The Journal of Reproduction and Development Supplement

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For an adequate functionality of corpus luteum (CL), luteal vasculature should be developed. Angiogenesis is controlled by mitogenic and angiostatic factors. Vascular endothelial growth factor (VEGF) is the main factor involved on equine CL angiogenesis. However, other factors like cytokines may participate on its regulation. The aim of the present work was to evaluate the role of: (i) tumor necrosis factor alpha (TNF), interferon gamma (IFN) and Fas Ligand (FasL) on mRNA expression of VEGF, VEGFR2, thrombospondin 1 (TSP1) and its receptor CD36 in equine luteal cells, throughout the luteal phase; and (ii) on VEGF protein expression in cells from mid luteal phase CL (mid CL). Besides, the effect of VEGF on mid CL secretory capacity (progesterone - P₄ and prostaglandin E₂ - PGE₂) was addressed. In the early CL, TNF increased mRNA expression of VEGF and VEGFR2 and reduced CD36, which was also decreased by IFN, FasL and TNF+IFN+FasL. In mid CL, TNF increased mRNA expression and protein of VEGF and reduced mRNA expression of CD36, FasL and TNF+IFN+FasL decreased VEGF protein expression. In the late CL, TNF and TNF+IFN+FasL reduced mRNA expression of VEGFR2 and TNF+IFN+FasL increased TSP1 and CD36. In mid CL VEGF (50 ng/ml) stimulated P₄ and PGE₂ secretion. The regulatory role of cytokines on angiogenic/antiangiogenic factors expression and the role of VEGF on luteal secretory capacity were shown, for the first time in the equine CL. The present study substantiates the importance of the immune system on vascular dynamics regulation and secretory function.

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In the present study, we determined the methylation status of CpG sites in the Dnmt1o 5-flanking region during spermatogenesis. DNA of germ cells during spermatogenesis (spermatogonia, spermatocyte, spermatide) was obtained by microdissection technique from testis of 9-week-old mice. During spermatogenesis, the methylation pattern within the CpGs sites in the Dnmt1o 5-flanking region was dramatically changed between stages, and the methylation at the CpGs in -993 bp, -984 bp and -590 to -53 in the 5-flanking region of the Dnmt1o gene was hypermethylated only at the spermatocytes stage, but hypomethylated in both spermatogonia and spermatid stages. Furthermore, the Dnmt1o 5-flanking region was full methylated in matured sperm. Intriguingly, we observed that there was significant non-CpG methylation in the spermatocytes during spermatogenesis. Moreover, during spermatogenesis, the low degree of the methylation level of CpG sites in spermatocytes increased to a higher degree in sperm, while the high ratio of the methylation of non-CpG sites in spermatocytes decreased. Therefore, germ cells in the spermatogenesis stages showed reciprocal methylation pattern between CpG and non-CpG in the 5-flanking region of Dnmt1o. Taken together, germ cells showed inverted methylation pattern between CpG and non-CpG sites in the Dnmt1o 5-flanking region, and the non-CpG methylation preferentially methylated in CpG poor region and associated with absence of methylated CpG in the Dnm1o 5-flanking region.

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