Effects of dietary supplementation of bacteriophage cocktail on health status of weanling pigs in a non-sanitary environment.

BACKGROUND: The study evaluated the effects of bacteriophage cocktail (BP) and ZnO administered during weaning time for piglets exposed to a non-sanitary environment. The bacteriophages were designed to eliminate Escherichia coli (K88, K99 and F41), Salmonella (typhimurium and enteritidis), and Clostridium perfreingens (types A and C). Forty 21-day-old crossbreed piglets were assigned to four treatments, including the PC (sanitary environment), NC (non-sanitary environment), BP (NC plus 108 pfu/kg BP), and ZO (NC plus 2,500 mg/kg ZnO). Piglets in the NC, BP and ZO were kept in a non-sanitary environment for 14 d, which was contaminated with the feces of infected pigs. RESULTS: Pigs in the BP and ZO treatments had a higher final body weight compared with the NC. The NC treatment showed the highest concentration of inflammatory cytokines including interleukin (IL)-1ß, IL-6 and tumor necrosis factor-α in the plasma. The administration of BP and ZO showed lower myeloperoxidase concentrations compared with the NC. The NC treatment showed a lower concentration of superoxide dismutase in serum compared with the PC. Among the treatments in non-sanitary environment, the NC treatment showed a higher concentration of malondialdehyde compared with the ZO. The PC treatment showed a lower concentration of butyric acid in the feces compared with the BP treatment. Among non-sanitary treatments, the villus height in the duodenum was greater in the BP and ZO compared with the NC. The lower abundance of Proteobacteria phylum was observed in the BP and PC treatments compared with the NC. The highest relative abundance of Eubacterium was recorded in the BP treatment. The abundance of Megasphaera and Schwartzia was higher in the NC pigs compared with the BP piglets. The abundance of Desulfovibrio was lower in the supplemented treatments (BP and ZO) compared with non-supplemented (NC and PC). The abundance of Cellulosilyticum genera was higher in the BP and ZO treatments rather than in the NC. The piglets in the NC treatment had the highest abundance of Escherichia-Shigella, followed by the PC and ZO treatments. CONCLUSION: In conclusion, these results suggest that the supplementation of bacteriophage cocktail could effectively control Proteobacteria phylum, Clostridium spp. and coliforms population and mitigated the adverse influences of weaning stress in piglets.

Calcium-binding proteins S100A8, S100A9, and S100A12: expression and regulation at the maternal-conceptus interface in pigs†.

Among the many calcium-binding proteins, S100A8, S100A9, and S100A12 play important roles in inflammation, innate immunity, and antimicrobial function, but their expression, regulation, and function at the maternal-conceptus interface in pigs are not fully understood. Therefore, we determined the expression and regulation of S100A8, S100A9, S100A12, and their receptor AGER at the maternal-conceptus interface in pigs. We found that S100A8, S100A9, and S100A12 mRNAs were expressed in the endometrium during the estrous cycle and pregnancy, with the greatest levels on Day (D) 12 of pregnancy, and AGER appeared at greater levels on D15 and D30 of pregnancy than on other days. The expression of S100A8, S100A9, and S100A12 was predominantly localized to epithelial cells in the endometrium, and they were detected in early-stage conceptus and later chorioallantoic tissues during pregnancy. AGER expression was localized to endometrial epithelial and stromal cells and chorionic epithelial cells. In endometrial explant tissues, the expression of S100A8, S100A9, and S100A12 was induced by estrogen, S100A8 by interleukin-1ß, and AGER by interferon-γ. We further found that on D12 of pregnancy, the expression of S100A8, S100A9, and S100A12 decreased significantly in the endometria of gilts carrying conceptuses derived from somatic cell nuclear transfer. These results indicate that the expression of S100A8, S100A9, and S100A12 is dynamically regulated in response to conceptus-derived signals at the maternal-conceptus interface, suggesting that S100A8, S100A9, and S100A12 could play a critical role in regulating endometrial epithelial cell function and conceptus implantation to support the establishment and maintenance of pregnancy in pigs.

Ovulatory upregulation of angiotensin-converting enzyme 2, a receptor for SARS-CoV-2, in dominant follicles of the human ovary.

OBJECTIVE: To determine the temporal expression of angiotensin-converting enzyme 2 (ACE2), a receptor for SARS-CoV-2, in dominant follicles throughout the periovulatory period in women and the regulatory mechanisms underlying ACE2 expression in human granulosa/lutein cells (hGLC). DESIGN: Experimental prospective clinical study and laboratory-based investigation. SETTING: University Medical Center and private in vitro fertilization center. PATIENT(S): Thirty premenopausal women undergoing surgery for tubal ligation and 16 premenopausal women undergoing in vitro fertilization. INTERVENTION(S): Administration of human chorionic gonadotropin (hCG) and harvesting of preovulatory/ovulatory follicles by timed laparoscopy, and collection of granulosa/lutein cells and cumulus cells at the time of oocyte retrieval. MAIN OUTCOME MEASURE(S): Expression and localization of ACE2 in granulosa cells and dominant follicles collected throughout the periovulatory period of the menstrual cycle and in hGLC using quantitative polymerase chain reaction, immunoblotting, and immunohistochemistry. RESULT(S): ACE2 expression (mRNA and protein) is up-regulated in human ovulatory follicles after administration of hCG. ACE2 expression was higher in cumulus cells than in granulosa cells. hCG increased the expression of ACE2 in primary hGLC cultures; the increase was inhibited by RU486 (an antagonist for progesterone receptor and glucocorticoid receptor) and CORT125281 (a selective glucocorticoid receptor antagonist), but not by AG1478 (an EGF receptor tyrosine kinase inhibitor) or by dexamethasone. CONCLUSION(S): The hormone-regulated expression of ACE2 in granulosa cells suggests a potential role of ACE2 in the ovulatory process. These data also imply the possible impact of COVID-19 on a vital cyclic event of ovarian function and thus on women's overall reproductive health. However, SAR-CoV-2 infection in ovarian cells in vivo or in vitro has yet to be determined.

The FOS/AP-1 Regulates Metabolic Changes and Cholesterol Synthesis in Human Periovulatory Granulosa Cells.

FOS, a subunit of the activator protein-1 (AP-1) transcription factor, has been implicated in various cellular changes. In the human ovary, the expression of FOS and its heterodimeric binding partners JUN, JUNB, and JUND increases in periovulatory follicles. However, the specific role of the FOS/AP-1 remains elusive. The present study determined the regulatory mechanisms driving the expression of FOS and its partners and functions of FOS using primary human granulosa/lutein cells (hGLCs). Human chorionic gonadotropin (hCG) induced a biphasic increase in the expression of FOS, peaking at 1 to 3 hours and 12 hours. The levels of JUN proteins were also increased by hCG, with varying expression patterns. Coimmunoprecipitation analyses revealed that FOS is present as heterodimers with all JUN proteins. hCG immediately activated protein kinase A and p42/44MAPK signaling pathways, and inhibitors for these pathways abolished hCG-induced increases in the levels of FOS, JUN, and JUNB. To identify the genes regulated by FOS, high-throughput RNA sequencing was performed using hGLC treated with hCG ± T-5224 (FOS inhibitor). Sequencing data analysis revealed that FOS inhibition affects the expression of numerous genes, including a cluster of genes involved in the periovulatory process such as matrix remodeling, prostaglandin synthesis, glycolysis, and cholesterol biosynthesis. Quantitative PCR analysis verified hCG-induced, T-5224-regulated expression of a selection of genes involved in these processes. Consistently, hCG-induced increases in metabolic activities and cholesterol levels were suppressed by T-5224. This study unveiled potential downstream target genes of and a role for the FOS/AP-1 complex in metabolic changes and cholesterol biosynthesis in granulosa/lutein cells of human periovulatory follicles.

Core Binding Factors are essential for ovulation, luteinization, and female fertility in mice.

Core Binding Factors (CBFs) are a small group of heterodimeric transcription factor complexes composed of DNA binding proteins, RUNXs, and a non-DNA binding protein, CBFB. The LH surge increases the expression of Runx1 and Runx2 in ovulatory follicles, while Cbfb is constitutively expressed. To investigate the physiological significance of CBFs, we generated a conditional mutant mouse model in which granulosa cell expression of Runx2 and Cbfb was deleted by the Esr2Cre. Female Cbfbflox/flox;Esr2cre/+;Runx2flox/flox mice were infertile; follicles developed to the preovulatory follicle stage but failed to ovulate. RNA-seq analysis of mutant mouse ovaries collected at 11 h post-hCG unveiled numerous CBFs-downstream genes that are associated with inflammation, matrix remodeling, wnt signaling, and steroid metabolism. Mutant mice also failed to develop corpora lutea, as evident by the lack of luteal marker gene expression, marked reduction of vascularization, and excessive apoptotic staining in unruptured poorly luteinized follicles, consistent with dramatic reduction of progesterone by 24 h after hCG administration. The present study provides in vivo evidence that CBFs act as essential transcriptional regulators of both ovulation and luteinization by regulating the expression of key genes that are involved in inflammation, matrix remodeling, cell differentiation, vascularization, and steroid metabolisms in mice.

Biological Evaluation of Hot-Melt Extruded Nano-selenium and the Role of Selenium on the Expression Profiles of Selenium-Dependent Antioxidant Enzymes in Chickens.

A study was conducted to determine the effect of dietary selenium (Se) concentration and source for broiler chickens on performance, nutrient digestibility, plasma Se, glutathione peroxidase (GPx) activity, and thiobarbituric acid reactive substances (TBARS). A total of 700 1-day-old broiler chicks were assigned to 7 diets with 20 birds per cage and 5 replicates per treatment. The experimental diets were fed for 32 days in 2 phases (phase 1, day 0 to 14 and phase 2, day 15 to 32). Treatments were as follows: control (without Se supplementation), sodium selenite (SeS; 0.15, 0.30, or 0.45 ppm), and hot-melt extruded sodium selenite (SeHME; 0.15, 0.30, or 0.45 ppm). There were significant linear responses (P < 0.01) for higher plasma Se concentration in SeS and SeHME treatments. Moreover, an increased (P < 0.01) Se concentration of plasma occurred in SeHME treatment compared with that in SeS treatment. The serum GPx analyses revealed that supplemental SeS and SeHME increased significantly the activity of GPx in the plasma in phase 1 (P < 0.05) and phase 2 (P < 0.05). There were significant linear (P < 0.01) responses of SeS and SeHME treatments for the expression of SelW, GPx1, GPx3, and GPx4 in the livers and spleens. In addition, SeHME showed an upregulated expression of GPx-4 in the livers (P < 0.01) and SelW in the spleens (P < 0.05) compared with SeS treatment. SeHME showed a lower TBARS on day 9. Moreover, a decreased (P < 0.01) TBARS occurred in SeS treatment compared with that in control treatment. In conclusion, SeHME can increase antioxidant activity and Se absorption, consequently being a more suitable source of Se than regular sodium selenite.

Analysis of interferon-γ receptor IFNGR1 and IFNGR2 expression and regulation at the maternal-conceptus interface and the role of interferon-γ on endometrial expression of interferon signaling molecules during early pregnancy in pigs.

It has long been known that pig conceptuses produce interferon-γ (IFNG) at the time of implantation, but the role of IFNG and its mechanism of action at the maternal-conceptus interface are not fully understood. Accordingly, we analyzed the expression and regulation of IFNG receptors IFNGR1 and IFNGR2 in the endometrium during the estrous cycle and pregnancy in pigs. Levels of IFNGR1 and IFNGR2 messenger RNA (mRNA) expression changed in the endometrium, with the highest levels during mid pregnancy for IFNGR1 and on Day 12 of pregnancy for IFNGR2. The expression of IFNGR1 and IFNGR2 mRNAs was also detected in conceptuses during early pregnancy and chorioallantoic tissues during mid to late pregnancy. IFNGR1 and IFNGR2 mRNAs were localized to endometrial epithelial and stromal cells and to the chorionic membrane during pregnancy. IFNGR2 protein was also localized to endometrial epithelial and stromal cells, and increased epithelial expression of IFNGR2 mRNA and protein was detectable during early pregnancy than the estrous cycle. Explant culture studies showed that estrogen increased levels of IFNGR2, but not IFNGR1, mRNAs, while interleukin-1ß did not affect levels of IFNGR1 and IFNGR2 mRNAs. Furthermore, IFNG increased levels of IRF1, IRF2, STAT1, and STAT2 mRNAs in the endometrial explants. These results in pigs indicate that IFNGR1 and IFNGR2 are expressed in a stage of pregnancy- and cell-type specific manner in the endometrium and that sequential cooperative action of conceptus signals estrogen and IFNG may be critical for endometrial responsiveness to IFNs for the establishment of pregnancy in pigs.

Puffing as a Novel Process to Enhance the Antioxidant and Anti-Inflammatory Properties of Curcuma longa L. (Turmeric).

Curcuma longa L. (turmeric) is used as a food spice; however, its strong taste restricts wider applications as a food ingredient despite its well-known health benefits. To develop an effective yet simple process for enhancing its antioxidant and anti-inflammatory activities, turmeric was gun-puffed at various pressures. Puffed turmeric exhibited an increase in its brown color and porous structures, indicating the occurrence of the Maillard reaction and vaporization during the process. Proximal analysis revealed that puffing did not alter the major constituents, although a very small decrease in crude fat extraction was observed under some circumstances. Total phenolic compounds in the extract were significantly increased after puffing, and subsequent assessment of antioxidant capacity, as determined using independent 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid) (ABTS), and ferric reducing antioxidant power (FRAP) assays, demonstrated enhanced antioxidant capacity in a puffing-pressure-dependent manner. Turmeric extract was further tested for the regulation of inflammatory responses in the murine macrophage RAW264.7 cell line. Suppression of pro-inflammatory cytokines interleukin (IL)-6 and tumor necrosis factor (TNF)-α in lipopolysaccharides (LPS)-induced macrophages was amplified using puffed-turmeric extracts compared to the control extract. Furthermore, macrophage-activation assessment revealed downregulated expression of inflammation-relevant cluster of differentiation (CD)80 and CD86 using puffed-turmeric extract in a puffing-pressure-dependent manner. However, expression of major histocompatibility complex (MHC)-II, which controls adoptive immunity, was not affected by treatment with any of the turmeric extracts. Overall, the current study demonstrated that puffing is a promising and simple method for enhancing the antioxidant and anti-inflammatory properties of turmeric.

Effects of hot melt extrusion processed nano-iron on growth performance, blood composition, and iron bioavailability in weanling pigs.

This study was conducted to investigate the effects of hot melt extrusion (HME) nano-iron as an alternative for the common ferrous sulfate on iron (Fe) bioavailability, growth performance, nutrient digestibility, intestinal morphology, and intestinal microbiota of weanling pigs. A total of 200 piglets (Landrace × Yorkshire × Duroc) were randomly allotted to seven treatments on the basis of initial body weight (BW) and sex. Treatments were the INO100 (100 ppm Fe as FeSO4), HME-Fe levels (50, 75, and 100 ppm nano-Fe as FeSO4). ORG100 (100 ppm Fe as iron methionine). In phase 1, the HME50 pigs showed the lowest Fe content in feed and feces. Plasma Fe concentration was increased in HME100 and ORG100 pigs. In phase 2, there were significantly lower concentration of Fe in feed and feces of HME50 pigs (p < 0.01). A lower Fe concentration in the plasma and liver were observed in HME50 pigs compared with HME100 pigs. Concentration of red blood cell (RBC) was the lowest (p < 0.01) for HME50 pigs. During phase 2, the HME100, HME75, and ORG100 pigs showed a higher RBC and hemoglobin values compared with HME50 pigs. Digestibility of gross energy (GE) and crude protein (CP) were significantly higher in HME100 pigs compared with HME50 pigs. There was an increased (p < 0.01) villus height in the duodenum and jejunum of HME100 pigs compared with HME50 pigs. It is concluded that dietary Fe does not improve growth performance of weanling pigs; however, increasing the dietary iron concentration in weanling piglets increased the RBC and hemoglobin. In addition, the potential ability of HME to be used at a lower level (HME75) was observed.

Changes in calcium levels in the endometrium throughout pregnancy and the role of calcium on endometrial gene expression at the time of conceptus implantation in pigs.

Calcium plays an essential role in regulating many cellular functions, including proliferation, differentiation, and apoptosis. In spite of its importance in the establishment and maintenance of pregnancy, changes in calcium levels at the maternal-conceptus interface during pregnancy and its action on endometrial gene expression are not well understood. Thus, we examined changes in calcium levels in the endometrium during pregnancy, calcium deposition at the maternal-conceptus interface during pregnancy, and the role of calcium on the expression of endometrial genes related to conceptus implantation during early pregnancy in pigs. The amounts of endometrial calcium increased during mid- to late pregnancy, and calcium deposition was mainly localized to endometrial and chorionic epithelial cells at the maternal-conceptus interface during pregnancy and conceptus tissues during early pregnancy. The amounts of total recoverable calcium in uterine flushings were greater on Day 12 of pregnancy than Day 12 of the estrous cycle, and estrogen increased absorption of calcium ions by endometrial tissues. Increasing endometrial calcium levels by treatment with A23187, a calcium ionophore, decreased the expression of the estrogen-responsive endometrial genes AKR1B1, ESR1, FGF7, IL1RAP, LPAR3, S100G, SPP1, and STC1 and increased the expression of genes related to prostaglandin synthesis and transport, namely PTGES, PTGS2, and SLCO5A1. These data suggest that calcium ions at the maternal-conceptus interface play a critical role in the establishment and maintenance of pregnancy in pigs by regulating the expression of endometrial genes involved in conceptus implantation, as well as the attachment of endometrial epithelial and conceptus trophectoderm/chorionic epithelial cells during pregnancy.

The endogenous hydrogen sulfide generating system regulates ovulation.

The generation of free-radicals such as nitric oxide has been implicated in the regulation of ovarian function, including ovulation. Tissues that generate nitric oxide typically generate another free-radical gas, hydrogen sulfide (H2S), although little is known about the role of H2S in ovarian function. The hypothesis of this study was that H2S regulates ovulation. Treatment with luteinizing hormone (LH) increased the levels of mRNA and protein of the H2S generating enzyme cystathionine γ-lyase (CTH) in granulosa cells of mice and humans in vivo and in vitro. Pharmacological inhibition of H2S generating enzymes reduced the number of follicles ovulating in mice in vivo and in vitro, and this inhibitory action was reversed by cotreatment with a H2S donor. Addition of a H2S donor to cultured mouse granulosa cells increased basal and LH-dependent abundance of mRNA encoding amphiregulin, betacellulin and tumor necrosis alpha induced protein 6, proteins important for cumulus expansion and follicle rupture. Inhibition of CTH activity reduced abundance of mRNA encoding matrix metalloproteinase-2 and -9 and tissue-type plasminogen activator, and cotreatment with the H2S donor increased the levels of these mRNA above those stimulated by LH alone. We conclude that the H2S generating system plays an important role in the propagation of the preovulatory cascade and rupture of the follicle at ovulation.

FOS, a Critical Downstream Mediator of PGR and EGF Signaling Necessary for Ovulatory Prostaglandins in the Human Ovary.

Context: Fos null mice failed to ovulate and form a corpus luteum (CL) even when given exogenous gonadotropins, suggesting that ovarian Fos expression is critical for successful ovulation and CL formation. However, little is known about FOS in the human ovary. Objectives: To determine the expression, regulation, and function of FOS in human periovulatory follicles. Design/Participants: Timed periovulatory follicles were obtained from normally cycling women. Granulosa/lutein cells were collected from in vitro fertilization patients. Main Outcome Measures: The in vivo expression after human chorionic gonadotropin (hCG) administration and in vitro regulation of FOS, JUN, JUNB, and JUND was evaluated at the mRNA and protein level. Binding of progesterone receptor (PGR) and FOS to their target genes was assessed by chromatin immunoprecipitation analyses. Prostaglandin E2 (PGE2) and progesterone were measured. Results: The expression of FOS, JUNB, and JUND drastically increased in ovulatory follicles after hCG administration. In human granulosa/lutein cell cultures, hCG increased the expression of FOS and JUN proteins. Inhibitors of PGR and epidermal growth factor (EGF) receptors reduced hCG-induced increases in the expression and phosphorylation of FOS. PGR bound to the FOS gene. A selective FOS inhibitor blocked hCG-induced increases in PGE2 and the expression of prostaglandin (PG) synthases and transporters (PTGES, SLCO2A1, and ABCC1). FOS bound to the promoter regions of these genes. Conclusions: The increase of FOS/activator protein 1 in human periovulatory follicles after hCG administration is mediated by collaborative actions of PGR and EGF signaling and critical for the upregulated expression of key ovulatory genes required for the rise in ovulatory PG in human granulosa cells.

Core Binding Factor ß Expression in Ovarian Granulosa Cells Is Essential for Female Fertility.

Core binding factor ß (CBFß) is a non-DNA-binding partner of all RUNX proteins and critical for transcription activity of CBF transcription factors (RUNXs/CBFß). In the ovary, the expression of Runx1 and Runx2 is highly induced by the luteinizing hormone (LH) surge in ovulatory follicles, whereas Cbfb is constitutively expressed. To investigate the physiological significance of CBFs in the ovary, the current study generated two different conditional mutant mouse models in which granulosa cell expression of Cbfb and Runx2 was reduced by Cre recombinase driven by an Esr2 promoter. Cbfbgc-/- and Cbfbgc-/- × Runx2gc+/- mice exhibited severe subfertility and infertility, respectively. In the ovaries of both mutant mice, follicles develop normally, but the majority of preovulatory follicles failed to ovulate either in response to human chorionic gonadotropin administration in pregnant mare serum gonadotropin-primed immature animals or after the LH surge at 5 months of age. Morphological and physiological changes in the corpus luteum of these mutant mice revealed the reduced size, progesterone production, and vascularization, as well as excessive lipid accumulation. In granulosa cells of periovulatory follicles and corpora lutea of these mice, the expression of Edn2, Ptgs1, Lhcgr, Sfrp4, Wnt4, Ccrl2, Lipg, Saa3, and Ptgfr was also drastically reduced. In conclusion, the current study provided in vivo evidence that CBFß plays an essential role in female fertility by acting as a critical cofactor of CBF transcription factor complexes, which regulate the expression of specific key ovulatory and luteal genes, thus coordinating the ovulatory process and luteal development/function in mice.

Vitamin D-metabolic enzymes and related molecules: Expression at the maternal-conceptus interface and the role of vitamin D in endometrial gene expression in pigs.

Vitamin D is a secosteroid hormone with many varied functions including regulation of blood calcium levels, cell proliferation, immunity, and reproduction in mammals. Vitamin D is activated by 25-hydroxylase (CYP2R1) and 1-alpha-hydroxylase (CYP27B1) and is degraded by 24-hydroxylase (CYP24A1). Vitamin D is transported by vitamin D-binding protein (group-specific component, GC) through the bloodstream and regulates cellular actions by binding to vitamin D receptor (VDR). In this study, we determined the expression and regulation of vitamin D-related molecules and the role of vitamin D at the maternal-conceptus interface in pigs. Vitamin D-metabolizing enzymes CYP2R1, CYP27B1, and CYP24A1, vitamin D binding protein GC, and vitamin D receptor VDR were expressed in the endometrium in a pregnancy stage-specific manner as well as in conceptus and chorioallantoic tissues during pregnancy. VDR protein was localized to endometrial and trophoblastic cells. Concentrations of calcitriol, the active form of vitamin D, in the endometrial tissues were higher during early pregnancy than in mid- to late pregnancy, while plasma concentrations of calcitriol were highest during late pregnancy. Furthermore, calcitriol affected the expression of several genes related to conceptus implantation, vitamin D metabolism, calcium ion regulation, PG metabolism, and calcium-binding proteins in endometrial tissue explants. These results show that CYP2R1, CYP27B1, CYP24A1, GC, and VDR were expressed at the maternal-conceptus interface, endometrial calcitriol levels were regulated during pregnancy, and calcitriol modulated the expression of endometrial genes, suggesting that calcitriol may play an important role in the establishment and maintenance of pregnancy by regulating endometrial function in pigs.

Analysis of cysteine-X-cysteine motif chemokine ligands 9, 10, and 11, their receptor CXCR3, and their possible role on the recruitment of immune cells at the maternal-conceptus interface in pigs.

Chemokines play critical roles in the establishment and maintenance of pregnancy in animals. Cysteine-X-cysteine motif chemokine ligand 9 (CXCL9), CXCL10, and CXCL11 are involved in recruiting immune cells by binding to their shared receptor, CXC receptor 3 (CXCR3), in a variety of tissues. This study examined the expression and regulation of chemokines CXCL9, CXCL10, and CXCL11, their receptor CXCR3, and their role at the maternal-conceptus interface in pigs. The endometrium expressed CXCL9, CXCL10, CXCL11, and CXCR3 stage specifically during pregnancy, with the greatest abundance on Day 15 of pregnancy. It was noted that their expression was primarily localized to stromal cells, endothelial cells, or vascular smooth muscle cells in the endometrium. Interferon-γ increased the abundance of CXCL9, CXCL10, CXCL11 mRNAs, but not CXCR3, in endometrial explants. Furthermore, recombinant CXCL9 (rCXCL9), rCXCL10, and rCXCL11 proteins increased migration of cultured peripheral blood mononuclear cells (PBMCs) in a dose-dependent manner. Recombinant CXCL9 and rCXCL10 caused migration of CD4+, CD8+, CD4+CD8+ T cells, and natural killer (NK) cells, and rCXCL11 increased migration of CD4+ T and NK cells in PBMCs. The present study demonstrated that interferon-γ-induced CXCL9, CXCL10, and CXCL11, and their receptor CXCR3 were expressed in the uterus in stage- and cell-type specific manners and increased the migration of T and NK cells, which showed the greatest endometrial infiltration on Day 15 of pregnancy. These results suggest that CXCL9, CXCL10, and CXCL11 may play an important role in the recruitment of immune cells into the endometrium during the implantation period in pigs.

Coordinated Regulation Among Progesterone, Prostaglandins, and EGF-Like Factors in Human Ovulatory Follicles.

Context: In animal models, the luteinizing hormone surge increases progesterone (P4) and progesterone receptor (PGR), prostaglandins (PTGs), and epidermal growth factor (EGF)-like factors that play essential roles in ovulation. However, little is known about the expression, regulation, and function of these key ovulatory mediators in humans. Objective: To determine when and how these key ovulatory mediators are induced after the luteinizing hormone surge in human ovaries. Design and Participants: Timed periovulatory follicles were obtained from cycling women. Granulosa/lutein cells were collected from in vitro fertilization patients. Main Outcome Measures: The in vivo and in vitro expression of PGR, PTG synthases and transporters, and EGF-like factors were examined at the level of messenger RNA and protein. PGR binding to specific genes was assessed. P4 and PTGs in conditioned media were measured. Results: PGR, PTGS2, and AREG expressions dramatically increased in ovulatory follicles at 12 to 18 hours after human chorionic gonadotropin (hCG). In human granulosa/lutein cell cultures, hCG increased P4 and PTG production and the expression of PGR, specific PTG synthases and transporters, and EGF-like factors, mimicking in vivo expression patterns. Inhibitors for P4/PGR and EGF-signaling pathways reduced hCG-induced increases in PTG production and the expression of EGF-like factors. PGR bound to the PTGS2, PTGES, and SLCO2A1 genes. Conclusions: This report demonstrated the time-dependent induction of PGR, AREG, and PTGS2 in human periovulatory follicles. In vitro studies indicated that collaborative actions of P4/PGR and EGF signaling are required for hCG-induced increases in PTG production and potentiation of EGF signaling in human periovulatory granulosa cells.

Characterization of interferon α and ß receptor IFNAR1 and IFNAR2 expression and regulation in the uterine endometrium during the estrous cycle and pregnancy in pigs.

Type I interferons (IFNs) bind to the heterodimeric receptor composed of IFN-α/ß receptor 1 (IFNAR1) and IFN-α and ß receptor 2 (IFNAR2) to transmit signals into the cell. It is well known that IFN-δ (IFND), a type I IFN, is secreted by the conceptus during early pregnancy in pigs. However, expression and regulation of IFNAR1 and IFNAR2 in the porcine uterine endometrium are not well understood. Thus, we analyzed the expression and regulation of IFNAR1 and IFNAR2 in the uterine endometrium during the estrous cycle and pregnancy and conceptus and chorioallantoic tissues during pregnancy in pigs. The IFNAR1 and IFNAR2 mRNAs were expressed in the uterine endometrium, and their levels on Day 12 of pregnancy were higher than those on Day 12 of the estrous cycle and highest during pregnancy. The IFNAR1 and IFNAR2 mRNAs were also expressed in conceptuses during early pregnancy, in chorioallantoic tissues during mid-to-term pregnancy, and in endometrial epithelial cells and chorionic membrane during mid-to-late pregnancy. The abundance of IFNAR1 and IFNAR2 mRNAs was increased by interleukin-1ß (IL1B), and the abundance of IFNAR2 was increased by estradiol in endometrial tissue explants. Thus, IFNAR1 and IFNAR2 mRNAs were expressed in the uterine endometrium during the estrous cycle and pregnancy in a pregnancy status- and stage-specific manner, and their expression was affected by estradiol and/or IL1B. These results suggest that endometrial and conceptus IFNAR1 and IFNAR2 may mediate the action of type I IFNs during the implantation period for the establishment and maintenance of pregnancy in pigs.

Analysis of Stage-Specific Gene Expression Profiles in the Uterine Endometrium during Pregnancy in Pigs.

The uterine endometrium plays a critical role in regulating the estrous cycle and the establishment and maintenance of pregnancy in mammalian species. Many studies have investigated the expression and function of genes in the uterine endometrium, but the global expression pattern of genes and relationships among genes differentially expressed in the uterine endometrium during gestation in pigs remain unclear. Thus, this study investigated global gene expression profiles using microarray in pigs. Diverse transcriptome analyses including clustering, network, and differentially expressed gene (DEG) analyses were performed to detect endometrial gene expression changes during the different gestation stages. In total, 6,991 genes were found to be differentially expressed by comparing genes expressed on day (D) 12 of pregnancy with those on D15, D30, D60, D90 and D114 of pregnancy, and clustering analysis of detected DEGs distinguished 8 clusters. Furthermore, several pregnancy-related hub genes such as ALPPL2, RANBP17, NF1B, SPP1, and CST6 were discovered through network analysis. Finally, detected hub genes were technically validated by quantitative RT-PCR. These results suggest the complex network characteristics involved in uterine endometrial gene expression during pregnancy and indicate that diverse patterns of stage-specific gene expression and network connections may play a critical role in endometrial remodeling and in placental and fetal development to establish and maintenance of pregnancy in pigs.

The strategy and method in modulating finger regeneration.

The tip of the human finger can regenerate if the amputation is distal to the nail bed, usually in young children. Studies in regeneration of rodent digits have shown that regeneration occurs if the amputation is distal to the mid-third phalanx for certain ages. The digit contains many different components, such as muscle, tendon, bone, skin, nerves and blood vessels, which must all be regrown in the proper location in order to restore functionality. The mechanism behind the complex healing/regeneration processes is still under investigation; however, improvements in injured finger regeneration have been gradually developing in animal models over the past few years. This review discusses a few strategies and methods to possibly enhance digit regeneration beyond current natural limits, focusing on aspects including scarless wound healing, cell-based treatments, tissue engineering and electrical stimulation.

Prostaglandin transporters ABCC4 and SLCO2A1 in the uterine endometrium and conceptus during pregnancy in pigs.

Prostaglandins (PGs) are involved in many reproductive activities including luteolysis, maternal recognition of pregnancy, endometrial gene expression, conceptus development, and parturition in domestic animals. However, mechanisms by which PGE2 and PGF2alpha are modulated in the uterine endometrium and expression of ABCC4 and SLCO2A1, responsible for efficient transport of PGs across the cell membrane, in the endometrium during the estrous cycle and pregnancy are not fully understood in pigs. Therefore, we determined expression of ABCC4 and SLCO2A1, genes involved in transport of PGE2 and PGF2alpha in the uterine endometrium during the estrous cycle and pregnancy in pigs. ABCC4 and SLCO2A1 mRNAs were expressed in the uterine endometrium, most abundantly on Day 12 of pregnancy and during late pregnancy. Expression of ABCC4 mRNA and protein was localized mainly to uterine luminal epithelial (LE) and glandular epithelial (GE) cells, and expression of SLCO2A1 mRNA and protein was expressed primarily in uterine LE and blood vessels. Expression of ABCC4 and SLCO2A1 mRNAs was also detected in conceptuses during early pregnancy. In addition, explant culture experiments showed that increasing doses of interleukin 1B (IL1B) with estrogen and progesterone increased levels of ABCC4 and SLCO2A1 mRNAs in the uterine endometrium. These results indicate that expression of genes responsible for transport of PGE2 and PGF2alpha are dynamically regulated in the uterine endometrium during pregnancy and that ABCC4 and SLCO2A1 play critical roles in supporting the establishment and maintenance of pregnancy by regulating PG transport at the maternal-fetal interface in pigs.