Adrenomedullin and Its Receptor Components in the Porcine Uterus and Peri-implantation Conceptuses

Adrenomedullin (ADM) is an evolutionarily conserved peptide hormone that regulates implantation, embryo spacing and placentation in humans and rodents. However, the potential roles of ADM in domestic animals, particularly pigs (as litter-bearing species), are not known. This study investigated expression patterns of ADM and its receptor components that include calcitonin receptor-like receptor (CALCRL), and receptor activity modifying protein (RAMP2) in porcine uteri and conceptuses (embryonic/fetus and its extra-embryonic membranes) during the peri-implantation period of pregnancy when 30–40% of embryonic death loss occurs. Gilts were observed for estrus and/or bred via artificial insemination at 12 and 24 h after onset of estrus (Day 0). On D 10, 11, 12, 13, 14, 15, or 16 of the estrous cycle and pregnancy (n=6 gilts per day and status), uteri were flushed with 20 ml sterile PBS (pH 7.2) after gilts were subjected to a midventral laparotomy. Pregnancy was confirmed by the presence of one or more morphologically normal conceptuses. Real-time quantitative RT-PCR analyses revealed that ADM mRNA in the endometrium was elevated for pregnant than for cyclic gilts between D 10 and 16 of pregnancy (day x status, P < 0.05). In cyclic gilts, endometrial ADM mRNA increased 9.0-fold (P < 0.05) between D 10 and 16; whereas it increased 58.1-fold (P < 0.01) in pregnant gilts between D 10 and 16. As ADM receptors, expression of mRNAs for CALCRL and RAMP2 also increased (P < 0.05) 13.4- and 5.5-fold, respectively, in the porcine conceptuses between D 10 and 16 of pregnancy. Further, ELISA analyses showed that total recoverable ADM in the uterine flushings was greater (status, P < 0.0001) for pregnant than for cyclic gilts in which ADM increased 173.8-fold (P < 0.0001) between D 10 and 16 of pregnancy. These results indicate that ADM may play functional roles in survival, growth and development of the porcine conceptus during the peri-implantation period of pregnancy.

New perspective on conceptus estrogens in maternal recognition and pregnancy establishment in the pig†

The proposed signal for maternal recognition of pregnancy in pigs is estrogen (E2), produced by the elongating conceptuses between days 11 to 12 of pregnancy with a more sustained increase during conceptus attachment and placental development on days 15 to 30. To understand the role of E2 in porcine conceptus elongation and pregnancy establishment, a loss-of-function study was conducted by editing aromatase (CYP19A1) using CRISPR/Cas9 technology. Wild-type (CYP19A1+/+) and (CYP19A1−/−) fibroblast cells were used to create embryos through somatic cell nuclear transfer, which were transferred into recipient gilts. Elongated and attaching conceptuses were recovered from gilts containing CYP19A1+/+ or CYP19A1−/− embryos on day 14 and 17 of pregnancy. Total E2 in the uterine flushings of gilts with CYP19A1−/− embryos was lower than recipients containing CYP19A1+/+ embryos with no difference in testosterone, PGF2α, or PGE2 on either day 14 or 17. Despite the loss of conceptus E2 production, CYP19A1−/− conceptuses were capable of maintaining the corpora lutea. However, gilts gestating CYP19A1−/− embryos aborted between days 27 and 31 of gestation. Attempts to rescue the pregnancy of CYP19A1−/− gestating gilts with exogenous E2 failed to maintain pregnancy. However, CYP19A1−/− embryos could be rescued when co-transferred with embryos derived by in vitro fertilization. Endometrial transcriptome analysis revealed that ablation of conceptus E2 resulted in disruption of a number biological pathways. Results demonstrate that intrinsic E2 conceptus production is not essential for pre-implantation development, conceptus elongation, and early CL maintenance, but is essential for maintenance of pregnancy beyond 30 days .

ROS-induced autophagy regulates porcine trophectoderm cell apoptosis, proliferation, and differentiation

Significant embryo loss remains a serious problem in pig production. Reactive oxygen species (ROS) play a critical role in embryonic implantation and placentation. However, the potential mechanism of ROS on porcine trophectoderm (pTr) cell fate during the peri-implantation period has not been investigated. This study aimed to elucidate the effects of ROS on pTr cell phenotypes and the regulatory role in cell attachment and differentiation. Herein, results showed that exogenous H2O2 inhibited pTr cell viability, arrested the cell cycle at S and G2/M phases, and increased cell apoptosis and autophagy protein light chain 3B and Beclin-1, whereas these effects were reversed by different concentrations of N-acetyl-l-cysteine (NAC) posttreatment. In addition, NAC abolished H2O2-induced autophagic flux, inhibited intracellular and mitochondrial ROS, and restored expression of genes important for mitochondrial DNA and biogenesis, cell attachment, and differentiation. NAC reversed H2O2-activated MAPK and Akt/mammalian target of rapamycin pathways in dose-dependent manners. Furthermore, analyses with pharmacological and RNA interference approaches suggested that autophagy regulated cell apoptosis and gene expression of caudal-related homeobox 2 and IL-1β. Collectively, these results provide new insights into the role of the ROS-induced autophagy in pTr cell apoptosis, attachment, and differentiation, indicating a promising target for decreasing porcine conceptus loss during the peri-implantation period.

118 DEEP SEQUENCING OF THE PORCINE ENDOMETRIAL TRANSCRIPTOME ON DAYS 12 AND 14 OF PREGNANCY

Establishment of pregnancy in mammals requires prolongation of luteal life span for sustained progesterone (P4) production. P4 stimulates secretory functions of the endometrium required for conceptus growth and development and is essential for endometrial receptivity. The porcine conceptus undergoes rapid differentiation and expansion of its trophoblastic membranes between Day 11 and Day 12 of gestation. With trophoblast elongation production of estrogen, the porcine embryonic pregnancy recognition signal, by the conceptus increases. A complex interplay of estrogen signaling and prostaglandin (PG) metabolism in the endometrium finally results in prevention of luteolysis. Conceptus attachment to the uterine surface epithelium starts around Day 14 of pregnancy, preceded by a pronounced vascularization at the implantation zones, initiating the epitheliochorial placentation. To characterize the complex transcriptome changes in the endometrium in the course of maternal recognition of pregnancy and initial placentation, deep sequencing of endometrial RNA samples of pregnant animals (n = 4) and corresponding non-pregnant controls (n = 4) was performed on Day 12 and Day 14 after ovulation using Illumina RNA-Seq (Illumina Inc., San Diego, CA, USA). Between 21 and 36 million 76-bp single-end reads were produced per sample. Reads were mapped with TopHat to the porcine genome assembly Sscrofa10.2 and relative expression values were calculated based on the National Center for Biotechnology Information’s gene annotation for the analysis of differential gene expression. Statistical analysis revealed more than 3500 differentially expressed genes (DEG) for Day 12 and almost 2500 DEG for Day 14 of pregnancy (DESeq, FDR 1%). Differential expression of selected genes was validated by the use of quantitative real-time PCR (RT-PCR). Comparison of the RNA-Seq data from Day 12 and Day 14 of pregnancy based on a comparison of the DEGs and of overrepresented functional categories revealed distinct differences in gene regulation, reflecting the different functions of the endometrium during these stages; that is, recognition of pregnancy on Day 12 and preparation for conceptus implantation on Day 14. The data from Day 14 was also compared to the published microarray data set from Day 14 of pregnancy (Østrup et al. 2010 Biol. Reprod.) and other sets of DEG by the use of gene set enrichment analysis (GSEA). Overall, a very good agreement with the results of the previous microarray study was found. Furthermore, a significant overlap with genes upregulated in bovine endometrium on Day 18 of pregnancy was found for the genes upregulated in porcine endometrium on Day 14. More than half of these genes were known interferon-regulated genes. In conclusion, RNA-Seq revealed distinct transcriptome changes on Day 12 and Day 14 of pregnancy in porcine endometrium associated with MRP and initiation of implantation.

Novel insights into the mechanisms of pregnancy establishment: regulation of prostaglandin synthesis and signaling in the pig

Ovarian progesterone induces essential changes leading to a temporary state of uterine receptivity for conceptus implantation. Estrogens secreted by the porcine conceptus on days 11 and 12 of pregnancy provide the initial signal for maternal recognition of pregnancy and maintenance of a functional corpus luteum (CL) for continued production of progesterone. As prostaglandins F2α(PGF2α) and E2(PGE2) exert opposing actions on the CL, a tight control over their synthesis and secretion is critical either for the initiation of luteolysis or maintenance of pregnancy. One of the supportive mechanisms by which conceptus inhibits luteolysis is changing PG synthesis in favor of luteoprotective PGE2. Conceptus PGE2could be amplified by PGE2feedback loop in the endometrium. In pigs, as in other species, implantation and establishment of pregnancy is associated with upregulation of expression of proinflammatory factors, which include cytokines, growth factors, and lipid mediators. The conceptus produces inflammatory mediators: interferon γ and interferon δ, interleukins IL1B and IL6, and PGs, which probably activate inflammatory pathways in the endometrium. The endometrium responds to these embryonic signals by enhancing further progesterone-induced uterine receptivity. Understanding the mechanisms of pregnancy establishment is required for translational research to increase reproductive efficiencies and fertility in humans and animals.