Steroid hormones in horses are biologically active compounds that are synthesized from cholesterol and play a significant role in various physiological processes. These hormones include glucocorticoids, mineralocorticoids, and sex steroids such as estrogens, androgens, and progestogens. They are involved in regulating metabolism, immune function, electrolyte balance, and reproductive functions. The levels of steroid hormones can be influenced by factors such as age, sex, stress, and disease states. Understanding their regulatory mechanisms and effects is essential for managing equine health and performance. This page compiles peer-reviewed research studies and scholarly articles that explore the synthesis, regulation, and physiological roles of steroid hormones in horses.
Satué K, Montesinos P, Muñoz A.In women and females of different species of laboratory animals, oestrogens stimulate the renin-angiotensin-aldosterone system (RAAS) by increasing tissue and circulating levels of angiotensinogen and renin during the preovulatory period. Progesterone and cortisol compete with aldosterone for mineralocorticoid receptors, which results in increased Na+ reabsorption during the postovulatory period. The purpose of the current research was to analyse the relationship of oestradiol-17β, progesterone and cortisol with RAAS in 23 mares during an oestrous cycle. During the preovulatory period, signif...
Ghosh S, Davis BW, Rosengren M, Jevit MJ, Castaneda C, Arnold C, Jaxheimer J, Love CC, Varner DD, Lindgren G, Wade CM, Raudsepp T.Disorders of sex development (DSD) and reproduction are not uncommon among horses, though knowledge about their molecular causes is sparse. Here we characterized a ~200 kb homozygous deletion in chromosome 29 at 29.7-29.9 Mb. The region contains genes which function as ketosteroid reductases in steroid hormone biosynthesis, including androgens and estrogens. Mutations in genes are associated with human DSDs. Deletion boundaries, sequence properties and gene content were studied by PCR and whole genome sequencing of select deletion homozygotes and control animals. Deletion analysis by PCR in ...
Waller CC, Weththasinghe SA, McClure L, Cawley AT, Suann C, Suann E, Sutherland E, Cooper E, Heather A, McLeod MD.Hemapolin (2α,3α-epithio-17α-methyl-5α-androstan-17β-ol) is a designer steroid that is an ingredient in several "dietary" and "nutritional" supplements available online. As an unusual chemical modification to the steroid A-ring could allow this compound to pass through antidoping screens undetected, the metabolism of hemapolin was investigated by an in vivo equine drug administration study coupled with GC-MS analysis. Following administration of synthetically prepared hemapolin to a thoroughbred horse, madol (17α-methyl-5α-androst-2-en-17β-ol), reduced and dihydroxylated madol (17α-me...
El-Sheikh Ali H, Legacki EL, Scoggin KE, Loux SC, Dini P, Esteller-Vico A, Conley AJ, Stanley SD, Ball BA.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 (3βHSD, CYP11A1, CYP17A1, CYP19...
Kirchmeier A, van Herwaarden AE, van der Kolk JH, Sauer FJ, Gerber V.This study describes steroid profiles in equine plasma before and after ACTH stimulation. In human medicine, other steroids have been shown to have a more pronounced reaction to an ACTH stimulation test than cortisol. This study aimed to determine if the same was true for the horse. A total of 11 clinically healthy horses were selected for this study. Ethylenediaminetetraacetic acid plasma samples were taken before and 60 min after stimulation with 1 μg/kg BW of synthetic ACTH administered intravenously. The samples were analyzed for cortisol, 11-deoxycortisol, 21-deoxycortisol, cortisone, ...
Viljanto M, Kicman AT, Walker CJ, Wolff K, Muir T, Hincks P, Biddle S, Scarth J.Boldenone (1-dehydrotestosterone) is an exogenous anabolic-androgenic steroid (AAS) but is also known to be endogenous in the entire male horse and potentially formed by microbes in voided urine, the gastrointestinal tract, or feed resulting in its detection in urine samples. In this study, equine fecal and urine samples were incubated in the presence of selected stable isotope labeled AAS precursors to investigate whether microbial activity could result in 1-dehydrogenation, in particular the formation of boldenone. Fecal matter was initially selected for investigation because of its high mic...
Fernandes CB, Ball BA, Loux SC, Boakari YL, Scoggin KE, El-Sheikh Ali H, Cogliati B, Esteller-Vico A.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 transc...
Aleman M, McCue PM, Chigerwe M, Madigan JE.Transient hypothalamic-pituitary-adrenal axis dysfunction occurs in critically ill foals with sepsis and neonatal maladjustment syndrome (NMS). Cortisol is the most commonly measured steroid. However, a complex interaction of various steroid compounds might play a role in pathophysiology of this disorder. Objective: To identify steroid compounds present at high concentrations at birth that rapidly and steadily decrease within the first 7 days of life in healthy foals and that might be supportive diagnosis of NMS and other neonatal disorders. Methods: Ten healthy neonatal Quarter Horse foals ...
Satué K, Fazio E, Ferlazzo A, Medica P.The hypothesis that a local serotonergic network might also exist in the follicle of mares remains poorly documented, with exception for humans and laboratory species. For this reason, the aim of the present study was to clarify this possibility, investigating intrafollicular serotonin concentrations of the cycling mare at ovulation time. Sixty ovaries collected from 30 clinically healthy mares of slaughterhouse meat production with clinically normal reproductive tracts after slaughtering were evaluated. Blood samples were taken prior to sacrifice. Follicles were classified in three categories...
Conley AJ, Ball BA.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 endometr...
Dembek KA, Johnson LM, Timko KJ, Minuto JS, Hart KA, Barr BS, Toribio RE.The hypothalamic-pituitary-adrenal axis regulates the response to sepsis-associated stress. Relative adrenal insufficiency or adrenocorticotropic hormone (ACTH):cortisol imbalance, defined as a poor cortisol response to administration of ACTH, is common and associated with death in hospitalized foals. However, information on other adrenal steroid response to ACTH stimulation in sick foals is minimal. Objective: To investigate the response of multiple adrenocortical steroids to administration of ACTH in foals. Methods: Hospitalized (n = 34) and healthy (n = 13) foals. Methods: In this prospecti...
Toribio RE.Neonatal encephalopathy (NE) and neonatal maladjustment syndrome (NMS) are terms used for newborn foals that develop noninfectious neurologic signs in the immediate postpartum period. Cerebral ischemia, hypoxia, and inflammation leading to neuronal and glial dysfunction and excitotoxicity are considered key mechanisms behind NE/NMS. Attention has been placed on endocrine and paracrine factors that alter brain cell function. Abnormal steroid concentrations (progestogens, neurosteroids) have been measured in critically ill and NE foals. In addition to supportive treatment, antimicrobials should ...
Legacki EL, Bal BA, Corbin CJ, Loux SC, Scoggin KE, Stanley SD, Conley AJ.The authors apologize for errors in Figure 6 of their article published in the October 2017 issue of Reproduction (vol 154 iss 4 pp 445–454). The authors explain that the addition of data (Figure 6) on steroid concentrations in the chorioallantois to their manuscript on fetal adrenal and fetal gonadal steroids during development of the equine fetus was made in response to reviewer comments. However, in compiling, summarizing and graphing the data, the wrong units were used in the final figure. The manuscript as published represents the data in Figure 6 as “ng/g”, when in fact they are ...
Legacki EL, Scholtz EL, Ball BA, Esteller-Vico A, Stanley SD, Conley AJ.Few studies have provided a longitudinal analysis of systemic concentrations of conjugated oestrogens (and androgens) throughout pregnancy in mares, and those only using immunoassay. The use of liquid chromatography tandem mass spectrometry (LC-MS/MS) will provide more accurate concentrations of circulating conjugated steroids. Objective: To characterise circulating concentrations of individual conjugated steroids throughout equine gestation by using LC-MS/MS. Methods: Longitudinal study and comparison of pregnant mares treated with vehicle or letrozole in late gestation. Methods: Sulphated oe...
Viljanto MJ, Kicman AT, Walker CJ, Parkin MC, Wolff K, Pearce CM, Scarth J.Boldenone is an anabolic-androgenic steroid that is prohibited in equine sports. Urine from the uncastrated male horse contains boldenone that is thought to be of endogenous origin and thus a threshold ('cut-off') concentration has been adopted internationally for free and conjugated boldenone to help distinguish cases of doping from its natural production. The testis is likely to be a source of boldenone. Qualitative analysis was performed on extracts of equine testicular homogenates (n = 3 horses) incubated non-spiked and in the presence of its potential precursors using liquid chromatog...
Genangeli M, Caprioli G, Cortese M, Laus F, Petrelli R, Ricciutelli M, Sagratini G, Sartori S, Vittori S.A new fast and easy analytical procedure for the simultaneous detection and quantification of 9 anabolic steroids (deslorelin, dexamethasone sodium phosphate, prednisolone, methylprednisolone, stanozolol, boldenone, nandrolone, dexamethasone isonicotinate and altrenogest) in horse urine for doping control have been developed by using the ultra-high-performance liquid chromatography coupled with tandem mass spectrometry technique (UHPLC-MS/MS). A total amount of 400 μl of sample was evaporated, restored and injected in the UHPLC-MS/MS. The proposed method was fully validated showing a recove...
Lindström H, Mazari AMA, Musdal Y, Mannervik B.Equine glutathione transferase A3-3 (EcaGST A3-3) belongs to the superfamily of detoxication enzymes found in all higher organisms. However, it is also the most efficient steroid double-bond isomerase known in mammals. Equus ferus caballus shares the steroidogenic pathway with Homo sapiens, which makes the horse a suitable animal model for investigations of human steroidogenesis. Inhibition of the enzyme has potential for treatment of steroid-hormone-dependent disorders. Screening of a library of FDA-approved drugs identified 16 out of 1040 compounds, which at 10 μM concentration afforded at ...
Valdez R, Cavinder CA, Varner DD, Welsh TH, Vogelsang MM, Ing NH.Glucocorticoids impair testosterone synthesis by an unknown mechanism. Stallions treated with the synthetic glucocorticoid dexamethasone had testes collected at 6 or 12 hours postinjection. The testicular expression of selected genes encoding nuclear receptors and steroidogenic enzymes was measured. At 6 hours, dexamethasone treatment decreased levels of NR0B2, NR4A1, NR5A1, and NR5A2 messenger RNAs (mRNAs) and NR5A2 mRNA levels remained depressed at 12 hours. In contrast, dexamethasone increased levels of NFKBIA mRNA at both time points. At 6 hours, dexamethasone did not alter levels ...
Held F, Ekstrand C, Cvijovic M, Gabrielsson J, Jirstrand M.Cortisol is a steroid hormone relevant to immune function in horses and other species and shows a circadian rhythm. The glucocorticoid dexamethasone suppresses cortisol in horses. Pituitary pars intermedia dysfunction (PPID) is a disease in which the cortisol suppression mechanism through dexamethasone is challenged. Overnight dexamethasone suppression test (DST) protocols are used to test the functioning of this mechanism and to establish a diagnosis for PPID. However, existing DST protocols have been recognized to perform poorly in previous experimental studies, often indicating presence of ...
Morgan RA, Keen JA, Homer N, Nixon M, McKinnon-Garvin AM, Moses-Williams JA, Davis SR, Hadoke PWF, Walker BR.Equine Cushing disease [pituitary pars intermedia dysfunction (PPID)] is a common condition of older horses, but its pathophysiology is complex and poorly understood. In contrast to pituitary-dependent hyperadrenocorticism in other species, PPID is characterized by elevated plasma ACTH but not elevated plasma cortisol. In this study, we address this paradox and the hypothesis that PPID is a syndrome of ACTH excess in which there is dysregulation of peripheral glucocorticoid metabolism and binding. In 14 horses with PPID compared with 15 healthy controls, we show that in plasma, cortisol levels...
Satué K, Marcilla M, Medica P, Ferlazzo A, Fazio E.Androgens modulate maternal ovarian activity, embryo implantation and correct placental development. The objective of this study was to establish reference values for testosterone (T), androstenedione (A4) and dehydroepiandrosterone (DHEA) concentrations in pregnant mares. A total of 30 healthy Spanish Purebred mares with an age range of 9.33 ± 3.31 years, were studied during the 11 months of gestation. T, A4 and DHEA concentrations were determined using EIA validated specifically for equines. T increased in the 2nd and 3rd month (P < 0.05), showing a plateau between the 4th and 6th m...
Wynn MAA, Ball BA, May J, Esteller-Vico A, Canisso I, Squires E, Troedsson M.The objectives of this study were to compare via liquid chromatography-tandem mass spectrometry (LC-MS/MS) progesterone (P4), 5α-dihydroprogesterone (DHP), allopregnanolone, 3β-hydroxy-5α-pregnan-20-one (3β5P), 20α-hydroxy-5α-pregnan-3-one (20α5P), 5α-pregnan-3β,20α-diol (βα-diol), and 5α-pregnan-3β,20β-diol (ββ-diol) concentrations in plasma of mares with experimentally-induced, ascending placentitis compared to gestationally age-matched control mares. Placentitis was induced via intracervical inoculation of Streptococcus equi spp. zooepidemicus between 260 and 280 days of ge...
Młodawska W, Grzesiak M, Kochan J, Nowak A.Steroidogenic activity in the equine ovary from birth to puberty has been poorly investigated. This study aimed to examine the capability of the ovarian follicles of prepubertal and pubertal fillies to produce steroid hormones and to evaluate the expression and cellular localization of androgen receptor (AR) in their ovaries. The ovaries of 6-18 month-old fillies were divided into two groups: prepubertal (PrP) - without preovulatory follicle (pF) and corpus luteum (CL), and ovulating/postpubertal (Ov/pB) - with pF and/or CL in at least one of the gonads. Adult mares (Me) were used as a control...
Müller V, Curcio BR, Toribio RE, Feijó LS, Borba LA, Canisso IF, Nogueira CEW.This study aimed to evaluate steroid hormones in foals born from mares treated for ascending placentitis with different combinations of trimethoprim-sulfamethoxazole (TMS), flunixin meglumine (FM), long-acting altrenogest (ALT) and estradiol cypionate (ECP) for ten consecutive days, starting two days after experimental induction of placentitis with Streptococcus zooepidemicus. Fourty-six pregnant mares and respective foals were assigned as healthy group (Control, n = 8) or treated groups as follows: TMS+FM (n = 8), TMS+FM+ALT (n = 8), TMS+FM+ALT+ECP (n = 6), TMS+FM+ECP (n = 6) and no...
Viljanto M, Hincks P, Hillyer L, Cawley A, Suann C, Noble G, Walker CJ, Parkin MC, Kicman AT, Scarth J.The use of testosterone and its pro-drugs, such as dehydroepiandrosterone (DHEA), is currently regulated in horseracing by the application of international testosterone thresholds. However, additional steroidomic approaches, such as steroid ratios, to distinguish overall adrenal stimulation from drug administrations and an equine biological passport for longitudinal steroid profiling of individual animals could be advantageous in equine doping testing. Thus, DHEA concentrations and related ratios (testosterone [T] to DHEA and DHEA to epitestosterone [E]) were assessed in the reference populati...
Choi TLS, Kwok KY, Kwok WH, Tsoi YYK, Wong JKY, Wan TSM.Anabolic and androgenic steroids (AAS) are banned substances in both human and equine sports. They are often administered intramuscularly to horses in esterified forms for the purpose of extending their time of action. The authors' laboratory has previously reported an UHPLC/HRMS method using quadrupole-Orbitrap mass spectrometer in full scan and parallel reaction monitoring (PRM) mode for the detection of 48 AAS and/or their esters in horse hair. However, two injections were required due to the long duty cycle time. In this paper, an UHPLC/HRMS method using multiplexed targeted MS mode was de...
Conley AJ, Scholtz EL, Legacki EL, Corbin CJ, Knych HK, Dujovne GD, Ball BA, Moeller BC, Stanley SD. and evidence indicates that the bioactive, 5α-reduced progesterone metabolite, 5α-dihydroprogesterone (DHP) is synthesized in the placenta, supporting equine pregnancy, but its appearance in early pregnancy argues for other sites of synthesis also. It remains unknown if DHP circulates at relevant concentrations in cyclic mares and, if so, does synthesis involve the non-pregnant uterus? Jugular blood was drawn daily from cyclic mares ( = 5). Additionally, ovariectomized mares (OVX) and geldings were administered progesterone (300 mg) intramuscularly. Blood was drawn before and after t...
Rink BE, Kuhl J, Esteves CL, French HM, Watson E, Aurich C, Donadeu FX.Mesenchymal stem or stromal cells (MSCs) play key roles in tissue homeostasis. In the cyclic equine endometrium, this may be regulated by changes in serum concentrations of sex steroid hormones. This study was designed to investigate the changes in endometrial expression of MSC markers during reproductive cycles in mares and the influence of sex steroid hormones on endometrial MSC proliferation in vitro. Endometrial biopsies were collected from pony mares at different reproductive stages (estrus; day 5 and 13 after ovulation; seasonal anestrus; 20 h and 7days post-partum; n = 5 per stag...
Satué K, Marcilla M, Medica P, Ferlazzo A, Fazio E.The objectives of this study were to establish reference values for serum concentrations of placental growth factor (PlGF) and haptoglobin (Hp), and to analyze whether the levels of oestrone sulphate (E1S) and progesterone (P4) are physiologically involved in the dynamic modifications of the above parameters in pregnant mares. A total of 30 healthy Spanish Purebred mares ranging in age 9.33 ± 3.31 years were studied during the 11 months of gestation. Serum concentrations of PlGF were detected by EIA, Hp using commercial Phase Haptoglobin assay and E1S and P4 levels through RIA. The serum ...
Galvão A, Wolodko K, Rebordão MR, Skarzynski D, Ferreira-Dias G.In the present report we describe the involvement of transforming growth factor B1 (TGF) in functional regression and structural luteolysis in the mare. Firstly, TGF and its receptors activin-like kinase (ALK) 5 and TGF receptor 2 were identified in corpus luteum (CL) steroidogenic, endothelial and fibroblast-like cells. Also, TGF and ALK5 protein expression were shown to be increased in Mid-, and Late-CL (p < 0.05). Subsequently, using an in vitro model with Mid-CL cells, we studied the role of TGF on secretory activity and cell viability. Cell treatment with TGF decreased progesterone...
SHORT RV.A comparison has been made between the steroids present in equine follicular fluid and luteal tissue. Follicular fluid was found to contain progesterone, 17α-hydroxyprogesterone, androstenedione, epitestosterone, 19-norandrostenedione, oestrone, oestradiol-17β and 6α-hydroxyoestradiol-17β, in confirmation of previous studies. On the other hand, luteal tissue contained large amounts of progesterone and 20α-hydroxypregn-4-en-3-one, and a small quantity of 17α-hydroxyprogesterone. No C18 or C19 steroids could be detected.
These findings are therefore interpreted as evidence in favour of ...
Hart KA, Dirikolu L, Ferguson DC, Norton NA, Barton MH.To compare daily endogenous cortisol production rate and the pharmacokinetics of an i.v. bolus of hydrocortisone between neonatal foals and adult horses. Methods: 10 healthy full-term 2- to 4-day-old foals and 7 healthy adult horses. Methods: Blood samples were collected from each horse every 15 to 20 minutes for 24 hours for determination of 24-hour mean cortisol concentration. Afterward, dexamethasone (0.08 mg/kg) was administered i.v. to suppress endogenous cortisol production. Twelve hours afterward, hydrocortisone sodium succinate (1.0 mg/kg) was administered as a rapid i.v. bolus and ser...
Hoffsis GF, Murdick PW, Tharp VL, Ault K.The plasma concentration of cortisol and corticosterone in the normal horse was determined by competitive protein-binding analysis (radiostereoassay). The mean plasma concentration of the 2 steroids in resting horses was 5.12 μg./ml., with standard deviation of 1.67 μg. There were no significant differences in the mean values according to age, sex, and pregnancy. Seemingly, diurnal variations in concentrations of the steroids existed in horses; values were high in the morning and low in the evening. Plasma values were increased in horses given corticotropin (acth) and were decreased in those...
Almeida J, Conley AJ, Mathewson L, Ball BA.In the mammalian testis, Leydig cells are primarily responsible for steroidogenesis. In adult stallions, the major endocrine products of Leydig cells include testosterone and estrogens. 3β-hydroxysteroid dehydrogenase/Δ(5)-Δ(4)-isomerase (3βHSD) and 17α-hydroxylase/17,20-lyase (P450c17) are two key steroidogenic enzymes that regulate testosterone synthesis. Androgens produced by P450c17 serve as substrate for estrogen synthesis. The aim of this study was to investigate localization of the steroidogenic enzymes P450c17, 3βHSD, and P450arom and to determine changes in expression during dev...
Galvão A, Valente L, Skarzynski DJ, Szóstek A, Piotrowska-Tomala K, Rebordão MR, Mateus L, Ferreira-Dias G.Regulation of immune-endocrine interactions in the equine endometrium is not fully understood. The aims of the present study were to: (1) investigate the presence of tumour necrosis factor alpha (TNF), interferon gamma (IFNG), Fas ligand (FASLG) and their receptors in the mare endometrium throughout the oestrous cycle; and (2) assess endometrial secretory function (prostaglandins), angiogenic activity and cell viability in response to TNF, oestradiol (E2), progesterone (P4) and oxytocin (OXT). Transcription of TNF and FASLG mRNA increased during the early and late luteal phase (LP), whereas IF...
Chan GH, Ho EN, Leung DK, Wong KS, Wan TS.The use of anabolic androgenic steroids (AAS) is prohibited in both human and equine sports. The conventional approach in doping control testing for AAS (as well as other prohibited substances) is accomplished by the direct detection of target AAS or their characteristic metabolites in biological samples using hyphenated techniques such as gas chromatography or liquid chromatography coupled with mass spectrometry. Such an approach, however, falls short when dealing with unknown designer steroids where reference materials and their pharmacokinetics are not available. In addition, AASs with fast...
Tait AD, Santikarn S, Allen WR.The 5,7-dienes, 3 beta-hydroxy-5,7-pregnadien-20-one and 3 beta-hydroxy-5,7-androstadien-17-one were extracted from fetal horse gonads and purified by solvent partition, thin-layer chromatography and high performance liquid chromatography. The isolated steroids were identified by comparison with the synthetic steroids using ultraviolet and mass spectroscopy and by gas chromatography-mass spectroscopy. The identification of these compounds as endogenous steroids, together with the data on their biosynthesis reported previously, support the proposal that in the fetal horse gonad there is a 5,7-d...
Rambags BP, van Tol HT, van den Eng MM, Colenbrander B, Stout TA.Progesterone and oestrogen play essential roles in the maintenance of pregnancy in eutherian mammals and are thought to exert their effects on the developing conceptus indirectly, via the endometrium. In some species, early embryos have themselves been shown to express steroid receptors, thereby suggesting that reproductive steroids may also influence embryonic development directly. The aim of this study was to determine whether early intrauterine equine conceptuses express either the classical intracellular progesterone (PR) and oestrogen receptors (ERalpha and ERbeta) or the more recently ch...
Hedberg Y, Dalin AM, Forsberg M, Lundeheim N, Sandh G, Hoffmann B, Ludwig C, Kindahl H.The mare is the only non-primate species known to display estrous signs after ovariectomy and adrenal hormones have been implicated as a possible cause. Moreover, in several species, estradiol seems to have a stimulatory effect on the hypothalamic-pituitary-adrenal axis. The aim of the present study was to compare the effect of ACTH (tetracosactide) on pertinent hormones [cortisol, progesterone, androstenedione, testosterone (intact and ovariectomized mares) and estradiol (ovariectomized mares only)] in intact mares in estrus with the same mares after ovariectomy (n=5). Blood samples were coll...
Weidolf LO, Lee ED, Henion JD.Sulfoconjugated anabolic steroids were separated by micro-bore high-performance liquid chromatography. The eluent was introduced into the atmospheric pressure ion source of the triple-quadrupole mass spectrometer via an ion spray liquid chromatograph/mass spectrometer interface operated in the negative ion mode. The limit of detection was 10 pg on-column by selected ion monitoring of the molecular ion and the response increased linearly over a concentration range of 2.4 orders of magnitude. Following work-up by a liquid-solid extraction procedure of equine urine samples, full-scan daughter ion...
Legacki EL, Corbin CJ, Ball BA, Wynn M, Loux S, Stanley SD, Conley AJ.Mammalian pregnancies need progestogenic support and birth requires progestin withdrawal. The absence of progesterone in pregnant mares, and the progestogenic bioactivity of 5α-dihydroprogesterone (DHP), led us to reexamine progestin withdrawal at foaling. Systemic pregnane concentrations (DHP, allopregnanolone, pregnenolone, 5α-pregnane-3β, 20α-diol (3β,20αDHP), 20α-hydroxy-5α-dihydroprogesterone (20αDHP)) and progesterone) were monitored in mares for 10days before foaling (n=7) by liquid chromatography-mass spectrometry. The biopotency of dominant metabolites was assessed using luci...
Betteridge KJ.Carl Hartman's title of 47 years ago is invoked in tribute to his first recovery of a bovine embryo 30 years before that, and his legacy of an emphasis on the value of descriptive and comparative studies in reproductive biology. The horse's qualification as a farm animal has waned since those times but, in a conference understandably dominated by research in ruminants and pigs, there are lessons to be learned from some peculiarities of equine embryonic development. Morphological and physiological features of the conceptus and its interaction with its environment during the first month of pregn...
Hasan M, Siegmund W, Oswald S.Cytochrome P450 3A (CYP) enzymes are involved in the elimination of many drugs and are known to be regulated by several environmental factors. Thus, it was the aim of this study to develop and validate an analytical method allowing estimation of the hepatic CYP3A enzyme activity using the 4-hydroxycholesterol to cholesterol ratio as an endogenous biomarker in serum. Both compounds were isolated from the biological matrix by liquid-liquid extraction using n-hexane after saponification with ethanolic sodium methoxide solution (2M) to cleave the steroids from their esterified forms without any ki...
Lindström H, Peer SM, Ing NH, Mannervik B.Glutathione transferases (GSTs) comprise a superfamily of enzymes prominently involved in detoxication by making toxic electrophiles more polar and therefore more easily excretable. However some GSTs have developed alternative functions. Thus, a member of the Alpha class GSTs in pig and human tissues is involved in steroid hormone biosynthesis, catalyzing the obligatory double-bond isomerization of Δ-androstene-3,17-dione to Δ-androstene-3,17-dione and of Δ-pregnene-3,20-dione to Δ-pregnene-3,20-dione on the biosynthetic pathways to testosterone and progesterone. The human GST A3-3 is the ...
Arkoun B, Gautier C, Delalande C, Barrier-Battut I, Guénon I, Goux D, Bouraïma-Lelong H.Among mammals, the stallion produces the largest amount of testicular estrogens. These steroid hormones are produced mainly by Leydig and Sertoli cells in the testis and also in the epididymis. Their role in horse testicular physiology and their ability to act on spermatozoa are still unknown. In order to determine if spermatozoa are targets for estrogens, the presence of estrogen receptors in mature ejaculated spermatozoa has been investigated. The presence of a single isoform of ESR1 (66kDa) and ESR2 (61kDa) was found by Western-blot analysis in samples from seven stallions. Confocal analysi...
Watson ED, Al-Zi'abi MO.The mare is a seasonal breeder and undergoes a period of ovarian transition in spring between winter anoestrus and cyclicity. During spring transition LH concentrations are low and many mares have successive large anovulatory follicular waves which reach the size of preovulatory follicles. Follicular angiogenesis is essential for growth and health of preovulatory follicles. The aim of the present study was to investigate the morphology and vascularity of transitional anovulatory follicles. On gross inspection, the wall of transitional follicles was visibly less well vascularized than that of p...
Ousey JC.The endocrine profiles in the periparturient mares are dominated by increasing concentrations of progestagens and decreasing oestrogens. These hormones are produced by precursors from the foetus, metabolized by the placenta and act primarily on the maternal uterus. The circulating concentrations of hormones in maternal plasma, generally, represent a small proportion of those metabolized by the foetus and utero-placental tissues. There is clear evidence that the foetal hypothalamo-pituitary-adrenal (HPA) axis initiates the process of foetal maturation and the hormonal cascade which culminates i...
Gerstenberg C, Allen WR, Stewart F.Previous studies showed a dramatic increase in EGF gene expression in the endometrial glands of pregnant mares around day 40 after ovulation. To investigate how the steroid hormones of pregnancy might regulate this expression, in situ hybridization was used to monitor the levels of EGF mRNA in endometrial biopsies obtained from seasonally anoestrous or ovariectomised mares given exogenous progesterone and oestrogen, alone or in combination, for up to 46 days. Biopsies were also taken from mares during the non-pregnant cycle, during normal pregnancies and pregnancies compromised by endometrial ...
Galvão A, Wolodko K, Rebordão MR, Skarzynski D, Ferreira-Dias G.In the present report we describe the involvement of transforming growth factor B1 (TGF) in functional regression and structural luteolysis in the mare. Firstly, TGF and its receptors activin-like kinase (ALK) 5 and TGF receptor 2 were identified in corpus luteum (CL) steroidogenic, endothelial and fibroblast-like cells. Also, TGF and ALK5 protein expression were shown to be increased in Mid-, and Late-CL (p < 0.05). Subsequently, using an in vitro model with Mid-CL cells, we studied the role of TGF on secretory activity and cell viability. Cell treatment with TGF decreased progesterone...
Legacki EL, Corbin CJ, Ball BA, Scoggin KE, Stanley SD, Conley AJ.Steroidogenic enzymes in placentas shape steroid hormone profiles in the maternal circulation of each mammalian species. These include 3β-hydroxysteroid dehydrogenase/Δ5-4 isomerase (3βHSD) 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 a...
Fitzgerald CCJ, Hedman R, Uduwela DR, Paszerbovics B, Carroll AJ, Neeman T, Cawley A, Brooker L, McLeod MD.The study of urinary phase II sulfate metabolites is central to understanding the role and fate of endogenous and exogenous compounds in biological systems. This study describes a new workflow for the untargeted metabolic profiling of sulfated metabolites in a urine matrix. Analysis was performed using ultra-high-performance liquid chromatography-high resolution tandem mass spectrometry (UHPLC-HRMS/MS) with data dependent acquisition (DDA) coupled to an automated script-based data processing pipeline and differential metabolite level analysis. Sulfates were identified through -means clustering...
Kerban A, Boerboom D, Sirois J.The time- and gonadotropin-dependent regulation of steroidogenic acute regulatory protein (StAR) has not been characterized in vivo in preovulatory follicles of large monoovulatory species or sexually mature animals. The objectives of this study were to clone equine StAR and describe the regulation of its messenger RNA (mRNA) in equine follicles after the administration of an ovulatory dose of hCG. The screening of an equine follicle complementary DNA (cDNA) library with a mouse StAR cDNA probe revealed two forms of equine StAR that differ only in the length of their 3'-untranslated region (3'...
Pereira GR, Lorenzo PL, Carneiro GF, Ball BA, Bilodeau-Goeseels S, Kastelic J, Pegoraro LM, Pimentel CA, Esteller-Vico A, Illera JC, Granado GS....The objectives of this study were to evaluate the effects of equine growth hormone (eGH) on nuclear and cytoplasmic maturation of equine oocytes in vitro, steroid production by cumulus cells, and expression and subcellular localization of eGH-receptors (eGH-R) on equine ovarian follicles. Cumulus-oocyte complexes (COCs) were recovered by aspirating follicles <30 mm in diameter from abattoir-derived ovaries. The COCs were morphologically evaluated and randomly allocated to be cultured in either a control maturation medium or supplemented with 400 ng/mL eGH, for 30 h at 38.5°C in air with 5%...
McDowell KJ, Adams MH, Adam CY, Simpson KS.Two experiments were performed to determine changes in the abundance of oestrogen and progesterone receptor (ER alpha and PR) mRNAs in equine endometrium during the oestrous cycle and early pregnancy, and under the influence of exogenous steroids. In Expt 1, endometrial biopsies were obtained from non-mated mares during oestrus and at days 5, 10 and 15 after ovulation, and from pregnant mares at days 10, 15 and 20 after ovulation. There were overall effects of day on the abundance of ER alpha (P = 0.0001) and PR (P = 0.0014) mRNAs. The amount of ER alpha mRNA decreased at day 10 of pregnancy, ...
Houghton E, Holtan D, Grainger L, Voller BE, Rossdale PD, Ousey JC.Radioimmunoassay (RIA) and gas chromatography-mass spectrometry (GC-MS) were used to determine plasma progestagen concentrations in the normal and premature foal. Radioimmunoassay provides a profile of plasma progestagens with respect to time but, due to the non-specific nature of the technique and without prior chromatographic purification, quantitative data based on RIA analysis must be interpreted with caution. In contrast, the greater specificity of GC-MS allows identification of specific plasma progestagens and measuring of multiple analytes in a single analysis. Both techniques demonstra...
Raeside JI, Christie HL, Renaud RL, Waelchli RO, Betteridge KJ.Because estradiol (E(2)) production by the early equine conceptus is considered crucial to the establishment of pregnancy, the amounts of E(2), estrone (E(1)), and their sulfates (E(2)S, E(1)S) were measured by RIA in yolk-sac fluid of 63 conceptuses collected by transcervical lavage over the period of 11-26 days after ovulation. Amounts increased significantly with age of conceptus, especially for E(1)S. Then, the metabolism of E(2), which may be highly relevant for its action, was examined in the conceptus and endometrium over the period when the conceptus ceases to migrate within the uterus...
Galvao AM, Ramilo DW, Skarzynski DJ, Lukasik K, Tramontano A, Mollo A, Mateus LM, Ferreira-Dias GM.Proapoptotic factor Fas ligand (FASL) and its cell surface receptor FAS are tumor necrosis factor superfamily members that trigger apoptosis in different cell types. However, their influence on luteal steroidogenesis is not clearly understood. The aim of the present work was to determine (i) the presence of the cytokine FASL and its receptor FAS in the mare's corpus luteum (CL) throughout the luteal phase, as well as (ii) the influence of FASL alone, or together with the cytokines tumor necrosis factor alpha (TNF) and interferon gamma (IFNG), on equine luteal cell production of luteotrophic an...
Legacki EL, Scholtz EL, Ball BA, Stanley SD, Berger T, Conley AJ.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 ...
