Jeffrey V. May

Basic fibroblast growth factor as a regulator of ovarian granulosa cell differentiation: a novel non-mitogenic role

The role of basic fibroblast growth factor (bFGF) in ovarian granulosa cell differentiation was investigated in vitro. To this end, use was made of a primary culture of rat granulosa cells the differentiation of which was monitored by the acquisition of aromatase activity. Concurrent treatment with highly purified bFGF (10 ng/ml) produced a significant (P less than 0.05), albeit reversible inhibition (88 +/- 6%) of FSH (but not basal)-supported aromatization. Although independent of the FSH dose employed and of cell density, bFGF-attenuated aromatase activity proved dose-dependent, with a projected minimal effective dose of 0.11 +/- 0.03 ng/ml (7.5 +/- 2 pM), and an apparent median inhibitory dose of 0.63 +/- 0.09 ng/ml (43 +/- 6 pM). Unaccounted for by alterations in granulosa cell number, plating efficiency, or viability, the ability of bFGF to attenuate FSH hormonal action proved partly attributable to site(s) of action distal, rather than proximal to cAMP generation. Taken together, these observations indicate that the cytodifferentiative and replicative actions of bFGF in the granulosa cell can be dissociated, and lend additional support to the prospect that bFGF, possibly of intraovarian origin, may play a role in granulosa cell differentiation in the course of their ontogeny.

INTERACTIONS BETWEEN HORMONES AND GROWTH FACTORS IN THE REGULATION OF GRANULOSA CELL DIFFERENTIATION IN VITRO

Evidence from studies in vitro supports the concept that growth factors modulate endocrine-dependent differentiative processes in follicle development. Based upon results from granulosa cell culture systems, it is suggested that the study of growth factors and their regulatory mechanisms (endocrine, paracrine, autocrine control) could perhaps be generalized to other areas concerned with the regulation of steroid secretion such as placental physiology, the regulation of fetal gonads and puberty, secondary steroid metabolism and steroid-secreting tumors.

The effect of plating density on granulosa cell growth and differentiation in vitro

The extent of FSH-mediated LH/hCG receptor induction and of basal and FSH-stimulated progesterone production by porcine granulosa cells in vitro, in serum-containing medium, is directly related to the plating density. Relative to pre-culture levels, low- and high-density cultures of cells routinely exhibited 1-2- and 10-11-fold increases in [125I]iodo-hCG binding, respectively. Monolayer growth, i.e. cell division, as measured by increases in cell protein or DNA content, was inversely related to plating density. This density-directed inverse relationship between growth and differentiation did not appear to be coupled under the conditions utilized. Whereas monolayer growth was dependent upon the cell surface density, i.e. the number of cells per unit surface area, differentiation was dependent upon cell concentration, i.e. cells per unit volume of medium. Cells plated at low density in medium containing 10% serum exhibited 50% less [125I]iodo-hCG binding than cells in 5% serum (P less than 0.025). Conversely, cells plated at high density exhibited a 14% increase (P less than 0.025) in binding at the higher serum level. Thus, it appears that the extent of differentiation depends upon the capacity of cells to neutralize serum inhibition which in turn is dependent upon the cell concentration. Serum neutralization by granulosa cells is an FSH-dependent process. Conditioned medium derived from insulin-treated, high-density cultures did not facilitate optimum LH/hCG receptor induction in low-density cultures. Conditioned medium from cultures treated with insulin plus FSH, however, facilitated LH/hCG receptor induction in low-density cultures to the same extent as obtained in high-density cultures. The enhancement by FSH-conditioned medium cannot be attributed to residual FSH or to dilution of serum components during the preparation of the conditioned medium. The phenomena of serum-attenuated granulosa cell differentiation in vitro, and of a density-dependent reversal of this process, may have regulatory implications in vivo since follicular fluid contains many serum components and since the granulosa cell complement is an important determinant of follicle maturation.

A model system for the biochemical study of luteinizing hormone/chorionic gonadotropin receptor synthesis

A model system for the biochemical study of LH/CG receptor synthesis has been developed. Culture conditions for porcine granulosa cells were adapted that maximized the selective induction of LH/CG receptors by cAMP-inducing stimuli with an elimination of background LH/CG receptor appearance. It was found that the addition of FSH (1.5 micrograms/ml) or cholera toxin (10 ng/ml) 1 day after plating resulted in optimal induction of the LH/CG receptor (20-60 pg [125I]CG bound/micrograms DNA 72 h after addition) with virtually no LH/CG receptor appearance in the absence of added stimuli. Later additions of FSH or cholera toxin required insulin (1.0 microgram/ml) which alone caused background LH/CG receptor appearance in the absence of any additional stimuli. Furthermore, insulin increased the general rate of cellular protein synthesis, whereas FSH or cholera toxin each decreased it. Thus, the use of FSH or cholera toxin, without insulin, may enable one to detect the synthesis of the LH/CG receptor by metabolic labeling techniques where background protein synthesis has been lowered.