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Infertility and subfertility represent major problems in domestic animals and humans, and the majority of embryonic loss occurs during the first month of gestation that involves pregnancy recognition and conceptus implantation. The critical genes and physiological pathways in the endometrium that mediate pregnancy establishment and success are not well understood. In study one, predominantly Angus heifers were classified based on fertility using serial embryo transfer to select animals with intrinsic differences in pregnancy loss. In each of the four rounds, a single in vitro-produced, high-quality embryo was transferred into heifers on Day 7 postestrus and pregnancy was determined on Days 28 and 42 by ultrasound and then terminated. Heifers were classified based on pregnancy success as high fertile (HF), subfertile (SF), or infertile (IF). In study two, fertility-classified heifers were resynchronized and bred with semen from a single high-fertility bull. Blood samples were collected every other day from Days 0 to 36 postmating. Pregnancy rate was determined on Day 28 by ultrasound and was higher in HF (70.4%) than in heifers with low fertility (36.8%; SF and IF). Progesterone concentrations in serum during the first 20 days postestrus were not different in nonpregnant heifers and also not different in pregnant heifers among fertility groups. In study three, a single in vivo-produced embryo was transferred into fertility-classified heifers on Day 7 postestrus. The uteri were flushed on Day 14 to recover embryos, and endometrial biopsies were obtained from the ipsilateral uterine horn. Embryo recovery rate and conceptus length and area were not different among the heifer groups. RNA was sequenced from the Day 14 endometrial biopsies of pregnant HF, SF, and IF heifers (n = 5 per group) and analyzed by edgeR-robust analysis. There were 26 differentially expressed genes (DEGs) in the HF compared to SF endometrium, 12 DEGs for SF compared to IF endometrium, and three DEGs between the HF and IF endometrium. Several of the DEG-encoded proteins are involved in immune responses and are expressed in B cells. Results indicate that preimplantation conceptus survival and growth to Day 14 is not compromised in SF and IF heifers. Thus, the observed difference in capacity for pregnancy success in these fertility-classified heifers is manifest between Days 14 and 28 when pregnancy recognition signaling and conceptus elongation and implantation must occur for the establishment of pregnancy.

The developmental program of the embryo displays a plasticity that can result in long-acting effects that extend into postnatal life. In mammals, adult phenotype can be altered by changes in the maternal environment during the preimplantation period. One characteristic of developmental programming during this time is that the change in adult phenotype is often different for female offspring than for male offspring. In this paper, we propose the hypothesis that sexual dimorphism in preimplantation programming is mediated, at least in part, by sex-specific responses of embryos to maternal regulatory molecules whose secretion is dependent on the maternal environment. The strongest evidence for this idea comes from the study of colony-stimulating factor 2 (CSF2). Expression of CSF2 from the oviduct and endometrium is modified by environmental factors of the mother, in particular seminal plasma and obesity. Additionally, CSF2 alters several properties of the preimplantation embryo and has been shown to alleviate negative consequences of culture of mouse embryos on postnatal phenotype in a sex-dependent manner. In cattle, exposure of preimplantation bovine embryos to CSF2 causes sex-specific changes in gene expression, interferon-τ secretion and DNA methylation later in pregnancy (day 15 of gestation). It is likely that several embryokines can alter postnatal phenotype through actions directed towards the preimplantation embryo. Identification of these molecules and elucidation of the mechanisms by which sexually-disparate programming is established will lead to new insights into the control and manipulation of embryonic development.

Sex affects function of the developing mammalian embryo as early as the preimplantation period. There were two goals of the current objective. The first was to determine the degree and nature of differences in gene expression between female and male embryos in the cow at the morula stage of development. The second objective was to determine whether DKK1, a molecule known to alter differentiation of the blastocyst, would affect gene expression differently for female and male morulae. In Experiment 1, female and male embryos were treated with DKK1 at Day 5 after insemination. Morulae were harvested 24 h after treatment, pooled in groups of 20 for microarray analysis and RNA subjected to analysis of gene expression by microarray hybridization. There were 662 differentially expressed genes between females and males and 128 of these genes had a fold change ≥ 1.5 between the two sexes. Of the genes upregulated in females, 49.5% were located in the X chromosome. Functional analysis predicted that cell survival was greater in female embryos. Experiment 2 involved a similar design except that transcripts for 12 genes previously reported to be affected by sex, DKK1 or the interaction were quantified by quantitative polymerase chain reaction. Expression of all genes tested that were affected by sex in experiment 1 was affected in a similar manner in Experiment 2. In contrast, effects of DKK1 on gene expression were largely not repeatable in Experiment 2. The exception was for the Hippo signaling gene AMOT, which was inhibited by DKK1. In Experiment 3, embryos produced by fertilization with unsorted sperm were treated with DKK1 at Day 5 and abundance of transcripts for CDX2, GATA6, and NANOG determined at Days 5, 6 and 7 after insemination. There was no effect of DKK1 on expression of any of the three genes. In conclusion, female and male bovine embryos have a different pattern of gene expression as early as the morula stage, and this is due to a large extent to expression of genes in the X chromosomes in females. Differential gene expression between female and male embryos is likely the basis for increased resistance to cell death signals in female embryos and disparity in responses of female and male embryos to changes in the maternal environment.

Abstract Background While initially sensitive to heat shock, the bovine embryo gains thermal resistance as it progresses through development so that physiological heat shock has little effect on development to the blastocyst stage by Day 5 after insemination. Here, experiments using 3’ tag digital gene expression (3’DGE) and real-time PCR were conducted to determine changes in the transcriptome of morula-stage bovine embryos in response to heat shock (40 degrees C for 8 h) that could be associated with thermotolerance. Results Using 3’DGE, expression of 173 genes were modified by heat shock, with 94 genes upregulated by heat shock and 79 genes downregulated by heat shock. A total of 38 differentially-regulated genes were associated with the ubiquitin protein, UBC. Heat shock increased expression of one heat shock protein gene, HSPB11, and one heat shock protein binding protein, HSPBP1, tended to increase expression of HSPA1A and HSPB1, but did not affect expression of 64 other genes encoding heat shock proteins, heat shock transcription factors or proteins interacting with heat shock proteins. Moreover, heat shock increased expression of five genes associated with oxidative stress (AKR7A2, CBR1, GGH, GSTA4, and MAP2K5), decreased expression of HIF3A, but did not affect expression of 42 other genes related to free radical metabolism. Heat shock also had little effect on genes involved in embryonic development. Effects of heat shock for 2, 4 and 8 h on selected heat shock protein and antioxidant genes were also evaluated by real-time PCR. Heat shock increased steady-state amounts of mRNA for HSPA1A (PHSP90AA1 (PSOD1 or CAT. Conclusions Changes in the transcriptome of the heat-shocked bovine morula indicate that the embryo is largely resistant to effects of heat shock. As a result, transcription of genes involved in thermal protection is muted and there is little disruption of gene networks involved in embryonic development. It is likely that the increased resistance of morula-stage embryos to heat shock as compared to embryos at earlier stages of development is due in part to developmental acquisition of mechanisms to prevent accumulation of denatured proteins and free radical damage.