Single-nuclei RNA sequencing (snRNA-seq) uncovers trophoblast cell types and lineages in the mature bovine placenta.

Ruminants have a semi-invasive placenta, which possess highly vascularized placentomes formed by maternal endometrial caruncles and fetal placental cotyledons and required for fetal development to term. The synepitheliochorial placenta of cattle contains at least two trophoblast cell populations, including uninucleate (UNC) and binucleate (BNC) cells that are most abundant in the cotyledonary chorion of the placentomes. The interplacentomal placenta is more epitheliochorial in nature with the chorion developing specialized areolae over the openings of uterine glands. Of note, the cell types in the placenta and cellular and molecular mechanisms governing trophoblast differentiation and function are little understood in ruminants. To fill this knowledge gap, the cotyledonary and intercotyledonary areas of the mature day 195 bovine placenta were analyzed by single nuclei analysis. Single-nuclei RNA-seq analysis found substantial differences in cell type composition and transcriptional profiles between the two distinct regions of the placenta. Based on clustering and cell marker gene expression, five different trophoblast cell types were identified in the chorion, including proliferating and differentiating UNC and two different types of BNC in the cotyledon. Cell trajectory analyses provided a framework for understanding the differentiation of trophoblast UNC into BNC. The upstream transcription factor binding analysis of differentially expressed genes identified a candidate set of regulator factors and genes regulating trophoblast differentiation. This foundational information is useful to discover essential biological pathways underpinning the development and function of the bovine placenta.

Single-cell insights into development of the bovine placenta†.

A central determinant of pregnancy success is proper development of the conceptus (embryo/fetus and associated extraembryonic membranes including the placenta). Although the gross morphology and histology of the bovine placenta have been well studied, the cellular and molecular mechanisms regulating placenta development and trophoblast differentiation and function remain essentially undefined. Here, single-cell transcriptome (scRNA-seq) analysis was performed on the day 17 bovine conceptus and chorion of day 24, 30, and 50 conceptuses (n = 3-4 samples per day) using the 10X Genomics platform. Bioinformatic analyses identified cell types and their ontogeny including trophoblast, mesenchyme, and immune cells. Loss of interferon tau-expressing trophoblast uninucleate cells occurred between days 17 and 30, whereas binucleate cells, identified based on expression of placental lactogen (CSH2) and specific pregnancy-associated glycoprotein genes (PAGs), first appeared on day 24. Several different types of uninucleate cells were present in day 24, 30, and 50 samples, but only one (day 24) or two types of binucleate cells (days 30 and 50). Cell trajectory analyses provided a conceptual framework for uninucleate cell development and binucleate cell differentiation, and bioinformatic analyses identified candidate transcription factors governing differentiation and function of the trophoblasts. The digital atlas of cell types in the developing bovine conceptus reported here serves as a resource to discover key genes and biological pathways regulating its development during the critical periods of implantation and placentation.

Insights into the lipidome and primary metabolome of the uterus from day 14 cyclic and pregnant sheep†.

In ruminants, conceptus elongation requires the endometrium and its secretions. The amino acid, carbohydrate, and protein composition of the uterine lumen during early pregnancy has been defined in sheep; however, a comprehensive understanding of metabolomic changes in the uterine lumen is lacking, particularly with respect to lipids. Here, the lipidome and primary metabolome of the uterine lumen, endometrium, and/or conceptus was determined on day 14 of the estrous cycle and pregnancy. Lipid droplets and select triglycerides were depleted in the endometrium of pregnant ewes. In contrast, select ceramides, diglycerides, and non-esterified fatty acids as well as several phospholipid classes (phosphatidylcholine, phosphatidylinositol, phosphatidylglycerols, and diacylglycerols) were elevated in the uterine lumen of pregnant ewes. Lipidomic analysis of the conceptus revealed that triglycerides are particularly abundant within the conceptus. Primary metabolite analyses found elevated amino acids, carbohydrates, and energy substrates, among others, in the uterine lumen of pregnant ewes. Collectively, this study supports the hypothesis that lipids are important components of the uterine lumen that govern conceptus elongation and growth during early pregnancy.

Characterization and regulation of extracellular vesicles in the lumen of the ovine uterus†.

Secretions of the endometrium are vital for peri-implantation growth and development of the sheep conceptus. Extracellular vesicles (EVs) are present in the uterine lumen, emanate from both the endometrial epithelia of the uterus and trophectoderm of the conceptus, and hypothesized to mediate communication between those cell types during pregnancy establishment in sheep. Size-exclusion chromatography and nanoparticle tracking analysis determined that total EV number in the uterine lumen increased from days 10 to 14 of the cycle but was lower on days 12 and 14 of pregnancy in sheep. Intrauterine infusions of interferon tau (IFNT) did not affect total EV number in the uterine lumen. Quantitative mass spectrometric analyses defined proteins and lipids in EVs isolated from the uterine lumen of day 14 cyclic and pregnant sheep. In vitro analyses found that EVs decreased ovine trophectoderm cell proliferation and increased IFNT production without effects on gene expression as determined by RNA-seq. Collective results support the idea EVs impact conceptus growth during pregnancy establishment via effects on trophectoderm cell growth.

Prostaglandin-endoperoxide synthase 2 is not required for preimplantation ovine conceptus development in sheep.

Conceptus development and elongation is required for successful pregnancy establishment in ruminants and is coincident with the production of interferon τ (IFNT) and prostaglandins (PGs). In both the conceptus trophectoderm and endometrium, PGs are primarily synthesized through a prostaglandin-endoperoxide synthase 2 (PTGS2) pathway and modify endometrial gene expression and thus histotroph composition in the uterine lumen to promote conceptus growth and survival. Chemical inhibition of PG production by both the endometrium and the conceptus prevented elongation in sheep. However, the contributions of conceptus-derived PGs to preimplantation conceptus development remain unclear. In this study, CRISPR-Cas9 genome editing was used to inactivate PTGS2 in ovine embryos to determine the role of PTGS2-derived PGs in conceptus development and elongation. PTGS2 edited conceptuses produced fewer PGs, but secreted similar amounts of IFNT to their Cas9 control counterparts and elongated normally. Expression of PTGS1 was lower in PTGS2 edited conceptuses, but PPARG expression and IFNT secretion were unaffected. Content of PGs in the uterine lumen was similar as was gene expression in the endometrium of ewes who received either Cas9 control or PTGS2 edited conceptuses. These results support the idea that intrinsic PTGS2-derived PGs are not required for preimplantation embryo or conceptus survival and development in sheep.

Progesterone effects on extracellular vesicles in the sheep uterus.

Progesterone (P4) acts via the endometrium to promote conceptus growth and implantation for pregnancy establishment. Many cells release extracellular vesicles (EVs) that are membrane-bound vesicles of endosomal and plasma membrane origin. In sheep, endometrial-derived EVs were found to traffic to the conceptus trophectoderm. Thus, EVs are hypothesized to be an important mode of intercellular communication by transferring select RNAs, proteins, and lipids between the endometrium and conceptus. Electron microscopy analysis found that the endometrial luminal and glandular epithelia were the primary source of EVs in the uterus of cyclic sheep. Size exclusion chromatography and nanoparticle tracking analysis (NTA) found that total EV number in the uterine lumen increased from day 10 to 14 in cyclic sheep. Next, ewes were ovariectomized and hormone replaced to determine effects of P4 on the endometrium and EVs in the uterine lumen. Transcriptome analyses found that P4 regulated 1611 genes and nine miRNAs in the endometrium. Total EV number in the uterine lumen was increased by P4 treatment. Small RNA sequencing of EVs detected expression of 768 miRNAs and determined that P4 regulated seven of those miRNAs. These studies provide fundamental new information on how P4 influences endometrial function to regulate conceptus growth for pregnancy establishment in sheep.

Extracellular vesicles: Novel regulators of conceptus-uterine interactions?

This review focuses on extracellular vesicles (EV) in the uterus and their potential biological roles as mediators of conceptus-uterine interactions essential for implantation and pregnancy establishment. Growing evidence supports the idea that EV are produced by both the endometrium and conceptus during pregnancy. Exosomes and microvesicles, collectively termed EV, mediate cell-cell communication in other tissues and organs. EV have distinct cargo, including lipids, proteins, RNAs, and DNA, that vary depending on the cell of origin and regulate processes including angiogenesis, adhesion, proliferation, cell survival, inflammation, and immune response in recipient cells. Molecular crosstalk between the endometrial epithelium and the blastocyst/conceptus, particularly the trophectoderm, regulates early pregnancy events and is a prerequisite for successful implantation. Trafficking of EV between the conceptus and endometrium may represent a key form of communication important for pregnancy establishment. Increased understanding of EV in the uterine environment and their physiological roles in endometrial-conceptus interactions is expected to provide opportunities to improve pregnancy success.

Creating ω-3 Fatty-Acid-Enriched Chicken Using Defatted Green Microalgal Biomass.

This study was to create an ω-3 (n-3) fatty-acid-enriched chicken product using defatted green microalgae (DGA, Nannochloropsis oceanica) biomass out of biofuel research. Hatching Ross broiler chicks were fed a corn-soybean meal diet containing 0 (control), 2, 4, 8, or 16% DGA for 6 weeks (n = 6 cages/diet). The DGA inclusion resulted in a linear (p < 0.001) increase in total n-3 fatty acids, eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA) in plasma, liver, breast, and thigh at weeks 3 and 6. The increase in the breast EPA + DHA by the 16% DGA diet reached 60-fold (p < 0.0001) over the control. The 8 and 4% DGA diets elevated (p < 0.05) liver mRNA levels of Δ-9 (88%) and Δ-6 (96) desaturases. In conclusion, 8-16% of the DGA can be added in diets for broilers to produce a n-3 fatty-acid-enriched chicken meat.