Global Transcriptomic Profiling of Bovine Endometrial Immune Response In Vitro. I. Effect of Lipopolysaccharide on Innate Immunity

The dysregulation of endometrial immune response to bacterial lipopolysaccharide (LPS) has been implicated in uterine disease and infertility in the postpartum dairy cow, although the mechanisms are not clear. Here, we investigated whole-transcriptomic gene expression in primary cultures of mixed bovine epithelial and stromal endometrial cells. Cultures were exposed to LPS for 6 h, and cellular response was measured by bovine microarray. Approximately 30% of the 1006 genes altered by LPS were classified as being involved in immune response. Cytokines and chemokines (IL1A, CX3CL1, CXCL2, and CCL5), interferon (IFN)-stimulated genes (RSAD2, MX2, OAS1, ISG15, and BST2), and the acute phase molecule SAA3 were the most up-regulated genes. Ingenuity Pathway Analysis identified up-regulation of many inflammatory cytokines and chemokines, which function to attract immune cells to the endometrium, together with vascular adhesion molecules and matrix metalloproteinases, which can facilitate immune cell migration from the tissue toward the uterine lumen. Increased expression of many IFN-signaling genes, immunoproteasomes, guanylate-binding proteins, and genes involved in the intracellular recognition of pathogens suggests important roles for these molecules in the innate defense against bacterial infections. Our findings confirmed the important role of endometrial cells in uterine innate immunity, whereas the global approach used identified several novel immune response pathways triggered by LPS in the endometrium. Additionally, many genes involved in endometrial response to the conceptus in early pregnancy were also altered by LPS, suggesting one mechanism whereby an ongoing response to infection may interfere with the establishment of pregnancy

Impact of endotoxemia on ovarian signaling and function

The ovary is the female reproductive organ responsible for producing gametes and sex steroid hormones. There are many stages throughout the estrous cycle at which perturbations could affect quality of the oocyte. Heat stress (HS) is detrimental to fertility, and swine are inherently susceptible due to lack of functional sweat glands. During times of HS, blood flow is redirected to the periphery, creating a hypoxic environment within the gastrointestinal tract, culminating in compromised intestinal integrity and the influx of lipopolysaccharide (LPS) into circulation. This influx of LPS into circulation is known as endotoxemia and can arise from a myriad of physiological states, such as HS, and off-feed events, as well as obesity, diabetes, and alcoholism. Alterations to follicular growth and development incurred during endotoxemia could delay or inhibit ovulation of a viable oocyte, thus compromising potential future embryos. We hypothesized that the ovary is responsive to circulating LPS and that endotoxemia upregulates toll-like receptor 4 (TLR4) pathways, as well disrupt insulin signaling and steroidogenesis in the ovary. To test this central hypothesis, various animal models; pre- and post-pubertal gilts, as well as lactating cows, were exposed to HS and/or LPS to perturb intestinal integrity and/or mimic chronic endotoxemia. We analyzed mRNA and protein abundance in hepatic and ovarian tissue via qRT-PCR, western blotting, and immunohistochemistry, as well as quantification of insulin and LPS-binding protein, 17β-estradiol, and progesterone via ELISAs. Overall, our data demonstrates that the TLR4 pathway is activated by endotoxin in both gilts and lactating cows, and could be partially culpable for infertility that arises as a consequence of HS or infection. Interestingly, lactating cows become tolerant to exponentially increasing exposures to LPS in terms of dominant follicular growth and development. Taken together, our discoveries further delineate effects of LPS on ovarian signaling and function. These alterations, or lack thereof, are important to understand in order to facilitate development of mitigation strategies to ameliorate infertility in the future

Investigating the ovarian response to endotoxemia

The ovary is responsible for production of steroid hormones and gametes in the female. It is endowed with a finite pool of primordial follicles, established in utero, that serve as the source of oocytes during a female’s reproductive lifespan. Alterations to ovarian function and signaling can result in disruptions to steroid hormone production, follicle activation, and oocyte quality. The degree of disruption is dependent on the developmental or estrous cycle stage of the animal as well as the duration of the perturbation. Inflammation can have deleterious effects on ovarian signaling and function, and can arise from multiple sources, such as endotoxemia, or increased levels of lipopolysaccharide (LPS) in blood. One major source of LPS is the intestine, where stresses such as hypoxia due to heat stress, high fat diets, or obesity can induce increased intestinal permeability and allow passage of LPS from the intestinal lumen into circulation. LPS signaling via its receptor, toll-like receptor 4 (TLR4), may induce inflammation in the ovary, potentially compromising oocyte and follicular quality, resulting in reduced fertility. This thesis investigated the central hypothesis that increases in circulating LPS would alter ovarian signaling and function as evidenced by activation of the TLR4, phosphatidylinositol 3 kinase (PI3K), and steroidogenic pathways. To test this hypothesis, we utilized three animal model-LPS exposure schemes in pigs: post-pubertal chronic LPS infusion, post-pubertal heat stress (HS), and pre-pubertal high fat diet (HFD). We analyzed ovarian protein and mRNA abundance via western blotting and qRT-PCR, as well as quantification of 17β-estradiol, progesterone, and LPS binding protein concentrations via ELISAs. Overall, our findings demonstrate that the ovary is responsive to LPS as demonstrated by increased ovarian TLR4 in an LPS infusion but not HFD model. We also discovered alterations to the PI3K and steroidogenic pathways in our HFD model not seen with the other exposure paradigms, indicating a potential effect of developmental age or an alternate effector mechanism in these pigs. Taken together, these data demonstrate the alterations in ovarian function due to different exposures to LPS in isolation or combination with other physiological changes. This understanding is vital to the development of amelioration strategies to reduce infertility

The CD14 functional gene polymorphism -260 C>T is not involved in either the susceptibility to Chlamydia trachomatis infection or the development of tubal pathology

BACKGROUND: The functional polymorphism -260 C>T in the LPS sensing TLR4 co-receptor CD14 gene enhances the transcriptional activity and results in a higher CD14 receptor density. Individuals carrying the T/T genotype also have significantly higher serum levels of soluble CD14. The T allele of this polymorphism has recently been linked to Chlamydia pneumoniae infection. We investigated the role of the CD14 -260 C>T polymorphism in the susceptibility to and severity (defined as subfertility and/or tubal pathology) of C. trachomatis infection in Dutch Caucasian women. METHODS: The different CD14 -260 C>T genotypes were assessed by PCR-based RFLP analysis in three cohorts: 1) A cohort (n = 576) of women attending a STD clinic, 2) a cohort (n = 253) of women with subfertility, and 3) an ethnically matched control cohort (n = 170). The following variables were used in the analysis: In cohort 1 the CT-DNA status, CT IgG serology status, self-reported symptoms and in cohort 2, the CT IgG serology status and the tubal status at laparoscopy. RESULTS: In the control cohort the CC, CT and TT genotype distribution was: 28.2%, 48.2%, and 23.5% respectively. No differences were found in the overall prevalence of CD14 -260 genotypes (28.1%, 50.7%, and 21.2%) in cohort 1 when compared to the control cohort. Also no differences were observed in women with or without CT-DNA, with or without serological CT responses, with or without symptoms, or in combinations of these three variables. In subfertile women with tubal pathology (cohort 2, n = 50) the genotype distribution was 28.0%, 48.0%, and 24.0% and in subfertile women without tubal pathology (n = 203), 27.6%, 49.3% and 23.2%. The genotype distribution was unchanged when CT IgG status was introduced in the analyses. CONCLUSION: The CD14 -260 C>T genotype distributions were identical in all three cohorts, showing that this polymorphism is not involved in the susceptibility to or severity of sequelae of C. trachomatis infection

Modulation of the maternal immune system by the pre-implantation embryo

Background: A large proportion of pregnancy losses occur during the pre-implantation period, when the developing embryo is elongating rapidly and signalling its presence to the maternal system. The molecular mechanisms that prevent luteolysis and support embryo survival within the maternal environment are not well understood. To gain a more complete picture of these molecular events, genome-wide transcriptional profiles of reproductive day 17 endometrial tissue were determined in pregnant and cyclic Holstein-Friesian dairy cattle

Effect of age, body condition, pregnancy and lactation on circulating leptin concentrations in beef cattle

A series of experiments were conducted to evaluate the potential role of leptin in bovine reproduction. In Experiment 1, mean circulating leptin concentrations of postpartum cows were not affected by exogenous dexamethasone treatments. In Experiment 2, mean leptin concentrations were not correlated with female age or body weight but were positively correlated with body condition scores of beef cattle. Leptin concentrations were higher in 1 year old heifers (8.9 ng/ml) compared with 2 year old cows (6.0 ng/ml), but heifer leptin concentrations were not different than 4 to 6 year old cows (8.0 ng/ml) and cows ≥7 years of age (10.5 ng/ml). Mean leptin concentrations were negatively correlated with age in heifers and cows ≤2 years of age and positively correlated with age in cows \u3e3 years of age. In Experiment 3, there were no differences in mean leptin concentrations for 56 days starting 14 days following AI among 2-year old and 3-year old cows pregnant to AI (1.2 ng/ml), the clean-up bulls (1.2 ng/ml) and nonpregnant females (2.2 ng/ml) after a 60-day breeding season. Plasma leptin concentrations were lower for lactating cows (1.0 ng/ml) compared with nonlactating cows (2.1 ng/ml). Female age did not affect circulating leptin concentrations. In Experiment 4, oviduct and uterine epithelial cells from mid-luteal phase females stained positive for the long form of the leptin receptor, and uterine biopsies revealed intense staining for the long form of the leptin receptor on the luminal side of the uterine endometrium. Bovine blastocysts stained positive for the long form of the leptin receptor in the trophoblast cells. In Experiment 5, addition of leptin to culture medium at 0, 100 and 1,000 ng/ml did not affect the percentage embryos developing to the blastocyst stage. Also, leptin did not affect the ratio of blastocysts:8- to 16-cell embryos among the 0 ng/ml treatment group, the 100 ng/ml treatment and the 1,000 ng/ml treatment groups. Results indicate that in the beef cow, the release of leptin and subsequent role(s) of leptin in reproductive processes are likely different than those that have been reported for mice, rats and humans

Investigating impacts of altered central metabolism on ovarian function

Mammalian reproduction is highly dependent on the ovary. Ovarian health is crucial for the health of the female and the offspring. Observations of the timing of puberty and ovarian function reveal a major role for the metabolic status of the female on reproductive function. Many metabolic signals such as leptin, insulin, and LPS influence ovarian function as they participate in metabolic stress. All of these signals cross pathways at PI3K. Obesity, the accumulation of excessive fat, is an international health concern of wide prevalence. It causes an increase in circulating leptin, insulin, and LPS, and is associated with numerous reproductive disorders. Here, we investigated the effects of increasing systemic leptin during gestation on the offspring, and observed that gestational hyperleptinemia can alter the ovarian capacity to metabolize toxicants later in the offspring\u27s life. Heat stress is a condition of increased core temperature, and it impedes the reproductive performance of production animals during summer, making it an increased threat to food security, climate change only worsening it. Heat stress causes increased circulating insulin and LPS. We heat stressed gilts cyclically after synchronizing their estrous cycles, and observed decreased phosphorylation of AKT and increased TLR4 abundance in their ovaries. This demonstrates that, during heat stress, ovarian TLR4 signaling is upregulated and that the ovary might be initiating a stress response as indicated by reduced AKT phosphorylatio

Identification of genes induced by the conceptus in the bovine endometrium during the pre-implantation period

An intact embryo-maternal communication in the pre-implantation period is particularly critical for establishment of pregnancy and early embryonic losses have been identified as the major cause of reproductive failure in cattle. Thus, to gain deeper insight into this complex embryo-maternal crosstalk, a combination of subtracted cDNA libraries and cDNA array hybridization was applied to identify mRNAs differentially regulated genes in the bovine endometrium by the presence of a conceptus. One cDNA library was constructed according the suppression subtractive hybridization method (Diatchenko et al., 1996) with minor modifications; a second cDNA library was constructed of subtracted cDNA purchased from the vertis Biotechnologie AG. As biological model endometrial tissue samples of monozygotic twins (generated by embryo splitting) collected at day 18 of gestation were used, which is a unique possibility to eliminate genetic variability as a factor potentially affecting the results of gene expression analyses. Array hybridization was carried out using 33P-labeled cDNA probes obtained from five monozygotic twin pairs. Sequence analysis revealed 87 different genes or mRNAs, respectively, which displayed a difference in signal intensity of 2.0 fold or more in at least four out of five twin pairs. Eighty genes corresponded to genes with known or inferred function, either the bovine gene or the human orthologue. For 7 mRNAs a match with bovine ESTs was obtained only. For nine selected genes the expression in the bovine endometrium was quantified by the use of quantitative real-time RT-PCR to verify the results obtained by array hybridization and to perform more precise quantitative measurements for these genes. Overall, the results of array hybridization and real-time RT-PCR correlated very well. Almost half of the identified genes are known to be stimulated by type I interferons reflecting the response to IFNt, which is the pregnancy recognition signal in ruminants. Of particular interest among the interferon stimulated genes is ISG15, one of the most markedly upregulated genes in the present study, which is hypothesized to stabilize intracellular endometrial proteins through conjugation processes. For the ISG15ylation system mRNAs of four potential components (IFITM1, IFITM3, HSXIAPAF1, and DTX3L) were found in addition to ISG15 and UBE1L, and in situ hybridization revealed similar mRNA expression patterns of these genes. It is therefore suggested, that modification of endometrial proteins through ISG15ylation plays a fundamental role in the IFNt signaling. A classification of the identified genes according to their assignment to Gene Ontologies revealed the orchestrated interaction of various processes and mechanisms with regard to the preparation of the maternal endometrium for embryonic implantation. As particular interesting, genes were identified involved in modulation of the maternal immune system at the humoral and cellular level, cell adhesion, cell communication, regulation of transcription, cell differentiation, cell growth, and cell proliferation. These findings underline that an intense embryo-maternal dialogue takes place during the pre-implantation period, which culminates in a receptive endometrium prepared for implantation of the conceptus. To conclude, this is the first study of its kind for cattle in the pre-implantation stage of embryonic development and revealed the orchestrated upregulation of genes important for embryonic implantation during the pre-implantation period in the bovine endometrium. The presented results provide new starting points for detailed investigations of the embryo-maternal dialogue by which the endometrium is prepared for conceptus attachment

Identification of Gene Expression Changes Associated With Uterine Receptivity in Mice

The mouse is a widely used animal model for studying human reproduction. Although global gene expression changes associated with human uterine receptivity have been determined by independent groups, the same studies in the mouse are scarce. The extent of similarities/differences between mice and humans on uterine receptivity at the molecular level remains to be determined. In the present study, we analyzed global gene expression changes in receptive uterus on day 4 of pregnancy compared to non-receptive uterus on day 3 of pregnancy in mice. A total of 541 differentially expressed genes were identified, of which 316 genes were up-regulated and 225 genes were down-regulated in receptive uterus compared to non-receptive uterus. Gene ontology and gene network analysis highlighted the activation of inflammatory response in the receptive uterus. By analyzing the promoter sequences of differentially expressed genes, we identified 12 causal transcription factors. Through connectivity map (CMap) analysis, we revealed several compounds with potential anti-receptivity activity. Finally, we performed a cross-species comparison against human uterine receptivity from a published dataset. Our study provides a valuable resource for understanding the molecular mechanism underlying uterine receptivity in mice