Gene functional research using polyethylenimine-mediated in vivo gene transfection into mouse spermatogenic cells

Aim： To study polyethylenimine （PEI）-mediated in vivo gene transfection into testis cells and preliminary functional research of spermatogenic cell-specific gene NYD-SP12 using this method. Methods： PEI/DNA complexes were introduced into the seminiferous tubules of mouse testes using intratesticular injection. Transfection efficiency and speciality were analyzed on the third day of transfection with fluorescent microscopy and hematoxylin staining. The long-lasting expression of the GFP-NYD-SP12 fusion protein and its subcelluar localization in spermatogenic cells at different stages were analyzed with fluorescent microscopy and propidium iodide staining. Results： With the mediation of PEI, the GFP-NYD-SP12 fusion gene was efficiently transferred and expressed in the germ cells （especially in primary spermatocytes）. Transfection into Sertoli cells was not observed. The subcellular localization of the GFP-NYD-SP2 fusion protein showed dynamic shifts in spermatogenic cells at different stages during spermatogenesis. Conclusion： PEI can efficiently mediate gene transfer into spermatocytes. Thus, it might be useful for the functional research of spermatogenic-cell specific genes such as the NYD-SP12 gene. In our gtudy, the NYD-SP12 protein was visualized and was involved in the formation of acrosome during spermatogenesis. Our research will continue into the detailed function of NYD-SP12 in spermatocytes. （Asian J Androl 2006 Jan; 8： 53-59）

Simulated Microgravity Conditions and Carbon Ion Irradiation Induce Spermatogenic Cell Apoptosis and Sperm DNA Damage

Objective To investigate the effect of simulated microgravity and carbon ion irradiation （CIR） on spermatogenic cell apoptosis and sperm DNA damage to the testis of male Swiss Webster mice, and assess the risk associated with space environment. Methods Sperm DNA damage indicated by DNA fragmentation index （DFI） and high DNA stainability （HDS） was measured by sperm chromatin structure assay （SCSA）. Apoptosis of spermatogenic cells was detected by annexin V-propidium iodide assay. Bax （the expression levels of p53） and proliferating cell nuclear antigen （PCNAI were measured by immunoblotting; p53 and PCNA were located by immunohistology. Results HDS, DFI, apoptosis index, and the expression levels of p53 and Bax were detected to be significantly higher in the experimental groups （P〈0.05） compared with those in the control group, however, the PCNA expression varied to a certain degree, p53- and PCNA- positive expression were detected in each group, mainly in relation to the spermatogonic cells and spermatocytes. Conclusion The findings of the present study demonstrated that simulated microgravity and CIR can induce spermatogenic cell apoptosis and sperm DNA damage. Sperm DNA damage may be one of the underlying mechanisms behind male fertility decline under space environment. These findings may provide a scientific basis for protectint~ astronauts and space traveler＇s health and safety.

LM23 is a novel member of the Speedy/Ringo family at the ：rossroads of life and death of spermatogenic cell

LM23 is a gene specifically expressed in the testis of Rattus norvegicus, as previously reported by our laboratory. The aim of the study is to further investigate the biological function of LM23. Several bioinformatic tools were utilized, including PROSITE and BLAST. To determine the subcellullar localization of LM23, a polyclonal antibody specific for LM23 was generated via the immunization of rabbits. The LM23gene was cloned from rat testis tissue, and LM23 protein was expressed in Escherichia co/i. The biological function of LM23 was analyzed with microarray analysis and immunohistochemistry, using a rat model of LM23 gene knockdown. The results suggested that LM23 belongs to the Speedy/Ringo family. LM23 regulated the GI/S and G2/M transitions of the cell cycle during spermatogenesis. Downregulation of the LM23gene during spermatogenesis could lead to the activation of both the Fas-FasL pathway and the mitochondrial pathway. These novel findings indicate that LM23 has a diverse array of functions that are important in both the life and death of the spermatogenic cell.

Effects of Local Testicular Heating on Bcl-2 and Bax Protein Expression in Spermatogenic Cells in Rats

Objective To investigate the expression of Bcl-2 and Bax in spermatogenic cells induced by local testicular heating in rats Methods Forty adult male SD rats were divided into experimental group and control group at random. According to day 0.5, 1, 3 and 6 after local testicular heating, each group was divided into 4 subgroups： experimental subgroup （n=6） and control subgroup （n=4）. The expression of Bcl-2 and Bax in the spermatogenic cells was detected on day 0.5, 1, 3 and 6 after heat exposure by using immunohistochemistry. Results Compared with control groups, the ratio of positive cells and content of Bcl-2 positive cells significantly decreased in all experimental subgroups （P〈0.01）. The content of Bax positive cells increased in all experimental subgroups （P〈0.01）, the ratio of positive cells which had no significant difference （P〉0.05） except 6 d group decreased （P〈0.01 ）. Redistribution of Bax from a cytoplasmic to perinuclear or nuclear localization could be observed after heating. Conclusions Expression of Bcl-2 would decrease and Bax would increase with redistribution in spermatogenic cells in rats after heating. The change of Bcl-2 and Bax expression in spermatogenic cells would be correlated with the spermatogenic cell apoptosis induced by heating.

Study on the Regulation of Bcl-2 Gene on Rat Spermatogenic Cells Apoptosis in Transcription Level

Objective To detect the change of Bcl 2 gene expression in the apopototic process of spermatogenic cells in rat with vasoligation and vasostomy, and to find out the relationship between the transcription of Bcl 2 and the apoptosis of spermatognic cells Materials & Methods Sixty adult male Sprague Dawley rats in 3 groups were operated with vasoligation and vasostomy. Then hybridization in situ with hypersensitive Bcl 2 RNA probe was used to detect the change of Bcl 2 mRNA. Results The transcription of Bcl 2 gene in spermatogenic cells was obviously inhibited in the vasoligation group compared with that in the control group (P<0.05), and the transcription in the vasostomy group showed no difference from that of the control group. Conclusion Bcl 2 gene has an anti apoptotic effect in rats with vasostomy, and there was a transcriptional regulation of Bcl 2 gene in rat spermatogenic cell during the period of pre vasoligation to post vasoligation and to post vasosotomy.

A Study on Location of Ureaplasma Urealyticum in Spermatogenic Cells

A study on the location of Ureaplasma Urealyticum (UU) in spermatogenic cells in seminal fluid from infertile males was made using rabbit anti-UU antibodies labelled with horse peroxidase (HRP). The results showed that the positive rates of UU in spermatogenic cells were 39.8% (113/284) and 42.2% (35/83)in the infertile males and the males whose wives had history of habitual abortion, respectively. The positive rates of UU were 45.8% (38/83) and 31.6% (30/95)in the patients with positive and negative anti-sperm antibodies, respectively. A high positive rate of UU (73.3%) was found in the infertile males with oligospermia (<20×109/L). The medicines of tetracyclines, such as minomycin, could effectively inhibit the growth of UU, thus converting the UU-positive into negative and improving the patients'fertility.

Cuproptosis mediates copper-induced testicular spermatogenic cell death

Cuproptosis,a novel mechanism of programmed cell death,has not been fully explored in the context of spermatogenic cells.Thisstudy investigated the potential involvement of cuproptosis in spermatogenic cell death using a mouse model of copper overload.Sixty male Institute of Cancer Research(ICR)mice were randomly divided into four groups that received daily oral gavage withsodium chloride(control)or copper sulfate(CuSO_(4))at 50 mg kg^(−1),100 mg kg^(−1),or 200 mg kg^(−1),for 42 consecutive days.Micesubjected to copper overload exhibited a disruption in copper homeostasis.Additionally,significant upregulated expression of keycuproptosis factors was accompanied by a significant rise in the rates of testicular tissue cell apoptosis.Immunohistochemicalanalysis revealed the presence of ferredoxin 1(Fdx1)in Sertoli cells,Leydig cells,and spermatogenic cells at various stages oftesticular development,and the Fdx1-positive staining area was significantly increased in copper-overloaded mice.Mitochondrialdysfunction and decreased adenosine triphosphate levels were also observed,further implicating mitochondrial damage undercuproptosis.Further analyses revealed pathological lesions and blood−testis barrier destruction in the testicular tissue,accompaniedby decreased sperm concentration and motility,in copper-overloaded mice.In summary,our results indicate that copper-overloadedmice exhibit copper homeostasis disorder in the testicular tissue and that cuproptosis participates in spermatogenic cell death.These findings provide novel insights into the pathogenic mechanisms underlying spermatogenic cell death and provide initialexperimental evidence for the occurrence of cuproptosis in the testis.

Urokinase Receptor Gene Expression Inhibited by siRNA Cocktails in Co-culture of Spermatogenic Cells with Sertoli Cells from 3-Week-Old Rat

Objective To study the functions of urokinase receptor （uPAR） during spermatogenesis by adding targeted siRNA cocktails to the co-culture of Sertoli cell with spermatogenic cells. Methods The seminiferous tubule samples from SD rats aged 20-23 d were digested with collagenase for 15-20 rain, then were cut into smaU fragments. Tubular fragments were digested with collagenase again for 5 -10 min, then gently resuspended in F12/ DMEM supplemented with vitamins and hormones. Cell samples were seeded in plate and cultured at 32 ℃ for 2 weeks. After 48 h, siRNA cocktails at a concentration of 15 nmol/L or 30 nmol/L were introduced into this cocultured Sertoli/spermatogenic cells. After then for 48 h, expression of uPAR mRNA was analyzed by real-time RT-PCR. Results Some spermatogenic cells took place morphologic changes and generated an obvious flagellum. The expression levels of uPAR mRNA silencing with siRNA cocktails at a concentration of 15 nmol/L or 30 nmol/L were significantly lower than those in nontransfected group （P〈0.05）; the inhibition rate was 63.5% or 76.7%, respectively. Conclusion The meiosis and spermiogenesis could take place in this culture system, and the siRNA cocktails can effectively inhibit the gene expression of uPAR in co-cultured Sertoli/spermatogenic cells.

Quantitative (stereological) study of incomplete spermatogenic suppression induced by testosterone undecanoate injection in rats

Aim: To evaluate the key lesions in spermatogenesis suppressed partially by testosterone undecanoate (TU) treatment. Methods: Adult male SD rats were treated with vehicle or TU (19 mg/kg) injection (i.m.) every 15 days for 130 days. The numbers of all types of cells (nuclei) in the seminiferous tubules and the interstitial tissue were estimated using a contemporary stereological tool, the optical disector. Results: In response to TU treatment, the numbers of non-type B spermatogonia, type B spermatogonia and late elongated spermatids per testis were reduced to 51 %, 66 % and 14 % of the controls, respectively. The conversion ratios from type B spermatogonia to early spermatocytes and pachytene spermatocytes were not significantly affected and the ratios to the later germ cell types fell to 51 % - 65 % of the controls. Less than 1.0 % of immature round spermatids were seen sloughing into the tubule lumen, 4.0 % of elongated spermatids retained in the seminiferous epithelium, and about half of the elongated spermatid nuclei appreciably malformed. Leydig cells were atrophied but their number and the peritubular myoid cell number per testis were unchanged. Conclusion: Double inhibition of spermatogenesis (i.e. inhibition at spermiation and spermatogonial conversion to type B spermatogonia), a scenario seen in the monkey and human following gona-dotrophin withdrawal, was not sufficiently effective for a complete spermatogenic suppression in the rat after TU treatment, probably due to ineffective inhibition of the Leydig cell population and therefore the intra-testicular testosterone levels.

Percoll fractionation of adult mouse spermatogonia improvesgerm cell transplantation

Aim: To isolate and transplant germ celis from adult mouse testes for transplantation. Methods: In order to distinguish transplanted celis from endogenous celis of recipients, donor transgenic mice expressing green fluores cent protein (GFP) were used. Germ celis 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 previ ously treated with busulfan at 5 weeks of age to remove the endogenous spermatogenic celis. Results: Twenty vveeks 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 celis were transplanted without concentration the success rate was zero (0/9). Conclusion: Germ celis 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.

Disruption of ectoplasmic specializations between Sertoli cells and maturing spermatids by anti-nectin-2 and anti-nectin-3 antibodies

Aim： To understand the biological functions of the ectoplasmic specializations between Sertoli cells and maturing spermatids in seminiferous epithelia. Methods： In order to disrupt the function of the ectoplasmic specializations, nectin-2, which is expressed at the specialization, was neutralized with anti-nectin-2 antibody micro-injected into the lumen of the mouse seminiferous tubule. Anti-nectin-3 antibody was also micro-injected into the lumen in order to neutralize nectin-3, which is expressed at the specialization. Results： The actin filaments at the specialization disappeared, and exfoliation of maturing spermatids was observed by electron microscopy. Conclusion： Nectin-2 was neutralized by anti-nectin-2 antibody and nectin-3 was neutralized by anti-nectin-3 antibody, respectively. Inactivated nectin-2 and nectin-3 disrupted the nectin-afadin-actin system, and finally the actin filaments disappeared. As a result, the specialization lost the holding function and detachment of spermatids was observed. One of the functions of the specialization seems to be to hold maturing spermatids until spermiation.

Bazi Bushen alleviates reproductive aging in aged male mice

Bazi Bushen(BZBS),a traditional Chinese medicine(TCM),has demonstrated therapeutic efficacy in testicular dysfunction within D-galactose and NaNO_(2)mouse models.This study aimed to ascertain if BZBS could also mitigate the decline in testicular function associated with natural aging.Therefore,male aged mice were employed to evaluate the preventive effects of BZBS on male reproductive aging.This was achieved by assessing sex hormone production,testicular histomorphology,and spermatogenesis.Relative to the untreated aged control group,BZBS administration elevated the levels of sex hormones and spermatocyte populations and preserved normal testicular structure in aged mice.Notably,spermatogenesis was maintained.Further analyses,including malondialdehyde(MDA)assays and real-time PCR,indicated that BZBS diminished testicular oxidative stress and the inflammatory burden.Corroborating these findings,mice treated with BZBS exhibited reductions in the populations of senescent and apoptotic cells within the seminiferous tubules,suggesting alleviated cellular damage.In contrast,we observed that rapamycin,a drug known for its longevity benefits,induced excessive testicular apoptosis and did not decrease lipid peroxidation.Collectively,our results highlight BZBS’s promising clinical potential in counteracting male reproductive aging,underlining its mechanisms of action.