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Mass spectrometry (MS) approaches were used herein to identify metabolites and proteins in uterine flushings (UF) that may contribute to nourishing the conceptus. Ovine uteri collected on Day 12 of the estrous cycle (n = 5 ewes exposed to vasectomized ram) or Days 12 (n = 4), 14 (n = 5), or 16 (n = 5) of pregnancy (bred with fertile ram) were flushed using buffered saline. Metabolites were extracted using 80% methanol and profiled using ultraperformance liquid chromatography (LC) tandem mass spectrometry. The proteome was examined by digestion with trypsin, followed by the analysis of peptides with LC-MS/MS. Metabolite profiling detected 8510 molecular features of which 9 were detected only in UF from Day 14–16 pregnant ewes that function in fatty acid transport (carnitines), hormone synthesis (androstenedione like), and availability of nutrients (valine). Proteome analysis detected 783 proteins present by Days 14–16 of pregnancy in UF, 7 of which are as follows: annexin (ANX) A1, A2, and A5; calcium-binding protein (S100A11); profilin 1; trophoblast kunitz domain protein 1 (TKDP); and interferon tau (IFNT). These proteins function in endocytosis, exocytosis, calcium signaling, and inhibition of prostaglandins (annexins and S100A11); protecting against maternal proteases (TKDP); remodeling cytoskeleton (profilin 1); and altering uterine release of prostaglandin F2 alpha as well as inducing IFNT-stimulated genes in the endometrium and the corpus luteum (IFNT). Identifying metabolites and proteins produced by the uterus and conceptus advances our understanding of embryo/maternal signaling and provides insights into possible the causes of reproductive failure. Summary Sentence Discovery of metabolites and proteins in uterine flushings contributes to understanding how the conceptus communicates with the endometrium during early pregnancy in sheep.

Antral follicle count (AFC) and anti-Müllerian hormone (AMH) concentrations are reflective for ovarian reserve and have been associated with improved reproductive performance in cattle. Key events for regulation of uterine receptivity are orchestrated by progesterone. As progesterone concentrations are greater in animals with high than low AFC, we tested the hypothesis, if the resulting improved uterine environment will lead to improved conceptus elongation and endometrial response to interferon tau. For four years, 10 heifers with lowest and highest AFC, respectively, were selected from 120 heifers. Reproductive tracts and blood samples for progesterone and AMH analysis were collected after synchronization and insemination. For a recovered conceptus, length was determined, and interferon tau (IFNT) transcript abundance was analyzed. Endometrial transcript abundance of interferon-stimulated gene 15 (ISG15) and oxytocin receptor (OXTR) were analyzed. Progesterone concentrations did not differ between low and high AFC groups (P = 0.1). A difference in conceptus length was not observed. Endometrial abundance of ISG15 did not differ between pregnant low and high AFC heifers. Abundance of OXTR was greater in open low AFC than open high AFC heifers (P < 0.01). Interaction of AMH and pregnancy status was determined, with greater AMH in pregnant than open high AFC heifers (P < 0.05). Improved uterine environment in high vs. low AFC heifers did not result in longer conceptuses or improved endometrial response. As the increase in OXTR transcript abundance was only detected in low AFC heifers, reported differences in reproductive performance might be associated with earlier initiation of luteolysis. Summary Sentence Up-regulation of endometrial oxytocin receptor transcription is a prerequisite for the initiation of luteolysis and occurs later in heifers with increased ovarian reserve indicating temporal differences in the initiation of luteolysis. Graphical Abstract

A major unresolved issue is how the uterus influences infertility and subfertility in cattle. Serial embryo transfer was previously used to classify heifers as high-fertile (HF), subfertile (SF), or infertile (IF). To assess pregnancy loss, two in vivo-produced embryos were transferred into HF, SF, and IF heifers on day 7, and pregnancy outcome was assessed on day 17. Pregnancy rate was substantially higher in HF (71%) and SF (90%) than IF (20%) heifers. Elongating conceptuses were about twofold longer in HF than SF heifers. Transcriptional profiling detected relatively few differences in the endometrium of nonpregnant HF, SF, and IF heifers. In contrast, there was a substantial difference in the transcriptome response of the endometrium to pregnancy between HF and SF heifers. Considerable deficiencies in pregnancy-dependent biological pathways associated with extracellular matrix structure and organization as well as cell adhesion were found in the endometrium of SF animals. Distinct gene expression differences were also observed in conceptuses from HF and SF animals, with many of the genes decreased in SF conceptuses known to be embryonic lethal in mice due to defects in embryo and/or placental development. Analyses of biological pathways, key players, and ligand–receptor interactions based on transcriptome data divulged substantial evidence for dysregulation of conceptus–endometrial interactions in SF animals. These results support the ideas that the uterus impacts conceptus survival and programs conceptus development, and ripple effects of dysregulated conceptus–endometrial interactions elicit loss of the postelongation conceptus in SF cattle during the implantation period of pregnancy.

Conceptus estrogens and prostaglandins have long been considered the primary signals for maternal recognition of pregnancy (MRP) in the pig. However, loss-of-function studies targeting conceptus aromatase genes (CYP19A1 and CYP19A2) and prostaglandin–endoperoxide synthase 2 (PTGS2) indicated that conceptuses can not only signal MRP without estrogens or prostaglandins but can maintain early pregnancy. However, complete loss of estrogen production leads to abortion after day 25 of gestation. Although neither conceptus estrogens nor prostaglandins had a significant effect on early maintenance of corpora lutea (CL) function alone, the two conceptus factors have a biological relationship. To investigate the role that both conceptus estrogens and prostaglandins have on MRP and maintenance of pregnancy, a triple loss-of function model (TKO) was generated for conceptus CYP19A1, CYP19A2, and PTGS2. In addition, a conceptus CYP19A2–/– model (A2KO) was established to determine the role of placental estrogen during later pregnancy. Estrogen and prostaglandin synthesis were greatly reduced in TKO concept uses which resulted in a failure to inhibit luteolysis after day 15 of pregnancy despite the presence of conceptuses in the uterine lumen. However, A2KO placentae not only maintained functional CL but were able to maintain pregnancy to day 32 of gestation. Despite the loss of placental CYP19A2 expression, the allantois fluid content of estrogen was not affected as the placenta compensated by expressing CYP19A1 and CYP19A3, which are normally absent in controls. Results suggest conceptuses can signal MRP through production of conceptus PGE or stimulating PGE synthesis from the endometrium through conceptus estrogen. Failure of conceptuses to produce both factors results in failure of MRP and loss of pregnancy. Summary Sentence Triple knockout of conceptus CYP19A1/CYP19A2/PTGS2 results in failure to prevent luteolysis during early pregnancy in the pig. Graphical Abstract

A major unresolved issue in the cloning of mammals by somatic cell nuclear transfer (SCNT) is the mechanism by which the process fails after embryos are transferred to the uterus of recipients before or during the implantation window. We investigated this problem by using RNA sequencing (RNA-seq) to compare the transcriptomes in cattle conceptuses produced by SCNT and artificial insemination (AI) at day (d) 18 (preimplantation) and d 34 (postimplantation) of gestation. In addition, endometrium was profiled to identify the communication pathways that might be affected by the presence of a cloned conceptus, ultimately leading to mortality before or during the implantation window. At d 18, the effects on the transcriptome associated with SCNT were massive, involving more than 5,000 differentially expressed genes (DEGs). Among them are 121 genes that have embryonic lethal phenotypes in mice, cause defects in trophoblast and placental development, and/or affect conceptus survival in mice. In endometria at d 18, <0.4% of expressed genes were affected by the presence of a cloned conceptus, whereas at d 34, ∼36% and <0.7% of genes were differentially expressed in intercaruncular and caruncular tissues, respectively. Functional analysis of DEGs in placental and endometrial tissues suggests a major disruption of signaling between the cloned conceptus and the endometrium, particularly the intercaruncular tissue. Our results support a \"bottleneck\" model for cloned conceptus survival during the periimplantation period determined by gene expression levels in extraembryonic tissues and the endometrial response to altered signaling from clones.

In vitro embryo production (IVP) in cattle has gained worldwide interest in recent years, but the efficiency of using IVP embryos for calf production is far from optimal. This review will examine the pregnancy retention rates of IVP embryos and explore causes for pregnancy failures. Based on work completed over the past 25 yr, only 27% of cattle receiving IVP embryos will produce a live calf. Approximately 60% of these pregnancies fail during the first 6 wk of gestation. When compared with embryos generated by superovulation, pregnancy rates are 10% to 40% lower for cattle carrying IVP embryos, exemplifying that IVP embryos are consistently less competent than in vivo-generated embryos. Several abnormalities have been observed in the morphology of IVP conceptuses. After transfer, IVP embryos are less likely to undergo conceptus elongation, have reduced embryonic disk diameter, and have compromised yolk sac development. Marginal binucleate cell development, cotyledon development, and placental vascularization have also been documented, and these abnormalities are associated with altered fetal growth trajectories. Additionally, in vitro culture conditions increase the risk of large offspring syndrome. Further work is needed to decipher how the embryo culture environment alters post-transfer embryo development and survival. The risk of these neonatal disorders has been reduced by the use of serum-free synthetic oviductal fluid media formations and culture in low oxygen tension. However, alterations are still evident in IVP oocyte and embryo transcript abundances, timing of embryonic cleavage events and blastulation, incidence of aneuploidy, and embryonic methylation status. The inclusion of oviductal and uterine-derived embryokines in culture media is being examined as one way to improve the competency of IVP embryos. To conclude, the evidence presented herein clearly shows that bovine IVP systems still must be refined to make it an economical technology in cattle production systems. However, the current shortcomings do not negate its current value for certain embryo production needs and for investigating early embryonic development in cattle.

Conceptus expansion throughout the uterus of mammalian species with a noninvasive epitheliochorial type of placentation is critical establishing an adequate uterine surface area for nutrient support during gestation. Pig conceptuses undergo a unique rapid morphological transformation to elongate into filamentous threads within 1 h, which provides the uterine surface to support development and maintain functional corpora lutea through the production of estrogen. Conceptus production of a unique interleukin 1β, IL1B2, temporally increases during the period of trophoblast remodeling during elongation. CRISPR/Cas9 gene editing was used to knock out pig conceptus IL1B2 expression and the secretion of IL1B2 during the time of conceptus elongation. Trophoblast elongation occurred on day 14 in wild-type (IL1B2+/+) conceptuses but did not occur in ILB2-null (IL1B2−/−) conceptuses. Although the morphological transition of IL1B2−/− conceptuses was inhibited, expression of a number of conceptus developmental genes was not altered. However, conceptus aromatase expression and estrogen secretion were decreased, indicating that IL1B2 may be involved in the spatiotemporal increase in conceptus estrogen synthesis needed for the establishment of pregnancy in the pig and may serve to regulate the proinflammatory response of endometrium to IL1B2 during conceptus elongation and attachment to the uterine surface.

Establishment and maintenance of pregnancy in the pig is a complex process that relies on conceptus regulation of the maternal proinflammatory response to endometrial attachment. Following elongation, pig conceptuses secrete interferon gamma (IFNG) during attachment to the endometrial luminal epithelium. The objective here was to determine if conceptus production of IFNG is important for early development and establishment of pregnancy. CRISPR/Cas9 gene editing and somatic cell nuclear transfer technologies were used to create an IFNG loss-of-function study in pigs. Wild-type (IFNG+/+) and null (IFNG–/–) fibroblast cells were used to create embryos through somatic cell nuclear transfer. IFNG expression was not detected in IFNG–/– conceptuses on either day 15 or day 17 of pregnancy. Ablation of conceptus IFNG production resulted in the reduction of stromal CD3+ and mast cells, which localized to the site of conceptus attachment on day 15. The uteri of recipients with IFNG–/– conceptuses were inflamed, hyperemic and there was an abundance of erythrocytes in the uterine lumen associated with the degenerating conceptuses. The endometrial stromal extracellular matrix was altered in the IFNG–/– embryo pregnancies and there was an increased endometrial mRNA levels for collagen XVII (COL17A1), matrilin 1 (MATN1), secreted phosphoprotein 1 (SPP1), and cysteine-rich secretory protein 3 (CRISP3), which are involved with repair and remodeling of the extracellular matrix. These results indicate conceptus IFNG production is essential in modulating the endometrial proinflammatory response for conceptus attachment and survival in pigs. Summary sentence Ablation of IFNG in the pig conceptus causes conceptus degeneration and endometrial inflammation.

The emerging paradigm in the immunology of pregnancy is that implantation of conceptuses does not progress in an immunologically suppressed environment. Rather, the endometrium undergoes a controlled inflammatory response during implantation as trophectoderm of elongating and implanting pig conceptuses secrete the pro-inflammatory cytokine interferon gamma (IFNG). Results of this study with pigs revealed: (1) accumulation of immune cells and apoptosis of stromal cells within the endometrium at sites of implantation during the period of IFNG secretion by conceptuses; (2) accumulation of proliferating cell nuclear antigen (PCNA)-positive T cells within the endometrium at sites of implantation; (3) significant increases in expression of T cell co-signaling receptors including programmed cell death 1 (PDCD1), CD28, cytotoxic T-lymphocyte associated protein 4 (CTLA-4), and inducible T cell co-stimulator (ICOS), as well as chemokines CXCL9, 10, and 11 within the endometrium at sites of implantation; (4) significant increases in T cell co-signaling receptors, PDCD1 and ICOS, and chemokine CXCL9 in the endometrium of cyclic gilts infused with IFNG; and (5) identification of CD4+ (22.59%) as the major T cell subpopulation, with minor subpopulations of CD8+ (1.38%), CD4+CD25+ (1.08%), and CD4+CD8+ (0.61%) T cells within the endometrium at sites of implantation. Our results provide new insights into the immunology of implantation to suggest that trophectoderm cells of pigs secrete IFNG to recruit various subpopulations of T cells to the endometrium to contribute to a controlled inflammatory environment that supports the active breakdown and restructuring of the endometrium in response to implantation of the conceptus. Summary Sentence Pig conceptus IFNG recruits T cells to the endometrium to contribute to a tightly controlled inflammatory environment that supports the active breakdown and restructuring of the endometrium in response to implantation of the conceptus.

Decidualization is a critical event for the blastocyst implantation, placental development and fetal growth and the normal term. In mice, the embryo implantation to the uterine epithelial would trigger the endometrial stromal cells to differentiate into decidual stromal cells. However, decidualization in women takes place from the secretory phase of each menstrual cycle and continues to early pregnancy if there is conceptus. Deficient decidualization is often associated with pregnancy specific complications and reproductive disorders. Dramatic changes occur in the gene expression profiles during decidualization, which is coordinately regulated by steroid hormones, growth factors, and molecular and epigenetic mechanisms. Recently, emerging evidences showed that epigenetic modifications, mainly including DNA methylation, histone modification, and non-coding RNAs, play an important role in the decidualization process via affecting the target genes’ expression. In this review, we will focus on the epigenetic modifications in decidualization and open novel avenues to predict and treat the pregnancy complications caused by abnormal decidualization.