ABSTRACT
To explore the functions of human ribonuclease 9 (RNase 9), we constructed a mammalian fusion expression vector pcDNA-hRNase9, prepared recombinant human RNase 9-His fusion protein from HEK293T cells and determined its N-terminal amino acid sequences. According to the determined mature protein, recombinant human RNase 9 was prepared in E. coli. Ribonucleolytic activity and antibacterial activity of recombinant human RNase 9 were detected, and the distribution of human RNase 9 on tissues and ejaculated spermatozoa and in vitro capacitated spermatozoa were analyzed via indirect immunofluorescence assay. The results showed that recombinant human RNase 9 did not exhibit detectable ribonucleolytic activity against yeast tRNA, but exhibited antibacterial activity, in a concentration/time dependent manner, against E. coli. Immunofluorescent analyses showed that the predicted human RNase 9 was present throughout the epididymis, but not present in other tissues examined, and human RNase 9 was also present on the entire head and neck regions of human ejaculated spermatozoa and in vitro capacitated spermatozoa. These results suggest that human RNase 9 may play roles in host defense of male reproductive tract.
Subject(s)
Adult , Humans , Male , Young Adult , Amino Acid Sequence , Anti-Infective Agents , Metabolism , Blotting, Western , Cloning, Molecular , Enzyme-Linked Immunosorbent Assay , Epididymis , Escherichia coli , Genetic Vectors , Molecular Sequence Data , Recombinant Fusion Proteins , Chemistry , Metabolism , Ribonuclease, Pancreatic , Metabolism , Ribonucleases , Chemistry , Metabolism , Seminal Plasma Proteins , Chemistry , Metabolism , Spermatozoa , Metabolism , TestisABSTRACT
The epididymis is an important organ in the male genital system, which is responsible for the maturation, transportation and storage of spermatozoa. The proper function of the epididymis is closely related with its robust physiological metabolism, and free radicals are inevitably produced as a consequence. An excess of free radicals would lead to the oxidative stress of the epididymis, damage the sperm membrane and DNA, seriously affect sperm maturation and result in male infertility. This article reviews the mechanism of epididymal oxidative stress in energy metabolism and inflammatory reaction as well as the roles of superoxide dismutase, catalase, glutathione peroxidase, indoleamine 2, 3 dioxygenase, glutathione and thioredoxin in the antioxidant process, offering a new insight into the prevention, diagnosis and treatment of male infertility.
Subject(s)
Animals , Humans , Male , Rats , Antioxidants , Epididymis , Metabolism , Infertility, Male , Oxidative StressABSTRACT
Sperm maturation in the epididymis is regulated by changes of luminal ion concentration and processing of sperm surface membrane by several glycosidases and proteases, and the actions of the proteases are controlled by protease inhibitors present in specific areas of the epididymis. WFDC-type serine protease inhibitors that are highly expressed in the epididymis play an important role in natural immunity and male reproduction. This paper gives an overview of the structure and function of the protein and its application prospects in the development of drugs for male reproductive tract infection and immunocontraception.
Subject(s)
Humans , Male , Anti-Infective Agents , Therapeutic Uses , Contraceptive Agents , Epididymal Secretory Proteins , Metabolism , Epididymis , Chemistry , Metabolism , Serine Proteinase Inhibitors , Genetics , Metabolism , Therapeutic UsesABSTRACT
<p><b>OBJECTIVE</b>To explore the effect of basic fibroblast growth factor 1 (bFGF1) during embryonic development on hematopoiesis, to study the expression of FGF1, vascular endothelial growth factor receptor (KDR), CD133, CD34 and transcription factors Ihh, SCL, GATA-1, GATA-2 and PU. 1 in the yolk sac, and to learn about the role and relationships of FGF1, hematopoietic cells and transcription factors during embryonic hematopoiesis.</p><p><b>METHODS</b>10 microm sections and total RNA were prepared from 107 human embryos aged 3-12 weeks. Immunohischemical SP staining and RT-PCR were performed.</p><p><b>RESULTS</b>The yolk sac blood islands of human 3 approximately 12 weeks embryos consisted of peripheral vascular endothelial cells and central hematopoietic cells. The expression of FGF1 was firstly found in visceral mesoderm around periphery of yolk sac blood island at day 16, while was little inside it. KDR was not or lowly expressed and CD34 and CD133 were not expressed then. The expression increased, gray value decreased and staining enhanced at day 21. Strong staining of CD34+, CD133+ and KDR+ cells were found in blood island and mesoderm at day 30, their gray values changed from 156 +/- 16, 173 +/- 18 and 160 +/- 14 to 53 +/- 7, 52 +/- 6 and 69 +/- 8 respectively. FGF1 expression was strong positive, the gray value declined dramatically from 161 +/- 13 to 40 +/- 5. Some positive cells formed vessel-like structure along the periphery of blood island. Moderate expression of CD34+, CD133+, KDR+ cells increased at day 45, the cells aggregated into mass in blood island and FGF1+ cells did the same in blood island, while little in mesoderm. Its gray valve was increased. After 7 weeks, CD133+, KDR+, CD34+ cells significantly decreased their gray values increased, the staining became week. FGF1 was weakly expressed in yolk sac and its gray value increased to 179 +/- 22. RT-PCR showed Ihh, SCL, GATA-1 and GATA-2 were expressed at different time in yolk sac. PU. 1 were not expressed at day 16, and then expressed.</p><p><b>CONCLUSIONS</b>The hematopoietic properties of yolk sac may be dependent on signaling through FGF receptors and FGF1 plays an important role in hematopoietic stem cell homeostasis. The FGF pathway regulates primitive hematopoiesis by modulating transcription factors such as Gata1 expression level and activity.</p>
Subject(s)
Humans , Embryo, Mammalian , Metabolism , Physiology , Fibroblast Growth Factor 1 , Metabolism , Hematopoiesis , In Vitro Techniques , Yolk Sac , Metabolism , PhysiologyABSTRACT
<p><b>OBJECTIVE</b>To study the expression and distribution of KDR, VEGF and CD34 in yolk sac and liver of human embryo at different development stage.</p><p><b>METHODS</b>Yolk sacs and livers of 15 human embryos were analyzed by the immunohistochemical SP kits for the expression of KDR, VEGF and CD34.</p><p><b>RESULTS</b>KDR, VEGF and CD34 were all expressed in yolk sacs and livers of the embryos. In the intermediate liver group, the grey value of KDR and VEGF were 103.8 +/- 6.1 and 96.4 +/- 6.3, respectively, stronger than that in the late liver group which were 90.4 +/- 6.0 and 87.4 +/- 6.3, respectively (P < 0.05). A positive correlation between the levels of KDR and VEGF was observed (P < 0.05).</p><p><b>CONCLUSION</b>The expression of KDR and CD34 in yolk sac and liver of embryo suggests the presence of hemangioblast in these organs. Interaction of KDR and VEGF might relate to survival, proliferation, migration and differentiation of hemangioblasts.</p>
Subject(s)
Humans , Embryo, Mammalian , Metabolism , Immunohistochemistry , Liver , Embryology , Metabolism , Vascular Endothelial Growth Factor A , Vascular Endothelial Growth Factor Receptor-2 , Yolk Sac , MetabolismABSTRACT
<p><b>OBJECTIVE</b>To provide materials for the study of the function of ESC42 protein specifically expressed in the human epididymis.</p><p><b>METHODS</b>The ESC42 gene was amplified from the human epididymis cDNA library by PCR and then cloned into prokaryotic expression vector pGEX-4T-1, expressed and purified by recombinant DNA techniques. The specificity of ESC42 protein was identified by Western blot and MALDI-TOF-MS. The database was searched by Ms-Fit.</p><p><b>RESULTS</b>The recombinant plasmid expressed a Mr 38 x 10(3) fusion protein in E. coli at a level of 30% of the total protein, and the purity was as high as 99%. The ESC42 protein was identified by ESC42 monoclonal antibody and its molecular weight was 11 978.12, tested by MALDI-TOF-MS. The peptide mass fingerprint analysis showed that the coverage rate of the sequence reached 48% with 100% matching. The motif scan in Prosite database reveal that ESC42 belonged to the beta-defensin family and had antibacterial activity.</p><p><b>CONCLUSION</b>Obtaining high purity of rhESC42 protein may lay a foundation for the study of its functions.</p>
Subject(s)
Animals , Humans , Male , Mice , Amino Acid Sequence , Antibodies, Monoclonal , Allergy and Immunology , Cloning, Molecular , Defensins , Genetics , Allergy and Immunology , Epididymis , Metabolism , Escherichia coli , Genetics , Gene Library , Mice, Inbred BALB C , Plasmids , Genetics , Recombinant Fusion Proteins , Allergy and ImmunologyABSTRACT
<p><b>OBJECTIVE</b>To investigate whether the -344T/C polymorphism of CYP11B2 gene is associated with essential hypertension in the Hans in Shandong province.</p><p><b>METHODS</b>Plasma renin activity (PRA) and plasma aldosterone concentration (PAC) were measured with radioimmunoassays; the hypertensives were classified as low-renin and normal- or high-renin group by PAC/PRA ratio. -344T/C polymorphism was determined by polymerase chain reaction-restricted fragment length polymorphism (PCR-RFLP) in controls and hypertensives.</p><p><b>RESULTS</b>No significant differences were found in genotype distribution or allele frequency between groups of control and primary hypertension or between groups of control and normal- or high-renin hypertension. The C allele frequency in low-renin hypertension group was significantly higher than that in normotensives and normal- or high-renin hypertension group (P < 0.05).</p><p><b>CONCLUSION</b>These results suggest that -344T/C polymorphism of CYP11B2 gene may be associated with low-renin essential hypertension in the Han nationality in Shandong province.</p>