Clinical, pathologic, and epidemiologic features of nocardioform placentitis in the mare.

Placentitis is the leading cause of infectious abortion in the horse and contributes to roughly 19% of all abortions in the United States. A type of placental infection, nocardioform placentitis (NP) is associated with gram-positive branching actinomycetes localized within the ventral body of the feto-maternal interface to create a lymphoplasmacytic mucoid lesion. While the etiology of this disease is poorly described, this placental infection continues to cause episodic abortions in addition to weak and/or growth retarded neonates. The goal of the present study was to perform a comprehensive analysis of pregnancies associated with a nocardioform-affected placenta and make inferences into the epidemiology of this elusive disease. To do so, 264 mares were enrolled in the study, with 145 as having suspected disease (n = 145; NP) either based on pregnancy-related complications or postpartum placental evaluation, while an additional 119 were enrolled as healthy pregnancies (n = 119; CON). Following diagnosis as either NP or CON based on gross and histopathology at the University of Kentucky Veterinary Diagnostic Laboratory, information was gathered on the mares and neonates for comparisons between diseased and healthy pregnancies. Clinically, a significant portion of diseased mares had clinical indications of NP, including premature mammary gland development, thickening of the placenta noted on transrectal ultrasonography, and separation between the chorioallantois and endometrium noted on abdominal ultrasonography, while vulvar discharge was not commonly noted. Additionally, NP was correlated with increased mare age, decreased gestational length, and decreased neonatal weight, although neonatal IgG and WBC were comparable to CON. Incidence of NP was not correlated with last breeding date, pre- and post-breeding therapeutics, parity, prophylactic medications, or housing. Additionally, NP did not affect postpartum fertility. While NP was associated with a poor neonatal outcome (abortion and/or growth retarded neonate), this did not appear to be influenced by the bacteria isolated (Amycolatopsis spp. vs. Crossiella equi), and mares diagnosed with NP do not appear to be infectious to other pregnant mares nor have repetitive years of the disease. Interestingly, lesion size was positively correlated with last breeding date, as mares bred later in the breeding season correlating with a larger placental lesion. In conclusion, while the etiology of NP continues to elude researchers, the epidemiology of this disease has gained clarity, providing inferences into the management of suspect mares.

Serum amyloid A, Serum Amyloid A1 and Haptoglobin in pregnant mares and their fetuses after experimental induction of placentitis.

Serum amyloid A (SAA) and Haptoglobin (Hp) are acute phase proteins, produced during inflammation, such as placentitis. In horses, SAA and SAA1 are protein coding genes. Objectives were to analyze SAA and Hp concentrations and relative abundance of SAA, SAA1 and Hp mRNA transcript in maternal and fetal tissues after experimental induction of placentitis or mares of a control group. Serum Amyloid A family proteins were in marked abundance in the stroma of the endometrium and chorioallantois associated with inflammatory cells. Maternal plasma SAA concentrations were greater (P = 0.01) in mares with experimentally induced placentitis compared to those of the control group. Maternal Hp from the groups were not different, but fetal Hp concentrations of mares with experimentally induced placentitis were greater (P = 0.02). Maternal plasma SAA and Hp concentrations were greater than fetal plasma concentrations in mares with experimentally induced placentitis (P < 0.05). Relative abundance of SAA mRNA transcript was greater in the maternal, fetal liver and chorioallantois of mares with experimentally induced placentitis (P < 0.05) compared to those in the control group. Interestingly, relative abundance of SAA1 mRNA transcript was greater in the chorioallantois of mares with experimentally induced placentitis (P < 0.05). The SAA and Hp concentrations, therefore, were greater in mares with induced placentitis. Furthermore, relative abundance of SAA1 mRNA transcript is specifically greater in the chorioallantois of mares with placentitis, which warrants further studies to elucidate the immunological response of SAA1 in the chorioallantois of mares with placentitis.

Alterations of Circulating Biomarkers During Late Term Pregnancy Complications in the Horse Part I: Cytokines.

Equine abortions are attributed to both infectious and noninfectious causes. Clinical extrapolations are often made from the experimental model for ascending placentitis towards other causes of fetal compromise, including various markers of inflammation, including the cytokines IL-2, 5, IL-6, IL-10, IFNγ, and TNF. It is unknown if these cytokine changes are noted under field conditions, or if they increase preceding other pregnancy related complications. To assess this, Thoroughbred mares (n = 702) had weekly blood obtained beginning in December 2013 and continuing until parturition. Fetal membranes were submitted to the UKVDL for complete gross and pathologic assessment and classified as either ascending placentitis (n = 6), focal mucoid placentitis (n = 6), idiopathic abortion (n = 6) or control (n = 20). Weekly serum samples were analyzed via immunoassay for concentrations of IL-2, IL-5, IL-6, IL-10, IFNγ, and TNF. For both focal mucoid placentitis and ascending placentitis, an increase (P < .05) in the concentrations of IL-2, IL-5, IL-6, IL-10, IFNγ, and TNF was noted preceding parturition in comparison to controls. Cytokine profiles preceding idiopathic abortion did not differ from controls. In conclusion, serum cytokines may be considered potential biomarkers for the prediction of placental infection, while no changes in cytokine profiles were noted when noninfectious causes of abortion occurred. Additionally, this is the first study to report an increase in cytokines during the disease process of focal mucoid placentitis, the etiology of which includes Nocardioform placentitis.

The imbalance of the Th17/Treg axis following equine ascending placental infection.

Ascending placentitis is a leading cause of abortion in the horse, but adaptive immune response to this disease is unknown. To evaluate this, sub-acute placentitis was experimentally-induced via trans-cervical inoculation of S. zooepidemicus, and endometrium and chorioallantois was collected 8 days later (n = 6 inoculated/n = 6 control). The expression of transcripts relating to Th1, Th2, Th17, and Treg maturation was assessed via RNASeq. IHC of transcription factors relating to each subtype in the same tissues (Th1: TBX21, Th2: GATA3, Th17: IRF4, Treg: FOXp3). An immunoassay was utilized to assess circulating cytokines (Th1: IFNg, IL-2; Th2: IL-4, IL-5; Th17: IL-17, IL-6; Treg: IL-10, GM-CSF). An increase in Th1 and Th17-related transcripts were noted in the chorioallantois, although no alterations were seen in the endometrium. Th2 and Treg-related transcripts altered in a dysregulated manner, as some transcripts increased in expression while others decreased. Immunolocalization of Th1, Th2, and Th17 cells was increased in diseased chorioallantois, while no Treg cells were noted in the diseased tissue. Secreted cytokines relating to Th1 (IFNg, IL-2), Th17 (IL-6), Th2 (IL-5), and Treg (IL-10) populations increased in maternal circulation eight days after inoculation. In conclusion, the Th1/Th17 response to ascending placentitis occurs primarily in the chorioallantois, indicating the adaptive immune response to occur in fetal derived placental tissue. Additionally, ascending placentitis leads to an increase in the helper T cell populations (Th1/Th17/Th2) while decreasing the Treg response. This increase in Th17-related responses alongside a diminishing Treg-related response may precede or contribute to fetal demise, abortion, or preterm labor.

New insights in equine steroidogenesis: an in-depth look at steroid signaling in the placenta.

Steroid production varies widely among species, with these differences becoming more pronounced during pregnancy. As a result, each species has its own distinct pattern of steroids, steroidogenic enzymes, receptors, and transporters to support its individual physiological requirements. Although the circulating steroid profile is well characterized during equine pregnancy, there is much yet to be explored regarding the factors that support steroidogenesis and steroid signaling. To obtain a holistic view of steroid-related transcripts, we sequenced chorioallantois (45 days, 4 months, 6 months, 10 months, 11 months, and post-partum) and endometrium (4 months, 6 months, 10 months, 11 months, and diestrus) throughout gestation, then looked in-depth at transcripts related to steroid synthesis, conjugation, transportation, and signaling. Key findings include: 1) differential expression of HSD17B isoforms among tissues (HSD17B1 high in the chorioallantois, while HSD17B2 is the dominant form in the endometrium) 2) a novel isoform with homology to SULT1A1 is the predominant sulfotransferase transcript in the chorioallantois; and 3) nuclear estrogen (ESR1, ESR2) and progesterone (PGR) expression is minimal to nonexistant in the chorioallantois and pregnant endometrium. Additionally, several hypotheses have been formed, including the possibility that the 45-day chorioallantois is able to synthesize steroids de novo from acetate and that horses utilize glucuronidation to clear estrogens from the endometrium during estrous, but not during pregnancy. In summary, these findings represent an in-depth look at equine steroid-related transcripts through gestation, providing novel hypotheses and future directions for equine endocrine research.

Transcriptomic analysis reveals the key regulators and molecular mechanisms underlying myometrial activation during equine placentitis†.

The key event in placentitis-induced preterm labor is myometrial activation with the subsequent initiation of labor. However, the molecular mechanisms underlying myometrial activation are not fully understood in the mares. Therefore, the equine myometrial transcriptome was characterized during placentitis (290.0 ± 1.52 days of GA, n = 5) and the prepartum period (330 days of GA, n = 3) in comparison to normal pregnant mares (289.8 ± 2.18 days of GA, n = 4). Transcriptome analysis identified 596 and 290 DEGs in the myometrium during placentitis and the prepartum period, respectively, with 138 DEGs in common. The placentitis DEGs included eight genes (MMP1, MMP8, S100A9, S100A8, PI3, APOBEC3Z1B, RETN, and CXCL2) that are exclusively expressed in the inflamed myometrium. Pathway analysis elucidated that inflammatory signaling, Toll-like receptor signaling, and apoptosis pathways dominate myometrial activation during placentitis. The prepartum myometrium was associated with overexpression of inflammatory signaling, oxidative stress, and 5-hydroxytryptamine degradation. Gene ontology enrichment analysis identified several chemoattractant factors in the myometrium during placentitis and prepartum period, including CCL2, CXCL1, CXCL3, and CXCL6 in common. Upstream regulator analysis revealed 19 potential upstream regulators in placentitis dataset including transcription regulators (E2F1, FOXM1, HIF1A, JUNB, NFKB1A, and STAT1), transmembrane receptors (FAS, ICAM1, SELP, TLR2, and TYROBP), growth factors (HGF and TGFB3), enzymes (PTGS2 and PRKCP), and others (S100A8, S100A9, CD44, and C5AR1). Additionally, three upstream regulators (STAT3, EGR1, and F2R) were identified in the prepartum dataset. These findings revealed the key regulators and pathways underlying myometrial activation during placentitis, which aid in understanding the disease and facilitate the development of efficacious therapies.

Steroid synthesis and metabolism in the equine placenta during placentitis.

Equine placentitis is associated with alterations in maternal peripheral steroid concentrations, which could negatively affect pregnancy outcome. This study aimed to elucidate the molecular mechanisms related to steroidogenesis and steroid-receptor signaling in the equine placenta during acute placentitis. Chorioallantois (CA) and endometrial (EN) samples were collected from mares with experimentally induced placentitis (n = 4) and un-inoculated gestationally age-matched mares (control group; n = 4). The mRNA expression of genes coding for steroidogenic enzymes (3ßHSD, CYP11A1, CYP17A1, CYP19A1, SRD5A1, and AKR1C23) was evaluated using qRT-PCR. The concentration of these enzyme-dependent steroids (P5, P4, 5αDHP, 3αDHP, 20αDHP, 3ß-20αDHP, 17OH-P, DHEA, A4, and estrone) was assessed using liquid chromatography-tandem mass spectrometry in both maternal circulation and placental tissue. Both SRD5A1 and AKR1C23, which encode for the key progesterone metabolizing enzymes, were downregulated (P < 0.05) in CA from the placentitis group compared to controls, and this downregulation was associated with a decline in tissue concentrations of 5αDHP (P < 0.05), 3αDHP (P < 0.05), and 3ß-20αDHP (P = 0.052). In the EN, AKR1C23 was also downregulated in the placentitis group compared to controls, and this downregulation was associated with a decline in EN concentrations of 3αDHP (P < 0.01) and 20αDHP (P < 0.05). Moreover, CA expression of CYP19A1 tended to be lower in the placentitis group, and this reduction was associated with lower (P = 0.057) concentrations of estrone in CA. Moreover, ESR1 (steroid receptors) gene expression was downregulated (P = 0.057) in CA from placentitis mares. In conclusion, acute equine placentitis is associated with a local withdrawal of progestins in the placenta and tended to be accompanied with estrogen withdrawals in CA.

Alterations in T cell-related transcripts at the feto-maternal interface throughout equine gestation.

INTRODUCTION: The tolerance of pregnancy by the maternal immune system is balanced between recognition and protection. In the human this is controlled by balancing helper T cell populations (Th1, Th2) in addition to immune suppression from the regulatory arm (Tregs), but this has not been evaluated in the horse. METHODS: RNA sequencing was performed on chorioallantois and endometrium of mares at 120, 180, 300 and 330 days of gestation (n = 4/stage), as well as 45-day chorioallantois (n = 4) and diestrus endometrium (n = 3). Transcripts were selected for relativity to Th1, Th2, or Treg-associated. qPCR and immunohistochemistry were used to confirm the results of select differentially expressed genes. RESULTS: In the endometrium, Th1 transcripts were highest in the diestrus mare and decreased as gestational length progressed. In contrast, Th2 transcripts were upregulated in comparison to the diestrus mare and highest in mid gestation. Treg transcripts were found increased in comparison to the diestrus mare, but decreased prepartum. In the chorioallantois no Th1 transcripts changed. The majority of Th2 transcripts increased from 45 to 300 days gestation, and then decreased prepartum. Treg-related transcripts trended down in the chorioallantois from 45 days to 120 days gestation, followed by an upregulation to 300 days and a secondary decline prepartum. DISCUSSION: The mare experiences a complex and evolving immune profile within the tissues of the feto-maternal interface. This consists of a balance between the Th1 and Th2 response, and a dynamic Treg response that is hypothesized to regulate overall events within the immune system.

Uterine cervix as a fundamental part of the pathogenesis of pregnancy loss associated with ascending placentitis in mares.

Anatomical and molecular changes in the cervical barrier in women are a fundamental part of the pathogenesis of pregnancy loss associated with chorioamnionitis. However, there is little information regarding changes in the cervix associated with ascending infection in pregnant mares. To better characterize morphological and molecular changes in the cervix during placentitis, we examined full thickness histology and mRNA expression for a number of inflammatory and endocrine factors in the mucosa and stroma of the cervix of mares (n = 5) after experimental induction of placentitis via transcervical inoculation with Streptococcus equi ssp zooepidemicus at approximately 290d of gestation. Gestationally age-matched mares (n = 4) served as controls. Target transcripts included steroid receptors (PGR, ESR1 and 2), OXTR, prostaglandins synthases and receptors (PTGS1, PTGS2, PGES, PGFS, PTGER2 and PTGER4), cytokines (IL1b, IL6, CLCX8, IL10 and TNFα) and acute phase proteins (SAA). Histologically, a marked modification in the cervical epithelia and stroma was characterizing cervicitis. Additionally, the mRNA expression of IL1ß, IL6, CXCL8, SAA and PTGS2 was greater (P < 0.05) in both mucosa and stroma of the inoculated mares; whereas TNFα, IL10 and PGES were upregulated (P < 0.05) only in the cervical mucosa. Progesterone receptor, ESR1 and PTGER4 were upregulated in the cervical stroma of placentitis mares. In conclusion, the cervical response to placentitis was characterized by an upregulation of inflammatory cytokines that was accompanied by induction of PTGS2 and PGES. Further, receptors known to be associated with relaxation of the cervix in other species (ESR1 and PTGER4) were upregulated in the cervical stroma of placentitis mares. These findings indicate that the cervix is not only a physical barrier but that it has an active role in the pathogenesis of ascending placentitis.

Relationship between anti-Müllerian hormone and fertility in the mare.

The objectives of this study were to evaluate; 1) the stability of measured serum anti-Müllerian hormone (AMH) concentrations in samples after multiple freeze-thaw cycles, 2) the repeatability of AMH concentrations within mares during the same breeding season as well as across breeding seasons, and 3) the relationship between serum AMH concentrations and fertility (measured as first cycle pregnancy rates) in thoroughbred mares. For the first aim, AMH concentrations (n = 9) were examined across four freeze-thaw cycles with no significant change in measured AMH concentrations. For the second aim, serum AMH concentrations (n = 12) were examined over three successive estrous cycles and over two successive breeding seasons and AMH levels were significantly correlated for individual animals within (r; 0.71-82) or across breeding seasons (r = 0.81). For the third aim, Thoroughbred mares (n = 419) on farms in central Kentucky had blood samples taken during estrus. Pregnancy was determined with transrectal ultrasonography at Days 13-18 after mating and ovulation, and pregnancy outcome was recorded as open, pregnant or twins. The relationships between mare age, serum AMH concentrations and the interaction of age and AMH with pregnancy outcome was examined by nominal logistic regression, and the relationship between serum AMH concentrations and mare age, pregnancy outcome and the interaction of age and pregnancy outcome was examined by ANOVA. Data in this study were then stratified according to quartiles into lower (25%), mid-50% (second and third quartiles combined - 50%) and upper (25%) quartiles for age and serum AMH concentration for further analysis by logistic regression. There were significant effects of mare age and pregnancy outcome, but not their interaction on serum AMH concentrations which were higher (P = 0.04) in pregnant than in open mares (0.65 ± 0.03 vs 0.55 ± 0.04 ng/mL, respectively). Likewise, logistic regression revealed significant effect of mare age and AMH but not their interaction on pregnancy outcome on the first mated cycle. Mares in the lower AMH quartile were more likely to be open at Day 13-18 than mares in the middle (odds ratio (OR) = 1.87)=13 or upper quartile (OR = 2.62) for AMH concentrations. Mares in the mid-50% (OR = 3.91) or upper (OR = 4.97) age quartile were more likely to be open at Day 13-18 compared to mares in the young age quartile. Based upon a Chi-squared analysis, the proportion of pregnant mares differed across age quartiles (P < 0.0001) and was greater (P < 0.05) in the young mare quartile. The proportion of pregnant mares tended to differ across AMH quartile (P = 0.1), and when adjusted for age quartile using the Cochran-Mantel-Haenszel Test, the proportion of pregnant mares differed (P < 0.05) across AMH quartile. In conclusion, mares with peripheral AMH concentrations in the lowest quartile had lower fertility compared to mares in the mid-50% or upper quartile.

The anti-inflammatory effect of exogenous lactoferrin on breeding-induced endometritis when administered post-breeding in susceptible mares.

The deposition of semen into the uterus of the horse induces a transient innate immune response that lasts 24-36 h in the normal mare. There exists a subset of mares that are unable to resolve this inflammation in a timely manner, and are classified as susceptible to the disease of persistent breeding-induced endometritis (PBIE). Lactoferrin is a protein of interest as a potential therapeutic for this persistent inflammation due to its anti-inflammatory and bactericidal properties. The addition of human recombinant lactoferrin (hrLF) to the insemination dose was previously shown to suppress mRNA expression of the pro-inflammatory cytokine tumor necrosis factor (TNF)-α at 6 h after insemination, but no studies have shown the effect of lactoferrin when infused post-breeding. Therefore, the objectives of this study were to (1) assess the safety of intra-uterine infusion of hrLF, (2) evaluate the effect of intrauterine infusion of hrLF post-breeding as a modulator of the immune response to breeding in the susceptible mare, and (3) determine the most effective concentration of hrLF. For the first experiment four normal mares received an intrauterine infusion of 500 µg/mL hrLF resuspended in 10 mL lactated Ringer's solution (LRS) and heart rate, rectal temperature, respiration, and endometrial quality were evaluated. For the second experiment, six mares classified as susceptible to PBIE were bred during estrus with 500 × 106 progressively motile sperm comprised of the ejaculates from two stallions, which were centrifuged over Androcoll-E to remove seminal plasma. Each insemination dose was resuspended in 30 mL LRS. Six hours after breeding, a 1L LRS uterine lavage was performed prior to treatments. Four treatments were administered over four consecutive estrous cycles in randomized order of: 10 mL LRS (vehicle control), 50 µg/mL hrLF resuspended in 10 mL LRS, 250 µg/mL hrLF resuspended in 10 mL LRS, and 500 µg/mL hrLF resuspended in 10 mL LRS. Twenty-four hours after breeding the mares were evaluated via transrectal ultrasonography for fluid retention. A low volume uterine lavage (250 mL LRS) was performed and the effluent was evaluated for polymorphonuclear neutrophils (PMNs). Finally, an endometrial biopsy was obtained for qPCR analysis of selected inflammatory cytokines. Lactoferrin had no significant overall effect on vital signs or endometrial quality. The addition of hrLF (50 µg/mL, 250 µg/mL, 500 µg/mL) did not significantly affect the amount of fluid detected post-breeding, but suppressed the ratio of PMNs to epithelial cells at all three concentrations compared to controls. In addition, all three concentrations of hrLF increased the mRNA expression of the anti-inflammatory cytokine interleukin-1 receptor antagonist (IL-1RN), while the 50 µg/mL dose significantly suppressed mRNA expression of the pro-inflammatory cytokine interferon gamma (IFNγ). In conclusion, the infusion of hrLF post-breeding was found to modulate the inflammatory response to breeding in the mare, and appears to be most effective at the 50 µg/mL concentration.

Characterization of the cervical mucus plug in mares.

The cervical mucus plug (CMP) is believed to play an integral role in the maintenance of pregnancy in the mare, primarily by inhibiting microbial entry. Unfortunately, very little is known about its composition or origin. To determine the proteomic composition of the CMP, we collected CMPs from mares (n = 4) at 9 months of gestation, and proteins were subsequently analyzed by nano-LC-MS/MS. Results were searched against EquCab2.0, and proteomic pathways were predicted by Ingenuity Pathway Analysis. Histologic sections of the CMP were stained with H&E and PAS. To identify the origin of highly abundant proteins in the CMP, we performed qPCR on endometrial and cervical mucosal mRNA from mares in estrus, diestrus as well as mares at 4 and 10 m gestation on transcripts for lactotransferrin, uterine serpin 14, uteroglobin, uteroferrin, deleted in malignant brain tumors 1 and mucins 4, 5b and 6. Overall, we demonstrated that the CMP is composed of a complex milieu of proteins during late gestation, many of which play an important role in immune function. Proteins traditionally considered to be endometrial proteins were found to be produced by the cervical mucosa suggesting that the primary source of the CMP is the cervical mucosa itself. In summary, composition of the equine CMP is specifically regulated not only during pregnancy but also throughout the estrous cycle. The structural and compositional changes serve to provide both a structural barrier as well as a physiological barrier during pregnancy to prevent infection of the fetus and fetal membranes.

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.

Expression and localization of cysteine-rich secretory protein-3 (CRISP-3) in the prepubertal and postpubertal male horse.

The seminal plasma protein, cysteine-rich secretory protein-3 (CRISP-3), has been correlated with increased fertility and first-cycle conception rates, and has been suggested to be involved in the modulation of polymorphonuclear neutrophil and phagocytosis of spermatozoa during the inflammatory response to breeding in the horse. Previous research demonstrated that equine CRISP-3 is located in both the ampulla of the vas deferens and the seminal vesicles. However, this was done with nonquantitative laboratory techniques. In humans and rodents, CRISP-3 has been described as an androgen-dependent protein, but the effect of androgens on the expression of CRISP-3 has not been investigated in the horse. The objectives of this study were to (a) confirm and quantify the expression of CRISP-3 in the male equine reproductive tract, (b) describe the localization of CRISP-3 within the specific tissues which express it, and (c) determine if expression of CRISP-3 increases after puberty. We hypothesized that expression of CRISP-3 would be expressed in both the ampulla of the vas deferens and the seminal vesicles, and expression would increase after puberty. Tissues were collected postmortem from three prepubertal colts (<6 months) and six postpubertal stallions (>3 years). Tissue samples were collected from the ampulla of vas deferens, seminal vesicles, bulbourethral gland, prostate gland, testis, as well as the cauda, corpus, and caput aspects of the epididymis. Quantitative real-time polymerase chain reaction and immunohistochemistry (IHC) were performed using an equine-specific CRISP-3 designed primer and monocolonal antibody. A mixed linear additive model was used to compare mRNA expression between age groups, and significance was set to P < 0.05. There was a significant interaction between maturity and tissue type (P < 0.0001). Expression of CRISP-3 mRNA was found primarily in the ampulla of vas deferens with lesser expression in the seminal vesicles. Expression of CRISP-3 was higher in the postpubertal stallion when compared with the prepubertal colt for the ampulla (P < 0.0001) and seminal vesicles (P = 0.0013). IHC showed that equine CRISP-3 is primarily located in the glandular aspects of both the ampulla of vas deferens and the seminal vesicles, with staining concentrated in the cytoplasm of the epithelial cells that surrounded the glands of the mucosa. CRISP-3 was only observed in the postpubertal male horse suggesting that puberty plays a role in the activation of equine CRISP-3 expression.

Equine 5α-reductase activity and expression in epididymis.

The 5α-reductase enzymes play an important role during male sexual differentiation, and in pregnant females, especially equine species where maintenance relies on 5α-reduced progesterone, 5α-dihydroprogesterone (DHP). Epididymis expresses 5α-reductases but was not studied elaborately in horses. Epididymis from younger and older postpubertal stallions was divided into caput, corpus and cauda and examined for 5α-reductase activity and expression of type 1 and 2 isoforms by quantitative real-time polymerase chain reaction (qPCR). Metabolism of progesterone and testosterone to DHP and dihydrotestosterone (DHT), respectively, by epididymal microsomal protein was examined by thin-layer chromatography and verified by liquid chromatography tandem mass spectrometry (LC-MS/MS). Relative inhibitory potencies of finasteride and dutasteride toward equine 5α-reductase activity were investigated. Pregnenolone was investigated as an additional potential substrate for 5α-reductase, suggested previously from in vivo studies in mares but never directly examined. No regional gradient of 5α-reductase expression was observed by either enzyme activity or transcript analysis. Results of PCR experiments suggested that type 1 isoform predominates in equine epididymis. Primers for the type 2 isoform were unable to amplify product from any samples examined. Progesterone and testosterone were readily reduced to DHP and DHT, and activity was effectively inhibited by both inhibitors. Using epididymis as an enzyme source, no experimental evidence was obtained supporting the notion that pregnenolone could be directly metabolized by equine 5α-reductases as has been suggested by previous investigators speculating on alternative metabolic pathways leading to DHP synthesis in placenta during equine pregnancies.

Molecular changes in the equine follicle in relation to variations in antral follicle count and anti-Müllerian hormone concentrations.

REASONS FOR PERFORMING STUDY: The wide variation in circulating anti-Müllerian hormone (AMH) concentrations between mares is attributed to differences in antral follicle count (AFC) which may reflect follicular function. There are few data regarding variations in AFC and associated regulatory factors for AMH in the equine follicle during follicular development. OBJECTIVES: To examine molecular and hormonal differences in the equine follicle in relation to variations in AFC and circulating AMH concentrations during follicular development and to identify genes co-expressed with AMH in the equine follicle. STUDY DESIGN: Observational study. METHODS: Plasma AMH concentrations and AFC were determined in 30 cyclic mares. Granulosa cells, theca cells and follicular fluid were recovered from growing (n = 17) or dominant follicles (n = 13). The expression of several genes, known to be involved in folliculogenesis and steroidogenesis, was examined using real-time reverse transcriptase polymerase chain reaction and immunohistochemistry. Intrafollicular oestradiol and AMH concentrations were determined by immunoassay. RESULTS: Within growing follicles, the expression of AMH, AMHR2, ESR2 and INHA in granulosa cells was positively correlated with AFC and plasma AMH concentrations. In addition, the expression of ESR1 and FSHR was positively associated with plasma AMH concentrations. No significant associations were detected in dominant follicles. Furthermore, there was no association between AMH or oestradiol concentrations in follicular fluid and variations in AFC. Finally, the expression of AMH and genes co-expressed with AMH (AMHR2, ESR2 and FSHR) in granulosa cells as well as intrafollicular AMH concentrations decreased during follicular development while intrafollicular oestradiol concentrations increased and were inversely related to intrafollicular AMH concentrations. CONCLUSIONS: This study indicates that variations in AFC and circulating AMH concentrations are associated with molecular changes in the growing equine follicle.

The interrelationship between anti-Müllerian hormone, ovarian follicular populations and age in mares.

REASONS FOR PERFORMING STUDY: Anti-Müllerian hormone (AMH) is a granulosa-cell-derived glycoprotein, which plays an important inhibitory role during folliculogenesis. Concentrations of AMH are highly correlated with antral follicle counts (AFCs) in other species, which in turn are related to follicular reserve. Relatively little is known about AMH and AFC in the mare. OBJECTIVES: To determine plasma AMH concentrations and AFCs in mares of different ages, to measure the repeatability of AMH concentrations and AFCs within and across oestrous cycles and to assess the relationship between plasma AMH concentrations and AFCs with regard to mare age and follicle size. STUDY DESIGN: An observational study examining the relationship between AMH, AFC and age in 45 mares. METHODS: Young (3-8 years), middle-aged (9-18 years) and old mares (19-27 years) were examined by transrectal ultrasonography over 2 or 3 oestrous cycles. Plasma AMH concentrations and AFCs were determined, and antral follicles were classified by size into different groups. RESULTS: Plasma AMH concentrations varied widely between mares within similar age groups. Antral follicle counts were significantly lower in old mares than in young and middle-aged mares, and AMH concentrations were significantly lower in old than in middle-aged mares. A positive relationship was detected between AFC and AMH, and this relationship varied by mare age with a strong correlation in older mares (ρ = 0.86; P<0.0001), a moderate correlation in middle-aged mares (ρ = 0.60; P = 0.01) and no correlation in young mares (ρ = 0.40, P<0.4). The AMH concentrations were significantly related to the number of antral follicles between 6 and 20 mm in diameter, and the repeatability of AFCs and AMH concentrations was high within and between oestrous cycles. CONCLUSIONS: Our findings indicate that the relationship between AMH and AFC varies across age groups, and concentrations of AMH might be a better reflection of reproductive age than calendar age.

Attempts to induce nocardioform placentitis (Crossiela equi) experimentally in mares.

REASONS FOR PERFORMING STUDY: Nocardioform placentitis in horses is poorly understood, and the development of an experimental model would be of help in understanding the pathogenesis of the disease. OBJECTIVES: To investigate whether (1) intrauterine inoculation of Crossiela equi during the periovulatory period or (2) i.v., oral or intranasopharyngeal inoculation of C. equi during midgestation would result in nocardioform placentitis, and (3) before and after mating endometrial swabs present evidence of nocardioform placentitis-associated organisms (C. equi or Amycolatopsis spp.). METHODS: In Study I, mares (n = 20) received an intrauterine inoculation of C. equi 24 h after artificial insemination. Endometrial swabs were obtained 24 h post inoculation for PCR analysis. In Study II, pregnant mares (at 180-240 days of gestation) were inoculated with C. equi by intranasopharyngeal (n = 5), oral (n = 4) or i.v. (n = 4) routes. Sixty contemporaneous pregnant mares maintained on the same farm served as control animals. In Study III, privately owned Thoroughbred mares (n = 200) had endometrial swabs collected before and within 24-48 h after mating for detection of nocardioform microorganisms. RESULTS: In Study I, C.equi was identified by PCR in 3 of 20 mares following intrauterine inoculation. Pregnancy was established in 19 of 20 treated mares. There were 2 embryonic losses and one abortion at 177 days of gestation (undetermined cause). Sixteen mares delivered a normal foal and placenta. In Study II, one mare (oral inoculation) aborted at 200 days of gestation (unidentified cause). The remaining mares delivered a normal foal and placenta. In Study III, none of the mares yielded positive endometrial PCR for nocardioform microorganisms. CONCLUSIONS: We were unable to induce nocardioform placentitis, and there was no evidence of nocardioform microorganisms in endometrial swabs of broodmares before or after mating. These findings suggest that nocardioform placentitis is not induced simply via the presence of nocardiform actinomycetes and that route, insufficient duration of exposure and dose may play a role in the development of disease. Additional predispositions may also be involved in the development of nocardioform placentitis.

Expression of receptors for ovarian steroids and prostaglandin E2 in the endometrium and myometrium of mares during estrus, diestrus and early pregnancy.

The objective of this study was to compare expression of estrogen receptor alpha (ER-α), ß (ER-ß), progesterone receptor (PR), as well as prostaglandin E2 type 2 (EP2) and 4 (EP4) receptors in the equine myometrium and endometrium during estrus, diestrus and early pregnancy. Tissues were collected during estrus, diestrus, and early pregnancy. Transcripts for ER-α (ESR1), ER-ß (ESR2), PR (PGR), EP2 (PTGER2) and EP4 (PTGER4) were quantified by qPCR. Immunohistochemistry was used to localize ER-α, ER-ß, PR, EP2 and EP4. Differences in transcript in endometrium and myometrium were compared by the ΔΔCT method. Expression for ESR1 (P<0.05) tended to be higher during estrus than diestrus in the endometrium (P=0.1) and myometrium (P=0.06). In addition, ESR1 expression was greater during estrus than pregnancy (P<0.05) in the endometrium and tended to be higher in estrus compared to pregnancy in the myometrium (P=0.1). Expression for PGR was greater (P<0.05) in the endometrium during estrus and diestrus than during pregnancy. In the myometrium, PGR expression was greater in estrus than pregnancy (P=0.05) and tended to be higher during diestrus in relation to pregnancy (P=0.07). There were no differences among reproductive stages in ESR2, PTGER2 and PTGER4 mRNA expression (P>0.05). Immunolabeling in the endometrium appeared to be more intense for ER-α during estrus than diestrus and pregnancy. In addition, immunostaining for PR during pregnancy appeared to be more intense in the stroma and less intense in glands and epithelium compared to estrus and diestrus. EP2 immunoreactivity appeared to be more intense during early pregnancy in both endometrium and myometrium, whereas weak immunolabeling for EP4 was noted across reproductive stages. This study demonstrates differential regulation of estrogen receptor (ER) and PR in the myometrium and endometrium during the reproductive cycle and pregnancy as well as abundant protein expression of EP2 in the endometrium and myometrium during early pregnancy in mares.

Characterization of prostaglandin E2 receptors (EP2, EP4) in the horse oviduct.

Biological effects of prostaglandin E2 are mediated via one of four receptors designated EP1, EP2, EP3 and EP4 which are encoded by separate genes. In general, EP1 and EP3 induce smooth muscle contraction whereas EP2 and EP4 induce smooth muscle relaxation. The objective of the current study was to characterize the expression of the genes for PGE2 receptors (EP2 and EP4) in the horse oviduct based upon immunohistochemistry (IHC) and quantitative PCR (qPCR). Oviductal tissue was collected from mares at estrus (n=5), at 5 days post-ovulation (n=4), and from prepubertal mares (n=5). Isthmic and ampullar regions of the oviduct were fixed for IHC or preserved for RNA isolation. Prostaglandin E2 receptors EP2 and EP4 were strongly expressed by the luminal epithelium of both the isthmic and ampullar regions of the horse oviduct with a lesser immuno-expression noted within the smooth muscle in both regions of the oviduct. Based upon qPCR, relative amounts of EP2 or EP4 mRNA did not differ across estrous cycle stage or from prepubertal mares. However, across region and estrous cycle stage, relative amount of EP2 was greater (P<0.05) than EP4, and relative amount of EP2 mRNA was greater (P<0.001) in the ampullar compared with the isthmic oviduct. The relative roles of these receptors in regulating oviduct function in the mare remains to be determined.