OBJECTIVETo study the effects of experimental varicocele (VC) on the serum testosterone (T) and intratesticular testosterone in adolescent rats.

METHODSA VC rat model was established by partial ligation of the left kidney vein in 20 SD rats, and another 20 were included in a sham operation group as controls. At 4 and 8 weeks, the concentrations of the serum T and intratesticular T were measured by radioimmunoassay (RIA). The testis tissues were homogenized and the extract liquid taken for RIA.

RESULTSCompared with the controls, the level of serum T declined at 4 and 8 weeks in the VC group, but not significantly (P > 0.05), so was that of bilateral intratesticular T at 4 weeks, and with statistical significance at 8 (P < 0.01).

CONCLUSIONWithin 8 weeks, experimental VC could reduce the level of bilateral intratesticular T, but not that of serum T. Varicocele could damage Leydig cells.

Animals ; Leydig Cells ; Male ; Rats ; Rats, Sprague-Dawley ; Testis ; metabolism ; Testosterone ; blood ; metabolism ; Varicocele ; metabolism

OBJECTIVETo investigate the mechanism of serum testosterone reduction, its relationship with metabolism, changes in the number and morphology of Leydig cells and endocrine function in aging male rats.

METHODSThe levels of serum total testosterone (tT), LH, FSH, HDL, LDL, TG, TC, Glu, INS, IRG and LP were determined in young (9 mo) and aging rats (12, 15, 18 and 21 mo), with 6 in each group. The morphological changes of Leydig cells were observed under the microscope. The concentrations of testosterone secreted from the cultured Leydig cells with the stimulation of hCG and Forskolin were assayed. The apoptosis rates of Leydig cells were detected by TUNEL. The visceral fat was isolated and weighed, and the Lee's index calculated. All the above indexes were recorded and compared among different age groups.

RESULTSThe aging rats showed a significant decrease in the levels of serum tT and TSI ([1.26 +/- 0.65] ng/ml and [0.07 +/- 0.65] ng/mIU) as compared with the young rats ([3.24 +/- 0.38] ng/ml and [0.21 +/- 0.01] ng/mIU) (P < 0.01). Obvious differences were found in the morphology of Leydig cells among different age groups. The T secretion of Leydig cells at 24, 48 and 72 h in aging rats was markedly decreased (P < 0.05) while their TUNEL positive rate remarkably increased in the aging rats (17.36% +/- 1.31%) compared with the young ones (7.02% +/- 1.05%) (P < 0.05). There were statistically significant differences between the young and aging rats in all the biochemical parameters including IRG, HDL, LDL, TG, TC and visceral fat content (P < 0.05), except the levels of serum Glu, INS and LP (P > 0.05).

CONCLUSIONThe serum T level and secreting capacity of Leydig cells are significantly lower in aging rats than in young ones, and the metabolic parameters undergo regular changes with the decreasing level of serum T. The reduction of testosterone in aging male rats may be associated with the decreased secreting capacity and number of Leydig cells and declined function of the pituitary.

Aging ; Animals ; Leydig Cells ; cytology ; metabolism ; secretion ; Male ; Rats ; Rats, Sprague-Dawley ; Testosterone ; blood ; metabolism

OBJECTIVETo explore the characteristics and distribution of GATA-4 in the testis of male mice.

METHODSParaffin sections were obtained from the testes of 24 male B6SJLF1/J mice, aged 0 day (n = 6), 2 weeks (n = 6), 4 weeks (n = 6) and 6 weeks (n = 6), and the expressions of GATA-4 in the testis were observed by the immunohistochemical ABC method and DAB visualization at different times.

RESULTSPositive expressions of GATA4 were found in the Sertoli cells and Leydig cells of all the mice, but significantly higher in the 4- and 6-week-old than in the 0-day and 2-week-old groups (P < 0.01). And they were also observed in the germ cells of the 4- and 6-week-old mice, significantly higher in the latter than in the former (P < 0.01).

CONCLUSIONGATA-4 exists in the testis of male mice, which has provided a morphological base for sex determination and differentiation and hormone regulation in the testis.

Animals ; Cell Differentiation ; GATA4 Transcription Factor ; metabolism ; Germ Cells ; metabolism ; Leydig Cells ; metabolism ; Male ; Mice ; Sertoli Cells ; metabolism ; Testis ; cytology ; metabolism

OBJECTIVETo investigate the expression of human epididymal secretary protein 2 isoform human epididymal protein 2beta1(HE2beta1) in the testis and epididymis of adolescent male rats along with its significance.

METHODSImmunohistochemical staining was used to detect the expression and localization of HE2beta1 in the testis and epididymis of 15 adolescent SD rats.

RESULTSHE2beta1 immunoreactive staining was detected in the testis and epididymis. In the epithelia of the epididymal duct, HE2beta1 expressed mainly in the supranuclear region of the principle cells and the basement membrane of some epithelial cells; there were no immunostaining in the n clear cells, halo cells and basal cells. The immunopositive reaction was detected, weak in the distal caput, strong in the proximal, middle corpus and the cauda, but negative in the initial segment. Immunopositive results of HE2beta1 were also observed in some of the nuclei of spermatogonia and Sertoli cells with negatively-stained cytoplasm.

CONCLUSIONImmunohistochemical staining is a fairly sensitive method for detecting HE2beta1 expression. The localization and expression level of HE2beta1 in the genital duct of adolescent male rats exhibited a region- and cell-specific expression pattern, which suggests that HE2beta1 may play an important role in spermatogenesis, maturation and epididymal epithelial innate defense mechanisms.

Animals ; Antigens, Surface ; biosynthesis ; Epididymis ; metabolism ; Glycopeptides ; biosynthesis ; Humans ; Immunohistochemistry ; Leydig Cells ; metabolism ; Male ; Rats ; Rats, Sprague-Dawley ; Testis ; metabolism

The expression of osteopontin (OPN) in boar testis was studied. Western blot analysis detected 66- and 32-kDa OPN immunopositive bands in the testes of adult boars. In postnatal piglets, the 66-kDa OPN band was detected in the testes, but not the 32-kDa band. In the newborn testis, OPN immunostaining was seen in gonocytes and in some supporting cells in the seminiferous tubules, as well as in interstitial Leydig cells. In the adult boar testis, OPN immunoreactivity was detected in seminiferous tubules with varying intensities. Intense OPN immunostaining was seen in the residual bodies and acrosomes in the spermatids while, occasionally, OPN immunostaining was seen in spermatogonia and various stage of spermatocytes but in few Sertoli cells in the seminiferous tubules. In addition, Leydig cells in adult boars were weakly immunostained with OPN. These findings suggest that OPN is detected in the majority of germ cells and is involved in spermatogenesis in boar testis.
Animals ; Animals, Newborn ; Blotting, Western/veterinary ; Immunohistochemistry/veterinary ; Leydig Cells/metabolism ; Male ; Osteopontin/*biosynthesis ; Sertoli Cells/metabolism ; Spermatogenesis/physiology ; Spermatozoa/metabolism ; Swine/*metabolism ; Testis/cytology/*metabolism

Hedgehog signaling pathway plays an important role in regulating the development of endocrine glands. Desert hedgehog (Dhh) has become a recent focus for its regulation of testis development, especially of Leydig cells. Dhh, as a Sertoli cell product, regulates the proliferation and differentiation of Leydig cell lineage and functions to secrete testosterone through a paracrine signaling mechanism. Testosterone, as the most important sex hormone of the male, plays a critical role in testis development, spermatogenesis and maintenance of normal masculinization. Therefore, normal Dhh signaling pathway ensures normal spermatogenic function. Researches on the Hedgehog signaling pathway in the testis have a potential significance for studying the pathophysiological mechanisms of androgen deficiency and dyszoospermia, as well as for the clinical treatment of these diseases.
Cell Differentiation ; Cell Proliferation ; Hedgehog Proteins ; metabolism ; Humans ; Leydig Cells ; cytology ; Male ; Signal Transduction ; Spermatogenesis ; Testis ; cytology ; metabolism

OBJECTIVETo observe the effects of TGF-beta on the expression of connexin43 (Cx43) and Cx43-mediated gap junctional intercellular communication (GJIC) in rat Leydig cells, and investigate the association of its effects on Leydig cells with its ability of changing GJIC.

METHODSPrimarily cultured purified Leydig cells were divided into a blank control group, a positive control group (treated with the GJIC inhibitor Carbenoxolone), and four TGF-beta1 groups (treated with TGF-beta1 at the concentration of 1, 2, 5 and 10 ng/ml, respectively, for 20 hours). The localization and expression of Cx43 were detected by immunofluorescence and Western blot, and the changes in GJIC analyzed by FRAP assay.

RESULTSCx43 was expressed as scattered bright spots in the cytoplasm and membrane of Leydig cells. TGF-beta1 significantly elevated the expression of Cx43 in the cytoplasm, but caused no evident change in the membrane. Western blot showed an evident increase in the phosphorylation of Cx43 with the increased concentration of TGF-beta1 as compared with that of the blank control group (P < 0.05). After 20 hours of treatment with TGF-beta1 at 5 ng/ml, the fluorescence intensity of Leydig cells was markedly reduced (P < 0.01), with a mean fluorescence recovery rate of merely (43.58 +/- 1.87)%.

CONCLUSIONTGF-beta1 could significantly down-regulate GJIC between adjacent Leydig cells, and this inhibitory effect may be achieved by promoting the expression of Cx43 in the cytoplasm and elevating the phosphorylation of Cx43.

Animals ; Cell Communication ; drug effects ; Cells, Cultured ; Connexin 43 ; metabolism ; Gap Junctions ; drug effects ; metabolism ; Leydig Cells ; drug effects ; metabolism ; Male ; Phosphorylation ; Rats ; Transforming Growth Factor beta1 ; pharmacology

OBJECTIVETo study the differentially expressed proteins in the process of annexin 5 stimulating testosterone secretion in cultured rat Leydig cells.

METHODSPrimary rat Leydig cells were cultured in vitro and treated with annexin 5 at the concentration of 1 nmol/L for 24 hours, and the cell proteins were extracted to be compared by two-dimensional gel electrophoresis (2-DE). The differentially expressed protein spots were selected to be analyzed by mass spectrometry.

RESULTSWe obtained electrophoresis profiles with high resolution and reproducibility, found 50 differentially expressed protein spots, and identified 36 by mass spectrometry, of which 23 were overexpressed and 13 underexpressed in the Leydig cells treated with annexin 5.

CONCLUSIONDifferentially expressed protein profiles were established in the process of annexin 5 stimulating testosterone secretion in cultured rat Leydig cells, and identified the key role of these proteins in testosterone secretion. Our findings might be helpful to illuminate the mechanism of annexin 5 regulating testosterone secretion in rat Leydig cells.

Animals ; Annexin A5 ; pharmacology ; Cells, Cultured ; Electrophoresis, Gel, Two-Dimensional ; Leydig Cells ; drug effects ; metabolism ; Male ; Mass Spectrometry ; Proteins ; metabolism ; Proteome ; analysis ; Rats ; Rats, Sprague-Dawley ; Testosterone ; secretion

OBJECTIVETo construct a directional differentiation model from mouse embryonic stem cells into leydig-like cells in vitro.

METHODSMouse ES-D3 cells were transfected with plasmid containing steroidogenic factor 1 (SF-1) gene, then treated with RA and 8Br-cAMP, while the cells transfected with empty plasmid were used as the negative controls. The morphology of leydig-like cells differentiated from ES-D3 cells was observed with light microscopy. The expression levels of StAR, P450scc and 3β-HSD were detected by RT-PCR, Western Blot and fluorescence microscopy analysis in leydig-like cells derived from the ES cells.

RESULTSES-D3 cells were transfected with plasmid containing SF-1 gene successfully, and SF-1 was expressed 24 h after transfection. The SF-1-transfected ES-D3 cells were induced by RA and 8Br-cAMP to differentiate into leydig-like cells. The differentiated cells showed spindle shape with tentacles, which expressed the specific protein marker for leydig cells 3β-HSD1 and P450scc. Meanwhile, in these leydig-like cells, the expression of StAR increased compared with control group. 3β-HSD1, P450scc and StAR were not detected in negative control group.

CONCLUSIONWhen the ES-D3 cells are transfected with SF-1 plasmid and then treated with RA and 8Br-cAMP, the cells are able to differentiate into leydig-like cells, indicating that the model of directional differentiation of ES cells into leydig-like cells has been constructed successfully.

Animals ; Cell Differentiation ; drug effects ; genetics ; Cell Line ; Embryonic Stem Cells ; cytology ; metabolism ; Leydig Cells ; cytology ; metabolism ; Male ; Mice ; Steroidogenic Factor 1 ; genetics ; Transfection

OBJECTIVETo investigate the effects of di-(2-ethylhexyl) phthalate (DEHP) on the expressions of Caspase-3 and Caspase-9 genes in rat Leydig cells and the apoptosis of the cells in vitro.

METHODSLeydig cells were isolated from male SD rats, primarily cultured and treated with DEHP at a low (10 nmol/L), a medium (50 nmol/L) and a high dose (100 nmol/L) for 24 hours. Then the mRNA expressions of Caspase-3 and Caspase-9 genes in the Leydig cells were detected by real time PCR, their protein expressions determined by Western blot, and the apoptosis of the Leydig cells measured by flow cytometry.

RESULTSCompared with the DMSO control group, the low-, medium- and high-dose DEHP groups showed significantly upregulated expressions of Caspase-3 mRNA (1.69 +/- 0.38 vs 3.82 +/- 0.39, 6.91 +/- 0.40 and 15.47 +/- 0.40, P < 0.05), Caspase-3 protein (0.18 +/- 0.0.09 vs 0.32 +/- 0.10, 0.61 +/- 0.08 and 0.89 +/- 0.09, P < 0.05), Caspase-9 mRNA (2.24 +/- 0.41 vs 5.16 +/- 0.43, 9.61 +/- 0.45 and 19.22 +/- 0.43, P < 0.05) and Caspase-9 protein (0.26 +/- 0.07 vs 0.40 +/- 0.08, 0.68 +/- 0.09 and 0.96 +/- 0.08, P < 0.05), as well as increased apoptosis rate of Leydig cells (4.36 +/- 1.11 vs 7.52 +/- 1.09, 12.72 +/- 1.10 and 24.59 +/- 1.11, P < 0.05), all in a dose-dependent manner.

CONCLUSIONDEHP can induce the apoptosis of rat Leydig cells by activating the apoptosis Caspase pathway, and consequently affect the function of Leydig cells.

Animals ; Apoptosis ; drug effects ; Caspase 3 ; metabolism ; Caspase 9 ; metabolism ; Diethylhexyl Phthalate ; toxicity ; Leydig Cells ; drug effects ; metabolism ; Male ; Rats ; Rats, Sprague-Dawley