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.

Estrogens Regulate Placental Angiogenesis in Horses.

A sufficient vascular network within the feto-maternal interface is necessary for placental function. Several pregnancy abnormalities have been associated with abnormal vascular formations in the placenta. We hypothesized that growth and expansion of the placental vascular network in the equine (Equus caballus) placenta is regulated by estrogens (estrogen family hormones), a hormone with a high circulating concentration during equine gestation. Administration of letrozole, a potent and specific inhibitor of aromatase, during the first trimester (D30 to D118), decreased circulatory estrone sulfate concentrations, increased circulatory testosterone and androstenedione concentrations, and tended to reduce the weight of the fetus (p < 0.1). Moreover, the gene expression of CYP17A1 was increased, and the expression of androgen receptor was decreased in the D120 chorioallantois (CA) of letrozole-treated mares in comparison to that of the control mares. We also found that at D120, the number of vessels tended to decrease in the CAs with letrozole treatment (p = 0.07). In addition, expression of a subset of angiogenic genes, such as ANGPT1, VEGF, and NOS2, were altered in the CAs of letrozole-treated mares. We further demonstrated that 17ß-estradiol increases the expression of ANGPT1 and VEGF and increases the angiogenic activity of equine endothelial cells in vitro. Our results from the estrogen-suppressed group demonstrated an impaired placental vascular network, suggesting an estrogen-dependent vasculogenesis in the equine CA during the first trimester.

Steroidogenic Enzyme and Steroid Receptor Expression in the Equine Accessory Sex Glands.

The expression pattern and distribution of sex steroid receptors and steroidogenic enzymes during development of the equine accessory sex glands has not previously been described. We hypothesized that equine steroidogenic enzyme and sex steroid receptor expression is dependent on reproductive status. Accessory sex glands were harvested from mature stallions, pre-pubertal colts, geldings, and fetuses. Expression of mRNA for estrogen receptor 1 (ESR1), estrogen receptor 2 (ESR2), androgen receptor (AR), 3ß-Hydroxysteroid dehydrogenase/Δ5-4 isomerase (3ßHSD), P450,17α hydroxylase, 17-20 lyase (CYP17), and aromatase (CYP19) were quantified by RT-PCR, and protein localization of AR, ER-α, ER-ß, and 3ßHSD were investigated by immunohistochemistry. Expression of AR, ESR2, CYP17, or CYP19 in the ampulla was not different across reproductive statuses (p > 0.1), while expression of ESR1 was higher in the ampulla of geldings and fetuses than those of stallions or colts (p < 0.05). AR, ESR1 and ESR2 expression were decreased in stallion vesicular glands compared to the fetus or gelding, while AR, ESR1, and CYP17 expression were decreased in the bulbourethral glands compared to other glands. ESR1 expression was increased in the prostate compared to the bulbourethral glands, and no differences were seen with CYP19 or 3ß-HSD. In conclusion, sex steroid receptors are expressed in all equine male accessory sex glands in all stages of life, while the steroidogenic enzymes were weakly and variably expressed.

Equine granulosa cell tumours among other ovarian conditions: Diagnostic challenges.

BACKGROUND: Granulosa cell tumours (GCT) are the most common ovarian tumours in mares. While the classical presentation may not represent diagnostic challenges, diagnosis is not easy in the early stages. OBJECTIVES: Illustrate the variability in the presentation and serum biomarkers associated with ovarian abnormalities in the mare. STUDY DESIGN: Retrospective case series. METHODS: Nonclassical cases of GCTs and other ovarian conditions were identified and behaviour, GCT endocrine results, palpation and ultrasonographic findings are described and the diagnostic value of each is discussed. RESULTS: Mares in this case series with GCTs had been presenting clinical signs ranging from no behavioural changes to behaviours including aggression, stallion-like and inability to work under saddle. Hormonal profiles of endocrinologically functional GCTs can be erratic and unpredictable. The clinical form and ultrasonographic appearance may also vary with time from an initially enlarged/anovulatory follicular structure that later develops a multicystic 'honeycomb' appearance. Mares with GCTs can also present with persistent anovulatory follicles or apparent luteal tissue that are unresponsive to treatment. If both ovaries are of relatively normal size and symmetry, but hormonal biomarkers are markedly increased (AMH >10 ng/mL, inhibin B and/or testosterone >100 pg/mL; 0.37 nmol/L), it is likely that a functional GCT is present. Still, it can be a challenge to decide which ovary to remove. Post-surgical endocrine testing can be helpful, especially if histopathology is not performed or a GCT is not found. MAIN LIMITATIONS: Cases limited to 14. CONCLUSIONS: Granulosa cell tumours present with a wide variety of clinical signs that do not fit what is commonly described as 'classic'. Only if AMH, testosterone and inhibin B concentrations are markedly increased, and there is an abnormally enlarged ovary, the diagnosis of a GCT is more confident. In the presence of normal size ovaries, normal hormonal biomarkers and abnormal behaviour, it is more likely that the ovaries are not involved.

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.

Hormone-responsive organoids from domestic mare and endangered Przewalski's horse endometrium.

The endometrium, the inner uterine lining, is composed of cell layers that come in direct contact with an embryo during early pregnancy and later with the fetal placenta. The endometrium is responsible for signals associated with normal reproductive cyclicity as well as maintenance of pregnancy. In the mare, functionally competent in vitro models of the endometrium have not been successful. Furthermore, the ability to study various reproductive processes in vitro may allow critical evaluation of signaling pathways involved in the reproductive diseases of animals that cannot be handled frequently, such as various wildlife species. Here we report the establishment of organoids, 3D structures, derived from fresh and frozen-thawed equine endometrium (Equus ferus caballus and E. f. przewalskii). Although organoids from domestic mares responded to exogenous hormonal stimuli, organoids from Przewalski's horse failed to respond to exogenous hormones. The present study represents a 'first' for any large animal model or endangered species. These physiologically functional organoids may facilitate improved understanding of normal reproductive mechanisms, uterine pathologies, and signaling mechanisms between the conceptus and endometrium and may lead to the development of novel bioassays for drug discovery.

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.

Equine hydrallantois is associated with impaired angiogenesis in the placenta.

INTRODUCTION: Hydrallantois is the excessive accumulation of fluid in the allantoic cavities during the last trimester of pregnancy, leading to abdominal wall hernias, cardiovascular shock, abortion, and dystocia. It has been postulated that hydrallantois is associated with structural and/or functional changes in the chorioallantoic membrane. In the present study, we hypothesized that angiogenesis is impaired in the hydrallantoic placenta. METHOD: Capillary density in the hydrallantoic placenta was evaluated in the chorioallantois via immunohistochemistry for Von Willebrand Factor. Moreover, the expression of angiogenic genes was compared between equine hydrallantois and age-matched, normal placentas. RESULTS: In the hydrallantoic samples, edema was the main pathological finding. The capillary density was significantly lower in the hydrallantoic samples than in normal placentas. The reduction in the number of vessels was associated with abnormal expression of a subset of angiogenic and hypoxia-associated genes including VEGF, VEGFR1, VEGFR2, ANGPT1, eNOS and HIF1A. We believe that the capillary density and the abnormal expression of angiogenic genes leads to tissue hypoxia (high expression of HIF1A) and edema. Finally, we identified a lower expression of genes associated with steroidogenic enzyme (CYP19A1) and estrogen receptor signaling (ESR2) in the hydrallantoic placenta. DISCUSSION: Based on the presented data, we believe that formation of edema is due to disrupted vascular development (low number of capillaries) and hypoxia in the hydrallantoic placenta. The edema leads to further hypoxia and consequently, causes an increase in vessel permeability which leads to a gradual increase in interstitial fluid accumulation, resulting in an insufficient transplacental exchange rate and accumulation of fluid in the allantoic cavity.

Alteration of the mare's immune system by the synthetic progestin, altrenogest.

PROBLEM: Progestins are immunomodulatory in a variety of species. In the horse, the most commonly administered synthetic progestin is altrenogest (ALT), but its effect on the immune system of the non-pregnant mare is unknown. METHODS: Peripheral blood mononuclear cells (PBMCs) from diestrous mares were incubated with varying concentrations of progesterone (P4) or ALT to assess intracellular production of IFNγ and the expression of select cytokines. Additionally, ten mares received either ALT or VEH daily utilizing a switchback design beginning on the day of ovulation and continuing for 7 days. Circulating PBMCs and endometrial biopsies were obtained to assess the production and expression of the same cytokines. RESULTS: In vitro, both P4 and ALT caused a dose-dependent decrease in intracellular IFNγ in PBMCs. P4 caused a dose-dependent decrease in the expression of IFNγ, IL-10 and IL-4, while ALT caused an increase in the expression of IL-6 and IL-1ß in PBMCs. In vivo, ALT suppressed the intracellular levels of IFNγ in PBMCs on d6. While control mares experienced a decrease in IL-1ß expression from d0 to d6, ALT-treated mares did not. In the endometrium, ALT increased the expression of IL-1RN and IFNγ in comparison with VEH-treated mares. CONCLUSION: P4 and ALT appear to alter the immune system of the non-pregnant mare both systemically in addition to locally within the endometrium. Further research is necessary to determine the pathways through which this synthetic progestin functions on the immune system of the horse, and the consequences it may have.

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.

Steroidogenic enzyme activities in the pre- and post-parturient equine placenta.

Steroidogenic enzymes in placentas shape steroid hormone profiles in the maternal circulation of each mammalian species. These include 3ß-hydroxysteroid dehydrogenase/Δ5-4 isomerase (3ßHSD) and 17α-hydroxylase/17,20-lyase cytochrome P450 (P450c17) crucial for progesterone and androgen synthesis, respectively, as well as aromatase cytochrome P450 (P450arom) that converts Δ4-androgens to estrogens. 5α-reductase is another important enzyme in equine placentas because 5α-dihydroprogesterone (DHP) sustains pregnancy in the absence of progesterone in the second half of equine pregnancy. DHP and its metabolites decline dramatically days before foaling, but few studies have investigated placental enzyme activity before or at parturition in mares. Thus, key enzyme activities and transcript abundance were investigated in equine placentas at 300 days of gestation (GD300) and post-partum (term). Equine testis was used as a positive control for P450c17 activity. Substrates were incubated with microsomal preparations, together with enzyme inhibitors, and products were measured by liquid chromatography tandem mass spectrometry or radiometric methods (aromatase). Equine placenta expressed high levels of 3ßHSD, 5α-reductase and aromatase, and minimal P450c17 activity at GD300 compared with testis (600-fold higher). At foaling, 3ßHSD and aromatase activities and transcript abundance were unchanged but 5α-reductase (and P450c17) was no longer detectable (P < 0.05) and transcript was decreased. Trilostane inhibited 3ßHSD significantly more in testis than placenta, suggesting possible existence of different 3ßHSD isoforms. Equine placentas have significant capacity for steroid metabolism by 5α-reductase, 3ßHSD and aromatase but little for androgen synthesis lacking P450c17. Declining pre-partum 5α-reduced pregnane concentrations coincide with selective loss of placental 5α-reductase activity and expression at parturition in horses.

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.

Endocrine changes, fetal growth, and uterine artery hemodynamics after chronic estrogen suppression during the last trimester of equine pregnancy.

Equine pregnancy is characterized by very high circulating concentrations of estrogens. The physiological roles of estrogens during equine gestation are largely unknown, although some studies suggest a role in the regulation of uterine artery hemodynamics and a relationship between low circulating estrogen concentrations and late pregnancy loss. The objectives of this experiment were to evaluate the effects of estrogen suppression on uterine artery hemodynamics and on pregnancy outcome. Estrogen synthesis was suppressed using letrozole, a potent aromatase inhibitor. Twelve pregnant mares were randomly assigned to a control (n = 6) or treatment (n = 6; 500 mg letrozole orally every 4 days) group with treatment starting at 240 days of gestation and continuing until parturition. Weekly serum samples were analyzed to determine testosterone, dehydroepiandrosterone sulfate, estradiol, estrone sulfate, progestins, and prostaglandin F2α metabolite concentrations. Ultrasonographic examinations were performed biweekly and measurements included uterine artery hemodynamics (diameter, pulsatility, and resistance indices), fetal growth using the diameter of the fetal eye, and placental evaluation using the combined thickness of the uterus and placenta. At parturition, gestational length, foal weight, and neonatal viability were determined. Letrozole suppressed estrogen synthesis during gestation by approximately 90% compared to control values. This large reduction in circulating estrogens had no effect on uterine artery hemodynamics, normal placental development, maintenance of pregnancy, or neonatal viability. However, neonates from letrozole-treated mares had lower (P < 0.05) birth weights than controls, suggesting that estrogens may play a role in fetal growth that is not mediated through regulation of uterine blood flow.

Biological Functions and Clinical Applications of Anti-Müllerian Hormone in Stallions and Mares.

Anti-Müllerian hormone (AMH) plays a major role in sexual differentiation, Leydig cell differentiation, and folliculogenesis. In addition, AMH has clinical value in equine practice. In stallions, AMH can serve as an endocrine marker for equine cryptorchidism and as an immunohistochemical marker for Sertoli cell tumors. Considering that AMH is also an ovarian specific product, intact mares can be differentiated from ovariectomized mares. Peripheral AMH concentrations reflect the follicular population in mares, and therefore, are useful in the assessment of ovarian reserve and reproductive life-span of aged mares. Last, AMH is particularly suitable as a diagnostic marker for equine granulosa cell tumors.

The dynamic steroid landscape of equine pregnancy mapped by mass spectrometry.

Liquid chromatography-tandem mass spectrometry (LC-MS/MS) allowed comprehensive analysis of various steroids detectable in plasma throughout equine gestation. Mares (n=9) were bled serially until they foaled. Certain steroids dominated the profile at different stages of gestation, clearly defining key physiological and developmental transitions. The period (weeks 6-20) coincident with equine chorionic gonadotropic (eCG) stimulation of primary corpora lutea and subsequent formation of secondary luteal structures was defined by increased progesterone, 17OH-progesterone and androstenedione, all Δ4 steroids. The 5α-reduced metabolite of progesterone, dihydroprogesterone (DHP) paralleled progesterone secretion at less than half the concentration until week 12 of gestation when progesterone began to decline but DHP concentrations continued to increase. DHP exceeded progesterone concentrations by week 16, clearly defining the luteo-placental shift in pregnane synthesis from primarily ovarian to primarily placental. The period corresponding to the growth of fetal gonads was defined by increasing dehydroepiandrosterone and pregnenolone (Δ5 steroids) concentrations from week 14, peaking at week 34 and declining to term. Metabolites of DHP (including allopregnanolone) dominated the steroid profile in late gestation, some exceeding DHP by weeks 13 or 14 and near term by almost tenfold. Thus Δ4 steroids dominated during ovarian stimulation by eCG, inversion of the ratio of progesterone: DHP (increasing 5α-pregnanes) marked the luteo-placental shift, Δ5 steroids defined fetal gonadal growth and 5α-reduced metabolites of DHP dominated the steroid profile in mid- to late-gestation. Comprehensive LC-MS/MS steroid analysis provides opportunities to better monitor the physiology and the progress of equine pregnancies, including fetal development.

Pregnancy without progesterone in horses defines a second endogenous biopotent progesterone receptor agonist, 5α-dihydroprogesterone.

One of the most widely accepted axioms of mammalian reproductive biology is that pregnancy requires the (sole) support of progesterone, acting in large measure through nuclear progesterone receptors (PRs) in uterine and cervical tissues, without which pregnancy cannot be established or maintained. However, mares lack detectable progesterone in the latter half of pregnancy. Instead of progesterone, several (mainly 5α-reduced) pregnanes are elevated and have long been speculated to provide progestational support in lieu of progesterone itself. To the authors' knowledge, evidence for the bioactivity of a second potent endogenously synthesized pregnane able to support pregnancy in the absence of progesterone has never before been reported. The 5α-reduced progesterone metabolite dihydroprogesterone (DHP) was shown in vivo to stimulate endometrial growth and progesterone-dependent gene expression in the horse at subphysiological concentrations and to maintain equine pregnancy in the absence of luteal progesterone in the third and fourth weeks postbreeding. Results of in vitro studies indicate that DHP is an equally potent and efficacious endogenous progestin in the horse but that the PR evolved with increased agonistic potency for DHP at the expense of potency toward progesterone based on comparisons with human PR responses. Sequence analysis and available literature indicate that the enzyme responsible for DHP synthesis, 5α-reductase type 1, also adapted primarily to metabolize progesterone and thereby to serve diverse roles in the physiology of pregnancy in mammals. Our confirmation that endogenously synthesized DHP is a biopotent progestin in the horse ends decades of speculation, explaining how equine pregnancies survive without measurable circulating progesterone in the last 4 to 5 mo of gestation.

Effects of a GnRH cytotoxin on reproductive function in peripubertal male dogs.

Methods for long-term or permanent disruption of reproductive function via nonsurgical techniques are needed for a variety of species, including companion animals. In a previous study, we demonstrated the ability of a cytotoxin (pokeweed antiviral protein-PAP) conjugated to d-Lys(6)-GnRH, to disrupt reproductive function in adult male dogs. The objective of the present study was to examine the ability of a d-Lys(6)-GnRH-PAP conjugate to disrupt reproductive function in peripubertal male dogs. Peripubertal male dogs (n=15; approximately 16-32 weeks old) were treated with d-Lys(6)-GnRH-PAP as follows: dogs (n=7; Group I) received GnRH-PAP (0.1 mg/kg SQ) with a second treatment (0.25 mg/kg) 20 weeks later. An additional group (n=3; Group II) of peripubertal dogs was treated with GnRH-PAP (0.25 mg/kg) twice (20 weeks apart). Control dogs (n=5) received d-Lys(6)-GnRH analog (0.0045 mg/kg SQ) without PAP. Efficacy was assessed by monitoring testis size, serum concentrations of testosterone and LH, as well as LH release subsequent to a GnRH (5 microg/kg) stimulus. Dogs in Group I (n=5) that did not respond to the initial two treatments were given a third GnRH-PAP injection (0.25 mg/kg), 12 months after the initial treatment. The initial GnRH-PAP treatment in peripubertal male dogs did not affect testis growth, LH release or serum testosterone concentrations; however, administration of a higher dose of GnRH-PAP after puberty resulted in a marked and rapid decline in testis size, serum testosterone concentrations and LH responsiveness to GnRH stimulation in 9 of 10 dogs. Suppression of reproductive function was maintained in treated dogs for 18-50 weeks; four dogs had suppression of reproductive activity through the end of the study. In conclusion, GnRH-PAP given after puberty markedly suppressed reproductive activity. Due to variability in the response and duration of suppression after treatment with GnRH-PAP, more research is required to determine its efficacy for nonsurgical sterilization of the male dog.

Evaluation of Coomassie blue staining of the acrosome of equine and canine spermatozoa.

OBJECTIVE: To evaluate Coomassie blue staining of the acrosome of equine and canine spermatozoa. SAMPLE POPULATION: Spermatozoa of 5 mixed-breed male dogs and 3 Thoroughbred stallions. PROCEDURE: Various proportions of intact and acrosome-damaged spermatozoa were fixed in 2% phosphate-buffered formaldehyde or 4% paraformaldehyde, smeared onto glass slides, and stained with Coomassie blue stain. Acrosomal status (damaged vs intact) was also assessed by use of flow cytometry after staining with fluorescein isothiocyanate-conjugated Pisum sativum agglutinin (FITC-PSA) and propidium iodide. Comparisons were made between percentages of expected and observed acrosome-intact spermatozoa in different proportions of live and flash-frozen samples; the percentages of acrosome-intact spermatozoa as determined by use of Coomassie blue staining and flow cytometry were also compared. RESULTS: Strong correlations were found between the expected and observed distributions of acrosome-intact spermatozoa when fixed in 4% paraformaldehyde (r2 = 0.93 and 0.89 for canine and equine spermatozoa, respectively) as well as between Coomassie blue-stained cells and those stained with FITC-PSA and assessed by use of flow cytometry (r2 = 0.96 and 0.97 for canine and equine spermatozoa, respectively). However, in canine samples that were fixed in 2% phosphate-buffered formaldehyde, these correlations were weak. CONCLUSIONS AND CLINICAL RELEVANCE: Staining with Coomassie blue stain was a simple and accurate method to evaluate the acrosome in equine and canine spermatozoa after fixation in 4% paraformaldehyde. This assay should be useful in routine evaluation of semen samples from these species.

Reactive oxygen species and cryopreservation promote DNA fragmentation in equine spermatozoa.

The objective of this study was to examine the effect of reactive oxygen species (ROS) and cryopreservation on DNA fragmentation of equine spermatozoa. In experiment 1, equine spermatozoa were incubated (1 hour, 38 degrees C) according to the following treatments: 1) sperm alone; 2) sperm + xanthine (X, 0.3 mM)-xanthine oxidase (XO, 0.025 U/mL); 3) sperm + X (0.6 mM)-XO (0.05 U/mL); and 4) sperm + X (1 mM)-XO (0.1 U/mL). In experiment 2, spermatozoa were incubated (1 hour, 38 degrees C) with X (1 mM)-XO (0.1 U/mL) and either catalase (200 U/mL), superoxide dismutase (SOD, 200 U/mL), or reduced glutathione (GSH, 10 mM). Following incubation, DNA fragmentation was determined by the single cell gel electrophoresis (comet) assay. In experiment 3, equine spermatozoa were cryopreserved, and DNA fragmentation was determined in fresh, processed, and postthaw sperm samples. In experiment 1, incubation of equine spermatozoa in the presence of ROS, generated by the X-XO system, increased DNA fragmentation (P <.005). In Experiment 2, the increase in DNA fragmentation associated with X-XO treatment was counteracted by the addition of catalase and GSH but not by SOD, suggesting that hydrogen peroxide and not superoxide appears to be the ROS responsible for such damage. In experiment 3, cryopreservation of equine spermatozoa was associated with an increase (P <.01) in DNA fragmentation when compared with fresh or processed samples. This study indicates that ROS and cryopreservation promote DNA fragmentation in equine spermatozoa; the involvement of ROS in cryopreservation-induced DNA damage remains to be determined.