Several culture systems such as tissue and organ culture system, seminiferous tubules culture system, co-culture systems of Sertoli cells and germ cells for male germ cell differentiation studies in vitro have been established. The importance of the blood-testis barrier and the polarity of testicular cells have been emphasized recently. And then the bicameral chambers culture system that can mimic the compartmental structure of testes and the calcium algmate culture system that can provide three-dimensional support have been primarily set up. These culture systems are powerful tools for facilitating the understanding to the spermatogenesis. The entire meiotic part of spermatogenesis in some animals has been achieved in vitro by different laboratories, but little is known about the regulation mechanism of the meiotic step of spermatogenesis in detail. This review focuses on the present studies on the differentiation of male germ cells, especially the meiotic step of the differentiation.
Cell Differentiation ; Humans ; Male ; Seminiferous Tubules ; cytology ; Sertoli Cells ; cytology ; Spermatogenesis ; Spermatozoa ; cytology

OBJECTIVETo establish a stable recipient mouse model for stem cell transplantation into seminiferous tubules and improve the traditional techniques for transplantation.

METHODSSixty male ICR mice were equally divided into Groups A, B, C and D, and injected with Busulfan at 15 mg/kg, 30 mg/kg, 40 mg/kg and 0 mg/kg, respectively. The survival rate was recorded every day, and the testis weight and spermatogenesis of testicular tubules were determined at 4, 8 and 12 weeks after the injection. We improved the stem cell transplantation technique and designed a new transplantation device, which connected the nozzle end, syringe and puncture needle by a three-way joint. The nozzle end was used for tentative injection, and the syringe for drawing and then injecting the cell suspension.

RESULTSOnly one mouse in Group C died after the injection. At 4 weeks after Busulfan treatment, the testis weight decreased apparently in Groups A, B and C, with significant differences from D (P < 0.05). The differences remained significant at 8 weeks (P < 0.05), except between Groups A and D (P > 0.05), but at 12 weeks none of the first three groups showed any significant difference from Group D (P > 0.05). At 4 and 8 weeks, the rate of hollow seminiferous tubules was < 50% in Group A and > 50% in Groups B and C, and almost returned to normal at 12 weeks, with no significant differences among the three groups (P > 0.05), but it remained unchanged in Group D. The improved transplantation device increased the success rate (> 90%), lowered the donor cell loss (< 50 microl cell suspension needed for both testes) and shortened the process ( < 10 min for one testis).

CONCLUSIONIntraperitoneal injection of Busulfan at 30 mg/kg is suitable for the establishment of the recipient mouse model of stem cell transplantation. The improved transplantation device and methods help promote the efficiency and success rate of the transplantation operation.

Animals ; Disease Models, Animal ; Male ; Mice ; Mice, Inbred ICR ; Seminiferous Tubules ; cytology ; Stem Cell Transplantation ; methods ; Testis

OBJECTIVETo investigate the ectopic grafts of mouse testicular cells by observing the reconstruction of seminiferous tubules, colonization of spermatogenic cells and spermatogenesis using immunodeficient mice as recipients.

METHODSThe testes of newborn male ICR mice were digested to obtain single cell suspension. The cells were then mixed with matrigel and subcutaneously grafted into the dorsal region of the male nude mice. The mice were castrated after the operation and the grafts were dissected from 5 of the nude mice at 4, 6, 8 and 10 weeks, respectively. The success rates of transplantation and the graft diameters were calculated, and the structure of the reconstituted seminiferous tubules, colonization of the germ cells and spermatogenesis were observed by HE staining and immunohistochemistry.

RESULTSAll the mice recipients survived after the testicular cell transplantation. Within 10 weeks after the operation, tissue masses could be observed, with the diameter increased from (3.91 +/- 0.71) mm at 4 weeks to (6.69 +/- 0.50) mm. Neovascularization was detected at the surface of the masses and seminiferous tubule structures found in the grafts. The germ cells that developed from spermatogonia to round spermatids were observed, but with no sperm in the tubules. Germ cells, Sertoli cells and Leydig cells were identified by immunochemical detection of Mvh, Gata4 and P450Scc in the grafts at 8 weeks.

CONCLUSIONSeminiferous tubules could be ectopically reconstructed from suspension of neonatal mouse testicular cells. Ectopic grafting provided a preferable model for the studies on testis tissue engineering and interactions between testicular cells during testicular development and spermatogenesis.

Animals ; Male ; Mice ; Mice, Inbred BALB C ; Mice, Inbred ICR ; Mice, Nude ; Seminiferous Tubules ; cytology ; Sertoli Cells ; cytology ; transplantation ; Spermatids ; cytology ; Spermatogenesis ; Testis ; cytology ; transplantation ; Transplantation, Heterologous

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

OBJECTIVETo determine whether testosterone-induced intra-testicular testosterone withdrawal and therefore spermatogenic impairment is associated with looser arrangement of spermatogenic cells in rats.

METHODSAdult male SD rats received intramuscular injection of testosterone undecanoate at 19 mg/(kg x 15 d) for 130 days, and then testicular tissue blocks were obtained for the preparation of methacrylate resin-embedded sections and observation of the changes in testicular histology.

RESULTSApart from such changes as impaired spermiogenesis and spermiation, apparently looser arrangement of spermatogenic cells was seen in 11.5% of the seminiferous tubule profiles, with radial cracks (empty spaces) running towards the tubule lumen being formed between lines, bundles or groups of spermatogenic cells (mainly spermatids and spermatocytes).

CONCLUSIONLooser arrangement of spermatogenic cells is one of the key histological changes resulting from intra-testicular testosterone withdrawal in rats.

Animals ; Male ; Rats ; Rats, Sprague-Dawley ; Seminiferous Tubules ; cytology ; drug effects ; Spermatogenesis ; drug effects ; Testis ; cytology ; drug effects ; pathology ; Testosterone ; administration & dosage ; adverse effects

OBJECTIVETo find the expression of specific genes related to the meiosis of germ cells during spermatogenesis in the rat testis.

METHODSSegments of seminiferous tubules were obtained from the adult male SD rats, at stages XIII - I of meiosis, and the interstitial cells of the same testis were isolated under the stereomicroscope. The total RNAs of stages XIII - I segments and the testicular interstitial cells were extracted respectively, and mRNA differential display RT-PCR (DDRT-PCR) was conducted. The obtained cDNA fractions were purified and recovered, the reverse dot blot hybridization, sub-clones and screens of blue/white dots performed, and the results of sub-clones were identified by restriction endonuclease EcoR I digestion.

RESULTSSixteen differential cDNA fractions were obtained through primary DDRT-PCR, 7 from stages XIII - I segments and 9 from the testicular interstitial cells. Another 11 were selected for further screening by reverse dot blot hybridization, their size ranging from 200 to 500 bp, of which 6 were from the stages XIII - I segments of seminiferous tubules and the other 5 from the rat testicular interstitial cells. All of the 11 cDNA fractions were sub-cloned and screened by blue/white dots.

CONCLUSIONSpecifically expressed differential cDNA fractions can be obtained and primarily identified from testicular interstitial cells and the seminiferous tubules, which, as the sequence tags of the testicular meiotic expression, deserve further investigation.

Animals ; Expressed Sequence Tags ; Gene Expression Regulation, Developmental ; Leydig Cells ; cytology ; Male ; Meiosis ; genetics ; Rats ; Rats, Sprague-Dawley ; Reverse Transcriptase Polymerase Chain Reaction ; Seminiferous Tubules ; cytology ; Spermatogenesis ; genetics

AIMTo evaluate the effect of a protein synthesis inhibitor cycloheximide on arresting activity in spermatogenesis and sperm count in male rats.

METHODSThe study used seminiferous tubule (ST) segments from adult rats cultured in vitro with or without cycloheximide to condition culture media, which have been concentrated, size fractioned (30-50 kDa) and administered 7 days to adult rats by intraperitoneal injections. The effects on testicular and epididymal weights, spermatogenesis and epididymal sperm count were determined.

RESULTSThe fraction (30-50 kDa), named arresting, obtained from the culture without cycloheximide decreased testicular and epididymal weights (P<0.01) and reduced the epididymal sperm count significantly. Study of the spermatogenic cycle by transillumination showed spermatogenic arrest at stage VII in rats treated with arresting compared to that observed in controls. The length of stage VII in the group receiving the seminiferous tubules culture media with cycloheximide (30-50 KDa CHX-STCM fraction) was similar to control.

CONCLUSIONThe difference in the effect may be the result of the presence or absence of arresting, a protein secreted by the tubules.

Animals ; Arrestin ; biosynthesis ; Culture Media, Conditioned ; Cycloheximide ; pharmacology ; Epididymis ; cytology ; drug effects ; growth & development ; Male ; Protein Synthesis Inhibitors ; pharmacology ; Rats ; Seminiferous Epithelium ; cytology ; drug effects ; physiology ; Seminiferous Tubules ; metabolism ; Sperm Count ; Testis ; drug effects ; growth & development

AIMTo observe the effect of tamoxifen citrate on spermatogenesis and tubular morphology in rats.

METHODSThe effect of tamoxifen citrate i.g. at doses of 400 and 800 mg.kg(-1).day(-1) in 0.1 mL olive oil for 30 days on seminiferous tubular morphology, seminiferous epithelial diameter (STD), epithelial height (SEH), epididymal sperm count and percent abnormal sperm were evaluated at day 1, 12 and 36 after treatment. Controls were given the vehicle.

RESULTSThe higher dose resulted in tubular atrophy on day 31. The STD, SEH and sperm count were decreased and the abnormal spermatozoa increased in a dose-dependent manner with the maximal effect on day 36.

CONCLUSIONTamoxifen citrate induces tubular shrinkage and atrophy and sperm abnormality at a dose-dependent manner.

Animals ; Epithelial Cells ; cytology ; drug effects ; Male ; Rats ; Seminiferous Tubules ; anatomy & histology ; drug effects ; Sperm Count ; Spermatogenesis ; drug effects ; physiology ; Tamoxifen ; pharmacology

Fragments of testis tissue from immature animals grow and develop spermatogenesis when grafted onto subcutaneous areas of immunodeficient mice. The same results are obtained when dissociated cells from immature testes of rodents are injected into the subcutis of nude mice. Those cells reconstitute seminiferous tubules and facilitate spermatogenesis. We compared these two methods, tissue grafting and cell-injection methods, in terms of the efficiency of spermatogenesis in the backs of three strains of immunodeficient mice, using neonatal porcine testicular tissues and cells as donor material. Nude, severe combined immunodeficient (SCID) and NOD/Shi-SCID, IL-2Rgammacnull (NOG) mice were used as recipients. At 10 months after surgery, the transplants were examined histologically. Both grafting and cell-injection methods resulted in porcine spermatogenesis on the backs of recipient mice; the percentage of spermatids present in the transplants was 67% and 22%, respectively. Using the grafting method, all three strains of mice supported the same extent of spermatogenesis. As for the cell-injection method, although SCID mice were the best host for supporting reconstitution and spermatogenesis, any difference from the other strains was not significant. As NOG mice did not show any better results, the severity of immunodeficiency seemed to be irrelevant for supporting xeno-ectopic spermatogenesis. Our results confirmed that tubular reconstitution is applicable to porcine testicular cells. This method as well as the grafting method would be useful for studying spermatogenesis in different kinds of animals.
Animals ; Cell Transplantation ; methods ; Injections ; Male ; Mice ; Mice, Inbred BALB C ; Mice, Inbred NOD ; Mice, Nude ; Mice, SCID ; Organ Size ; Seminiferous Tubules ; cytology ; physiology ; transplantation ; Spermatids ; cytology ; transplantation ; Spermatogenesis ; physiology ; Subcutaneous Tissue ; surgery ; Swine ; Tissue Transplantation ; methods ; Transplantation, Heterologous ; methods

AIMTo evaluate the effect of tamoxifen citrate on male reproductive system of rat.

METHODSGroups of male rats were gavaged with tamoxifen at doses of 200 mg x kg(-1) x d(-1), 400 mg x kg(-1) x d(-1) or 800 mg x kg(-1) x d(-1) in 0.1 mL olive oil for 10 consecutive days. Controls were treated with 0.1 mL olive oil. Rats were anesthetized and killed on d 3, d 15 or d 35 after the last dose. Testes were collected, processed for paraffin embedding, sectioned at 5 microm thickness, stained with HE and analyzed microscopically.

RESULTSThere was a dose-dependent increase in the occurrence of seminiferous tubular distortion with germinal cell sloughing. The highest dose increased the number of multinucleated giant cells on d 3 and d 15.

CONCLUSIONTamoxifen citrate induces multinucleated giant cells and germinal epithelial sloughing in a dose-dependent manner and these changes are detrimental to male fertility.

Animals ; Antineoplastic Agents, Hormonal ; toxicity ; Dose-Response Relationship, Drug ; Giant Cells ; drug effects ; ultrastructure ; In Vitro Techniques ; Male ; Rats ; Rats, Wistar ; Seminiferous Tubules ; cytology ; drug effects ; Tamoxifen ; toxicity ; Testis ; cytology ; drug effects