperm suspension, and determined the expression of the proliferating cell nuclear antigen (PCNA), the mRNA expressions of CyclinA1, CyclinB1, VASA and p53, and the protein expressions of p53 and phosphorylated p53 in the testis by immunohistochemistry, qRT-PCR and Western blot, respectively., Results: In comparison with the controls, the mice treated with EB showed significantly decreased testicular indexes (P <0.05), no sperm in the sperm suspension or epididymal tubes, remarkably reduced numbers of spermatogonia, primary spermatocytes and Sertoli cells (P <0.05), down-regulated expression of PCNA (P <0.05) and mRNA expressions of CyclinA1, CyclinB1, PCNA and VASA in the seminiferous tubules (P <0.05), but a dose-dependent increase of the p53 level (P <0.05). Western blot revealed markedly higher levels of p53 protein expression and phosphorylation in the EB than in the control group (P <0.05) and even higher in the 10 mg/kg than in the 5 mg/kg EB group (P <0.05)., Conclusions: EB inhibited the proliferation of spermatogenic cells by down-regulating the expressions of cell cycle-related factors in a dose-dependent manner, which might contribute to abnormal proliferation of spermatogenic cells in the testis of infertile male mice.

Objective: To investigate the effects of cynomorium songaricum (CS) decoction on the testis weight, serum testosterone level, and sperm parameters of rats with oligoasthenospermia (OAS), explore its action mechanism of improving the proliferation of undifferentiated spermatogonial cells, and provide some experimental and theoretical evidence for the development of new Chinese drugs for OAS., Methods: Thirty 8-week-old male SD rats were randomly divided into five groups of equal number: blank control, model control, high-dose CS, medium-dose CS, and low-dose CS. OAS models were established by intraperitoneal injection of cyclophosphamide and, a month later, treated intragastrically with normal saline or CS at 2, 1, and 0.5 g per kg of the body weight per day, all for 4 weeks. Then, the testes of the animals were harvested to obtain the testicular weight, sperm concentration and motility, and the level of serum testosterone (T), detect the expressions of the transcription factor 1 (Oct4), Thy-1 cell surface antigen (Thy1), promyelocytic leukemia zinc finger (PLZF), KIT proto-oncogene receptor tyrosine kinase (C-kit) and glial cell-derived neurotrophic factor (GDNF) in the testis tissue of the rats in the low-dose CS group by real-time PCR., Results: The testis weights in the blank control, model control, high-dose CS, medium-dose CS, and low-dose CS groups were (1.52±0.06), (1.55±0.06), (1.43±0.30), (1.35±0.40) and (1.34±0.04) g, respectively, not significantly different in the blank and model controls from those in the CS groups (P>0.05). The visual field sperm count per 10 HP was significantly increased in the high-, medium-, and low-dose CS groups (202±20, 196±5 and 216±25) as compared with the blank and model controls (200±15 and 134±30) (P<0.05). The mRNA expressions of the Oct4, Thy1, PLZF and GDNF genes were remarkably higher in the low-dose CS group than in the controls (P<0.05), but that of the C-kit gene showed no significant difference from the latter (P>0.05). The visual field sperm motility per 10 HP was markedly increased in the blank control (［52.1±5.5］%), model control (［38.1±2.5］%), high-dose CS (［59.1±9.5］%), medium-dose CS (［58.7±9.5］%), and low-dose CS (［49.6±1.0］%) groups, and so was the level of serum testosterone (［190±87.5］, ［82.5±25.8］, ［229±75.6］, ［331±86.7］ and ［185±82.4］ mmol/L), both remarkably higher in the CS groups than in the model controls (P<0.05) but with no statistically significant difference between the CS groups and the blank controls (P>0.05)., Conclusions: CS can significantly improve sperm concentration, sperm motility and serum T level in OAS rats, probably by inducing the expression of GDNF in the rat Sertoli cells, promoting the proliferation of undifferentiated spermatogonial cells, and enhancing spermatogenesis.

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.

Objective: To investigate the regulation of T3 on the maldeveloped gonocytes (Go) and the expression of NCAM in Flutamide-induced cryptorchidism SD rat., Methods: Postnatal day (PD) 1 and PD20 cryptorchidism SD rat induced by Flu were treated with 15 microg/(kg x d) T3 by subcutaneous injections. Incidence of the cryptorchidism, histology were evaluated. And the expression of NCAM was detected by immunohistochemisty and RT-PCR., Results: The incidence of cryptorchidism in the group of Flu-induced offsprings treated by 15 microg/(kg x d) T3 from PD1 was 25% (8/32), which was lower than that of Flu group 40.9% (9/22). No Go and NCAM expression were observed in the seminiferous tubules. NCAM mRNA expression in Flu-induced PD13, PD20 testes was up-regulated when compared with control (P < 0.01, P < 0.05), while after treated with 15 microg/ (kg x d) T3 the NCAM expression was significantly down-regulated compared to that of non T3 treated group (P < 0.01), and was not significantly different from that of control (P > 0.05)., Conclusion: The incidence of Flu-induced cryptorchidism can be decreased by T3 treatment after birth. T3 may contribute to the down-regulation of the NCAM expression, and reduction or elimination of the remainded gonocytes in crytorcidism seminiferous tubules.

Objective: To research the effect of fluorine on the expression of Fas protein, then study the mechanism of male reproductive toxicity induced by fluoride on molecular level., Methods: Thirty Wistar male rats were divided into control group, low-dose group and high-dose group. The NaF dosage for every group were 0,2 and 4g/L. The content of NaF in testis was measured by using fluorine selective electrode. Changes of testosterone and Fas protein were observed using the methods of radioimmunoassay, in situ hybridization. In addition, we observed the quality of spermatozoa., Results: The testis fluoride content of two fluorine treatment groups were higher than that of control group (P < 0.05), and had a dose-dependent effect. The level of testosterone, the number and the livability of the spermatozoon in fluorotic groups were lower than those of control rats (P < 0.05), and the above indexes decreased with the incrase of dosage. The expression of Fas in spermatogenic cells and the sperm aberration of each fluorotic group were higher than control group (P < 0.05), both of them increased with the increase of dosage., Conclusion: Fluorin could reduce the level of serum testosterone, then activated the Fas/FasL system, which caused damage to the reprodutive system.

Objective: To 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., Method: Forty 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)., Conclusion: One 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.

Objective: To study the effects of growth factors on the survival and proliferation of human spermatogonial stem cells (SSCs) in vitro., Methods: SSCs 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., Results: The survival time and proliferation rate of the SSCs were significantly increased in the treated groups as compared with the control (P < 0.05)., Conclusion: The growth factors SCF, LIF and bFGF can promote the survival and proliferation of SSCs in vitro.

Objective: To perform a preliminary study on the dysfunction of spermatogonial stem cells (SSCs) induced by cyclophosphamide., Methods: According 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., Results: There 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)., Conclusion: Cyclophosphamide does not significantly induce SSCs apoptosis. It may be more important to interfere the proliferation and differentiation of SSCs.

Objective: To observe the relationship between spermatogenic cell apoptosis and serum estradiol level in rats exposed to fluoride., Methods: A total of 30 male Wistar rats were allocated into six groups randomly. The six experimental groups were 28-day control group, 28-day low-dose fluoride treatment group, 28-day high-dose fluoride treatment group, 38-day control group, 38-day low-dose fluoride treatment group, and 38-day high-dose fluoride treatment group. The fluorosis model was acquired by subcutaneous injection of NaF solution. The content of NaF in testis was measured by using fluorine selective electrode. The serum estradiol level was radioimmunochemically detected. And the apoptotic spermatogenic cells were quantitatively measured by TUNEL., Results: The content of NaF in testis and the ratio of apoptotic spermatogenic cell in fluoride treatment groups significantly increased with increased experimental dosage and prolonged experimental period (P < 0.05). Meanwhile, the serum estradiol level significantly decreased (P < 0.05), which was negatively correlated with the content of NaF in testis as well as the ratio of apoptotic spermatogenic cell (P < 0.05)., Conclusion: Excessive fluoride could lead disturbance to serum estradiol level during some range of dose and time, which is an important factor to spermatogenic cell apoptosis.

Objective: To evaluate the effects of administration of perfluorooctane sulfonate(PFOS) on spermiogenesis function of male rats., Methods: 36 male rats were randomly divided into 4 groups, which received 0, 0.5, 1.5, 4.5 mg x kg(-1) PFOS by food intake per day for 65 days. The testicular and epididymal viscera coefficients, the number, motility and deformity of sperm were examined. The activities of lactate dehydrogenase isoenzyme-x (LDHx), sorbitol dehydrogenase (SDH) and the generation of malonyldialdehyde (MDA) in the testes were also measured., Results: The viscera coefficients did not show any significant change ( P > 0.05) while the body weight and weight of testis decreased ( P < 0.05) in treated rats compared with the corresponding control group animals. In 1.5,4.5 mg x kg(-1) PFOS treated rats there were significant decreases in the sperm count (P < 0.05) and the mean activities of LDHx and SDH whereas obvious increases in the rate of sperm deformity ( P < 0.05). In 4.5 mg x kg(-1) PFOS group the generation of MDA increased (P < 0.05) while the motility of sperm reduced (P < 0.05) with respect to the control value., Conclusion: It suggested that PFOS could elicit the impairment of sperm production and maturation of male rats.

Objective: To observe the effect of acrylonitrile on the spermatogenesis in mice., Methods: 50 male pubertal mice and 25 male adult mice were exposed to acrylonitrile (1.25, 2.5, 5 mg/kg) by intraperitoneal injection for 5 days, and detected the DNA content and cell period of spermatogenic cells by flow cytometry after 35 days., Results: (1) The percentage of 1C cell in the exposed pubertal mice groups was lower than in the control group, respectively. The percentage of 1C cell in the exposed adult mice groups was lower than in the control group, respectively (P < 0.05). The percentage of apoptotic cell in pubertal and adult mice was higher than in the control group, respectively (P < 0.05). (2) The percentage of cells in S-phase in the exposed pubertal mice groups was higher than in the control group, respectively (P < 0.05), G0/G1 phase were lower(P < 0.05). The percentage of cells in G2/M phase in the exposed adult mice groups was higher than in the control group, respectively(P < 0.05), G0/G1 phase was lower(P < 0.05)., Conclusion: There are restrained effects of acrylonitrile on the spermatogenesis in mice.

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.

Objectives: To study the change of telomerase activity in rat spermatogonia when the telomerase RNA was enclosed, and reactivity of the change to cytokine(SCF, TGF-beta 1)., Methods: The antisense oligonucleotides(PS-ASON) of telomerase was transfected into proliferating spermatogonia in vitro with the liposomes as the vector. Then the cytokine, stem cell factor (SCF) or transforming growth factor-beta 1(TGF-beta 1), was added. The proliferative activity of the spermatogonia was determined before and after the inhibition by MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide]assay. The change of telomerase activity was detected by telomeric repeat amplification protocol (TRAP)., Results: 1 mumol/L PS-ASON obviously downregulated the telomerase activity and inhibited spermatogonia proliferation. When the inhibition was over, the activity recovered to some extent(P < 0.01). Growth factors can regulate the spermatogonia after inhibition, SCF may improve the activity of telomerase and the proliferation of spermatogonia. Adversely, TGF-beta 1 may inhibit the recovery of telomerase activity., Conclusions: To inhibit spermatogonia telomerase activity antisensely can limit the proliferation of spermatogonia efficiently, which was regulated by cytokine. This method might be a new and efficient way in male birth control.

To confirm the claims that the procarbazines (PCZ) effect on stem spermatogonia can be overcome by gonadal steroid hormones,mature,LBNF1 male rats were implanted with silastic capsules containing testosterone (T 200 mm) + estradiol (E 50 mm). The silastic capsules of control and TE groups were removed 24 hours after the PCZ injection and the rats were sacrificed 9 weeks later. The functional status of the testis and the hormonal parameters at the time of the injection of PCZ were measured and correlated with the degree of protection ,as measured by the recovery of sperm counts. The results indicated that testosterone and estradiol does protect spermatogenesis from PCZ intoxification. To determine whether the protection is specific for stem spermatogonia, we also measured body weight loss 3-4 days, lymphocyte counts 1 day, and spermatocyte count 9 days after PCZ treatment. There were no differences in the dose response curves of these three assays between the hormone and sham treated rats. The lack of protection indicates that the hormonal protection of stem spermatogonia was not due to the alterations in either systemic or testicular pharmacokinetics of spermatocyte. We conclude that A3 spermatogonia and preleptotene spermatocyte are sensitive to PCZ and there is no evidence of hormonal protection for the differentiated spermatogonia and preleptotene spermatocyte.