Journal of Clinical Endocrinology & Metabolism, Vol 71, 26-33, Copyright © 1990 by Endocrine Society

ARTICLES

Proliferating human granulosa-lutein cells in long term monolayer culture: expression of aromatase, cholesterol side-chain cleavage, and 3 beta-hydroxysteroid dehydrogenase

JM McAllister, JI Mason, W Byrd, JM Trant, MR Waterman and ER Simpson
Cecil H. and Ida Green Center for Reproductive Biology Sciences, University of Texas Southwestern Medical Center, Dallas 75235-9051.

The development of long term culture conditions with which to study the regulation of expression of aromatase, cholesterol side-chain cleavage enzyme, and 3 beta-hydroxysteroid dehydrogenase (3 beta HSD) in human granulosa-lutein cells is described in this report. Conditions have been established for the dispersal, growth, freezing, and storage of functional human granulosa cells isolated from preovulatory follicles of women undergoing laparoscopy for gamete intrafallopian tube transfer and in vitro fertilization procedures. Optimal growth conditions for human granulosa-lutein cells were determined by plating cells at a low density and testing the capacity of a variety of culture conditions to support growth. A combination of fetal bovine serum (FBS), horse serum, and the serum substitute UltroSer G was found to increase cell number to maximal levels, 8- to 10-fold higher than with sera alone. Human granulosa-lutein cells grown under these conditions had a doubling rate of 36-40 h and were morphologically distinct from human theca interna cells grown under similar conditions. Human granulosa-lutein cells treated with forskolin retracted and rounded up, whereas cultures of human ovarian theca interna cells or human fibroblasts treated similarly did not retract. Human granulosa-lutein cells were grown for successive passages and transferred to serum-free medium containing forskolin, LH, hCG, or cholera toxin. Addition of these agents resulted in a time- and dose-dependent increase in aromatase activity and progesterone secretion. In these studies FSH treatment was found not to increase aromatase activity. In a study of the time course of 3 beta HSD activity in the absence of forskolin under serum-free conditions, it was found that 3 beta HSD activity increased 3-fold during the 72-h treatment period. Forskolin-stimulated 3 beta HSD activity also increased in a time-dependent manner, with levels in treated cells 3- fold higher than those in control cells. Northern analysis performed on total RNA obtained from forskolin- or hCG-stimulated granulosa-lutein cells confirmed that the increase in aromatase activity was associated with a corresponding increase in levels of mRNA specific for aromatase cytochrome P-450. Levels of mRNA encoding cholesterol side-chain cleavage cytochrome P-450 were similarly increased in cells treated with forskolin compared with unstimulated values at each of the time points investigated. Under serum-free conditions in the absence of stimulation, the 3.4-kilobase band of aromatase cytochrome P-450 mRNA was detectable.(ABSTRACT TRUNCATED AT 400 WORDS)

This article has been cited by other articles:

				J. Simard, M.-L. Ricketts, S. Gingras, P. Soucy, F. A. Feltus, and M. H. Melner Molecular Biology of the 3{beta}-Hydroxysteroid Dehydrogenase/{Delta}5-{Delta}4 Isomerase Gene Family Endocr. Rev., June 1, 2005; 26(4): 525 - 582. [Abstract] [Full Text] [PDF]

				J. W. Kim, N. Peng, W. E. Rainey, B. R. Carr, and G. R. Attia Liver Receptor Homolog-1 Regulates the Expression of Steroidogenic Acute Regulatory Protein in Human Granulosa Cells J. Clin. Endocrinol. Metab., June 1, 2004; 89(6): 3042 - 3047. [Abstract] [Full Text] [PDF]

				Y. Nishi, T. Yanase, Y.-M. Mu, K. Oba, I. Ichino, M. Saito, M. Nomura, C. Mukasa, T. Okabe, K. Goto, et al. Establishment and Characterization of a Steroidogenic Human Granulosa-Like Tumor Cell Line, KGN, That Expresses Functional Follicle-Stimulating Hormone Receptor Endocrinology, January 1, 2001; 142(1): 437 - 445. [Abstract] [Full Text] [PDF]

				L. Devoto, L. K. Christenson, J. M. McAllister, A. Makrigiannakis, and J. F. Strauss III Insulin and insulin-like growth factor-I and- II modulate human granulosa-lutein cell steroidogenesis: enhancement of steroidogenic acute regulatory protein (StAR) expression Mol. Hum. Reprod., November 1, 1999; 5(11): 1003 - 1010. [Abstract] [Full Text] [PDF]

				V. L. Nelson, R. S. Legro, J. F. Strauss III, and J. M. McAllister Augmented Androgen Production Is a Stable Steroidogenic Phenotype of Propagated Theca Cells from Polycystic Ovaries Mol. Endocrinol., June 1, 1999; 13(6): 946 - 957. [Abstract] [Full Text]

				B. C. J. M. Fauser and A. M. van Heusden Manipulation of Human Ovarian Function: Physiological Concepts and Clinical Consequences Endocr. Rev., February 1, 1997; 18(1): 71 - 106. [Abstract] [Full Text]

				P. Vigano, B. Gaffuri, G. Ragni, A. M. Di Blasio, and M. Vignali Intercellular Adhesion Molecule-1 is Expressed on Human Granulosa Cells and Mediates Their Binding to Lymphoid Cells J. Clin. Endocrinol. Metab., January 1, 1997; 82(1): 101 - 105. [Abstract] [Full Text] [PDF]