This Article Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Copyright Permission Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by GARRETT, W. M. Articles by ANDERSON, L. D. Search for Related Content PubMed PubMed Citation Articles by GARRETT, W. M. Articles by ANDERSON, L. D.

Androgen Metabolism by Porcine Granulosa Cells during the Process of Luteinization in Vitro: Identification of 19-Oic-Androstenedione as a Major Metabolite and Possible Precursor for the Formation of C₁₈ Neutral Steroids^*

Department of Anatomy, University of Maryland School of Medicine Baltimore, Maryland 21201;
Children’s Hospital, Oakland Research Institute Oakland, California 94609

Address all correspondence and requests for reprints to: Dr. Larry D. Anderson, Department of Anatomy, University of Maryland School of Medicine, 655 West Baltimore Street, Baltimore, Maryland 21201.

Abstract

The present studies were conducted to define the pathway(s) by which androstenedione is metabolized in porcine granulosa cells (pGC) and determine whether metabolism of this steroid is affected by in vitro luteinization. pGC isolated from large preovulatory follicles were cultured for up to 2 days, in the presence of 5 µM unlabeled or [4-¹⁴C]-labeled androstenedione. Metabolism of androstenedione was assessed by HPLC, using in-line liquid scintillation detection. Metabolite identification was confirmed by gas chromatography-mass spectrometry of HPLC fractions isolated from medium conditioned by granulosa cells (pGCCM) cultured for 48 h in the presence of unlabeled androstenedione. The metabolites identified were 19- oic-androstenedione (3,17-dioxo-4-androsten-19-oic acid), 19- hydroxytestosterone, 19-hydroxyandrostenedione, 19-nor-testosterone, an estrenolone of as yet unproven stereoisomerism, 5(10)-estrene-3β,17β-diol, 17β-estradiol, testosterone, and 19-nor-androstenedione. Results indicate that 19-nor-androstenedione is artifactually derived from 19-oic-androstenedione as a result of degradation in storage and during isolation.

After metabolite identification, studies of the time course of androstenedione metabolism by pGC during in vitro luteinization were conducted. 17β-Estradiol and 19-oic-androstenedione were the predominant metabolites, and accumulation of these steroids was virtually identical. Production of these metabolites was maximal during the first 12 h of culture. The accumulation of 5(10)-estrene-3β,17β-diol and 19-nor-testosterone was maximal at 48 h of culture, with 5(10)-estrene-3β,17β-diol consistently accumulating in greater concentrations than 19-nor-testosterone. Aromatase activity of pGC was negligible from 36β48 h of culture, as demonstrated by minimal accumulation of 17β- estradiol during this period of culture. The accumulation of 19-oic-androstenedione, 5(10)-estrene-3β,17β-diol, and 19-nor-testosterone was also negligible during this latter time period, suggesting that their formation is associated with aromatase.

From these results, pGC from preovulatory follicles undergoing luteinization in vitro lose the ability to convert androstenedione to estrogens. The formation of 19-oic-androstenedione, shown here for the first time, parallels the formation of 17β- estradiol, and this acidic steroid is proposed to be a product of aromatase. As reported in previous studies, pGC do produce Ci8 neutral steroids from exogenous androstenedione. The production of these steroids requires an active aromatase to produce their immediate precursor, which is here hypothesized to be 19- oic-androstenedione. However, their maximal production does not commence until aromatase activity has declined, and it is hypothesized that their production depends on modifications in steroid metabolism associated with luteinization. (Endocrinology 129: 2941β2950, 1991)

Footnotes

^* This work was supported in part by NIH Grants HD-17179 and HD-08834 (to L.D.A.) and DK-34400 (to C.H.L.S.). This work was performed in partial fulfillment of the requirements for a Doctorate of Philosophy (W.M.G.).

Received June 13, 1991.

This article has been cited by other articles:

				Y. Hong, B. Yu, M. Sherman, Y.-C. Yuan, D. Zhou, and S. Chen Molecular Basis for the Aromatization Reaction and Exemestane-Mediated Irreversible Inhibition of Human Aromatase Mol. Endocrinol., February 1, 2007; 21(2): 401 - 414. [Abstract] [Full Text] [PDF]

				C. J. Corbin, S. M. Mapes, J. Marcos, C. H. Shackleton, D. Morrow, S. Safe, T. Wise, J. J. Ford, and A. J. Conley Paralogues of Porcine Aromatase Cytochrome P450: A Novel Hydroxylase Activity Is Associated with the Survival of a Duplicated Gene Endocrinology, May 1, 2004; 145(5): 2157 - 2164. [Abstract] [Full Text] [PDF]

				C. J. Corbin, J. M. Trant, K. W. Walters, and A. J. Conley Changes in Testosterone Metabolism Associated with the Evolution of Placental and Gonadal Isozymes of Porcine Aromatase Cytochrome P450 Endocrinology, November 1, 1999; 140(11): 5202 - 5210. [Abstract] [Full Text]