Proteome of the early embryo-maternal dialogue in the cattle uterus

We analyzed embryo-maternal interactions in the bovine uterus on day 8 of development. Proteomic profiles were obtained by two-dimensional difference gel electrophoresis from 8 paired samples of uterine fluid (UF) from the same animal with and without embryos in the uterus. Results were contrasted with UF obtained after artificial insemination. We detected 50 differential protein spots (t test, p < 0.05). Subsequent protein characterization by nano-LC-ESI-MS/MS enabled us to identify 38 proteins, obtaining for first time the earliest evidence of involvement of the down-regulated NFkB system in cattle as a pregnancy signature pathway. Embryos enhanced the embryotrophic ability of UF and decreased uterine protein, while blood progesterone was unaltered. Twinfilin, hepatoma-derived growth factor, and synaptotagmin-binding cytoplasmic RNA interacting protein have not previously been identified in the mammalian uterus. TNFα and IL-1B were localized to embryos by immunocytochemistry, and other proteins were validated by Western blot in UF. Glycosylated-TNFα, IL-1B, insulin, lactotransferrin, nonphosphorylated-peroxiredoxin, albumin, purine nucleoside phosphorylase, HSPA5, and NFkB were down-regulated, while phosphorylated-peroxiredoxin, annexin A4, and nonglycosylated-TNFα were up-regulated. The embryonic signaling agents involved could be TNFα and IL-1B, either alone or in a collective dialogue with other proteins. Such molecules might explain the immune privilege during early bovine development

79 a dimorphic response to early male and female embryos in the bovine uterus

Sexual dimorphism has been reported in early mammalian embryos. However, it is unknown whether in utero signalling at early stages differs between male and female embryos. In this work, we used bovine embryos produced with sex-sorted spermatozoa to analyse embryo-maternal interactions measured as changes in uterine fluid (UF). Male (M) or female (F), Day-5 in vitro-produced embryos (E) (n=23-51) were non-surgically transferred into the uteri of well-nourished heifers (body condition score=3 in a scale 0-5). All recipients (n=8) received male and female embryos within non-consecutive oestrous cycles (4 recipients with male embryos first and 4 with female embryos first). On Day 8, embryos and their corresponding diluted MUF and FUF were recovered. Proteins were extracted from a first-flushed fraction of 45mL PBS containing protease inhibitor, while flushing continued for embryo recovery. Data were analysed by ANOVA and Duncan's test. Total embryo recovery rates (RR) tended to differ (P=0.06) between ME and FE (18.6±2.5 vs 27.7±3.3). However, blastocysts RR (11.6±1.7 vs 14.2±2.3; P=0.56) and flushed volume RR (57.8±2.9 vs 60.9±2.3) did not vary between ME and FE. Recoverable protein was lower in FUF than MUF (9.0±1.2 vs 13.2±1.5μg/100μL [P<0.05] and 2580±102 vs 3450±131μg total [P<0.001], respectively). Proteomic profiles were obtained in concentrated UFs by 2-D fluorescence difference gel electrophoresis and protein characterisation by nano-LC-ESI-MS/MS. After dialyzation against SOFaaci, factors ≥3kDa contained in MUF and FUF were used in culture (1mgmL(-1)) with Day-5 male and female embryos in a 2×2 factorial design. Blastocyst development, cell counts and caspase-3 positive embryonic cells were analysed in 5 replicates. FUF and MUF differed in 41 protein spots (t-test; P<0.05), out of which 35 proteins were identified. Up-regulated proteins (n=34; in FUF) represented an increased carbohydrate metabolism activity combined with anti-stress responses, involving the NFkB system, insulin and oestradiol. PARK7, a protein not previously identified in the bovine uterus is also diferentially expressed in FUF and MUF. MUF+ME tended to show (P<0.06) higher expansion rates in vitro than MUF+FE, FUF+FE and FUF+ME (51.4±5.2 vs 30.0±5.2, 24.5±5.7 and 35.7±5.7, respectively). Trophoblast cell counts tended to be higher (P<0.10) in MUF+ME (98.7±9.5) than in FUF+FE (85.7±10.6) and MUF+FE (81.0±9.8). In the inner cell mass, caspase-positive cells percentage in MUF+ME (9.8±1.5) differed (P<0.03) from FUF+FE (15.6±1.5) (groups omitted did not show significant differences). Embryonic sex is maternally detectable at early stages, leading to a favourable uterine environment specifically induced by males, but not by females. This could be associated with a sex-selection mechanism for male embryos in well-nourished females

79 a dimorphic response to early male and female embryos in the bovine uterus

Sexual dimorphism has been reported in early mammalian embryos. However, it is unknown whether in utero signalling at early stages differs between male and female embryos. In this work, we used bovine embryos produced with sex-sorted spermatozoa to analyse embryo-maternal interactions measured as changes in uterine fluid (UF). Male (M) or female (F), Day-5 in vitro-produced embryos (E) (n=23-51) were non-surgically transferred into the uteri of well-nourished heifers (body condition score=3 in a scale 0-5). All recipients (n=8) received male and female embryos within non-consecutive oestrous cycles (4 recipients with male embryos first and 4 with female embryos first). On Day 8, embryos and their corresponding diluted MUF and FUF were recovered. Proteins were extracted from a first-flushed fraction of 45mL PBS containing protease inhibitor, while flushing continued for embryo recovery. Data were analysed by ANOVA and Duncan's test. Total embryo recovery rates (RR) tended to differ (P=0.06) between ME and FE (18.6±2.5 vs 27.7±3.3). However, blastocysts RR (11.6±1.7 vs 14.2±2.3; P=0.56) and flushed volume RR (57.8±2.9 vs 60.9±2.3) did not vary between ME and FE. Recoverable protein was lower in FUF than MUF (9.0±1.2 vs 13.2±1.5μg/100μL [P<0.05] and 2580±102 vs 3450±131μg total [P<0.001], respectively). Proteomic profiles were obtained in concentrated UFs by 2-D fluorescence difference gel electrophoresis and protein characterisation by nano-LC-ESI-MS/MS. After dialyzation against SOFaaci, factors ≥3kDa contained in MUF and FUF were used in culture (1mgmL(-1)) with Day-5 male and female embryos in a 2×2 factorial design. Blastocyst development, cell counts and caspase-3 positive embryonic cells were analysed in 5 replicates. FUF and MUF differed in 41 protein spots (t-test; P<0.05), out of which 35 proteins were identified. Up-regulated proteins (n=34; in FUF) represented an increased carbohydrate metabolism activity combined with anti-stress responses, involving the NFkB system, insulin and oestradiol. PARK7, a protein not previously identified in the bovine uterus is also diferentially expressed in FUF and MUF. MUF+ME tended to show (P<0.06) higher expansion rates in vitro than MUF+FE, FUF+FE and FUF+ME (51.4±5.2 vs 30.0±5.2, 24.5±5.7 and 35.7±5.7, respectively). Trophoblast cell counts tended to be higher (P<0.10) in MUF+ME (98.7±9.5) than in FUF+FE (85.7±10.6) and MUF+FE (81.0±9.8). In the inner cell mass, caspase-positive cells percentage in MUF+ME (9.8±1.5) differed (P<0.03) from FUF+FE (15.6±1.5) (groups omitted did not show significant differences). Embryonic sex is maternally detectable at early stages, leading to a favourable uterine environment specifically induced by males, but not by females. This could be associated with a sex-selection mechanism for male embryos in well-nourished females