WNT4 Gene and Protein Expression in Endometrial Cancer and Its Significance.

BACKGROUND: The inappropriate action of WNT4 and estrogens affects uterine homeostasis and function, and may lead to endometrial cancer (EC). OBJECTIVE: The aim was to evaluate the alterations of WNT4 gene expression and WNT4 protein immunoreactivity (Ir) in EC, considering tumor characteristics, the clinicopathological association and estrogen dependence. METHODS: WNT4 mRNA levels were compared between benign (control) endometrium (n = 8) and endometroid EC (EEC) and non-endometroid EC (non-EEC) samples (n = 28) using the real-time PCR technique. The WNT4-Ir and ERα-Ir were evaluated by immunohistochemistry (IHC). WNT4 mRNA gene and WNT4-Ir were correlated with clinicopathological and blood morphological parameters. Overall survival (OS) was assessed. The bioanalysis was utilized to study WNT4 expression in large patient cohort (n = 549). RESULTS: WNT4 gene expression was decreased in EC samples (specifically in EEC but not in non-EEC) compared to the control. The WNT4 gene expression was also decreased in EC samples categorized by the tumor characteristics. There was no statistical difference in WNT4-Ir or ERα-Ir between the control and EC. There was no correlation between OS and WNT4 gene expression and WNT4-Ir. Bioanalysis showed that WNT4 and ESR1 gene expression alterations tended to be mutually exclusive. An alteration in WNT4 expression was found in different histological tumor types in a large group of EC patients. CONCLUSIONS: There is a great need to evaluate the molecular background of EC. Our study suggests that the WNT4 gene has the potential to be a marker of functional estrogen signaling in EEC.

Embryonic signals mediate extracellular vesicle biogenesis and trafficking at the embryo-maternal interface.

BACKGROUND: Extracellular vesicles (EVs) are membrane-coated nanoparticles secreted by almost all cell types in living organisms. EVs, as paracrine mediators, are involved in intercellular communication, immune response, and several reproductive events, including the maintenance of pregnancy. Using a domestic animal model (Sus scrofa) with an epitheliochorial, superficial type of placentation, we focused on EV biogenesis pathway at the embryo-maternal interface, when the embryonic signaling occurs for maternal recognition and the maintenance of pregnancy. RESULTS: Transmission electron microscopy was used during early pregnancy to visualize EVs and apocrine and/or merocrine pathways of secretion. Immunofluorescent staining localized proteins responsible for EV biogenesis and cell polarization at the embryo-maternal interface. The expression profiles of genes involved in biogenesis and the secretion of EVs pointed to the possible modulation of endometrial expression by embryonic signals. Further in vitro studies showed that factors of embryonic origin can regulate the expression of the ESCRT-II complex and EV trafficking within endometrial luminal epithelial cells. Moreover, miRNA-mediated rapid negative regulation of gene expression was abolished by delivered embryonic signals. CONCLUSIONS: Our findings demonstrated that embryonic signals are potent modulators of ESCRT-dependent EV-mediated secretory activity of the endometrium during the critical stages of early pregnancy. Video Abstract.

The molecular dialog between the conceptus and maternal tissues that takes place prior to and during implantation is slowly becoming better known. The need for better understanding of one of life's founding stages is even greater in light of the observation that, not only in our species, many embryos fail to implant, both in natural conception and following assisted reproductive techniques. Although implantation strategies differ among eutherian mammals, the initial stages of apposition and adhesion are common and are a foundation for successful pregnancy. In early pregnancy, as the embryo arrives in the uterus, intensive communication between the embryo and mother begins. Among the wide range of cell-to-cell communication strategies, there is one, relatively recently discovered, governed by extracellular vesicles, small membranous vesicles that contain cell-specific collections of proteins, lipids, and genetic material. The present study was undertaken to answer the question of how signaling molecules released by cells participating in the embryo-maternal dialog contribute to extracellular vesicle-mediated cell-to-cell communication. Our results shed new light on the role of hormones, non-coding RNAs, and extracellular vesicles in the early stages of mammalian pregnancy, which are contributing to species reproductive success.

miR-125b-5p impacts extracellular vesicle biogenesis, trafficking, and EV subpopulation release in the porcine trophoblast by regulating ESCRT-dependent pathway.

Intercellular communication is a critical process that ensures cooperation between distinct cell types at the embryo-maternal interface. Extracellular vesicles (EVs) are considered to be potent mediators of this communication by transferring biological information in their cargo (e.g., miRNAs) to the recipient cells. miRNAs are small non-coding RNAs that affect the function and fate of neighboring and distant cells by regulating gene expression. Focusing on the maternal side of the dialog, we recently revealed the impact of embryonic signals, including miRNAs, on EV-mediated cell-to-cell communication. In this study, we show the regulatory mechanism of the miR-125b-5p ESCRT-mediated EV biogenesis pathway and the further secretion of EVs by trophoblasts at the time when the crucial steps of implantation are taking place. To test the ability of miR-125b-5p to influence the expression of genes involved in the generation and release of EV subpopulations in porcine conceptuses, we used an ex vivo approach. Next, in silico and in vitro analyses were performed to confirm miRNA-mRNA interactions. Finally, EV trafficking and release were assessed using several imaging and particle analysis tools. Our results indicated that conceptus development and implantation are accompanied by changes in the abundance of EV biogenesis and trafficking machinery. ESCRT-dependent EV biogenesis and the further secretion of EVs were modulated by miR-125b-5p, specifically impacting the ESCRT-II complex (via VPS36) and EV trafficking in primary porcine trophoblast cells. The identified miRNA-ESCRT interplay led to the generation and secretion of specific subpopulations of EVs. miRNA present at the embryo-maternal interface governs EV-mediated communication between the mother and the developing conceptus, leading to the generation, trafficking, and release of characteristic subpopulations of EVs.

Atretic preovulatory follicles could be precursors of ovarian lutein cysts in the pig.

Ovarian cysts contribute to reduced reproductive performance in pigs. Unfortunately, the mechanism of lutein cysts formation remains unknown. Here, we compared the endocrine and molecular milieus of intact, healthy preovulatory follicles (PF), gonadotropin (eCG/hCG)-induced healthy and atretic-like PF, as well as gonadotropin-provoked and spontaneous ovarian cysts in gilts. Several endocrine and molecular indicators and microRNA were compared in walls of PF and cysts. Intact and healthy PF, showed high estradiol/androstendione and low progesterone levels associated with CYP17A1, HSD17B1, and CYP19A1 elevation and reduced StAR/HSD3B1 protein expression. In contrast, low estradiol/androstendione and high progesterone concentrations, accompanied by decreased CYP17A1, HSD17B1, CYP19A1 and increased HSD3B1 protein abundance, appeared in atretic-like PF, gonadotropin-induced and spontaneous cysts. High progesterone receptor (PGR) protein abundance was maintained in intact and healthy PF, while it dropped in atretic-like PF, gonadotropins-induced and spontaneous cysts. The atretic PF showed high level of TNFα compared to healthy PF. In conclusion, follicular lutein cysts could be recruited from atretic-like PF with lost estrogenic milieu and inability to ovulate. Ovulatory cascade was presumably disrupted by a low PGR and high TNFα levels associated with earlier luteinization of follicular walls. These results suggest a novel mechanism of lutein ovarian cysts development in pigs and, perhaps, other species.

Extracellular vesicles: Focus on peri-implantation period of pregnancy in pigs.

The establishment of cell-to-cell communication between the endometrium and the developing embryo is the most important step in successful mammalian pregnancy. Close interaction between the uterine luminal epithelium and trophoblast cells requires triggering timely molecular dialog for successful maternal recognition of pregnancy, embryo implantation, and placenta development. Quite recently, extracellular vesicles (EVs) carrying unique molecular cargo emerged as evolutionarily conserved mediators of cell-to-cell communication during early pregnancy. To date, the presence of EVs at the embryo-maternal interface has been demonstrated in numerous mammals, including domestic livestock, such as pigs. However, few studies have focused on revealing the mechanism of EV-mediated crosstalk between developing early embryos and receptive endometrium. Over the past years, it has appeared that understanding the role of EVs in mammalian reproduction can substantially improve our understanding of the biological challenges of successful reproductive performance. This review describes current knowledge of EVs, specifically in relation to the peri-implantation period in pigs, characterized by common features of embryo implantation and high embryonic mortality in mammals.

Attainment of Sexual Maturity and Gonadotropin Priming in Gilts Determine Follicular Development, Endocrine Milieu and Response to Ovulatory Triggers.

The routine procedure of estrous cycle synchronization in pigs allows for the use of gonadotropins to stimulate ovarian activity. The applied protocols of eCG and hFSH priming similarly affected development of ovarian follicles in two classes 3−6 mm and >6 mm of diameter, however, the number of small follicles (<3 mm) was 2-fold higher in hFSH- than in eCG-primed prepubertal gilts. The attainment of sexual maturity increased concentration of estradiol, testosterone and androstenedione in the follicular fluid of hFSH/eCG-primed gilts, however, prostaglandin E2 and F2α metabolite increased in mature hFSH- and eCG-primed gilts, respectively. The maturity increased mRNA and/or protein expression of key steroidogenic enzymes, prostaglandin synthases or luteinizing hormone receptors in follicular walls. Both hormonal primers played a moderate role in affecting expression of steroidogenic enzymes in follicular walls. In vitro studies showed higher estradiol production in r-hLH (p = 0.04)- and r-hCG (p = 0.049)-stimulated follicular walls of mature gilts than in prepubertal hFSH-primed gilts. Both ovulatory triggers decreased the abundance of LHCG/FSH mRNA receptors in follicular walls, which mimic downregulation of these receptors by a preovulatory LH surge, confirmed in vivo. These data revealed the importance of sexual maturity in the protection of the estrogenic environment, and the selective, moderate role of eCG and FSH in the activation of steroidogenic enzymes in preovulatory follicles.

Early steps of embryo implantation are regulated by exchange of extracellular vesicles between the embryo and the endometrium.

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 the 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.

Application of two-dimensional difference gel electrophoresis to identify protein changes between center, margin, and adjacent non-tumor tissues obtained from non-small-cell lung cancer with adenocarcinoma or squamous cell carcinoma subtype.

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.

miR-26a-5p and miR-125b-5p affect trophoblast genes and cell functions important during early pregnancy†.

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.

Comparative Analysis of Bacterial Cellulose Membranes Synthesized by Chosen Komagataeibacter Strains and Their Application Potential.

This article presents a comparative analysis of bacterial cellulose membranes synthesized by several strains of the Komagataeibacter genus in terms of their specific physical, physico-chemical, and mechanical properties. Herein, the aim was to choose the most suitable microorganisms producing cellulosic materials with the greatest potential for the fabrication of bio-inspired nanocomposites. The selection was based on three main steps, starting from the evaluation of BNC biosynthetic efficiency with and without the addition of ethanol, followed by the assessment of mechanical breaking strength, and the physical parameters (compactness, structural integrity, appearance, and thickness) of the obtained biological materials. Ultimately, based on the performed screening procedure, three efficiently growing strains (K. hansenii H3 (6Et), K. rhaeticus K4 (8Et), and Komagataeibacter sp. isolated from balsamic vinegar (12Et)) were chosen for further modifications, enabling additional cellulose functionalization. Here, supplementation of the growth medium with five representative polymeric compounds (citrus/apple pectin, wheat starch, polyvinyl alcohol, polyethylene glycol) led to significant changes in BNC properties, especially dye loading abilities, mechanical strength, and water adsorption/retention capacities. The resulting nanocomposites can be potentially useful in various fields of medicine and industry, and in the future, they may become a practical and cost-effective competitor against commercial biomaterials currently available on the market.

The Influence of Intravaginal Gestagens Treatment on the Morphological Features and Endometrial Steroid Hormone Receptors Content during Anestrus Type II in Dairy Cattle.

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.

Profile for mRNA transcript abundances in the pig endometrium where inflammation was induced by Escherichia coli.

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.

Endocrine and molecular milieus of ovarian follicles are diversely affected by human chorionic gonadotropin and gonadotropin-releasing hormone in prepubertal and mature gilts.

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.

Prolonged Effect of Seminal Plasma on Global Gene Expression in Porcine Endometrium.

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-γ 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.

Bacterial Nanocellulose-A Biobased Polymer for Active and Intelligent Food Packaging Applications: Recent Advances and Developments.

The aim of this review is to provide an overview of recent findings related to bacterial cellulose application in bio-packaging industry. This constantly growing sector fulfils a major role by the maintenance of product safety and quality, protection against environmental impacts that affect the shelf life. Conventional petroleum-based plastic packaging are still rarely recyclable and have a number of harmful environmental effects. Herein, we discuss the most recent studies on potential good alternative to plastic packaging-bacterial nanocellulose (BNC), known as an ecological, safe, biodegradable, and chemically pure biopolymer. The limitations of this bio-based packaging material, including relatively poor mechanical properties or lack of antimicrobial and antioxidant activity, can be successfully overcome by its modification with a wide variety of bioactive and reinforcing compounds. BNC active and intelligent food packaging offer a new and innovative approach to extend the shelf life and maintain, improve, or monitor product quality and safety. Incorporation of different agents BNC matrices allows to obtain e.g., antioxidant-releasing films, moisture absorbers, antimicrobial membranes or pH, freshness and damage indicators, humidity, and other biosensors. However, further development and implementation of this kind of bio-packaging will highly depend on the final performance and cost-effectiveness for the industry and consumers.

Altrenogest affects the development and endocrine milieu of ovarian follicles in prepubertal and mature gilts†.

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.

Pentraxin-3 mediates prosurvival actions of interferon tau in bovine luteinized granulosa cells.

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.

MiRNAs in the Peri-Implantation Period: Contribution to Embryo-Maternal Communication in Pigs.

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.

Clustered microRNAs: The molecular mechanism supporting the maintenance of luteal function during early pregnancy.

MicroRNAs (miRNAs) are recognized as the important regulators of ovarian function. However, little is known about the hormonal regulation of miRNA expression and the role of the specific miRNA-mRNA interactions in corpus luteum. Therefore, the present study was undertaken to determine: (a) the expression of miRNAs in the corpus luteum in early pregnancy vs regression; (b) the effect of conceptus and uterine signals in the expression of selected miRNAs; and (c) the role of specific miRNA-mRNA interactions in the molecular changes and secretory function of the corpus luteum in the pig. The results showed that the majority of miRNAs differentially expressed in the corpus luteum in early pregnancy vs regression belong to independent clusters (eg, miR-99b, miR-532), which are highly conserved among different animal species. The main conceptus signal in the pig (17ß-estradiol) elevated the luteal expression of the miR-99b cluster and lowered the expression of NR4A1 and AKR1C1, the genes involved in corpus luteum regression. Furthermore, the delivery of miR-99b cluster mimics to luteal tissue concomitantly decreased NR4A1 and AKR1C1 expression and enhanced progesterone secretion. The present study demonstrated that conceptus signals can support the maintenance of luteal function during pregnancy by clustered miRNA-stimulated pathways, governing the expression of genes involved in luteal regression.

Ectromelia virus suppresses expression of cathepsins and cystatins in conventional dendritic cells to efficiently execute the replication process.

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.