The interaction between seminal plasma, sperm, and endometrium in inter- and intra-species breeding in equids.

In brief: In some instances, extra-species breeding in equids is more successful than intraspecies breeding; however, little is known about the immunomodulatory effect of donkey semen and seminal plasma on the mare's endometrium. This study compared the mare uterine inflammatory response during extra- and intraspecies breeding. Abstract: Anecdotal experience suggests horse mares have less post-breeding inflammation and better fertility when bred with donkeys. This study aimed to compare the post-breeding inflammatory response of mares exposed to donkey and horse semen and seminal plasma and evaluate the proteome and metabolome of donkey and horse sperm and seminal plasma. Uterine edema, intrauterine fluid accumulation, polymorphonuclear neutrophils on cytology, and concentrations of progesterone, and pro- and anti-inflammatory cytokines (IL1A, IL1B, IL4, IL6, CXCL8, IL10) were assessed pre- and post infusion of semen and seminal plasma (donkey and horse). The metabolome and proteome were analyzed by LC-MS/MS. Mare cycles bred with horse semen had a greater progesterone concentration than those bred with donkey semen at 8 days post ovulation (P = 0.046). At 6 h post infusion, the inflammatory response due to the donkey semen tended to be lower (P = 0.074). Donkey seminal plasma had anti-inflammatory properties compared to horse semen and seminal plasma, as determined by fewer neutrophils on uterine cytology (P < 0.05). Horse semen resulted in greater concentrations of IL6 and lesser concentrations of IL1B (P < 0.05). PGE1, PGE3, and lactoferrin concentrations were significantly more abundant in donkey sperm and seminal plasma. Prostaglandins play an important role in immunomodulation and might contribute to the response triggered in interspecies breeding. In conclusion, breeding horse mares with donkey semen induces similar post-breeding endometritis as observed with horse semen. Donkey seminal plasma results in a lower post-infusion inflammatory response compared to other combinations in the immediate post-breeding.

Tumor necrosis factor signaling during equine placental infection leads to pro-apoptotic and necroptotic outcomes.

Ascending placentitis is the leading cause of abortion in the horse. The pleiotropic cytokine tumor necrosis factor (TNF) is an upstream regulator of this disease, but little is understood regarding its function in pregnancy maintenance or placental infection. To assess this, RNA sequencing was performed on chorioallantois and endometrium of healthy pregnant mares at various gestational lengths (n = 4/gestational age), in addition to postpartum chorioallantois, and diestrus endometrium to assess expression of TNF, TNFR-1, and TNFR-2. Additionally, ascending placentitis was induced via trans-cervical inoculation of S. equi spp. zooepidemicus in pregnant mares (n = 6 infected / n = 6 control) and tissues and serum were collected to evaluate TNF-related transcripts. IHC was performed to confirm protein localization of TNFR-1 and TNFR-2. In healthy pregnancy, TNFR-1 appears to be the predominant TNF-related receptor. Following induction of disease, TNF concentrations increased in maternal serum, but expression did not alter at the tissue level. While both TNFR-1 and TNFR-2 increased following induction of disease, alterations in downstream pathways indicate that TNFR-1 is the dominant receptor in ascending placentitis, and is primarily activated within the chorioallantois, with minimal signaling occurring within the endometrium. In conclusion, TNF appears to be involved in the pathophysiology of ascending placentitis. An increase in this cytokine during disease progression is believed to activate TNFR-1 within the chorioallantois, leading to various pro-apoptotic and necroptotic outcomes, all of which may signal for fetal demise and impending abortion.

Transcriptional and Histochemical Signatures of Bone Marrow Mononuclear Cell-Mediated Resolution of Synovitis.

Osteoarthritis (OA) may result from impaired ability of synovial macrophages to resolve joint inflammation. Increasing macrophage counts in inflamed joints through injection with bone marrow mononuclear cells (BMNC) induces lasting resolution of synovial inflammation. To uncover mechanisms by which BMNC may affect resolution, in this study, differential transcriptional signatures of BMNC in response to normal (SF) and inflamed synovial fluid (ISF) were analyzed. We demonstrate the temporal behavior of co-expressed gene networks associated with traits from related in vivo and in vitro studies. We also identified activated and inhibited signaling pathways and upstream regulators, further determining their protein expression in the synovium of inflamed joints treated with BMNC or DPBS controls. BMNC responded to ISF with an early pro-inflammatory response characterized by a short spike in the expression of a NF-ƙB- and mitogen-related gene network. This response was associated with sustained increased expression of two gene networks comprising known drivers of resolution (IL-10, IGF-1, PPARG, isoprenoid biosynthesis). These networks were common to SF and ISF, but more highly expressed in ISF. Most highly activated pathways in ISF included the mevalonate pathway and PPAR-γ signaling, with pro-resolving functional annotations that improve mitochondrial metabolism and deactivate NF-ƙB signaling. Lower expression of mevalonate kinase and phospho-PPARγ in synovium from inflamed joints treated with BMNC, and equivalent IL-1ß staining between BMNC- and DPBS-treated joints, associates with accomplished resolution in BMNC-treated joints and emphasize the intricate balance of pro- and anti-inflammatory mechanisms required for resolution. Combined, our data suggest that BMNC-mediated resolution is characterized by constitutively expressed homeostatic mechanisms, whose expression are enhanced following inflammatory stimulus. These mechanisms translate into macrophage proliferation optimizing their capacity to counteract inflammatory damage and improving their general and mitochondrial metabolism to endure oxidative stress while driving tissue repair. Such effect is largely achieved through the synthesis of several lipids that mediate recovery of homeostasis. Our study reveals candidate mechanisms by which BMNC provide lasting improvement in patients with OA and suggests further investigation on the effects of PPAR-γ signaling enhancement for the treatment of arthritic conditions.

Interleukin-6 pathobiology in equine placental infection.

PROBLEM: Ascending placentitis is the leading cause of abortion in the horse. Interleukin (IL)-6 is considered predictive of placental infection in other species, but little is understood regarding its role in the pathophysiology of ascending placentitis. METHOD OF STUDY: Sub-acute ascending placentitis was induced via trans-cervical inoculation of S zooepidemicus, and various fluids/serum/tissues collected 8 days later. Concentrations of IL-6 were detected within fetal fluids and serum in inoculated (n = 6) and control (n = 6) mares. RNASeq was performed on the placenta (endometrium and chorioallantois) to assess transcripts relating to IL-6 pathways. IHC was performed for immunolocalization of IL-6 receptor (IL-6R) in the placenta. RESULTS: IL-6 concentrations increased in allantoic fluid following inoculation, with a trend toward an increase in amniotic fluid. Maternal serum IL-6 was increased in inoculated animals, while no changes were noted in fetal serum. mRNA expression of IL-6-related transcripts within the chorioallantois indicates that IL-6 is activating the classical JAK/STAT pathway, thereby acting as anti-inflammatory, anti-apoptotic, and pro-survival. The IL-6R was expressed within the chorioallantois, indicating a paracrine signaling pathway of maternal IL-6 to fetal IL-6R. CONCLUSION: IL-6 plays a crucial role in the placental response to induction of sub-acute equine ascending placentitis, and this could be noted in amniotic fluid, allantoic fluid, and maternal serum. Additionally, IL-6 is acting as anti-inflammatory in this disease, potentially altering disease progression, impeding abortion signals, and assisting with the production of a viable neonate.

The Effect of Mycobacterium Cell Wall Fraction on Histologic, Immunologic, and Clinical Parameters of Postpartum Involution in the Mare.

Maintaining yearly foal production is important for the economic success of the broodmare, and this requires breeding to occur as quickly postpartum as possible. The initial postpartum estrus occurs within 5-20 days postpartum, whereas the uterus is still undergoing repair from tissue alterations during pregnancy and parturition, a process known as involution. Attempts have been made to hasten this process, but with minimal success. Mycobacterium cell wall fraction (MCWF) is an immunomodulator that has been shown to reduce bacterial growth and alter aspects of the immune response to breeding, but it is unknown if MCWF hastens the process of involution. Therefore, the objectives of this study were to (1) investigate the effect of MCWF on tissue remodeling, (2) assess the effect of MCWF on the local immune system of the uterus, and (3) determine the optimal treatment interval needed for these processes to occur. We hypothesize that repeated treatments of MCWF postpartum will hasten the process of involution. To study this, 16 pregnant mares of mixed breeds were evaluated postpartum. Control mares (n = 4) received 1.5 mL lactated Ringer's solution intravenously on Day 1 (Day 0 = day of parturition) postpartum and again on Day 7, whereas treated mares either received 1.5 mL Settle intravenously on Day 1 and Day 7 (TX1; n = 6) or 1.5 mL Settle intravenously on Day 1 and then every 3 days until ovulation was detected (TX2; n = 6) and then evaluated until 15 days postpartum. Mares were assessed every 3 days for clinical, immunologic, and histologic parameters. Clinical parameters were assessed with transrectal ultrasonography and included ovarian activity, uterine fluid retention, and measurement of the uterine diameter, in addition to endometrial culture. Immunologic parameters included endometrial biopsies for quantitative polymerase chain reaction for expression of various cytokines (interleukin [IL]-1ß, IL-1RN, IL-4, IL-6, IL-8, IL-10, tumor necrosis factor [TNF], interferon [IFN]-γ, and granulocyte-macrophage colony-stimulating factor) in addition to endometrial cytology. Formalin-fixed endometrial biopsies were histologically assessed for the retention of microcaruncles, dilation of endometrial glands, and inflammation of the mucosa, stratum compactum, and spongiosum. Statistics were performed using SAS 9.4, using a mixed model for repeated measures with mare and treatment as a random effect. All post-hoc analysis was done using a Tukey's honestly significant difference test. Involution was considered complete by Day 15 postpartum in all mares, and the day postpartum had a significant effect on almost all parameters investigated, indicating the immunologic process of involution. Treatment with MCWF decreased the magnitude of bacterial growth in addition to time to negative culture. In addition, MCWF increased the expression of IL-1ß, IFNγ, and TNF. Although minimal treatment effect was noted histologically, a decrease in mucosal inflammation was seen in MCWF-treated mares. In conclusion, involution appears to be influenced by the immune system. In addition, MCWF appears to have a bactericidal effect on the postpartum mare, and this may be because of an increase in proinflammatory cytokines. It is unknown if this bactericidal property will improve fertility on the first estrous cycle postpartum, and future studies are needed to determine this.

Biomarkers for placental disease in mares.

Placentitis is an important cause of abortion, stillbirth, and neonatal death in horses. The diagnosis of placentitis is based on occurrence of clinical signs (premature mammary gland development and vulvar discharge) and ultrasonography of the caudal placental pole. However, early and subtle cases can be missed. In the last few years, several studies have provided objective means of diagnosing placentitis in mares with single or serial measurements of blood markers. Among the markers evaluated the steroids produced by the fetoplacental unit have been shown to change in association with placentitis. Mares with chronic placentitis have an increase in peripheral progestogens; however, mares acutely infected will display a reduction in peripheral concentrations of progestogens. Estradiol-17ß (free- and conjugated form) concentrations are drastically reduced in plasma of mares with placentitis. Acute-phase proteins, particularly serum amyloid A, are increased in plasma of mares suffering from placentitis, and this increase is due to endometrial and chorioallantoic secretions, and minimally from the fetus. Alpha-fetoprotein, a protein expressed in the fetoplacental unit, was shown to be increased in plasma of mares suffering from placentitis. A plephora of microRNA have been identified in plasma and tissues of mares undergoing experimentally induced placentitis, but have not been tested in spontaneous cases. Unique proteomic signatures were found in the fetal fluids of mares undergoing experimentally induced ascending placentitis, making the fetal fluids potentially useful to diagnose placentitis in mares. However, currently the lack of use of transabdominal fetal fluid sampling prevents wide use of the fetal fluids as diagnostic techniques. This manuscript aimed to discuss recent discoveries regarding biomarkers for placentitis in mares.

Small RNA (sRNA) expression in the chorioallantois, endometrium and serum of mares following experimental induction of placentitis.

Intrauterine infection and inflammation remain a major cause of preterm labour in women and mares, with little known about small RNA (sRNA) expression in tissue or circulation. To better characterise placental inflammation (placentitis), we examined sRNA expression in the endometrium, chorioallantois and serum of mares with and without placentitis. Disease was induced in 10 mares via intracervical inoculation of Streptococcus equi ssp. zooepidemicus, either with moderate or high levels of inoculum; three uninoculated gestationally matched mares were used as controls. Matched chorioallantois and endometrium were sampled in two locations: Region 1, gross inflammation near cervical star with placental separation and Region 2, gross inflammation without placental separation. In Region 1, 26 sRNAs were altered in chorioallantois, while 20 were altered in endometrium. Within Region 2, changes were more subdued in both chorioallantois (10 sRNAs) and endometrium (two sRNAs). Within serum, we identified nine significantly altered sRNAs. In summary, we have characterised the expression of sRNA in the chorioallantois, the endometrium and the serum of mares with experimentally induced placentitis using next-generation sequencing, identifying significant changes within each tissue examined. These data should provide valuable information about the physiology of placental inflammation to clinicians and researchers alike.

A comparison of progesterone assays for determination of peripheral pregnane concentrations in the late pregnant mare.

During the latter half of gestation in mares, there is a complex milieu of pregnanes in peripheral blood. Progesterone concentrations are often assessed by immunoassay during late gestation as a measure of pregnancy well-being; however, interpretation of results is complicated by the numerous cross-reacting pregnanes present in high concentrations during late gestation. Further, many mares are supplemented with an exogenous progestin, altrenogest, which may also cross-react with existing assays and further confound interpretation. The objectives of this study were: 1) to compare differences in pregnane concentrations determined with four immunoassays compared to LC-MS/MS and 2) to assess cross-reactivity observed with the same immunoassays, specifically considering pregnenolone (P5), progesterone (P4), 5α-dihydroprogesterone (DHP), allopregnanolone, and altrenogest. Blood samples from four healthy mares in late gestation were evaluated by immunoassay and by LC-MS/MS. Measured immuno-reactive progesterone (ir-progesterone) concentrations differed (p < 0.0001) between immunoassays, although results were highly correlated (r = 0.85-1.0; p < 0.001). Measured ir-progesterone concentrations by immunoassay were linearly associated (r2 = 0.68-0.76; p < 0.001) with concentrations of P5, P4, DHP, and allopregnanolone determined by LC-MS/MS. There was no detectable cross-reaction of altrenogest in any immunoassay, but varying degrees of cross-reactivity was observed with other pregnanes analyzed. These data confirm ir-progesterone concentrations during late gestation vary depending upon the assay used and the cross-reactivity to other pregnanes present in late gestation, although the synthetic progestin altrenogest did not affect the results of any immunoassay tested.

Sex-steroid receptors, prostaglandin E2 receptors, and cyclooxygenase in the equine cervix during estrus, diestrus and pregnancy: Gene expression and cellular localization.

The cervix is a dynamic structure that undergoes dramatic changes during the estrous cycle, pregnancy and parturition. It is well established that hormonal changes, including estrogens, progestogens and prostaglandins, regulate the expression of key proteins involved in cervical function. The arachidonic acid cascade is important in the remodeling and relaxation of the cervix in the days preceding parturition. Despite the complexity of this mechanism, regulation of cervical function has received little study in the mare. Therefore, the objective of this study was to compare the expression of estrogen receptor α (ESR1) and ß (ESR2), progesterone receptor (PGR), prostaglandin E2 type 2 (PTGER2) and type 4 (PTGER4) receptors as well as cyclooxygenase-1 (PTGS1) and -2 (PTGS2) in the equine cervical mucosa and stroma during estrus, diestrus and late pregnancy using qPCR. Immunohistochemistry was used to localize ESR1, ESR2, PGR, PTGER2 and PTGER4 receptors in these regions of the cervix. Relative mRNA expression of ESR1 and PGR was greater during estrus and diestrus than in late pregnancy in both the mucosa and stroma of the cervix. Expression of PTGER2 was highest in the cervical stroma during late pregnancy compared to either estrus or diestrus. Moreover, PTGS1 expression in mucosa and PTGS2 in stroma was greater during late pregnancy compared with estrus, but not diestrus. Immunostaining for ESR1, ESR2, PGR, PTGER2 and PTGER4 was consistently detected in the nucleus and cytoplasm of epithelium of the endocervix as well as the smooth muscle cytoplasm of the cervix in all stages evaluated. Immunolabeling in smooth muscle nuclei was detected for ESR1 and PGR in estrus, diestrus and late pregnancy, and for ESR2 in estrus and late pregnancy stages. The changes noted in late gestation likely reflect preparation of the equine cervix for subsequent parturition.

Regulation of axonemal motility in demembranated equine sperm.

Equine in vitro fertilization is not yet successful because equine sperm do not effectively capacitate in vitro. Results of previous studies suggest that this may be due to failure of induction of hyperactivated motility in equine sperm under standard capacitating conditions. To evaluate factors directly affecting axonemal motility in equine sperm, we developed a demembranated sperm model and analyzed motility parameters in this model under different conditions using computer-assisted sperm analysis. Treatment of ejaculated equine sperm with 0.02% Triton X-100 for 30 sec maximized both permeabilization and total motility after reactivation. The presence of ATP was required for motility of demembranated sperm after reactivation, but cAMP was not. The calculated intracellular pH of intact equine sperm was 7.14 ± 0.07. Demembranated sperm showed maximal total motility at pH 7. Neither increasing pH nor increasing calcium levels, nor any interaction of the two, induced hyperactivated motility in demembranated equine sperm. Motility of demembranated sperm was maintained at free calcium concentrations as low as 27 pM, and calcium arrested sperm motility at much lower concentrations than those reported in other species. Calcium arrest of sperm motility was not accompanied by flagellar curvature, suggesting a failure of calcium to induce the tonic bend seen in other species and thought to support hyperactivated motility. This indicated an absence, or difference in calcium sensitivity, of the related asymmetric doublet-sliding proteins. These studies show a difference in response to calcium of the equine sperm axoneme to that reported in other species that may be related to the failure of equine sperm to penetrate oocytes in vitro under standard capacitating conditions. Further work is needed to determine the factors that stimulate hyperactivated motility at the axonemal level in equine sperm.