114 BOVINE EMBRYO-MATERNAL RECOGNITION MAY OCCUR EARLIER IN AI THAN IN EMBRYO TRANSFER (ET)

Abstract

Embryo-maternal recognition is known to be mediated by the trophoblast and/or its secretory product during the preimplantation period. However, it was recently proposed that embryo-maternal recognition (along with the maternal immune system) could be activated by zona pellucida-degradation products including oligosaccharide chains from very early stage embryos (Fujiwara et al. 2009 J. Reprod. Immunol. 81, 1–8). If this is true, we predict that mammalian mothers recognise the presence of an embryo immediately after fertilization in vivo. The purpose of the present study was to compare the expressions of immune tolerance genes in bovine endometrial-luminal lymphoid (ELL) cells collected from animals after AI or embryo transfer (ET). ELL cells were collected from recipient cows inseminated with frozen–thawed semen on Day 0 (=oestrus, n = 8) and from cows that received a transferred embryo on Day 7 (n = 8) of the oestrous cycle. On Days 9 and 11, ELL cells ipsilateral to the corpus luteum were flushed non-surgically from four cows of each group using Ringer's solution (500 mL). To eliminate large cell clusters of endometrium and the embryo (blastocyst), the flushing medium was passed through an embryo collection filter (EmCon filter, 75 μm stainless steel screen). A single viable blastocyst was collected from each recipient cow. The recovered flushing medium containing ELL cells was centrifuged. The ELL cells were suspended in PBS and separated by Lympholyte-H (Cedarlane, Ontario, Canada) gradient centrifugation. Using RT-real-time PCR, transcripts for four immune tolerance genes in ELL cells from AI or ET examined were transforming growth factor-β (TGF-β), macrophage-colony stimulating factor (M-CSF), interleukin-10 (IL-10), and forkhead box P3 (Foxp3). Data were normalized to that of an endogenous control (glyceraldehyde-3-phosphate dehydrogenase) and analysed using ANOVA followed by Scheffe′s test. Transcripts for TGF-β, M-CSF, IL-10, and Foxp3 were detected in all RT-samples derived from ELL cells on Days 9 and 11 of the oestrous cycle. Transcript levels of TGF-β, M-CSF and IL-10 in the AI group were the same as those of the ET group (P > 0.05), although the transcript level of Foxp3, a master regulator of the function of regulatory T-cells, in the AI group was significantly higher than that of the ET group on Days 9 and 11 (P < 0.05). Furthermore, the transcript level of Foxp3 in the ET group on Day 11 was significantly higher than that of the ET group on Day 9 (P < 0.05). These results indicate that the embryo-maternal recognition in AI occurs earlier than in ET, and suggest that cows recognise an embryo earlier than those generally believed.