Erin Legacki

The dynamic steroid landscape of equine pregnancy mapped by mass spectrometry.

Liquid chromatography-tandem mass spectrometry (LC-MS/MS) allowed comprehensive analysis of various steroids detectable in plasma throughout equine gestation. Mares (n=9) were bled serially until they foaled. Certain steroids dominated the profile at different stages of gestation, clearly defining key physiological and developmental transitions. The period (weeks 6-20) coincident with equine chorionic gonadotropic (eCG) stimulation of primary corpora lutea and subsequent formation of secondary luteal structures was defined by increased progesterone, 17OH-progesterone and androstenedione, all Δ4 steroids. The 5α-reduced metabolite of progesterone, dihydroprogesterone (DHP) paralleled progesterone secretion at less than half the concentration until week 12 of gestation when progesterone began to decline but DHP concentrations continued to increase. DHP exceeded progesterone concentrations by week 16, clearly defining the luteo-placental shift in pregnane synthesis from primarily ovarian to primarily placental. The period corresponding to the growth of fetal gonads was defined by increasing dehydroepiandrosterone and pregnenolone (Δ5 steroids) concentrations from week 14, peaking at week 34 and declining to term. Metabolites of DHP (including allopregnanolone) dominated the steroid profile in late gestation, some exceeding DHP by weeks 13 or 14 and near term by almost tenfold. Thus Δ4 steroids dominated during ovarian stimulation by eCG, inversion of the ratio of progesterone: DHP (increasing 5α-pregnanes) marked the luteo-placental shift, Δ5 steroids defined fetal gonadal growth and 5α-reduced metabolites of DHP dominated the steroid profile in mid- to late-gestation. Comprehensive LC-MS/MS steroid analysis provides opportunities to better monitor the physiology and the progress of equine pregnancies, including fetal development.

Progestin withdrawal at parturition in the mare

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 luciferase reporter assays. Stable transfected Chinese hamster ovarian cells expressing the equine progesterone receptor (ePGR) were transfected with an MMTV-luciferase expression plasmid responsive to steroid agonists. Cells were incubated with increasing concentrations (0-100nM) of progesterone, 20αDHP and 3α,20βDHP. The concentrations of circulating pregnanes in periparturient mares were (highest to lowest) 3α,20βDHP and 20αDHP (800-400ng/mL respectively), DHP and allopregnanolone (90 and 30ng/mL respectively), and pregnenolone and progesterone (4-2ng/mL). Concentrations of all measured pregnanes declined on average by 50% from prepartum peaks to the day before foaling. Maximum activation of the ePGR by progesterone occurred at 30nM; 20αDHP and 3α,20βDHP were significantly less biopotent. At prepartum concentrations, both 20αDHP and 3α,20βDHP exhibited significant ePGR activation. Progestogenic support of pregnancy declines from 3 to 5days before foaling. Prepartum peak concentrations indicate that DHP is the major progestin, but other pregnanes like 20αDHP are present in sufficient concentrations to play a physiological role in the absence of DHP. The authors conclude that progestin withdrawal associated with parturition in mares involves cessation of pregnane synthesis by the placenta.

Equine fetal adrenal, gonadal and placental steroidogenesis

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

Steroid regulation of early postnatal development in the corpus epididymidis of pigs

Development of the epididymis including blood-epididymal barrier formation is not required until sperm reach the epididymis peripuberally. Regulation of this development in the early postnatal period is largely unknown. The current objectives were to evaluate potential roles of endogenous estrogen and androgen signaling during early development of the corpus epididymidis and to determine the timing of formation of the blood-epididymal barrier in the pig. Effects of endogenous steroids were evaluated using littermates treated with vehicle, an aromatase inhibitor (letrozole) to reduce endogenous estrogens, an estrogen receptor antagonist (fulvestrant) or an androgen receptor antagonist (flutamide). Phosphorylated histone 3 immunohistochemistry was used to identify proliferating epithelial cells. Lanthanum nitrate and electron microscopy were used to analyze formation of the blood barrier in the corpus epididymidis. Reducing endogenous estrogens increased the number of proliferating corpus epithelial cells at 6 and 6.5 weeks of age compared with vehicle-treated boars (P<0.01 and P<0.001 respectively). Blocking androgen receptors did not alter proliferation rate at 6.5 weeks of age. Although barrier formation was similar between 6 and 6.5 weeks of age in vehicle-treated animals, intercellular barriers increased in letrozole-treated littermates at 6.5 weeks of age. Fulvestrant treatment, which should mimic aromatase inhibition for regulation through ESR1 and ESR2 signaling but potentially stimulate endogenous estrogen signaling through the G protein-coupled estrogen receptor (GPER), had the opposite effect on aromatase inhibition. These responses in conjunction with the presence of GPER in the corpus epididymidis suggest early corpus epididymal development is regulated partially by GPER.

Porcine Sertoli Cell Proliferation after Androgen Receptor Inactivation

ABSTRACT Sertoli cell proliferation in neonatal boars is potentially androgen dependent. Hence, the immediate objective was to evaluate effects of androgen receptor-mediated signaling on the first wave of Sertoli cell proliferation. The experimental design employed littermate pairs of boars with one member assigned to receive a daily oral dose of flutamide, an androgen receptor antagonist, beginning at 1 wk of age and the littermate the canola oil vehicle. Experiment 1 examined the response at 6.5 wk of age after completion of the first wave of Sertoli cell proliferation, and experiment 2 examined the response at 11 wk of age after initiation of the second wave of Sertoli cell proliferation. Experiment 3 was designed to evaluate initial responses at 2, 3, or 4 wk of age. Additional littermates from four of the litters evaluated at 2 wk of age were hemicastrated at 8 days of age. Testis weight increased approximately 50% in the flutamide-treated boars compared with vehicle-treated littermates (P = 0.01) by 6.5 wk of age. Approximately 80% more Sertoli cells/testis were present in flutamide-treated boars at 6.5 wk of age compared with their vehicle-treated littermates (P < 0.01). Animals that were hemicastrated at 8 days of age had more Sertoli cells/testis than their intact littermates at 2 wk of age (P < 0.01), but flutamide inhibited the hemicastration response. Androgen receptor antagonism during postnatal Sertoli cell proliferation increases Sertoli cell numbers, as does hemicastration, but receptor antagonism initially inhibits Sertoli cell proliferation induced by hemicastration.

Steroidogenic enzyme activities in the pre- and post-parturient equine placenta

Steroidogenic enzymes in placentas shape steroid hormone profiles in the maternal circulation of each mammalian species. These include 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 and its metabolites decline dramatically days before foaling, but few studies have investigated placental enzyme activity before or at parturition in mares. Thus, key enzyme activities and transcript abundance were investigated in equine placentas at 300 days of gestation (GD300) and post-partum (term). Equine testis was used as a positive control for P450c17 activity. Substrates were incubated with microsomal preparations, together with enzyme inhibitors, and products were measured by liquid chromatography tandem mass spectrometry or radiometric methods (aromatase). Equine placenta expressed high levels of 3βHSD, 5α-reductase and aromatase, and minimal P450c17 activity at GD300 compared with testis (600-fold higher). At foaling, 3βHSD and aromatase activities and transcript abundance were unchanged but 5α-reductase (and P450c17) was no longer detectable (P < 0.05) and transcript was decreased. Trilostane inhibited 3βHSD significantly more in testis than placenta, suggesting possible existence of different 3βHSD isoforms. Equine placentas have significant capacity for steroid metabolism by 5α-reductase, 3βHSD and aromatase but little for androgen synthesis lacking P450c17. Declining pre-partum 5α-reduced pregnane concentrations coincide with selective loss of placental 5α-reductase activity and expression at parturition in horses.

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

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

Ovine placental steroid synthesis and metabolism in late gestation

Abstract Steroid synthesis is required for pregnancy maintenance and for parturition, but comparatively little is known about the major metabolic routes that influence circulating concentrations. Dietary intake changes progesterone and estradiol concentrations in pregnant ewes but whether this reflects placental synthesis is unknown. Progesterone metabolism by 5alpha-reduction is a major metabolic route in other species and can influence the onset of parturition. Therefore, studies were conducted to (1) determine placental enzyme activity, progesterone, and estradiol measured by immunoassay in late gestation ewes on low-, moderate-, and high-nutritional planes, (2) to assess the significance of 5alpha-reduction of progesterone in determining progesterone concentrations in late gestation ewes (gestation day 145) given finasteride to inhibit 5alpha-reductase metabolism. In the second experiment, steroid profiles were examined comprehensively in blood and tissues by liquid chromatography tandem mass spectrometry for the first time in this species. Dietary intake altered progesterone and estradiol serum concentrations but without correlated changes in placental 3beta-hydroxysteroid dehydrogenase, 17alpha-hydroxylase/17,20-lyase cytochrome P450 or aromatase activity. 5alpha-reduced pregnane metabolites were identified in ewes at 145 days of gestation, but concentrations were lower than those of progesterone. Finasteride inhibited 5alpha-reduced progesterone metabolism but did not impact serum progesterone concentrations in these ewes. We conclude that (1) diet-induced changes in serum progesterone and estradiol concentrations are not likely a result of altered placental synthesis of sex steroid but most likely by their metabolism, and (2) metabolism by 5α-reduction is not a major determinant of systemic progesterone concentrations in late gestation ewes. Summary Sentence In pregnant sheep, diet affects systemic progesterone and estradiol concentrations but not by altering placental synthesis, and although pregnane metabolism includes 5α-reduction, it is not an important determinant of progesterone concentration.