124 EXPRESSION OF PLURIPOTENCY MARKERS IN GENITAL RIDGES OF OVINE FOETUSES

Abstract

Fundamental problems of primordial germ cell (PGC) allocation, characterization, lineage restriction, and differentiation have been extensively studied in the mouse. Historically, these cells can be identified using criteria as alkaline phosphatase, but also by the expression of other pluripotency markers suchas OCT4, Nanog, cKIT, SSEA1, SSEA4. Much less is known about PGC inother mammals. The objective ofthis study wasto describe, for the first time, the expression of multiple pluripotency markers in the genital ridges of ovine fetuses evaluating the binding of antibodies for stem cell surface markers (SSEA-1 and EMA-1) and the expression of Oct4, Nanog, and Sox2 genes. Ovine fetuses (24 and 27 days) were collected from slaughtered ewes and transported in a PBS solution at 4°C, to be processed as follows: Immunohistochemistry: 4 foetuses for each age were frozen in OCT TissueTek freezing compound and stored at -80°C. Tissue blocks were cut in 6-μm sections, placed on slides, and immediately prepared for indirect immunofluorescent staining. Section were fixed in 4% paraformaldehyde (10 min) and incubated overnight with SSEA-1 (1:100) and EMA-1 (1:50) antibodies (Hybridoma bank). Antibodies were detected with fluorescently-labelled goat anti-mouse secondary antibodies (1:100 dilution; Sigma) in 15% goat serum in DPBS for 1 h at room temperature. After washing, slides were mounted using ProLong antifade mounting medium (Molecular Probes). Gene expression: total RNA was extracted from fetal genital ridge samples with Trizol™ (Invitrogen). Any contaminating DNA was removed with RNase-free DNase I (Invitrogen). First strand cDNA synthesis was performed with SuperScript™ III RNase H-Reverse Transcriptase (Invitrogen) in the presence of RNase inhibitor (Promega). Real Time PCR amplification was performed in a BioRad iCycler™, with Platinum SYBR Green qPCR Super Mix UDG (Invitrogen). The relative quantification of the target genes was calculated with the 2-ddCt method (Livak and Schmittgen 2001 Methods 25, 402-408), after normalization against β-actin expression levels. Fragment identity was confirmed by sequencing. Our results indicate that the cortical region of the genital ridges is positive to stem cell surface markers. A larger number of positive cells was recorded in Day 24 fetuses with respect to Day 27 ones. The SSEA-1 and EMA-1 showed identical expression patterns. Similar levels of Oct4 and Nanog mRNA were detected in the genital ridges at both stages, whereas Sox2 displayed a lower abundance at Day 27. In conclusion, ovine PGC can be recognized by stem cell surface antigens and by the expression of Oct4, Nanog, and Sox2 pluripotency markers, as in mouse. The combined biomolecular and immunohistochemical approach provides a useful tool to follow the ovine PGC during in vitro culture to derive pluripotent stem cells in this species.