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Purification and characterisation of two porcine hypothalamic fractions with LH-releasing activity: evidence for a single LH and FSH-releasing hormone.

Two fractions with LH releasing activity were obtained from porcine hypothalami by Enzmann et al. (1972). These fractions were designated E 57 and E 203. By comparison with pure natural LH-RH and synthetic LH-RH, E 203 was identified as purified natural LH-RH. E 57, which has only 25% of the activity of E 203, was further evaluated for its biological and immunological activity. E 57 has the same ratio of LH/FSH-releasing activity as natural LH-RH. The binding to a specific LH-RH antiserum is identical with that of natural LH-RH and E 203. It is concluded, that E 57 is an artifact of isolation derived from natural LH-RH. These findings can be considered as additional evidence for the identity of the hypothalamic LH and FSH releasing hormone, since both gonadotrophins are stimulated by the two hypothalamic fractions.

Animals↗

Biologically active luteinizing hormone (LH) in plasma: II. Comparison with immunologically active LH levels throughout the human menstrual cycle.

The levels of biologically active luteinizing hormone were determined by an in vitro bioassay method in plasma samples collected daily over a complete menstrual cycle from 12 menstruating women. These cycles were normal according to a number of criteria, including daily plasma levels of oestradiol, 17-hydroxyprogesterone and progesterone. Immunoreactive LH was estimated in the same 12 cycles by a radio-immunoassay (RIA) procedure (HCG-RIA) using an HCG antiserum and iodinated HCG. The 2nd IRP of HMG was selected as standard although significant deviations from parallelism were found with 7 out of the 12 plasma pools studied. The use of the 1st IRP of human pituitary gonadotrophins (FSH and LH (ICSH)) for bioassay (herafter HPG-1st IRP) as standard in this system resulted invariably in invalid assays, due to lack of parallelism. Immunoreactive LH was also measured in 8 of the 12 cycles by a RIA procedure (HLA-RIA) using a human LH antiserum and iodinated human LH of pituitary origin. Results are expressed in terms of the HPG-1st IRP. The plasma levels of biologically and immunologically active LH were qualitatively similar throughout the menstrual cycle. However, the LH levels measured by the bioassay invariably exceeded those estimated by the RIA procedures. The biological to immunological (B/I) ratio over the entire menstrual cycle (312 comparisons) was 5.5 with 95% confidence limits at 5.2 and 5.8 when the HCG-RIA system was employed. Using the HLH-RIA system (208 comparisons), the corresponding ratio was 6.4 (6.0:6.9). When regression lines were calculated using the bioassay results as the independent variable and the RIA results as the dependent variable, the 95% confidence limits of the regression lines did not include the origin. Furthermore,, in keeping with the high B/I ratios, the slopes of the two regression lines and their conficence limits differed markedly from unity. It is concluded that although qualitatively similar profiles were observed between the biological and immunological activities throughtout the menstrual cycle, two aspects require further attention. Firstly, the elevated B/I ratios together with the behaviour of the dose-effect lines obtained with different standards in the various RIA systems suggest that presently available reference standard preparations of pituitary and/or urinary origin are not suitable for the assay of LH in human plasms. Secondly, from the regression analyses of the biological and immunological activities it is infered that the RIA methods detect immunological activity which is not associated with biological activity. If so, the validity of these RIA procedures for specifically measuring low levels of biologically active LH in plasma may be in question.

Adult↗

Pituitary responsiveness to synthetic LH-RH and pituitary LH content at various reproductive stages in the sheep.

In sheep the basal concentration of LH in jugular vein plasma was significantly higher during the first 50 days of gestation in late pregnancy or at parturition. The pituitary response to a single i.v. injection of 200 microng synthetic LH-RH was determined at different stages of gestation and compared with that of anoestrous and cyclic sheep. Pituitary response to LH-RH decreased progressively with advancing gestation: by 56 days after mating the response had declined to 35% and by parturition to 14% of the value in anoestrous sheep. The pituitary response to LH-RH increased after parturition and the pattern of recovery differed in non-lactating and lactating sheep. By 63 days postpartum the response to LH-RH in non-lactating and lactating animals had returned to values similar to those in sheep during anoestrus and sheep during the luteal phase of the oestrous cycle. A decrease in pituitary responsiveness during pregnancy was associated with a decrease in pituitary content of LH. The quantity of LH released in response to a standard injection of LH-RH was linearly related to pituitary LH content.

Animals↗

Plasma LH concentrations after single or double injections of synthetic LH-RH in dairy cows.

Milked Friesian dairy cows were given 1 or 2 i.v. injections of synthetic LH-RH. The second of 2 injections of LH-RH given 1.5 h apart induced a significantly greater release of LH than did the first. The response to this injection was also significantly greater than that caused by injections given 6 h apart. There was no significant difference between the LH responses to the two injections given 6 h apart. The total LH release produced by the two injections of 100 microgram synthetic LH-RH given 1.5 h apart was significantly greater than that produced by single injection of 200 microgram synthetic LH-RH. It is concluded that there is a short-term increase in the responsiveness of the bovine pituitary gland to LH-RH after an initial LH-RH stimulus.

Animals↗

Stimulation of pituitary and testicular functions with LH-RH agonist or pulsatile LH-RH treatment in the rhesus monkey during the non-breeding season.

Out-of-season male rhesus monkeys were used to compare the effectiveness of pulsatile treatment with LH-RH and administration of LH-RH agonist on testicular function. Treatment with LH-RH agonist (1.0 microgram Hoe 766/day) for 11 weeks resulted in partial stimulation of pituitary and testicular functions. The pituitary LH response to the agonist increased during treatment. Testosterone levels were stimulated to within the normal range and 2 of the 4 treated monkeys produced ejaculates, but sperm counts were below normal. Pulsatile treatment with LH-RH (100 ng every 96 min for 7 days alternating with LH-RH agonist treatment for 7 days) in 2 monkeys induced full testicular activity after 7 weeks. Ejaculations were induced at a time when the rhesus monkey is normally sexually inactive. Seminal characteristics at the end of treatment were similar to values in the normal breeding season. In samples collected from one monkey over a 24-h period before treatment there were no LH spikes and very low testosterone levels. During pulsatile LH-RH treatment distinct LH and testosterone spikes occurred, comparable to those in the breeding season.

Animals↗

LH release and luteal function in post-partum acyclic ewes after the pulsatile administration of LH-RH.

The administration of LH-RH in a pulsatile regimen (100 ng i.v./h for 48 h) to acyclic ewes 26-30 days post partum increased plasma LH concentrations, and both the frequency and amplitude of plasma LH pulses. In 12/14 ewes these increases were followed by plasma LH surges similar to the preovulatory surges observed in 10 control cyclic ewes. Subsequent luteal function in the post-partum ewes was deficient. Plasma progesterone was detected in 7/12 post-partum ewes showing plasma LH surges. The concentrations were lower (1.3 +/- 0.2 ng/ml) and detected for shorter periods (3-10 days) than in cyclic ewes (2.4 +/- 0.2 ng/ml, 12/15 days). In the post-partum ewes the increases in plasma LH concentrations before the LH surge were higher but of shorter duration than in the cyclic ewes. The inadequate luteal function in the post-partum ewes could therefore have been due to inappropriate LH stimulation of the ovary before the LH surge.

Animals↗

GnRH pulse frequency determines LH pulse amplitude by altering the amount of releasable LH in the pituitary glands of ewes.

We have measured the size of the releasable pools of LH and FSH in the pituitary glands of ovariectomized ewes in which the pituitary was isolated surgically from the hypothalamus. The ewes were given GnRH pulses (250 ng) every hour (N = 3) or every 2 h (N = 3) for 1 week and then given a high dose GnRH infusion (0.5 micrograms/min) for 4 h. Blood samples were collected to characterize the LH and FSH responses to the GnRH pulses and infusion. The LH, but not FSH, responses to the individual GnRH pulses were pulsatile and the amplitudes of the LH pulses were greater in the sheep receiving pulses every 2 h. The sheep receiving hourly pulses showed lower LH responses to the high-dose infusions than did the sheep receiving pulses every 2 h. These data indicate a relationship between the amplitude of LH pulses and the size of the releasable pool of LH in the pituitary gland. As the frequency of GnRH pulses is decreased the amplitude of the LH responses is increased in direct proportion to the size of the releasable LH pool.

Animals↗

Patterns of secretion of GnRH, LH and FSH during the postovulatory period in mares: mechanisms prolonging the LH surge.

To study the mechanisms responsible for the unusually slow decline of the ovulatory LH surge in mares, secretion patterns of GnRH, LH and FSH were monitored in pituitary venous blood collected every 2 or 5 min for 10.5-18.0 h from five mares on the third (n = 4) or fifth day after ovulation (first sampling period). To determine the effectiveness of progesterone negative feedback, mares were then given a luteolytic dose of a prostaglandin analogue (PGF2 alpha) and pituitary venous sampling (every 2 or 5 min for 16 h) recommenced 20-22 h later (second sampling period). During the declining arm of the LH surge, large peaks (detected by the Cluster algorithm) of concurrent LH and FSH secretion occurred infrequently, with four peaks being detected in a combined sampling period of 75 h. Outside the peaks, LH or FSH secretion continued (as assessed by a pituitary to jugular-venous concentration ratio > or = 1.25) during 46% +/- 13 or 40% +/- 10, respectively, of the sampling period. GnRH immunoactivity was detected during each spontaneous gonadotrophin peak, but at other times was generally at assay sensitivity. After PGF2 alpha, plasma progesterone fell (ng ml-1, mean +/- SEM; first sampling period: 8.6 +/- 0.8; second; 2.0 +/- 0.3; P = 0.001) and the frequency of LH (P < 0.05) and FSH (P < 0.02) peaks rose, with 28 peaks detected for each hormone in a total of 80 h sampling. Peaks in LH were smaller during the second period, with decreases observed in maximum (P = 0.027) and mean (P = 0.025) secretion rates. Maximum GnRH secretion rate during peaks also declined (P = 0.010); however, the decrement (-30 +/- 6%) was less than that in maximum LH secretion rate (-82 +/- 5%; P = 0.040), suggesting that other factors contribute to the reduced LH peak amplitude. In summary, gonadotrophin peak frequency during the downswing of the surge in mares is slow, as in the midluteal phase, and the slow rate of decline in peripheral gonadotrophin concentrations is due, at least in part, to continued secretion between pulses. Moreover, progesterone negative feedback is highly effective in early dioestrus, in that lessening it without complete removal markedly accelerates gonadotrophin pulse frequency.

Animals↗

Effects of treatment with LH releasing hormone before the early increase in LH secretion on endocrine and reproductive development in bull calves.

Between 6 and 20 weeks of age an early increase in LH secretion has been reported in Hereford bull calves. Delaying this early increase in LH secretion delays testicular development. This study was designed to determine whether a premature increase in LH secretion during the early postnatal period enhances testicular development. Ten age- and body weight-matched Hereford bull calves were divided into two groups. One group (n = 5) received 200 ng LH releasing hormone (LHRH) i.v. every 2 h for 14 days, between 4 and 6 weeks of age. On the basis of blood samples taken every 15 min for 10 h, mean serum LH and testosterone concentrations and LH pulse frequency were increased by LHRH treatment (P < 0.05). Serum concentrations of FSH were not significantly influenced by treatment (P > 0.05). In treated animals at 24 weeks of age, mean serum testosterone concentrations and LH pulse amplitude were increased (P < 0.05). The concentrations of spermatozoa in electroejeculates collected at 52 weeks of age were greater in LHRH-treated compared with control calves. Testicular growth was enhanced by LHRH treatment and histological evaluation of the testis at 54 weeks of age showed increased spermatogenesis and also larger numbers of Sertoli cells per tubule cross-section as a result of LHRH treatment. We conclude that treatment with LHRH before the early increase in LH secretion altered testicular development and suggest that the early increase in LH secretion in bull calves may be critical for initiating and regulating the progression of reproductive maturation.

Animals↗

Anaesthesia with alphaxalone plus alphadolone acetate blocks the oestrogen-stimulated LH surge and impairs pulsatile LH secretion in ovariectomized female rats.

Two experiments were undertaken to assess further the action of the steroid anaesthetic alphaxalone upon LH secretion in chronically ovariectomized female rats. For the first experiment, 31 rats were given two injections of oestradiol benzoate (20 micrograms/100 g body weight), each 72 h apart, to stimulate an LH surge 6 h after the second injection. However, one group of seven rats was anaesthetized with alphaxalone throughout the 6-h period and a second group of eight was similarly anaesthetized only for the last 2 h of the 6-h period. The steroid-stimulated LH surge was blocked in both groups of rats anaesthetized with alphaxalone. The second experiment involved a comparison of pulsatile LH secretion in animals which were either unanaesthetized (n = 8) or anaesthetized with alpha xalone (n = 9). In six out of nine rats the anaesthetic did not affect the maximum or minimum plasma LH concentrations but significantly slowed the frequency at which LH pulses were measured. In the remaining three anaesthetized rats the drug blocked pulsatile LH secretion. The experiments confirm that some secretion of LH continues during alphaxalone anaesthesia but indicate also that the drug has a more deleterious action upon the oestrogen-stimulated LH surge than believed hitherto.

Anesthetics↗

Analysis of the ratio of biological to immunological LH secreted during the oestrogen-induced LH surge in the ewe.

Plasma concentrations of in-vitro biological and immunological LH were measured throughout the LH surge in cyclic ewes and in ovariectomized ewes treated i.m. with oestradiol benzoate. Both activities increased in parallel during the LH surge in both groups, although the ratio of biological to immunological activities (B/I ratio) was highest at the peak of the LH surge. The two activities were highly correlated (r = 0.86-0.92), with similar slopes from their regression analysis for the cyclic and ovariectomized groups (1.15 and 1.16 respectively). However, the intercepts of the regression lines did not pass through the origin, but intersected the y (radioimmunoassay) axis, suggesting that these serum samples contained immunoactivity not associated with LH bioactivity. In conclusion, an increase in the LH B/I ratio was observed during the LH surge in oestrogen-treated ovariectomized ewes and in cyclic ewes. This increase was not attributable to a change in the relationship between these two LH activities during the LH surge, but rather to the detection of bioinactive immunoactive material in plasma of unknown composition.

Animals↗

Effect of FSH, LH, LH-RH and arginine-vasotocin on the production of steroids, nonapeptide hormones and cGMP by rabbits granulosa cells isolated at different stages of reproductive cycle.

Granulosa cells isolated from ovaries of non-cycling, cycling and pregnant rabbits of the same age were cultured in vitro either without or with pFSH (1 micrograms/ml), bLH (1 IU/ml), LH-RH (25 ng/ml) or arginine-8-vasotocin (100 ng/ml). The production of immunoreactive progesterone, estradiol-17 beta, oxytocin, arginine-8-vasopressin and cGMP was analyzed. The gonadotropins did not show any significant effects on the cells isolated from non-cycling and cycling rabbits, but not from these of pregnant ones. LH-RH inhibited and vasotocin stimulated progesterone production. All hormones used stimulated estradiol release from cells of non-cycling rabbits, while in a case of cycling animals no change was found. In the cell from pregnant females the release of estradiol was enhanced after LH treatment only. The treatment with FSH and LH (but not with LH-RH or vasotocin) resulted in a remarkable rise of granulosa vasopressin surge irrespectively to the reproductive stage. Oxytocin production by granulosa cells incubated either without or with LH, LH-RH or vasotocin was undetectable. However, FSH strongly stimulated oxytocin release. FSH and in lesser extent, LH or LH-RH (but not vasotocin) activated granulosa cGMP production in the cells from cycling and pregnant (but not from non-cycling) animals. It was also found that, in contrast to other reproductive stages, basal progesterone release from the cells of pregnant rabbits was increased, while in a case of non-cycling animals the basal estradiol release was decreased and that of cGMP was increased.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

[Investigations on LH and FSH serum levels before and after application of synthetic LH-releasing-hormone in healthy men of different age].

50 male healthy volunteers of different age had measurements of plasma luteinizing hormone (LH) and follicle-stimulating hormone (FSH). Basal levels of LH and FSH as well as maximal release of both hormones after i.v. application of 25 mug of synthetical LH-releasing hormone have been done. Results demonstrate an increasing reserve capacity of LH and FSH with age. FSH/LH ratio is altered in favor of FSH with duration of life. Sensitivity of gonadotropin producing cells of adenohypophysis to LH-releasing hormone is diminished in old age that can be recognized from a diminution of the relative rise of serum LH and FSH after LH-RH application. Correlations and partial correlations of absolute and relative body weight with age and basal LH and FSH serum levels demonstrate that age and not body weight plays a main part for increasing gonadotropin serum values. Causative responsibility for the increased LH serum concentrations is a decrease of testosterone production with age. Data obtained in this study and reports presented in literature give no convincing explanation for the increase of FSH serum levels with age.

Adolescent↗

Serum FSH and LH levels in men after administration of a long-acting LH-RH preparation.

The effects of the natural gonadotropin releasing hormone (LH-RH) and of a long-acting analogue, desgly10-D-leu6-LH-RH ethylamide, on the serum FSH, LH, testosterone and oestradiol-17 beta levels were studied in healthy men. The basal value for FSH was 3.58 +/- 0.47 mU/ml (SE), for LH 6.33 +/- 1.19 mU/ml. Following an intravenous dose of LH-RH the values increased by 3.40 +/- 0.88 and 25.99 +/- 5.67 mU/ml, respectively. After intravenous administration of the long-acting analogue, the peak FSH exceeded the basal value by 12.89 +/- 6.95 mU/ml, while LH increased by 41.27 +/- 4.72 mU/ml. The effect of the analogue on FSH and LH lasted 693 min and 862 min, respectively. Neither preparation changed the serum testosterone or oestradiol-17 beta levels essentially. The results suggest that the long-acting variant of LH-RH is more suitable than the natural hormone for examination of the FSH and LH reserves in men.

Adolescent↗

The cAMP-Dependent induction of LH receptors in primary cultures of porcine granulosa cells is not due to the expression of an intracellular pool of LH receptors.

The present studies examined whether the increase in cell surface LH receptors in primary cultures of porcine granulosa cells after exposure to FSH or cholera toxin, agents that increase intracellular cAMP, is due to de novo synthesis of the receptor or to a cAMP-dependent translocation of an intracellular pool of LH receptors to the cell surface. LH receptor induction by FSH was fully inhibited by the addition of cycloheximide to the incubation media, but resumed after cycloheximide was removed. These data suggest that FSH-induced LH receptor appearance requires protein synthesis. However, to be confident that the inhibition of LH receptor appearance did not result from lack of transit of preformed receptors requiring a rapidly turning over pool of proteins, we assayed for possible latent receptors in the cell interior by extracting granulosa cells with Triton X-100. Under conditions which detected about 74% of LH receptors in cells exposed to cholera toxin, little [125]iodo-hCG-binding activity was detected in cells not exposed to a cAMP-promoting stimulus. These findings suggest that a preformed pool of LH receptors does not exist in untreated cells, and that the cAMP-mediated induction of LH receptors requires de novo synthesis of the receptor.

Animals↗

Estradiol suppression of luteinizing hormone (LH)/human chorionic gonadotropin receptors and LH-sensitive adenylyl cyclase without decreased adenosine 3',5'-monophosphate content in rabbit corpora lutea.

It has been observed that elevated concentrations of estradiol, the principle luteotropin in the rabbit, reduce LH-stimulated adenylyl cyclase activity in corpora lutea during midpseudopregnancy without suppressing serum and tissue progesterone concentrations. If LH modulates intraluteal cAMP levels in this species, this suggests that in the presence of exogenous estradiol, progesterone synthesis may be independent of cAMP. To test this possibility and to investigate the physiological significance of LH in regulating the rabbit corpus luteum, the concentrations of cAMP and progesterone and the activity of adenylyl cyclase were measured in luteal homogenates, the numbers of LH/hCG receptors were estimated in crude membrane preparations, and the concentrations of progesterone and estradiol were measured in serum on days 2-12 of pseudopregnancy in rabbits treated with or without estradiol. Throughout days 2-12 of pseudopregnancy, estradiol treatment increased LH-stimulated adenylyl cyclase activity on day 2, but reduced its activity after day 4 by up to 50%, reduced the number of LH/hCG receptors after day 2 by up to 50%, and had no effect on the activities of basal, epinephrine-stimulated, or NaF-stimulated adenylyl cyclase. Tissue cAMP levels were not altered by estradiol treatment, nor were serum progesterone concentrations (except for an increase on day 2). Since LH receptors and LH-stimulated adenylyl cyclase were both reduced by day 5 of pseudopregnancy without a concomitant decrease in luteal cAMP or serum progesterone concentrations, our data suggest that LH is not a physiological regulator of luteal cAMP or serum progesterone during days 4-12 of pseudopregnancy. We propose that basal adenylyl cyclase activity, which is not reduced by estradiol treatment, may play a more significant role than LH-stimulated adenylyl cyclase activity in regulating tissue cAMP levels and the progesterone synthetic capacity of the rabbit corpus luteum.

Adenylyl Cyclases↗

Expression of functional luteinizing hormone (LH) receptor and its messenger ribonucleic acid in bovine endometrium: LH augmentation of cAMP and inositol phosphate in vitro and human chorionic gonadotropin (hCG) augmentation of peripheral prostaglandin in vivo.

Bovine endometrium contains LH/hCG binding sites and LH increases endometrial production of prostaglandin H synthase-2 (PGHS-2) and prostaglandin synthesis. This study showed that uterine endometrium contained both LH receptor mRNA transcript and a 93-kDa immunoreactive protein that bound to anti-rat LH receptor antibody. LH receptor and its mRNA were expressed maximally in the endometrium of cows from the luteal phase compared to the follicular phase of the estrous cycle. Furthermore, there was a response shown when incubation of endometrial minces from both pre-estrus/estrus and luteal phase (but not post-ovulatory phase) with LH or oxytocin (20 ng/ml) that resulted in a significant (p<0.02) increase in cAMP and total inositol phosphates. When Day 15 cows were injected i.v. with 3000 units hCG, the increase in peripheral 13,14-dihydro-15-keto PGF(2alpha) was 2.5-fold higher than saline controls or oxytocin. We conclude that LH stimulates endometrial cAMP and total inositol phosphates which results in increased formation of uterine PGHS-2 similar to LH effect on ovarian PGHS-2. The increased 13,14-dihydro-15-keto PGF(2alpha) production induced in vivo by injections of hCG indicates that LH may have a reinforcing role in luteolysis.

Animals↗

Prolonged suppression of plasma LH levels in male rats after a single injection of an LH-RH agonist in poly(DL-lactide-co-glycolide) microcapsules.

The authors have examined the effects of a subcutaneous injection of the LH-RH agonist D-Trp6-LH-RH formulated in biodegradable poly(DL-lactide-co-glycolide) microcapsules on plasma levels of D-Trp6LH-RH, LH, and PRL in adult, gonadectomized male rats. Immunoreactive D-Trp6-LH-RH was detectable in the plasma of these animals at 1, 2, 3, and 4 weeks after injection. LH concentrations were greatly reduced 1 week after administering the D-Trp6-LH-RH microcapsule, continued to decrease during the following week, and remained suppressed until the end of the study, 6 weeks after the injection. Plasma PRL levels appeared elevated 1 to 2 weeks after the injection and suppressed thereafter, but these effects were significant only in animals rendered hyperprolactinemic by transplantation of an isologous pituitary under the renal capsule. These results demonstrate that an LH-RH agonist formulated in biodegradable microcapsules and administered as a subcutaneous injection can exert marked biologic effects in rats for at least 6 weeks. These findings also suggest that prolonged exposure to an LH-RH agonist may first produce stimulation, followed by an inhibition of PRL release from both in situ and ectopic pituitaries.

Animals↗