James I. Raeside

Synthesis of free and sulphoconjugated 16-androstene steroids by the Leydig cells of the mature domestic boar

This study examined the involvement of sulphoconjugation in the biosynthesis of the 16-androstene steroids in Leydig cells of the mature boar, since the formation of steroid sulphoconjugates can reduce the levels of these steroids that accumulate in fatty tissue. Leydig cells were purified from testes of mature male pigs and incubated with pregnenolone, or various individual 16-androstene steroids for 10 min, 1, 4 and 8h. Sulphoconjugated steroids were recovered by solid-phase extraction followed by solvolysis. Profiles of unconjugated and sulphoconjugated steroids were analysed by HPLC. Steroids present in the sulphoconjugated fractions were purified, derivatised as O-methoxime/trimethylsilyl ethers (MO-TMS), and subsequently identified using gas chromatography-mass spectrometry (GC-MS). The principal metabolite produced from incubations with pregnenolone, androstadienol, androstadienone and 5alpha-androstenone was 3beta-androstenol. 16-Androstene steroids that were sulphoconjugated included 5alpha-androstenone, 3beta-androstenol and 3alpha-androstenol. Approximately 70% of the total amount of each 16-androstene steroid was in its sulphoconjugated form after incubations for 4h or more. The finding that sulphoconjugated 5alpha-androstenone was present in large amounts suggests that this steroid may be converted from a 3-keto to a 3-enol form which is subsequently sulphoconjugated. These findings emphasise the need to consider the impact of sulphoconjugation of the 16-androstene steroids and their role in contributing to boar taint.

Estrogen Metabolism in the Equine Conceptus and Endometrium During Early Pregnancy in Relation to Estrogen Concentrations in Yolk-Sac Fluid

Abstract Because estradiol (E2) production by the early equine conceptus is considered crucial to the establishment of pregnancy, the amounts of E2, estrone (E1), and their sulfates (E2S, E1S) 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 E1S. Then, the metabolism of E2, 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. Eleven conceptuses collected mainly on Days 12, 15, and 18, with endometrial biopsy samples taken immediately thereafter, were used for steroid metabolic studies. Trophoblastic and endometrial tissues were incubated with [3H]-labeled E2 or E1, and with [14C]-E1 in one experiment. Steroids were recovered from the media by solid-phase extraction (SPE) and eluted separately as unconjugated and conjugated fractions. Conjugation increased from Day 12 for the trophoblast (more so by bilaminar than trilaminar tissues on Day 18) and was much greater for endometrium, with almost all as sulfoconjugates. HPLC profiles of free and sulfate fractions were obtained from a gradient of acetonitrile/water. Interconversion (E2 ⇌ E1) by trophoblast varied with development; it favored E2 in older conceptuses, more in bilaminar than trilaminar tissues. Some more polar products were also noted, with loss of tritium seen as [3H]2O at SPE, and confirmed by HPLC in a second system with authentic reference steroids. Almost all radioactivity in the endometrium was present as E2 in both free and sulfate fractions. It was concluded that local metabolism of E2 is quantitatively significant and may play an important role in the actions of the large amounts of estradiol produced by the early equine conceptus.

Suitability of epididymal and testicular ultrasonography and computerized image analysis for assessment of current and future semen quality in the ram.

Breeding soundness evaluation (BSE) is the primary assessment for determining the reproductive potential of male animals. This method, however, cannot be used to evaluate semen frequently or to predict future semen quality. Computerized analysis of ultrasonographic images provides information on histophysiological changes in male reproductive organs. We hypothesized that: (i) semen parameters would correlate with ultrasonographic characteristics of the distal region (cauda) of the epididymis and (ii) testicular ultrasound images and/or circulating testosterone concentration would predict future semen quality in the ram. Six adult rams underwent BSE and scrotal ultrasonography approximately 60 d apart (average duration of the spermatogenic cycle) both during the breeding (December and February) and non-breeding (June and August) seasons. An inverse correlation was found between pixel intensity (numerical pixel values) of the epididymes and percentage of sperm in semen with normal morphology (r = −0.46, P < 0.05). Pixel heterogeneity (standard deviation of pixel values) correlated negatively with percentage of sperm with normal morphology (r = −0.42, P < 0.05) and directly with percentage of spermatozoa with abnormal tails (r = 0.43, P < 0.05). Pixel heterogeneity of testicular parenchyma obtained approximately 60 d prior to semen evaluation inversely correlated with percentage of sperm with normal morphology (r = −0.73, P < 0.01) and sperm progressive motility (r = −0.76, P < 0.01), and directly with percentage of sperm with abnormal tails (r = 0.72, P < 0.01) and loose heads (r = 0.79, P < 0.01). We concluded that scrotal ultrasonography combined with computer-assisted analyses of epididymal and testicular echotexture in the ram was a valuable method for determining certain current and future semen parameters, respectively.

Estrogen metabolism by the equine embryo proper during the fourth week of pregnancy

Estrogen production by the trophoblast is considered important in early equine pregnancy and leads to high concentrations in yolk-sac (Y-S) fluid. The embryo proper is a potential site for their action. We examined estrogen metabolism in the embryo proper because some actions of estrogens are derived from locally formed metabolites. The embryo proper, as well as separated extraembryonic tissues, of conceptuses collected about day 25 of pregnancy, were incubated with (3)[H]-estrone (E(1)) and (3)[H]-estradiol (E(2)). Steroids were recovered from media by solid-phase extraction and eluted separately as unconjugated and conjugated fractions. Profiles of free and sulfo-conjugated fractions were obtained by HPLC. Some differences and similarities were noted for the embryo proper as compared to the extraembryonic tissues. No reduction of E(1) to E(2) was noted for the embryo proper and allantois, but some was seen with the bilaminar Y-S wall. Less conversion of E(2) to E(1) occurred in the embryo proper than in the extraembryonic tissues. Profiles for hydrolyzed sulfates from incubation of the embryo proper were very similar for both substrates, mainly with E(1) present. Thus, low levels of reductase and high levels of oxido- activities were apparent for the 17beta-hydroxysteroid dehydrogenase enzymes. Further evidence of an active role for the embryo proper was seen as minor, polar products, and an unknown compound eluting between E(2) and E(1). These findings show, for the first time, that the embryo proper can metabolize estrogens that are found in Y-S fluid - a function of potential significance at this stage in its development.

Identification of 5α-androstane-3β,17β-diol and 3β-hydroxy-5α-androstan-17-one sulfates as quantitatively significant secretory products of porcine leydig cells and their presence intesticular venous blood

By means of high performance liquid chromatography and gas chromatography-mass spectrometry it has been found that 5 alpha-androstane-3 beta,17 beta-diol sulfate and 3 beta-hydroxy-5 alpha-androstan-17-one sulfate (epiandrosterone) are major secretory steroids of the mature boar testes. These same compounds were similarly identified in culture media when porcine Leydig cells were incubated with androstenedione as substrate. In addition, they were seen as the principal secretory products when [3H]androstenedione and [3H]testosterone were used as substrates; and their presence was greatly reduced by an inhibitor of 5 alpha-reductase (N,N-diethyl,4-methyl-3-oxo-4-aza-5 alpha-androstane-17 beta-carboxamide). Greater quantities of 5 alpha-androstanediol than epiandrosterone were noted in all instances. These findings provide further evidence of the versatile activity of the boar testes in steroidogenesis.

5Alpha-Reduced Steroids Are Major Metabolites in the Early Equine Embryo Proper and Its Membranes

ABSTRACT Steroid production and metabolism by early conceptuses are very important for the establishment and maintenance of pregnancy in horses. Our earlier work suggested the possible formation of 5alpha-reduced steroids in equine conceptuses. We have now demonstrated the formation of 5alpha-reduced metabolites of androstenedione, testosterone, and progesterone by the embryo and its membranes. A total of 44 conceptuses were collected from 26 mares between 20 and 31 days of pregnancy. Tissues from the embryo proper and from the separated components of the conceptus (bilaminar and trilaminar trophoblast, allantois) were incubated with tritium-labeled substrates. 5Alpha-reduced metabolites (5alpha-dihydro- and 3beta,5alpha-tetrahydro- steroids) as radiolabeled products were identified from a series of chromatographic steps using four solvent systems for high-performance liquid chromatography. Use of a 5alpha-reductase inhibitor confirmed the metabolites were indeed 5alpha-reduced steroids. For the embryo, the only products from androstenedione were 5alpha-dihydroandrostenedione and 3beta,5alpha-tetrahydroandrostenedione, with no evidence of more polar metabolites; there was some 3beta,5alpha-tetrahydrotestosterone but no 5alpha-dihydrotestosterone from testosterone, and formation of androstenedione was followed by the production of 5alpha-dihydroandrostenedione and 3beta,5alpha-tetrahydroandrostenedione. The major 5alpha-reduced product from progesterone was 3beta,5alpha-tetrahydroprogesterone, with lesser amounts of 5alpha-dihydroprogesterone. For the membranes, reductions to tetrahydro, 5alpha-reduced steroids were prominent in most instances, but also present were considerable amounts of products more polar than the substrates. The well-recognized activity of some 5alpha-reduced steroids—for example, 5alpha-dihydrotestosterone in male sexual differentiation—provokes interest in their even earlier appearance, as seen in this study, and suggests a possible role for them in early embryonic development in horses and, more generally, in other species.

The presence of 19-norandrostenedione and its sulphate form in yolk-sac fluid of the early equine conceptus

C(18) neutral steroid formation by cytochrome P450 aromatase has been recorded for several equine and porcine tissues. High activity of P450 aromatase is reflected in the quantities of estrogens in yolk-sac (y-s) fluid of early equine conceptuses. In a previous study of y-s fluid we detected large amounts of androgens by radioimmunoassay (RIA), using an antiserum for androstenedione (A(4)). Here, we report that RIA, following chromatography, gave tentative identification of the major peak as norandrostenedione (19-norA) not as A(4). Furthermore, even greater quantities of 19-norA seemed to be present in y-s fluid as a sulphoconjugate, as noted from extraction, solvolysis, HPLC, followed by RIA. Confirmation of these unusual findings was attained after further purification with two HPLC systems and definitive identification by LC-MS with an authentic standard of 19-norA. Initial extraction of the steroid sulphate as a methylene-blue complex also yielded 19-norA suggesting that the 3-enol form had enabled sulphoconjugation. The biological significance of retention mainly as a sulphate is not known; however, the large amounts of 19-norA found in the fluid accords well with reports on the catalytic activity shown in vitro by the blastocyst isozyme of P450 aromatase in the pig and horse.

Isolation of 11β-hydroxylated androgens from testicular vein blood of the mature boar

The isolation of 11 beta-hydroxyandrostenedione and 11 beta-hydroxytestosterone from testicular vein blood of the mature male domestic pig is described. Blood was collected from veins and arteries on the surface of the testes of mature boars. Steroids were extracted from plasma with SEP-PAK C18 cartridges and recovered with acetonitrile. A separation of steroids was made by high performance liquid chromatography (HPLC) using acetonitrile/water (37/63; v/v), and fractions were collected manually with detection at 254 nm. Preliminary identification was based on comparison with the HPLC retention time of an authentic steroid standard. Final characterization was achieved by means of capillary gas chromatography-mass spectrometry (GC-MS). The findings record the first evidence for the secretion of C19-11-hydroxylated steroids by normal testes in a mammalian species.

Effect of a 5α-reductase inhibitor on the metabolism of 19-norandrogens by porcine leydig cells

Summary The metabolism of 19-norandrostenedione and [3H]19-nortestosterone was examined in porcine Leydig cell preparations in the absence, or presence, of a 4-aza-steroid inhibitor of 5α-reductase. Evidence for a major production of 5α-estrane-3β, 17β-diol and 3β-hydroxy-5α-estran-17-one, as sulfo-conjugated steroids, was obtained by HPLC and gas-chromatography/mass spectrometry. The 4-aza-steroid clearly reduced the formation of both of the above 5α-reduced products from the 19-norandrogens. From the HPLC profiles of the radioactive metabolites, it was also concluded that estrogen secretion was increased significantly by exposure of the cells to the 5α-reductase inhibitor.

Hydroxylation of 19-norandrogens by porcine leydig cells

The metabolism of 19-norandrogens by porcine Leydig cells was investigated. Non-radioactive 19-norandrostenedione (19-Nor A) and [3H]19-nortestosterone (19-Nor T) were used as substrates in incubations with cell preparations from mature male pigs. Steroid products were separated by reversed-phase HPLC and material in selected peaks was rechromatographed before attempts to identify them by GC-MS. Both 11 beta- and 6 beta-hydroxylated derivatives of 19-Nor A were found and a third product (11-oxo-19-Nor A) was tentatively identified. The profile of radioactive metabolites from [3H]19-Nor T also favours the view of a capacity for hydroxylation of 19-norandrogens by porcine Leydig cells. The significance of these findings together with our earlier report of direct 11 beta-hydroxylation of C19 steroids by such cells remains to be examined.