Jennifer H. Dorrington

Effects of FSH on gonadal functions.

Publisher Summary This chapter discusses the effects of follicle stimulating hormone (FSH) on gonadal functions. FSH regulates directly the metabolic activity of two cell types in the gonads―granulosa cells of the ovary and the Sertoli cells of the testis. At the onset of testicular development, the cortex involutes and the medulla proliferates. Sertoli cell progenitors originate from the epithelium of the gonad, penetrate into the medulla region, and associate with germ cells to give rise to sex cords. The incorporation of germ cells into the spermatic cords of the medulla is one of the earliest indications that the gonad is destined to become a testis. The cords differentiate into seminiferous tubules lined with immature Sertoli cells in close contact with the primitive germ cells. In the immature Sertoli cells, FSH stimulates the conversion of testosterone to estrogens and 5α-reduced androgens. FSH also stimulates the synthesis of ABP, thereby influencing the amount of testosterone and DHT that is specifically bound. In granulosa cells, FSH stimulates the conversion of testosterone to estrogens. In addition to acting as a substrate for estrogen synthesis, testosterone augments the effect of FSH on progesterone synthesis. A better understanding of the hormonal control of the metabolism and the secretion of Sertoli and granulosa cells may give insight into the defects that underlie infertility and, furthermore, may lead to the development of new methods for the control of gametogenesis.

Immunohistochemical detection of transforming growth factor-α in Leydig cells during the development of the rat testis

In this paper the localization of transforming growth factor alpha (TGF-alpha) is described in the rat testis at various stages throughout development, e.g. neonatal, prepubertal, and adult, in order to examine somatic cells and germinal cells at different stages of differentiation. This was done by immunoperoxidase staining using a monoclonal antibody that does not cross-react with epidermal growth factor (EGF). In sections of testes from neonatal rats, intense staining was present in Leydig cells. In the cells of the seminiferous tubules the staining was faint or undetectable. At the time when many mesenchymal cells differentiate into Leydig cells in the 21-day-old rat, TGF-alpha was visualized in most but not all of the identifiable Leydig cells. In interstitial cell cultures derived from 21-day-old rats, the majority of the Leydig cells contained TGF-alpha, but in a proportion of the Leydig cells TGF-alpha was undetectable. No staining was apparent in Sertoli cells and germ cells in seminiferous tubules or in Sertoli cell cultures derived from 21-day-old rats. Under these in vitro conditions it was found that peritubular-myoid cells also possessed TGF-alpha immunoreactivity. In the adult testis all Leydig cells stained positively for TFG-alpha, whereas no staining was found in the cells of the seminiferous tubules. Treatment of adult rats with ethylene-1,2-dimethane-sulfonate (EDS) resulted in the destruction of Leydig cells and the loss of all positively stained for TGF-alpha.(ABSTRACT TRUNCATED AT 250 WORDS)

FSH induction of aromatase in cultured rat granulosa cells measured by a radiometric assay.

The effects of FSH on the aromatase activity of rat granulosa cells in culture were studied by measuring the stereospecific transfer of 3H from [1,2,6,7-3H]testosterone or [1 beta-3H]testosterone into 3H2O. The use of 3H2O release as a specific assay for aromatization in granulosa cells was validated by various means. 2 days after plating, cultures of granulosa cells released only minimal amounts of 3H2O from [1 beta-3H]testosterone during a 3-h incubation, whereas cells which had been treated with FSH, or with (Bu)2cAMP plus 3-isobutyl-1-methylxanthine (MIX), from the time of plating released considerable amounts of 3H2O into the culture medium. The release of 3H2O from [1 beta-3H]testosterone by cultured cells was inhibited by the aromatizable androgens, 19-hydroxytestosterone and 19-hydroxyandrostenedione, indicating the specificity of the method for aromatization. In order to study the mechanism by which FSH enhanced the release of 3H2O, optimal conditions for aromatization by cell-free sonicates were determined. Optimal aromatase activity was achieved by incubating cell-free sonicates at 37 degrees C in the presence of O2 in 20 mM phosphate buffer (pH 7.4) containing 5 mM dithiothreitol, 20 mM MgCl2, 0.5 mM NADP+, 20 mM glucose 6-phosphate and 2 U/ml glucose-6-phosphate dehydrogenase. A concentration of 0.25 microM testosterone and 0.1 microCi tritium was used in the standard assay. Under these conditions the assay was linear for 1 h with up to 150 microgram protein from granulosa cells having maximal aromatase activity. When cells in culture were stimulated with purified FSH for 48 h from the time of plating, the aromatase activity in cell-free sonicates increased in a dose-dependent fashion. The ED50 for Sairams FSH S-1528 C2 was 12 ng/ml. It was concluded from these studies that FSH increases estrogen levels primarily by inducing an active aromatase rather than by influencing secretion or availability of substrate or cofactor for the aromatization reaction.

The effect of gonadotrophins on the 3′,5′-AMP levels of seminiferous tubules

Abstract The addition of follicle-stimulating hormone (FSH) to isolated tubules from hypophysectomized rats was shown to increase the level of adenosine 3′,5′-monophosphate (3′,5′-AMP). In contrast, luteinizing hormone (LH) exerted no effect in this system. The results presented are consistent with the concept that FSH exerts a direct effect upon cells within the seminiferous tubule, possibly on Sertoli cells, whereas the effects of LH on spermatogenesis are primarily due to the stimulation of androgen production by the interstitial cells of the testis.

Immunohistochemical localization of transforming growth factor-β1 and -β2 during follicular development in the adult rat ovary

The transforming growth factors-β (TGF-β) affect the metabolic activities of each of the cell types in the ovary. In vitro studies using immature rat ovaries have shown the expression of TGF-β1 and/or TGF-β2 mRNA in thecal/interstitial cells and in granulosa cells (Mulheron and Schomberg, 1990; Mulheron et al., 1991). To obtain information on the localization of TGF-β1 and TGF-β2 in the rat ovary in vivo, we have examined the immunohistochemical staining using antibodies specific for either TGF-β1 orTGF-β2. In the adult ovary the immunostaining for TGF-β1 was intense, whereas the staining for TGF-β2 was faint. The pattern of immunostaining for TGF-β1 and TGF-β2 remained constant in the interstitial cell compartment and was not affected by the stage of the oestrous cycle. Since the interstitium surrounds follicles at all stages of development we conclude that TGF-β is not actively involved in regulating the progression of follicles at discrete stages. At the time of antrum formation in the follicle, intense staining for TGF-β1 was observed in thecal cells. Around the preovulatory stage of development, TGF-β1 and TGF-β2 immunoreactivity was also found in the granulosa cells. In the corpus luteum, intense staining for TGF-β1 was found in some areas, whereas other areas were negative. Weak to moderate staining for TGF-β2 was observed. In conclusion, all major cell types show strong immunostaining for TGF-β1 and less intense staining for TGF-β2, confirming an autocrine/paracrine role for TGF-βs in the regulation of ovarian cell growth and function.

Intraovarian regulation of follicular development

Abstract Pituitary gonadotrophins are required for normal follicular growth and development; the proliferation of granulosa cells, however, cannot be attributed to the direct actions of these hormones. Since polypeptide growth factors are known to have mitogenic actions on granulosa cells, the possibility that the surrounding thecal cells may be a source of growth factors that are physiologically important in regulating granulosa cell proliferation was examined in the bovine ovary. In support of this hypothesis bovine thecal cells in culture were shown secrete products that stimulated [ 3 H]thymidine incorporation into bovine granulosa cell DNA, anwiled to an increase in cell number. To characterize the growth-promoting factor thecal cell conditioned medium was concentrated and the peptides fractionated on a Bio-Gel P-60 column in 1.0 M atic acid. Fractions were tested for their ability to stimulate [ 3 H]thymidine incorporation into bovine granulosa cell DNA. Growth-promoting activity was located in the 6–9 K and the 16 K molecular weight fractions. The peak fractions inhibited FSH-induced aromatase activity in rat granulosa cells, indicating the presence of EGF-like or TGF-α-like peptides. A specific radioimmunoassay for TGF-α indicated that the active fractions that promoted both growth and aromatase activity contained TGF-α. An immunohistochemical approach as also used to localize TGF-α in the bovine ovary throughout the various stages of folliular, development. Immunoperoxidase staining was most intense in the theca of follicles at the discrete physiological stages known to show rapid granulosa cell growth. Staining intensity for TGF-α declined in large pre-ovulatory follicles, coincident with the known decline in granulosa cell mitosis. Northern analysis of bovine tneca mRNA showed the presence of the TGF-α mRNA transcript. A growth inhibitory activity was observed in the fractions with a molecular weight of approx. 25 K. The inhibitory activity had the molecular weight of TGF-β, and TGF-β was found to inhibit [ 3 H]thymidine incorporation into bovine granulosa cell DNA. The presence of TGF-β-like activity was also induced by the stimulation of FSH-induced aromatase activity in rat granulosa cells. In suppossedly, bovine thecal cells secrete both TGF-α and TGF-β in vitro. As shown in the bioassay, the TGF-α-like and the TGF-β-like factors have pronounced effects on the proliferation of bovine granulosa cells; TGF-α promotes cell growth whereas TGF-β inhibits cell growth. The relative concentrations of TGF-α and TGF-β may determine the rates of proliferation of granulosa cells during follicular development.

Site at which FSH regulates estradiol-17β biosynthesis in Sertoli cell preparations in culture

Sertoli cells were isolated from testes of 20-day-old rats and were maintained in primary culture. The ability of these cells to synthesise estradiol-17beta from a variety of exogenous substrates, progesterone, testosterone,androstenedione, 19-hydroxyandrostenedione and 19-hydroxytestosterone in the presence and absence of follicle-stimulating hormone (FSH) was examined. In the presence of each of the substrates alone for 24 h the rate of estradiol-17beta synthesis was very low. FSH (NIH-FSH-S11, 5 mug/ml) stimulated estradiol-17beta synthesis 75-fold when added to medium containing testosterone (5 X 10(-7)M) but caused only marginal stimulation when added to medium containing progesterone (5 X 10(-7) M). Both FSH and dibutyryl cyclic AMP (bu2cAMP) stimulated the conversion of each of the substrates, androstenedione, 19-hydroxyandrostenedione and 19-hydroxytestosterone to estradiol-17beta, and the effects were similar to those observed in the presence of testosterone. These data indicate that, under the culture conditions employed, progesterone is not an effective substrate for conversion to estradiol-17beta by Sertoli cells. Estradiol-17beta synthesis was stimulated by FSH in the presence of the C19 steluences the conversion of androgens to estrogens, either directly or indirectly, at the aromatisation step (i.e. the conversion of 19-hydroxylated androgens to estrogens).

Aromatase activity in granulosa cells: regulation by growth factors.

This study describes the effects of insulin, insulin-like growth factor 1 (IGF1), and epidermal growth factor (EGF) on the aromatase activity of granulosa cells isolated from immature rat ovaries. None of the growth factors alone influenced the basal level of aromatase activity, but did modulate follicle-stimulating hormone (FSH)-induced aromatase activity. Insulin and IGF1 augmented the action of a sub-optimal concentration of FSH (5 ng/mL) on aromatase activity in a dose-dependent manner. In contrast, EGF (1-10 ng/mL) was effective in inhibiting aromatase activity maximally stimulated by FSH. Since insulin and IGF1 had opposing actions to those of EGF on FSH-induced aromatase activity, we examined the interactions between the growth factors. EGF inhibited the actions of both FSH and insulin on aromatase activity. Both IGF1 and EGF increased the [3H]thymidine incorporation into the DNA of bovine granulosa cells in vitro, IGF1 being a more potent mitogen. Whereas EGF inhibited the actions of IGF1 on aromatase activity, it did not inhibit the effects of IGF1 on the growth of granulosa cells. In summary, growth factors influence both the differentiation and growth of granulosa cells, and may be important regulators of follicular development.

Actions of growth factors in the follicle

In this paper we have examined the possibility that soluble factors produced by the thecal and granulosa cells may be essential local modulators of follicular development. The observations that insulin could influence both the growth and the differentiation of granulosa cells were important in establishing the concept that peptides could act as amplifiers of the actions of gonadotrophins. Insulin alone did not influence aromatase activity significantly but acted synergistically with FSH to augment aromatase activity in rat granulosa cells. Unlike aromatase activity, insulin alone was able to significantly stimulate 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) activity, the maximum level achieved approaching that obtained with high concentrations of FSH. To determine if insulin could influence other parameters of granulosa cell function in addition to steroidogenesis, we measured a component of extracellular matrix, fibronectin, previously shown to be inhibited by FSH. Treatment with insulin independently inhibited the increase in fibronectin secretion observed in control cultures. Also, insulin alone was able to stimulate quiescent bovine granulosa cells to incorporate [3H]thymidine into DNA under serum-conditions. The concentration of insulin required in these experiments was higher than physiological levels suggesting that other insulin-like growth factors may be involved. Our work and that of others has shown that IGF1 can mimic the actions of insulin and is effective at lower concentrations. A possible source of IGF1 production in the follicle was sought initially by collecting rat granulosa cell conditioned medium, and assessing biological activity and immunoreactivity. Conditioned medium augmented the actions of FSH on aromatase activity and alone stimulated 3 beta-HSD, indicating the presence of insulin-like bioactivity. A positive reaction on immunoblots using specific antiserum confirmed the presence of immunoreactive IGF1. Conditioned medium from thecal cells contained a growth-promoting activity (TcGF) that did not augment FSH-induced aromatase activity. The production of growth factors locally within the follicle may represent the self-amplifying mechanism that enables the dominant follicle to complete its developmental program and ovulate.

Metabolism of testosterone by preparations from the rat testis

Abstract Seminiferous tubules isolated from immature and adult rats were incubated with [ 14 C] testosterone. Androstanediol and androstenedione were the major metabolites; dihydrotestosterone and androsterone were produced in lesser amounts. Cell suspensions of spermatocytes prepared from tubules of immature rats formed dihydrotestosterone as the major metabolite of testosterone. Smaller amounts of androstanediol were formed and no androsterone was detectable. The results show that spermatocytes in common with androgen responsive tissues have the capacity to metabolize testosterone to 5α-reduced products.