Age diminishes the testicular steroidogenic response to repeated intravenous pulses of recombinant human LH during acute GnRH-receptor blockade in healthy men

Testosterone (Te) concentrations fall gradually in healthy aging men. Postulated mechanisms include relative failure of gonadotropin-releasing hormone (GnRH), luteinizing hormone (LH), and/or gonadal Te secretion. Available methods to test Leydig cell Te production include pharmacological stimulation with human chorionic gonadotropin (hCG). We reasoned that physiological lutropic signaling could be mimicked by pulsatile infusion of recombinant human (rh) LH during acute suppression of LH secretion. To this end, we studied eight young (ages 19-30 yr) and seven older (ages 61-73 yr) men in an experimental paradigm comprising 1) inhibition of overnight LH secretion with a potent selective GnRH-receptor antagonist (ganirelix, 2 mg sc), 2) intravenous infusion of consecutive pulses of rh LH (50 IU every 2 h), and 3) chemiluminometric assay of LH and Te concentrations sampled every 10 min for 26 h. Statistical analyses revealed that 1) ganirelix suppressed LH and Te equally (> 75% median inhibition) in young and older men, 2) infused LH pulse profiles did not differ by age, and 3) successive intravenous pulses of rh LH increased concentrations of free Te (ng/dl) to 4.6 +/- 0.38 (young) and 2.1 +/- 0.14 (older; P < 0.001) and bioavailable Te (ng/dl) to 337 +/- 20 (young) and 209 +/- 16 (older; P = 0.002). Thus controlled pulsatile rh LH drive that emulates physiological LH pulses unmasks significant impairment of short-term Leydig cell steroidogenesis in aging men. Whether more prolonged pulsatile LH stimulation would normalize this inferred defect is unknown.     

Glucose ingestion acutely lowers pulsatile LH and basal testosterone secretion in men

Chronic hyperglycemia inhibits the male gonadal axis. The present analyses test the hypothesis that acute glucose ingestion also suppresses LH and testosterone (T) secretion and blunts the LH-T dose-response function. The design comprised a prospectively randomized crossover comparison of LH and T secretion after glucose vs. water ingestion in a Clinical Translational Research Center. The participants were healthy men (n = 57) aged 19-78 yr with body mass index (BMI) of 20-39 kg/m(2). The main outcome measurements were deconvolution and LH-T dose-response analyses of 10-min data. LH-T responses were regressed on glucose, insulin, leptin, adiponectin, age, BMI, and CT-estimated abdominal visceral fat. During the first 120 min after glucose ingestion, for each unit decrease in LH concentrations, T concentrations decreased by 86 (27-144) ng/dl (r = 0.853, P < 0.001). Based upon deconvolution analysis, glucose compared with water ingestion reduced 1) basal (nonpulsatile; P < 0.001) and total (P < 0.001) T secretion without affecting pulsatile T output and 2) pulsatile (P = 0.043) but not basal LH secretion. By multivariate analysis, pulsatile LH secretion positively predicted basal T secretion after glucose ingestion (r = 0.374, P = 0.0042). In addition, the glucose-induced fall in pulsatile LH secretion was exacerbated by higher fasting insulin concentrations (P = 0.054) and attenuated by higher adiponectin levels (P = 0.0037). There were no detectable changes in the analytically estimated LH-T dose-response curves (P > 0.30). In conclusion, glucose ingestion suppresses pulsatile LH and basal T secretion acutely in healthy men. Suppression is influenced by age, glucose, adiponectin, and insulin concentrations.     

Analytical construct of reversible desensitization of pituitary-testicular signaling: illustrative application in aging

Luteinizing hormone (LH) administered in pharmacological amounts downregulates Leydig cell steroidogenesis. Whether reversible downregulation of physiological gonadotropin drive operates in vivo is unknown. Most of the analytical models of dose-response functions that have been constructed are biased by the assumption that no downregulation exists. The present study employs a new analytical platform to quantify potential (but not required) pulsatile cycles of LH-testosterone (T) dose-response stimulation, desensitization, and recovery (pulse-by-pulse hysteresis) in 26 healthy men sampled every 10 min for 24 h. A sensitivity-downregulation hysteresis construct predicted marked hysteresis with a median time delay to LH dose-response inflection within individual T pulses of 23 min and with median T pulse onset and recovery LH sensitivities of 1.1 and 0.10 slope unit, respectively (P < 0.001). A potency-downregulation model yielded median estimates of one-half maximally stimulatory LH concentrations (EC(50) values) of 0.66 and 7.5 IU/l for onset and recovery, respectively (P < 0.001). An efficacy-downregulation formulation of hysteresis forecasts median LH efficacies of 20 and 8.3 ng·dl(-1)·min(-1) for onset and offset of T secretory burst, respectively (P = 0.002). Segmentation of the LH-T data by age suggested greater sensitivity, higher EC(50) (increased LH potency), and markedly (2.7-fold) attenuated LH efficacy in older individuals. Each of the three hysteresis models yielded a marked (P < 0.005) reduction in estimated model residual error compared with no hysteresis. In summary, model-based analyses allowing for (but not requiring) reversible pituitary-gonadal effector-response downregulation are consistent with a hypothesis of recurrent, brief cycles of LH-dependent stimulation, desensitization, and recovery of pulsatile T secretion in vivo and an age-associated reduction of LH efficacy. Prospective studies would be required to prove this aging effect.     

Age-specific changes in the regulation of LH-dependent testosterone secretion: assessing responsiveness to varying endogenous gonadotropin output in normal men

Pulsatile and thus total testosterone (Te) secretion declines in older men, albeit for unknown reasons. Analytical models forecast that aging may reduce the capability of endogenous luteinizing hormone (LH) pulses to stimulate Leydig cell steroidogenesis. This notion has been difficult to test experimentally. The present study used graded doses of a selective gonadotropin releasing hormone (GnRH)-receptor antagonist to yield four distinct strata of pulsatile LH release in each of 18 healthy men ages 23-72 yr. Deconvolution analysis was applied to frequently sampled LH and Te concentration time series to quantitate pulsatile Te secretion over a 16-h interval. Log-linear regression was used to relate pulsatile LH secretion to attendant pulsatile Te secretion (LH-Te drive) across the four stepwise interventions in each subject. Linear regression of the 18 individual estimates of LH-Te feedforward dose-response slopes on age disclosed a strongly negative relationship (r = -0.721, P < 0.001). Accordingly, the present data support the thesis that aging in healthy men attenuates amplitude-dependent LH drive of burst-like Te secretion. The experimental strategy of graded suppression of neuroglandular outflow may have utility in estimating dose-response adaptations in other endocrine systems.     

Age-related decline of plasma bioavailable testosterone in adult men

Plasma bioavailable and total testosterone (T), gonadotropins (FSH, LH) and prolactin (PRL) were determined in 70 ambulatory men subdivided into 3 groups according to age: group I (n = 22; age 20-35 yr), group II (n = 22; age: 36-50 yr) and group III (n = 26; age 51-70 yr). Bioavailable T levels declined significantly with age (r = -0.42; P less than 0.01) while those of total T decreased less significantly (r = -0.28; P less than 0.05). In addition, the decrease of bioavailable T occurred earlier. FSH was shown to increase with age (r = 0.41; P less than 0.01) whereas LH and PRL were not found to change significantly. Bioavailable T was correlated with total T (r = 0.25; P less than 0.05) and inversely correlated with FSH (r = -0.26; P less than 0.05). No correlation could be demonstrated between LH and either bioavailable or total T. In view of the age-related increase of sex hormone binding globulin, a fact generally observed in the literature, bioavailable T may be considered a more reliable index than total T for the evaluation of T production. Thus it may be concluded that the early decrease of bioavailable T in ambulatory men not known to have any pathology or any medication altering testicular function corresponds in fact to age-related decline of T secretion by the testes.     

Central infusion of recombinant ovine leptin normalizes plasma insulin and stimulates a novel hypersecretion of luteinizing hormone after short-term fasting in mature beef cows

The present studies tested the hypotheses that short-term fasting would reduce leptin gene expression and circulating concentrations of leptin and insulin in mature, ovariectomized, estradiol-implanted cows and that intracerebroventricular infusions of recombinant ovine leptin (oleptin) would attenuate reductions in insulin concentration and stimulate LH secretion. Ovariectomized cows were assigned to either control (normal fed; n = 6) or fasted (60 h of fasting; n = 7) groups and infused with 200 microg recombinant oleptin three times at hourly intervals on Day 2 (n = 6 per group). Fasting decreased plasma concentrations of insulin (P < 0.01) and leptin (P < 0.04) but, as expected, did not reduce plasma concentrations of glucose or any LH secretion variable. Central infusion of leptin on Day 2 increased (P < 0.01) plasma concentrations of leptin in both control and fasted groups. Concomitantly, leptin treatment increased plasma insulin (P < 0.01) and LH (P < 0.03) concentrations in fasted but not in control cows. Increases in overall mean and baseline concentrations of LH after leptin treatment were the result of an augmentation of the size of LH pulses. The effects of fasting on leptin gene expression and the potential diurnal effects on circulating leptin were examined in a group of cows (n = 12) not treated with leptin. Fasting for 60 h reduced (P < 0.001) leptin gene expression by 30%, and no diurnal effects on circulating leptin were observed. These results indicate that although short-term fasting does not reduce the frequency or amplitude of LH pulses or the concentration of LH in mature cows, this nutritional perturbation clearly sensitizes both the hypothalamic-pituitary axis and endocrine pancreas to exogenous leptin, which in these experiments resulted in heightened secretion of both LH and insulin.     

Maintenance or stimulation of steroidogenic enzymes and testosterone production in rat Leydig cells by continuous and pulsatile infusions of luteinizing hormone during passive immunization against gonadotrophin-releasing hormone.

The importance of the pulsatility of luteinizing hormone (LH) secretion in maintaining key enzymes in the testosterone biosynthetic pathway in Leydig cells was studied using rats in which LH secretion was suppressed by passive immunization against gonadotropin-releasing hormone (GnRH) and replaced by continuous or pulsatile i.v. infusions of exogenous LH, all delivering the same daily dose of the hormone (300 ng per 100 g NIDDK-ovine LH-24). Continuous infusions (12.5 ng per 100 g h-1) were compared with infusions of 1 min pulses every 2 h (25 ng per 100 g) and every 4 h (50 ng per 100 g). After 5 days of treatment in vivo with sheep anti-GnRH serum (or normal sheep serum) and LH (or vehicle), Leydig cells were purified and assayed in vitro for maximum production of testosterone stimulated by human chorionic gonadotrophin (hCG) and supported by 25-hydroxycholesterol and for the activities of cholesterol side-chain cleavage, delta 5-3 beta-hydroxysteroid dehydrogenase-delta 5-4-isomerase (3 beta-HSD-isomerase) and 17 alpha-hydroxylase. Relative contents of cholesterol side-chain cleavage and 17 alpha-hydroxylase were also quantified by western and immunoblotting analysis. Activity of 3 beta-HSD-isomerase was reduced by about 40% by anti-GnRH treatment and was increased by all LH regimens in anti-GnRH-treated animals, with no consistent pattern in the effects of the different LH regimens. Results for testosterone-producing capacity and the other two enzymes differed in several respects. Treatment with anti-GnRH serum markedly reduced basal, hCG-stimulated and 25-hydroxycholesterol-supported testosterone production (by 80-90%) and the activities of cholesterol side-chain cleavage (about 80%) and 17 alpha-hydroxylase (about 65%). Infusion of exogenous LH in any of the regimens tested prevented these changes or increased the activities to values greater than those in normal serum-treated controls. Differences in immunodetectable contents of the two enzymes generally paralleled those in enzyme activities. There was a consistent trend in the effects of LH replacement regimens on these parameters of steroidogenic activity: continuous infusions were more effective than pulses at 2 h intervals and these in turn were more effective than pulses at 4 h intervals, suggesting that the frequency of LH exposure is more important than the amplitude of individual exposures in maintaining Leydig cell steroidogenic function.(ABSTRACT TRUNCATED AT 400 WORDS)     

Dynamic testosterone responses to near-physiological LH pulses are determined by the time pattern of prior intravenous LH infusion

The long-lived glycoprotein hormone, human chorionic gonadotropin (hCG), downregulates testosterone (T) biosynthesis in vitro and in vivo in animals and humans. The degree to which short-lived pulses of pituitary luteinizing hormone (LH) do so, particularly at physiological concentrations, is not known. We test the hypothesis that continuous LH infusion compared with bolus injections of LH every 1 h or every 2 h overnight downregulates T secretory responses to a subsequent fixed template of three consecutive intravenous pulses of a physiological amount of recombinant human (rh) LH (triple stimulus). Nineteen healthy men ages 18-49 yr each underwent four separate randomly ordered overnight gonadotropin-releasing hormone-receptor antagonist treatments with superimposed intravenous infusions of saline or rhLH (1-h pulses, 2-h pulses, or continuously). Each 12-h infusion protocol was followed by the triple rhLH-pulse stimulus the next morning. During the triple stimulus, basal (nonpulsatile) as well as total (basal plus pulsatile) T secretion was higher after overnight 2- and 1-h rhLH pulses than after continuous rhLH or saline delivery. Approximate entropy, a probabilistic measure of feedforward-induced irregularity of T concentration time series, was higher after 1-h rhLH pulses than after continuous rhLH. Analytical estimation of pulsatile rhLH-T dose-response measures revealed higher T secretory sensitivity and greater rhLH potency (lower EC(50)) after exposure to 1-h than 2-h rhLH pulses. Collectively, these data indicate that in vivo dynamics of LH-stimulated T secretion under standardized conditions in men depend on the prior time mode of LH delivery in the bloodstream.     

Short-term fasting leads to inhibition of responsiveness to LH-stimulated testosterone secretion in the adult male bonnet monkey

A variety of stressors including fasting profoundly inhibit reproductive function in mammals. Although the effect of short-term fasting on gonadotropic axis is well established, the direct effects of fasting on gonads have not been reported. The objectives of the present experiments were to examine the effect of short-term fasting on circulating luteinizing hormone (LH) and testosterone (T) secretion, and to determine the responsiveness of testis to exogenous recombinant human (rh) LH treatment in male bonnet monkeys. In addition, an experiment was carried out to examine whether brief inhibition of endogenous LH secretion causes alteration in testicular responsiveness. Adult male monkeys were fasted for 1 day for examining the circulating endocrine hormone concentrations and challenged with rhLH injection 1 day after fasting. Food withdrawal for 1 day resulted in significant (P<0.05) decrease in LH, T and increase in cortisol concentrations. Surprisingly, T secretion in response to direct stimulation of Leydig cells by LH was not observed in fasted monkeys. In fed monkeys, treatment with Antide (a specific gonadotropin releasing hormone receptor antagonist to inhibit pituitary LH secretion) for 1 day did not compromise T secretion stimulated by rhLH, suggesting that loss of responsiveness of testis to exogenous LH treatment in fasted monkeys was not because of interruption in pituitary LH stimulation of the testis. The results indicate that short-term fasting in adult male monkeys cause inhibition of LH and T secretion, and inhibition of responsiveness of testis to LH stimulation.     

Effect of the increase of steroid binding plasma levels after passive immunization against testosterone on the control of luteinizing hormone (LH) secretion in ovariectomized underfed dairy heifers.

The ability of passive immunization against testosterone to increase sex steroid binding levels in plasma and thus to overcome the negative feedback of oestradiol-17 beta (E2) on LH secretion in underfed heifers was investigated. Dairy heifers were ovariectomized and divided in 3 groups: high energy diet (H group, n = 4), low energy diet (L group, n = 3) and low energy diet + E2 implants (LE2 group, n = 4). Twenty-four h before injection of bovine immunoglobulins, the mean concentrations of LH were not different between H and L groups. LH baseline was lower (0.8 vs 1.1 ng/ml, P less than 0.03) and the median number of LH pulses was higher (10 vs 5, P less than 0.03) in H than in L group. E2 markedly decreased (P less than 0.01) the mean and basal concentrations of LH (0.27 ng/ml), and number of LH pulses (0) in the LE2 group (P less than 0.05). After injection of anti-testosterone immunoglobulins in the L group, mean and basal LH concentrations tended to decrease. The median number of LH pulses in the L group rose 8 days after immunization (5 vs 7 on day -1 and day +8, P less than or equal to 0.05). Amplitude of pulses tended to decrease after injection (P = 0.08). In the LE2 group, the mean concentration and baseline of LH were not affected by passive immunization against testosterone, while pulses of LH appeared at day +1 and rose (P = 0.07) at day +8 after immunization with 3.5 pulses.(ABSTRACT TRUNCATED AT 250 WORDS)     

Pituitary and Leydig cell function in boars actively immunized against gonadotrophin-releasing hormone

Crossbred boars were (a) immunized against GnRH conjugated to human serum globulin (200 micrograms GnRH-hSG) in Freund's adjuvant at 12 weeks of age and boosted at weeks 18 and 20 (N = 10), (b) served as controls and received hSG only in adjuvant (N = 10), or castrated at weaning (N = 10). At 24 weeks of age (immediately before slaughter), the boars were challenged with saline or pig LH (1 microgram/10 kg body weight). After slaughter, fresh testicular fragments were incubated with pig LH (0.05 and 0.2 ng/2 ml medium) to assess the effects of immunization on Leydig cell function. Pituitary contents of LH and FSH, and testicular LH receptor content were also measured. The results indicated that plasma LH and testosterone concentrations, pituitary LH content, testicular LH receptor content, testis and sex accessory organ weights were significantly reduced in GnRH-immunized boars compared to hSG-adjuvant controls. However, plasma and pituitary FSH content were not affected by high antibody titres generated against GnRH. The testicular testosterone response to exogenous LH in vivo and in vitro was significantly reduced (P less than 0.05) in GnRH-immunized boars. These results indicate that active immunization against GnRH impairs pituitary and Leydig cell functions in boars.     

Secretion patterns of luteinizing hormone in growing mithuns (Bos frontalis)

To characterize the luteinizing hormone (LH) secretion patterns in growing mithun (Bos frontalis), a semi-wild ruminant, six female mithuns (1 year old; BW: 145.5 kg) were maintained in a semi-intensive system. Plasma progesterone (P(4)) level was measured in twice-a-week samples collected for six weeks to assess ovarian status. This was followed by a frequent sampling period. Blood samples collected at 15 min intervals for 9 h were assayed for plasma LH. Luteinizing hormone patterns consisted of pulses of varying amplitudes. Luteinizing hormone pulses occurred at an average rate of 0.54/h ( approximately 5 pulses/9 h). The rate did not differ among mithuns. The mean plasma LH levels was correlated with body weight (r=0.82; p<0.05) and pulse amplitude (r=0.87; p<0.01). Neither the LH amplitude nor the frequency was affected by time (p>0.05). The mean plasma P(4) concentration was 0.37 ng/ml. In conclusion, we demonstrated a pulsatile nature of LH secretion in growing mithuns. In addition, the mean plasma LH level and LH amplitude were positively correlated with body weight. It appears that in contrast to cattle, five LH pulses per nine hours recorded in mithuns were not an indication of approaching puberty.     

Effects of intermittent pulsatile infusion of luteinizing hormone-releasing hormone on dihydrotestosterone-suppressed gonadotropin secretion in castrate rams

The feedback effects of dihydrotestosterone (DHT) on gonadotropin secretion in rams were investigated using DHT-implanted castrate rams (wethers) infused with intermittent pulsatile luteinizing hormone-releasing hormone (LHRH) for 14 days. Castration, as anticipated, reduced both serum testosterone and DHT but elevated serum LH and follicle-stimulating hormone (FSH). Dihydrotestosterone implants raised serum DHT in wethers to intact ram levels and blocked the LH and FSH response to castration. The secretory profile of these individuals failed to show an endogenous LH pulse during any of the scheduled blood sampling periods, but a small LH pulse was observed following a 5-ng/kg LHRH challenge injection. Dihydrotestosterone-implanted wethers given repeated LHRH injections beginning at the time of castration increased serum FSH and yielded LH pulses that were temporally coupled to exogenous LHRH administration. While the frequency of these secretory episodes was comparable to that observed for castrates, amplitudes of the induced LH pulses were blunted relative to those observed for similarly infused, testosterone-implanted castrates. Dihydrotestosterone was also shown to inhibit LH and FSH secretion and serum testosterone concentrations in intact rams. In summary, it appears that DHT may normally participate in feedback regulation of LH and FSH secretion in rams. These data suggest androgen feedback is regulated by deceleration of the hypothalamic LHRH pulse generator and direct actions at the level of the adenohypophysis.     

Effect of transport on pulsatile LH release in ovariectomized ewes with or without prior steroid exposure at different times of year

The initial aim of the present study was to test whether the stress of transport suppresses LH pulsatile secretion in ewes. In a pilot experiment in the late breeding season, transport resulted in an unexpected response in three out of five transported, ovariectomized ewes pretreated with oestradiol and progesterone. Before transport, seasonal suppression of LH pulses had occurred earlier than anticipated, but LH pulsatility suddenly restarted for the period of transport. This finding was reminiscent of unexplained results obtained in ovariectomized ewes infused centrally with high doses of corticotrophin-releasing hormone after pretreatment with low doses of oestradiol with or without progesterone. Hence, an additional aim of the present study was to examine whether these latter results with corticotrophin-releasing hormone could be reproduced by increasing endogenous corticotrophin-releasing hormone secretion by transport. Subsequent experiments used groups of at least eight ovariectomized ewes at different times of the year with or without prior exposure to steroids to assess whether these unexpected observations were associated with season or the prevailing endocrine milieu. In the mid-breeding season, transport for 4 h in the absence of steroid pretreatment for 8 months reduced LH pulse frequency from 7.5 +/- 0.3 to 6.3 +/- 0.4 pulses per 4 h (P < 0.05) and LH pulse amplitude from 2.6 +/- 0.5 to 1.8 +/- 0.3 ng ml-1 (P < 0.05). Similarly, in the mid-breeding season, 34 h after the cessation of pretreatment with oestradiol and progesterone, transport suppressed LH pulse frequency from 6.1 +/- 0.4 to 5.5 +/- 0.3 pulses per 4 h (P < 0.05) with a tendency of effect on amplitude (6.2 +/- 2.7 to 2.61 +/- 0.6 ng ml-1; P = 0.07; note the large variance in the pretransport data). During mid-anoestrus, evidence of a suppressive effect of transport was only observed on LH pulse amplitude (4.7 +/- 0.6 versus 3.0 +/- 0.5 pulses per 4 h; P < 0.05) in ovariectomized ewes that had not been exposed to ovarian steroids for 4 months. Repetition of the pilot experiment with 12 ewes during the transition into anoestrus resulted in one ewe with LH pulses seasonally suppressed but increased by transport; 11 ewes had a distinct pulsatile LH pattern which was decreased by transport in six ewes. In anoestrus, there was no effect of transport on LH pulse frequency or amplitude in intact ewes, or those ovariectomized 2-3 weeks previously, with or without prior oestradiol and progesterone treatment. However, basal concentrations of cortisol were greater in anoestrus than in the breeding season, and the increment in cortisol during transport was similar in anoestrus and the breeding season but greater during the transition into anoestrus (P < 0.05). Progesterone concentrations increased from 0.31 +/- 0.02 ng ml-1 before transport to 0.48 +/- 0.05 ng ml-1 during the second hour of transport (P < 0.05). In conclusion, transport reduced LH pulse frequency and amplitude in ovariectomized ewes that had not been exposed to exogenous steroids for at least 4 months. In most animals, the previously observed increase in LH pulsatility induced by exogenous CRH was not reproduced by increasing endogenous CRH secretion by transport. However, in four ewes, transport did increase LH pulsatility, but only during the transition into anoestrus in ewes with seasonally suppressed LH profiles after withdrawal of steroid pretreatment.     

Spontaneous and GnRH-induced pulsatile LH and testosterone release in pubertal, adult and aging male beagles

Baseline concentrations of LH and testosterone (T) in blood, their pulses, and LH and T response to GnRH (5mug/kg) treatment were compared in 19 sexually sound male beagles and in 2 sexually dysfunctional dogs. The intact beagles were allocated to 4 groups according to age, which ranged from pubertal 7-mo-old animals to 11-yr-old adults. Baseline concentrations of LH and T were measured every 15 min for a period of 6 h and for a further 3 h following challenge with GnRH. Both LH and T were released in a pulsatile fashion with a wide range of pulse frequency and amplitude. The time intervals between the LH and T pulses ranged from 30 to 60 min, with no significant difference between groups. However, LH concentrations were significantly higher (P<0.01) and T values were markedly lower in the 7-mo-old pubertal dogs than in the other age groups. Following GnRH administration, LH peaked within 15 to 30 min in all the animals, with a significantly higher increase occurring in the pubertal group (P < 0.05). Peak T values occurred 15 to 105 min after the LH peaks, with no clear increases occurring in the pubertal dogs. In the 2 sexually dysfunctional animals, LH levels increased following GnRH treatment; however, T values remained extremely low both before and after treatment, indicating loss of Leydig cell function.     

The effect of the presence and pattern of luteinizing hormone stimulation on ovulatory follicle development in sheep

The aim of this study was to examine the role of LH on the growth of the large preovulatory follicle and its secretion of hormones in sheep. Ewes with ovarian autotransplants were treated with GnRH-antagonist at the time of luteal regression and different LH regimes applied for 60-66 h before administration of an ovulatory stimulus (hCG). In Experiment 1 (N = 24; n = 8), ewes received either no LH or constant or pulsatile infusion of LH at the same dose (1.25 microg/h). In Experiment 2 (N = 12, n = 6), LH was constantly infused at a rate of 1.25 microg or 2.5 microg oLH/h. In Experiment 1, animals receiving either pulsatile or constant LH exhibited increases in estradiol and inhibin A secretion (P < 0.001) and a depression in FSH (P < 0.001) that resembled the normal follicular phase. Similarly in Experiment 2, doubling the dose of LH resulted in a two-fold increase in ovarian estradiol secretion (P < 0.05) but no other changes. All animals receiving LH, regardless of the pattern of stimulation, ovulated and established a normal luteal phase. In contrast, no LH treatment resulted in constant immuno-active LH without pulses, unchanged FSH and inhibin A concentrations (P < 0.05), and basal estradiol secretion (P < 0.001). Morphologically normal large antral follicles were observed in this group and although corpora lutea formed in response to hCG, progesterone profiles were abnormal. In conclusion, these results suggest that LH is an essential requirement for normal ovulatory follicle development and subsequent luteal function and show that a pulsatile mode of LH stimulation is not required by ovulatory follicles.     

Modulation of luteinizing hormone pulse amplitude by the frequency of luteinizing hormone-releasing hormone stimulation and by testosterone in castrated, hypothalamic-lesioned male rats

The frequency of spontaneous luteinizing hormone (LH) pulses is thought to be a direct result of the frequency of luteinizing hormone-releasing hormone (LHRH) pulses from the hypothalamus. By contrast, the amplitude of spontaneous LH pulses may be controlled by several factors other than the amplitude of LHRH pulses. We tested two hypotheses: 1) that LH pulse amplitude is determined in part by the frequency of LHRH pulses of constant magnitude, and 2) that testosterone (T) exerts a direct feedback effect on the pituitary gland to regulate LH pulse amplitude. Gonadal feedback was eliminated by castrating adult male rats (n = 20). Endogenous LHRH secretion was eliminated by lesioning the medial basal hypothalamus. Serum LH levels (0.19 +/- 0.04 ng/ml RP-2, mean +/- SEM) and T levels (0.15 +/- 0.02 ng/ml), measured several weeks after hypothalamic lesioning, confirmed the hypogonadotropic hypogonadal state of the animals. During a 8-h period, unanesthetized, unrestrained animals were injected with 40-ng pulses of LHRH via catheters into the jugular vein, and blood samples for LH measurement were drawn at 10-min intervals. The LHRH pulse interval was 20 min during the first 4 h in all animals. The pulse interval was doubled to 40 min in half of the animals (n = 10) during the next 4 hours; in the other 10 animals, the pulse interval was maintained constant at 20 min throughout the study. Within both of these groups, one-half of the animals (n = 5) were infused with T to achieve a physiological level of T in serum (2.46 +/- 0.36 ng/ml at 4 h), while the other half received vehicle.(ABSTRACT TRUNCATED AT 250 WORDS)     

Alterations in pituitary gland sensitivity in ram lambs to physiological doses of gonadotrophin-releasing hormone (GnRH), after divergent selection based on the luteinizing hormone response to a pharmacological GnRH challenge.

Divergent selection in 10-week-old Finn-Dorset ram lambs was based on the luteinizing hormone (LH) response to a pharmacological dose of GnRH (5 micrograms). After eight generations of selection, the LH responses of the two lines (low and high) to GnRH differed by a factor of five. This study investigates the pituitary sensitivity of the two lines to exogenous GnRH. Initially, two pilot studies were performed: one to determine the range of doses of GnRH which would stimulate LH pulses of similar amplitude to those seen endogenously, and the other to confirm that sodium pentobarbitone prevents pulsatile LH secretion in prepubertal ram lambs. The results indicated that barbiturate anaesthesia suppressed pulsatile LH secretion in castrated and intact ram lambs. A model system was therefore constructed in 18 10-week-old intact ram lambs (high n = 7, low n = 11), whereby endogenous pulsatile LH secretion was prevented by sodium pentobarbitone anaesthesia and the amplitudes of LH pulses produced in response to different doses of exogenous GnRH could be measured. The GnRH dose-response curves demonstrated that there was a five-fold difference in the sensitivity of the pituitary glands of the two lines to stimulation with GnRH. The projected minimum concentration of GnRH required to produce a measurable pulse of LH was 4.75 ng for the high-line animals and 26.6 ng for the low-line animals. The results indicated that the low-line animals required five times more GnRH than the high-line lambs to stimulate LH pulses of similar amplitude (high line 43.67 ng; low line 206.55 ng). These results demonstrate that selection has produced two lines of sheep which differ in the control of LH secretion at the level of the hypothalamus-pituitary gland.     

Effect of the peripubertal pattern of LH and FSH secretion on in-vitro oestradiol-17 beta secretion and follicular growth within juvenile rat ovaries

Juvenile rat ovaries were placed in perfusion culture and exposed to (1) no gonadotrophin, (2) tonic NIH-FSH (200 ng RP-1 equiv./ml) or (3) NIH-FSH + NIH-LH pulses (2/h, amplitude = 80 ng RP-1 equiv./ml). After 3 h of perifusion, the ovaries were prepared for histological analysis and the perifusate assayed for oestradiol-17 beta. Since the NIH-FSH preparation is contaminated with LH, a second experiment was conducted using recombinant bovine LH and FSH. Ovaries were perifused for 3 h with (1) no hormones, (2) recombinant FSH (200 ng/ml) or (3) recombinant FSH plus 25 ng recombinant LH/ml. NIH-FSH alone increased the number of mid-size antral follicles (P less than 0.05) and decreased the number of small antral follicles (P less than 0.05). Pulsatile LH in the presence of FSH increased the number of mid-size antral follicles without reducing the number of small antral follicles. Studies with recombinant FSH and LH demonstrated that both FSH and LH are necessary to stimulate follicles to grow, indicating that the growth-promoting property of the NIH-FSH is due to the presence of both FSH and LH. Regardless of whether NIH or recombinant gonadotrophins were used, follicular growth was induced without increasing oestradiol secretion. These results demonstrate that enhanced oestradiol secretion is not essential for the induction of follicular growth, while both LH and FSH are necessary to stimulate small antral follicles to grow into mid-size antral follicles.(ABSTRACT TRUNCATED AT 250 WORDS)