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The most critical stage of pregnancy is embryo implantation, which relies on the synchronized developmental capacity of the embryo and uterine receptivity to implantation. In early pregnancy, conceptus and uterus release several factors enabling successful implantation and placentation. Molecules involved in embryo-maternal crosstalk include, but are not limited to, hormones, growth factors, and cytokines. The discovery of microRNAs (small non-coding RNAs regulating gene expression) has revolutionized our understanding of many biological processes, including pregnancy. To date, numerous miRNAs have been detected in different species during pregnancy, both at the endometrial and embryonic sites. Thus, microRNAs are considered important regulators of early pregnancy events. Here, we report miR-26a-5p and miR-125b-5p effects on human and pig trophoblast cell function. Both microRNAs change the level of several genes and proteins important for proper embryo development. Moreover, miR-26a-5p stimulates porcine trophoblast proliferation and has a negative impact on its affinity to laminin. However, miR-125b-5p decreases porcine trophoblast cell migration. Our studies suggest that miR-26a-5p and miR-125b-5p can affect early pregnancy functions by regulating genes and processes important for proper conceptuses’ development and progression through the implantation process.

Different strategies are used to meet optimal reproductive performance or manage reproductive health. Although exogenous human chorionic gonadotropin (hCG) and gonadotropin-releasing hormone (GnRH) agonists (A) are commonly used to trigger ovulation in estrous cycle synchronization, little is known about their effect on the ovarian follicle. Here, we explored whether hCG- and GnRH-A-induced native luteinizing hormone (LH) can affect the endocrine and molecular milieus of ovarian preovulatory follicles in pigs at different stages of sexual development. We collected ovaries 30 h after hCG/GnRH-A administration from altrenogest and pregnant mare serum gonadotropin (eCG)-primed prepubertal and sexually mature gilts. Several endocrine and molecular alternations were indicated, including broad hormonal trigger-induced changes in follicular fluid steroid hormones and prostaglandin levels. However, sexual maturity affected only estradiol levels. Trigger- and/or maturity-dependent changes in the abundance of hormone receptors (FSHR and LHCGR) and proteins associated with lipid metabolism and steroidogenesis (e.g., STAR, HSD3B1, and CYP11A1), prostaglandin synthesis (PTGS2 and PTGFS), extracellular matrix remodeling (MMP1 and TIMP1), protein folding (HSPs), molecular transport (TF), and cell function and survival (e.g., VIM) were observed. These data revealed different endocrine properties of exogenous and endogenous gonadotropins, with a potent progestational/androgenic role of hCG and estrogenic/pro-developmental function of LH.

In early pregnancy, as the embryo arrives in the uterus, intensive communication between the embryo and uterus begins. Hundreds of molecules are known to be involved, but despite numerous findings, full understanding of the complexity of the embryo–maternal dialog remains elusive. Recently, extracellular vesicles, nanoparticles able to transfer functionally active cargo between cells, have emerged as important players in cell–cell communication, and as such, they have gained great attention over the past decade also in reproductive biology. Here we use a domestic animal model (Sus scrofa) with an epitheliochorial, superficial type of placentation because of its advantage in studding uterine luminal fluid extracellular vesicles. We show that during early pregnancy, the uterine lumen is abundant with extracellular vesicles that carry a plethora of miRNAs able to target genes involved in embryonic and organismal development. These extracellular vesicles, upon the delivery to primary trophoblast cells, affect genes governing development as well as cell-to-cell signaling and interactions, consequently having an impact on trophoblast cell proliferation, migration, and invasion. We conclude that exchange of a unique population of extracellular vesicles and their molecular cargo at the maternal–embryo interface is the key to the success of embryo implantation and pregnancy.

Pentraxin 3 (PTX3), a multimeric glycoprotein, is implicated in various biological functions. PTX3 was shown to be elevated in the corpus luteum (CL) of early pregnant ewes; however, its role in sheep or other ruminants’ CL during this reproductive stage or how it is regulated remain unknown. Here we explored the role of PTX3 and its relationship with interferon-tau (IFNT; the pregnancy recognition signaling molecule during early pregnancy in domestic ruminants) in bovine luteinized granulosa cells (LGCs). IFNT robustly elevated PTX3 expression in bovine LGCs, and significantly stimulated its expression in luteal endothelial cells, along with CL slices; yet, LGCs were the most responsive and sensitive among these luteal models. ALK2/ALK3/ALK6 kinase inhibitor, dorsomorphin, dose-dependently inhibited basal and IFNT-elevated PTX3 expression in LGCs. In contrast, ALK4/5/7 inhibitor, SB431542, did not alter basal and TGFB1-induced PTX3. We found that recombinant human PTX3 itself moderately but significantly increases LGC numbers. Because PTX3 is highly expressed in bovine LGCs, we next examined the impact of lowering endogenous PTX3 levels with siRNA. PTX3 silencing decreased the viable cell numbers and reversed IFNT actions on cell viability, percentage of proliferating cells, and on two key survival/death genes: BIRC5 encoding surviving protein, and FASL – a death-inducing signal. Interestingly, thrombospondin-1, a known luteal proapoptotic factor, was inversely related to PTX3 in LGCs. Together, these findings suggest a novel role for PTX3 during early pregnancy, as mediator of IFNT prosurvival actions supporting CL maintenance during this reproductive stage.

Background: Gestagens are the most widely used therapy in anestrus type II. The aim of this research is to evaluate the effectiveness of the vaginal progesterone inserts therapy in anestrus type II in cows. Methods: The study was conducted on 33 cows. Progesterone (PR) and estrogen (ER) receptors expression in endometrium was assessed on a molecular level based on mRNA tissue expression. Additionally, blood 17ß-estradiol and progesterone levels were evaluated. Results: A decrease in mRNA expression of A and B PR and ER α was noted in treated and untreated animals. In the treated group, an increase of ERß mRNA expression was observed, while a decreased was found in untreated animals. There was increased PR, ERα and ß expression in endometrial tissue in treated cows, and decreased expression of these factors in untreated cows. In the treated group, recurrence of ovarian cyclicity was noted in 52% of animals and pregnancy was obtained in 34.8% of them, while in the untreated group, recurrence did not occur. In the control group, spontaneous recurrence of ovarian cyclicity was not observed. An increase of PR expression was correlated with increased proliferation of endometrial cells. Conclusions: It seems likely that the endometrium is well developed and ready for placentation after removing the exogenous source of progesterone and preventing the recurrence of cyclicity of ovaries.

Uterine inflammation is a common reproductive disorder in domestic animals, leading to disturbances in many reproductive processes and economic losses. More information on inflammatory pathways, however, is needed to understand mechanisms of uterine inflammation. The aim of the study was to investigate transcriptomic profiles of the pig endometrium affected by inflammation. On day 3 of the estrous cycle (day 0 = initial day of study), saline or Escherichia coli suspension were injected into uterine horns. In endometrial tissues collected 8 days later, microarray analysis results indicated there were 189 differentially abundant mRNA transcripts (DEGs, 95 in relatively greater and 94 in lesser abundance) after saline injections compared with samples where there was severe acute inflammation. Relative abundance of mRNA transcripts for proteins assigned to inflammatory response, movement of phagocytes, quantity of phagocytes, leukocyte migration and adhesion of immune cells and many other functions related to inflammation were different in the Escherichia coli-treated endometrium than in samples from gilts treated with saline. Among others, S100A9, SLC11A1, CCL15, CCL3L3, CCR1, CD48, CD163, THBS1, KIT, ITGB3, JAK3 and NFKB2 mRNA transcripts were in relatively greater abundance and there were those in relatively lesser abundance including IL24, FGG, SST, CXCL16 and CREB. In this study, for the first time, there was detection of alterations in the transcriptome of the inflamed pig endometrium which may be an important finding for maintaining uterine homeostasis and functions. Results form the basis for future studies focusing on regulation of uterine inflammation in animals and women.

Circulating, non-coding RNAs, such as microRNAs (miRNAs) have been proposed to be powerful pathophysiological indicators of pregnancy in animals and humans. Since their discovery, it is known that miRNAs can take part in numerous biological processes, including cell proliferation and differentiation during early embryonic development and establishment of pregnancy. Our recent studies have indicated that maternal blood can carry miRNAs reported previously at the embryo-maternal interface in pigs. To expand the scope of our research, we tested the hypothesis that miRNAs previously identified in conceptuses, trophoblasts, endometrium and uterine lumen-derived extracellular vesicles (EVs) collected before Day 20 of pregnancy can show reproductive status-dependent profiles in the serum of cyclic and pregnant crossbred pigs. Custom-designed TaqMan arrays, multiplex real-time reverse transcription (RT)-PCR and real-time RT-PCR allowed us to identify a number of reproductive status-dependent miRNAs in serum samples collected from pigs during the estrous cycle or pregnancy (Days 16 and 20). We found that serum samples were enriched with miRNAs involved in processes important during the estrous cycle and early pregnancy, e.g. cell sensitivity and viability, angiogenesis, embryonic cell proliferation and differentiation. Further validation revealed different abundance of ssc-miR-143-3p and ssc-miR-125b in pregnant and non-pregnant animals and correlation of ssc-miR-125b levels with litter size. In addition, analyzed serum samples contained both EVs and Argonaute2 proteins, which are known to be involved in miRNA transportation and intercellular communication. In summary, we identified several circulating miRNAs that differ in abundance between cyclic and pregnant animals and could serve as potential indicators of reproductive status in pigs during breeding management.

Lung cancer is responsible for the most cancer-related mortality worldwide and the mechanism of its development is poorly understood. Proteomics has become a powerful tool offering vital knowledge related to cancer development. Using a two-dimensional difference gel electrophoresis (2D-DIGE) approach, we sought to compare tissue samples from non-small-cell lung cancer (NSCLC) patients taken from the tumor center and tumor margin. Two subtypes of NSCLC, adenocarcinoma (ADC) and squamous cell carcinoma (SCC) were compared. Data are available via ProteomeXchange with identifier PXD032736 and PXD032962 for ADC and SCC, respectively. For ADC proteins, 26 significant canonical pathways were identified, including Rho signaling pathways, a semaphorin neuronal repulsive signaling pathway, and epithelial adherens junction signaling. For SCC proteins, nine significant canonical pathways were identified, including hypoxia-inducible factor-1α signaling, thyroid hormone biosynthesis, and phagosome maturation. Proteins differentiating the tumor center and tumor margin were linked to cancer invasion and progression, including cell migration, adhesion and invasion, cytoskeletal structure, protein folding, anaerobic metabolism, tumor angiogenesis, EMC transition, epithelial adherens junctions, and inflammatory responses. In conclusion, we identified several proteins that are important for the better characterization of tumor development and molecular specificity of both lung cancer subtypes. We also identified proteins that may be important as biomarkers and/or targets for anticancer therapy.

Seminal plasma (SP) deposited in the porcine uterine tract at the time of mating is known to elicit an initial response that is beneficial for pregnancy outcome. However, whether SP has any long-term effect on alterations in endometrial molecular and cellular processes is not known. In this study, using microarray analyses, differential changes in endometrial transcriptome were evaluated after Day 6 of SP-infusion (6DPI) or Day 6 of pregnancy as compared to corresponding day of estrous cycle. Both, pregnancy and SP induced significant changes in the endometrial transcriptome and most of these changes were specific for a particular group. Functional analysis of differentially expressed genes (DEGs) using Ingenuity Pathway Analysis revealed that inhibition in immune response was affected by both pregnancy and SP infusion. Long-term effects of SP included differential expression of genes involved in inhibition of apoptosis, production of reactive oxygen species and steroid biosynthesis, and activation of processes such as proliferation of connective tissue cells and microvascular endothelial cells. Moreover, interleukin-2 and interferon-&gamma, was identified to be responsible for regulating expression of many DEGs identified on 6DPI. The present study provides evidence for the long-term effects of SP on porcine endometrium that can be beneficial for pregnancy success.

Altrenogest with gonadotropins is commonly used to synchronize the estrous cycle, but it can also lead to follicular cyst formation, especially in prepubertal gilts. Here, we aimed to investigate how maturity and altrenogest treatment affect the development, endocrine milieu, and molecular control of ovarian follicles. Crossbred prepubertal and mature gilts were challenged or not (control) with altrenogest, and ovaries were collected in the morning on the first day of behavioral estrus. In prepubertal gilts, altrenogest decreased the percentage of primordial and atretic small follicles, but increased large antral follicles when compared with controls. In mature gilts, altrenogest reduced the percentage of primary follicles and elevated the total number of antral follicles. Maturity affected the estradiol level in the follicular fluid of preovulatory follicles, luteinizing hormone (LH)-stimulated cyclic adenosine monophosphate (cAMP) generation, and LH receptor messenger RNA (mRNA) expression in granulosa. Moreover, cytochrome P45017A1 (CYP17A1) mRNA levels in the theca layer were affected and correlated with follicular androstendione and estradiol concentration. Altrenogest negatively affected follicular fluid progesterone concentration and decreased levels of prostaglandin (PG) E2 in prepubertal gilts and PGF2alpha metabolite in mature gilts. LH-stimulated cAMP release in granulosa cells of mature gilts as well as human chorionic gonadotropin- and forskolin-induced cAMP were also affected. In addition, altrenogest downregulated CYP17A1 mRNA in the prepubertal theca layer and PGF2alpha synthase expression in the granulosa and theca layer of mature gilts. To the best of our knowledge, this is the first study to report multiple effects of maturity and altrenogest on the endocrine milieu and molecular regulations governing ovarian follicle development in gilts.

MicroRNAs (miRNAs) constitute a large family of noncoding RNAs, approximately 22 nucleotides long, which function as guide molecules in RNA silencing. Targeting most protein-coding transcripts, miRNAs are involved in nearly all developmental and pathophysiological processes in animals. To date, the regulatory roles of miRNAs in reproduction, such as fertilization, embryo development, implantation, and placenta formation, among others, have been demonstrated in numerous mammalian species, including domestic livestock such as pigs. Over the past years, it appeared that understanding the functions of miRNAs in mammalian reproduction can substantially improve our understanding of the biological challenges of successful reproductive performance. This review describes the current knowledge on miRNAs, specifically in relation to the peri-implantation period when the majority of embryonic mortality occurs in pigs. To present a broader picture of crucial peri-implantation events, we focus on the role of miRNA-processing machinery and miRNA−mRNA infarctions during the maternal recognition of pregnancy, leading to maintenance of the corpus luteum function and further embryo implantation. Furthermore, we summarize the current knowledge on cell-to-cell communication involving extracellular vesicles at the embryo−maternal interface in pigs. Finally, we discuss the potential of circulating miRNAs to serve as indicators of ongoing embryo−maternal crosstalk.

Circulating miRNAs were proposed to be indicators of normal or complicated pregnancies. Based on this knowledge and our recent transcriptomic approach showing expression of miRNAs in the porcine endometrium, conceptuses and uterine extracellular vesicles during pregnancy, we have hypothesized that signs of ongoing local embryo-maternal crosstalk involving miRNAs can be detected in the circulation of pregnant gilts as early as a few days after maternal recognition of pregnancy. By applying several molecular biology techniques that differ in dynamic range and precision in maternal serum of Day 16 pregnant pigs, we were able to show for the first time increased levels of several miRNAs, previously reported to be expressed in either conceptuses and extracellular vesicles (miR-26a and miR-125b) or pregnant endometrium (miR-23b). Our results clearly showed that real-time RT-PCR and digital PCR are the most reliable methods, being able to detect small-fold changes of low-abundant circulating miRNAs. Further validation in a separate group of gilts confirmed an increase in miR-23b and miR-125b levels. In silico analyses identified pregnancy-related biological processes and pathways affected by these miRNAs. Target prediction analysis revealed hundreds of porcine transcripts with conserved sites for these miRNAs, which were classified into signaling pathways relevant to pregnancy. We conclude that a unique set of miRNAs can already be observed in the circulation of pigs during the first weeks of pregnancy, as a result of the initiation of embryo-maternal communication.

Porcine conceptuses secrete pregnancy-recognition signals (estrogens, including estradiol-17β) that inhibit luteolysis, thereby prolonging progesterone production by corpora lutea. The supportive mechanism by which the conceptus also inhibits luteolysis is by shifting endometrial prostaglandin (PG) synthesis to luteoprotective PGE2. Progesterone stimulates endometrial production of factors that are essential for conceptus development. Priming the uterus by progesterone and loss of progesterone receptors from the uterine epithelium by D1ay 10-12 after estrus are key for achieving endometrial receptivity for implantation. Conceptus implantation involves a series of events, many resembling the inflammatory reaction, that are greatly influenced by cytokines, growth factors, and prostaglandins. We herein present a novel, dual role for PGF2α in corpora lutea that depends on the acquisition of luteolytic sensitivity, based on the knowledge that PGF2α triggers pathways involved in luteolysis during the estrous cycle or/and may have an alternative function in maintaining progesterone synthesis during pregnancy. We also point out a new role for PGF2α that, together with PGE2, can act as embryonic signal mediators. PGF2α, which until recently was considered undesirable for promoting pregnancy, is now known to stimulate conceptus-maternal interactions and angiogenesis in the endometrium. This function is in line with other important prostaglandin functions, such as stimulating adhesion of trophoblasts (PGE2, PGI2) as well as endometrial vascular functions and trophoblast cell proliferation (PGI2). Finally, microRNAs have emerged as important post-transcriptional regulators of gene function, adding a new area of investigation that may enhance understanding of conceptus-endometrial interactions.

Inhibition of luteolysis and establishment of pregnancy in pigs results from oestrogen secretion by the conceptuses and requires progesterone produced by the corpus luteum (CL). An integral part of maternal recognition of pregnancy in the pig is the redirection of prostaglandin (PG) F2alpha secretion from endocrine (blood) to exocrine (uterus) direction and an increase of PGE2 synthesis in both the endometrium and conceptus. Uterine and conceptus PGE2 synthases play an integrated role in establishing the PGE2:PGF2alpha ratio necessary for luteal maintenance. The luteolytic or luteotrophic changes in the CL are synchronised with the release of maternal pituitary and ovarian hormones. The presence of uterine oxytocin (OT) and luteinising hormone (LH) receptors are important for the luteolytic effect of PGF2alpha. Conceptus oestrogen secretion coincides with autocrine and paracrine dialogue between the multiple conceptuses and uterine biological compounds and their receptors in trophoblast and endometrium.

Background Cathepsins are a group of endosomal proteases present in many cells including dendritic cells (DCs). The activity of cathepsins is regulated by their endogenous inhibitors – cystatins. Cathepsins are crucial to antigen processing during viral and bacterial infections, and as such are a prerequisite to antigen presentation in the context of major histocompatibility complex class I and II molecules. Due to the involvement of DCs in both innate and adaptive immune responses, and the quest to understand the impact of poxvirus infection on host cells, we investigated the influence of ectromelia virus (ECTV) infection on cathepsin and cystatin levels in murine conventional DCs (cDCs). ECTV is a poxvirus that has evolved many mechanisms to avoid host immune response and is able to replicate productively in DCs. Results Our results showed that ECTV-infection of JAWS II DCs and primary murine GM-CSF-derived bone marrow cells down-regulated both mRNA and protein of cathepsin B, L and S, and cystatin B and C, particularly during the later stages of infection. Moreover, the activity of cathepsin B, L and S was confirmed to be diminished especially at later stages of infection in JAWS II cells. Consequently, ECTV-infected DCs had diminished ability to endocytose and process a soluble antigen. Close examination of cellular protein distribution showed that beginning from early stages of infection, the remnants of cathepsin L and cystatin B co-localized and partially co-localized with viral replication centers (viral factories), respectively. Moreover, viral yield increased in cDCs treated with siRNA against cathepsin B, L or S and subsequently infected with ECTV. Conclusions Taken together, our results indicate that infection of cDCs with ECTV suppresses cathepsins and cystatins, and alters their cellular distribution which impairs the cDC function. We propose this as an additional viral strategy to escape immune responses, enabling the virus to replicate effectively in infected cells. Electronic supplementary material The online version of this article (10.1186/s12866-019-1471-1) contains supplementary material, which is available to authorized users.

In order to characterize the transition of the corpora lutea (CL) from acquisition of luteolytic sensitivity to rescue of luteal function: i) the expression of 38 factors associated with steroids, prostanoids, and angiogenic systems and ii) concentrations of the main hormones responsible for maintenance of CL function in cyclic and pregnant pigs were examined. Additionally, the effect of prostaglandin (PG) E2 and F2α on luteal function during the estrous cycle and pregnancy was evaluated in vitro. Significantly up-regulated gene expression was revealed in CL collected on day 14 of the estrous cycle (CYP19A1, ESR2, PTGS2, HIF1A, and EDN1) and on days 12–14 of pregnancy (SCARB1, PGRMC1, STAR, HSD3B1, NR5A1, PTGFR, PTGER4, and VEGFA). Elevated concentrations of estradiol-17β and PGE2 occurred in CL on days 12 and 14 of pregnancy respectively, while an increased intraluteal PGF2α content was noted on day 14 of the estrous cycle. Both PGs increased the synthesis of progesterone by cultured luteal slices obtained on day 14 of pregnancy, in contrast to the action of PGF2α on the corresponding day of the estrous cycle. PGE2 stimulated cAMP production via PTGER2 and PTGER4, while PGF2α elevated the content of CREB in cultured luteal slices from CL of pregnant pigs. In silico analysis showed that infiltration of lymphocytes and apoptosis of microvascular endothelium were activated in CL on day 12 of the estrous cycle vs pregnancy. Summarizing, an abundance of E2 and PGE2 during pregnancy regulates specific pathways responsible for steroidogenesis, the prostanoid signaling system and angiogenesis during rescue from luteolysis in porcine CL.

The brown adipocyte phenotype (BAP) in white adipose tissue (WAT) is transiently induced in adult mammals in response to reduced ambient temperature. Since it is unknown whether a cold challenge can permanently induce brown adipocytes (BAs), we reared C57BL/6J (B6) and AxB8/PgJ (AxB8) mice at 17 or 29°C from birth to weaning, to assess the BAP in young and adult mice. Energy balance measurements showed that 17°C reduced fat mass in the preweaning mice by increasing energy expenditure and suppressed diet-induced obesity in adults. Microarray analysis of global gene expression of inguinal fat (ING) from 10-day-old (D) mice indicates that expression at 17°C vs. 29°C was not different. Between 10 and 21 days of age, the BAP was induced coincident with morphologic remodeling of ING and marked changes in expression of neural development genes (e.g., Akap 12 and Ngfr). Analyses of Ucp1 mRNA and protein showed that 17°C transiently increased the BAP in ING from 21D mice; however, BAs were unexpectedly present in mice reared at 29°C. The involution of the BAP in WAT occurred after weaning in mice reared at 23°C. Therefore, the capacity to stimulate thermogenically competent BAs in WAT is set by a temperature-independent, genetically controlled program between birth and weaning.—Chabowska-Kita, A., Trabczynska, A., Korytko, A., Kaczmarek, M. M., Kozak, L. P. Low ambient temperature during early postnatal development fails to cause a permanent induction of brown adipocytes.

The new corpora lutea (CLs) in pigs are formed from the preovulatory follicles after the luteinizing hormone (LH) surge. However, total autonomy and independence of CLs from LH up to Day 12 of cycle has recently been questioned. Transformation of estrous cycle CL to CL of pregnancy initiated by embryonic signals requires not only the cessation of prostaglandin F2 (PGF2α) supply to the luteal tissue but also needs the CL to overcome luteolytic acquisition and/or changing its sensitivity to PGF2αduring Days 12–14 of pregnancy. The luteolytic cascade is prevented by inhibition of lymphocyte infiltration and leucocyte recruitment, limitation of cell apoptosis, upregulation of pregnancy-associated genes and an enhanced antiluteolytic role of PGE2. Our ‘two-signal switch hypothesis’ highlights the importance ofpostPGF2αand PGE2receptor signaling pathways activation in CLs during luteolysis and rescue. The ‘luteolytic switch’ involves increased expression of many regression mediators and activation of thepostPTGFR signaling pathway. The ‘rescue switch’ initiated by embryonic signals – estradiol 17β and PGE2– inducespostPTGER2/4 pathway, turning the ‘luteolytic switch’ off and triggering activity of genes responsible for CL maintenance. In mid and late pregnancy, CLs are maintained by LH and the synergistic action of metabolic hormones. This paper provides an outline of recent views on CL regression, rescue and maintenance during pregnancy in pigs that conflict with previous paradigms and highlights new findings regarding the actions of prostaglandins, role of microRNAs (miRNA) and immune system and signaling pathways governing the life cycle of porcine CL.

Interferon-tau (IFNT), a maternal recognition of pregnancy (MRP) signals in domestic ruminants, suppresses the release of luteolytic pulses of uterine prostaglandin F2a (PGF2a), thus extending the corpus luteum (CL) life span. We hypothesized that IFNT also exerts anti-luteolytic actions in bovine CL. To examine the direct effects of IFNT on bovine CL, luteal slices and enriched luteal endothelial cells (LECs) were utilized. We found that recombinant ovine IFNT (roIFNT) markedly elevates interferon-associated genes (STAT1, STAT2 and IRF9) and interferon-stimulated genes (ISGs: MX2, ISG15 and OAS1Y) in both models. Furthermore, IFNT time-dependently induced STAT1 phosphorylation in LECs without affecting total STAT1. roIFNT-stimulated viable LECs numbers and the knockdown of protein inhibitor of activated STAT1 (PIAS1) abolished this effect, suggesting that PIAS1 may mediate the proliferative effect of IFNT. IFNT significantly downregulated luteolytic genes such as TGFB1, thrombospondin-1 (THBS1), endothelin-1 (EDN1) and serpin family E member-1 (SERPINE1) in LECs. However, less robust effects were observed in luteal slices. Moreover, PGF2a alone induced THBS1, SERPINE1 and EDN1 mRNA in CL slices whereas in the presence of IFNT, THBS1 and SERPINE1 stimulation was abolished. Collectively, these results indicate that IFNT acts via STAT1- IRF9-dependent and independent pathways and affects diverse luteal functions. Most interestingly, this study suggests the existence of an anti-luteolytic effect of IFNT in bovine CL, namely, inhibiting key PGF2a-induced luteolytic genes. The proliferative effect of IFNT may constitute an additional mechanism that promotes luteal cell survival, thus, extending the luteal life span during early pregnancy in cows.

Endothelial progenitor cells (EPCs) represent a small population of blood cells (5-40 cells/mm3), with an ability to differentiate into endothelial cells that form the lining of the blood vessels and contribute to postnatal angiogenesis. Abundant evidence shows that recruitment of EPCs from the bone marrow, the monocyte/macrophage lineage and the organs facilitate the endothelial regeneration and repair. Changes in the number of EPCs were observed in both, chronic kidney and cardiovascular diseases. Thus, these cells were tested for usage in diagnosis and therapy. In this paper, we review the current knowledge on the EPC biology and contribution of these cells to the kidney and cardiovascular diseases.

Establishment of pregnancy in pigs requires continuous function of corpora lutea and endometrial preparation for embryo implantation. Progesterone regulates expression of many proteins necessary for endometrial remodelling and embryo-maternal communications. Attaining the uterine receptivity involves progesterone priming and loss of progesterone receptors in the uterine epithelium before days 10-12 after oestrus. Spermatozoa and oocytes in oviduct alter secretion of specific proteins that exert beneficial effect on gametes and embryos. Moreover, an appropriate leucocyte activation and maintenance of delicate cytokine balance within the oviduct and uterus are important for early pregnancy. This early local immune response is rather mediated by seminal plasma components. These components also influence prostaglandin (PG) synthesis in the oviduct that is important for gamete and embryo transport. Pregnancy establishment requires the biphasic pattern of oestrogen secretion by conceptuses on days 11-12 and 15-30. Conceptus affects lipid signalling system consisting of prostaglandins and lysophosphatic acid. PG synthesis is changed by conceptus signals in favour of luteoprotective PGE(2) . Additionally, existence of PGE(2) positive feedback loop in the endometrium contributes to increased PGE(2) /PGF(2α) ratio during the peri-implantation period. PGE(2) through endometrial PGE(2) receptor (PTGER2) elevates the expression of enzymes involved in PGE(2) synthesis. Higher PGE(2) secretion in uterine lumen coincides with the elevated expression of HOXA10 transcription factor critical for implantation. A stable adhesion between conceptus and endometrium requires reduction in mucin-1 on the apical surface of epithelium and integrin activation by extracellular matrix proteins. Furthermore, growth factors, cytokines and its receptors are involved in embryo-maternal interactions.

The homeobox A (HOXA) family of genes is responsible for segmental development of the female reproductive tract during embryogenesis. However, HOXA10 has been shown to be essential not only for uterus development, but also for implantation. Persistent expression and steroid-dependent regulation of this gene has been demonstrated in adult human, primate, murine and canine uteri. Moreover, HOXA10-dependent expression of prostaglandin H synthase-2 (PGHS-2), a key enzyme in prostaglandin production, has been previously detected. The role of the HOXA10 gene in the porcine uterus is not well established. Therefore, the present studies were undertaken to 1) examine the effect of E(2) and P(4) on HOXA10 mRNA and protein content in the endometrium collected on day 9 of the estrous cycle and 2) determine the PGHS-2 protein expression and PGE(2) and PGF(2α) secretion from endometrial tissue in response to steroid treatment. Endometrial explants collected from mature gilts on day 9 of the estrous cycle were incubated with E(2) (1-100 nM), P(4) (10-1000 nM) or E(2) (10 nM) and P(4) (100 nM) for 24 h. E(2) alone or E(2) in the presence of P(4) increased HOXA10 mRNA expression in the endometrium (P0.05). The HOXA10 protein level was upregulated in response to E(2), P(4) and both steroids administered simultaneously (P0.05). Moreover, E(2) and P(4) stimulated PGHS-2 protein expression in cultured endometrial explants. PGE(2), but not PGF(2α), secretion increased in the presence of E(2) (P0.05). However, the release of both prostaglandins was decreased after treatment of endometrial explants with the highest dose of P(4) (P0.01). These results demonstrate that E(2) and P(4) are important regulators of HOXA10 gene expression in the adult porcine endometrium during the mid-luteal phase of the estrous cycle. Additionally, the similar profiles of endometrial HOXA10 and PGHS-2 expression in the presence of E(2) and P(4) indicate that both genes are simultaneously regulated by steroids in the porcine uterus.

The mechanism by which mice, exposed to the cold, mobilize endogenous or exogenous fuel sources for heat production is unknown. To address this issue we carried out experiments using 3 models of obesity in mice: C57BL/6J+/+ (wild-type B6) mice with variable susceptibility to obesity in response to being fed a high-fat diet (HFD), B6. Ucp1-/- mice with variable diet-induced obesity (DIO) and a deficiency in brown fat thermogenesis and B6. Lep-/- with defects in thermogenesis, fat mobilization and hyperphagia. Mice were exposed to the cold and monitored for changes in food intake and body composition to determine their energy balance phenotype. Upon cold exposure wild-type B6 and Ucp1-/- mice with diet-induced obesity burned endogenous fat in direct proportion to their fat reserves and changes in food intake were inversely related to fat mass, whereas leptin-deficient and lean wild-type B6 mice fed a chow diet depended on increased food intake to fuel thermogenesis. Analysis of gene expression in the hypothalamus to uncover a central regulatory mechanism revealed suppression of the Npvf gene in a manner that depends on the reduced ambient temperature and degree of exposure to the cold, but not on adiposity, leptin levels, food intake or functional brown fat., Author Summary Current knowledge does not provide a clear, definite view of central mechanisms controlling energy balance upon cold-activated thermogenesis. Here we show that upon cold exposure lean mice maintain body composition but increase food intake to fuel thermogenesis, whereas cold-exposed mice with DIO utilize endogenous fat stores and then transition to increased food intake as body composition approaches that of the lean controls. Using knockout mice with leptin and Ucp1 gene deficiency our study indicates that the relative energy utilization from food intake and endogenous energy reserves to maintain body temperature during cold exposure is independent of both leptin action and brown fat-linked thermogenesis. Using a combination of genetic and biological approaches, we demonstrate that Npvf gene expression in the hypothalamus is regulated by changes in ambient temperature in a manner independent of the nutritional status of the mouse.

Tightly coordinated, reciprocal embryo-maternal interactions affect gene expression during early pregnancy. Recently, microRNAs (miRNAs) have emerged as new players in the fine tuning of embryo development and implantation in mammals via posttranscriptional gene regulation mechanisms. Here, we integrated transcriptomic and computational approaches to profile miRNAs and miRNA synthesis and transport-related genes at different developmental stages of porcine conceptuses and trophoblast during early pregnancy in the pig. Using semiquantitative RT-PCR, we examined mRNA levels of 10 genes encoding proteins involved in miRNA synthesis and transport: DROSHA, DGCR8, XPO5, DICER1, TARBP2, TNRC6A, AGO1, AGO2, AGO3, and AGO4. Custom, multispecies microarrays were used to profile miRNAs. Prediction algorithms of miRNA-mRNA interactions allowed identification of target transcripts for the analyzed miRNAs. These included VEGF, LIF, PTGS2, and IL-6R, known to be crucial components of embryo-maternal interactions in the pig. Two selected miRNAs, miR-26a and miR-125b, were tested for the presence in the extracellular vesicles isolated from uterine luminal flushings during pregnancy. Results of in vitro study demonstrated that miRNAs, such as miR-125b, can regulate expression of genes crucial for embryo development and implantation in porcine endometrial luminal epithelial cells. For the first time, expression profiles of miRNAs and related genes in porcine conceptuses and trophoblast during maternal recognition of pregnancy and embryo implantation in the pig were described. Altogether, our results indicate potential roles of these small, noncoding RNAs in the early development of embryos and embryo-maternal cross-talk during early pregnancy in the pig.

Tightly coordinated, reciprocal embryo-maternal interactions affect gene expression during early pregnancy. Recently, microRNAs (miRNAs) have emerged as new players in the fine tuning of embryo development and implantation in mammals via posttranscriptional gene regulation mechanisms. Here, we integrated transcriptomic and computational approaches to profile miRNAs and miRNA synthesis and transport-related genes at different developmental stages of porcine conceptuses and trophoblast during early pregnancy in the pig. Using semiquantitative RT-PCR, we examined mRNA levels of 10 genes encoding proteins involved in miRNA synthesis and transport: DROSHA, DGCR8, XPO5, DICER1, TARBP2, TNRC6A, AGO1, AGO2, AGO3, and AGO4. Custom, multispecies microarrays were used to profile miRNAs. Prediction algorithms of miRNA-mRNA interactions allowed identification of target transcripts for the analyzed miRNAs. These included VEGF, LIF, PTGS2, and IL-6R, known to be crucial components of embryo-maternal interactions in the pig. Two selected miRNAs, miR-26a and miR-125b, were tested for the presence in the extracellular vesicles isolated from uterine luminal flushings during pregnancy. Results of in vitro study demonstrated that miRNAs, such as miR-125b, can regulate expression of genes crucial for embryo development and implantation in porcine endometrial luminal epithelial cells. For the first time, expression profiles of miRNAs and related genes in porcine conceptuses and trophoblast during maternal recognition of pregnancy and embryo implantation in the pig were described. Altogether, our results indicate potential roles of these small, noncoding RNAs in the early development of embryos and embryo-maternal cross-talk during early pregnancy in the pig.

Introduction of semen into the female reproductive tract may induce molecular and cellular changes facilitating conception and pregnancy. Because prostaglandins (PGs) and appropriate vascularization of the endometrium are crucial for pregnancy success, the effect of seminal plasma (SP) on PG synthesis and angiogenesis was investigated. Gilts at estrus received an infusion of 100 ml of either SP or PBS (control). Uterine horns were collected on Days 1, 3, 5, and 10 after each treatment. Concentrations of PGE2, PGF2alpha , and PGFM were measured in the uterine lumen and endometrial tissue. Expression of PG synthesis pathway enzymes and angiogenic factors, infiltration of immune cells, and vascular bed development were assessed. One day after SP infusion, the PGE2:PGF2alpha ratio in the uterine lumen was lower than in controls. In endometrial tissue, however, PGE2 levels and the PGE2:PGF2alpha ratio were elevated on Day 10. PG-endoperoxide synthase expression in the endometrium was up-regulated on Day 1 and decreased on Day 5 after SP treatment compared to that in controls. PGF2alpha synthase levels were decreased on Day 5 and 10 in SP-treated animals when compared to controls. SP-induced vascular bed development was apparent shortly before the time corresponding to maternal recognition of pregnancy in the pig. Together, these data indicate that the porcine uterus can be sensitized shortly after SP exposure to evoke prolonged effects on PG synthesis and angiogenesis in the endometrium, persisting over the course of the prereceptive phase. Thus, SP can affect uterine receptivity and embryo-maternal interactions in pigs through locally direct and/or indirect mechanisms.

Several factors participate in regulation of growth and development as well as angiogenesis of the uterus during pregnancy, and hence little is known about the role of hormonal regulation of vascular endothelial growth factor (VEGF)-receptor system expression. This study has examined the effect of insulin-like growth factor-I (IGF-I), relaxin (RLX), oxytocin (OT) and prostaglandin (PG) E(2), on VEGF secretion and VEGF-receptor system mRNA expression in the porcine endometrial stromal cells. IGF-I and RLX were identified as the most effective inducers of VEGF secretion and mRNA expression. Although PGE(2) stimulated VEGF secretion and VEGF164 mRNA expression, OT inhibited both secretion and mRNA expression of VEGF. When tested for VEGF receptors (R), all factors failed to affect their mRNA expression. Media conditioned by stromal cells collected after IGF-I and RLX treatment significantly increased endothelial cell proliferation and this effect was blocked by soluble VEGFR-1. These data suggest that during early pregnancy IGF-I, RLX and PGE(2) can affect VEGF expression in the endometrium and therefore may support uterine and embryo development, implantation and pregnancy.

Luteinizing hormone/human chorionic gonadotropin (LH/hCG) receptors are widely expressed in gonadal cells, however, the presence of these receptors has also been demonstrated in several other non-gonadal female and male tissues. The expression level of non-gonadal LH/hCG receptors is much lower than in gonads, although their expression is regulated by similar mechanisms and they also exert biological effects using similar signaling pathways. Hormonally regulated LH/hCG receptor expression in the oviduct suggests that LH could be involved in the regulation of its contraction, gametes/embryos transport and synchronization of the fertilization. One of the major roles of the myometrial LH/hCG receptors may also be the stimulation of growth and maintenance of the uterine relaxation during pregnancy. In pigs, LH seems to be one of the pleiotropic factors which influence the endometrial prostaglandin F(2alpha) synthesis and initiation of the luteolysis. The LH/hCG receptor expression in several cancer cells provides new possibilities for developing new strategies for targeted cancer therapy based on lytic LH/hCG conjugates.

Changes in both the morphology and the steroidogenic activity of porcine ovaries denervated surgically on day 12 of the oestrous cycle were studied. Neurectomy of the plexus and the superior ovarian nerves caused a dramatic reduction in the number (or even a disappearance) of dopamine-β-hydroxylase- and/or neuropeptide tyrosine-immunoreactive nerve terminals. On day 20 of the subsequent oestrous cycle, the number of small follicles increased (P < 0.01) and that of large follicles decreased (P < 0.05) in the denervated ovaries, as compared to the controls. Neurectomy led to a decrease in the level of progesterone (P 4 ; P < 0.001) and androstenedione (A 4 ; P < 0.01) in the fluid from small follicles, A 4 (P < 0.001) and testosterone (T; P < 0.05) in the fluid from medium-sized follicles, as well as in the content of all these steroids in the fluid from large-sized follicles (P < 0.001 for P 4 and P < 0.05 for A 4 and T). Denervation also caused a decrease in the content of A 4 (P < 0.01) and T (P < 0.001) in the wall of follicles. Neurectomy resulted in a significant increase in the immunoexpression of cholesterol side-chain cleavage cytochrome P450 in the follicles and a decrease of 3β-hydroxysteroid dehydrogenase. After denervation, plasma levels of LH, P 4 , A 4 , T, oestrone and oestradiol-17β were lower (P < 0.05–0.001) on the particular days of the study than in the control group. Our data revealed that the denervation of ovaries during the middle luteal phase of the oestrous cycle in gilts caused distinct changes in both the morphology and the steroidogenic activity of the organ, confirming an important role of the peripheral nervous system in the control of the gonad in this species.

The mechanism by which mice, exposed to the cold, mobilize endogenous or exogenous fuel sources for heat production is unknown. To address this issue we carried out experiments using 3 models of obesity in mice: C57BL/6J+/+ (wild-type B6) mice with variable susceptibility to obesity in response to being fed a high-fat diet (HFD), B6. Ucp1-/- mice with variable diet-induced obesity (DIO) and a deficiency in brown fat thermogenesis and B6. Lep-/- with defects in thermogenesis, fat mobilization and hyperphagia. Mice were exposed to the cold and monitored for changes in food intake and body composition to determine their energy balance phenotype. Upon cold exposure wild-type B6 and Ucp1-/- mice with diet-induced obesity burned endogenous fat in direct proportion to their fat reserves and changes in food intake were inversely related to fat mass, whereas leptin-deficient and lean wild-type B6 mice fed a chow diet depended on increased food intake to fuel thermogenesis. Analysis of gene expression in the hypothalamus to uncover a central regulatory mechanism revealed suppression of the Npvf gene in a manner that depends on the reduced ambient temperature and degree of exposure to the cold, but not on adiposity, leptin levels, food intake or functional brown fat.

Endometrial prostaglandins (PGs) and the PGE2/PGF2α ratio play an important role in regulating the estrous cycle and establishment of pregnancy. The enzymes downstream of cyclooxygenase-2 may determine the PGE2/PGF2α ratio in the porcine uterus. Thus, we have cloned porcine PGF synthase (PGFS) and microsomal PGE synthase-1 (mPGES-1) and characterized their expression in porcine endometrium during the estrous cycle and early pregnancy. PGFS and mPGES-1 amino acid sequences possessed a high degree (>67% and >77%, respectively) of identity with the other mammalian homologs. There was little modulation of mPGES-1 throughout the estrous cycle; however, PGFS expression was highly up-regulated in endometrium around the time of luteolysis. During early pregnancy, PGFS at the protein level showed a time-dependent increase (low on d 10–13, intermediate on d 14–23, and high on d 24–25). In pregnancy, expression of mPGES-1 was intermediate on d 10–11 and low on d 14–17 and then increased after d 22, reaching the maximum on d 24–25. Immunohistochemistry showed localization of PGFS and mPGES-1 proteins mainly in luminal and glandular epithelium. Concluding, the spatiotemporal expression of PGFS throughout the estrous cycle indicates an involvement of PGFS in regulating luteolysis in the pig. The comparison of endometrial PGFS and mPGES-1 expression on d 10–13 of the estrous cycle and pregnancy suggest a supportive role of these enzymes in determining the increase of uterine PGE2/PGF2α ratio during maternal recognition of pregnancy. Moreover, high expression of both PG synthases after initiation of implantation may indicate their significant role in placentation.

Synchronization and superovulation are commonly used to obtain large numbers of embryos for experimental and practical purposes. This study compared the number, quality, and in vitro development of embryos recovered from gilts following single or double estrus synchronization and superovulation. Prepubertal gilts from the single synchronization group were injected with 1500 I.U. PMSG and 1000 I.U. hCG 72 h later. The double synchronized group of gilts was treated with 750 I.U. PMSG and 500 I.U. hCG 72 h later. After 17 days, 1500 I.U. PMSG followed by 1000 I.U. hCG was administered. Five days after insemination embryos were recovered and cultured for 6 days. Both single and double hormonal stimulation schedules resulted in recovery of elevated numbers of embryos (28.4 and 23.4 vs. 11.3; p

Extended exposure of progesterone and conceptus estrogen influences the vascular compartment of the uterus and expression of many factors, such as prostaglandins (PGs), growth factors, extracellular matrix and adhesion molecules, cytokines and transcription factors. One of the supportive mechanisms by which the conceptus inhibits luteolysis is by changing PG synthesis in favor of luteoprotective PGE2. Alteration in PG synthesis may result from increased PGE synthase (mPGES-1) expression in the trophoblast and endometrium on days 10-13 of pregnancy with simultaneous down-regulation of PGF synthase (PGFS) and prostaglandin 9-ketoreductase (CBR1). Conceptus and endometrial, rather than luteal, synthesis of PGE2, is involved in the process of maternal recognition of pregnancy. However, complex (direct and indirect) actions of estrogen on the CL, including decreased luteal VEGF soluble receptor on day 12 of pregnancy, are important for luteal maintenance. Moreover, conceptus signals affect another lipid signaling component - lysophosphatidic acid receptor (LPA3), as well as HoxA10 and Wnt in the endometrium, to create the appropriate uterine environment for establishment of pregnancy and implantation.