Specific binding of luteinizing hormone to leydig tumor cells

A radioimmunoassay was used to detect luteinizing hormone (LH) bound to washed Leydig tumor cells. Tumor cell suspensions were incubated with LH at 37° and washed repeatedly by centrifugation with isotonic 0.9% NaCl solution. The tumor cells contained large quantities of LH even after they were washed sufficiently to produce a 106-fold dilution of unbound LH. Six washings (106-fold dilution) were no more effective in removing LH from the cells than three washings (103-fold dilution). Binding was not influenced by the temperature at which the cells were washed. The extent of LH binding was related to the number of cells, with approximately 5300 ± 960 molecules of LH bound per cell. LH binding was also proportional to the same concentrations of LH which produced a steroidogenic dose response curve. The binding constant of 1.5 × 10-8 m was considered to be higher than that expected for nontumorous tissues. Tumor cells bound more LH than did erythrocytes or thymocytes under the same conditions

Cessation of steroidogenesis in leydig cell tumors after removal of luteinizing hormone and adenosine cyclic 3',5'-monophosphate

Luteinizing hormone (LH), but not follicle-stimulating hormone or prolactin, was shown to enhance steroid synthesis of Leydig tumor cells in vitro. Adenosine cyclic 3',5'-monophosphate (cAMP) duplicated the effect of LH. Removal of LH from the medium within 1 hour of incubation by washing the cells had no effect on the rate of steroid synthesis previously stimulated by LH. Under these conditions, addition of LH antiserum was required to reduce steroid synthesis. In contrast, removal of cAMP by washing the tumor cells caused a rapid termination of the previously induced steroidogenesis. Cycloheximide reduced the steroid synthesis initiated by LH. These results suggest that (a) steroidogenesis may be controlled by short lived factors (proteins), (b) LH may be required continually to elevate cAMP levels to maintain steroid synthesis at stimulated rates, and (c) that LH is probably bound to the tumor cells

Effect of luteinizing hormone antiserum on progesterone and 20α-dihydroprogesterone secretion in the pregnant rat

The effect of a single injection of luteinizing hormone (LH) antiserum on ovarian progesterone and 20α-dihydroprogesterone in day-8 and day-15 pregnant rats was studied. Within 24 h of an injection of LH antiserum, progesterone secretion was reduced by 80% in day-8 and by 25% in day-15 pregnant rats. The 20α-dihydroprogesterone levels after antiserum treatment were markedly increased in rats which were 8 days pregnant but reduced in rats which were 15 days pregnant. The free cholesterol content of the ovary did not change after antiserum injection but the cholesteryl ester content markedly increased. It is thus apparent that neutralization of endogenous LH resulted in a significant reduction in the progesterone secretion of the corpus luteum of the pregnant rat. The significance of these results is discussed

Progestin secretion by the ovary in lactating rats: effect of LH-antiserum, LH and prolactin

In order to understand the control mechanism of progestin secretion during lactation, progesterone (P) and 20α-hydroxypregn-4- en-3-one (20α-OH-P) were determined in ovarian venous blood collected from rats nursing 2 or 6 pups at various stages of lactation in controls, and rats given LH antiserum, LH and prolactin. Generally, P secretion rate increased gradually to reach a peak on Day 12 of lactation and fell sharply on Day 16. Secretion rate of 20α-OH-P decreased as that of P increased and rose sharply on Day 16. On Day 8, P secretion averaged 13.5 μg/30 min in rats nursing 6 pups and was significantly higher than that in rats nursing 2 pups (7.2 μg/30 min). In rats nursing 6 pups LH antiserum reduced P secretion on Day 8 to the level of 2 pup control group. No difference in P secretion was seen between control and LH antiserum groups on Day 16 when P secretion was extremely low (0.6 μg/30 min). LH antiserum also reduced 20α-OH-P secretion in the 6 pup group on Days 4, 8 and 16. Treatment with LH antiserum did not interfere with lactation judged by normal increases in body weight of pups. LH increased P secretion in 2 pup group on Day 8 and in 6 pup group on Day 16. Prolactin treatment did not result in an increase in P and 20α-OH-P secretions on Days 4 and 8, but it reduced significantly the secretion of 20α-OH-P on Day 16. Thus, it was shown that LH is also involved in progestin secretion, during lactation, when the secretory activity is elevated, and that over-all progestin secretion in lactating rats is a reflection of the amount of trophic hormones secreted by the pituitary in response to the strength of suckling stimulus

Role of endogenous primate LH in maintaining corpus luteum function of the monkey

Antiserum to human chorionic gonadotropin (Ayerst) was prepared by administering it in Freund's complete adjuvant to rabbits. Contaminating non-specific antibodies were absorbed with dilute normal human serum and a kaolin-acetone extract prepared from urine of children. Cross-reactivity of this material with human LH or monkey pituitary extract was established using the Ouchterlony double diffusion and competitive binding test. Biological testing involved the capacity of this material to neutralize the ovulation inducing activity of human LH and monkey pituitary extract. It was also able to compete with these two preparations in a modified radioimmunoassay using 125I-labeled human LH. This LH specific antibody (CG A/S) was then injected (2 ml/day) on days 15 through 18 or 15 and 16 into normally cycling Macaca jascicularis. Blood samples were drawn on alternate days beginning on day 9 during the experiment and the serum assayed for progesterone. Progesterone values increased due to ovulation and were subsequently reduced by the injected CG A/S. Those animals receiving only 2 injections had progesterone levels which rebounded to normal levels upon withdrawal of injections. However, 4 injections prevented this rebound and animals so treated had menstrual cycles reduced by 5 to 9 days. The conclusion drawn from these observations is that LH has an important role in the maintenance of the continued function of the subhuman primate corpus luteum

Studies with antisera to luteinizing hormone in vivo and in vitro on luteal steroidogenesis and enzyme regulation of cholesteryl ester turnover in rats

The effect of antiserum to luteinizing hormone (LH) on progesterone and 20α-dihydroprogesterone output and on the enzymes regulating luteal cholesteryl ester turnover was measured in pseudopregnant rats to provide information on the role of LH in regulating luteal function. Progesterone synthesis was reduced when antiserum was added directly to an incubation system of luteinized ovarian slices from animals which had received intravenous saline or 10 μg LH. Steroidogenesis was stimulated in vitro when LH was previously given in vivo, and also on incubating luteinized ovaries from animals treated with antiserum. Treatment in vivo with antiserum alone, however, increased progesterone and 20α-dihydroprogesterone synthesis in vitro but this appeared to be an effect of incubation since in a separate experiment peripheral serum levels of both progesterone and 20α-dihydroprogesterone were reduced after antiserum treatment. Ovarian levels of cholesteryl ester and cholesterol were increased after antiserum treatment in vivo. The level of ovarian cholesteryl esterase was reduced to only 10% of the control level 24 h after antiserum treatment and in this period the level of cholesteryl ester synthetase increased 1.5 times. From these results LH appears to play a direct role in regulating the activity of enzymes controlling cholesteryl ester turnover and thereby regulates the availability of cholesterol for conversion to steroids

Metabolism of free and esterified cholesterol by Leydig-cell tumour mitochondria

1. Experiments were designed to localize intracellularly the enzymes and sterol substrates required for steroidogenesis in Leydig-cell tumours. Subcellular fractions were prepared by differential centrifugation of tumour homogenates. Both free and esterified cholesterol were associated primarily with the fractions sedimenting at 1400g(av.) and the lipid layer floating on the surface of the isolation tubes; they were not found in the mitochondria, where the conversion of cholesterol into pregnenolone occurred. 2. Hydrolysis of esterified cholesterol was required before it could be oxidized to pregnenolone. 3. An enzyme capable of hydrolysing cholesterol esters was located external to the mitochondria. 4. Mitochondria were subfractionated by allowing them to swell in 0.02m-phosphate buffer (pH7.2) and separating the inner and outer membranes by sedimentation in sucrose gradients. The outer membrane, identified by its content of monoamine oxidase, contained most of the cholesterol associated with the mitochondria. The inner membrane, identified by its content of succinate dehydrogenase, contained the cholesterol side-chain-cleaving enzyme and very little cholesterol. 5. Accumulation of sterols by the mitochondria was studied by incubating this fraction with labelled free and esterified cholesterol suspended in lipid-free bovine serum albumin. Two phases of cholesterol accumulation were observed. The first phase, requiring 10–15min, was independent of the incubation temperature, and was inhibited by the presence of bovine serum albumin in the incubation medium. The second phase of accumulation was independent of the serum albumin content of the medium but was inhibited by low incubation temperature. 6. Esterified cholesterol was not accumulated by the mitochondria after the initial rapid binding phase. 7. The findings suggest that cholesterol was not rapidly accumulated by the mitochondrial fraction in vitro and that mechanisms may be required to facilitate cholesterol transport into mitochondria in intact tumour cells during the periods in which steroidogenesis is stimulated maximally

Influence of luteinizing hormone and adenosine 3′:5′-cyclic monophosphate on the metabolism of free and esterified cholesterol in mouse Leydig-cell tumours

1. Male C57B1/6J mice bearing Leydig-cell tumours known to synthesize steroids in response to luteinizing hormone (LH) were given intravenous injections of [1,2-(3)H]cholesterol (50–100μCi per animal). Single-cell suspensions were prepared from the tumours 5–9 days after the injection of [(3)H]cholesterol and were incubated at 37°C in foetal calf serum supplemented with 50mm-Tris–HCl, pH7.4. At various times after the start of incubation cells were collected by filtration of portions of the suspension and their sterols analysed. Within 10min after LH (5μg/ml) or 3′:5′-cyclic AMP (20mm) was added to the cell suspensions an increased conversion of ester cholesterol into free cholesterol could be demonstrated. 2. To observe this rapid effect of LH it was necessary to incubate the cells for 60min before addition of hormone. 3. The specific radioactivity of testosterone produced was approximately equal to that of the intracellular cholesterol regardless of the presence or absence of LH. 4. The amount of free cholesterol produced in response to LH was far greater than that needed for steroid synthesis. 5. Free cholesterol, but not esterified cholesterol, was released into the incubation medium linearly with time and this release was unaffected by LH. LH may stimulate steroidogenesis in part by increasing the concentration of free cholesterol within the cell

The role of FSH and LH in the initiation of ovulation in rats and hamsters: a study using rabbit antisera to ovine FSH and LH

The relative roles of FSH and LH in ovulation induction in immature and adult cycling rats and hamsters have been evaluated. Both heterologous purified pituitary hormones and homologous crude pituitary extracts have been used as ovulatory stimuli in immature animals primed with PMSG. Well-characterized FSH and LH antisera have been used in the above model systems to achieve specific neutralization of FSH and LH. The present study revealed that LH is the physiological trigger needed for induction of ovulation in both rats and hamsters and FSH cannot, by itself, induce ovulation in the total absence of LH