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 PLZF^highc-KIT^pos in A₁ spermatogonia, while A₂-A₄-differentiating spermatogonia were PLZF^lowc-KIT^pos. Additionally, this procedure was used to examine asymmetrically expressing GFRA1 and PLZF clones, asymmetric A_pr 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.
Animals ; Cell Differentiation ; Fluorescent Antibody Technique ; Gene Expression Regulation/genetics* ; Glial Cell Line-Derived Neurotrophic Factor Receptors/genetics* ; Immunohistochemistry ; Male ; Mice ; Mice, Inbred C57BL ; Promyelocytic Leukemia Zinc Finger Protein/genetics* ; Proto-Oncogene Proteins c-kit/genetics* ; Seminiferous Tubules/cytology* ; Spermatogenesis ; Spermatogonia/metabolism* ; Testis/cytology* ; Tissue Fixation ; Transcription Factors/genetics*

Objective: To investigate the role of pH2AX in the reversibility of mouse testicular reproductive function impaired by single heat stress. METHODS: Twenty-four C57 male mice were randomly divided into heat stress and control groups and immersed in water at 43℃ and 25℃, respectively, for 15 minutes. At 1, 7, and 14 days of heat exposure, all the mice were sacrificed and their testis tissues collected for determining the apoptosis of the germ cells by TUNEL and measuring the expression level of the pH2AX protein by immunohistochemistry and Western blot. RESULTS: The highest percentage of apoptotic cells were found in the seminiferous tubules of the mice in the heat stress group on the 1st day of the exposure and almost no apoptosis was observed at 7 and 14 days. The pH2AX protein was expressed in the nuclei of the basement membrane of adjacent seminiferous tubules. Compared with the control group, the expression of pH2AX was significantly increased on the 1st day of exposure (0.47 ± 0.02 vs 1.61 ± 0.04, P <0.01), then decreased at 7 days (0.85 ± 0.03) in comparison with that on the 1st day (P <0.01), and again elevated at 14 days (1.72 ± 0.02) as compared with either those at 1 and 7 days (P <0.01) or that of the control (P <0.01). CONCLUSIONS Heat stress causes dynamic changes of the pH2AX expression in the testis of the mouse, which are associated with heat stress-induced proliferation and division of the testicular spermatogenic cells.
Animals ; Apoptosis ; Blotting, Western ; Heat Stress Disorders ; complications ; Histones ; metabolism ; Hot Temperature ; Immunohistochemistry ; In Situ Nick-End Labeling ; Male ; Mice ; Mice, Inbred C57BL ; Random Allocation ; Seminiferous Tubules ; cytology ; Spermatozoa ; cytology ; metabolism ; Testis ; Time Factors

OBJECTIVETo study on the expression patterns of proteins associated with cell junctions in the developing mouse testes.

METHODThe expression levels of reproductive related cell lines spermatogonia cell line GC1 spg, spermatocyte cell line GC2 spg, leydig cell line TM3, and sertoli cell line TM4, primary sertoli cells, and 1-6-week mouse testes were analyzed using Western blot.

RESULTSThe sertoli cell junction-associated membrane proteins adhesion molecule A, Occludin and Claudin, and the sertoli-germ cell junction-associated membrane proteins junctional adhesion molecule C, Nectin-3, and E-cadherin were stage-specific in the seminiferous tubules in the mouse testes. The adaptor proteins associated with cell juctions zonula occludens-1, zonula occludens-2, Afadin, Β-catenin, and CD2-associated protein were not stage-specific in the seminiferous tubules in the mouse testes.

CONCLUSIONSIn the seminiferous tubules in the mouse testes, the membrane proteins associated with cell junctions are stage-specific. However, the expressions of adaptor proteins associated with cell junctions do not obviously change.

Adaptor Proteins, Signal Transducing ; metabolism ; Animals ; Cdh1 Proteins ; metabolism ; Cell Adhesion Molecules ; metabolism ; Cell Line ; Cytoskeletal Proteins ; metabolism ; Humans ; Intercellular Junctions ; metabolism ; Male ; Membrane Proteins ; metabolism ; Mice ; Microfilament Proteins ; metabolism ; Nectins ; Seminiferous Tubules ; cytology ; metabolism ; Sertoli Cells ; cytology ; Testis ; cytology ; Zonula Occludens-1 Protein ; metabolism ; Zonula Occludens-2 Protein ; metabolism ; beta Catenin ; metabolism

OBJECTIVETo investigate the effects of single heat stress treatment on spermatogenic cells in mice.

METHODSWe randomly divided 36 C57 male mice into a control and a heat stress treatment group and submerged the lower part of the torso in water at 25 °C and 43 °C, respectively, both for 15 minutes. At 1, 7, and 14 days after treatment, we obtained the testicular organ indexes, observed the changes in testicular morphology by HE staining, and determined the location and expression levels of the promyelocytic leukemia zinc finger (PLZF) and synaptonemal comlex protein-3 (SCP-3) in the testis tissue by immunohistochemistry and Western blot.

RESULTSThe testicular organ index was significantly lower in the heat stress treatment than in the control group (P < 0.05). Compared with the controls, the heat shock-treated mice showed loosely arranged spermatogenic cells scattered in the seminiferous tubules at 1 day after heat stress treatment, atrophied, loosely arranged and obviously reduced number of spermatogenic cells at 7 days, and relatively closely arranged seminiferous tubules and increased number and layers of spermatogenic cells at 14 days. The number of SCP-3 labelled spermatocytes obviously decreased in the heat stress-treated animals at 1 and 7 days and began to increase at 14 days. The PLZF protein expression was significantly reduced in the heat stress treatment group at 1 day as compared with that in the control (0.19 ± 0.12 vs 0.64 ± 0.03, P < 0.01), but elevated to 0.77 ± 0.02 at 7 and 14 days, even remarkably higher than in the control animals (P < 0.01).

CONCLUSIONHeat stress treatment can induce short-term dyszoospermia in mice, which can be recovered with the prolonged time after treatment.

Animals ; Blotting, Western ; Hot Temperature ; Immunohistochemistry ; Male ; Mice ; Nuclear Proteins ; metabolism ; Promyelocytic Leukemia Protein ; Seminiferous Tubules ; cytology ; Spermatocytes ; cytology ; pathology ; Testis ; metabolism ; Transcription Factors ; metabolism ; Tumor Suppressor Proteins ; metabolism

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

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

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

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

AIMTo assess proliferative and apoptotic potential of the seminiferous epithelium cells in relation to Sertoli cell maturation in newborn rats under the influence of estradiol, follicle stimulating hormone (FSH) or both agents given together.

METHODSFrom postnatal day (PND) 5 to 15 male rats were daily injected with 12.5 microg of 17beta-estradiol benzoate (EB) or 7.5 IU of human purified FSH (hFSH) or EB + hFSH or solvents (control). On postnatal day 16, autopsy was performed. Sertoli cell maturation/function was assessed by morphometry. Proliferation of the seminiferous epithelium cells was quantitatively evaluated using immunohistochemical labeling against proliferating cell nuclear antigen and apoptosis using the TUNEL method.

RESULTSAlthough EB inhibited Sertoli cell maturation and hFSH was not effective, a pronounced acceleration of Sertoli cell maturation occurred after EB + hFSH. Whereas hFSH stimulated Sertoli cell proliferation, EB or EB + hFSH inhibited Sertoli cell proliferation. All treatments significantly stimulated germ cell proliferation. Apoptosis of Sertoli cells increased 9-fold and germ cells 2-fold after EB, and was not affected by hFSH but was inhibited after EB + hFSH.

CONCLUSIONAt puberty, estradiol inhibits Sertoli cell maturation, increases Sertoli and germ cell apoptosis but stimulates germ cell proliferation. Estradiol in synergism with FSH, but neither of the hormones alone, accelerates Sertoli cell maturation associated with an increase in germ cell survival. Estradiol and FSH cooperate to induce seminal tubule maturation and trigger first spermatogenesis.

Animals ; Animals, Newborn ; Apoptosis ; drug effects ; Cell Differentiation ; drug effects ; Cell Proliferation ; drug effects ; Dose-Response Relationship, Drug ; Estradiol ; pharmacology ; Follicle Stimulating Hormone ; pharmacology ; Male ; Rats ; Rats, Wistar ; Seminiferous Tubules ; cytology ; drug effects ; Sexual Maturation ; drug effects ; physiology ; Spermatogenesis ; drug effects ; physiology ; Spermatozoa ; cytology ; drug effects ; Testis ; cytology ; drug 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