Immunoassayable LH Research Articles

In vitro exposure of frog (Rana pipiens) pituitaries to gonadotropin-releasing hormone results in a persistent stimulation of gonadotropin biosynthesis.

To further investigate the action of gonadotropin-releasing hormone (GnRH) on gonadotropin (Gth) secretion and synthesis, pituitaries from frogs, Rana pipiens, were incubated in the presence or absence of 100 ng/ml GnRH for 12 h. GnRH was then removed from the medium and glands were labeled with 35S-methionine either 12, 24, or 36 h after the conclusion of GnRH exposure. Immunoassayable LH and FSH (iLH and iFSH; both labeled and nonlabeled hormone) were determined by separate radioimmunoassays. Newly synthesized (radiolabeled) Gth was quantified by immunoprecipitation. When GnRH was present in the medium, it stimulated the secretion of both iLH and iFSH (compared to unexposed glands). Following withdrawal of the secretagogue, iLH and iFSH secretion diminished until they returned to control levels (within ca. 12 h). Previous exposure to GnRH enhanced the biosynthesis of Gth; this elevation was detectable up to 36 h after removal of the secretagogue. The magnitude of the elevation in Gth synthesis did not diminish with time, as secretion had, but remained constant. Gth synthesis was comparable in glands that had been exposed to GnRH 12 h and those exposed to the secretagogue 36 h prior. Thus, the GnRH-induced elevation in Gth secretion and synthesis can be dissociated; the elevation in synthesis is more chronic than the elevation in secretion.

Luteinizing hormone synthesis in cultured fetal human pituitary cells exposed to gonadotropin-releasing hormone.

To investigate whether GnRH regulates LH synthesis during human development, pituitary cells from second trimester fetuses were incubated with [35S]methionine ([35S]met) and [3H]glucosamine ([3H]gln) for 48 h with 0, 10(-9), and 10(-7) mol/L GnRH. Immunoassayable (i) LH was measured in media and cellular lysates, and dual label scintillation analysis was used to quantitate incorporation of radiolabeled precursors into cells, trichloroacetic acid-precipitable proteins, and immunoprecipitated LH subjected to electrophoresis. Exposure of cells to GnRH did not affect cellular uptake or incorporation of precursors into proteins, but specifically increased total (secreted plus cellular) LH synthesis. Both GnRH concentrations significantly increased iLH release and enhanced secreted and cellular [3H]gln-LH. The secretion of [35S] met-LH was stimulated only by 10(-7) mol/L GnRH. The proportion of newly synthesized LH that was secreted and the 3H/35S ratio of secreted and cellular LH were uninfluenced by GnRH. Although basal LH synthesis was not sex dependent, total iLH content and GnRH-stimulated LH translation were greater in cells from females than in those from males. Therefore, GnRH regulates LH synthesis by second trimester fetal human gonadotrophs without influencing the proportion of total radiolabeled LH that is secreted. The existence of a sex difference in total iLH content and GnRH-stimulated LH translation is consistent with the sexual dimorphism in pituitary LH content occurring during human development.

Leydig cell tumor estrogen secretion: Suppression by a gonadotropin releasing hormone agonist

A 35-year-old patient and a 48-year-old patient with a benign feminizing Leydig cell tumor were treated just before orchiectomy with a GnRH agonist, respectively buserelin, administered subcutaneously for 7 days (500 micrograms/8 h) and intranasally for the following 7 days (400 micrograms/8 h), and long acting détryptoréline in a single im injection of 3.75 mg. After 10 days of treatment, breast pain was relieved. In the first patient, serum immunoassayable FSH and LH first rose and reached a peak by the first day, then decreased to nearly basal values; they unexpectedly rose again and the second peak was as high as the first one; they again decreased just before orchiectomy. The bioassayable to immunoassayable LH ratio rose and reached a peak on day 3; then it decreased and reached a nadir before orchiectomy. In the second patient, after the initial increase, FSH and LH decreased regularly with no subsequent increase. In both patients, an increase in plasma testosterone (T) and estradiol (E2) followed the first gonadotropin peak, then T and E2 decreased progressively and reached castration levels. It was thus possible to strongly inhibit both E2 tumoral secretion and T secretion. Our findings suggest that E2 tumoral secretion inhibition by GnRH agonists might be proposed as an alternative treatment to surgery, i.e. for patients who refuse orchiectomy or for whom surgery is contraindicated.

Human serum LH inhibitor(s): behaviour and contribution to in vitro bioassay of LH using dispersed mouse Leydig cells

The present study aimed at investigating the nature and causes of non-parallelism in testosterone responses to serial dilutions of peripheral serum and standard LH preparations in the mouse Leydig cell in vitro bioassay of LH. Immunoadsorption with monoclonal antibody to the beta-subunit of LH was used to obtain LH-free serum; the procedure removed more than 98% of the immunoassayable LH. When a constant amount of the LH-free serum was added to standard dilutions, the bioassay dose-response curves to serum dilutions, the standards became parallel, i.e. the well-known source of error of this assay system was eliminated. When standard curves prepared in medium and LH-free serum (final concentration 10%) were compared, no effect of the serum was found on basal cAMP and testosterone production. However, the LH-stimulated testosterone and cAMP production were suppressed by serum by a rather constant factor of 40%. Mild heating (60 degrees C, 15 min) or treatment with dextran-coated charcoal, but not other extraction, was able to eliminate the inhibitory activity of the LH-free serum. Binding studies demonstrated that [125I]HCG interaction with mouse Leydig cell homogenates was inhibited by LH-free serum in a fashion indicative of reduced LH receptor number, but not of reduced binding affinity. In conclusion, these data show that human serum contains LH inhibitor(s) which affect the LH-receptor interaction and LH stimulated testosterone production in mouse Leydig cell in vitro. The effect is marked in serum concentration over 1.5% and it shows only minor variation between individual sera. This source of error can be effectively removed from the LH in vitro bioassay by using LH-free serum for preparation of dilutions of LH standards.

Prolonged duration of gonadotropin inhibition by a third generation GnRH antagonist.

The dose-response effects of a single administration of Nal-Lys-GnRHant (antagonist) on serum LH and FSH concentrations were compared to the effects of Nal-Glu-GnRHant in monkeys. Twenty ovariectomized monkeys were divided into four sc treatment groups: a) 1.0 mg/kg Nal-Glu-GnRHant; or Nal-Lys-GnRHant at b) 0.3; c) 1.0; d) 3.0 mg/kg. Each monkey received vehicle (propylene glycol/water, 1:1) on day 0, followed by an antagonist preparation on day 11. Serum LH and FSH were measured by RIA; serum LH was also measured by in vitro bioassay. The short-term effects were similar among the four treatment groups. Typically, serum LH declined (p less than 0.05) within 4 to 8 h, achieving maximal reduction by 24 h. Serum FSH levels declined more slowly, but were significantly reduced by 24 h (p less than 0.05). Recovery during the study interval to pretreatment control values occurred in only two groups: a) Nal-Glu-GnRHant (1.0 mg/kg) by day 4 post-treatment and b) Nal-Lys-GnRHant (0.3 mg/kg) by day 2 post-treatment. Monkeys receiving 1.0 or 3.0 mg/kg Nal-Lys-GnRHant had a prolonged inhibition of serum LH and FSH levels. In all animals, serum FSH and LH returned to control levels within 2 months. The duration of gonadotropin inhibition was also prolonged when the Nal-Lys-GnRHant was administered iv. In contrast, Nal-Glu-GnRHant reduced serum LH and FSH for 3 days or less in all monkeys. The serum bioassayable LH levels paralleled those of immunoassayable LH. The prolonged inhibition of gonadotropin secretion following Nal-Lys-GnRHant distinguishes its action from those of previous GnRH antagonists and make this compound of great interest for clinical investigations.

Periovulatory hormonal dynamics: relationship of immunoassayable gonadotropins and ovarian steroids to the bioassayable luteinizing hormone surge in rhesus monkeys.

The relationship between ovarian steroids and LH during the midcycle gonadotropin surge is controversial. Recent demonstration of temporal and quantitative differences in immunoassayable LH (I-LH) and bioassayable LH (B-LH) at midcycle have further clouded this issue. To evaluate the relationship of I-LH, FSH, and ovarian steroids to the onset of the midcycle B-LH surge, blood samples were obtained from five chronically catheterized rhesus monkeys at 2-h intervals for 5-6 days. The plasma was assayed for FSH, LH, 17 beta-estradiol (E2), progesterone (P4), and 17 alpha-hydroxyprogesterone (17-OHP) by RIA and for LH by a rat interstitial cell testosterone in vitro bioassay. The initiation of the B-LH surge served as time zero (to) for the temporal analysis of changes in plasma hormone levels. The I-LH and FSH surges were initiated 6.4 +/- 2.2 h (mean +/- SEM) and 5.2 +/- 1.9 h, respectively, after the onset of the B-LH surge. Although the duration of the ascending limb of the surge was similar for B-LH, I-LH, and FSH, the mean +/- SEM total duration of the B-LH surge (34.5 +/- 3.5 h) was significantly longer (P less than 0.025) than those of I-LH (24.4 +/- 5.0 h) and FSH (27.6 +/- 2.3 h). Before the onset of the B-LH surge (to - 4 h), the ratio of B-H to I-LH was unity; however, during the acme of gonadotropin secretion (to + 12-16 h), the B-LH to I-LH ratio approached 6:1. Doubling times for B-LH, I-LH, and FSH were similar during the ascending phase of the surge. Plasma E2 concentrations increased continuously from to - 40 h to to, with a mean +/- SEM doubling time of 32.6 +/- 4.7 h. Peak E2 concentrations occurred within 6 h after the onset of the B-LH surge. Plasma P4 concentrations began to increase at to - 6 h in four monkeys and at to in one monkey. Plasma P4 concentration plateaued from to to to + 24 h, then increased rapidly, with a mean +/- SEM doubling time of 20.5 +/- 2.9 h. Although there were significant individual variations in plasma 17-OHP concentrations, a definite increase in 17-OHP occurred by to - 10 h, and peak concentrations occurred at to + 1 h.(ABSTRACT TRUNCATED AT 400 WORDS)

Hormonal effects of gonadotropin-releasing hormone (GnRH) agonist in the human male. III. Effects of long term combined treatment with GnRH agonist and androgen.

Chronic treatment with agonist analogs of GnRH results in reversible oligospermia in man, but leads to impotence and decreased libido due to a concomitant fall in serum testosterone (T) concentrations. We, therefore, assessed the effects of combined treatment with a potent GnRH agonist and T on gonadotropins and spermatogenesis in normal men, anticipating that addition of androgen would prevent agonist-induced changes in libido. Seven normal men were treated with 200 micrograms of the GnRH agonist D-(Nal2)6GnRH (GnRH-A), sc, daily for 16 weeks. In addition, 200 mg T enanthate were administered every 2 weeks for the entire 16-week treatment period. Basal LH, FSH, and T concentrations were measured every week during a 5-week control period, daily on treatment days 0, 1-10, 14, 18, 22, 26, and 28, every week thereafter until day 56, and every 2 weeks thereafter for the remainder of the treatment and recovery phases. Detailed analysis of LH and FSH over the 24-h period was performed by multiple blood sampling on days 0, 1, 10, 28, 56, 84, and 112. Semen analyses were performed every week during the control phase and every 2 weeks during the treatment and recovery phases. The mean sperm count declined by 83%, to a nadir of 16.6 +/- 6.2 (+/- SEM) million/ml. One subject had no significant decrease in sperm count. Azoospermia was not achieved in any subject. Basal serum LH concentrations, after an early phase of stimulation, declined to near baseline by day 14. However, basal, 24-h integrated serum LH concentrations, and 24-h urinary LH excretion were not significantly lowered by combined treatment. Bioassayable serum LH concentrations, however, declined significantly from 20.4 +/- 6.3 to 4.5 +/- 0.5 mIU/ml, and the bioassayable to immunoassayable LH ratio decreased from 2.1 +/- 1.0 to 0.7 +/- 0.1 after 16 weeks of GnRH-A treatment. Basal and 24-h integrated FSH concentrations, after an initial period of stimulation, declined progressively to baseline by days 5-6 and were significantly below baseline by day 112. Serum T concentrations did not fall into the hypogonadal (less than 250 ng/dl) range in any subject at any time during the treatment period. After discontinuation of treatment, LH, FSH, and sperm counts returned to normal in all subjects. Thus, single daily injection of GnRH-A and T failed to predictably induce azoospermia in normal men over the 16-week treatment period.(ABSTRACT TRUNCATED AT 400 WORDS)

The late pubertal cascade in perimenarchial monkeys: onset of asymmetrical ovarian estradiol secretion and bioassayable luteinizing hormone release.

Changes in ovarian function and pituitary gonadotropin secretion were studied in perimenarchial rhesus monkeys. Even in the premenarchial interval, a modest degree of asymmetrical ovarian estradiol secretion was evident. A progression toward marked asymmetry of ovarian function continued after menarche, culminating in ovulatory menstrual cycles with intermenstrual intervals of approximately 28 days. Among postmenarchial monkeys manifesting five or fewer episodes of overt uterine bleeding, no ovulations were detected despite estradiol elevations similar to those of adults in the midfollicular phase. Soon thereafter, among individuals usually having more than 10 menses, the first ovulations were likely to be achieved. The initiation of estrogen-positive feedback, driving the surge modes of gonadotropin secretion, was accompanied by the onset of a striking disparity between bioassayable vs. immunoassayable LH in the circulation. In this report we describe a cascade of late pubertal events including: 1) the gradual establishment of cyclic asymmetrical ovarian estrogen secretion in the perimenarchial interval, 2) increased pituitary responsiveness to GnRH, 3) quantitative and qualitative changes in the pulsatile secretion of pituitary gonadotropins, and 4) an enhancement of bioassayable LH secretion, especially during the preovulatory surge.

Diethylstilbestrol reversal of gonadotropin patterns in infant rhesus monkeys

The role of sex steroids in the sexual differentiation of primates is poorly understood. The present study was designed to examine the early endocrine effects of pre and postnatal exposure of rhesus monkeys to DES and to assess the efficacy of the model to study the impact of currently used therapeutic steroids on fetal development. Pregnant female rhesus were given DES orally (0.1 or 1.0 mg/kg/day) on a sugar cube from days 25–150 of gestation. Infants of non-treated mothers were administered DES in orange-flavored drink until weaning, after which the dose was placed on a sugar cube. Dose levels examined were 0.5 and 50.0μg/kg/day from days 1–365 with day of birth designated as Day 0. Control infants were run concurrently. Blood samples (2 ml) for gonadotropin assay were obtained from all groups on Days 4 and 14 after birth, at 14-day intervals until Day 84 and at monthly intervals thereafter. Serum FSH and LH were determined by radioimraunoassay. In controls, FSH levels rose at birth and fell to low, stable levels at 4–5 months of age in male and female infants. The greatest postnatal peak in FSH was found in females ( P < 0.05) which declined to a nadir at a much slower rate than in males. Immunoassayable LH levels were similar in infant male and female controls. In males prenatally exposed to DES, FSH remained elevated for at least the first 18 months of life in contrast to the fall at 4 months seen in controls. Immunoassayable LH tended to rise after 4 months of age in male and female infants exposed in utero to the highest treatment dose of DES, but did not in the controls. DES administered during the first year of life caused an unexpected increase in FSH in males throughout the treatment period after which levels declined. In females DES suppressed FSH for the first 4 months of life. This was followed by a reversal from suppressed to elevated levels after 4 months of age until dosing was discontinued. LH levels were not affected in males by postnatal exposure to DES. In females LH levels were greater than controls for the first 4 months of treatment, subsequently returning to normal. These studies have demonstrated unequivocally that exposure of rhesus monkeys to DES either in utero or during the first year of life causes profound changes in postnatal endocrine function. Thus. the rhesus infant could serve as a model for predicting the long-term reproductive effects of in utero or postnatal exposure to hormonally-active drugs and environmental agents.

Pulsatile pituitary gonadotropin secretion during maturation of the dominant follicle in monkeys: estrogen positive feedback enhances the biological activity of LH.

In the follicular phase of the menstrual cycle, pulsatile patterns of LH and FSH secretion in monkeys change during maturation of the dominant follicle. At the preovulatory surge, the most striking event is the prodigious elevations of bioassayable LH, rising up to 50-fold within 24 h. Principally, establishment of the surge is due to marked enhancement of the amplitude of LH secretory pulses. In contrast, LH and FSH measured by RIA enter, in parallel, the surge modes of secretion approximately 5 h later than bioassayable LH and rise more slowly; the B:I ratio may reach 10:1. This same disparity between bioassayable versus immunoassayable LH was induced in castrate monkeys under estrogen positive feedback stimulation. We conclude that the preovulatory estrogen surge promotes the secretion of an LH molecule(s) having enhanced biological activity.

Presence of Immunoreactive Luteinizing Hormone in the Rat Forebrain

This study demonstrates the presence of immunoreactive LH in rat brain by radioimmunoassay and immunocytochemistry. High levels of radioimmunoassayable LH were identified in the hypothalamus, while significant but lesser quantities were found in the amygdala, septal area, preoptic area, thalamus, caudate nucleus, and hippocampus. Correlative immunocytochemistry localized immunopositive fibers in hypothalamic and several extrahypothalamic brain structures. Immunoreactive cell bodies were seen in colchicine-treated rats in the arcuate nucleus of the hypothalamus. Gel chromatography of hypothalamic extracts revealed that immunoassayable LH coeluted with LH standard and rat pituitary extracts. Possible mechanisms related to the origin and functional neuronal LH are discussed.

Hormonal measurement in rat anterior pituitary cell cultures: Loss of immunoreactive LH counteracted by fetal calf serum and bacitracin

Immunoassayable LH in media samples from rat anterior pituitary cell cultures declines during storage and only 20% of the LH remains after 4 weeks at -20°C. The LH loss was not due to bacterial contamination or to damage to the hormone from repeated freezing and thawing. SDS-PAGE of 125IrLH in media samples showed greater recovery of 125IrLH when 1 mM bacitracin or 2% fetal calf serum were present in the medium. The ratio of intact: subunit 125IrLH was unchanged by the presence of bacitracin or fetal calf serum indicating that the loss of immunoreactive LH was not due to dissociation of intact hormone. LH appears to be irreversibly altered in stored culture media, a process which can be prevented by the addition of bacitracin or fetal calf serum to the media prior to storage. The use of either substance allows accurate and reproducible measurement of LH released from pituitary cells in culture.

Effects of synthetic gonadotropin-releasing hormone on incorporation of radioactive glucosamine and amino acids into luteinizing hormone and total protein by rat pituitaries in vitro.

The influence of synthetic gonadotropin-releasing hormone (GnRH) on incorporation of glucosamine and amino acids into LH and total protein was investigated. Rat pituitaries were incubated in vitro with radioactive precursors for 4 h at 37 C. Labeled LH was isolated by immunoprecipitation with specific anti-LH-beta serum. Total LH in the medium and pituitary glands was measured by radioimmunoassay. GnRH significantly stimulated incorporation of [3H]glucosamine into LH in pituitaries from immature male, intact female, and ovariectomized rats. Pituitaries from ovariectomized rats synthesized more [3H]glucosamine-labeled LH than those from intact adult rats. GnRH enhanced release of [3H]glucosamine-LH into medium in pituitaries of both adult intact and ovariectomized rats. The response to GnRH was greatly reduced when pituitaries were exposed to cold shock before the 4 h incorporation period. In contrast to its effect on incorporation of [3H]glucosamine into LH, GnRH had either no or little effect, depending on experimental conditions, on uptake of [3H]glucosamine by pituitaries. Incorporation of [3H]glucosamine into total protein was not affected by GnRH treatment. GnRH increased release of either [3H]leucine- or [3H]alanine-labeled LH into medium, but had no significant effect on incorporation of labeled amino acids into total LH in the system. GnRH modified neither uptake nor incorporation of amino acids into total protein. GnRH significantly stimulated the release of cold LH into medium under all conditions, but the total amount of immunoassayable LH in the system was not detectably modified by GnRH treatment.

LH and its subunits in human pituitary, serum and urine.

Human postmenopausal serum and urine, and saline extracts of human pituitaries were gel-filtered on Sephadex G-100. The eluted fractions were radioimmunoassayed using 3 specific assays for native hLh, hLHalpha and hLHbeta. The elution profile of immunoreactive components in postmenopausal urine exhibited 2 peaks of activity corresponding to those demonstrated for 125I-labeled hLH and hLHalpha. No LHbeta was demonstrated. The immunoreactive components of post-menopausal serum eluted identically to 125I-LH, LHalpha and LHbeta. In addition, a peak of immunoreactive LH material was observed in the pre-LH region of the elution profile, distinctly separate from the void volume. The percent of the total immunoassayable serum LH contained in this peak varied. In elution profiles of all pituitary extracts, LH, LHalpha and LHbeta elution peaks corresponding to the respective labeled preparations were observed. An apparent excess of free alpha subunit was noted. All three antisera demonstrated immunoassayable LH material in the void volume. This large species contained a higher proportion of material reacting in the anti-hLHalpha system than did native hLH. Examination of the void volume material by electron microscopy indicated membrane material, but no ribosomes. Chemical assay failed to detect RNA. When void volume material was subjected to conditions which are known to (a) disrupt membrane continuity and (b) dissociate the noncovalently bonded subunits of native LH, this large species of LH failed to dissociate into smaller molecular species. These studies reveal: (1) an excess of free alpha subunit in the pituitary, serum and urine; (2) a large species of LH in the pituitary which upon analysis does not appear to be simple aggregation of LH, MEMBRANE ENTRAPPED NATIVE LH, OR RIBOSOMAL BOUND LH.

Reproductive function in the rat following selective destruction of afferent fibres to the hypothalamus from the limbic system

A technique has been developed for the routine bilateral surgical interruption of the stria terminalis and fimbria in the rat at a site sufficiently remote to preclude direct damage to the preoptic area or hypothalamus and has been used to study the effects of such interruption on reproductive function. In the male rat such lesions led to a slight hypersecretion of LH in intact animals and a slight reduction in the rise of immunoassayable LH in the plasma after castration. In immature female rats both the time of vaginal opening and of first ovulation were advanced without any increase in the rate of somatic growth. In the adult female the early effect of the lesion was to block ovulation in that cycle when made at proestrus; delayed pseudopregnancies were not observed. When the lesion was made at the metoestrous stage of the cycle an immediate pseudopregnancy resulted provided that the fimbria on one side was completely destroyed and there was at least some damage to the hippocampus on the other side; the degree of damage to the stria terminalis had no influence on this response. Following these acute responses the lesioned females resumed regular oestrous cycles but became pseudopregnant following sterile mating and could become pregnant, deliver and rear healthy litters of normal size. After exposure to constant light, lesioned animals showed persistent vaginal and behavioural oestrus and some ovulated after mating. The compensatory ovarian hypertrophy after unilateral ovariectomy and the elevated plasma LH levels after ovariectomy were comparable to the changes seen in control rats. Oestrogen alone reduced the plasma LH levels in ovariectomized animals; subsequent administration of a second dose of oestrogen or progesterone resulted in an increase in LH levels, though this was not as striking as in control animals. The progesterone induced rise was not preceded by a decrease in LH concentration as it was in the controls. Various types of more extensive hypothalamic deafferentation which include the stria terminalis and fimbria result in abnormalities of reproductive function that were not seen in our animals in which these tracts alone were destroyed. We conclude that the destruction of other fibres must be held responsible for the abnormalities which follow more extensive deafferentation.