ABSTRACT
<p><b>OBJECTIVE</b>To investigate the methylation Oct4 in orientation induced differentiation in bone marrow mesenchymal stem cells</p><p><b>METHODS</b>Mice BMSCs were isolated and purified from bone marrow by adherent culture,and then identified by morphology and immunocytochemistry.Mouse osteoblastic cells were cultured by bone fragments inoculation,and then identified by alkaline phosphatase(AKP)staining and alizarin red staining.BMSCs were induced to differentiate into osteoblasts in vitro. Indirect immunofluorescence staining and reverse transcription polymerase chain reaction(RT PCR)were used to detect the expressions of Oct4 in BMSCs before and after induction.The methylation status of Oct4 gene in mouse BMSCs was explored by a methylation specific PCR before and after induction</p><p><b>RESULTS</b>The isolated mice BMSCs massively proliferated in vitro and formed cell colones with uniform morphology.Positive expressions of CD29,cKit,and CD44 and negative expression of CD34 were found in the isolated cells.After 10 days[DK]'[DK] induction,both AKP and the alizarin red were positive in cells and osteoblastic cells isolated from mice skull bones.The indirect immunoinfluorescence staining and RT-PCR also showed that the Oct4 expression in the directed differentiation of mouse BMSCs was down-regulated.The CpG island of Otc4 gene promoter in mouse BMSCs became methylated during the induced differentiation.</p><p><b>CONCLUSIONS</b>Mice BMSCs and osteoblasts were successfully cultured in vitro in this studyOct4 may be involved in the maintenance of adult stem cell pluripotency.The down regulated expression of Oct4 gene in mouse BMSCs during the directed differentiation may contribute to the methylation of CpG island in Otc4 gene promoter.</p>
Subject(s)
Animals , Mice , Bone Marrow Cells , Cell Biology , Cell Differentiation , Cells, Cultured , CpG Islands , DNA Methylation , Mesenchymal Stem Cells , Cell Biology , Octamer Transcription Factor-3 , Metabolism , Osteoblasts , Cell Biology , Promoter Regions, GeneticABSTRACT
<p><b>AIM</b>To investigate the spatial and temporal expression of germ cell nuclear factor (GCNF) in mouse and rat epididymis during postnatal period.</p><p><b>METHODS</b>The epididymal sections from different postnatal days were stained for GCNF by the indirect immunofluorescence technique and digital photographs were taken by a Carl Zeiss confocal microscope.</p><p><b>RESULTS</b>GCNF was first detected on day 12 in mouse epididymis and day 14 in rat epididymis. The highest expression of GCNF was observed on day 35 in both mouse and rat epididymis. In adults, GCNF exhibited a region-specific expression pattern, i.e., it was expressed predominantly in the initial segment, caput and proximal corpus of rat epididymis and was abundant in the proximal corpus of mouse epididymis. GCNF could be found in the nuclei of the principal, apical, narrow, clear and halo cells.</p><p><b>CONCLUSION</b>GCNF may play an important role in epididymal differentiation and development and in sperm maturation.</p>
Subject(s)
Animals , Male , Mice , Rats , Aging , DNA-Binding Proteins , Epididymis , Chemistry , Fluorescent Antibody Technique, Indirect , Mice, Inbred BALB C , Microscopy, Confocal , Nuclear Receptor Subfamily 6, Group A, Member 1 , Rats, Sprague-Dawley , Receptors, Cytoplasmic and Nuclear , Tissue DistributionABSTRACT
<p><b>AIM</b>To assess the spatial and temporal expression of germ cell nuclear factor (GCNF) in male mouse germ cells during postnatal development and in sperm before and after capacitation.</p><p><b>METHODS</b>The indirect immunofluorescence method with anti-GCNF antiserum was used to investigate the GCNF expression in mice at day 8, 10, 14, 17, 20, 28, 35, 70, and 420 after birth and in sperm before and after capacitation.</p><p><b>RESULTS</b>With the proceeding of spermatogenesis, GCNF was first detected in the nuclei of spermatogonia and a few early stage primary spermatocytes at day 8, which was increased gradually at day 10 to 14 inclusive. From day 17 to day 20, the GCNF was concentrated in round spermatids, while both spermatogonia and early stage primary spermatocytes became GCNF negative. From day 28 until day 420, strong GCNF expression was shown in round spermatids and pachytene spermatocytes, while spermatogonia, early primary spermatocytes and elongating spermatids were all GCNF negative. In addition, it was also found that GCNF was localized on the acrosomal cap region of spermatozoa and there was a big change in GCNF expression during capacitation, from 98 % GCNF positive before capacitation to about 20 % positive following capacitation. The localization of GCNF in caput and cauda spermatozoa was similar.</p><p><b>CONCLUSION</b>GCNF may play important roles in spermatogenesis, capacitation and fertilization.</p>