Parental bias in expression and interaction of genes in the equine placenta.

Most autosomal genes in the placenta show a biallelic expression pattern. However, some genes exhibit allele-specific transcription depending on the parental origin of the chromosomes on which the copy of the gene resides. Parentally expressed genes are involved in the reciprocal interaction between maternal and paternal genes, coordinating the allocation of resources between fetus and mother. One of the main challenges of studying parental-specific allelic expression (allele-specific expression [ASE]) in the placenta is the maternal cellular remnant at the fetomaternal interface. Horses (Equus caballus) have an epitheliochorial placenta in which both the endometrial epithelium and the epithelium of the chorionic villi are juxtaposed with minimal extension into the uterine mucosa, yet there is no information available on the allelic gene expression of equine chorioallantois (CA). In the current study, we present a dataset of 1,336 genes showing ASE in the equine CA (https://pouya-dini.github.io/equine-gene-db/) along with a workflow for analyzing ASE genes. We further identified 254 potentially imprinted genes among the parentally expressed genes in the equine CA and evaluated the expression pattern of these genes throughout gestation. Our gene ontology analysis implies that maternally expressed genes tend to decrease the length of gestation, while paternally expressed genes extend the length of gestation. This study provides fundamental information regarding parental gene expression during equine pregnancy, a species with a negligible amount of maternal cellular remnant in its placenta. This information will provide the basis for a better understanding of the role of parental gene expression in the placenta during gestation.

Paternally expressed retrotransposon Gag-like 1 gene, RTL1, is one of the crucial elements for placental angiogenesis in horses†.

RTL1 (retrotransposon Gag-like 1) is an essential gene in the development of the human and murine placenta. Several fetal and placental abnormalities such as intrauterine growth restriction (IUGR) and hydrops conditions have been associated with altered expression of this gene. However, the function of RTL1 has not been identified. RTL1 is located on a highly conserved region in eutherian mammals. Therefore, the genetic and molecular analysis in horses could hold important implications for other species, including humans. Here, we demonstrated that RTL1 is paternally expressed and is localized within the endothelial cells of the equine (Equus caballus) chorioallantois. We developed an equine placental microvasculature primary cell culture and demonstrated that RTL1 knockdown leads to loss of the sprouting ability of these endothelial cells. We further demonstrated an association between abnormal expression of RTL1 and development of hydrallantois. Our data suggest that RTL1 may be essential for placental angiogenesis, and its abnormal expression can lead to placental insufficiency. This placental insufficiency could be the reason for IUGR and hydrops conditions reported in other species, including humans.

Equine cervical remodeling during placentitis and the prepartum period: a transcriptomic approach.

Cervical remodeling is a critical component in both term and preterm labor in eutherian mammals. However, the molecular mechanisms underlying cervical remodeling remain poorly understood in the mare. The current study compared the transcriptome of the equine cervix (cervical mucosa (CM) and stroma (CS)) during placentitis (placentitis group, n = 5) and normal prepartum mares (prepartum group, n = 3) to normal pregnant mares (control group, n = 4). Transcriptome analysis identified differentially expressed genes (DEGs) during placentitis (5310 in CM and 907 in CS) and during the normal prepartum period (189 in CM and 78 in CS). Our study revealed that cervical remodeling during placentitis was dominated by inflammatory signaling as reflected by the overrepresented toll-like receptor signaling, interleukin signaling, T cell activation, and B cell activation pathways. These pathways were accompanied by upregulation of several proteases, including matrix metalloproteinases (MMP1, MMP2, and MMP9), cathepsins (CTSB, CTSC, and CTSD) and a disintegrin and metalloproteinase with thrombospondin type 1 motifs (ADAMTS1, ADAMTS4, and ADAMTS5), which are crucial for degradation of cervical collagens during remodeling. Cervical remodeling during placentitis was also associated with upregulation of water channel-related transcripts (AQP9 and RLN), angiogenesis-related transcripts (NOS3, ENG1, THBS1, and RAC2), and aggrecan (ACAN), a hydrophilic glucosaminoglycan, with subsequent cervical hydration. The normal prepartum cervix was associated with upregulation of ADAMTS1, ADAMTS4, NOS3 and THBS1, which might reflect an early stage of cervical remodeling taking place in preparation for labor. In conclusion, our findings revealed the possible key regulators and mechanisms underlying equine cervical remodeling during placentitis and the normal prepartum period.

Transcriptomic analysis of equine chorioallantois reveals immune networks and molecular mechanisms involved in nocardioform placentitis.

Nocardioform placentitis (NP) continues to result in episodic outbreaks of abortion and preterm birth in mares and remains a poorly understood disease. The objective of this study was to characterize the transcriptome of the chorioallantois (CA) of mares with NP. The CA were collected from mares with confirmed NP based upon histopathology, microbiological culture and PCR for Amycolatopsis spp. Samples were collected from the margin of the NP lesion (NPL, n = 4) and grossly normal region (NPN, n = 4). Additionally, CA samples were collected from normal postpartum mares (Control; CRL, n = 4). Transcriptome analysis identified 2892 differentially expressed genes (DEGs) in NPL vs. CRL and 2450 DEGs in NPL vs. NPN. Functional genomics analysis elucidated that inflammatory signaling, toll-like receptor signaling, inflammasome activation, chemotaxis, and apoptosis pathways are involved in NP. The increased leukocytic infiltration in NPL was associated with the upregulation of matrix metalloproteinase (MMP1, MMP3, and MMP8) and apoptosis-related genes, such as caspases (CASP3 and CASP7), which could explain placental separation associated with NP. Also, NP was associated with downregulation of several placenta-regulatory genes (ABCG2, GCM1, EPAS1, and NR3C1), angiogenesis-related genes (VEGFA, FLT1, KDR, and ANGPT2), and glucose transporter coding genes (GLUT1, GLUT10, and GLUT12), as well as upregulation of hypoxia-related genes (HIF1A and EGLN3), which could elucidate placental insufficiency accompanying NP. In conclusion, our findings revealed for the first time, the key regulators and mechanisms underlying placental inflammation, separation, and insufficiency during NP, which might lead to the development of efficacious therapies or diagnostic aids by targeting the key molecular pathways.

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.

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.

Equine placentitis is associated with a downregulation in myometrial progestin signaling†.

The current study aimed to elucidate the mechanisms underlying myometrial activation during equine placentitis related to progestogens and the progesterone receptor signaling pathways. Placentitis was induced via intracervical inoculation with Streptococcus equi ssp zooepidemicus in mares at approximately 290 days of gestation (placentitis group; n = 6) with uninoculated gestationally matched mares as controls (n = 4). Mares in the placentitis and control groups were euthanized, and myometrial samples were collected from two regions: region 1-parallel to active placentitis lesion with placental separation in placentitis group (P1) or caudal pole of the placenta in control group (C1); and region 2-parallel to apparently normal placenta without separation in placentitis group (P2) or uterine body in control group (C2). In the current study, SRD5A1 and AKR1C23, which encode for the key P4 metabolizing enzymes, were downregulated in P1 in comparison to C1, C2, and P2, and this was associated with a decline (P < 0.05) in 5αDHP, allopregnanolone (3αDHP), and 20αDHP in P1 in comparison to C1. Further, myometrial expression of PR was downregulated (P < 0.05) in P1 in comparison to C1 and P2, and this was associated with activation of the inflammatory cascade as reflected by significant upregulation of IL-1ß and IL-8 in P1 in comparison to C1, C2, and P2, and supported by increased tissue leukocytes in P1 in comparison to C1. In conclusion, equine placentitis is associated with a localized withdrawal of progestins and a downregulation of the PR in the myometrium concomitant with upregulation of inflammatory cytokines and subsequent myometrial activation.

Steroids in the establishment and maintenance of pregnancy and at parturition in the mare.

Historically, studies on the endocrinology of pregnancy and parturition in horses have made major contributions of relevance to mammals in general. Recent use of liquid chromatography mass spectrometry, measuring multiple steroid hormones simultaneously in blood, foetal and placental tissues throughout normal gestation, and in mares with experimentally induced placentitis, has advanced our current understanding of many of the unusual strategies seen during gestation and at foaling. This includes the stimulation of luteal steroidogeneisis by equine chorionic gonadotropin (eCG) from the endometrial cups, resulting in additional androgen and oestrogen secretion. Progesterone declines as the endometrial cups and eCG disappears, replaced by 5α-dihydroprogesterone (DHP), a potent equine progesterone receptor (PR) agonist, as the chorioallantoic placenta develops. Placental steroidogenesis thereafter is influenced by foetal pregnenolone and dehydroepiandrosterone secretion, providing substrate for 5α-pregnane and oestrogen synthesis, an unusual example of a 'foeto-placental unit'. Foetal gonadal dehydroepiandrosterone fuels placental oestrone sulphate secretion, peaking at higher concentrations in mares than any other species known, declining steadily thereafter to term. Additional 5α-reduced (DHP) metabolites increase from mid-gestation to peak concentrations 3-5 days before foaling, declining prepartum, most likely as a result of selective loss of placental SRD5A1 (5α-reductase) expression and activity. Similar changes occur in mares with experimentally induced placentitis, which is also associated with a decreased ratio of equine PR-B:PR-A in myometrium, suggesting that progestin withdrawal is both systemic (pregnanes) and local (receptor-dependent) in mares. In addition, some steroids detected during equine pregnancy by immuno-assay are not detected by mass spectrometry, further illustrating the immense value of this technology.

Fetal-fluid proteome analyses in late-term healthy pregnant mares and in mares with experimentally induced ascending placentitis.

Characterisation of fetal fluids in healthy and disease states of pregnant mares can help to unravel the pathophysiology and to identify putative markers of disease. Thus, this study aimed to compare the protein composition of: (1) amniotic and allantoic fluids of healthy mares obtained immediately after euthanasia and (2) allantoic fluid harvested via centesis before and after experimental induction of placentitis via transcervical inoculation of Streptococcus equi ssp zooepidemicus in healthy mares. Fetal fluids were analysed with a high-throughput proteomic technique after in-gel digestion. Statistical comparisons were performed following normalisation of peptide spectral match. Global normalisation was performed to calculate relative expression. There were 112 unique proteins present in both allantoic and amniotic fluids. There were 13 and 29 proteins defined as amniotic- or allantoic-specific respectively that were present in at least two fluid samples. Another 26 proteins were present in both amniotic and allantoic fluids. Panther DB functional classification grouped fetal-fluid proteins as transfer carriers, signalling molecules, receptors, immunity, hydrolase, enzymes, membrane traffic, cytoskeleton, cell adhesion, calcium binding and extracellular matrix. Experimentally induced placentitis resulted in 10 proteins being upregulated and 10 downregulated in allantoic fluid. Newly identified proteins and changes in the fetal-fluid proteome provide clues about the physiology of pregnancy and pathogenesis of placentitis.

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.

Extraction of RNA from formalin-fixed, paraffin-embedded equine placenta.

Archived formalin-fixed, paraffin-embedded (FFPE) samples represent a valuable resource for the determination of gene expression for physio/pathological conditions. In the present study, we validated a protocol for the extraction of RNA from FFPE samples collected from healthy and diseased equine placenta. The quality and quantity of the extracted RNA from the FFPE and matching RNAlater™-preserved samples and expression levels of common housekeeping genes and reference microRNAs were evaluated. Precision of the expression data was evaluated by comparing relative expression of CYP19A1 and HSD3B1 in FFPE and RNAlater™ samples. The median RNA concentration recovered from FFPE samples was 316.8 ng/mm3 of tissue (ranging between 61.6 and 917.4 ng/mm3 ), average RNA integrity number was 2.3 ± 0.9 (mean ± standard deviation), and 84% of samples had RNA fragments longer than 200 nucleotides (DV200 ). RNA concentrations and CT values for GAPDH, ACTB, miR-8908a and miR-369 in FFPE samples were significantly correlated (r = -0.8, -0.7, -0.4 and -0.4, respectively; p < 0.001). Expression pattern of normalized CYP19A1 and HSD3B1 in paired FFPE and RNAlater™ samples was significantly correlated (r = 0.97 for CYP19A1 and HSD3B1; p < 0.001). This study demonstrates that RNA can be extracted from FFPE equine placental tissue and used for downstream transcriptomic analysis. Similar RNA expression patterns were obtained using RNAlater™ and FFPE tissue samples.

Kinetics of the chromosome 14 microRNA cluster ortholog and its potential role during placental development in the pregnant mare.

BACKGROUND: The human chromosome 14 microRNA cluster (C14MC) is a conserved microRNA (miRNA) cluster across eutherian mammals, reported to play an important role in placental development. However, the expression kinetics and function of this cluster in the mammalian placenta are poorly understood. Here, we evaluated the expression kinetics of the equine C24MC, ortholog to the human C14MC, in the chorioallantoic membrane during the course of gestation. RESULTS: We demonstrated that C24MC-associated miRNAs presented a higher expression level during early stages of pregnancy, followed by a decline later in gestation. Evaluation of one member of C24MC (miR-409-3p) by in situ hybridization demonstrated that its cellular localization predominantly involved the chorion and allantoic epithelium and vascular endothelium. Additionally, expression of predicted target transcripts for C24MC-associated miRNAs was evaluated by RNA sequencing. Expression analysis of a subset of predicted mRNA targets showed a negative correlation with C24MC-associated miRNAs expression levels during gestation, suggesting the reciprocal control of these target transcripts by this miRNA cluster. Predicted functional analysis of these target mRNAs indicated enrichment of biological pathways related to embryonic development, endothelial cell migration and angiogenesis. Expression patterns of selected target mRNAs involved in angiogenesis were confirmed by RT-qPCR. CONCLUSION: This is the first report evaluating C24MC kinetics during pregnancy. The findings presented herein suggest that the C24MC may modulate angiogenic transcriptional profiles during placental development in the horse.

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.

Inhibition of 5α-reductase alters pregnane metabolism in the late pregnant mare.

In the latter half of gestation in the mare, progesterone concentrations decline to near undetectable levels while other 5α-reduced pregnanes are elevated. Of these, 5α-dihydroprogesterone and allopregnanolone have been reported to have important roles in either pregnancy maintenance or fetal quiescence. During this time, the placenta is necessary for pregnane metabolism, with the enzyme 5α-reductase being required for the conversion of progesterone to 5α-dihydroprogesterone. The objectives of this study were to assess the effects of a 5α-reductase inhibitor, dutasteride on pregnane metabolism (pregnenolone, progesterone, 5α-dihydroprogesterone, 20α-hydroxy-5α-pregnan-3-one, 5α-pregnane-3ß,20α-diol and allopregnanolone), to determine circulating dutasteride concentrations and to assess effects of dutasteride treatment on gestational parameters. Pregnant mares (n = 5) received dutasteride (0.01 mg/kg/day, IM) and control mares (n = 4) received vehicle alone from 300 to 320 days of gestation or until parturition. Concentrations of dutasteride, pregnenolone, progesterone, 5α-dihydroprogesterone, 20α-hydroxy-5α-pregnan-3-one, 5α-pregnane-3ß,20α-diol, and allopregnanolone were evaluated via liquid chromatography-tandem mass spectrometry. Samples were analyzed as both days post treatment and as days prepartum. No significant treatment effects were detected in pregnenolone, 5α-dihydroprogesterone, 20α-hydroxy-5α-pregnan-3-one, 5α-pregnane-3ß,20α-diol or allopregnanolone for either analysis; however, progesterone concentrations were increased (P < 0.05) sixfold in dutasteride-treated mares compared to control mares. Dutasteride concentrations increased in the treated mares, with a significant correlation (P < 0.05) between dutasteride concentrations and pregnenolone or progesterone concentrations. Gestational length and neonatal outcomes were not significantly altered in dutasteride-treated mares. Although 5α-reduced metabolites were unchanged, these data suggest an accumulation of precursor progesterone with inhibition of 5α-reductase, indicating the ability of dutasteride to alter progesterone metabolism.

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.

Equine fetal adrenal, gonadal and placental steroidogenesis.

Equine fetuses have substantial circulating pregnenolone concentrations and thus have been postulated to provide significant substrate for placental 5α-reduced pregnane production, but the fetal site of pregnenolone synthesis remains unclear. The current studies investigated steroid concentrations in blood, adrenal glands, gonads and placenta from fetuses (4, 6, 9 and 10 months of gestational age (GA)), as well as tissue steroidogenic enzyme transcript levels. Pregnenolone and dehydroepiandrosterone (DHEA) were the most abundant steroids in fetal blood, pregnenolone was consistently higher but decreased progressively with GA. Tissue steroid concentrations generally paralleled those in serum with time. Adrenal and gonadal tissue pregnenolone concentrations were similar and 100-fold higher than those in allantochorion. DHEA was far higher in gonads than adrenals and progesterone was higher in adrenals than gonads. Androstenedione decreased with GA in adrenals but not in gonads. Transcript analysis generally supported these data. CYP17A1 was higher in fetal gonads than adrenals or allantochorion, and HSD3B1 was higher in fetal adrenals and allantochorion than gonads. CYP11A1 transcript was also significantly higher in adrenals and gonads than allantochorion and CYP19 and SRD5A1 transcripts were higher in allantochorion than either fetal adrenals or gonads. Given these data, and their much greater size, the fetal gonads are the source of DHEA and likely contribute more than fetal adrenal glands to circulating fetal pregnenolone concentrations. Low CYP11A1 but high HSD3B1 and SRD5A1 transcript abundance in allantochorion, and low tissue pregnenolone, suggests that endogenous placental pregnenolone synthesis is low and likely contributes little to equine placental 5α-reduced pregnane secretion.

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.

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.

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.

Transcriptomic analysis of the chorioallantois in equine premature placental separation.

BACKGROUND: Equine premature placental separation (PPS) is poorly understood and represents an important risk factor for fetal/neonatal hypoxia. OBJECTIVES: To examine transcriptomic changes in the chorioallantois (CA) from mares with clinical PPS compared with the CA from normal foaling mares. Differential gene expression was determined and gene ontology as well as molecular pathways related to PPS were characterised. STUDY DESIGN: Retrospective case: control study. METHODS: CA were collected from Thoroughbred mares with a clinical history of PPS (n = 33) and from control Thoroughbred mares (n = 4) with normal parturition for examination of transcriptional changes in the placenta associated with PPS. Transcriptomic changes in the villous CA near the cervical star were determined by Illumina® sequencing and subsequent bioinformatic analysis. PPS samples were divided by k-means clustering, and differentially expressed genes (DEGs) in each PPS cluster were identified by comparing to controls. Shared DEGs between PPS clusters were used for gene ontology analysis and pathway analysis. RESULTS: A total of 1204 DEGs were identified between PPS and control. Gene ontology revealed extracellular matrix (ECM) and cell adhesion, and pathway analysis revealed fatty acid, p-53, hypoxia and inflammation. Eleven key regulator genes of PPS including growth factors (IGF1, TGFB2, TGFB3), transcription factors (HIF1A, JUNB, SMAD3), and transmembrane receptors (FGFR1, TNFRSF1A, TYROBP) were also identified. MAIN LIMITATIONS: The use of clinical history of PPS, in the absence of other criteria, may have led to misidentification of some cases as PPS. CONCLUSIONS: Transcriptomic analysis indicated that changes in ECM and cell adhesion were important factors in equine PPS. Key predicted upstream events include genes associated with hypoxia, inflammation and growth factors related to the pathogenesis of equine PPS.