Kristine D. Dahl

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.

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.

Gonadotropin requirements of the developing follicle.

OBJECTIVE To evaluate follicular FSH and LH requirements during suppression of endogenous gonadotropins with the GnRH antagonist Nal-Glu and whether LH-like activity could be supplied by administering subcutaneous hCG. DESIGN Randomized clinical trial. PARTICIPANTS Thirty-two normally cycling females in the late follicular phase (dominant follicle mean diameter > or = 14 mm). INTERVENTION Twelve normal women were randomized to receive 150 IU IM FSH with or without 75 IU SC hCG; 11 subjects were randomized to receive 225 IU FSH with or without 50 IU SC hCG; 9 women received 150 or 225 IU IM hMG. Subjects returned the next day for repeat blood sample and ultrasound. RESULTS Continued follicular maturation, as evidenced by rising E2 levels, correlated with serum immunoactive and bioactive FSH levels and was unrelated to bioactive LH-hCG. Two hundred twenty-five international units of exogenous FSH consistently supported follicular maturation. There was a similar increase in mean follicular diameter in women with an E2 rise versus those with a plateau or fall. In subjects receiving SC mini-dose hCG, serum bioactive LH-hCG levels were increased significantly and were similar to levels before Nal-Glu. CONCLUSIONS During administration of a GnRH-a, the maturing follicle appears to require only FSH support. In markedly hypogonadotropic women, mini-dose hCG may be a more practical alternative to recombinant LH to promote normal follicle maturation.

Vasoactive intestinal peptide stimulates plasminogen activator activity by cultured rat granulosa cells and cumulus-oocyte complexes

Vasoactive Intestinal Peptide (VIP), originally considered to be a gut hormone, has recently been found to increase estrogen and progesterone production by ovarian granulosa and luteal cells. Because several studies indicate that granulosa cells and oocytes are capable of producing plasminogen activators, we have studied the effects of VIP on plasminogen activator activity in cultured granulosa cells and cumulus-oocyte complexes collected from the ovaries of hypophysectomized, estrogen-treated immature rats. Using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) followed by a fibrin overlay technique to assess plasminogen activator activity, we observed that treatment with VIP stimulated the secretion of tissue-type plasminogen activator (tPA), but not urinary-type plasminogen activator (uPA), in a dose-dependent manner by cultured granulosa cells as well as by cumulus-oocyte complexes, but not by denuded oocytes. However, preparation of cumulus-free oocytes from cumulus-oocyte complexes which had previously been treated with VIP indicated substantial increases in tPA activity within the oocyte. The actions of VIP on tPA activity in granulosa cells were specific, because other closely related peptides (PHM-27 and glucagon) were ineffective. These effects of VIP, in addition to the previously observed effects on steroidogenesis, suggest that VIP may be an important regulator of ovarian function.

Use of the granulosa cell aromatase bioassay for measurement of bioactive follicle-stimulating hormone in urine and serum samples of diverse species.

Ovarian steroids and growth factors are intragonadal modulators which augment a key endpoint of follicle-stimulating hormone (FSH) action in granulosa cells: the induction of aromatase activity. Studies of these paracrine hormones that enhance FSH-stimulated estrogen biosynthesis by cultured rat granulosa cells, have led to the development of a sensitive and specific in vitro bioassay for FSH. This newly developed granulosa cell aromatase bioassay (GAB) allows for the measurement of bioactive FSH levels in serum and urine of humans and animals with various physiological and pathological conditions. These studies have demonstrated that the GAB assay is useful in detecting possible changes in the molecular forms of FSH. The adaptation of this method for urine samples allows for the measurement of bio-FSH levels in situations where venipuncture is not practical or in species for which specific radioimmunoassays are not available.

Effects of diverse mammalian and nonmammalian gonadotropins in a rat granulosa cell bioassay for follicle-stimulating hormone.

The biopotencies of pituitary gonadotropins purified from a marsupial (kangaroo), two avian (ostrich and turkey), a reptile (turtle), an amphibian (bullfrog), and two fish (sturgeon and teleost) species were examined using an in vitro rat granulosa cell bioassay for follicle-stimulating hormone (FSH). Treatment of cultured granulosa cells with increasing concentrations of gonadotropin preparations from these species resulted in dose-dependent increases in estrogen production from negligible amounts to maximal levels of approximately 2-29 ng/culture. The relative biopotencies of these FSH preparations from most potent to least potent were in the order of human greater than ostrich greater than turkey greater than kangaroo greater than turtle greater than sturgeon greater than bullfrog greater than teleost with ED50 values of human 8.7 ng/well; ostrich 10.5 ng/well; turkey 22.5 ng/well; kangaroo 58.2 ng/well; turtle 62.5 ng/well; sturgeon 260 ng/well; bullfrog 750 ng/well; teleost greater than 1000 ng/well. In contrast, luteinizing hormone (LH) preparations were considerably less effective for ostrich, turkey, kangaroo, turtle, and bullfrog, being six-, five-, three-, and twofold less potent than FSH preparations for the same species, demonstrating the specificity of this assay for FSH. An LH preparation from bullfrog was unable to significantly stimulate estrogen production below 500 ng/ml. Thus, the present in vitro bioassay (GAB) using rat granulosa cells provides a sensitive and specific assay for measuring FSH activities of gonadotropins from diverse mammalian and nonmammalian species.

The induction of premature luteolysis in normal women—follicular phase luteinizing hormone secretion and corpus luteum function in the subsequent cycle***

Women with luteal phase deficiency have been shown to have an increased frequency of luteinizing hormone pulses in the early follicular phase of the menstrual cycle. Because progesterone is known to modulate luteinizing hormone secretion, it has been hypothesized that the decreased progesterone secretion in a previous luteal phase deficiency cycle could lead to the abnormal luteinizing hormone secretory pattern in the ensuing early follicular phase. With the possibility that the higher luteinizing hormone pulse frequency might lead to another deficient luteal phase, it becomes conceivable that luteal phase deficiency could be self-perpetuating. To test this hypothesis, luteal phase deficiency was induced in six normal women by decreasing luteinizing hormone support of the corpus luteum with a gonadotropin-releasing hormone antagonist Nal-Glu, administered twice daily beginning in the midluteal phase after a control cycle. During the antagonist-treated luteal phase, each subject met the predetermined criteria for induced luteal phase deficiency: a 33% or greater decrease in integrated progesterone from the control cycle and an integrated progesterone level less than 100 ng/ml per day. Luteinizing hormone secretion patterns were determined by frequent blood sampling performed every 10 minutes for 12 hours in the early follicular phase of the control cycle and the cycle after antagonist administration. Daily luteal progesterone levels were measured in the control, treatment, and posttreatment cycles. Each volunteer served as her own control. Standard parameters were compared between the control and posttreatment pulse studies in the early follicular phase: (1) luteinizing hormone pulse frequency was 9.5 +/- 1.0 vs 10.0 +/- 0.9 pulses/12 hours, control vs posttreatment, respectively, p = 0.5; (2) luteinizing hormone pulse amplitude was 11.0 +/- 1.3 vs 12.0 +/- 2.2 ng/ml, p = 0.6; and (3) luteinizing hormone mean level was 19.4 +/- 2.3 vs 22.2 +/- 3.3 ng/ml, p = 0.1. Corpus luteum function was also compared between the control and posttreatment cycles. Luteal phase length was 13.7 +/- 0.6 vs 12.7 +/- 0.6 days, p = 0.08. Integrated progesterone values were 136.9 +/- 12.9 vs 130.5 +/- 11.3 ng/ml per day, p = 0.5. Therefore no discernible abnormalities in early follicular luteinizing hormone secretions or corpus luteum secretion of progesterone occurred after an induced luteal phase deficiency cycle.(ABSTRACT TRUNCATED AT 400 WORDS)

Chapter 9 Mechanism of action of FSH in the ovary

Publisher Summary This chapter discusses the mechanisms underlying the follicle-stimulating hormone (FSH) stimulation of ovarian-cell differentiation and maturation. FSH is necessary for the stimulation of preovulatory ovarian estrogen production and the initiation of follicle maturation. FSH is a glycoprotein hormone which contains two noncovalently linked dissimilar subunits. The gonadotropic hormones, including those from the placenta, consist of a common subunit designated α and a hormone-specific β subunit. The primary structure of FSH consists of the polypeptide backbone and oligosaccharide moieties. FSH molecules from all species including those from lower vertebrates contain carbohydrate units covalently linked to the polypeptide. Removal of this sugar residue by neuraminidase treatment decreases the in vivo biological activity of FSH because of its rapid elimination from the circulation, but its in vitro biological response is retained. In contrast, removal of the majority of carbohydrate moieties by specific enzymes or chemical treatment decreases the bioactivity of the molecules. FSH molecules secreted by the anterior pituitary interact with specific receptors in the gonadal cells. The FSH action in granulosa cells is enhanced by cotreatment with phosphodiesterase inhibitors that minimize cyclic AMP (cAMP) breakdown.

The Significance of a Single Serum LH Measurement in Women With Cycle Disturbances: Discrepancies Between Immunoreactive and Bioactive Hormone Estimates

OBJECTIVE We evaluated the significance of single serum LH estimates (as assessed by radiometric assay (IRMA) and Leydig cell in-vitro bioassay (BIO)) for the diagnosis of polycystic ovary syndrome (PCOS) in women with infertility and cycle abnormalities. DESIGN Hormonal and clinical comparisons between subgroups were made based on classification according to (a) rigid clinical and endocrine (excluding LH) characteristics of PCOS, (b) elevated IRMA-LH concentrations, (c) BIO-LH levels. In addition, androgen modulation of LH biopotency was studied in these patients. PATIENTS Ninety-nine women presenting at our infertility Unit with oligo/amenorrhoea. MEASUREMENTS AND RESULTS Of the total study group, 35 women were diagnosed positive as PCOS and 42 showed elevated IRMA-LH levels. Only 51% (n = 18) of PCOS patients showed elevated IRMA-LH levels, and in PCOS significantly higher levels of BIO-LH, androstenedione, oestrone, and BIO/IRMA-LH ratios were found as compared to non-PCOS patients. In the group with elevated IRMA-LH only 43% (n = 18) of subjects were diagnosed as PCOS, and no difference in BIO/IRMA-LH ratios was found. With increasing BIO-LH levels the probability of PCOS rises sharply (P < 0.001), whereas this probability is of only marginal significance (P < 0.06) for IRMA-LH. In the total study group a correlation is observed between serum testosterone (T) levels and IRMA-LH (r = 0.47), and BIO-LH (r = 0.51) concentrations. This correlation is absent comparing serum T and BIO/IRMA-LH ratios (r = 0.15). CONCLUSIONS Results presented in this study indicate that (1) women with infertility and oligo/amenorrhoea classified based on signs of PCOS or IRMA-LH levels, exhibit different clinical and endocrine characteristics, (2) only 51% of PCOS women exhibit elevated IRMA-LH concentrations, and only 43% of women with elevated IRMA-LH were diagnosed as PCOS, (3) IRMA-LH levels are a poor predictor of PCOS, whereas the predictive value of BIO-LH is better, (4) elevated BIO/IRMA-LH ratios in PCOS are dependent on alterations in BIO-LH, rather than IRMA-LH concentrations, and (5) no correlation was observed between serum T levels and BIO/IRMA-LH ratios.

Naturally Occurring Antihormones: Secretion of FSH Antagonists by Women Treated with a GnRH Analog

Follicle-stimulating hormone (FSH) is a glycoprotein essential for gonadal development and steroidogenesis. Recent studies suggest that deglycosylation of FSH results in the formation of antagonistic proteins that are capable of binding to gonadal receptors but that are devoid of bioactivity. Treatment of hypogonadal women with an antagonist of gonadotropin-releasing hormone substantially decreased serum FSH bioactivity with minimal changes in immunoreactivity. Chromatofocusing and size fractionation of the serum samples indicated the secretion of immunoreactive FSH isoforms that are devoid of bioactivity but that are capable of blocking FSH action in ovarian granulosa cells. These findings provide the first demonstration of naturally occurring circulating antihormones. These FSH antagonists may play an important role in the physiology and pathophysiology of the gonads.