Expression Of Oct4,Vasa,Stra8 And Scp3 In The Bone Marrow Stem Cells Of The Adult Female Rats In Vitro

Objectives:The aim is to investigate whether adult FBMSCs are able to express germ stem cell marker gene Oct4 and Vasa mRNA , differentiate into Stra8 (stimulated by retinoic acid gene 8, a specific expression gene in mammalian embryonic ovarian germ cell's transition from mitosis to meiosis i.e. the meiosis initiating gene) expression cells and the meiosis–specific gene Scp3(synaptonemal complex protein 3) expression cells in vitro.Methods:FBMSCs were separated from adult female SD rat bone marrow. All experiments were performed using FBMSCs from the 3nd passage adherent cell fraction. FBMSCs were cultured in DMEM supplemented with 15% fetal bovine serum(FBS) with 10-5M all-trans retinoic acid(ATRA group) or without ATRA(control group) for 8 days; RT-PCR was performed to detect the expression of germ stem cell marker gene Oct4 and Vasa mRNA , the meiosis initiating gene Stra8 mRNA and the meiosis–specific gene Scp3 mRNA; The resultant PCR products of Oct4 and Stra8 were sequenced in both directions;Immunocytochemistry staining was utilized to examine the expression of Scp3 protein in FBMSCs.Results:1. In ATRA group, some of the the FBMSCs were small or large in size and round or spherical in shape and had several small cells around the large and round cell. In control group, the FBMSCs still kept undifferentiated relatively elongated or spindle-shaped cells2. The FBMSCs of both ATRA group and control group expressed germ stem cell marker gene Oct4 and Vasa mRNA and the meiosis initiating gene Stra8 mRNA by RT-PCR from 1 to 8 days after induction differentiation. The sequence of the PCR product of Oct4 was matched 99% with the sequence of Oct4 in Genebank and the sequence of the PCR product of Stra8 was matched 100%.3. The FBMSCs of ATRA group expressed the meiosis–specific gene Scp3 mRNA by RT-PCR from 8 to 15 days after Stra8 mRNA and Vasa mRNA expression. But the FBMSCs in control group did not detect the expression of Scp3 mRNA.4. Some of the FBMSCs in ATRA group were Scp3 positive on 10, 15 and 29 day after culture with ATRA by immunocytochemistry. The cells were middle and round and were found in small clusters. Following longer in culture(from day 10 to day 29), the number of Scp3 positive cells gradually increased and the cells became larger. In control group, a few Scp3 positive cells began appearance on 15 day.Conclusions:1. The adult FBMSCs expressed germ stem cell marker gene Oct4 and Vasa mRNA and the meiosis initiating gene Stra8 in the ATRA group and the control group cultured in high FBS concentration. Stra8 is not only expressed in embryonic germ cells of ovary, but also can be expressed in adult FBMSCs. These results suggest that Adult FBMSCs contain pluripotent stem cells that could differentiate into oocyte.2. The expression of meiosis–specific gene Scp3 mRNA and protein were detected in the FBMSCs of ATRA group after Stra8 mRNA and Vasa mRNA expression (from 8 to 15 day). The FBMSCs in control group were not able to detecte the expression of Scp3 mRNA by RT-PCR, but were able to detecte the expression of Scp3 protein from 15 day by immunocytochemistry. It is possible that the number of the survival cells in control group was too small to obtain the enough total mRNA needed by RT-PCR in longer culture period in our culture condition.3. ATRA can accelerate the expression of Scp3 protein in the FBMSCs. The Scp3 is a component of the synaptonemal complex, a meiosis-specific protein structure essential for synapsis of homologous chromosomes during meiotic prophase I. The result demonstrate that some of the adult FBMSCs are able to develop and differentiate into meiotic prophase I oocyte-like cells.