Identification of novel candidate factor SNX5 specific for pregnancy failure in Holstein heifers

Pregnancy loss predominantly occurs during periods between blastocyst hatching and conceptus (embryo plus extraembryonic membranes) implantation to the endometrium in cattle. Insufficient biochemical communication between conceptus and endometrium has been suspected as the primary cause for early embryonic losses. If molecules regulating this communication were identified, molecular mechanisms associated with early pregnancy success or loss could be better understood. To identify novel factors as detection markers of non-pregnant or females undergoing embryonic loss, blood sera from embryo-transferred heifers on day 7 (day 0 = day of estrus) were collected on day 17, 20, or 22, which were subjected to metabolome and global proteome iTRAQ analyses. On each sample, the metabolome analysis partly divided serum components into pregnant or not. In the iTRAQ analysis, heatmap analysis with 25 unique proteins separated into pregnant or not on day 20 or 22. Furthermore, receiver operating characteristic curve (ROC) analysis identified five candidate proteins detecting non-pregnant heifers, of which SNX5 in day 22 sera had the highest area under the curve (AUC), 0.983. We also detected SNX5 in day 22 sera from non-pregnant heifers using western blotting. These results suggest that high SNX5 in day 22 sera could predict early pregnancy loss in heifers.

Genomic Effects of Arginine on Cell Behavior of Conceptus Trophectoderm in Ungulates

Hatched ungulate (e.g., pigs, sheep and other ruminants) blastocysts undergo dramatic morphological transitions from spherical to tubular to filamentous forms to conceptuses (embryo/fetus and associated extraembryonic membranes) before implantation. L-Arginine (Arg), a conditionally essential amino acid, is required for this process to activate the mTOR cell signaling pathway to induce proliferation of both porcine and ovine conceptus trophectoderm cells. However, the genomic effects of arginine on trophectoderm cells is unknown. RNA-seq was used for a comparative transcriptome analysis of porcine and ovine trophectoderm cells to further understand effects of Arg on regulation of metabolism in trophectoderm cells. An established porcine trophectoderm (pTr) cell line isolated from D12 porcine conceptuses, as well as an established ovine trophectoderm (oTr) cell line isolated from D15 ovine conceptuses were used to determine response to Arg at the physiological concentration of 0.2 mM in a 48-h culture. In pTr cells, a total of 2,723 differentially expressed genes (DEG; 1,482 up and 1,241 down) were identified in response to Arg. In oTr cells, a total of 5,369 DEG (2,819 up and 2,550 down) were detected. Comparison analyses showed that the Arg-treated pTr and oTr transcriptomes share 873 common DEG (273 up and 342 down). Canonical pathway analyses identified the top enriched pathways in both pTr and oTr cells, including activation of actin cytoskeleton signaling, adrenomedullin signaling, and IGF-1 signaling; and inhibition of cell cycle G2/M checkpoint regulation, and p53 signaling. In response to Arg, pathways associated with cholesterol biosynthesis, and estrogen-mediated S-phase entry were exclusively activated in the pTr cells; whereas interferon signaling, ephrin receptor signaling, and integrin signaling were specifically activated in the oTr cells. Results from this study advance understanding of mechanisms responsible for elongation of ovine and porcine conceptuses and enable the rational design of future experiments.

New Insights Into the Role of β-NGF/TrKA System in the Endometrium of Alpacas During Early Pregnancy

One striking reproductive feature in South American camelids is that more than 90% of gestations are established in the left uterine horn (LUH). This phenomenon could be related to a differential vascular irrigation of the LUH. An increase of vascularization in llama endometrium was observed after systemic administration of Beta Nerve Growth Factor (β-NGF), a neurotrophin present in the uterus and placenta of various mammals that is involved in pregnancy development. We hypothesized that the β-NGF signaling pathway is related to embryo implantation in the LUH in camelids. The aim of this study was to characterize the spatial expression of β-NGF and its high-affinity receptor, TrKA, between LUH and right uterine horn (RUH) of non-pregnant (NP) and early pregnant alpacas (15 and 30 days of gestation, 15 and 30P, respectively). In addition, β-NGF, TrKA, and Vascular Endothelium Growth Factor A (VEGFA) temporal gene expression patterns and counting of blood vessels were evaluated among groups. The β-NGF and TrKA were localized in the luminal, glandular, and vascular epithelium of the alpaca uterus and in the embryonic membranes of the 30-days-old conceptus. β-NGF and TrKA immunosignal were stronger in 15P females than that of NP and 30P. In addition, TrKA signal was higher in the LUH luminal epithelium of NP and 15P alpacas than that of NP-RUH and 15P-RUH. β-NGF mRNA relative abundance was higher in the 30P-RUH than that of NP-RUH; whereas TrKA mRNA abundance only differed between 15P-RUH and NP-LUH. VEGFA mRNA relative abundance was higher in NP females compared to the LUH of 15P and 30P alpacas, and lower to their right counterparts. The number of vessels per field was higher in 15P than that of 30P. A positive correlation was observed between the number of vessels per field and β-NGF immunosignal in 15P-LUH. In contrast, the area occupied by vessels was higher in 30P alpacas than of NP and 15P females. The changes of β-NGF/TrKA expression pattern in the peri-implantation endometria between LUH and RUH and their localization in the extraembryonic membranes support the implication of the neurotrophin during implantation and pregnancy development in South American Camelids.