Xiao-Chi Jia

Granulosa cells as hormone targets : the role of biologically active follicle-stimulating hormone in reproduction

Publisher Summary This chapter describes that with recent advances in cellular and molecular biology, significant progress has been made to elucidate the effect of diverse hormones at the ovarian granulosa cells. Follicle-stimulating hormone (FSH) is the primary regulator of granulosa cell differentiation. The mechanisms by which FSH induces the expression of multiple genes associated with feedback actions, ovulation, steroidogenesis, and other differentiated functions are studied. The chapter highlights the reception, action, and local synthesis of various growth factors and further explains the paracrine and autocrine roles of these growth factors in the modulation of FSH actions at the granulosa cells. The granulosa cell aromatase bioassay (GAB) allows measurement of serum levels of bioactive FSH in diverse species. The characterization of bioactive FSH molecules secreted by eukaryotic cell lines transfected with human FSH genes provides clinically useful reagents for stimulating follicle maturation and spermatogenesis.

Sensitive in vitro Bioassay for the Measurement of Serum Follicle-Stimulating Hormone

A sensitive in vitro bioassay for measuring serum follicle-stimulating hormone (FSH) levels has been developed based on the stimulation of estrogen production by cultured rat granulosa cells. In the presence of androstenedione, FSH stimulated estrogen production in a dose-dependent manner. Cell sensitivity to FSH was further enhanced by the addition of diethylstilbestrol, a phosphodiesterase inhibitor, insulin, and human chorionic gonadotropin. Although the inclusion of 4% gonadotropin-free serum from hypophysectomized rats inhibited granulosa cell responsiveness, pretreatment of serum with polyethylene glycol (12%) substantially eliminated the serum interfering effect without loss of FSH activity. In normal male and female rats, serum bioactive FSH levels were low, while castration increased these levels 2.6-and 5.7-fold, respectively. These increases were suppressed by treatment with testosterone propionate or estradiol benzoate. This in vitro assay allows the measurement of circulating bioactive FSH and provides a convenient tool to advance studies on the role of FSH in the neuroendocrine control of gonadal maturation and reproductive cycles.

Granulosa cell aromatase bioassay for follicle-stimulating hormone

Publisher Summary Follicle-stimulating hormone (FSH) is required for the maturation of ovarian follicles and testicular tubules during pubertal development. This chapter describes the granulosa cell aromatase (estrogen synthetase) bioassay (GAB) and its applications to the measurement of FSH bioactivities. The extreme sensitivity of the present in vitro bioassay allows for the measurement of circulating or urinary levels of bioactive FSH. Measurement of serum and urine levels of bioactive FSH should provide insight regarding the role of FSH in various physiological, pharmacological, and pathophysiological conditions. Because rat granulosa cells respond to FSH preparations from different species, this in vitro assay also provides valuable information on FSH levels in diverse animal species, including those that lack a specific radioimmunoassay. Because of its low sensitivity, the classic Steelman–Pohley FSH bioassay cannot be used to measure serum FSH levels. For urine samples, up to 1 liter of urine is extracted for the in vivo bioassay.

Synergistic effect of glucocorticoids and androgens on the hormonal induction of tissue plasminogen activator activity and messenger ribonucleic acid levels in granulosa cells

Tissue-type plasminogen activator (tPA) is secreted by rat granulosa cells in response to treatment with activators of protein kinase A (follitropin, FSH), protein kinase C (gonadotropin-releasing hormone, GnRH) and tyrosine kinase (epidermal growth factor, EGF). Because steroid hormones have been shown to enhance the gonadotropin stimulation of ovarian differentiation, we investigated the effects of steroid hormones, alone or together with various kinase activators, on tPA activities and mRNA levels in cultured rat granulosa cells. Treatment of cells with dexamethasone (DEX; a glucocorticoid agonist) or R1881 (an androgen agonist) caused an increase in tPA secretion and mRNA levels. In addition, the stimulation of tPA activity and mRNA levels by FSH (50 ng/ml) was synergistically enhanced by cotreatment with DEX or R1881 in a time-dependent manner with 2.8- and 1.6-fold increase at 9 h after incubation as compared to cells treated with FSH alone. In contrast, treatment with diethylstilbestrol had no effect on tPA levels. Furthermore, tPA activity and mRNA levels induced by GnRH and EGF were also increased by cotreatment with DEX or R1881 as compared with cells treated with GnRH or EGF alone. Likewise, the stimulation of tPA mRNA levels by dibutyryl cAMP, a protein kinase A activator, and phorbol myristate acetate (PMA), a protein kinase C activator, was enhanced by cotreatment with DEX or R1881. These results demonstrate that glucocorticoid and androgen enhance tPA secretion and mRNA levels stimulated by FSH, GnRH and EGF in granulosa cells. The rat granulosa cells provide a useful model for studying the mechanism of regulation of tPA gene expression by steroid hormones.

Forskolin and phosphodiesterase inhibitors stimulate rat granulosa cell differentiation

The effect of forskolin (an adenyl cyclase activator) and 1-methyl-3-isobutylxanthine (MIX, a phosphodiesterase inhibitor) on granulosa cell steroidogenesis and LH receptor formation was studied in vitro. Granulosa cells from immature hypophysectomized, estrogen-treated rats were cultured for 2-3 days in androstenedione-supplemented media in the absence or presence of FSH or forskolin (10(-7)-10(-4) M). Some cultures were also treated with forskolin with or without MIX (0.125-1.0 mM) or theophylline (1.25-10 mM). Forskolin (3 X 10(-6)-10(-4) M) stimulated the production of estrogen, progesterone, 20 alpha-hydroxypregn-4-en-3-one (20 alpha-OH-P) and cAMP in a dose-related manner to levels similar to or higher than that elicited by FSH alone. Similarly, forskolin and FSH both increased LH/hCG receptor content in cultured granulosa cells, although forskolin was only 50% as effective as FSH. Treatment with MIX alone increased basal levels of cAMP, accompanied by elevations of estrogen and progestin biosynthesis without affecting LH/hCG receptor content. In contrast, theophylline treatment only increased cAMP and progestin accumulation. Furthermore, MIX potentiated the stimulatory effects of forskolin and FSH on cAMP and progestin production. In contrast, MIX inhibited FSH- and forskolin-stimulated estrogen production. Thus, activation of adenyl cyclase and inhibition of cAMP breakdown in the cultured rat granulosa cells enhance steroidogenesis and LH receptor formation, reinforcing the concept that cAMP is a (but may not be the only) second messenger in the hormonal regulation of granulosa cell differentiation.

The Ovarian Granulosa Cell as a Follicle-Stimulating Hormone Target Tissue

The ovarian follicle is the basic functional unit of the ovary, and is comprised of an outer layer of theca cells, separated by a basement membrane from the inner layer of granulosa cells, which in turn surround the oocyte-cumulus cell complex. In response to the pituitary gonadotropins, follicle-stimulating hormone (FSH), luteinizing hormone (LH), and prolactin (PRL), the follicle is able to differentiate with regard to steroidogenesis and hormone receptor induction, as well as to produce a fertilizable ovum and adequate corpus luteum. The key anterior pituitary glycoprotein in the initiation of follicular maturation and granulosa cell differentiation is FSH. During reproductive life, FSH is released in an orchestrated cyclic fashion subject to endocrine control.

Regulation of LH and FSH Receptor Gene Expression in the Ovary and Testis

The action of gonadotropins is mediated through binding of these glycoprotein hormones to specific receptors on gonadal cells. In the testis, follicle stimulating hormone receptors (FSH-R) are located in Sertoli cells, while luteinizing hormone receptors (LH-R) are found exclusively in Leydig cells (reviewed in 1). In the ovary, granulosa cells contain the receptor for FSH, while granulosa, theca, and luteal cells contain LH-R (reviewed in 2). The binding of the respective ligand to the specific cell surface receptor initiates the transduction of the signal from the extracellular space into the cells, with resultant activation of specific genes (reviewed in 3).