Contents lists available at ScienceDirect General and Comparative Endocrinology journal homepage: www.elsevier.com/locate/ygcen Comparative analysis of steroids in cyclic and pregnant killer whales, beluga whales and bottlenose dolphins by liquid chromatography tandem mass spectrometry Erin L. Legacki a,1 , Todd R. Robeck b , Karen J. Steinman b , Alan J. Conley a, ⁎ a Department of Population Health & Reproduction, School of Veterinary Medicine, University of California, Davis, CA 95616, USA b SeaWorld Parks and Entertainment, Inc., SeaWorld and Busch Gardens Species Preservation Laboratory, San Diego, CA 92109, USA ARTICLE INFO Keywords: Cetacean Steroids Pregnancy Non-pregnant Pregnanes Androgens ABSTRACT There exists a surprising diversity in the physiology and endocrinology of pregnancy among mammals in both the source (luteal/placental) and metabolism of progesterone. To evaluate the possible diversity of steroid metabolism within toothed cetaceans, we investigated 5α-reduced progesterone metabolites and androgens in cyclic (luteal phase) and pregnant captive killer whales, belugas and bottlenose dolphins (n = 5/species) bled longitudinally in early, mid- and late pregnancy (0.16, 0.50 and 0.85 fractions of 535, 464 and 380 gestation days, respectively). Mid-luteal samples were also collected. Serum was analyzed by liquid chromatography tandem-mass spectrometry as previously validated for (among others) progesterone, 20αOH-progesterone (20αOHP), 5α-dihydroprogesterone (DHP), several additional 5α-reduced metabolites and androgens (dehy- droepiandrosterone, androstenedione and testosterone). The predominant mid-luteal pregnanes were: proges- terone, belugas; progesterone and 20αOHP, dolphins; allopregnanolone (3α-DHP) and progesterone, killer whales. Progesterone was 2-4-fold higher in early pregnancy than mid-luteal samples but decreased thereafter. The predominant metabolite, 3β,20α-dihydroprogesterone (3β,20α-DHP; 40–80 ng/ml) was higher in mid- and late-than early gestation in all 3 species. Concentrations of 20αOHP and 3β,20α-DHP were similar at mid- gestation but 20αOHP declined in late-gestation in killer whales, and 20αOHP was lower than 3β,20α-DHP in belugas and dolphins throughout gestation. Other 5α-reduced metabolites, DHP, 3α-DHP and 20α-DHP, were far lower throughout pregnancy (< 10 ng/ml). DHP and 3α-DHP decreased from early to mid-gestation in belugas, but changed little in killer whales and dolphins. These data suggest that progesterone metabolism is relatively conserved among these cetacean species. As in equine pregnancies, 3β,20α-DHP is the major metabolite, in- creasing at the expense of progesterone as pregnancy progresses. Androstenedione and testosterone also in- creased detectably in mid- to late-gestation in these species. The tissue source remains unknown, but proges- terone metabolism during gestation in these cetaceans is similar to horses and, together with androgens, may be reliable biomarkers of pregnancy. 1. Introduction Adaptation of terrestrial and semi-aquatic ancestral mammals to an entirely aquatic or marine existence has been accompanied by much studied morphological transitions and gene evolution among extant cetaceans including whales, dolphins and porpoises (Gingerich et al., 2001; Mancia, 2018; McGowen et al., 2014; Milinkovitch et al., 1993; Montgelard et al., 1997; Price et al., 2005; Thewissen et al., 2001). Much less is appreciated concerning cetacean reproduction in general or the endocrinology of pregnancy in particular, which in all mammals is considered to be dependent on progesterone secretion by corpora lutea or the placenta (Short, 1956; Short, 1960). However, progesterone is not the sole, physiologically significant, progestin in all mammalian species. In horses, progesterone disappears after 180–200 days of pregnancy which was hypothesized to be supported instead by the 5α- reduced metabolite of progesterone, 5α-dihydroprogesterone (DHP) (Holtan et al., 1991). Progesterone is an excellent substrate for the 5α- reductase enzyme, better than testosterone, suggesting that these en- zymes might be adapted more for female than for male reproduction (Scholtz et al., 2014). In recent in vivo and in vitro studies (Scholtz et al., https://doi.org/10.1016/j.ygcen.2019.113273 Received 21 May 2019; Received in revised form 10 September 2019; Accepted 12 September 2019 ⁎ Corresponding author. E-mail address: ajconley@ucdavis.edu (A.J. Conley). 1 Current address: National Institute of Standards and Technology, Charleston, SC. General and Comparative Endocrinology 285 (2020) 113273 Available online 13 September 2019 0016-6480/ © 2019 Elsevier Inc. All rights reserved. T