120 EXPRESSION OF TUMOR NECROSIS FACTOR (TNF) AND ITS RECEPTOR TNFR2 IN BOVINE ENDOMETRIUM AND EMBRYOS DURING THE EARLY DEVELOPMENT

Abstract

Tumor necrosis factor alpha (TNF), a pleiotropic cytokine that could be involved in early embryo-maternal interactions (Muñoz et al. 2012 J. Proteome Res. 11, 751–766), binds to receptors TNFR1 and TNFR2. The TNFR2 mediates apoptotic and survival processes (Fischer et al. 2011 Cell. Signal. 23, 161–170) and its expression is hormonally regulated (Okuda et al. 2010 Mol. Cell. Endocrinol. 330, 41–48). In this work we analyzed the expression of TNFR2 by Western blot (WB) and immunocytochemistry (ICQ) and its co-localization with TNF by ICQ in bovine embryos and endometrium. Heifers that were transferred with multiple in vitro produced (IVP) embryos (n = 3) or sham transferred (n = 3) on Day 5 to horn ipsilateral to the corpus luteum were slaughtered on Day 8. Embryos were flushed and endometrial samples were collected from caruncular and intercaruncular regions in the middle and cranial horn thirds. Endometrial samples and Day 8 IVP embryos were subjected to ICQ, and the immunostaining pattern of TNFR2 and TNF was examined by confocal microscopy. Endometrial samples were also subjected to WB. Expression of TNRF2 was quantified by densitometry (immunoblots) and blind assessment (immunostaining). Data were analyzed using the GLM procedure of SAS Version 9.2 (SAS Institute Inc., Cary, NC, USA) and REGWQ test for means. Trophectoderm (TF) cells from blastocysts and uterine epithelial and stromal cells showed TNFR2 expression. TNF and TNRF2 were predominantly co-localised in embryos and endometrial samples, although occasionally they were detected independently. The presence of embryos increased TNFR2 in the basal glandular epithelia (P ≤ 0.05). Moreover, TNFR2 was higher in the intercaruncular than in the caruncular luminal epithelium (P = 0.07). The presence of embryos did not affect TNFR2 expression between cranial and middle horn thirds. However, the TNFR2 low-molecular-weight isoform (Lmw) in the caruncles and in the middle third of the uterine horn tended to increase in the presence of embryos (P ≤ 0.1). Interestingly, TNFR2 Lmw was more abundant in the middle caruncular region than in other endometrial regions (P < 0.05). Our findings suggest that TNF can mediate embryo-maternal communication in the uterus, acting both in the embryonic and maternal sides. In addition, although implantation does not begin in ruminants until elongation is complete, early bovine embryos seem to show an ability to interact with caruncles.

Project AGL2009-10059 (MICINN). M. Muñoz, A. Balseiro, B. Trigal, and E. Correia are sponsored by RYC08-03454, Contrato de Investigación para Doctores grant from INIA, Cajastur, and FPU (AP2009-5265), respectively.