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

Action Mechanism of Inhibin α-Subunit on the Development of Sertoli Cells and First Wave of Spermatogenesis in Mice

Inhibin is an important marker of Sertoli cell (SC) activity in animals with impaired spermatogenesis. However, the precise relationship between inhibin and SC activity is unknown. To investigate this relationship, we partially silenced both the transcription and translation of the gene for the α-subunit of inhibin, Inha, using recombinant pshRNA vectors developed with RNAi-Ready pSIREN-RetroQ-ZsGreen Vector (Clontech Laboratories, Mountain View, Calif). We found that Inha silencing suppresses the cell-cycle regulators Cyclin D1 and Cyclin E and up-regulates the cell-cycle inhibitor P21 (as detected by Western blot analysis), thereby increasing the number of SCs in the G1 phase of the cell cycle and decreasing the amount in the S-phase of the cell cycle (as detected by flow cytometry). Inha silencing also suppressed Pdgfa, Igf1, and Kitl mRNA levels and up-regulated Tgfbrs, Inhba, Inhbb, Cyp11a1, Dhh, and Tjp1 mRNA levels (as indicated by real-time polymerase chain reaction [PCR] analysis). These findings indicate that Inha has the potential to influence the availability of the ligand inhibin and its antagonist activin in the SC in an autocrine manner and inhibit the progression of SC from G1 to S. It may also participate in the development of the blood–testis barrier, Leydig cells, and spermatogenesis through its effect on Dhh, Tjp1, Kitl, and Pdgfa. Real-time PCR and Western blot analyses of Inha, Inhba, and Inhbb mRNA and Inha levels over time show that Inha plays an important role in the formation of round spermatid during the first wave of spermatogenesis in mice

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

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

Effect of HCG antiserum on ovulation and corpus luteum formation in the monkey (Macaca fascicularis)

Considerable evidence is available from recent literature (1,2,3) that ovulation can be induced in the Simian primates by exogenous administration of LH or HCG. It has also been shown, using radioimmunoassay, that a surge of LH is released into the plasma of the intact cycling monkey just prior to ovulation (4,5). An attempt has been made here to neutralize this endogenous LH surge by giving HCG A/S and thereby study the role of LH in inducing ovulation and formation of a functional corpus luteum in the monkey. Antiserum (A/S) to HCG (Ayerst) was raised essentially according to methods described earlier (6) by administering to three New Zealand rabbits repeated injections of HCG in Freund's complete adjuvant (Difco) over a period of several months. The antiserum was absorbed with 1:10 diluted normal human serum and a Kaolin-acetone extract prepared from the urine of nursery school children. The cross-reactivity of this antiserum with human pituitary LH (HLH) and monkey pituitary LH (MPE, an extract of lyophilized pooled monkey pituitaries), was established both by the Ouchterlony double diffusion test and the ovulation inhibition test. The latter test consisted in administering to 22-day-old Charles River female rats 12 1U of PMS followed 48 hours later by an i.p. injection of 2.5 μg of HLH or 1-2 mg of MPE. Antiserum in graded doses was administered by i.p. route 1-2 hours prior to the LH injection. The number of ova shed were counted the next morning according to the method of Ying and Meyer (7). One ml of HCG A/S could neutralize in this test the response of up to 50 μg of HLH or 3 mg of MPE

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

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