Embryonic Sex Induces Differential Expression of Proteins in Bovine Uterine Fluid Enrique Gó mez,* ,† Jose ́ N. Caamañ o, † Fernando J. Corrales, ‡ Carmen Díez, † Eva Correia-A ́ lvarez, † David Martín, † Beatriz Trigal, † Susana Carrocera, † María I. Mora, ‡ Jairo Pello-Palma, § Juan F. Moreno, ∥ and Marta Muñ oz † † Centro de Biotecnología Animal−SERIDA, Camino de Rioseco 1225, La Olla−Deva, 33394 Gijó n, Asturias, Spain ‡ Centro de Investigació n Mé dica Aplicada (CIMA), Avda Pío XII 55, 31008 Pamplona, Navarra, Spain § Facultad de Química, Departamento de Química Física y Analítica, Universidad de Oviedo, Laboratorio 166, 33006 Oviedo, Asturias, Spain ∥ Sexing Technologies, Navasota, Texas, United States * S Supporting Information ABSTRACT: The bovine endometrium recognizes early embryos and reacts differently depending on the developmental potential of the embryo. However, it is unknown whether the endometrium can distinguish embryonic sex. Our objective was to analyze sexual dimorphism in the uterus in response to male and female embryos. Differentially expressed (DE) proteins, different levels of hexoses, and other embryotrophic differences were analyzed in uterine fluid (UF). Proteomic analysis of day-8 UF recovered from heifers after the transfer of day-5 male or female embryos identified 23 DE proteins. Regulated proteasome/immunoproteasome protein sub- units indicated differences in antigen processing between UF carrying male embryos (male-UF) or female embryos (female-UF). Several enzymes involved in glycolysis/gluconeogenesis and antioxidative/antistress responses were up-regulated in female-UF. Fructose concentration was increased in female-UF versus male-UF, while glucose levels were similar. In vitro cultures with molecules isolated from male-UF were found to improve male embryo development compared to female embryos cultured with molecules isolated from female-UF. We postulated that, in vivo, male embryos induce changes in the endometrium to help ensure their survival. In contrast, female embryos do not appear to induce these changes. KEYWORDS: bovine, embryo, sex, uterus, dimorphism, proteomics ■ INTRODUCTION As gestation progresses in mammals, the trophectoderm (TE) becomes a functional placenta that regulates exchanges between the embryo and the maternal blood. However, during oviductal and early uterine development, the mother and embryo enter into direct molecular “cross-talk”, which includes different endometrial reactions depending on the potential of the embryo to develop to term and beyond. 1,2 This dialogue may affect the outcome of the pregnancy and have consequences in adulthood. 3−5 In the cattle uterus, day-8 embryos overcome pro- inflammatory conditions by down-regulation of the nuclear factor kappa-B system (NFκB; 6 ). This mechanism has also been described in pig embryos. 7 In rats, increased NFκB expression in the uterus is detrimental for embryonic development. 5 These experiments using pigs, 7 rats, 5 and cattle 6 were performed with multiple embryos in the uterus. The presence of tens of embryos led to detectable changes in bovine uterine fluid (UF) as determined by proteome analysis. 6 However, in cattle, a species that normally carries only one or two embryos, no studies have detected changes induced by embryos in endometrial gene expression before day 15. 8,9 Therefore, early endometrial responses to zona-enclosed embryos seem to be conserved among polytocus and monotocus species, despite the difference between cattle and pigs in the signaling molecules used for maternal recognition of pregnancy. 10 During the cow preimplantation period, after embryonic genome activation and before X-chromosome inactivation, 11 both X chromosomes are active, with higher expression of X- linked and autosomal X-linked regulated genes in female embryos. 12 Thus, male and female embryos differ not only in their chromosomal complement but also in their epigenetic status 13 and transcriptional activity. 12,14−16 Sexually dimorphic transcription affects metabolism 17−19 and pregnancy recog- Received: September 5, 2012 Article pubs.acs.org/jpr © XXXX American Chemical Society A dx.doi.org/10.1021/pr300845e | J. Proteome Res. XXXX, XXX, XXX−XXX