EVIDENCES OF PRIMORDIAL GERM CELL MARKERS IN ADULT LAGOSTOMUS MAXIMUS OVARY ? AN ENDEMIC MAMMAL OF SOUTH AMERICA

FRAUNHOFFER NICOLAS A.; MEILERMAN ABUELAFIA ANALIA.; INSERRA PABLO IF.; LEOPARDO NOELIA P.; VITULLO ALFREDO DANIEL.

Boston

Encuentro; International Society for Stem Cell Research 11th Annual Meeting; 2013

International Society for Stem Cell Research

BACKGROUND: A central dogma of mammalian reproductive biology is that females are born with a finite, non-renewing pool of germ cells, all of them arrested in meiosis I (oocytes) and enclosed within follicles. In 2004, studies in mice challenged this dogma. It was concluded that the adult mouse ovary retains rare female germ cells or oogonia stem cells (OSCs) that can potentially generate oocytes de novo, a situation already described in prosimian since the 1970s´. More recently, in 2012, Tilly and coworkers identified and isolated OSCs from ovaries of reproductive-age women. Whether or not OSCs remain active in the adult mammalian ovary is a matter of current debate with studies showing evidence for and against oogenesis de novo. The South American plains vizcacha, L. maximus, is a hystricognathe rodent that shows the highest ovulation rate described for a mammal. Females release 400 to 800 oocytes in each estrus cycle accompanied by a considerable number of non-ovulated oocytes remaining inside luteinizing follicles. Moreover, oogenesis doesn´t stop during gestation; an ovulatory process takes place at mid-gestation with massive formation of secondary corpora lutea, and reproductive life spans over 10 years. In such a dynamic ovary, oogenesis de novo may well account for the permanent rebuilding of tissue organization and germ cell supply. OBJECTIVE: To search for OSCs throughout the reproductive cycle in the adult ovary of L. maximus by expression analysis of primordial germ line markers. MATERIAL AND METHODS: This study was reviewed and authorized by the institutional Committee on the Use and Care of Experimental Animals. A total of 25 adult ovaries belonging to non-pregnant ovulating females (OF; N=5); females at early-(EG; N=5), mid-(MG; N=5) and late-gestation (LG; N=5), and post-partum females (PP; N=5) were studied. All samples were processed by immunohistochemistry, immunofluorescence, western blot (WB) and real time PCR (qPCR) for DDX4, IFITM-3, STELLA, BLIMP-1, DAZ-L and OCT-4. RESULTS: Pluripotency marker OCT-4 was strongly detected in surface epithelium and clusters of cells inside the ovary, especially in LG and PP ovaries, and confirmed by western blot and qPCR. Germ cell markers DDX4 and DAZ-L were expressed in cytoplasm of oocytes contained in primordial follicles in all groups. Both germ cell markers were observed in clusters of oocytes in PP period. IFITM-3 and STELLA expression had a membrane and cytoplasmic localization, respectively, and were associated especially with the surface epithelium and the tunica albuginea. The WB and qPCR showed a slight increase in LG and PP ovaries. BLIMP-1 showed a nuclear distribution in some cells associated to the surface epithelium. It was confirmed by WB and qPCR. CONCLUSION: These results support the existence of potential OSCs in the mature ovary of L. maximus by showing cells expressing pluripotency and germ cell-specific markers associated to the surface epithelium and in cell clusters inside the ovary. It is worth to note that cells expressing OSCs markers are organized in clusters especially in LG and PP females which need to reorganize the ovarian reserve for the next reproductive season.