[Inhibitory effect of interleukin-1 beta on follicle stimulating hormone (FSH) induced estrogen production by cultured rat granulosa cells].

Interleukin-1 beta (IL-1 beta), one of the polypeptide lymphokines released in response to antigen, toxins, injury or inflammation by nearly all cell types, has multiple systemic effects. In the present study the effect of IL-1 beta on follicle stimulating hormone (FSH)-induced estrogen production in primary culture was investigated. Granulosa cells obtained from immature estrogen-treated female rats were cultured for 3 days with increasing doses of FSH (1-30 ng/ml) with or without increasing doses of IL-1 beta (2-20 U/ml). The FSH stimulated estrogen production is dose-dependent, whereas IL-1 beta alone did not affect estrogen biosynthesis. In contrast, simultaneous treatment with IL-1 beta caused a dose-dependent inhibition of FSH action. This inhibitory effect of IL-1 beta was evident 48 h after the treatment. Furthermore, IL-1 beta inhibited forskolin (10(-5) mmol/L) and (Bu)2 cAMP (10(-2) mmol/L)-stimulated estrogen production, indicating a post-cyclic AMP site of action. The present study suggests that IL-1 beta is a potent modulator of granulosa cell steroidogenesis. Decreased estrogen formation may contribute to the follicle atresia and the impaired reproductive functions during injury and inflammation.

Recombinant follicle-stimulating hormone induces ovulation and tissue plasminogen activator expression in hypophysectomized rats.

Ovulation in mammals is preceded by surges of the two pituitary gonadotropins, LH and FSH. Although previous studies have shown that purified FSH induces ovulation when administered to hypophysectomized rats, proof that FSH has inherent ovulatory potential is lacking because all FSH preparations have varying degrees of residual LH. To determine if FSH alone can induce ovulation, we generated LH-free recombinant FSH (RCFSH) by culturing eukaryotic cells transfected with the human common alpha- and FSH beta-subunit genes. Immature hypophysectomized rats were implanted with estrogen and then primed with PMSG (15 IU, sc). Fifty-two hours later, either RCFSH or hCG was injected (sc) to induce ovulation. A dose-dependent increase in the ovulation rate was stimulated by RCFSH, reaching 100% ovulation at 18 IU/rat, comparable to that achieved with 12 IU hCG. The maximum number of oocytes ovulated per ovary was similar for both groups. Ovulation induced by either RCFSH or hCG was time dependent and associated with a periovulatory increase in the ovarian activity and message levels of tissue-type plasminogen activator, a protease important in the preovulatory degradation of the follicle wall. Because PMSG has inherent LH-like activity in rats, we also implanted hypophysectomized rats with a minipump (sc) that released RCFSH (4 IU/day) to induce follicle growth. Fifty-two hours later, a single sc injection of a surge dose (20 IU) of RCFSH also induced ovulation, further indicating the ability of FSH alone to induce both follicle growth and ovulation. To test whether FSH can also induce ovulation in adult animals, rats were hypophysectomized on proestrous morning and treated with increasing doses of RCFSH (ip) to induce ovulation. At 7.8 IU RCFSH, all rats ovulated, with about 10 oocytes/rat. These results demonstrate that RCFSH is capable of inducing ovulation in hypophysectomized immature and adult rats, with associated increases in ovarian tissue-type plasminogen activator gene expression. Thus, FSH may be involved in follicular rupture in addition to its role in follicle recruitment and maturation. The preovulatory surges of both LH and FSH may represent a protective mechanism to ensure an optimal ovulatory stimulus. The present finding also serves as the basis to formulate new ovulation induction protocols.

Gonadotroph adenomas in men produce biologically active follicle-stimulating hormone.

Gonadotroph adenomas may exhibit qualitative and quantitative defects in gonadotropin biosynthesis and secretion. Hypersecretion of immunoreactive FSH dimers by these adenomas occurs frequently; however, it has not been known whether this FSH is biologically active. Using the granulosa cell aromatase bioassay and a highly specific immunoradiometric assay for FSH, we studied the serum bioactivity and bio- to immunoactivity (B/I) ratios of 14 men with FSH-secreting adenomas and compared these values to those of 11 age-matched normal men. In addition, three adenoma patients received TRH (400 micrograms, iv). The mean basal serum FSH level (international units per L), as measured by both bio- and immunoassays, and the FSH B/I ratios were significantly higher (P less than 0.02, by Kolmogorov-Smirnov test) in the adenoma patients than in normal men (mean +/- SEM; adenoma patients: bioactivity, 68.8 +/- 10.4; immunoreactivity, 34.8 +/- 13.7; B/I ratio, 3.4 +/- 0.6; normal men: bioactivity, 5.8 +/- 1.2; immunoreactivity, 6.4 +/- 0.8; B/I ratio, 0.90 +/- 0.1). Both bio- and immunoactive FSH rose after TRH injection, resulting in maintenance of the B/I (mean +/- SEM; pre-TRH: bio-FSH, 63.7 +/- 22.4; immuno-FSH, 28.0 +/- 14.1; B/I ratio, 2.8 +/- 1.2; post-TRH: bio-FSH, 125.6 +/- 42.7; immuno-FSH, 45.8 +/- 21.8; B/I ratio, 3.5 +/- 1.6). When gonadotroph adenoma cells from three separate patients were cultured and their conditioned media (n = 3) studied, relatively large amounts of both bio- and immuno-FSH were detected. Furthermore, the major isoelectric profile of bio-FSH (pH 4.9-3.0) in the conditioned medium from two such adenomas was shown by chromatofocusing to be comparable to that of purified human pituitary FSH (pH 5.2-3.6). We conclude that gonadotroph adenomas in men secrete FSH that is biologically active, both basally and in response to TRH.

In vitro and in vivo bioactivity of recombinant human follicle-stimulating hormone and partially deglycosylated variants secreted by transfected eukaryotic cell lines.

Recent studies have shown that Chinese hamster ovary (CHO) cells transfected with the FSH subunit genes secrete bioactive FSH. Here, we determined the in vitro and in vivo bioactivity of recombinant FSH produced by CHO mutant cells deficient in the glycosylation enzyme N-acetylglucosamine transferase-I (NAGT-), resulting in glycoproteins with asparagine-linked (GlcNAc)2(Mannose)5 oligosaccharides, or mutant cells defective in sialic acid transport into the Golgi (ST-). In the latter, glycoproteins are secreted lacking terminal sialic acids. Determination of in vitro bioactivity, using the granulosa cell aromatase bioassay, indicated that both FSH variants are as active as FSH secreted by the wild type (WT) cells and purified pituitary FSH. Also, these normal and variant forms of FSH are equipotent in a radioligand receptor assay using rat testis membranes. However, the variant FSH molecules are more basic than the WT FSH as determined using a chromatofocusing column (pI: wild type 3.6-5.0, NAGT- greater than 7.0, ST- approximately 6.0 and greater than 7.0). Injection of immature estrogen-treated rats with WT FSH induced high aromatase activity in their granulosa cells whereas treatment with either one of the FSH variants was ineffective; the lack of in vivo activity of the FSH variants was correlated with rapid clearance of these molecules in serum. Thus, recombinant human FSH produced by cells deficient in NAGT-I or defective in sialic acid transport retains normal receptor binding and in vitro bioactivity, but exhibits minimal in vivo activity and a shortened half-life when compared to WT FSH, indicating the important role of terminal sugars for FSH action in vivo.

Epidermal growth factor stimulates tissue plasminogen activator activity and messenger ribonucleic acid levels in cultured rat granulosa cells: mediation by pathways independent of protein kinases-A and -C.

Recent reports suggest that epidermal growth factor (EGF) or related peptides may act as local hormones to regulate granulosa cell differentiation. While FSH and GnRH are known to stimulate accumulation of tissue-type plasminogen activator (tPA) mRNA in granulosa cells, studies using nonovarian cells have shown stimulation of tPA by EGF. In this study, the effect of EGF and its structural analog transforming growth factor-alpha (TGF alpha) on ovarian tPA mRNA and activity was investigated. Granulosa cells obtained from immature estrogen-treated rats were cultured with FSH or increasing doses of EGF or TGF alpha before analysis of tPA activity using sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by a fibrin overlay technique. Like FSH and GnRH, EGF and TGF alpha stimulated the secretion of tPA activity in a dose- and time-dependent manner (onset, 12 h; maximum, 48 h). Northern blot hybridization of total RNA using a rat cRNA probe for tPA showed the accumulation of a 22S species mRNA in cells treated with EGF or TGF alpha, but not with nerve growth factor, suggesting increased expression of the tPA gene. Furthermore, slot blot hybridization of RNA from these cells confirmed a time-dependent increase in tPA mRNA preceding that in enzyme activity. Cotreatment of a saturating dose of EGF with phorbol myristate acetate (PMA) or GnRH resulted in additive increases in both tPA enzyme activity and mRNA levels. In addition, pretreatment with PMA desensitized the cells to subsequent treatment with PMA or GnRH, but did not diminish EGF-induced tPA mRNA, suggesting that EGF acts through a pathway independent of protein kinase-C. Also, extracellular cAMP levels did not increase with EGF treatment in the presence or absence of a phosphodiesterase inhibitor, suggesting the lack of involvement of the protein kinase-A pathway. Suppression of protein synthesis by cycloheximide inhibited the induction of tPA mRNA by EGF, whereas similar treatment resulted in the superinduction of tPA mRNA in FSH-treated cells, suggesting that EGF and FSH do not share the same pathway. These results suggest that EGF and TGF alpha induce tPA mRNA and activity in granulosa cells through a pathway independent of protein kinases-A (FSH) and -C (GnRH and phorbol ester), providing an interesting model for future elucidation of the molecular mechanism involved in tPA gene expression.

Inhibitory actions of interleukin-1 beta on steroidogenesis in primary cultures of neonatal rat testicular cells.

Interleukin-1 is an important cytokine produced by activated macrophages. Because macrophages have been localized in the testis and interleukin-1 bioactivity has been observed in the testis, the potential effect of interleukin-1 on gonadotropin-stimulated androgen production was investigated using primary cultures of neonatal rat testis cells. Cells were incubated for 3 days before change of medium and treatment with human chorionic gonadotropin and interleukin-1. After 3 additional days medium testosterone and progesterone levels were determined. Human chorionic gonadotropin treatment (0.30-30 micrograms/l) of cultured cells stimulated testosterone production dose-dependently with a maximum increase greater than 18-fold over control values. Although interleukin-1 treatment alone did not affect testosterone production, the concomitant addition of interleukin-1 beta (0.5-5 X 10(3) U/l) caused a dose-dependent decrease of human chorionic gonadotropin action, with 50% inhibition occurring at 1.4 X 10(3) U/l (0.6 X 10(-11) mol/l; N = 5 experiments). Interleukin-1 beta also inhibited forskolin- and dibutyryl cAMP-stimulated testosterone production. The suppression of human chorionic gonadotropin-induced testosterone production by testis cells was accompanied by increased (greater than 3-fold) progesterone levels. Moreover, the conversion of exogenously added androgen precursors (progesterone and 17 alpha-hydroxyprogesterone) to testosterone by human chorionic gonadotropin-stimulated cells was suppressed by interleukin-1 beta suggesting that the activity of the 17 alpha-hydroxylase enzyme may be decreased.(ABSTRACT TRUNCATED AT 250 WORDS)

Expression of biologically active human follitropin in Chinese hamster ovary cells.

To study the structure-function relationships of follitropin (FSH), we expressed the hormone in a heterologous cell system. A genomic clone bearing a 3.7-kilobase FSH beta insert containing the entire coding sequence was transfected alone or together with the alpha subunit gene into Chinese hamster ovary cells and stable lines expressing either FSH beta or FSH dimer were selected. Pulse-chase experiments revealed that, when transfected alone FSH beta was very slowly secreted similar to lutropin beta and thyrotropin beta but unlike choriogonadotropin beta which is efficiently secreted. However, cotransfection of the FSH beta and alpha subunit genes resulted in "rescue" of the beta subunit and rapid secretion of dimer. These data support the hypothesis that the glycoprotein hormones of pituitary origin have determinants for secretion that differ from those on the placental hormone, choriogonadotropin. Recombinant FSH stimulated steroidogenesis comparable to purified human FSH isolated from pituitaries in an in vitro rat granulosa cell assay and appears more homogeneous by chromatofocusing. Human FSH produced by this cell line provides a source of bioactive FSH for experimental and clinical use.

Increased metabolic heat production following chronic alpha-methyldopa therapy in hypertensives.

The objective of this study was to measure metabolic heat production in mild-moderate hypertensives when given chronic alpha-methyldopa for control of hypertension and to compare this with hydrochlorothiazide. Four men and one woman, age range 29-55 years, euthyroid, and not previously on alpha-methyldopa, had their drugs replaced by placebo for 4 weeks, and then alpha-methyldopa or hydrochlorothiazide was randomized single blindly to patients. The dose of each drug was increased until the ambulatory diastolic blood pressure was less than 95 mmHg (1 mmHg = 133.322 Pa), and then the dose was maintained for 20-24 weeks. The two active drugs were then crossed over, and each medication was again titrated to attain diastolic normotension (less than 95 mmHg). Maintenance was for a comparable 20-24 weeks. At week 4 (end of placebo period), weeks 20-24 (for hydrochlorothiazide or alpha-methyldopa monotherapy), and again at week 40-48 (end of cross-over period), metabolic heat production (watts), heart rate (beats per minute), and blood pressure (millimetres of Hg) were measured in a resting sitting position for 1 h in a whole body calorimeter (air temperature 28 degrees C and a dew point temperature less than 5 degrees C) for 15 experimental sessions. In four out of five patients, metabolic heat production was significantly (P less than 0.05) higher when patients were treated with alpha-methyldopa. We speculate from our data that alpha-methylnorepinephrine, the major metabolite of alpha-methyldopa, may be thermogenic and that this should be considered when the drug is prescribed.

An appraisal of antihypertensive efficacy and adverse reactions with two drug regimens: enalapril maleate as part of triple therapy compared to conventional triple therapy in moderate to severe hypertension.

A randomized, double-blind, parallel treatment trial was carried out in 24 patients with moderate to severe hypertension to compare the effectiveness and tolerance of two treatment regimens in reducing and maintaining supine diastolic blood pressure below 90 mmHg. Patients in Group I received 10 to 40 mg enalapril maleate per day with the addition of 50 mg hydrochlorothiazide per day and then 250 to 1000 mg alpha-methyldopa per day, if necessary. Patients in Group II received 50 mg hydrochlorothiazide per day with the addition of 80 to 240 mg propranolol and then 100 to 200 mg hydralazine per day, if necessary. Apart from the hydrochlorothiazide dosage which was fixed, the dosage of the other active drugs was titrated incrementally until the target blood pressure level was achieved. Blood pressures, heart rate and body weight were monitored at 2-weekly intervals during 26 weeks of active therapy. In Group I, blood pressure control was achieved and maintained with enalapril alone in 9 patients, 2 patients required double therapy and 1 patient triple therapy. In Group II, 9 patients required double therapy, 2 triple therapy, and only 1 patient received monotherapy. Supine and erect blood pressure control was comparable in both groups. There was, however, a significant decrease in supine heart rate in patients in Group II. More importantly, 8 of the 12 patients in Group II experienced non-life threatening adverse reactions (4 were hypokalaemic and required supplementary potassium, 2 had cold hands and feet, 1 man had sexual dysfunction and 1 acute gout) and no adverse reactions were reported by Group I patients.

Separation of essential hypertensive patients based on blood pressure responses after the withdrawal of antihypertensive agents by step-wise discriminant analysis.

Thirty-five patients with uncomplicated hypertension were observed for blood pressure behaviour after prolonged antihypertensive medication withdrawal. Twenty-three patients (Group 1) remained normotensive (systolic less than 140 mmHg, diastolic less than 95 mmHg) for over 60 weeks compared to 12 patients (Group 2) who became hypertensive again during a 4-week placebo period. Discriminant analysis was performed on 31 clinical and laboratory variables measured before therapy to separate any discriminating factors for the two groups. Four variables maximized the separation of Group 1 from Group 2 patients. In descending order, these were serum sodium (p less than 0.001), mean corpuscular volume (p less than 0.01), serum albumin (p less than 0.01), and body weight (p less than 0.05). Using these four variables 73.9% of patients in Group 1 and 75% of patients in Group 2 were classified correctly into their respective groups. It is suggested that this observation may be useful in the development of new therapeutic regimens for patients with uncomplicated essential hypertension, for example by predicting those patients who may respond favourably to intermittent therapy or even to its withdrawal.

Prolonged normotension following cessation of therapy in uncomplicated essential hypertension.

The blood pressure levels of 17 men and 7 women aged 27-65 years with uncomplicated essential hypertension were studied during and after withdrawal of antihypertensive therapy. Medication during the treatment phase included diuretics alone (13 patients), diuretic-nondiuretic combinations (5 patients), and nondiuretics alone (6 patients). Blood pressures were followed: greater than 48 weeks (with therapy) vs greater than 48 weeks (without therapy). SBP rose significantly (P less than 0.05) after therapy withdrawal but remained, for all but 6 of the 24, within the normotensive range (SBP mean +/- S.D. 125.2 +/- 11.1 mm Hg vs 134.9 +/- 13.3 mm Hg, and erect 87.4 +/- 9.3 mm Hg vs 131.2 +/- 12.0 mm Hg). DBP was unchanged (DBP mean +/- S.D., supine 83.8 +/- 8,0 mm Hg vs 85.2 +/- 6.2 mm Hg, and erect 87.4 +/- 9.3 mm Hg vs 86.7 +/- 6.6 mm Hg, P = 0.174). The blood pressures of 6 patients (25%) rose to levels requiring medication (SBP greater than 140 mm Hg. DBP greater than 95 mm Hg) between weeks 49 and 60. Salt intake was low (less than 100 mmol 24 h-1) and body weight remained stable (mean 75.4 +/- 12.4 kg vs 77.2 +/- 13.0 kg). These observations my indicate a role for intermittent antihypertensive therapy or quantitative reduction of medications, in the control of uncomplicated essential hypertension, provided that careful follow-up is available.

Comparison of antihypertensive efficacy: propranolol versus oxprenolol in accelerated hypertension.

An open, randomized crossover study was carried out in 8 patients with accelerated hypertension to compare the antihypertensive efficacy of oxprenolol with that of propranolol. Dosage was titrated for each patient to achieve normotensive levels and was in the range of 40 to 320 mg twice daily. The same mg dosage was maintained for the alternative drug after crossover at 32 weeks for the second treatment period. The results showed that both drugs had equivalent antihypertensive effect on the erect systolic and diastolic and supine systolic blood pressures. Oxprenolol, however, produced a significantly greater reduction in supine diastolic blood pressure than did propranolol. There were no significant changes in heart rate or any of the other clinical or laboratory parameters monitored during either treatment, and side-effects were remarkably infrequent.

Potassium conservation with amiloride/hydrochlorothiazide ("Moduret') in thiazide-induced hypokalaemia in hypertension.

A double-blind study was carried out in 24 hypertensive patients with thiazide-induced hypokalaemia (serum potassium less than 3.2 mmol/l) to compare the effects of treatment with an amiloride/hydrochlorothiazide combination or hydrochlorothiazide alone. The study was divided into three phases: (i) potassium repletion (Weeks 0 to 4) with oral potassium chloride (40 mmol/day), (ii) stabilization (Weeks 4 to 6) of normokalaemia, and (iii) active drug treatment (Weeks 6 to 14), patients being allocated at random to receive one or other of the two treatments. Dosage was 2 tablets per day of the 5 mg amiloride plus 50 mg hydrochlorothiazide combination or of 50 mg hydrochlorothiazide alone. The results showed that blood pressure control was comparable in both treatment groups but hydrochlorothiazide alone caused a statistically significant reduction in serum potassium levels compared to the drug combination. Apart from 1 patient who developed hypokalaemia on hydrochlorothiazide alone, no other side-effects of treatment were reported.