HF-0299

The Siberian sturgeon (Acipenser baerii) is a com.-important fish.As it is the females that produce the highly-prized caviar, genetic monosex cultures would be valuable.Before such populations can be produced, it will first be necessary to formally confirm whether this species has a ZZ/ZW sex-determination system.This discovery will in turn require the development of successful sex inversion and progeny testing approaches.This work is a first step in advancing this strategy.Our aim was to induce gonadal transdifferentiation using two steroids found naturally in fish -estradiol-17β (E2) and 11β-hydroxyandrostenedione (11β-And)- to feminize and masculinize genetic males and females, resp.Steroidal treatments were initiated at 3.5 mo of age, using 5μg/g body weight (b.w.) steroid injections; control fish received vehicle injections.Injections were given every 3 wk for 7 mo, concluding at 10.5 mo.Genetic and histol. tests were performed at 3.5, 6, 9, and 12 mo of age.All animals remained histol. undifferentiated at 3.5 mo.Control females showed signs of sex differentiation at 6 mo (columnar epithelium with distinct folds), and at 9 and 12 mo, control female and male gonads showed nests of oogonia and scattered spermatogonia, resp.The E2 treatment induced male-to-female gonad transdifferentiation in the genetically-sexed males at 6, 9, and 12 mo of age.In contrast, 11β-And failed to induce transdifferentiation of the female gonads.While some transient changes were observed, such as increased connective tissue between female germ cell clusters at 9 mo, the 12-mo-old fish displayed a normal female phenotype with no signs of sex inversion.Importantly, the exptl. E2 treatment produced live phenotypic females transdifferentiated from genetic males, with normal growth, feeding, and mobility as compared with control fish.Further experiments are needed to attempt masculinization of females, potentially with higher doses of natural androgens or other hormones and chems.

Mass spectrometric-based steroidomics is a valuable analytical approach that gives a comprehensive understanding of the interlinked steroid biosynthetic pathways. Here, we describe a rapid and versatile liquid chromatography-tandem mass spectrometry (LC-MS/MS) method designed to accurately quantify endogenous steroids in human serum. Sample preparation involved liquid-liquid extraction with methyl tert-butyl ether (MTBE) from 180 µL serum. The targeted steroids for quantification included androgens: dehydroepiandrosterone (DHEA), androstenedione (A4), testosterone (T), dihydrotestosterone (DHT), 11-oxyandrogens: 11β-hydroxy-androstenedione (11OHA4), 11-keto-androstenedione (11KA4), 11β-hydroxy-testosterone (11OHT), 11-keto-testosterone (11KT), progestogens: 17α-hydroxy-progesterone (17OHP4), progesterone (P4), 11β-hydroxy-progesterone (11OHP4), 11-keto-progesterone (11KP4), mineralocorticoids: aldosterone, corticosterone, and glucocorticoids: 11-deoxycortisol, cortisol, and cortisone. The lower limits of quantification (LLOQ) were 0.05 ng/mL for A4, T, 11KA4, P4, and cortisone, 0.1 ng/mL for DHT, 11OHA4, 11OHT, 11KT, 17OHP4, 11OHP4, 11KP4, corticosterone, aldosterone, 11-deoxycortisol, and cortisol, and 0.5 ng/mL for DHEA. Accuracy, precision, reproducibility, and recovery fell within acceptable limits for bioanalytical method validation. Using serum samples from 29 premenopausal women in different menstrual phases, we demonstrated the clinical utility of our method, which showed sufficient sensitivity to reliably quantify all targeted steroids at levels typically found in circulation, except for 11OHP4 and 11KP4.