The cellular communication network factor (CCN) family proteins regulate many biological events such as angiogenesis, tumor growth, placentation, implantation, and embryogenesis. The expression and function of CCN1, CCN2, and CCN3 at the maternal-conceptus interface are established in humans and rodents, but little is known about the role of CCN4 to CCN6 in the reproductive organs in any other species. Several studies in transcriptome analysis in pigs have shown that the expression of CCN4 and CCN6 increases in the endometrium during early pregnancy. However, their expression, regulation, and function in the endometrium throughout the estrous cycle and pregnancy have not been fully understood in pigs. Thus, we determined the expression, localization, and regulation of CCN4 and CCN6 during the estrous cycle and at the maternal-conceptus interface in pigs. We found that the levels of CCN4, but not CCN6, changed during the estrous cycle. The levels of CCN4 were greater during mid- to late pregnancy than in the early stage, and the levels of CCN6 were greatest on Day 15 of pregnancy. CCN4 and CCN6 were detected in conceptus tissues during early pregnancy and in chorioallantoic tissues during the later stage of pregnancy. CCN4 mRNA was mainly localized to epithelial cells, CCN6 mRNAs to epithelial and stromal cells in the endometrium. In endometrial explant cultures, CCN4 expression was increased by progesterone, and CCN6 expression by interferon-γ. These results suggest that CCN4 and CCN6 may play roles in the establishment and maintenance of pregnancy by regulating the endometrial epithelial cell functions in pigs.

Secretory leukocyte protease inhibitor (SLPI), also known as neutrophil elastase and cathepsin-G protease inhibitor, functions in protection of epithelial cells from proteases. SLPI is expressed and secreted by many mucosal tissues, including lungs, seminal vesicles and cervix in women. SLPI plays an important role in protection of endometrial epithelial cells during pregnancy from degradation by degradation by proteases derived from trophoblast at the maternal-conceptus interface. In pigs, SLPI mRNA is known to be expressed in endometrial tissues, but the expression of SLPI in the endometrium throughout the estrous cycle and pregnancy has not been determined. Therefore, we analyzed the expression and regulation of SLPI mRNA in the endometrium throughout the whole stages of the estrous cycle and pregnancy in pigs. We obtained endometrial tissues from gilts on Days 0 (day of estrus), 3, 6, 9, 12, 15, and 18 of the estrous cycle and on Days 10, 12, 15, 30, 60, 90, and 114 of pregnancy. Real-time RT-PCR analysis showed that the expression of SLPI mRNA in the endometrium increases during midt-o late pregnancy. During the estrous cycle, levels of SLPPI mRNA in estrus and proestrus were higher than those in diestrus and metestrus. In situ hybridization analysis showed that SLPI mRNA was specifically localized to the glandular epithelial cells in the endometrium during pregnancy with strong signal intensity during mid-to late pregnancy. SLPI mRNA was not detectable in conceptus tissues on Days 12 and 15 of pregnancy, but SLPI mRNA was expressed in chorioallantoic tissues during mid-to term pregnancy with increasing levels toward term pregnancy. To determine the effects of steroid hormones, estrogen and progesterone, on the expression of SLPI mRNA, endometrial explant tissues from immature pigs were treated with increasing doses of estradiol-17β (E2) and progesterone (P4). Increasing doses of E2 and P4 increased the expression of SLPI mRNA in endometrial tissues. These results showed that SLPI was expressed in the endometrium in a pregnancy stage-and cell type-specific manner and the expression of SLPI was regulated by E2 and P4 in endometrial tissues, suggesting that SLPI may play an important role in regulating the endometrial epithelial cell function during mid-to late pregnancy in pigs. Further analysis to determine the roles of SLPI at the maternal-conceptus interface is still needed.

Caspases are a family of cysteine protease enzymes composed of more than 10 members that play essential roles in apoptosis and inflammation. It has been reported that caspases play a critical role in regulating apoptosis at the maternal-conceptus interface in many species. However, the expression and regulation of caspases have not been determined in the endometrium in pigs. Therefore, we analyzed the expression, localization, and regulation of caspases in the endometrium during the estrous cycle and pregnancy in pigs. Real-time RT-PCR analysis showed that caspases were expressed in the endometrium during the estrous cycle and pregnancy. The expression of CASP6, CASP7, and CASP8 during the estrous cycle and CASP3, CASP6, CASP7, and CASP10 changed during pregnancy. Levels of CASP3 mRNA in the endometrium were higher on Day 12 of pregnancy than the estrous cycle and levels of CASP7 mRNAs were highest on Day 15 of estrous cycle and pregnancy. Immunohistochemistry analysis showed that CASP3 protein was localized to endometrial epithelial cells on Days 12 and 15 the estrous cycle and pregnancy, but cleaved CASP3 was localized only to luminal epithelial (LE) cells on Days 12 and 15 of pregnancy in the endometrium. CASP7 protein was localized to endometrial LE cells only on Day 15 of pregnancy. CASP3, CASP6, CASP7, CASP8, and CASP10 mRNAs were detectable in conceptus on D12 and D15 of pregnancy, and chorioallantoic tissues expressed CASP3, CASP6, CASP7, CASP8, CASP8, and CASP10 with increasing levels toward term pregnancy, except CASP3 mRNA. The effect of steroid hormones and interleukin-1βß (IL1B) on CASP3 expression and the effect of interferon-γ(IFNG) on CASP7 expression was determined by endometrial explant cultures and we found that CASP3 expression was increased by IL1B and CASP7 expression was increased by IFNG in a dose-dependent manner. These results showed that caspases were expressed in the endometrium during the estrous cycle and pregnancy in a stage- and/or pregnancy-specific dependent manner and some caspases were regulated by IL1B or IFNG in the endometrial tissues, suggesting that caspase may play an important role in regulating apoptosis for the establishment and maintenance of pregnancy at the maternal-conceptus interface in pigs.

Pro-inflammatory cytokines, interleukin-1β (IL1B), IL6, and tumor necrosis factor-alpha (TNF), are known to play important roles in regulating the endometrial function in the uterus during the estrous cycle and pregnancy in several species. However, the expression and function of these cytokines and their receptors in the uterine endometrium during the estrous cycle have not been studied in pigs. Thus, this study determined the expression and regulation of IL1B, IL6, TNF and their respective receptors, IL1R1, IL1RAP, IL6R, GP130, TNFRSF1A, and TNFRSF1B during the estrous cycle in pigs. To analyze levels of each gene expression in the uterine endometrium we obtained from endometrial tissues on Days 0, 3, 6, 9, 12, 15, and 18 of the estrous cycle. Real-time RT-PCR analysis showed that levels of IL1B, IL1RAP, IL6R, GP130, TNF, TNFRSF1A, and TNFRSF1B mRNAs were highest on Day 15 or 18 of the estrous cycle, which corresponds to the proestrus period. Levels of IL1R1 were highest on Day 0, while levels of IL6 were biphasic with high levels on Day 6 and Day 15. The abundance of IL1B, IL6, IL6R, and TNF mRNAs was decreased by progesterone, while levels of GP130 were increased by progesterone in endometrial tissue explants. These results showed that expression of pro-inflammatory cytokines and their receptors changed stage-specifically during the estrous cycle and regulated by progesterone in the uterine endometrium in pigs, suggesting that these pro-inflammatory cytokines may be involved in the regulation endometrial function during the estrous cycle in pigs.

The migration, adhesion, and proliferation of conceptuses during pregnancy are tightly controlled processes that are mediated by various factors including cytokines, growth factors, and hormones. Among many factors, chemokines play key roles in lymphocyte trafficking, cellular proliferation, vascularization, and embryogenesis in many mammalian species. Especially, it has been shown that C-X-C chemokine ligand 12 (CXCL12) plays an important role in early pregnancy by promoting trophoblast invasion, proliferation, and differentiation through its receptor, C-X-C chemokine receptor 4 (CXCR4) in humans. However, expression and function of CXCL12 in the uterine endometrium during pregnancy have not been well studied in pigs. Thus, we determined expression of CXCL12 and its receptor, CXCR4, in the uterine endometrium during the estrous cycle and pregnancy in pigs. We obtained endometrial tissues from gilts on day (D) 12 and D15 of the estrous cycle and D12, D15, D30, D60, D90, and D114 of pregnancy, conceptus tissues from D12 and D15 of pregnancy, and chorioallantoic tissues from D30, D60, D90, and D114 of pregnancy. Real-time RT-PCR analysis showed that levels of CXCL12 and CXCR4 mRNAs changed in the uterine endometrium during pregnancy. Levels of CXCL12 and CXCR4 mRNAs on D15 of pregnancy were higher than those on D15 of the estrous cycle. After D15 of pregnancy levels of CXCL12 and CXCR4 mRNAs gradually decreased toward term of pregnancy, and CXCL12 and CXCR4 were expressed in the chorioallantoic tissues during the mid- to late pregnancy. CXCL12 and CXCR4 mRNAs were expressed in chorioallantoic tissues during mid- to late pregnancy, and RT-PCR analysis showed that CXCL12 and CXCR4 mRNAs were detectable in conceptus on D12 and D15 of pregnancy. Immunohistochemistry showed that CXCL12 proteins were localized to endometrial luminal and glandular epithelial cells during the estrous cycle and pregnancy, and to chorionic epithelial cells during mid- to late pregnancy. Abundance of CXCL12 mRNAs, but not CXCR4, in the uterine endometrium was increased by the treatment of IFNG. These results showed that CXCL12 and CXCR4 were expressed in the uterine endometrium, conceptus, and chorioallantoic tissues and IFNG increased endometrial CXCL12 expression in pigs, suggesting that CXCL12 and its receptor may play a key role in regulation of the establishment and maintenance of pregnancy by affecting the conceptus development in pigs. [supported by the Next Generation BioGreen 21 Program (#PJ01110301), Rural Development Administration]

S100As are calcium-binding proteins with two EF-hand calcium-binding motifs. In several studies, S100A proteins are described to play important roles in pro-inflammatory responses including damage-associated molecular pattern (DAMP) signaling and in the establishment of pregnancy. However, the role of S100As have not been determined in the uterine endometrium during the estrous cycle in pigs. Thus, this study was performed to investigate expression and regulation of S100A8, S100A9, and S100A12 in the uterine endometrial tissues during the estrous cycle in pigs. Real-time RT-PCR analysis showed that S100A8, S100A9, and S100A12 mRNAs were expressed in the uterine endometrium during the estrous cycle with higher levels on days 15 and 18 of the estrous cycle than other days of cycle. To investigate the effects of steroid hormones, estradiol (E2) and progesterone (P4), on expression of S100A8, S100A9, and S100A12 mRNAs, endometrial tissue explants from immature pigs were treated with steroid hormones. Levels of S100A8, S100A9, and S100A12 were increased by the treatment of P4, and the increased levels of S100A8, S100A9, and S100A12 by P4 were not inhibited by the treatment of progesterone receptor antagonist, RU486. However, levels of S100A8, S100A9, and S100A12 were decreased by treatment of MEK inhibitor, U0126. These results exhibited that S100As were expressed in the uterine endometrium during the estrous cycle in a cyclic stage-specific manner, and their expression was affected by P4. These suggest that S100As may play an important role in endometrial function during the proestrous period of the estrous cycle in pigs. [Supported by the Next Generation Biogreen 21 program (#PJ01119103), Rural Development Administration, and by Korea Research Foundation (#2015R1D1A1A01058356)]

This study was to investigate effect of progesterone (P4) on prostaglandin (PG) synthases and plasminogen activators (PAs) system in bovine endometrium during estrous cycle. Endometrium tissues were collected from bovine uterus on follicular and luteal phase and were incubated with culture medium containing 0 (Control), 0.2, 2, 20 and 200 ng/ml P4 for 24 h. The PGF2α synthase (PGFS), PGE2 synthase (PGES), cyclooxygenase-2 (COX-2), urokinase PA (uPA), and PA inhibitors 1 (PAI-1) mRNA in bovine endometrium were analyzed using reverse transcription PCR and PA activity was measured using spectrophotometry. In results, COX-2 was higher at 2 ng/ml P4 group than control group in luteal phase (p<0.05), but, it did not change in follicular phase. Contrastively, PGES was significantly increased in 2 ng/ml P4 group compared to control group in follicular phase, but there were no significant differ among the treatments in luteal phase. uPA was no significant difference between P4 treatment groups and control group in both of different phase. PAI-1 was decreased in 20 ng/ml P4 group compared to control group in follicular phase (p<0.05). PA activity was decreased in 2 ng/ml P4 group compared to other groups in follicular and luteal phase (p<0.05). In conclusion, we suggest that P4 may influence to translation and post-translation process of PG production and PA activation in bovine endometrium.

This research was investigated the relationship between the number of the transferable embryos and estrus expression rate, BCS (Body Condition Score), which affect the nutritional state of the cow, in Holstein donor cows. CIDRs were inserted into the vaginas of twenty two head of Holstein cows, regardless of estrous cycle. Superovulation was induced using folliclar stimulating hormone (FSH). For artificial insemination, donor cows were injected with and estrus was checked about 48 hours after the injection. Then they were treated with 4 straws of semen 3 times, with 12-hour intervals. Embryos were collected by a non-surgical method 7 days after the first artificial insemination. When BCS was 2.5, the total number of collected ova was 7.3 + 1.9, which is significantly lower (p<0.05) than the numbers 15.4 + 2.8 and 15.4 + 2.1 that were obtained when BCSs were 2.75 and 3.0, respectively. Whereas the numbers of transferable embryos were 5.2 + 1.4 when BCS was 2.5, which was smaller than the numbers 6.0 + 2.1 and 8.5 + 1.8 that were obtained when BCSs were 2.75 and 3.0, respectively; however, the differences were not significant. As for estrus induction rate, the cow groups whose BCSs were 2.75 and 3.0 showed 100.0% and 95.0%, respectively. Whereas the cow group whose BCS was 2.5 showed 57.1%, and the differences were significant (p< 0.05). As for estrous expression rate, the cow groups whose BCSs were 2.5, 2.75 and 3.0 showed 100.0%, 100.0% and 85.7%, respectively; however, the differences were not significant. According to the result of this research, it is considered that the total number of collected ova and the number of transferable embryos will be affected by the nutritional state before and after in vivo embryo production and superovulation treatment, and that although the mechanism is not clear, poor stockbreeding management and nutritional level would cause the decrease of ovum recovery rate and the number of transferable embryos in high-producing cows. On the other hand, diverse researches on the superovulation treatment method that is suitable for high-producing Holstein donor cows would contribute to preventing ovarian cyclicity disorder, as well as to the early multiplication of cows with superior genes by increasing the utilization value of donor cows.

Na+/K+-ATPase, an energy-transducing ion pump, is responsible for maintenance of relatively high concentrations of potassium ions but low concentrations of sodium ions in the cell by transport of these ions across the plasma membrane and participates in transport of various nutrients including glucose, amino acids. and ions. Na+/K+-ATPase consists of α, β, and FXYD subunits, but only α and β subunits are needed for basic functions. FXYD subunit is an auxiliary protein for αβ complex of Na+/K+-ATPase. Our recent study has shown that α (ATP1A1-4) and β (ATP1B1-3) subunits of Na+/K+-ATPase are expressed in the uterine endometrium during the estrous cycle and pregnancy in pigs. In this study, we further determined expression of FXYD (FXYD1-7) subunits of Na+/K+-ATPase in the uterine endometrium during the estrous cycle and pregnancy in pigs. Real-time RT-PCR analysis showed that mRNAs for all subtypes of FXYD subunit were expressed in the uterine endometrium during the estrous cycle and pregnancy in a pregnancy status- and stage-specific fashion. In situ hybridization analysis exhibited that transcripts of all subtypes of FXYD subunit were primarily localized to luminal (LE) and glandular epithelia (GE) during the estrous cycle and early pregnancy and to chorionic membrane (CM) during mid to term pregnancy. RT-PCR analysis showed that FXYD subunits were expressed in conceptuses on D12 and D15 of pregnancy. These results indicate that all subtypes of FXYD subunit are expressed in the uterine endometrium and conceptuses during the estrous cycle and pregnancy in a pregnancy status- and stagespecific manner. These suggest that FXYD may be involved in the establishment and maintenance of pregnancy by regulating the activity of Na+/K+-ATPase in nutrient transport at the maternal-fetal interface in pigs. * This work was supported by the Next Generation BioGreen 21 program (#PJ007997), RDA and the National Research Foundation (NRF #2010-0012304) funded by the Korean Government, Republic of Korea.

The objective of this study was to investigate the relationship between estrous expression, body condition score (BCS), blood urea nitrogen (BUN) and number of transferable embryos for the purpose of improving reproductive performance in blood of Hanwoo donors. Sixty, at random stages of the estrous cycle, received a CIDR. Four days later, the animals were superovulated with a total of 28AU FSH (Antorin, 2AU=1 ml) administered twice daily in constant doses over 4 days. On the 3th administration of FSH, CIDR was withdrawn and 25 mg PGF2α was administered. Cows were artificially inseminated twice after estrous detection at 12 hr intervals. The cows received 100 μg GnRH at the time of 1nd insemination. Embryos were recovered 7 or 8 days after the 1st insemination. The estrous inducement rate and estrous expression rate were significantly lower for cows with BCS below 2.25 than for cows with BCS above 2.25. There was 50.0% of rate of mounting in cows with BCS below 2.25 whereas the rate of mounting was markedly increased in cows with BCS above 2.25 (94.1% and 89.5% for BCS 2.25～2.75 and BCS above 2.75 cows, respectively). Cows with BCS ＜2.25, 2.25～2.75 and ≥2.75 had number of transferable embryos of 4.5±0.7, 5.9±1.8 and 5.6±2.3 respectively.

Decorin (DCN) is a member of small leucine‐grich proteoglycans which are ubiquitous components of the extracellular matrix. It regulates many physiological processes, such as matrix formation, collagen fibrillogenesis, angiogenesis, cancer growth, and cardiovascular diseases. It has been shown that DCN is expressed in the uterus during pregnancy and modulates implantation and decidualization for the establishment and maintenance of pregnancy in mice and humans. Expression of DCN in the uterine endometrium during pregnancy has not been investigated in pigs. Thus, this study investigated expression of DCN in the uterine endometrium during the estrous cycle and pregnancy in pigs. Uterine endometrial tissues were from day (D) 12 and 15 of the estrous cycle and D12, D15, D30, D60, D90, and D114 of pregnancy. Northern blot and real‐gtime RT‐gPCR analyses showed that expression of DCN mRNA was detected throughout the estrous cycle and pregnancy with the highest levels during mid pregnancy. In situ hybridization analysis showed that DCN mRNA was localized to both luminal and glandular epithelia during the estrous cycle and pregnancy and also to chorionic membrane during mid pregnancy in pigs. To determine whether endometrial expression of DCN was affected by the somatic cell nuclear transfer (SCNT) procedure, DCN mRNA levels in the uterine endometrium from gilts with SCNT embryos on D30 of pregnancy were compared with those from gilts with normal embryos using real‐gtime RT‐gPCR analysis. The result showed that DCN mRNA levels in the uterine endometrium were not significantly different between gilts with normal embryos and SCNT embryos. These results suggest that DCN may play an important role for endometrial tissue remodeling during mid pregnancy, and DCN expression is not affected by the SCNT procedure at the early stage of pregnancy in pigs.

Lysophosphatidic acid (LPA), a simple phospholipid-derived mediator implicated in diverse biological actions, acts through the specific G-protein coupled receptors, LPA receptor (LPAR) 1～5. Our previous study showed that LPAR3 is expressed in the uterine endometrium in a cell type- and stage-specific manner and LPA via LPAR3 increases PTGS2 expression in the uterine endometrium during the period of implantation. Although LPAR3 is considered to be predominant LPA receptor in the uterine endometrium, other LPA receptors might play a role to mediate LPA functions in the uterine endometrium during pregnancy. Among LPARs, we investigated expression of LPAR1 during the estrous cycle and pregnancy in this study. Uterine endometrial tissue samples were collected from day (D) 12 and D15 of the estrous cycle and from D12, D15, D30, D60, D90 and D114 of pregnancy. Northern blot analysis determined that LPAR1 mRNA was constitutively expressed in the uterine endometrial tissues during the estrous cycle and pregnancy of all stages. Analysis by immunoblotting revealed that LPAR1 proteins were present in the porcine uterine endometrium during the estrous cycle and pregnancy. Immunohistochemical experiments demonstrated that LPAR1 protein was localized to endometrial epithelium and stromal cell, specifically to nuclei of these cell types. Results in this study show that LPAR1 is constitutively expressed in the uterine endometrium during the estrous cycle and pregnancy. These results suggest that LPA via LPAR1 may play a role in the uterine endometrial function throughout pregnancy in pigs.