Exploring the human chorionic gonadotropin induced steroid secretion profile of mouse Leydig tumor cell line 1 by a 20 steroid LC-MS/MS panel

Flaminia Fanelli; Matteo Magagnoli; Marco Mezzullo; Monica Lispi; Silvia Limoncella; Alessia Tommasini; Carla Pelusi; Daniele Santi; Manuela Simoni; Uberto Pagotto; Livio Casarini

doi:10.1016/j.jsbmb.2023.106270

Exploring the human chorionic gonadotropin induced steroid secretion profile of mouse Leydig tumor cell line 1 by a 20 steroid LC-MS/MS panel

J Steroid Biochem Mol Biol. 2023 May:229:106270. doi: 10.1016/j.jsbmb.2023.106270. Epub 2023 Feb 9.

Authors

Affiliations

¹ Endocrinology research group, Center for Applied Biomedical Research, Department of Medical and Surgical Sciences, Alma Mater Studiorum University of Bologna, Italy. Electronic address: flaminia.fanelli2@unibo.it.
² Endocrinology research group, Center for Applied Biomedical Research, Department of Medical and Surgical Sciences, Alma Mater Studiorum University of Bologna, Italy.
³ International Ph.D. School in Clinical and Experimental Medicine (CEM), University of Modena and Reggio Emilia, Modena, Italy; Global Medical Affair, Merck KGaA, Darmstadt, Germany.
⁴ Unit of Endocrinology, Department of Biomedical, Metabolic and Neural Sciences, University of Modena and Reggio Emilia. Ospedale di Baggiovara, Via P. Giardini 1355, 41126 Modena, Italy.
⁵ Endocrinology research group, Center for Applied Biomedical Research, Department of Medical and Surgical Sciences, Alma Mater Studiorum University of Bologna, Italy; Endocrinology and Prevention and Care of Diabetes Unit, IRCCS Azienda Ospedaliero-Universitaria di Bologna, Italy.
⁶ Unit of Endocrinology, Department of Biomedical, Metabolic and Neural Sciences, University of Modena and Reggio Emilia. Ospedale di Baggiovara, Via P. Giardini 1355, 41126 Modena, Italy; Unit of Endocrinology, Department of Medical Specialties, Azienda Ospedaliero-Universitaria of Modena. Ospedale di Baggiovara, Via P. Giardini 1355, 41126 Modena, Italy.
⁷ Unit of Endocrinology, Department of Biomedical, Metabolic and Neural Sciences, University of Modena and Reggio Emilia. Ospedale di Baggiovara, Via P. Giardini 1355, 41126 Modena, Italy; Unit of Endocrinology, Department of Medical Specialties, Azienda Ospedaliero-Universitaria of Modena. Ospedale di Baggiovara, Via P. Giardini 1355, 41126 Modena, Italy; Center for Genomic Research, University of Modena and Reggio Emilia, Via G. Campi 287, 41125 Modena, Italy.
⁸ Unit of Endocrinology, Department of Biomedical, Metabolic and Neural Sciences, University of Modena and Reggio Emilia. Ospedale di Baggiovara, Via P. Giardini 1355, 41126 Modena, Italy; Center for Genomic Research, University of Modena and Reggio Emilia, Via G. Campi 287, 41125 Modena, Italy.

PMID: 36764496
DOI: 10.1016/j.jsbmb.2023.106270

Abstract

The canonical androgen synthesis in Leydig cells involves Δ5 and Δ4 steroids. Besides, the backdoor pathway, eompassing 5α and 5α,3α steroids, is gaining interest in fetal and adult pathophysiology. Moreover, the role of androgen epimers and progesterone metabolites is still unknown. We developed a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for measuring 20 steroids and used it to investigate the steroid secretion induced by human chorionic gonadotropin (hCG) in the mouse Leydig tumor cell line 1 (mLTC1). Steroids were extracted from 500 µL supernatants from unstimulated or 100 pM hCG-exposed mLTC1 cells, separated on a Luna C8 100 × 3 mm, 3 µm column, with 100 µM NH4F and methanol as mobile phases, and analyzed by positive electrospray ionization and multiple reaction monitoring. Sensitivity ranged within 0.012-38.0 nmol/L. Intra-assay and inter-assay imprecision were < 9.1% and 10.0%, respectively. Trueness, recovery and matrix factor were within 93.4-122.0, 55.6-104.1 and 76.4-106.3%, respectively. Levels of 16OH-progesterone, 11-deoxycortisol, androstenedione, 11-deoxycorticosterone, testosterone, 17OH-progesterone, androstenedione, epitestosterone, dihydrotestosterone, progesterone, androsterone and 17OH-allopregnanolone were effectively measured. Traces of 17OH-dihydroprogesterone, androstanediol and dihydroprogesterone were found, whereas androstenediol, 17OH-pregnenolone, dehydroepiandrosterone, pregnenolone and allopregnanolone showed no peak. hCG induced an increase of 80.2-102.5 folds in 16OH-progesterone, androstenedione and testosterone, 16.6 in dihydrotestosterone, 12.2-27.5 in epitestosterone, progesterone and metabolites, 8.1 in 17OH-allopregnanolone and ≤ 3.3 in 5α and 5α,3α steroids. In conclusion, our LC-MS/MS method allows exploring the Leydig steroidogenesis flow according to multiple pathways. Beside the expected stimulation of the canonical pathway, hCG increased progesterone metabolism and, to a low extent, the backdoor route.

Keywords: Androgen; Backdoor pathway; Leydig cells; Liquid chromatography – tandem mass spectrometry; Progesterone.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Animals
Cell Line, Tumor
Chorionic Gonadotropin* / pharmacology
Chromatography, Liquid / methods
Gonadal Steroid Hormones* / analysis
Gonadal Steroid Hormones* / metabolism
Humans
Leydig Cells* / drug effects
Leydig Cells* / metabolism
Male
Mice
Tandem Mass Spectrometry / methods

Substances

Chorionic Gonadotropin
Gonadal Steroid Hormones