Differential Effect of Trilostane on the Progestin Milieu in the Pregnant Mare W
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Differential effect of trilostane on the progestin milieu in the pregnant mare W. E. Schutzer, J. L. Kerby and D. W. Holtan lDeparlment of Animal Sciences, and 2College of Pharmacy, Oregon State University, Corvallis, OR 97331, USA Trilostane, a competitive inhibitor of 3\g=b\-hydroxysteroiddehydrogenase, was administered intravenously to pregnant mares (n = 3) between day 277 and day 282 of gestation to determine its effect on the progestin milieu. In addition, placental tissue from mares at mid-gestation (150\p=n-\300days) (n = 4) were exposed to either deuterium-labelled pregne- nolone alone or deuterium-labelled pregnenolone and trilostane to examine the effect of trilostane on placental metabolism of pregnenolone. Blood samples were collected from indwelling jugular catheters at frequent intervals for 48 h after infusion. Both plasma samples and incubation media were quantitatively analysed, after solid phase extraction and steroid derivitization, for concentrations of eight different progestin metabolites using gas chromatography and mass spectrometry. Trilostane infusion differentially affected the progestin milieu in vivo without inducing abortion. Forty-five minutes after infusion, maternal plasma concentrations of pregnenolone, 5-pregnene-3\g=b\,20\g=b\-diol,5\g=a\-pregnane\x=req-\ 3\g=b\,20\g=b\-dioland 3\g=b\-hydroxy-5\g=a\-pregnan-20-oneincreased (P < 0.05) and remained high for 37 h. Concentrations of 5\g=a\-pregnane-3,20-dione,20\g=a\-hydroxy-5\g=a\-pregnan-3-oneand 5\g=a\-pregnane-3\g=b\,20\g=a\-dioldecreased 15 min after infusion (P < 0.05), yet 1.5 h after infusion, concentrations had increased and remained high until 37 h after infusion. Trilostane inhibited the conversion of pregnenolone to progesterone in vitro (P < 0.001) while mediating an increase (P < 0.05) in concentrations of 5\g=a\-pregnane-3,20-dioneand 3\g=b\-hydroxy-5\g=a\-pregnan-20-one.These observations demonstrate that the pregnant mare possesses unique steroid hormone metabolic activity and suggests that another steroid, perhaps 5\g=a\-pregnane-3,20-dioneand not progesterone, is the important steroid precursor for the other progestin metabolites found in circulating plasma. Introduction concentrations of other metabolites increase throughout pregnancy (Holtan et al, 1975). Another progestin metabolite, as identified with The mare possesses unique steroid hormone metabolic activity 20a-hydroxy-5a-pregnan-3-one (20 -5 ), gas during mid- and late-gestation. In contrast to other mammals, chromatography/mass spectrometry (GC/MS), can reach con¬ where circulating progesterone concentrations are generally centrations of pg ml"1 near parturition, (Holtan et al, 1991). and structural details raised throughout pregnancy, equine maternal concentrations The systematic names, abbreviations of of progesterone begin to decrease at about day 125 of the progestins that are discussed throughout this text, are Table gestation and are undetectable, or very low, near day 200 of given in I. gestation (term = 340 days). Furthermore, eight distinct, In common with other species, the equine placenta prominent progestin metabolites have been identified through¬ contains 3ß-hydroxysteroid-dehydrogenase (3ß-HSD) activity; metabolizes out pregnancy in plasma from pregnant mares (Holtan et al, the placenta pregnenolone to progesterone and has 1991). In addition, the mare is unusual in that ovariectomy as (Ainsworth and Ryan, 1969; Schutzer Holtan, 1996). It been that in mares is early as at day 50 of gestation has no adverse effect on thus suggested maintenance of pregnancy pregnancy maintenance (Holtan et al, 1979; Knowles et al, controlled primarily by progestins of a placental source (Moss To and Silver 1994) indicating that pregnancy is maintained henceforth by an et al, 1979). examine this possibility, Fowden extra-luteal source of progestin. One progestin metabolite, (1987) examined the effect of epostane, a 3ß-HSD competitive 5a-pregnane-3,20-dione, (5a-DHP), is detectable in the inhibitor, on plasma progesterone concentrations and on the mares near treatment peripheral circulation as early as day 10 (Atkins et al, 1976) and duration of gestation in term. Although decreased immunoreactive-progesterone, it did not cause *Current address: Division of Sciences, Primate Reproductive Oregon Regional abortion. In contrast, abortion was induced when Research Center, Beaverton, OR 97006, USA. pregnant Correspondence. rhesus monkeys, rats, sheep, swine, cows or humans were Received 5 January 1996. exposed to similar or lower doses of a 3ß-HSD competitive Downloaded from Bioscientifica.com at 09/25/2021 04:51:46AM via free access Table 1. Systematic names, abbreviations and structural details of steroids Systematic name Abbreviati! Ri R2 5-Pregnenes 3ß-hydroxy-5-pregnen-20-one Pregnenolone ß-OH = O 5-pregnen-3ß,20ß-diol Pregnenolone5-ßß ß-OH ß-OH 4-Pregnenes 4-pregnen-3,20-dione Progesterone = O = O 5a-Pregnanes 5a-pregnane-3,20-dione 5a-DHP = O = O 3ß-hydroxy-5a-pregnan-20-one 3ß-5 ß-OH = 20a-hydroxy-5a-pregnan-3-one 20 -5 = - 5o>pregnane-3ß,20ß-diol ßß-diol ß-OH ß-OH 5a-pregnane-3ß,20a-diol ß -diol ß-OH a-OH inhibitor in vivo (Schane et al, 1979; Creange et al, 1981; Winthrop Pharmaceuticals, Collegeville, PA) i.v. Trilostane was et al, 1982; Martin et al, 1987; et dissolved ml in St Taylor Crooij al, 1988). (90 mg ~ ) dimethyl sulfoxide (Sigma, Louis, Likewise, inhibition of 3ß-HSD in vitro stopped the conversion MO) and then this solution was added to a mixture made up of of pregnenolone to progesterone in dispersed sheep placental 75% (v/v) propylene glycol (Sigma) and 5% (v/v) Tween-80 cells (Power and Challis, 1987). (Sigma). Seven days later, two of the mares received vehicle It is important to recognize that Fowden and Silver (1987) alone and served as controls. Catheters (16 gauge 5.75 in; measured immunoreactive progestins and not progesterone Angiocath; Désuet Medical, Sandy, UT) were placed into itself; all of the progestin metabolites crossreact to some extent the jugular vein using an aseptic technique. Three blood with 'progesterone-specific' antibodies in radioimmunoassays. samples were taken at intervals of 15 min. Over a 15 min Therefore, in this study, which examined how inhibition of period, trilostane was infused. Immediately after the infusion, 3ß-HSD affects individual progestins, GC/MS was applied blood samples were taken every 15 min for 2 h, every 30 min which measures quantitatively concentrations of each specific for 2 h, once an hour for 4 h and then every 12 h for 3 days. progestin metabolite. Blood was collected into heparinized tubes (Vacutainer; Trilostane, a 3ß-HSD competitive inhibitor similar in Becton Dickson, Rutherford, NJ) and immediately centrifuged function to epostane (Potts et al, 1978; Ledger et al, 1985), was at 1000 g for 15 min. Plasma was stored at 20°C until — infused intravenously into mares (n = 3) at mid-gestation to analysis. examine its effect on the progestin milieu. By examining trilostane-mediated alterations of individual progestin concen¬ trations, metabolic activity, biological relevance and steroidal Experimental procedures in vitro of the found in maternal circulation can precursors progestins Homogenous regions of placentae, without the chorionic be established. The specific role of the placenta in steroid girdle or endometrial cells, were collected at random from metabolism, and therefore pregnancy maintenance, was also light horse mares (n = 4; approx. 500 at a local abattoir. investigated by using trilostane in vitro. kg) The gestation ages of the fetuses as estimated by measure¬ ment of the crown—rump length (Ginther, 1992) were 150 days, 250 days, 300 days and 320 days. The placentae were Materials and Methods separated manually from the uteri and immediately placed on ice. After transport to the laboratory, 1 g portions of each Animals placenta were finely minced and placed in sterile 20 ml glass tubes with rubber stoppers. Five ml of sterile filtered and Three mares caballus; pregnant pony (Equus approximately gassed (95% 02:5% C02) TCM-199 with Hank's salts and 200 kg) of unknown pedigree were maintained in outdoor sodium bicarbonate (Sigma) were added. After suspension, paddocks and fed a ration which met appropriate husbandry the tubes were gassed, covered and mixed on their sides for standards. The duration of gestation of the mares at the time of 60 min at 37°C in an environmental shaker. After pre- infusion was: 282 days, 280 days, and 277 days. The animals in incubation for 60 min, the tubes were centrifuged at 1000 g the in vivo study had been used in a similar previously study for 10 min and the medium off. Fresh (see Schutzer and Holtan (1996) for details). poured gassed-medium (5 ml) was added; the tissue was resuspended and either 25 pg deuterium labelled (D4)-pregnenolone control or 25 pg Experimental procedures in vivo D4-pregnenolone plus 2.0 pmol of trilostane in DMSO were added to the medium. The flasks were incubated and mixed Mares (« = 3) received 5 g 3ß-HSD competitive inhibitor, at 37°C for 4 h on their side. After incubation, the tubes trilostane ((4a,5a,17ß)-4,5-epoxy-3,17-dihydroxyandrost-2- were centrifuged at 750 g at 5°C for 15 min and stored at ene-2-carbonitrile; WIN 24540; generously donated by Sterling 20°C until assay. — Downloaded from Bioscientifica.com at 09/25/2021 04:51:46AM via free access Progestin analysis increase in the concentration of plasma pregnenolone, the direct substrate for 2; inset). Likewise, concen¬ A Hewlett Packard (Wilmington, DE) 5890 GC interfaced 3ß-HSD (Fig. trations of 5-pregnene-3ß,20ß-diol (pregnenolone-ßß), ßß-diol with a 5971A MS was used for quantitative analysis on eight and 3ß-5 also increased as a result of trilostane treatment different endogenous progestins as described by Houghton 2).