Jerrold J. Heindel

Isolation and Culture of FSH Responsive Sertoli Cells

Methods for the isolation and culture of enriched populations of Sertoli cells from 20-60 day old rats are described. The identity of the Sertoli cells was verified by bright light and electron microscopy. Freshly isolated Sertoli cells specifically bound follicle stimulating hormone (FSH) but not luteinizing hormone (LH) and responded to FSH stimulation with dramatic increase in cyclic AMP level. Isolated Sertoli cells, maintained in culture for 11 days, showed no evidence of proliferation but retained their characteristic ultrastructural features and FSH binding ability. Incubation of cultured cells with FSH resulted in a significant stimulation of cyclic AMP and androgen binding protein (ABP). Since the freshly isolated or cultured cells were predominantly (greater than 80%) Sertoli cells, these results provide direct evidence that the Sertoli cells represent a primary target site for FSH activity in the testes. The culture method also provides a valuable in vitro model for the study of chronic effects of various agents on the Sertoli cell.

Effect of hypotonic treatment on sertoli cell purity and function in culture

SummaryCommonly used enzymic methods for the isolation of rat Seroli cells yield populations containing ∼15% germ cells. Although the germ cells become eliminated after several media changes, they could interferen with the use of Sertoli cells for critical studies during the first several days of culture. A brief treatment of Sertoli cell monolayer cultures with 20 mM Tris-HCl (pH 7.4) was found to eliminate most of the residual contaiminating germ cells. The duration of this treatment varied from 1.0 to 10 min, depending on cell denisty in the culture, the degree of germ cell contamination, and the age of animals used for Sertoli cell isolation. In a study of 95% pure 7-d Sertoli cell cultures, the hypotonic treatment did not alter the DNA or RNA content per dish or the incorporation of [3H]uridine into total and poly A+RNA. Also, the hypotonic treatment did not alter specific Sertoli cell functions, i. e., secretion of Sertoli cell factor (inhibin) and stimulation of cAMP levels by follicle stimuting hormone in 2-d cultures. Androgen receptor concentration per dish was also not changed. Changes in several general metabolic parameters observed after hypotonic treatment of 2-d cultures were attributed primarily to loss of contaiminating germ cells. Consequently, hypotonic treatment can be used to eliminate contaminating germ cells from the Sertoli cell cultures without apparent detrimental effects on a number of Sertoli cell biochemical parameters. This may be of considerable importance when the purity of Sertoli cells is critical for the interpretation of experimental data.

Identification and characterization of a β1-adrenergic receptor in the rat sertoli cell ☆ ☆☆

The beta 1-adrenergic receptor of rat Sertoli cells was characterized by measurement of the ability of adrenergic receptor agonists and antagonists to stimulate cAMP accumulation in Sertoli cells from 18-day-old rats. Epinephrine, norepinephrine and isoproterenol stimulate cAMP accumulation in Sertoli cells which is not additive with maximal doses of FSH and which is age-dependent, beta-antagonists, alprenolol, hydroxybenzylpindolol or propranolol inhibit isoproterenol-induced cAMP accumulation while alpha-adrenergic antagonists have no effect. Dobutamine and soterenol stimulated cAMP accumulation to a greater extent than albuterol and metoproterenol. Finally, the stimulatory effects of isoproterenol and zinterol are both more sensitive to inhibition by beta 1-antagonists than by beta 2-antagonists. Taken together these data indicate the presence of a beta 1-adrenergic receptor in Sertoli cells which is coupled to adenylate cyclase.

Hormone interactions in the Sertoli cells.

SummaryThe effects of follicle-stimulating hormone (FSH) and testosterone in rat Sertoli cells were investigated in vitro by means of isolated cell populations. The Sertoli cells selectively bind FSH, and respond to FSH stimulation with increased accumulation of endogenous cyclic AMP and secretion of androgen-binding protein (ABP). FSH binding and cyclic AMP response in the Sertoli cells change dramatically during sexual maturation. Cyclic AMP response decreases despite an increase in FSH-binding receptors per cell. Evidence has been provided for the existence of cytoplasmic and nuclear androgen receptors and chromatin acceptor-sites that specifically bind the androgen-receptor complex in the Sertoli cells. A model has been proposed for the hormonal interactions in the seminiferous tubule and the possible role of Sertoli cells in mediating the hormonal effects on spermatogenesis.

Culture and FSH Responses of Sertoli Cells Isolated from Sexually Mature Rat Testis

We have previously provided evidence that 125I-FSH specifically binds to various preparations of rat testis which contain Sertoli cells, e.g. isolated seminiferous tubules and organ cultures of testicular explants which have been depleted of most germinal elements and Leydig cells (1,2). On the other hand, isolated germinal cells, peritubular cells or interstitial cells show no binding of the labeled FSH. Subsequently, it was demonstrated that FSH also stimulates endogenous cAMP (3′5′-cyclic adenosine monophosphate) levels in the isolated seminiferous tubules and organ cultures, but has no effect on the isolated peritubular or interstitial cells and only a slight stimulatory effect on the germinal cells (3). These results provided suggestive evidence that of the various cell types composing the testis, only the Sertoli cells possess the FSH binding receptors and respond to FSH stimulation with increased levels of cAMP. Since these two parameters are considered to be the initial steps of gonadotropin action in the target cells (4), it appeared that the Sertoli cells must represent the primary target site for the biological action of FSH in the testis.

Adenylyl cyclase from a prolactin producing tumor cell: The effect of phenothiazines

Abstract An adenylyl cyclase stimulated by low concentrations of chlorpromazine was observed in homogenates of a clonal pituitary tumor cell line (GH 3 /C14) which releases prolactin and growth hormone. A half-maximal increase in activity of the GH 3 /C14 cyclase occurred in the presence of 0.5 × 10 −6 M chlorpromazine and a significant increase in activity was observed with a concentration of chlorpromazine as low as 10 −7 M. Several derivatives (7-methoxychlorpromazine, 7-hydroxychlorpromazine and 8-hydroxychlorpromazine) were found to mimic the stimulatory action of chlorpromazine on adenylyl cyclase, whereas chlorpromazine-5, N-dioxide was ineffective. Under the assay conditions used, sodium fluoride caused a four-fold increase in activity. However, dopamine at concentrations up to 2 × 10 −4 M was ineffective in stimulating or inhibiting the enzyme whether present alone or in combination with chlorpromazine. The ergot alkaloids, ergotamine and ergocryptine, blocked the stimulation of cyclase activity observed in the presence of chlorpromazine (10 −5 M). Homogenates of normal pituitaries showed no enhancement of adenylyl cyclase activity by chlorpromazine alone. However, when chlorpromazine was tested in the presence of 5′ guanylimidophosphate [GPP(NH)P], there was a significant increase in cyclase activity in the pituitary similar to that observed in the GH 3 /C14 preparation. These results suggest that hyperprolactinemia resulting as a side effect of phenothiazine treatment may be attributable to a direct action of these drugs to increase adenylyl cyclase activity in prolactin-producing cells of the anterior pituitary.

Effects of FSH on cyclic nucleotide accumulation in testes of rats of various ages.

The accumulation of cyclic AMP in rat testis in response to FSH is much greater in immature rat compared to adult. Addition of 3-isobutyl-1-methylxanthine (MIX), a potent in vitro inhibitor of cyclic nucleotide phosphodiesterase activity, potentiates the effects of FSH but does not restore the cyclic AMP response of adult rat to that of immature rat testis. The apparent substrate affinities for cyclic AMP and cyclic GMP hydrolysis are similar in immature and mature testis. Developmental changes in cyclic AMP and cyclic GMP phosphodiesterase specific activities fail to account for the reduced responsiveness of adult testis to FSH. These results suggest a defect in synthetic rather than hydrolytic mechanisms in FSH-responsive cells.