Study On Neo-Oogenesis In The Postnatal Rat Ovary

Recently several reports provided evidences that indicate ovarian surface epithelium(OSE) of juvenile and adult mice contains germline stem cells(GSCs) that sustain postnatal follicular renewal.But their reports have met with skepticism and doubts.Here we carried out some relevant researches in rat to examine whether GSCs and neo-oogenesis exist in adult rat ovaries.In order to identify germ cells in ovary,we prepared a mouse anti-RVLG polyclonal antibody.A 627-bp cDNA fragment of RVLG(rat vasa-like gene) was cloned and sequence analysis showed that it was 60-bp longer than that released in the GenBank(NM₀01077647).Aligment with the RVLG-containing genomic DNA sequence(NW₀01084795.1) indicated that 45-bp fragment is located at the 3' of exon 7,other 15-bp at the 5' of exon 9,both resulting from an alternative splicing of the RVLG pre-mRNA in its intron 7 and intron 8.To further study the alternative splicing pattern in the whole ORF,we cloned the full-length of RVLG cDNA.Sequence analysis revealed the existence of the 45-bp,but the other 15-bp is absent.The R VLG cDNA fragment was inserted into the prokaryotic expression vector pGEX-4T-1,and transformed into E.coli BL21(DE3) to express the GST-pRVLG fusion protein.The GST-pRVLG fusion protein was expressed in E.coli BL21 (DE3) at a high level which accounts for more than 10%of the total bacterial cellular protein.The mouse anti-rat RVLG polyclonal antibody was generated by immunizing KM mouse with purified GST-pRVLG fusion protein followed by celiacly injecting the mouse with S180 cells.The antibody recognizes RVLG protein specifically and its titer is about 1:20000.We analyzed the presence of GSCs in the ovarian surface epithelium(OSE) and the expression of meiotic marker genes,which are required for neo-oogenesis, in juvenile and young adult rat ovaries.No large ovoid cells were observed in the OSE,and the RVLG expressing,BrdU incorporating germ cells were not identified in the postnatal ovarian cortex by immunohistochemical analysis.Transcription of SCP1,SCP3 and SPO11,three early meiotic-specific and oogenesis-associated genes,was undetectable in postnatal rat ovaries using RT-PCR.The early meiocytes expressing meiotic entry markers such as DMC1,STRA8 and SCP3, were not observed using Western blotting and immunohistochemical methods in juvenile and young rat ovarian cortex.Taken together,we didn't observe proliferating germline cells and meiosis in the postnatal rat ovary.Thus our data didn't support the postulation that GSCs and neo-oogenesis may exist in the postnatal rat ovary.