96 AN IMMUNOHISTOCHEMICAL STUDY ON MARKERS OF PLURIPOTENCY AND DNA METHYLATION IN THE DEVELOPING PORCINE GERM LINE

Abstract

Reprogramming of the germ line genome is a fundamental molecular process involving DNA demethylation. This has been demonstrated in the mouse (Seki Y et al. 2005 Dev. Biol. 278, 440–458), but has not yet been studied in the pig. From a large collection of porcine embryos/fetuses 2 to 7 weeks post-insemination (p.i.), a total of 35 randomly selected specimens from the end of the second (n = 10), third (n = 10), fourth (n = 5), and seventh (n = 10) gestational week were selected for an immunohistochemical study on pluripotency markers and DNA methylation in the developing germ line. Intact embryos and isolated developing gonads were paraffin embedded, sectioned (5 to 15 μm) and evaluated for the expression of markers of pluripotency [OCT4 (sc-8628, Santa Cruz Biotech., Santa Cruz, CA), Nanog (500-P236, PeproTech EC, Rocky Hill, NJ), and SOX2 (MAB2018, R&D Systems, Wiesbaden, Germany)], DNA methylation [5-methyl cytidine (ab10805, Abcam, Cambridge, MA)], and meiosis [SCP-3 (generous gift from C. Heyting)]. Heat-induced epitope retrieval (HIER) in an alkaline (pH 8.2) EDTA buffer (Shi SR et al. 2001 J. Histochem. Cytochem. 49, 931–937) and confocal laser scanning microscopy allowed for the evaluation of germ cells co-expressing OCT4 and 5-methyl cytidine. The expression of Nanog and SOX2 was found to be better visualized using HIER in an acidic (pH 6.0) citrate buffer. Isolated and clustered primordial germ cells (PGC) were identified by OCT4 labeling early during gastrulation in embryos around 2 weeks of age p.i. The amount of methylated DNA in PGC appeared similar to that in the nuclei of neighboring somatic cells at this stage. During colonization of the genital ridges, in embryos at the end of the third gestational week, this global DNA methylation status seemed to markedly decrease in PGC, remaining low in the gonadal maturing germ cells. Around onset of meiosis, in fetuses at the seventh gestational week, germ cells in 3 out of 5 female specimens studied had ceased to express markers of pluripotency. In contrast, such markers appeared to be retained in germ cells of male siblings. In conclusion, expression of pluripotency markers during porcine germ line development appears similar to what has been described in the mouse with expression ceasing at the beginning of meiosis in the female but not in the male fetus. Further, the timing of germ line DNA demethylation appears similar between the 2 species. In the mouse, PGC entering the genital ridges soon initiate meiosis, whereas in the pig, these events are separated by a 3-week period. The connection between porcine germ line pluripotency and DNA methylation status during the third to fourth week of development p.i. is presently being thoroughly investigated.