Secretion rates and short-term patterns of gonadotrophin-releasing hormone, FSH and LH throughout the periovulatory period in the mare

We have developed a non-surgical technique for long-term collection of pituitary venous blood which consists of slightly diluted hypophysial portal blood into which pituitary hormones have been secreted. In these experiments jugular and pituitary venous blood samples were collected from five unmedicated, ambulatory mares at 5-min intervals for 2-6 h on 11 occasions during the 6 days surrounding the ovulatory LH peak. Jugular blood only was collected from another five periovulatory mares without pituitary cannulae. The duration of oestrus was similar in mares with and without pituitary cannulae and all mares ovulated, showing that the procedure did not affect the reproductive axis. In all pituitary-cannulated mares the secretion of gonadotrophin-releasing hormone (GnRH), FSH and LH occurred almost continuously with broad, concurrent pulses of the three hormones superimposed upon this tonic background. Only 9% of the GnRH pulses appeared to be ineffective in inducing a rise in gonadotrophin levels. When measured in pituitary blood, gonadotrophin pulse frequency varied from 0.45 pulses/h early in the LH surge to 1.87 pulses/h at the time of ovulation. In contrast, mean pulse frequency measured in jugular blood did not exceed 1 pulse/h throughout the periovulatory period in cannulated or non-cannulated mares. The low amplitude of jugular pulses (less than 50% fractional increase) may have caused problems in identifying the pulses. In the two mares in which pituitary venous blood was sampled during more than one period before ovulation, GnRH secretion tended to be lower on the day of ovulation (day 0) than earlier in oestrus (ratio day 0:day -1; mare WV = 0.58, mare LS = 0.66), whereas LH secretion rate was higher on the day of ovulation (ratio day 0:day -1; mare WV = 1.54, mare LS = 6.68). These studies show that the painless and non-invasive collection of pituitary venous blood, which is possible only in horses, can provide a useful tool for studying hypothalamic-pituitary interactions under completely physiological conditions.     

LH and FSH responses to luteinizing-releasing hormone in normal,adult, human males

Time-response and dose-response relationships were established for the LH and FSH response to single intravenous bolus injections of synthetic LRH. Sixteen normal adult human males, ages 19–45 were studied, and 80 individual tests were performed using a broad range of LRH doses from 0–3000 μg. Serum gonadotropins were measured at frequent intervals ranging from ?30 to 180 min after LRH injection. The curves describing changes in blood LH and FSH with time were not superimposable at any dose level. The 50% decay time for the LH response showed a significant linear log-dose relationship to LRH.For LH, dose-response relationships existed for both, the peak LH release, and the total LH secretion (area under the time-response curve). No minimal or maximal effective doses were reached for LH within the dose range from 1 to 3000 μg LRH. The smallest dose (measured as peak LH concentration), that produced a response significantly different from saline injections, was 1.58 μg/subject; judged by total LH secretion (area under the time-response curve) the smallest dose producing a significant response was 2.39 μg/subject. No dose-response relationship existed for the peak FSH release. Judged by total FSH secretion (area under the time-response curve), the effects of LRH were dose related; the smallest effective dose was 20 μg LRH. As for LH, no maximal effective dose was reached within the dose range studied.The 95% confidence intervals for the peak responses to an intravenous bolus injection of 100 μg synthetic LRH were 400%–800% of the individual mean base line LH concentration, and 100%–200% of the individual mean base line FSH concentration. Serum testosterone did not change significantly in response to single bolus injections of LRH at any of the dose levels studied. The peak LH responses to any dose of LRH were positively correlated to the individual mean base line LH concentrations. No correlations were observed between the peak LH or FSH responses and age, race, marital status, body weight, or body surface area of the subjects.In a second series of studies, a 2-hr, constant infusion of synthetic LRH was administered at five different infusion rates (doses) to four normal adult human males for a total of 20 infusions. Serum LH and FSH responses were sustained at dose related levels characteristic of each individual during the entire LRH infusion period. In contrast to the lack of testosterone responses to bolus injections of LRH, serum testosterone rose significantly in response to all dose levels of LRH infusion. These are the most extensive pharmacologic studies of LRH-LH and LRH-FSH relations in man yet reported.     

Varying the patterns and concentrations of gonadotrophin-releasing hormone stimulation does not alter the ratio of LH and FSH released from perifused sheep pituitary cells

Our aim was to determine whether release of LH and FSH can be controlled differentially by the characteristics of applied signals of stimulatory gonadotrophin-releasing hormone (GnRH) alone, free of the effects of steroid feedback or other influences from the whole animal. The outputs of both gonadotrophins were significantly correlated (r approximately 0.90; P less than 0.0005) when samples of freshly dispersed sheep pituitary cells were perifused in columns for 7 h with medium containing a range of concentrations of GnRH in various patterns of pulses. Hormone released in response to the second, third and fourth pulses from every column was analysed in detail. Dose-response relationships for both LH and FSH were very similar when cells were stimulated with 5-8500 pmol GnRH/l in 5-min pulses every hour. When GnRH was delivered in pulses at a maximally stimulating level, the outputs of both hormones increased similarly with increasing inter-pulse intervals. Efficiency of stimulation (release of gonadotrophin/unit stimulatory GnRH) decreased (was desensitized) with increasing pulse duration in the same way for both hormones. Thus, varying the dose, interval and duration of GnRH pulses did not alter the proportions of LH and FSH released in the short-term from freshly dissociated cells. However, the same cell preparations released more LH relative to FSH when treated with maximally stimulating levels of GnRH for 3 h in the presence of 10% serum from a sheep in the follicular phase of its ovulatory cycle compared with charcoal-treated serum.(ABSTRACT TRUNCATED AT 250 WORDS)     

Gonadotrophin surge-attenuating factor attenuates in-vitro LH secretion induced by gonadotrophin-releasing hormone from cultured ovine pituitary cells only during the breeding season.

Primary cultures of ovine pituitaries from adult ewes were used to investigate aspects of gonadotrophin surge-attenuating factor (GnSAF) bioactivity in human follicular fluid (hFF) from superovulated women. During the autumn and first half of the winter, LH secretion induced by gonadotrophin-releasing hormone (GnRH) was markedly reduced (43.5 +/- 5.2% of control GnRH-induced LH secretion) by incubation for 48 h with steroid-free hFF. For the rest of the year, treatment with the same batch of steroid-free hFF resulted in non-significant reduction or stimulation of GnRH-induced LH secretion (71.3 +/- 13.2 to 117.8 +/- 11.2% of control GnRH-induced LH secretion). Incubation of pituitary cells for 48 h with oestradiol (1 pmol/l to 1 mumol/l), progesterone (1 pmol/l to 1 mumol/l) or oestradiol and progesterone combined (1 pmol/l to 1 mumol/l) in a two-way titration for 48 h had no significant effect on GnRH-induced LH secretion (83.4 +/- 7.6 to 110.6 +/- 5.0% of control secretion). Separating hFF into fractions of different molecular mass by ultrafiltration demonstrated that GnSAF bioactivity was present in a form 10-30 kDa in size. Incubation for 48 h with these fractions had no significant effect on basal FSH secretion but significantly attenuated GnRH-induced LH secretion during the autumn. The same fractions had little effect on GnRH-induced LH secretion from pituitary cells collected during the summer. We conclude that ovine pituitaries display at least partial reduction in sensitivity to GnSAF outside the breeding season.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of gonadotrophin-releasing hormone agonist-induced suppression of LH and FSH on follicle growth and corpus luteum function in the ewe

Continuous infusion of a gonadotrophin-releasing hormone (GnRH) agonist (buserelin) by osmotic minipump from day 1 of the luteal phase in five Welsh ewes resulted in a sustained suppression of plasma concentrations of FSH which increased three- to eightfold within 2 days after the end of infusion 29 days later. Plasma concentrations of LH increased three- to eightfold over the first 5 days of infusion and then became basal and non-pulsatile until 1 day after the end of infusion. Duration of the luteal phase and plasma concentrations of progesterone were not significantly different in control and treated ewes. Pulses of LH in control ewes were followed by increases in concentrations of progesterone in samples collected at 10-min intervals for 7 h on days 10 and 14 of the luteal phase. However, progesterone was also released in a pulsatile manner in the absence of LH pulses in both control and GnRH agonist-treated ewes. After natural luteolysis, no ovulation or corpus luteum function occurred in treated ewes up to 15 days after the end of treatment on day 29, even though oestrus, indicating follicular development and oestrogen secretion, had occurred 8-11 days after treatment ended. After 30 days of infusion the ovaries of GnRH agonist-treated ewes contained no follicles greater than 2.5 mm in diameter. In follicles of 1-2 mm in diameter the basal and LH-stimulated production of oestradiol and testosterone in vitro were similar in both control and GnRH agonist-treated ewes, and a similar proportion of these follicles was oestrogenic (greater than 370 mol oestradiol per follicle) in GnRH agonist-treated and control ewes. These results show (1) that progesterone secretion by the corpus luteum of the ewe can be sustained in the presence of basal concentrations but absence of pulsatile secretion of LH, and progesterone is released in a pulsatile manner whether or not LH pulses are present, (2) that follicular development beyond 2.5 mm in diameter in the ewe is dependent upon adequate stimulation by both LH and FSH and (3) that the continuous infusion of GnRH agonist is a simple method for providing reproducible suppression of LH and FSH and follicular development in the ewe to allow the study of gonadotrophin action on the ovary in vivo.     

Effect of the body condition of ewes on the secretion of LH and FSH and the pituitary response to gonadotrophin-releasing hormone

The effects of body fat content (body condition) of ewes on hypothalamic activity and gonadotrophin-releasing hormone (GnRH) secretion and on pituitary sensitivity to GnRH were investigated using Scottish Blackface ewes. Two groups of 12 ewes were fed so that they achieved either a high body condition score (2.98, S.E.M. = 0.046; approximately 27% of empty body weight as fat) or a low body condition score (1.94, S.E.M. = 0.031; approximately 19% of empty body weight as fat) by 4 weeks before the period of study. Thereafter, they were differentially fed so that the difference in mean condition score was maintained. Oestrus was synchronized, and on day 11 of the subsequent cycle half of the ewes of each group were ovariectomized. On day 12, the remaining ewes were injected (i.m.) with 100 micrograms prostaglandin F2 alpha analogue and ovariectomized 30 h later. Numbers of large ovarian follicles and corpora lutea present at ovariectomy were recorded. Blood samples were collected at 15-min intervals for 12 h on day 10 of the cycle (luteal phase) and at 10-min intervals from 24 to 30 h after prostaglandin injection (follicular phase). At days 2 and 7 after ovariectomy, samples were collected at 15-min intervals for 8 h and ewes were then injected with 10 micrograms GnRH and samples were collected for a further 3 h.(ABSTRACT TRUNCATED AT 250 WORDS)     

Regulation by oestradiol of serum levels of LH and FSH and pituitary levels of gonadotrophin-releasing hormone receptors after ovariectomy in the rat

We studied cyclic and ovariectomized, oestradiol treated rats to investigate whether oestradiol concentrations before ovariectomy determine the dynamics of the rises in serum levels of LH and FSH, and in pituitary gonadotrophin-releasing hormone (GnRH) receptors after ovariectomy. In cyclic rats, ovariectomy on metoestrus (oestradiol = 44 pmol/l), but not at midnight of dioestrus (oestradiol = 254 pmol/l) was followed by a rise in GnRH receptors 40 h later. Randomly cyclic females were ovariectomized under ether anaesthesia and treated with s.c. oestradiol implants for 2 days to provide serum levels spanning the physiological range. Exposure to lower oestradiol concentrations (48 or 74 pmol/l) resulted in significant rises in levels of LH and FSH, and in GnRH receptors earlier than exposure to higher (148 or 309 pmol/l) oestradiol levels (2 compared with 4 days). GnRH given for 24 h after removal of the implant was unable to induce a rise in GnRH receptors in any group, while LH and FSH responses to GnRH were proportional to the pretreatment levels of oestradiol. This suggests that the concentration of oestradiol before ovariectomy determines the subsequent dynamics of LH and FSH and of GnRH receptors through actions at the hypothalamic and pituitary level.     

Evidence for temporal coupling of growth hormone, prolactin, LH and FSH pulsatility overnight during normal puberty

The patterns of secretion of GH, LH, FSH and prolactin were determined over a single night (20.00-08.00 h; 15-min sampling) in 34 normal subjects (17 male, 17 female, aged 9.1-20.9 years). Plasma GH was measured by an immunoradiometric assay and LH, FSH and prolactin by radioimmunoassay in all samples. Data were analysed by Fourier transformation and cross-correlation after stationarization. The highest mean GH levels were noted in girls at Tanner stage 2/3 and in boys at stages 4/5. Prolactin levels were highest in girls at stage 4/5 and in boys at stage 2/3. LH and FSH showed a progressive rise by puberty stage in both sexes. The dominant pulse periodicities of GH and prolactin were 150-180 min in girls and 180 min in boys. LH and FSH pulse periodicity was around 90 min in early puberty and 180 min in later puberty in both sexes. LH and prolactin pulses showed a phase relationship with GH with a lag of 30-75 min (r = 0.32; P less than 0.001) and 30 min (r = 0.47; P less than 0.0001) respectively. Generally, LH and prolactin pulses were in phase (r = 0.42; P less than 0.0001) and there was a highly significant correlation (r = 0.64; P less than 0.0001) between FSH and LH pulsatility. Whereas mean overnight concentrations and pulse periodicity of the principal pituitary hormones varied between the sexes during early puberty, by the end of puberty a dominant pulse periodicity of around 150-180 min was established and there was remarkable temporal coupling of pulsatility.     

Overnight follicle stimulating hormone (FSH) and luteinizing hormone (LH) excretion in normal males.

FSH and LH in acetone precipitates of timed overnight urine collections were measured by radioimmunoassay. FSH and LH urinary excretion extrapolated to 24 h was determined in 147 boys 3-16 years and 20 normal men. FSH and LH (2nd IRP-HMG) progressively increased with age from 1.8 +/- 1.1 (FSH) and 2.1 +/- 1.2 (LH) IU/24 h for the 3-4 year age group to 8.6 +/- 3.2 (FSH) and 18.8 +/- 9.0 (LH) IU/24 h for the 15-16 year age group. FSH and LH excretion increased progressively with stage of sexual development. The integration of several hours of FSH and LH secretion and the accuracy, simplicity and convenience of collecting a single urine sample are advantages of the timed overnight urine collection which should make this technique extremely useful in the evaluation of gonadotropin function.     

Simultaneous measurement of luteinizing hormone (LH)-releasing hormone, LH, and follicle-stimulating hormone release in intact and short-term castrate rats

The temporal relationship between LHRH release and gonadotropin secretion as well as the effects of castration on LHRH release were investigated in conscious, freely moving male rats. LHRH release was measured in hypothalamic/median eminence perfusates, while levels of pituitary gonadotropins (LH, FSH) were determined in sequential blood samples obtained via atrial catheters. Twenty-four to 26 h before experiments, rats underwent sham surgery or castration. LHRH release in push-pull perfusates from both groups was pulsatile, and nearly all identified LH pulses (83.3%) were temporally associated with LHRH pulses. Of the fewer irregular FSH pulses that were observed, only 43.7% were temporally associated with LHRH pulses. Mean LHRH pulse amplitude and mean LHRH levels were not different in intact and castrate animals. The frequency of LHRH pulses was moderately increased in castrate rats (1.30 pulses/h) compared to that in intact animals (0.83 pulses/h), and this acceleration was accompanied by a significant increase in LH pulse frequency, pulse amplitude, and mean level. It was also noted that the number of silent LHRH pulses (those not associated with LH pulses) was dramatically reduced in castrate animals. Characteristics of gonadotropin release (pulse frequency, pulse amplitude, and mean level) were not significantly different in animals undergoing push-pull perfusion/bleeding procedures from those in rats not receiving push-pull cannula implants. We conclude from these studies that 1) LH pulses show a high concordance with LHRH pulses, providing evidence that the LHRH pulse generator operates as the neural determinant of LH pulses in male rats, 2) FSH secretion is not associated with LHRH release in an obvious and consistent manner, suggesting that LHRH/FSH relationships are not easily discerned in these animals or that a FSH-releasing factor distinct from the LHRH decapeptide may regulate FSH secretion, 3) a modest increase in LHRH pulse frequency occurs 24-30 h after castration, and 4) silent LHRH pulses occur with much greater regularity in intact than in castrate rats. The latter two observations suggest that both hypothalamic and intrapituitary sequelae of castration may be critically important in the development of postcastration increases in LH secretion and the negative feedback of gonadal steroids.     

Testosterone maintains pituitary and serum FSH and spermatogenesis in gonadotrophin-releasing hormone antagonist-suppressed rats

Groups of adult male rats were treated continuously for 30 days with either vehicle or the potent gonadotrophin-releasing hormone (GnRH) antagonist. (N-Ac-D-Nal(2)1,D-pCl-Phe2,D-Trp3,D-hArg(Et2)6,D-Ala10 )- GnRH (RS 68439; 35 micrograms/day). In addition, groups of vehicle- and antagonist-treated rats received s.c. testosterone implants sufficient to maintain serum testosterone concentrations 3.5- to 5-fold higher than those of vehicle-treated control rats. After 30 days of antagonist treatment serum LH, FSH and testosterone concentrations were at or below the detection limits of their respective assays and pituitary FSH content and GnRH receptor binding were reduced, relative to control animals, by 77 and 98% respectively. Testis weight in antagonist-treated rats was reduced by 75% and spermatogenesis was suppressed to an extent comparable to that observed in hypophysectomized rats. Testosterone, which caused a 40% reduction in serum FSH relative to control animals, prevented the antagonist-induced fall in both serum and pituitary FSH, but not GnRH receptors, below that observed in the vehicle plus testosterone-treated group. Furthermore, spermatogenesis in the antagonist plus testosterone-treated group was indistinguishable from that observed in control animals. It is concluded that testosterone is capable of maintaining serum and pituitary FSH levels in vivo, under conditions which presumably render the pituitary insensitive to hypothalamic GnRH.     

Effect of gonadotrophin-releasing hormone (GnRH) antagonist during the LH surge in normal women and during controlled ovarian hyperstimulation

OBJECTIVE: Several studies have suggested that GnRH is instrumental in triggering the LH surge. The present studies were performed to evaluate the effect of GnRH antagonist administration in women after the beginning of the LH surge. DESIGN: In study one, six normal cycling women were given a GnRH antagonist (20 mg Nal Glu s.c.) during an unstimulated cycle. Nal-Glu was administered when the LH level was higher than 10 U/l and associated with an oestradiol (E2) level higher than 730 pmol/l. In study two, a GnRH antagonist (3 mg Cetrorelix, ASTA-Medica, Frankfurt, Germany) was administered on day 8 of an IVF-ET cycle, in 157 women. Eighteen women among this cohort received the antagonist, when their LH level was higher than 10 U/l. RESULTS: In normal volunteers (study one), Nal-Glu was administered on day 13.7 +/- 1.4 (mean +/- SD) of the cycle when the LH level was 13.7 +/- 3.5 U/l with an E2 plasma level reaching 980 +/- 131 pmol/l. Twenty-four hours after administration of the antagonist, the LH surge had been interrupted in all subjects; it was postponed in three of the women, and abolished in the remaining three. LH levels fell by 68.5%, E2 by 42% and FSH by 53.2%. In study two, LH plasma levels 24 h after the antagonist administration fell by 94%. No premature ovulation occurred in any of the patients treated. Administering the antagonist before (n = 139) or during the LH surge (n = 18) made no statistically significant difference to the results of the IVF-ET attempt. CONCLUSIONS: Our results indicate that GnRH is required throughout the gonadotrophin surge in women, not only for the initiation but also for the maintenance of the LH surge. In addition, in our study, the suppression of the rise in LH, when the antagonist was given during the surge, had no detrimental impact on IVF-ET outcome. This suggests, if confirmed on a larger scale, that late follicular phase GnRH antagonist administration to prevent the LH surge in controlled ovarian hyperstimulation (COH) is a safe and useful treatment.     

Effect of level of food intake of ewes on the secretion of LH and FSH and on the pituitary response to gonadotrophin-releasing hormone in ovariectomized ewes

The effect of level of food intake on LH and FSH profiles and pituitary sensitivity to gonadotrophin-releasing hormone (GnRH) was investigated in two groups of 12 ovariectomized ewes. Ewes with a high intake (group H) had a mean daily intake (+/- S.E.M.) of 1.99 +/- 0.075 kg dry matter (DM)/head per day while ewes with a moderate intake (group M) consumed a mean of 1.02 +/- 0.021 kg DM/head per day. Ovaries were surgically removed from six ewes of each group on day 11 of the luteal phase and from the remainder 30 h after an injection of 100 micrograms prostaglandin analogue given on day 11 to induce luteolysis. During both the luteal phase and the follicular phase, mean LH and FSH concentrations and LH pulse frequencies and amplitudes were unaffected by the level of intake but mean plasma prolactin concentrations were higher (P less than 0.05) in group H than in group M ewes in the follicular phase. Mean LH and FSH concentrations at day 2 after ovariectomy were unaffected by treatment while mean prolactin concentrations were higher (P less than 0.05) in group H than in group M ewes. At day 7 after ovariectomy, mean LH and FSH concentrations were lower (P less than 0.05) in group H than in group M ewes although mean LH pulse frequencies and pulse amplitudes were not significantly affected by the level of intake at either time.(ABSTRACT TRUNCATED AT 250 WORDS)     

FSH causes a time-dependent stimulation of preovulatory follicle growth in the absence of pulsatile LH secretion in ewes chronically treated with gonadotrophin-releasing hormone agonist

The study investigated the relationship between the plasma concentration of FSH and the stimulation of preovulatory follicle growth in vivo in ewes chronically treated with the gonadotrophin-releasing hormone (GnRH) agonist buserelin (HOE 766). Welsh Mountain ewes with regular oestrous cycles were treated for 6 weeks with two discs implants placed s.c., each containing 5 mg of the agonist in a matrix of polyhydroxybutyric acid. Treatment with the agonist for 35 days produced a sustained suppression of the plasma concentration of FSH, stopped the pulsatile release of LH and prevented follicular development beyond 2.5 mm diameter. There was no difference between the total number of follicles greater than 1.0 mm diameter present in the ovaries of GnRH agonist-treated ewes and day 8 luteal phase control ewes. During the sixth week of agonist treatment ewes were infused with ovine FSH (6 micrograms NIADDK-oFSH-16/h) in the presence of only basal concentrations of LH. After 24, 48, 72 or 120 h of FSH infusion, the mean number of follicles greater than 1.0 mm diameters per ewe was not significantly different between treated and control animals. Infusion of FSH caused a time-dependent increase in (1) the number of follicles per ovary greater than 2.5 mm, (2) the mean diameter of these follicles and (3) the proportion of the large follicles which could be classified as oestrogenic (greater than 3.7 nmol oestradiol/follicle per h in vitro). Injection of human chorionic gonadotrophin (750 IU i.m.) after 120 h of FSH infusion caused the majority of these large follicles to ovulate and form apparently normal corpora lutea. These results indicate that, in the absence of pulsatile LH, FSH stimulates the growth of normal large oestrogenic follicles which, when stimulated, ovulate to produce viable corpora lutea.     

Release of LH and FSH after administration of synthetic LH-releasing hormone

To study the effect of synthetic leutenizing hormone-releasing hormone (LH-RH) follicle stimulating hormone-releasing hormone (FSH-RH) on the release of LH and FSH in the human being, a decapeptide, synthesized by the solid phase method, was injected into normal volunteers 21-36 years old. There were 3 untreated men, 2 untreated women, 3 men pretreated with ethinyl estradiol and 3 women pretreated with an oral contraceptive (lyndiol). The synthetic hormone ((pyro)Glu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2) caused an increase in serum LH values over the control (p less than .01) with no difference between men and women in the amount of LH released. FH was also significantly (p less than .01) increased but to lower levels than LH. Pretreatment with sex steroids led to a decrease in FSH values (p less than .10). Since the hormone is readily synthesized in large quantities, clinical studies are now possible.