The Immune Barrier of Porcine Uterine Mucosa Differs Dramatically at Proliferative and Secretory Phases and Could Be Positively Modulated by Colonizing Microbiota.

Endometrial immune response is highly associated with the homeostatic balance of the uterus and embryo development; however, the underlying molecular regulatory mechanisms are not fully elucidated. Herein, the porcine endometrium showed significant variation in mucosal immunity in proliferative and secretory phases by single-cell RNA sequencing. The loose arrangement and high motility of the uterine epithelium in the proliferative phase gave opportunities for epithelial cells and dendritic cells to cross talk with colonizing microbial community, guiding lymphocyte migration into the mucosal and glandular epithelium. The migrating lymphocytes were primarily NK and CD8+ T cells, which were robustly modulated by the chemokine signaling. In the secretory phase, the significantly strengthened mechanical mucosal barrier and increased immunoglobulin A alleviated the migration of lymphocytes into the epithelium when the neuro-modulation, mineral uptake, and amino acid metabolism were strongly upregulated. The noticeably increased intraepithelial lymphocytes were positively modulated by the bacteria in the uterine cavity. Our findings illustrated that significant mucosal immunity variation in the endometrium in the proliferative and secretory phases was closely related to intraepithelial lymphocyte migration, which could be modulated by the colonizing bacteria after cross talk with epithelial cells with higher expressions of chemokine.

Sex Matters: Physiological Abundance of Immuno-Regulatory CD71+ Erythroid Cells Impair Immunity in Females.

Mature erythrocytes are the major metabolic regulators by transporting oxygen throughout the body. However, their precursors and progenitors defined as CD71+ Erythroid Cells (CECs) exhibit a wide range of immunomodulatory properties. Here, we uncover pronounced sexual dimorphism in CECs. We found female but not male mice, both BALB/c and C57BL/6, and human females were enriched with CECs. CECs, mainly their progenitors defined as CD45+CECs expressed higher levels of reactive oxygen species (ROS), PDL-1, VISTA, Arginase II and Arginase I compared to their CD45- counterparts. Consequently, CECs by the depletion of L-arginine suppress T cell activation and proliferation. Expansion of CECs in anemic mice and also post-menstrual cycle in women can result in L-arginine depletion in different microenvironments in vivo (e.g. spleen) resulting in T cell suppression. As proof of concept, we found that anemic female mice and mice adoptively transferred with CECs from anemic mice became more susceptible to Bordetella pertussis infection. These observations highlight the role of sex and anemia-mediated immune suppression in females. Notably, enriched CD45+CECs may explain their higher immunosuppressive properties in female BALB/c mice. Finally, we observed significantly more splenic central macrophages in female mice, which can explain greater extramedullary erythropoiesis and subsequently abundance of CECs in the periphery. Thus, sex-specific differences frequency in the frequency of CECs might be imprinted by differential erythropoiesis niches and hormone-dependent manner.

A Comprehensive Profile of Chemokine Gene Expression in the Tissues of the Female Reproductive Tract in Mice.

Homeostatic leukocyte trafficking into and within the female reproductive tract (FRT) contributes to fertility and reproductive health. It is unclear how this process is regulated in the anatomically distinct reproductive tissues, or whether the genes involved are affected by cyclical changes in reproductive hormones. In tissues such as skin and intestine, mouse studies have defined evolutionarily conserved molecular mechanisms for tissue-specific homing, interstitial positioning, and leukocyte egress. Chemokine family members are invariably involved, with the chemokine expression profile of a tissue regulating leukocyte content. Reproductive tissues (ovary, vagina, cervix, uterine horn) of 8 week old virgin female C57BL/6 mice (n = 20) were collected, and expression of mRNA for leukocyte markers and chemokines conducted by qPCR. Lymphocytic and myeloid cell populations within the uterus, cervix, bone marrow and PALN from virgin C57BL/6 mice were determined by flow cytometric analysis. Variation in leukocyte content between reproductive tissues is evident, with the uterus and cervix containing complex mixtures of lymphocytes and myeloid cells. Twenty-six chemokine genes are expressed in the FRT, many by several component tissues, some preferentially by one. Most striking are Xcl1 and Ccl28, which are restricted to the uterus. Ccl20 and genes encoding CXCR2 ligands are primarily transcribed in cervix and vagina. Ovary shows the lowest expression of most chemokine genes, with the notable exception of Ccl21 and Ccl27. We also identify eight chemokines in the vagina whose expression fluctuates substantially across the oestrous cycle. These data reveal complex chemokine networks within the FRT, and provide a framework for future studies of homeostatic leukocyte trafficking into and within these tissues.Abbreviations: BM: bone marrow; DC: dendritic cell; DN: double negative; FRT: female reproductive tract; FSC: forward scatter; NK: natural killer; PALN: para-aortic lymph node; SSC: side scatter; Tregs: regulatory T cells.

Function of CCL5 in maternal-fetal interface of pig during early pregnancy.

Chemokines refer to chemoattractant cytokines, which have crucial functions in inflammation and immune responses in multiple cellular processes. In the present study, we described the potential role of porcine CCL5 in embryo implantation and fetal-maternal environment during early pregnancy. We first carried out phylogenetic analysis of porcine CCL5, and analyzed the cell specific localization of CCL5 and its receptor CCR3 in a kinetic approach within porcine estrous cycles and early gestation stage. In addition, CCL5 stimulated porcine uterine luminal epithelial (pLE) and porcine trophectoderm (pTr) cell proliferations, and cell cycle progressions via AKT and MAPK intracellular signaling tractions. Furthermore, CCL5 attenuated tunicamycin-induced endoplasmic reticulum (ER) stress signaling, and lipopolysaccharides-triggered inflammatory responses in pLE and pTr cells. Taken together, our study showed that CCL5 is involved in the placental development or promotes the placental development.

Female Mouse Model of Neisseria gonorrhoeae Infection.

Mouse models of infection are important tools in the study of infectious disease or host the development of products to prevent or treat infections. The estradiol-treated mouse model of Neisseria gonorrhoeae genital tract infection has proved to be a valuable system for determining the importance of gonococcal factors that mediate evasion of host innate effectors in vivo or host gonococcal adaptation to hormonally driven host factors in females. Examination of mechanisms that Neisseria gonorrhoeae uses to subvert the host immune response also has been greatly aided by this whole model system, as have studies on the consequence of antibiotic resistance mutations on gonococcal fitness in vivo and the search for new antibiotics to treat antibiotic-resistant infections. The strict human specificity of N. gonorrhoeae limits the ability of experimental murine infection to mimic human infection. However, in recent years, the development of transgenic mice and protocols for supplementing mice with human factors has improved animal modeling of gonorrhea. To date, however, because the mouse estrous cycle is much shorter than the human reproductive cycle, all reported gonorrhea mouse models require treatment with estradiol and antibiotics to maintain an estrus-like state and suppress the overgrowth of inhibitory commensal flora that occurs under the influence of estrogen to allow sustained N. gonorrhoeae infection. In this chapter, we detail the methods used to (1) prepare the mice for experimental infection with N. gonorrhoeae, (2) inoculate mice and quantitatively culture vaginal swabs for noncompetitive and competitive infection experiments, and (3) monitor the host innate immune response to infection.

Immune status during postpartum, peri-implantation and early pregnancy in cattle: An updated view.

Throughout the estrous cycle the mammalian endometrium undergoes morphological and functional changes that are essential for the establishment of pregnancy and proper ovarian and uterine functions. Among these changes, the most important are alterations in both inter- and intracellular signalling molecules, many of which modulate immune processes. In the endometrial tissue there are local innate (nonspecific) and adaptive (specific/acquired) response mechanisms which vary because of the endocrine status during the estrous cycle, pregnancy and postpartum period. Endometrial cells have responses that support the immune system by producing pro-inflammatory factors such as cytokines, sensors, effector molecules and chemokines. This response is important during gestation, pregnancy, and fetal growth, as well as in preventing infection, and immuno-rejection of the semi-allogeneic embryo. In dairy cows, both before and immediately after calving, there are marked changes in the values for hormonal and metabolic variables and the immune status is impaired. Thus, in several studies there has been assessment of the physiological and/or abnormal maternal immune changes and possible effects on dairy cow reproductive performance. The objective with this review is to summarize the novel information about the immune mechanisms involved during the postpartum period, subsequent peri-implantation period and pregnancy in dairy cows, and the possible effects on reproductive performance. This information provides for an enhanced understanding of the local and systemic immune responses associated with the metabolic and hormonal status of dairy cows, and alterations in the immune system of high producing cows and the possible effects on subsequent fertility.

The Role of Interleukin-10 in Mediating the Effect of Immune Challenge on Mouse Gonadotropin-Releasing Hormone Neurons In Vivo.

Immune challenge alters neural functioning via cytokine production. Inflammation has profound impact on the central regulation of fertility, but the mechanisms involved are not clearly defined. The anti-inflammatory cytokine interleukin (IL)-10 is responsible for balancing the immune response in the brain. To examine whether IL-10 has an effect on the function of the gonadotropin-releasing hormone (GnRH) neurons, we first examined the effect of immune responses with distinct cytokine profiles, such as the T cell-dependent (TD) and T cell-independent (TI) B-cell response. We investigated the effect of the TD and TI immune responses on ERK1/2 phosphorylation in GnRH neurons by administering fluorescein isothiocyanate/keyhole limpet hemocyanin (KLH-FITC) or dextran-FITC to female mice. Although dextran-FITC had no effect, KLH-FITC induced ERK1/2 phosphorylation in GnRH neurons after 6 d. KLH-FITC treatment increased the levels of IL-10 in the hypothalamus (HYP), but this treatment did not cause lymphocyte infiltration or an increase in the levels of proinflammatory cytokines. In IL-10 knock-out (KO) mice, KLH-FITC-induced ERK1/2 phosphorylation in the GnRH neurons was absent. We also showed that in IL-10 KO mice, the estrous cycle was disrupted. Perforated patch-clamp recordings from GnRH-GFP neurons, IL-10 immunohistochemistry, and in vitro experiments on acute brain slices revealed that IL-10 can directly alter GnRH neuron firing and induce ERK1/2 phosphorylation. These observations demonstrate that IL-10 plays a role in influencing signaling of GnRH neurons in the TD immune response. These results also provide the first evidence that IL-10 can directly alter the function of GnRH neurons and may help the maintenance of the integrity of the estrous cycle.

Innate immune genes in persistent mating-induced endometritis in horses.

Persistent mating-induced endometritis (PMIE) severely decreases fertility in horses. The aim of the present study was to evaluate differences between horses susceptible to PMIE and a control group in terms of the expression of selected immune response and effector genes, and the effects of oestrous cycle stage on this expression. Endometrial biopsies from 18 uterine samples of mares in the control group (eight in dioestrus, 10 in oestrus) and 16 PMIE-susceptible mares (four in dioestrus, 12 in oestrus) were analysed by quantitative real-time reverse transcription-polymerase chain reaction. Genes for pathogen recognition receptors Toll-like receptor 2 (TLR2) and NLR family CARD domain containing 5 (NLRC5), as well as tissue-specific inhibitor of metalloproteinase 1 (TIMP1), C-X-C motif chemokine ligand (CXCL) 9, CXCL10 and CXCL11 and uteroferrin were expressed at similar levels in the control group and in susceptible mares. Genes for C-C motif chemokine ligand 2 (CCL2) and the antimicrobial peptides secreted phospholipase A2 (sPLA2), lipocalin 2 and lactoferrin were all expressed at higher levels in susceptible compared with control mares. The expression of genes for the antimicrobial peptides equine ß-defensin 1 (EBD1), lysozyme (LYZ) and secretory leukoprotease inhibitor (SLPI) was also higher in susceptible than control mares. The diagnostic sensitivity of assays for EBD1, LYZ and SLP1 gene expression to detect susceptibility to PMIE was estimated to be 100%, 94% and 100% respectively, with specificities of 83%, 78% and 78% respectively. When all three tests were positive, the specificity increased to 94%, with an overall sensitivity of 94%. The present study has yielded insights into pathophysiological changes in mares susceptible to PMIE and identified robust diagnostic markers (EBD1, LYZ and SLPI) for susceptibility to this disease.

The effect of select seminal plasma proteins on endometrial mRNA cytokine expression in mares susceptible to persistent mating-induced endometritis.

In the horse, breeding induces a transient endometrial inflammation. A subset of mares are unable to resolve this inflammation, and they are considered susceptible to persistent mating-induced endometritis PMIE Select seminal plasma proteins cysteine-rich secretory protein-3 (CRISP-3) and lactoferrin have been shown to affect the innate immune response to sperm in vitro. The objective of this study was to determine whether the addition of CRISP-3 and lactoferrin at the time of insemination had an effect on the mRNA expression of endometrial cytokines in susceptible mares after breeding. Six mares classified as susceptible to PMIE were inseminated during four consecutive oestrous cycles with treatments in randomized order of: 1 mg/ml CRISP-3, 150 µg/ml lactoferrin, seminal plasma (positive control) or lactated Ringer's solution (LRS; negative control) to a total volume of 10 ml combined with 1 × 109 spermatozoa pooled from two stallions. Six hours after treatment, an endometrial biopsy was obtained for qPCR analysis of selected genes associated with inflammation (pro-inflammatory cytokines interleukin (IL)-1ß, IL-8, tumour necrosis factor (TNF)-α, interferon (INF)-γ, anti-inflammatory cytokines IL-1RN and IL-10, and inflammatory-modulating cytokine IL-6). Seminal plasma treatment increased the mRNA expression of IL-1ß (p = .019) and IL-8 (p = .0068), while suppressing the mRNA expression of TNF (p = .0013). Lactoferrin also suppressed the mRNA expression of TNF (p = .0013). In conclusion, exogenous lactoferrin may be considered as one modulator of the complex series of events resulting in the poorly regulated pro-inflammatory response seen in susceptible mares.

Characterization of cytological changes, IgA, IgG and IL-8 levels and pH value in the vagina of prepubertal and sexually mature Ellegaard Göttingen minipigs during an estrous cycle.

The pig is increasingly used as an advanced animal model of the genital tract in women and knowledge on the genital immune system is therefore needed. In this study, evaluation of vaginal smears revealed that almost no neutrophils or other leukocytes were present in the vaginal mucosa of prepubertal minipigs (n = 10). In sexually mature minipigs (n = 10), evaluated through an estrous cycle, there was an increase in number of mucosal neutrophils and other leukocytes during estrus. The level of total IgA on the vaginal mucosa increased during diestrus. The level of total IgG showed no significant changes through the cycle. The vaginal IgA level in the prepubertal minipigs was similar to the low estrus level in sexually mature minipigs, and the IgG level in prepubertal was similar to the stable level in the sexually mature minipigs.

Multi-parameter flow cytometric analysis of uterine immune cell fluctuations over the murine estrous cycle.

Investigating immune cell populations within various reproductive tissues commonly utilises flow cytometric methods. With advances in fluorophore technology and equipment capabilities, multiple cell types from a single tissue sample can be identified by using different combinations of cell surface markers to distinguish specific cell populations. Here a protocol optimized for mouse uterine tissue was used to show the proportional changes in dendritic cells, monocyte/macrophages, T and B cells, NK and NK T cells, and the granulocytes, neutrophils and eosinophils at each of the four stages of the estrous cycle. Importantly, we demonstrate that use of anti-SiglecF or assessment of FSC/SSC plots could be used to differentiate monocyte/macrophage and eosinophil populations that otherwise cannot be distinguished by use of the common combination of antibodies against F4/80 and CD11b. Our results clearly indicate that within the uterus a dynamic population of immune cells resides, with many cell types reaching peak abundance at estrus and metestrus phases of the cycle, consistent with their importance in the response to paternal antigens and/or pathogens encountered after insemination.

Low expression of IL-6 and TNF-α correlates with the presence of the nuclear regulators of NF-κB, IκBNS and BCL-3, in the uterus of mice.

The dynamic regulation of NF-κB activity in the uterus maintains a favorable environment of cytokines necessary to prepare for pregnancy throughout the estrous cycle. Recently, the mechanisms that directly regulate the NF-κB transcriptional activity in different tissues are of growing interest. IκBNS and BCL-3 are negative nuclear regulators of NF-κB activity that regulate IL-6 and TNF-α transcription, respectively. Both cytokines have been described as important factors in the remodeling of uterus for blastocyst implantation. In this work we analyzed in ICR mice the mRNA expression and protein production profile of IL-6, TNF-α, and their correspondent negative transcription regulators IκBNS or BCL-3 using real-time PCR, western blot and immunochemistry. We found that the expression of TNF-α and IL-6 was oscillatory along the estrous cycle, and its low expression coincided with the presence of BCL-3 and IκBNS, and vice versa, when the presence of the regulators was subtle, the expression of TNF-α and IL-6 was exacerbated. When we compared the production of TNF-α and IL-6 in the different estrous stages relating with diestrus we found that at estrus there is an important increase of the cytokines (p<0.05) decreasing at metestrus to reach the basal expression at diestrus. In the immunochemistry analysis we found that at diestrus BCL-3 is distributed all over the tissue with a barely detected TNF-α, but on the contrary, at estrus the expression of BCL-3 is not detected with TNF-α clearly observable along the tissue; the same phenomenon occur in the analysis of IκBNS and IL-6. With that evidence we suggest that the expression of TNF-α and IL-6 might be regulated through NF-κB nuclear regulators BCL-3 and IκBNS in the uterus of mice as has been demonstrated in other systems.

Resident Macrophages and Lymphocytes in the Canine Endometrium.

Resident immune cells play a major role in endometrial immunity and in tissue homoeostasis. This study aimed to analyse the distribution of macrophages, B and T lymphocytes (respectively, Mø, B-Lym and T-Lym) in the canine endometrium throughout the oestrous cycle and in late involution (at the proestrus stage post-parturition). An immunohistochemistry technique was used on samples from 50 post-pubertal healthy female dogs, of which five in late post-partum. The distribution of resident immune cells was analysed in three endometrial layers (superficial, intermediate and basal areas). Mø, B-Lym and T-Lym were demonstrated to reside in the endometrium in all the stages of the canine cycle; their numbers being considerably higher during late involution. T-Lym were scattered in the stroma or amidst the glandular epithelium, constituting the predominant immune cell population in anestrus and proestrus, but decreased in number at all other stages. Endometrial B-Lym remained fairly constant during the canine cycle, although its numbers were higher in late involution. Mø counts were higher during anestrus compared to the other stages, the cells being displaced into the superficial endometrial layer. Mø demonstrated the highest level in late involution samples, forming small aggregates below the surface epithelium. The number of immune cells was not normally distributed, suggesting the influence of individual factors, such as age or parity, not explored herein due to limited sample availability. Still, this study provides important information for the interpretation of endometrial biopsies in dogs and for the understanding of the increased susceptibility to uterine infection during dioestrus found in the bitch.

MRL/MpJ-Fas(lpr) mice show abnormalities in ovarian function and morphology with the progression of autoimmune disease.

The immune system is known to affect reproductive function, and maternal-fetal immune tolerance is essential for a successful pregnancy. To investigate the relationship between autoimmune disease and female reproductive function, we performed a comparative analysis of the ovarian phenotypes for C57BL/6 mice, autoimmune disease-prone MRL/MpJ (MRL/+) mice and congenic MRL/MpJ-Fas(lpr) (MRL/lpr) mice harboring a mutation in the Fas gene that speeds disease onset. Both MRL-background strains showed earlier vaginal opening than C57BL/6 mice. The estrous cycle became irregular by 6 and 12 months of age in MRL/lpr mice and mice of the other two strains, respectively. Histological analysis at 3 months revealed that the number of primordial follicles was smaller in MRL-background mice than in C57BL/6 mice after 3 months. In addition, MRL/lpr and MRL/+ mice displayed lower numbers of ovarian follicles and corpora lutea at 3 and 6 months, and 6 and 12 months, respectively, than that in age-matched C57BL/6 mice. MRL/lpr and MRL/+ mice developed ovarian interstitial glands after 3 and 6 months, respectively. In particular, MRL/lpr mice showed numerous infiltrating lymphocytes within the ovarian interstitia, and partially stratified ovarian surface epithelia with more developed microvilli than that observed in C57BL/6 mice at 6 months. No significant differences in serum hormone levels were observed between the strains. In conclusion, MRL/lpr mice display altered ovarian development, morphology and function consistent with the progression of severe autoimmune disease, as these findings are less severe in MRL/+ counterparts.

Disruption of estrous cycle homeostasis in mice with experimental autoimmune encephalomyelitis.

Multiple sclerosis (MS) is widely viewed as a prototypic human autoimmune disease involving proinflammatory T cells that induce lesions in the central nervous system (CNS) in response to myelin self proteins. Although the impact of sex hormones on MS is well recognized, the converse effects of autoimmunity on sex hormones are still unclear. The current study was designed to assess the impact of CNS autoimmunity on female reproductive physiology. In order to identify subtle hormonal disturbances as a result of autoimmunity, we analyzed the estrous cycle in SJL/J mice after active induction of experimental autoimmune encephalomyelitis (EAE), an animal model with substantial similarities to MS. Here we show that CNS autoimmunity significantly shortens the murine estrous cycle. This shortening of the estrous cycle is characterized by a dramatic decrease in the length of the metestrus-diestrus luteal phase partially offset by a highly significant but less dramatic elongation of the proestrus-estrus follicular phase of the uterine cycle. Thus, our study provides experimental evidence for a direct causal link between CNS autoimmunity and disruption of the homeostatic balance of the uterine cycle often observed in women with MS.

Unique temporal and spatial expression patterns of IL-33 in ovaries during ovulation and estrous cycle are associated with ovarian tissue homeostasis.

Ovaries are among the most active organs. Frequently occurring events such as ovulation and ovarian atresia are accompanied with tissue destruction and repairing. Critical roles of immune cells or molecules in those events have been well recognized. IL-33 is a new member of the IL-1 cytokine gene family. Recent studies suggest its roles beyond immune responses. We systemically examined its expression in ovaries for its potential roles in ovarian functions. During ovulation, a high level of IL-33 was transiently expressed, making it the most significantly upregulated immune gene. During estrous cycle, IL-33 expression levels fluctuated along with numbers of ovarian macrophages and atresia wave. Cells with nuclear form of IL-33 (nIL-33(+) cells) were mostly endothelial cells of veins, either in the inner layer of theca of ovulating follicles during ovulation, or surrounding follicles during estrous cycle. Changes in number of nIL-33(+) cells showed a tendency similar to that in IL-33 mRNA level during estrous cycle. However, the cell number sharply declined before a rapid increase of macrophages and a surge of atresia. The decline in nIL-33(+) cell number was coincident with detection of higher level of the cytokine form of IL-33 by Western blot, suggesting a release of cytokine form of IL-33 before the surge of macrophage migration and atresia. However, IL-33 Ab, either by passive transfer or immunization, showed a limited effect on ovulation or atresia. It raises a possibility of IL-33's role in tissue homeostasis after ovarian events, instead of a direct involvement in ovarian functions.

Characterization of the Th profile of the bovine endometrium during the oestrous cycle and early pregnancy.

Despite extensive research in the area of cow fertility, the extent to which the maternal immune system is modulated during pregnancy in cattle remains unclear. Therefore, the objective of the current study was to characterize the presence and response profile of B, T-helper (LTh), T- cytotoxic (LTc), gamma delta-T (γδT) and natural killer (NK) lymphocytes in terms of cell number, distribution and cytokine expression in bovine endometrial tissue to pregnancy. Endometrial tissue samples were collected from beef heifers on Days 5, 7, 13 and 16 of the estrous cycle or pregnancy. Samples were analysed by immunofluorescence to identify the presence and abundance of B-B7 (B-cells), CD4 (LTh), CD8 (LTc), γδT cell receptor (TCR) and CD335/NKp46 (NK cells) -positive immune cells. Quantitative real time PCR (QPCR) was carried out to analyse mRNA relative abundance of FOXP3 (a marker of regulatory T (Treg) cells) and a panel of immune factors, including MHC-I, LIF, Interleukins 1, 2, 6, 8, 10, 11,12A, IFNa and IFNG. Results indicate that B-B7+ cells are quite populous in bovine endometrial tissue, CD4+ and CD8+ -cells are present in moderate numbers and γδTCR+ and CD335+ cells are present in low numbers. Pregnancy affected the total number and distribution pattern of the NK cell population, with the most significant variation observed on Day 16 of pregnancy. Neither B lymphocytes nor T lymphocyte subsets were regulated temporally during the oestrous cycle or by pregnancy prior to implantation. mRNA transcript abundance of the immune factors LIF, IL1b, IL8 and IL12A, IFNa and IFNG, expression was regulated temporally during the estrous cycle and LIF, IL1b, IL-10, IL11, IL12A were also temporally regulated during pregnancy. In conclusion, the endometrial immune profile of the oestrous cycle favours a Th2 environment in anticipation of pregnancy and the presence of an embryo acts to fine tune this environment.

Dynamics of CD3⁺ T-cell distribution throughout the estrous cycle and gestation in the bovine endometrium.

T cells are the dominant lymphocytes in the endometrium and are considered to play a crucial role in implantation and in the maintenance of gestation through cytokine production and immune regulation. The mechanisms underlying immunoregulation at the feto-maternal interface are still obscure for this complex system. Understanding the role of T cells is a key factor in understanding the endometrial immune system. In this study, the distribution of endometrial CD3⁺ T cells in bovines was examined by immunohistochemical analysis. The estrous cycle and gestation was divided into 4 stages, and the number of CD3⁺-positive T cells was counted in each stage. CD3⁺ cells were found in the endometrium in significant numbers throughout the estrous cycle and were mostly located in the subepithelial area. The number of CD3⁺ cells significantly increased in the early and mid-luteal phases but decreased after implantation with the progression of gestation. No T cells were found in the placentome or specifically in the tissues near the fetus, including the trophoblastic area. In addition, very few T cells were found in stromal regions close to the myometrium of the endometrium. These findings suggest that downregulation of bovine endometrial CD3⁺ T-cell functions is closely related to the successful maintenance of gestation in a spatiotemporal manner.

The interleukin-1ß system in the corpora lutea of pigs during early pregnancy and the estrous cycle.

Expression of mRNAs encoding interleukin-1ß (IL-1ß), IL-1ß receptor I (IL-1RI), IL-1 receptor accessory protein (IL-1RAcP) and IL-1 receptor antagonist (IL-1Ra), as well as synthesis of IL-1ß and IL-1RI proteins, were examined in the corpus luteum (CL) during critical stages of CL activity on days 10-16 of pregnancy and 2-16 of the estrous cycle. Luteal cells were cultured in vitro with IL-1ß, and the effect on release of steroid hormones was determined. Expression of the IL-1ß system in the CL changed significantly during pregnancy and the estrous cycle. IL-1ß, IL-1RI, and IL-1Ra mRNA levels were elevated on days 12-13, whereas IL-1RAcP mRNA was increased on days 15-16 of pregnancy. In cyclic CL, expression of IL-1ß, IL-1RI, and IL-1RAcP mRNAs was increased on days 12-13. IL-1ß and IL-1RI protein were highest in the CL on days 10-11 and 8-11 of pregnancy and the estrous cycle. Luteal cells harvested from gravid and cyclic CL produced IL-1ß in vitro. IL-1ß increased progesterone and estradiol-17ß (E2) release by luteal cells on days 10-16 and 10-11 of pregnancy, respectively and on days 2-11 of the estrous cycle. IL-1ß decreased the level of E2 produced by regressed CL (days 15-16). Expression of the IL-1ß system in CL and IL-1ß secretion from luteal cells changed depending on the status of the CL. These data show that IL-1ß may be involved in intraluteal, luteotrophic regulation of CL functions in gravid and cyclic pigs.

The novel roles of neutrophils via opioid peptides: regulation of the estrous cycle and pain.

Neutrophils are excreted into the vaginal vault at metestrus during the estrous cycle, and this phenomenon has long been used to determine the phase of the estrous cycle. A much smaller number of neutrophils are also detected in the uterus and the ovary. Recently, we provided several lines of evidence supporting the notion that neutrophils infiltrate into the ovary to regulate the estrous cycle by opioid peptides. Upon inflammation, on the other hand, neutrophils infiltrate into the site of infection to suppress pain by opioid peptides. Thus, opioid peptides are key molecules by which neutrophils play a novel role in regulation of the pain and estrous cycle. In both cases, opioid peptides appear to be secreted by neutrophils stimulated with chemokines, such as MIP-2 and KC in mouse, corticotropin-releasing hormone and IL-1.