Objective: To explore the role of TGF-β1 in the proliferation and apoptosis of Sertoli cells and its effect on the expressions of tight junction-related proteins and genes in rats. METHODS: Rat Sertoli cells were isolated in vitro, primarily cultured, and divided into groups A (blank control), B (TGF-β1 receptor blocker), C (TGF-β1), and D (TGF-β1 + receptor blocker). The proliferation and apoptosis of the cells were detected by CCK-8 and flow cytometry, respectively. After establishment of the dual-chamber model for the primary culture of Sertoli cells, the trans-epithelia electrical resistance (TER) value was measured and the relative expressions of Occludin, ZO-1 and Claudin Ⅱ determined by RT-PCR and Western blot. RESULTS: The OD value of the proliferation of the Sertoli cells was markedly higher in group C than in groups A and D (0.79 ± 0.04 vs 0.66 ± 0.05 and 0.68 ± 0.02, P<0.05), with statistically significant differences among the four groups (F = 5.05, P <0.05). However, no remarkable difference with found among the four groups in the apoptosis rate of the cells (F = 1.13, P >0.05). The TER value was dramatically decreased in group C as compared with groups A and D (［176.37 ± 16.61］ vs ［281.42 ± 9.83］ and ［254.37 ± 13.55］ /cm2, P<0.01), with statistically significant differences among the four groups (F = 38.99, P<0.01). There were no remarkable differences among the four groups in the mRNA expressions of ZO-1 and Claudin Ⅱ (F = 0.49 and 0.93, P>0.05) or their protein expressions (F = 0.28 and 1.31, P>0.05). Both the mRNA and protein expressions of Occludin were markedly lower in group C than in A and D (P<0.01 and P<0.05), with statistically significant differences among the four groups (F = 6.86 and 6.87, P<0.01). CONCLUSIONS TGF-β1 can promote the proliferation of Sertoli cells in rats and act on the tight junction of the cells by regulating the expression of Occludin.
Animals ; Apoptosis ; Cell Proliferation ; Cells, Cultured ; Claudin-2 ; metabolism ; Male ; Occludin ; metabolism ; RNA, Messenger ; Rats ; Sertoli Cells ; cytology ; physiology ; Tight Junction Proteins ; metabolism ; Tight Junctions ; genetics ; metabolism ; Transforming Growth Factor beta1 ; physiology ; Zonula Occludens-1 Protein ; metabolism

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 isolate, identify and culture human spermatogonial stem cells (SSC) and then obtain purified and enriched human SSCs for research and application.

METHODSWe detected the expression of CD90 in the human testis using the immunofluorescence technique and isolated human testicular spermatogenic cells by two-step enzymatic digestion, followed by differential plating and magnetic-activated cell sorting (MACS) with CD90 as an SSC marker. Then we identified the isolated CD90-positive spermatogenic cells by RT-PCR and immunocytochemistry, and meanwhile cocultured them with Sertoli cells in SG medium in vitro.

RESULTSThe isolated CD90-positive cells showed a relatively homogeneous characteristic in size and morphology and expressed the genes specific for human SSCs, with high expressions (90.5%) of GFRA1, GPR125, and UCHL1. After coculture with Sertoli cells in the SG medium for 2 weeks, the isolated CD90-positive cells maintained a good activity.

CONCLUSIONCD90 can be regarded as a speci- fic marker for human SSCs and used to obtain highly enriched human SSCs by differential plating and MACS. Furthermore, the isolated human SSCs can be cultured in SG medium in vitro.

Adult Stem Cells ; cytology ; Biomarkers ; metabolism ; Cell Separation ; methods ; Cell Shape ; Cell Size ; Coculture Techniques ; Glial Cell Line-Derived Neurotrophic Factor Receptors ; metabolism ; Humans ; Immunohistochemistry ; Male ; Receptors, G-Protein-Coupled ; metabolism ; Sertoli Cells ; Spermatogonia ; cytology ; Testis ; metabolism ; Thy-1 Antigens ; isolation & purification ; metabolism ; Ubiquitin Thiolesterase ; metabolism

OBJECTIVETo investigate the impact of in utero exposure to di-n-butyl phthalate (DBP) on the apoptosis of testicular cells in the pubertal male rat offspring.

METHODSTen pregnant SD rats were randomly divided into a control and an experimental group to be treated intragastrically with olive oil (1 ml per day) and DBP (500 mg per kg of body weight per day) respectively between gestation days 12 and 19. At the pubertal age (postnatal day 45, PND 45), the testes of the male rat offspring were removed for observation of the cell structure under the transmission electron microscope and the development of different spermatogenetic cells by HE staining. The apoptosis of testicular cells was detected by the TUNEL method, the expressions of the apoptosis-regulating proteins Bcl-2, Bcl-XL, Bax and p53 were determined by immunohistochemistry and Western blot, and the data obtained were compared between the two groups by t-test.

RESULTSTransmission electron microscopy revealed increased apoptosis and vacuolization of testicular cells in the PND-45 rat offspring, HE staining showed markedly decreased numbers of different spermatogenetic cells, TUNEL manifested significantly increased apoptosis of testicular cells in the experimental group as compared with the control (12.00 ± 5. 22 vs 3.17 ± 1.47, P < 0.01), and immunohistochemistry and Western blot exhibited remarkably higher expressions of Bax and p53 in the former than in the latter group (P < 0.05).

CONCLUSIONIn utero exposure to DBP can increase the apoptosis of germ cells and Sertoli cells, induce the vacuolization of testicular cells, and significantly elevate the expressions of the apoptosis-promoting proteins Bax and p53 in the pubertal male rat offspring.

Animals ; Apoptosis ; Body Weight ; Dibutyl Phthalate ; adverse effects ; Female ; Immunohistochemistry ; In Situ Nick-End Labeling ; Male ; Pregnancy ; Prenatal Exposure Delayed Effects ; Rats ; Rats, Sprague-Dawley ; Sertoli Cells ; cytology ; pathology ; Spermatogenesis ; Testis ; cytology ; pathology ; Tumor Suppressor Protein p53 ; metabolism ; bcl-2-Associated X Protein ; metabolism

OBJECTIVETo establish an in vitro model of cultured mouse testis using rotary aerobic culture.

METHODSRotary aerobic incubation with optimized culture conditions was used for in vitro culture of mouse testis, and the morphology of the cultured testicular tissues was compared with that cultured in Transwell chambers. The changes in the testicular tissue structure were examined using HE staining, and the cell proliferation was assessed with BrdU staining. Testosterone concentrations in the culture medium were tested with radioimmunoassay and the expression of the functionally related proteins in the testis was detected using immunohistochemistry.

RESULTSThe testicular tissue cultured by optimized rotary aerobic culture presented with more intact histological structure with the size of the testis ranged from 0.3 to 0.8 mm(3). In the two culture systems, the prolifeation index of the spermatogonia increased and that of Sertoli cells decreased with time, and such changes in spermatogonia and Sertoli cell proliferation indices became statistically significant at 3 days (P<0.05) and 5 days (P<0.05) of culture, respectively, as compared with those at 1 day. The concentration of testoerone in the culture media decreased significantly with incubation time (P<0.05). At 3 days of culture, the protein expression of 3β-hydroxysteroid dehydrogenase, cytochrome P450 17α-hydroxylase and cholesterol side-chain cleavage enzyme was detected in Leydig cell cytoplasm and vimentin expression in Sertoli cell cytoplasm.

CONCLUSIONAn in vitro model of cultured mouse testis has been successfully established using rotary aerobic incubation.

17-Hydroxysteroid Dehydrogenases ; metabolism ; Animals ; Cholesterol Side-Chain Cleavage Enzyme ; metabolism ; Culture Media ; chemistry ; Leydig Cells ; cytology ; Male ; Mice ; Organ Culture Techniques ; Radioimmunoassay ; Sertoli Cells ; cytology ; Spermatogonia ; cytology ; Testis ; Testosterone ; chemistry ; Vimentin ; metabolism

OBJECTIVETo investigate the mechanism of di-2-ethylhexyl phthalate (DEHP) and cypermethrin (CYP) inducing gonadal dysgenesis in prepubertal male rats.

METHODSA total of 40 healthy 3-week-old specific pathogen-free male Sprague-Dawley rats were randomly and equally divided into four groups: control group (corn oil), DEHP group (500 mg/kg, dissolved in corn oil), CYP group (80 mg/kg, dissolved in corn oil), and combined exposure group (exposed to 500 mg/kg DEHP and 80 mg/kg CYP, dissolved in corn oil). Rats were treated by gavage administration once a day for 30 days. Twenty-four hours after the last exposure, the animals were sacrificed. The body weight and the wet weight of testis were determined, and the weight coefficient of testis was calculated. Radioimmunoassay was used to determine serum testosterone level. Ultrastructural-level histopathological changes of the testis were examined by transmission electron microscopy. The mRNA and protein expression of follicle stimulating hormone receptor (FSHR), androgen binding protein (ABP), inhibin beta-B (INHBB) and vimentin (VIM) were analyzed by real-time PCR and Western blot, respectively. Factorial design analysis of variance was used to compare differences between groups; interaction diagrams were used to determine the interaction between DEHP and CYP.

RESULTSCompared with those of the control group, the testis weights and testis coefficients of the DEHP, CYP, and combined exposure groups significantly decreased by 39.3-59.2%and 19.7-58.6%, respectively, and all exposure groups showed significant reductions in serum level of testosterone, ranging from 49.1% to 62.7% (P < 0.05 or P < 0.01). And all the exposure groups showed different levels of ultrastructural damages in the testes. Compared with that in the control group, the mRNA expression of FSHR, ABP, INHBB, and VIMin the DEHP group was down-regulated by 1.72, 2.64, 2.83 and 1.79 times, and their protein levels were significantly reduced by 65.2%, 53.7%, 70.1%, and 51.9% (P < 0.05 or P < 0.01). Significant decreases in mRNA expression of ABP (down 1.72 times) and INHBB (down 2.06 times) were observed in the CYP group, and their protein levels decreased by 38.3% and 49.7%, respectively (P < 0.05). The combined exposure to both DEHP and CYP resulted in big decreases in the mRNA levels of FSHR (down 1.62 times), ABP (down 2.00 times), INHBB (down 2.35 times), and VIM (down 1.54 times) and protein levels of FSHR (down 52.1%), INHBB (down 53.9%), and VIM (down 58.8%) (P < 0.05). Factorial design analysis of variance showed that the combination of two substances had an antagonistic effect on the expression of ABP and INHBB (P < 0.05).

CONCLUSIONDEHP and CYP, alone or combined, can lead to gonadal dysgenesis in prepubertal male rats. Both of them can disrupt functional mRNA and protein expression in Sertoli cells to certain levels. The combination of DEHP and CYP shows antagonistic effects, and DEHP has a stronger reproductive toxicity than CYP.

Animals ; Diethylhexyl Phthalate ; toxicity ; Gonadal Dysgenesis ; chemically induced ; Male ; Pyrethrins ; toxicity ; Rats ; Rats, Sprague-Dawley ; Sertoli Cells ; metabolism ; Testis ; cytology ; drug effects

OBJECTIVETo explore the mechanism of hyperthermia inducing infertility by observing the expression of glial cell line-derived neurotrophic factor (GDNF) in rat Sertoli cells cultured in vitro at different temperatures.

METHODSUsing combination enzyme digestion and selective adhesion, we isolated Sertoli cells from male Wistar rats and cultured them in vitro at different temperatures, followed by observation of the changes in their adhesion and morphology and identification by FasL immunohistochemical staining. We divided the Sertoli cells into a control group (35 degrees C) and four experimental groups (36 degrees C, 37 degrees C, 38 degrees C, and 39 degrees C), measured their proliferation by CCK-8, observed their morphology and structure by HE staining, and determined the expression of GDNF by RT-PCR, immunofluorescence and Western blot.

RESULTSSertoli cells were successfully isolated and in vitro-cultured, with a purity of (95.30 +/- 2.15)% (n = 10). The CCK-8 assay showed that the proliferation of the Sertoli cells was the highest at 36 degrees C, gradually decreasing with the temperature above 36 degrees C, and significantly inhibited at 39 degrees C (P < 0.01). Immunofluorescence revealed the expression of GDNF in the cytoplasm, with the highest fluorescence intensity at 36 degrees C. RT-PCR and Western blot exhibited a decreasing trend of the GDNF expression with the increasing temperature above 36 degrees C. There were statistically significant differences in the expression of GDNF between the control group and the four experimental groups (P < 0.01).

CONCLUSIONThe proliferation and GDNF expression of in vitro-cultured Sertoli cells differ significantly at different temperatures. At > 36 degrees C, the higher the temperature is, the lower the Sertoli cell proliferation and GDNF expression are. Our findings suggest that high temperature above 36 degrees C suppresses the function of Sertoli cells and may also damage spermatogenesis.

Animals ; Cells, Cultured ; Glial Cell Line-Derived Neurotrophic Factor ; metabolism ; Male ; Rats ; Rats, Wistar ; Sertoli Cells ; cytology ; metabolism ; Temperature ; Testis ; cytology

OBJECTIVETo evaluate the effects of the Chinese herbal formula Wuzi Yanzong Pill (, WYP) on the spermatogenesis and specific secretory functions of Sertoli cells in rat model and to investigate the underlying mechanism.

METHODSFive groups of male Sprague-Dawley rats including the control group, the model group, the low-dose WYP group, the medium-dose WYP group and the high-dose WYP group (5 in each group) were treated daily with vehicle, multiglycosides of Tripterygium wilfordii Hook f (GTW) either alone (20 mg/kg) or followed by WYP (0.5, 1.0, or 2.0 g/kg daily), respectively for 30 days. Serum levels of follicle-stimulating hormone (FSH), inhibin B (INHB) and testosterone (T) were evaluated using enzyme-linked immunosorbent assay. Androgen-binding protein (ABP) gene expression and transferrin (TF) protein expression in testis tissue specimens of all rats were determined using real-time reverse transcriptase polymerase chain reaction and Western blotting analysis, respectively. Histopathological alterations in the testis were determined using Johnsen's score.

RESULTSThe toxicity of GTW towards Sertoli cell secretory functions and spermatogenesis was accompanied by increased serum FSH concentrations and decreased INHB and T concentrations. Upregulated ABP mRNA levels, and decreased TF protein expression and Johnsen's scores were detected in the model group compared with the control group P<0.05 or P<0.01). Oral high-dose WYP administrations to GTW-treated rats effectively alleviated all of the GTW-induced changes in specific secretory functions of Sertoli cells (ABP, INHB and TF). Furthermore, serum T level and Johnsen's score of the testis increased greatly compared with the model group (P<0.01).

CONCLUSIONWYP has the ability to improve the spermatogenesis, possibly through modulating the secretory proteins expression of Sertoli cells.

Androgen-Binding Protein ; genetics ; metabolism ; Animals ; Blotting, Western ; Drugs, Chinese Herbal ; pharmacology ; Follicle Stimulating Hormone ; blood ; Gene Expression Regulation ; drug effects ; Inhibins ; blood ; Male ; RNA, Messenger ; genetics ; metabolism ; Rats ; Rats, Sprague-Dawley ; Sertoli Cells ; drug effects ; secretion ; Spermatogenesis ; drug effects ; Tablets ; Testis ; cytology ; metabolism ; Testosterone ; blood ; Transferrin ; metabolism

OBJECTIVE: To determine the effect of soy isoflavones on cell proliferation and the transcription levels of follicle-stimulating hormone receptor (FSHR), inhibin α (INHα), INHβB, androgen binding protein (ABP), transferrin (Tf) and vimentin in testis sertoli cells in SD rats. METHODS: Sertoli cells were cultured in vitro, exposed to daidzein at 0.03, 0.3, 3, and 30 μmol/L and genistein at 0.05, 0.5, 5 and 50 μmol/L, respectively. MTT was used to detect the proliferation of sertoli cells. Real-time PCR was used to detect the relative mRNA expressions of FSHR, INHα, INHβB, ABP, Tf and vimentin. RESULTS: Compared with control groups, cell proliferation and the relative mRNA expression levels of INHβB and ABP in the treated cells showed no significant alternation. The INHα mRNA expression levels were increased in 0.3 and 3 μmol/L Dai and 0.05 μmol/L Gen, while the mRNA expression levels of FSHR were downregulated in 30 μmol/L Dai and Gen at all concentrations. Tf mRNA expression levels were downregulated in 30 μmol/L Dai and 5 μmol/L and 50 μmol/L Gen, and the mRNA expression levels of vimentin were downregulated in 3 and 30 μmol/L Dai and 50 μmol/L Gen. CONCLUSION Soy Isoflavones may have potential detrimental effect on the male reproductive system, as they may impact the function of sertoli cells by downregulating the transcription levels of some important proteins.
Androgen-Binding Protein ; metabolism ; Animals ; Inhibin-beta Subunits ; metabolism ; Inhibins ; metabolism ; Isoflavones ; adverse effects ; Male ; RNA, Messenger ; Rats ; Rats, Sprague-Dawley ; Receptors, FSH ; metabolism ; Sertoli Cells ; drug effects ; Soybeans ; chemistry ; Testis ; cytology ; drug effects ; Transferrin ; metabolism

OBJECTIVETo investigate the effect of different doses of glyphosate on apoptosis and expressions of androgen-binding protein (ABP) and vimentin mRNA in mouse Sertoli cells.

METHODSPrimarily cultured mouse Sertoli cells incubated with different doses of glyphosate (60, 90, 120, 150 and 180 mg/L) for 24 h. The growth and morphological alterations in the cells were observed under inverted microscope, and the cell proliferation rate was evaluated withMTT assay. Hoechst 33342 staining was used to detect cell apoptosis after the treatment, and RT-PCR was performed to examine the changes in the expression of ABP and vimentin mRNAs.

RESULTSSertoli cells exposed to glyphosate showed a reduced cell volume, cell dissociation with occasional cell disruption. The proliferation of the exposed was suppressed with an increased rate of cell apoptosis and lowered expressions of ABP and vimentin mRNAs (P<0.05).

CONCLUSIONGLY can cause cellular damages, inhibit cell proliferation, induce cell apoptosis, and decrease expression of ABP and vimentin mRNAs in mouse Sertoli cells in vitro.

Androgen-Binding Protein ; genetics ; metabolism ; Animals ; Apoptosis ; drug effects ; Cell Proliferation ; drug effects ; Cells, Cultured ; Dose-Response Relationship, Drug ; Glycine ; administration & dosage ; analogs & derivatives ; toxicity ; Herbicides ; administration & dosage ; toxicity ; Male ; Mice ; RNA, Messenger ; metabolism ; Sertoli Cells ; cytology ; drug effects ; metabolism ; Vimentin ; genetics ; metabolism