FSHR and Premeiotic Marker STRA8 Expression of Adult Female Bone Marrow Stem Cells Induced by Retinoic Acid

One of the most devastating adverse effects of cancer treatments including bone marrow transplantation is damage to the reproductive system, which in young girls and women less than 40 years old is frequently associated with premature menopause and infertility, although adult bone marrow stem cells (BMSCs) are able to generate cells of all three germ layers under appropriate experimental conditions. These outcomes seem to be a result of cytotoxic effects on oocytes housed within the ovaries. Recent studies showed that female BMSCs (FBMSC) could differentiate into granulosa cells (GCs) and oocyte and form primordial follicles in ovary after FBMSCs transplantation and might have repaired chemotherapy-induced damage to the niche or the ovarian stroma. The formation of new primordial follicles in adult mammal ovaries is a controversial issue because some new studies have concluded that all offspring were derived from the recipient germ line after FBMSCs transplantation. To explore the effects of FBMSCs on female reproductive function, the experiment is to investigate whether adult female BMSCs are able to differentiate into Follicle-stimulating hormone receptor (FSHR, a GCs specific marker in the ovary cells) positive GCs and stra8 (stimulated by retinoic acid gene 8, a specific expression gene in mammalian germ cell’s transition from mitosis to meiosis) expression oogonia in vitro induced by all-trans retinoic acid (RA).

Methods: FBMSCs were separated from adult female SD rat bone marrow. FBMSCs were cultured in DMEM supplemented with 10% fetal bovine serum. All experiments were performed using FBMSCs from the 2nd passage adherent cell fraction. FBMSCs were cultured above medium with 10⁻⁶ M RA or without RA(control group). RT-PCR was performed to detect the expression of Stra8 mRNA and FSHR mRNA, Western blot and immunocytochemistry staining was utilized to examine the expression of FSHR in FBMSCs cultured with RA.

Results:

1. Some of the FBMSCs were large and round and had a oocyte-like cell appearance 7 days after induction culture.

2. Some FBMSCs were FSHR positive by immunocytochemistry 3 days after induction differentiation, the FSHR⁺ cells were small or middle and round and were found in small clusters. Following longer in culture(from day 5 to day 7), the most of FSHR⁺ cells gradually became larger. The FBMSCs also expressed FSHR mRNA by RT-PCR and FSHR protein by Western Blotting 7days after induction differentiation. In control group, the FBMSC still kept undifferentiated relatively elongated or spindle-shaped cells that they were FSHR negative.

3. The FBMSCs expressed Stra8 mRNA by RT-PCR 7days after induction differentiation, the RA-regulated gene specifically expressed in premeiotic germ cells and a marker of the stem cells transdifferentiated into germ cells.

In control group, the FBMSCs were stra8 negative. Recent reports indicated that Stra8 was expressed in embryonic germ cells of ovary in mice and was not expressed after birth in ovary, it implied that there was not oocyte production in postnatal ovary. Our results is not support the view and suggest that adult FBMSCs could be able to generate oocytes.

Conclusions: These results suggest that RA could induce FBMSC to differentiate into GC-like cells and oogonia-like cells in vitro. Stra8 is not only expressed in embryonic germ cells of ovary, but also can be expressed in adult FBMSC induced by RA. Adult FBMSCs contain pluripotent stem cells that could differentiate into germ cells and gonadal somatic cells. It is a point to investigate the function of GC-like cells derived from FBMSCs and the differentiation capable of oogonia-like cells derived from FBMSCs in vitro in a further step of the research work.