Perioestrous patterns of circulating LH,FSH, prolactin and oestradiol-17β in the gilt

Concentrations of luteinizing hormone (LH), follicle stimulating hormone (FSH) and prolactin (PRL) were measured in jugular blood and those of oestradiol-17β (E₂17β) in utero-ovarian blood. Samples were taken from five intact gilts every 15 min for 108 h starting between day 15 and day 18 of the oestrous cycle. In the late luteal/early follicular phase, high pulsatile LH secretion, close to one pulse per hour, was observed. This could be the stimulus necessary for the final maturation of the ovarian follicles.Thereafter, frequency and amplitude of pulses, and the baseline value, decreased and were low at least between 36 and 12 h before the preovulatory LH surge. PRL and FSH concentrations also declined. This was probably due to the increase of oestrogen secretion. As E₂17β concentrations were still high, the surge of LH which was accompanied by increase in FSH and PRL, occurred for approximately 13 to 20 h. While LH and PRL mean levels decreased, FSH concentrations continued to increase. Peaks of PRL were observed during the late luteal/early follicular phase and during the LH discharge. During the period of estrus, each exposure to the boar was immediately followed by one of these peaks, which could play a role in the sexual behavior of the gilt.     

Effects of oestradiol-17β and progesterone on total and nuclear-protein synthesis in epithelial and stromal tissues of the mouse uterus, and of progesterone on the ability of these tissues to bind oestradiol-17β

1. A method is described for separating uterine epithelium that is 80% pure and connective-tissue stroma that is 60% pure. This was used to study the effects of steroid hormones on total and nuclear-protein synthesis in these tissues. 2. Oestradiol-17beta given alone produces mitoses in the epithelium but not in the stroma. It stimulated incorporation in vitro of [(14)C]lysine into total protein, histones and acidic nuclear proteins to a greater extent in epithelium than stroma. Incorporation into acidic nuclear proteins was most markedly stimulated, reaching four to six times the normal value 4h after treatment, and then declining rapidly. This peak was only seen in epithelial preparations. 3. After pretreatment with progesterone, oestradiol-17beta has the reverse effect, producing mitoses only in stroma. Progesterone alone had no effect on the amounts or rates of incorporation of [(14)C]lysine into stromal nuclear proteins, but changes after oestradiol-17beta treatment were similar to those seen in epithelium with oestradiol-17beta alone. In the epithelium, progesterone alone depressed incorporation into histones and acidic nuclear proteins, but did not abolish the subsequent response to oestradiol-17beta. With this treatment there was a rapid, large and transient increase in incorporation into epithelial total protein not seen with oestradiol-17beta alone. 4. Progesterone had no qualitative effect on the distribution of specific oestrogen-binding proteins, as judged by sucrose-density-gradient centrifugation. However, progesterone treatment increased the uptake in vivo of [6,7-(3)H]oestradiol-17beta by stroma, and it is possible that this is important although the differences were not apparent after labelling in vitro.     

Spermine synthesis in the uterus of the ovariectomized rat in response to oestradiol-17β

We reported that spermidine and spermine pools in the uterus both doubled within 24h after oestradiol administration to castrated rats (Russell & Taylor, 1971). Now we have studied the enzymic synthesis of spermine (by spermidine-dependent S-adenosyl-l-methionine decarboxylase) and find that the activity of the enzyme(s) involved is elevated soon after hormone administration. Enzyme activity is increased within 4h and is five times that of controls within 24h. Cycloheximide or actinomycin D administered at the time of oestradiol injection completely blocked the increase in enzyme activity. The enzyme involved in spermine synthesis, S-adenosyl-l-methionine decarboxylase, with S-adenosyl-l-methionine and spermidine as required substrates, was partially purified on Sephadex and DEAE-cellulose columns. The decarboxylation of S-adenosyl-l-methionine could not be separated from the transfer of a propylamine moiety from the decarboxylated S-adenosyl-l-methionine to spermidine to form spermine. We were unable also to separate this system from the enzyme that formed spermidine when S-adenosyl-l-methionine and putrescine are used as substrates. Spermidine-stimulated S-adenosyl-l-methionine decarboxylase has an apparent half-life of 60min, identical with the half-life reported for putrescine-stimulated S-adenosyl-l-methionine decarboxylase. These results strongly suggest that the same enzyme(s) operate in the synthesis of both spermidine and spermine.     

Synchronous onset of oestradiol-17β secretion by Meishan conceptuses

The response of Meishan conceptuses to an exogenous precursor for oestradiol-17β biosynthesis was investigated in vitro, to determine whether gestational age or morphological stage of development elicit changes in hormone metabolism. Conceptuses were recovered on days 11, 12, 13 or 15 after the onset of oestrus and cultured for 6 hours at 37°C, in the presence or absence of testosterone. On days 12 and 13 after the onset of oestrus spherical conceptuses were recovered from some gilts, whereas others yielded elongated or filamentous conceptuses. All conceptuses recovered on day 15 after oestrus had elongated. The number of cells per individual conceptus increased from days 11 to 13 after the onset of oestrus (P < 0.001), as did conceptus surface area (P = 0.038). Supplementing culture media with testosterone, as a substrate for oestrogen biosynthesis, significantly increased conceptus oestradiol-17β secretion in vitro on days 12, 13 and 15, regardless of whether pre- or post-elongation conceptuses were cultured. However, on day 11 oestradiol-17β was only detected at significant concentrations in the culture media of four testosterone supplemented conceptuses and only one gilt produced conceptuses capable of secreting oestradiol-17β in the absence of testosterone. Therefore, the onset of conceptus oestradiol-17β secretion is apparently limited by the expression of aromatase enzymes that are activated synchronously, irrespective of the stage of morphological development, within Meishan litters. Once established, Meishan conceptus oestradiol-17β secretion in vitro is increased in the presence of exogenous testosterone.     

Androgen dynamics in vitro in the human prostate gland. Effect of oestradiol-17β

Normal, hyperplastic and adenocarcinomatous human prostatic tissue was perfused in vitro with radioactively labelled androstenedione, testosterone and 5alpha-dihydrotestosterone with and without added oestradiol-17beta. Various parameters of tissue-steroid relationship were measured at the steady state. When oestradiol (0.11 or 0.22mumol/l) was added to the perfusing medium, the entry of the steroids into the tissue and their metabolism was increased in the majority of the glands studied. The ;uptake' of all the steroids varied, in response to the addition of oestradiol, in both normal and adenocarcinomatous glands in a way differing from the response of hyperplastic glands. As a consequence, the tissue clearance of the steroids, particularly of androstenedione and testosterone, increased in normal and adenocarcinomatous glands in the presence of oestradiol, and decreased in the hyperplastic tissues. At a concentration 0.33mumol/l, oestradiol decreased the entry of the steroids in all the tissues studied, while the clearance of steroids tended to decrease. The significance of these findings in terms of the regulation of androgen dynamics in vivo in the normal and diseased human prostate, with particular regard to the response to oestrogen treatment, is discussed.     

Nuclear binding of oestradiol-17β and induction of protein synthesis in the rat uterus during postnatal development

1. The uterine response to a single injection of oestradiol-17beta during postnatal development of the rat was studied with respect to (i) nuclear binding of oestradiol-17beta; (ii) induction of the synthesis of a specific cytoplasmic protein (;induced protein' of Gorski); (iii) rate of incorporation of (3)H-labelled amino acids into total protein and into nuclear acid-soluble and acid-insoluble protein; and (iv) rate of [(3)H]thymidine incorporation into DNA. 2. Specific nuclear binding of oestradiol-17beta could be demonstrated even at birth. Administration of oestradiol-17beta in vivo caused a significant increase in the number of nuclear binding sites in rats aged 10 days or older. 3. A rapid method is described for the detection of the ;induced protein', based on cellulose acetate electrophoresis. Induction of this protein could be demonstrated at the age of 10, 15 and 20 days, but not in 5-day-old rats. 4. In 20-day-old rats the rate of (3)H-labelled amino acid incorporation into protein increased by 3h after oestradiol administration. Incorporation into the different protein fractions reached peak values asynchronously: at 3-4h for acid-insoluble nuclear protein, at 6h for total protein and at about 12h for acid-soluble protein. 5. Treatment with oestradiol failed to stimulate amino acid incorporation into protein in 5- or 10-day-old rats; at the age of 15 to 30 days the hormone caused a significant increase in incorporation into total protein and into both types of nuclear protein. 6. Since the capacity for nuclear binding of oestradiol and for synthesis of the induced protein is demonstrable in the rat uterus before it acquires the ability to respond to the hormone with enhanced general protein synthesis and DNA synthesis, it appears that nuclear binding and the synthesis of the induced protein may be necessary but not sufficient conditions for the trophic action of oestradiol.     

High-affinity binding of oestradiol-17β by cytosols from testis interstitial tissue, pituitary, adrenal, liver and accessory sex glands of the male rat

The specificity of the binding of oestradiol-17beta by cytoplasmic fractions of several tissues of the male rat was investigated. 1. Agar-gel electrophoresis, Sephadex chromatography, adsorption by dextran-coated charcoal and sucrose-gradient centrifugation were used to estimate the binding capacity and specificity. The four different methods all gave similar results for the capacity of the specific oestradiol-17beta-binding macromolecules in the testis. 2. The presence of a specific saturable binding protein with a sedimentation coefficient of 8S was demonstrated in liver, adrenal, pituitary, prostate, epididymis and testis interstitial tissue. The highest concentration of oestradiol-17beta-binding macromolecules was found in testis interstitial tissue (0.12pmol/mg of protein) and in the pituitary (0.075pmol/mg of protein). 3. The oestradiol-17beta receptor in the testis cytosol showed the characteristics of a protein with respect to Pronase treatment and temperature sensitivity. In competition experiments with different steroids the receptor showed a high affinity for oestradiol-17beta, a moderate affinity for diethylstilboestrol and oestradiol-17alpha and a low affinity for oestrone, oestriol, testosterone and 5alpha-dihydrotestosterone (17beta-hydroxy-5alpha-androstan-3-one). 4. The wide distribution of oestradiol-17beta receptors in the male rat is in apparent contradiction to the current concept of the specificity of steroid-hormone action. Further research is required to investigate a possible physiological meaning of the presence of specific receptors in the different tissues.     

C-rasH and ornithine decarboxylase are induced by oestradiol-17β in the mouse uterine luminal epithelium independently of the proliferative status of the cell

Oestradiol-17 β (E₂) treatment of the ovariectomized mouse results in a synchronised wave of cell proliferation in the uterine luminal epithelium. At the peak of DNA synthesis the mRNA level of the c-ras^H protooncogene and ornithine decarboxylase were significantly increased. Progesterone treatment completely inhibits the E₂ induced wave of DNA synthesis but does not greatly influence the level of these 2 mRNAs. Thus in the uterine luminal epithelium E₂ regulates the level of ornithine decarboxylase and c-ras^H independently of cell proliferation.     

Early effects of oestradiol-17β on the chromatin and activity of the deoxyribonucleic acid-dependent ribonucleic acid polymerases (I and II) of the rat uterus

Oestradiol-17β (1.0μg) was injected intravenously into ovariectomized rats. The earliest detectable hormonal response in isolated uterine nuclei was an increase (10–15min) in RNA polymerase II activity (DNA-like RNA synthesis), which reached a peak at 30min and then decreased to control values (by 1–2h) before displaying a second increase over control activity from 2 to 12h. The next response to oestradiol-17β was an increase (30–60min) in polymerase I activity (rRNA synthesis) and template capacity of the chromatin. The concentrations of acidic chromatin proteins did not begin to increase until 1h after injection of oestradiol-17β and histone concentrations showed no significant changes during the 8h period after administration. The early (15min) increase in RNA synthesis in `high-salt conditions' can be completely eliminated by α-amanitin, an inhibitor of the RNA polymerase II. The exact nature of this early increase in endogenous polymerase II activity remains to be determined, e.g. whether it is caused by the increased availability of transcribable DNA of the chromatin or via direct hormonal activation of the enzyme per se.     

Changes in the patterns of synthesis of ribonucleic acid species in immature rat uterus in response to oestradiol-17β

1. After treatment of immature rats with diethylstilboestrol, the wet weight and RNA content of uterine tissue increased rapidly, reaching a peak at 40hr. After an initial lag of a few hours, the acid-soluble ribose and protein contents also rose to maxima at 40hr. No increase in DNA content occurred until at least 24hr. after treatment. 2. The RNA from immature rat uterus isolated at various times up to 6hr. after administration of oestradiol-17beta was labelled by injecting [(3)H]uridine and [(3)H]guanosine intraperitoneally 30min. before the animals were killed. It was fractionated on columns of kieselguhr coated with methylated serum albumin and the radioactivity in fractions corresponding to transfer RNA, 7s RNA, ribosomal RNA, Q(1)-RNA, Q(2)-RNA and DNA-like RNA was determined. 3. The radioactivity of the whole RNA increased steadily for 6hr. after hormone treatment. The earliest changes occurred in the Q(1)-RNA (ribosomal RNA precursor), whereas at longer time-intervals the radioactivity of the ribosomal RNA, 7s RNA and transfer RNA increased by four- to five-fold. The radioactivity of the DNA-like RNA increased by about 50%, but only at the longer time-intervals. 4. It is concluded that one of the earliest changes in response to oestradiol treatment is a major increase in synthesis of ribosomal RNA followed later by a similar increase in synthesis of transfer RNA and by a much smaller increase in synthesis of DNA-like RNA. The change in synthesis of ribosomal RNA in immature rat uterus may represent one of the most important responses to oestradiol treatment.     

The synthesis of ribonucleic acid in immature rat uterus responding to oestradiol-17β

Stimulation of incorporation of labelled precursors into the RNA of immature rat uterus is an early result of oestradiol-17beta action. However, the extent of the increased incorporation varies with the mode of administration of the labelled precursors and with the weight of the rat. At the age and weight range normally used response is maximal at ten times control incorporation, 4h after the administration of 0.3mug or more of oestradiol-17beta. Under these conditions the stimulation of incorporation into the acid-soluble fraction is only 2-2.5-fold. When the purified RNA is separated on polyacrylamide gels the major increase in incorporation of radioactive precursor is found in rRNA and 4S RNA; the formation of the former has been followed from the 45S precursor. Preceding these events by at least 30min, however, is an increase in the incorporation of precursor into RNA species of very high molecular weight, which remained in the first few slices of the gel. The possible significance of these findings is discussed. The increased synthesis of rRNA in response to oestradiol-17beta is more strongly inhibited by actinomycin D than the synthesis of other RNA species. Cycloheximide, depending on time of administration and dosage, inhibits either RNA synthesis or the maturation of rRNA.     

A possible role for 5-phosphoribosyl 1-pyrophosphate in the stimulation of uterine purine nucleotide synthesis in response to oestradiol-17β

1. It has been reported that the rate of purine nucleotide synthesis de novo in the immature rat uterus is doubled at 6h after administration of oestradiol-17beta. The present work confirms an increased incorporation of glycine and adenine into uterine nucleotides between 2 and 6h after hormone treatment and investigates the mechanism of this response. 2. Activation of regulatory enzymes is unlikely to promote increased nucleotide synthesis: the activities of 5-phosphoribosyl 1-pyrophosphate amidotransferase (EC 2.4.2.14) and adenine phosphoribosyltransferase (EC 2.4.2.7) are the same in uterine extracts from control and oestrogen-treated rats. 3. Therefore it was proposed that oestradiol might promote an increased supply of a rate-limiting substrate. The low oestrogen-sensitive rate of AMP synthesis from adenine and endogenous 5-phosphoribosyl 1-pyrophosphate in the intact uterus compared with the high, oestrogen-insensitive rate in uterine extracts supplemented with 5-phosphoribosyl 1-pyrophosphate is evidence that the supply of 5-phosphoribosyl 1-pyrophosphate limits purine nucleotide formation and may increase after hormone treatment. This proposal is supported by the decrease in AMP synthesis in the whole tissue in the presence of guanine and 7-amino-3-(beta-d-ribofuranosyl)pyrazolo[3,4-d]pyrimidine (formycin). These compounds do not inhibit adenine uptake or adenine phosphoribosyltransferase activity, but they both decrease the availability of 5-phosphoribosyl 1-pyrophosphate, the former by promoting its utilization by hypoxanthine/guanine phosphoribosyltransferase (EC 2.4.2.8) and the latter by inhibiting its synthesis from ribose 5-phosphate and ATP by ribose 5-phosphate pyrophosphokinase (EC 2.7.6.1). 4. It is unlikely that the increased availability of 5-phosphoribosyl 1-pyrophosphate results from hormonal stimulation of ribose 5-phosphate formation. Methylene Blue and phenazine methosulphate both increase ribose 5-phosphate without altering the supply of 5-phosphoribosyl 1-pyrophosphate. 5. The activity of ribose 5-phosphate pyrophosphokinase is low in uterine extracts and increases rapidly in response to oestradiol. Therefore the hormonal activation of the routes of purine nucleotide synthesis both de novo and from preformed precursors may be due, at least in part, to an increased availability of the common rate-limiting substrate 5-phosphoribosyl 1-pyrophosphate, mediated by activation of ribose 5-phosphate pyrophosphokinase.     

Quantitative analysis of estradiol-17β-induced changes in the ultrastructure of the liver of the male zebrafish,Brachydanio rerio

Summary Hepatocytes of male zebrafish, Brachydanio rerio, were studied by means of light- and electron-microscopy, following a period of maximally 16 days of in-vivo treatment with estradiol-17. The responsiveness of the male hepatocytes to this female sex steroid was investigated by use of morphometric methods. The results of this investigation show that the responsiveness was most obvious between 2 and 16 days, as revealed by an increase in cell size, accompanied by a proliferation of the granular endoplasmic reticulum and the Golgi complex. In addition, accumulations of glycogen granules, which are characteristic of hepatocytes in untreated males, had disappeared and lipid droplets had accumulated. These experimentally induced changes in the morphology of the male hepatocyte closely resemble those described for the female hepatocyte during the sexual cycle. It is concluded that the hepatocytes of male zebrafish can be stimulated by estradiol-17 to produce vitellogenin and that in female zebrafish this steroid is a key sex hormone responsible for vitellogenin production by the liver during the natural sexual cycle.     

Regulation of polyribosome formation and protein synthesis in the uterus. Effect of administration of graded doses of oestradiol-17β and actinomycin D on the amino acid-incorporation activity in vitro and the cytoplasmic concentration in vivo of polyribosomes

1. The effects of graded doses of oestradiol-17beta and actinomycin D, administered separately or together, on the amino acid-incorporation activity in vitro and the cytoplasmic concentration in vivo of uterine polyribosomes are described. Preparations of polyribosomes isolated from uteri of ovariectomized adult rats were determined for cytoplasmic concentration in vivo and assayed for [(14)C]leucine-incorporation activity in the cell-free system, exactly as described by Teng & Hamilton (1967b). 2. A minimal dose of 10mug of oestradiol-17beta administered for 10h was found to increase, by about 100%, both the amino acid-incorporation activity in vitro and the cytoplasmic concentration in vivo of the polyribosomes. A minimal dose of 250mug of actinomycin D administered for 10h was found to inhibit, by about 50%, the incorporation activity in vitro of the polyribosomes. All doses of the inhibitor administered for 10h failed to alter the cytoplasmic concentration in vivo of the polyribosomes. 3. A dose of 10mug of oestradiol-17beta restored to the control value the inhibitory effect of a dose of either 50 or 125mug of actinomycin D on the activity in vitro of the polyribosomes, at 10h after treatment with the inhibitor and the hormone. In these experiments, there was an increase of 60-100% in the cytoplasmic concentration in vivo of the polyribosomes. 4. A dose of 125mug of actinomycin D, administered to animals along with 10mug of oestradiol-17beta for 6-36h, abolished the hormone-induced enhancement of the incorporation activity in vitro, but did not prevent an increase of about 200% in the cytoplasmic concentration in vivo of the polyribosomes. However, treatment with 750mug of the inhibitor abolished both stimulatory effects of the hormone. 5. The results reported indicate that the stimulatory effects of oestradiol-17beta in vivo on the number and activity of the cytoplasmic polyribosomes in the uterus of the ovariectomized rat have different sensitivities to actinomycin D, but the primary molecular mechanisms responsible for the results are unknown. The major conclusion drawn is that the formation and appearance in the cytoplasm of newly formed polyribosomes are important features of the early action of oestrogen in the uterus.     

Production,clearance rates and metabolic fate of estradiol-17β in the plasma of the laying hen

A single dose of tritiated estradiol-17β (³H-E₂β) was injected i.v. into 5 high egg producing White Leghorn hens, 31 weeks of age, at 19.2 ± 2.1 (mean ± S.D.) hr before oviposition. Blood (2 ml) was sampled at approximately 5 min intervals over 40 min. Whenever possible, metabolites were monitored and identified by the double isotope technique with the addition of the corresponding ¹⁴C-labelled standards to plasma prior to analysis. The metabolic half-life and clearance rate of ³H-E₂β in plasma were 10.9 ± 1.9 min and 118 ± 18 ml/min/kg body weight, respectively. The calculated production rate of E₂β at 19.2 hr before oviposition was 19.5 ± 5.7 ng/min based on the plasma level (93±22 pg/ml) measured at that time. The relative concentrations (% of plasma radioactivity) of the major metabolites isolated at 5.7 ± 0.6 min post injection were, in descending order: estradiol-17β-3-sulfate (E₂β-3S : 14.9 ± 2.7), estradiol-17α-3-sulfate (E₂α-3S; 5.7 ± 0.3), estrone (E₁; 4.6 ± 0.5), estrone sulfate (E₁S; 2.2 ± 0.5), and estradiol-17 α (E₂α; 1.2 ± 0.4). As time proceeded, the relative concentration of E₂α-3S gradually increased so that by 43.2 ± 1.0 min it became the most abundant identifiable metabolite (12.3 ± 1.1) followed by E₂β-3S (9.1 ± 1.7), E₂S (1.2 ± 0.6), E₁ (0.7 ± 0.4) and E₂α (0.3 ± 0.2). These findings are consistent with the view that one of the major pathways of E₂β metabolism in the circulation of the hen is via E₂β

E₂β?3S ?E₁S

E₂α-3S.     

Histone acetylation and hormone action. Early effects of oestradiol-17β on histone acetylation in rat uterus

The effect of oestradiol treatment on the acetylation of histones of the immature rat uterus has been studied. A 10mug dose of oestradiol causes a 70% increase at 5min and a 140% increase at 10min after administration in the labelling of the histone fraction F2+F3. No effect of oestradiol is seen on the labelling of histones F1 or acidic non-histone chromatin proteins. The oestradiol stimulation is seen in animals pretreated with either cycloheximide or actinomycin D. The stimulation of labelling caused by oestradiol is completely abolished by pretreatment of the animals with the anti-oestrogen, nafoxidine. The stimulation is given by lower doses of oestradiol, by stilboestrol and oestriol, but is not given by testosterone. These results suggest that stimulation of histone acetylation in the uterus is the earliest known effect of the hormone on its target tissue.     

Effect of estradiol-17 β on the cytology of the liver,gonads and pituitary,and on plasma electrolytes in the female freshwater eel

Summary Female freshwater eels injected with estradiol-17 (E2) for 15–78 days appear paler and secrete more mucus than controls. The resulting strongly opalescent blood plasma indicates that vitellogenin synthesis occurs in the liver, which shows a significant hypertrophy, an increased vacuolization (lipid material) and glycogen depletion. Plasma sodium is lowered, but calcium levels are considerably increased. The gonosomatic index increases (0.92±0.1 to a maximum of 2.21). Oocytes are enlarged, but the incorporation of vitellogenin remains discrete. Gonadotrophs (GTH cells), small and scarcely visible in the pituitary of control eels, are hypertrophied and contain numerous glycoprotein granules after E2-administration. E2 may act on the pituitary and/or hypothalamus via a positive feedback to induce gonadotrophin (GTH) synthesis; GTH release seems to be very limited as indicated by the ovarian response. The differentiation of GTH cells in eels treated with fish pituitary extracts is most probably due to secretion of E2 by the ovary, which reacts on the pituitary. Various hypotheses are considered to explain the low GTH release.Thyrotrophs, somatotrophs and prolactin cells of the pituitary are stimulated. In the pars intermedia, MSH and PAS-positive cells appear less active. A possible antidopaminergic effect of E2 is discussed.E2 administration constitutes a simple and economic technique to induce the synthesis of GTH and will facilitate the biochemical and biological study of the latter hormone in eels.     

Easy stereoselective synthesis of 5α-estrane-3β,17α-diol, the major metabolite of nandrolone in the horse

5α-Estrane-3β,17α-diol is the major metabolite of nandrolone in horse urine. The presence of 5α-estrane-3β,17α-diol in female and gelding urines is prohibited by Racing Rules and its natural presence in male urine led regulation authorities to establish a concentration threshold of 45 ng/mL. This paper describes a rapid, simple and stereoselective synthesis of 5α-estrane-3β,17α-diol, providing horseracing laboratories with an essential reference material for their antidoping performance.