The dynamic steroid landscape of equine pregnancy mapped by mass spectrometry

Abstract

Liquid chromatography-tandem mass spectrometry (LC-MS/MS) allowed comprehensive analysis of various steroids detectable in plasma throughout equine gestation. Mares (nZ9) 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 D4 steroids. The 5a-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 (D5 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 D4 steroids dominated during ovarian stimulation by eCG, inversion of the ratio of progesterone: DHP (increasing 5a-pregnanes) marked the luteo-placental shift, D5 steroids defined fetal gonadal growth and 5a-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

“…The LC-MS/MS method used was developed and described previously (Legacki et al 2016). Briefly, reverse-phase gradient separation was performed on an Agilent UHPLC C18 analytical column (2.1 × 50 mm, 1.8 µm ps) with two mobile phases delivered at 0.4 mL/min, an injection volume of 20 µL and a column temperature of 40°C.…”

“…In the first experiment, this cell line was transfected transiently with an MMTV-luciferase expression plasmid responsive to steroid agonists. Cells were grown for 24 h and then incubated with increasing concentrations (0-300 nM) of progesterone and two of the most abundant pregnane metabolites present in high concentrations in late gestation mares, 20αOH-DHP (20αDHP) and 5α-pregnan-3β,20α-diol (3β,20αDHP) (Legacki et al 2016). Luciferase expression was measured 48 h later to assess progestogenic bioactivity.…”

“…Further, we developed an in vitro bioassay and showed that DHP was as biopotent as progesterone in activating the equine progesterone receptor but not the human progesterone receptor (Scholtz et al 2014). These data proved that DHP alone is sufficient to support equine pregnancies, but sampling was infrequent in late gestation and events around parturition were not well characterized (Scholtz et al 2014, Legacki et al 2016.…”

“…Studies in women (Pieber et al 2001) and mice (Condon et al 2003, Mendelson & Condon 2005 revealed that birth is not invoked by a decline in systemic progesterone but involves a decrease in expression or function of the classic nuclear progesterone receptors (Kastner et al 1990) in target tissues (Conneely et al 2003, Mesiano et al 2011 such as myometrium. In horses, the endocrinology of pregnancy is more complex (Conley 2016, Legacki et al 2016) than other animals and the mechanisms initiating parturition are a matter of speculation (Thorburn 1993, Silver 1994, Conley & Neto 2008, Fowden et al 2008. When rigorous methods were applied, progesterone disappeared from the circulation and was absent throughout much of the second half of gestation in mares (Short 1959).…”

“…Previous attempts to show bioactivity in vitro were unsuccessful (Ousey et al 2000). Some recently developed methods measure multiple pregnanes and other steroids throughout pregnancy with accuracy and specificity (Legacki et al 2016) using liquid chromatography-tandem mass spectrometry (LC-MS/ MS). The method included pregnenolone, the universal steroid substrate, which was predicted to provide information on changes in the overall rate of pregnane synthesis at parturition.…”