Spatiotemporal endometrial transcriptome analysis revealed the luminal epithelium as key player during initial maternal recognition of pregnancy in the mare

During the period of maternal recognition of pregnancy (MRP) in the mare, the embryo needs to signal its presence to the endometrium to prevent regression of the corpus luteum and prepare for establishment of pregnancy. This is achieved by mechanical stimuli and release of various signaling molecules by the equine embryo while migrating through the uterus. We hypothesized that embryo’s signals induce changes in the endometrial gene expression in a highly cell type-specific manner. A spatiotemporal transcriptomics approach was applied combining laser capture microdissection and low-input-RNA sequencing of luminal and glandular epithelium (LE, GE), and stroma of biopsy samples collected from days 10–13 of pregnancy and the estrous cycle. Two comparisons were performed, samples derived from pregnancies with conceptuses ≥ 8 mm in diameter (comparison 1) and conceptuses ≤ 8 mm (comparison 2) versus samples from cyclic controls. The majority of gene expression changes was identified in LE and much lower numbers of differentially expressed genes (DEGs) in GE and stroma. While 1253 DEGs were found for LE in comparison 1, only 248 were found in comparison 2. Data mining mainly focused on DEGs in LE and revealed regulation of genes related to prostaglandin transport, metabolism, and signaling, as well as transcription factor families that could be involved in MRP. In comparison to other mammalian species, differences in regulation of genes involved in epithelial barrier formation and conceptus attachment and implantation reflected the unique features of equine reproduction at the time of MRP at the molecular level.

Involvement of the Interferon Tau Gene in Maternal Recognition of Gestation in Sheep

Objective: To describe the involvement of the interferon tau gene in the maternal recognition of pregnancy in sheep. Design/Methodology/Approach: A search and analysis of the scientific documents retrieved from the Web of Science and Scopus databases related to the functions of the interferon tau gene in the maternal recognition of pregnancy in sheep were conducted. Results: The interferon tau gene (IFNτ) participates in maternal recognition of pregnancy to avoid possible rejection of the embryo, and supports the secretion of progesterone involved in preparing the endometrium for implantation; it also inhibits myometrial motility to maintain pregnancy. IFNτ stimulates the transcription of so-called interferon-stimulated genes (ISGs), which are the effectors of cell-autonomous antiviral defense. One of the representative members of ISGs is the interferon 15-stimulated gene (ISG15) which regulates endometrial receptivity at implantation, as well as survival, growth and development of the conceptus. Study Limitations/Implications: Most embryonic losses occur between fertilization and maternal recognition of pregnancy. Understanding this issue is essential to understanding the possible causes of early pregnancy losses. Findings/Conclusions: Considerable progress has been made in the discovery of how the IFNτ and ISG15 genes act in maternal recognition of gestation in sheep

Bovine Embryonic Mortality with Special Reference to Mineral Deficiency, Heat Stress and Endocrine Factors: A Review

Embryonic Mortality is the major cause of reproductive and economic loss in cattle and Buffaloes. Embryonic Mortality is more common during the early than the late embryonic period, i.e., from day 8th to 16th at the hatching of blastocysts and initiation of elongation and commencement of implantation without affecting cycle lengths. Early embryonic mortality is a major source of embryonic and economic loss with mortality rate up to 40%. Embryonic mortality is also reported due to mineral deficiency and heat stress in cattle and buffaloes. Physical modifications of animal environment, nutritional management with Antioxidant, trace minerals and genetic development of breeds that are less sensitive to heat stress should be best solution. Embryonic death occurs at the time of maternal recognition of pregnancy, probably related to a failure of the Interferon tau (IFNι) secretory mechanism along with progesterone deficiency and luteal insufficiency. Recent research, both in terms of physiological mechanisms and pharmacological treatments has mostly focused on the period of maternal recognition of pregnancy or the anti-luteolytic effect. hCG/ GnRH /Progesterone supplementation have shown positive results. Supplementation of interferon as anti-luteolytic agent and supplementing Omega-3 has shown encouraging results. Ovarian examination, Animal history, blood/milk progesterone levels, PAG test and ultrasound appear to be the only practical tool presently available for diagnosis of embryonic mortality. This present review article is covering all the aspects of embryonic mortality with special reference totrace minerals, heat stress, hormonal impact and interferon tau.

Epigenetic Changes in Equine Embryos After Short-term Storage at Different Temperatures

Background: In embryos subjected to assisted reproductive techniques, epigenetic modifications may occur that can influence embryonic development and establishment of pregnancy. In horses, the storage temperature during transport of fresh embryos before transfer is a major concern. The aim of this study was to determine the effects of two storage temperatures (5˚C and 20˚C) on equine embryos, collected at day 7 after ovulation and stored for 24 hours, on the following characteristics: (i) morphological and histological development; (ii) expression of candidate genes associated with embryo growth and development, maternal recognition of pregnancy, methylation, and apoptosis; and (iii) gene-specific and global-DNA methylation. Embryos (n=80) were collected from Haflinger mares (n=15) on day 7 (n=60) or day 8 (n=20) after ovulation and assigned to 4 groups: day 7 control (E7F, fresh); day 7, stored for 24h at 5°C (E5C); day 7, stored for 24h at 20°C (E20C); and day 8 control (E8F, fresh 24h time control). The embryos and the storage medium from all groups were analyzed for: (i) medium temperature, pH, and lipid peroxidation (malondialdehyde; MDA), (ii), embryo morphology, mRNA expression, and DNA methylation (immunohistochemistry and gene-specific DNA methylation). Results: Temperature during storage (E5C and E20C) did not affect embryo size (382±47 and 553±99 µm, respectively). There were no changes in pH and MDA accumulation irrespective of group. The relative mRNA abundance of specific genes related to growth and development (POU5F1, SOX2, NANOG), maternal recognition of pregnancy (CYP19A1, PTGES2), DNA methylation (DNMT1, DNMT3A, DNMT3B), and apoptosis (BAX) in the E5C and E20C were either up or downregulated (P<0.05) when compared to controls (E7F and E8F). The global methylation status, even as 5mC and 5hmC immune expression were similar among treatment groups. The specific genes ESR1, NANOG, and DNMT1 were hypomethylated (P<0.001) in E20C embryos when compared to E8F (advanced stage). Conclusions: Therefore, our study demonstrates for the first time the gene-specific and global-DNA methylation status of fresh equine embryos collected on days 7 and 8 after ovulation. In addition, short-term storage, regardless of temperature, modified gene expression and methylation of genes involved in embryo development and may therefore compromise embryo viability and development after transfer.

Evaluation of interferon-stimulated gene 15 (ISG15) expression in blood neutrophils in beef cattle poisoned by Senecio spp.

ABSTRACT: This study aimed to assess liver damage and interferon-stimulated gene 15 (ISG15) blood expression as a consequence of embryonic signaling on maternal recognition of pregnancy in beef cattle presenting natural ingestion of Senecio spp. Epidemiological aspects, as the presence of the plant, associated to gamma glutamyl transferase (GGT) activity can be used as Senecio spp. poisoning diagnosis. Maternal recognition of pregnancy period occurs when the embryo secretes interferon tau (IFNT) to signal its presence to the mother and eventually extend corpus luteum (CL) lifespan. In our study, liver damage was determined by concentration serum GGT, cytological and histopathological examinations. Reproductive status was evaluated by concentration of progesterone, CL diameter and ISG15 mRNA expression on Day 19 following fixed-time artificial insemination (FTAI). Cows were categorized into two groups based on concentration of GGT: Group 1 (GGT<30U/L) and 2 (GGT>31U/L). No difference on body condition scores was observed. All the cows presented liver damage based on cytology and histopathological exams. Cows from the Group 1 had higher pregnancy rate, presenting larger CL diameter and greater concentration of progesterone. Interestingly, ISG15 mRNA expression had no difference between Groups 1 and 2, even presenting difference in pregnancy status. These findings suggest embryonic loss beyond Day 19. It suggests late embryonic mortality may be associated to liver insufficiency. In conclusion, liver injury and/or concentration of GGT does not alter ISG15 expression on blood neutrophils, however cows presenting lower concentration of GGT (<30U/L) had increased pregnancy status. Therefore, the concentration of GGT allow us to screen liver status and foresee a successful pregnancy in beef cattle.

Temporal changes in the corpus luteum during early pregnancy reveal regulation of pathways that enhance steroidogenesis and suppress luteolytic mechanisms†

Although rescue of the corpus luteum (CL) is required for pregnancy, luteal function during maternal recognition of pregnancy remains largely unexplored. CL were collected from pregnant cattle on days 14, 17, 20, and 23, to encompass the maternal recognition of pregnancy period. Next-generation sequencing was used to profile mRNA abundance during this time, while tandem mass spectrometry and nanostring technology were used to profile proteins and miRNA, respectively. A total of 1157 mRNA were differentially abundant, while 27 miRNA changed, and 29 proteins tended to change. mRNA that increased were regulators of interferon signaling and DNA repair, while those that decreased were associated with luteolytic processes, such as calcium signaling and matrix metallopeptidase (MMP) signaling, indicating inhibition of these processes. One of these, MMP12, was regulated by prostaglandin F2A in vitro. mRNA that were maximally abundant on day 20 were primarily associated with immune processes. Two of these, C-C motif chemokine ligand 1 and NFKB inhibitor alpha, were regulated by interferon tau in vitro. MiRNA that increased were predicted to inhibit phosphatidylinositol signaling, while those that decreased may be negative regulators of steroidogenesis. One protein that was greater on day 20 than on day 14 was aldehyde dehydrogenase 1 family member A1 (ALDH1A1), which synthesizes retinoic acid. Pharmacological inhibition of this enzyme, or of retinoic acid receptor signaling, led to suppression of progesterone production in vitro. Overall, these data indicate that there are changes in the CL of pregnancy that are important for continued luteal function.