OBJECTIVESTo study the expression of Smad1 and Smad5 in the testis of infertile rats with adenine-modeled kidney-yang deficiency and the pathological mechanism of infertility with kidney-yang deficiency, attempting to obtain experimental evidence for the prevention and treatment of male infertility.

METHODSForty-eight 60 d male SD rats were divided randomly into 6 groups with 8 in each: 7 d, 14 d and 21 d kidney-yang deficiency groups, and 7 d, 14 d and 21 d control groups. The experimental rats had been fed with adenine (300 mg/kg) and the expression levels of Smad1 and Smad5 were measured with immunohistochemical SABC method at the 7th, 14th and 21st day.

RESULTSSmad1 immunoreactivity was mainly located in the spermatogonia, spermatocytes and spermatids, and the reactive substance distributed in cytoplasm with negative nuclei. Sertoli cells and Leydig cells were negative. Compared with the control, the expression level of Smad1 was decreased significantly at the 21st day (P < 0.05), but with no significant difference at the 7th and 14th day (P > 0.05). Smad5 immunoreactivity was mainly located in the spermatogonia and spermatocytes, and the reactive substance distributed in cytoplasm with negative nuclei. Compared with the control, the expression level of Smad5 was not significantly different at the 7th day (P > 0.05). The expression of Smad5 was negative at the 14th and the 21st day.

CONCLUSIONThe weaker expression of Smad1 and no expression of Smad5 may be one of the pathological mechanisms of infertility with adenine-modeled kidney-yang deficiency.

Animals ; Infertility, Male ; metabolism ; pathology ; Male ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Smad1 Protein ; biosynthesis ; Smad5 Protein ; biosynthesis ; Testis ; metabolism ; Yang Deficiency ; metabolism ; pathology

OBJECTIVETo explore the effect of Bushen Tiaojing Recipe (BTR) on the expressions of drosophila mothers against decapentaplegic protein (Smadl), Smad5, Smad8, and Smad4 on human mural granulosa cells.

METHODSSixty-six patients undergoing in vitro fertilization-embryo transfer (IVF-ET) were randomly assigned to two groups in the ratio of 1:2, the treatment group and the control group. Twenty-three patients in the treatment group were treated with BTR and GnRHa/FSH/hCG, while forty-three patients in the control group were treated with GnRHa/FSH/hCG. The mRNA expressions of Smad1, Smad5, Smad8, and Smad4 on mural granulosa cells of the mature follicle were detected by real-time PCR on the ovum retrieval day. The expressions of Smad1, Smad5, Smad8, and Smad4 at the protein level were observed using cell immunofluorescence method.

RESULTSThe mRNA and protein expressions of Smadl in the granulosa cells were significantly higher in the treatment group than in the control group (P <0.05). There was no statistical difference in the mRNA and protein expressions of Smad5, Smad8, and Smad4 between the two groups.

CONCLUSIONThe mechanisms of BTR for improving the pregnancy rate and the ovarian functions might be correlated with up-regulating mRNA and protein expressions of Smadl of human mural granulosa cells.

Adult ; Drugs, Chinese Herbal ; pharmacology ; Female ; Granulosa Cells ; drug effects ; metabolism ; Humans ; Ovarian Follicle ; cytology ; Signal Transduction ; Smad1 Protein ; metabolism ; Smad4 Protein ; metabolism ; Smad5 Protein ; metabolism ; Smad8 Protein ; metabolism

AIMTo study the expression and regulation of Smad1, Smad2 and Smad4 proteins (intracellular signaling molecules of transforming growth factor-b family) in rat testis during postnatal development.

METHODSThe whole testes were collected from SD rats aged 3, 7, 14, 28 and 90 (adult) days. The cellular localization and developmental changes were examined by immunohistochemistry ABC method with the glucose oxidase-DAB-nickel enhancement technique. Quantitative analysis of the immunostaining was made by the image analysis system. The Smads proteins coexistence in the adult rat testis was tested by the double immune staining for CD14-Smad4 and Smad2-Smad4. The protein expression of Smad during rat testicular development was examined by means of Western blots.

RESULTSSmad1, Smad2 and Smad4 were present throughout testicular development. The immunostaining of Smad1 and Smad2 were present in spermatogenic cells. A positive immunoreactivity was located at the cytoplasm, but the nucleus was negative. Smad1 was immunolocalized at the d14, d28 and adult testes, while Smad2, at the d7, d14, d28 and adult testis. There was positive immunoreaction in the Sertoli cells and Leydig cells as well. The immunolocalization of Smad4 was exclusively at the cytoplasm of Leydig cells and the nuclei were negative throughout the testicular development. No expression was detected in the germ cells. The results of image and statistical analysis showed that generally the expression of Smad1, Smad2 and Smad4 in the testis tended to increase gradually with the growth of the rat.

CONCLUSIONThe present data provide direct evidences for the molecular mechanism of TGF-bgr action in rat testes during postnatal development and spermatogenesis.

Animals ; Blotting, Western ; DNA-Binding Proteins ; analysis ; biosynthesis ; Immunohistochemistry ; Male ; Rats ; Rats, Sprague-Dawley ; Signal Transduction ; physiology ; Smad Proteins ; Smad1 Protein ; Smad2 Protein ; Smad4 Protein ; Testis ; chemistry ; growth & development ; physiology ; Trans-Activators ; analysis ; biosynthesis

OBJECTIVETo explore whether strontium ranelate (Sr) promotes osteoblast lineage differentiation of rat bone mesenchymal stem cells (BMSCs) through the bone morphogenetic protein-2 (BMP-2)/Smad signaling pathway.

METHODSCultured rat BMSCs were exposed to different concentrations of Sr, noggin (an inhibitor of BMP-2) or Smad1 siRNA. The activity of alkaline phosphatase (ALP) in the exposed cells was detected by colorimetry, and the formation of mineralized nodules was observed with alizarin red staining. The expressions of phosphorylated (p) Smad1/5/8 and Runt-related transcription factor 2 (Runx2) in the cells were detected by Western blotting.

RESULTSExposure to Sr at 0.1 to 10 mmol/L for 1 h markedly increased the expression of p-Smad1/5/8 in the BMSCs, and the increment was the most obvious following 1 mmol/L Sr exposure. Preconditioning with 100 ng/ml noggin for 2 h inhibited Sr-induced up-regulation of p-Smad1/5/8 expressions. Exposure of the cells to 0.1 to 5 mmol/L Sr for 6 h significantly enhanced Runx2 expression, and the peak enhancement occurred following 1 mmol/L Sr exposure. Transfection of the BMSCs with Smad1 siRNA decreased the basal level of Smad1/5/8 protein expression, and also inhibited Sr-induced up-regulation of p-Smad1/5/8 and Runx2 expressions as well as Sr-induced enhancement of ALP activity and formation of mineralized nodules.

CONCLUSIONThe BMP-2/Smad pathway is involved in Sr-induced osteoblast differentiation of rat BMSCs.

Alkaline Phosphatase ; metabolism ; Animals ; Bone Marrow Cells ; cytology ; Bone Morphogenetic Protein 2 ; metabolism ; Cell Differentiation ; drug effects ; Cells, Cultured ; Mesenchymal Stromal Cells ; cytology ; Osteogenesis ; Rats ; Rats, Sprague-Dawley ; Signal Transduction ; Smad1 Protein ; metabolism ; Strontium ; pharmacology ; Thiophenes ; pharmacology

OBJECTIVETo study the role of antenatal glucocorticoid (dexamethasone and betamethasone) on bone morphogenetic protein (BMP) signal transduction of the rat fetal lungs.

METHODSFifteen pregnant rats were randomly divided into five groups: the rats treated with dexamethasone for 1 day (1D-DEX) or 3 days (3D-DEX), with betamethasone for 1 day (1D-BEX) or 3 days (3D-BEX) or with normal saline (control group), followed cesarean section on the 19th day of gestation. The mRNA levels of BMP4, BMPR-II, Smad1 and ATF-2 of fetal rat lungs were ascertained by reverse transcriptase polymerase chain reaction (RT-PCR). The expression of BMP4, BMPR-II, Smad1 and ATF-2 antigen expression in fetal lungs was assessed by immune histochemical staining. The expression of BMP4 and BMPR-II was determined by Western blot.

RESULTSThe levels of BMP4, BMPR-II and Smad1 mRNA expression were up-regulated in the 1D-BEX, 3D-BEX and 3D-DEX groups compared with those in the control group (P<0.05). The immune histochemiscal analysis showed that the expression of BMP4, BMPR-II, Phospho-Smad1 (pSmad1) and ATF-2 in the 1D-BEX, 3D-BEX and 3D-DEX groups was significantly higher than that in the control group (P<0.01). The results of Western blot demonstrated that the expression of BMP4 and BMPR-II protein increased significantly in the 1D-BEX, 3D-BEX and 3D-DEX groups when compared with the control group (P<0.01).

CONCLUSIONSBetamethasone and dexamethasone may play important roles in the regulation of BMP signal transduction in the rat fetal lungs. Up-regulation of BMP4, BMPR-II and Smad1 might be one of crucial factors for the glucocorticoid-induced maturity of fetal lungs.

Activating Transcription Factor 2 ; analysis ; genetics ; Animals ; Betamethasone ; pharmacology ; Bone Morphogenetic Protein 4 ; analysis ; genetics ; physiology ; Bone Morphogenetic Protein Receptors, Type II ; analysis ; genetics ; Dexamethasone ; pharmacology ; Female ; Fetus ; drug effects ; metabolism ; Lung ; drug effects ; metabolism ; Pregnancy ; Rats ; Rats, Sprague-Dawley ; Reverse Transcriptase Polymerase Chain Reaction ; Signal Transduction ; drug effects ; Smad1 Protein ; analysis ; genetics