In vivo bioactivities and clearance patterns of highly purified human luteinizing hormone isoforms

Previous studies have shown that highly purified isoforms of human pituitary LH exhibited a 20-fold range of in vitro bioactivities. The aim of this study was to determine the corresponding plasma half-lives, metabolic clearance rates (MCR), and in vivo bioactivities of these human (h) LH isoforms. Cannulated adult male rats were administered hLH isoforms as a bolus i.v. injection. For the half-life studies, blood was then serially collected over a 6-h period, and serum was assayed for hLH using a specific immunofluorometric assay. All hLH (n = 19) isoforms exhibited biexponential disappearance profiles with an initial fast half-life (t 1/2) for component A of 12.8 +/- 3.7 min, followed by a slow component B with t 1/2 of 58.9 +/- 4.4 min. The prevalence of component B in relation to component A increased significantly (r = 0.81, P < 0.001) over a 3-fold range when correlated with the sialic acid content of the isoform. Similarly, the MCR showed a significant correlation (r = 0.77, P < 0.001) with sialic acid content. The basis for the two t 1/2 components was then investigated. In the first experiment, rat plasma containing primarily component B was collected 90 min after hLH isoform administration and injected into a second animal. Only component B was observed with no evidence of component A, which indicates that the two t 1/2 components are not the product of the redistribution of the hLH isoform between body compartments. In the second experiment, component B was found to be dependent on sialic acid content, as desialylated hLH isoforms showed a rapid disappearance (t 1/2 = 8.6 +/- 3.1) with the component B proportion decreasing to < 10% of that of the nondesialylated control. This data indicates that sialic acid protects component B from rapid clearance. In addition, the proportion of the two components is dependent on sialic acid content, suggesting that the molecular location of the sialic acid on the carbohydrate moieties of hLH has a critical role in the clearance process. To determine the in vivo bioactivity of the hLH isoforms, an acute in vivo bioassay was developed in male rats. The assay was based on the hLH dose-dependent increase in total testosterone release in the same rat model as used in the plasma disappearance studies. Using the second International Standard (IS) hLH (0.3 IU-2.6 IU/kg) as standard, a linear dose-response of 24-h integrated serum testosterone levels was observed, with an index of precision of 0.11. Using this in vivo assay, a 16-fold range in in vivo bioactivities (3,200 to 51,100 IU/mg) was observed for 14 hLH isoforms. These in vivo bioactivities correlated with sialic acid content (r = 0.78, P < 0.001), MCR (r = 0.56, P < 0.05) and LH in vitro bioactivity (r = 0.75, P < 0.001) as determined using mouse Leydig cells in culture. Desialylation lead to over a 100-fold decrease in in vivo bioactivity of hLH. It is concluded that hLH isoforms are cleared in vivo by a two-component clearance mechanism, the proportion of which varies between isoforms and is dependent on sialic acid content of the isoform. These findings suggest that the molecular location of sialic acid on the hLH isoform is critical in defining the plasma disappearance of component B, whereas the mechanism of elimination of component A may well involve the hepatic GalNAc-sulphate receptor. Using an in vivo bioassay, the 16-fold difference in bioactivity between isoforms is attributed primarily to differences in their in vitro activity at the cellular level with a minor influence (< 2-fold) due to differences in in vivo clearance.     

Detection of epitopes on follicle-stimulating hormone and FSH-antiserum-induced suppression of bioactivity of follicle-stimulating hormone and luteinizing hormone

There are currently two major approaches to hormonal male contraception. One relies on testosterone (analogs) either alone or in combination with gonadotropin releasing hormone (GnRH) (analogs or immunizations), the other on immunizations against follicle-stimulating hormone (FSH). Theoretically, the latter method will suppress spermatogenesis whilst not interfering with libido. An absolute requirement is, however, that an anti-FSH vaccine does not include anti-luteinizing hormone (LH) antibodies (LH being responsible for the induction of testosterone which is necessary to maintain libido). In this report we show that when whole FSH is used for vaccination, in most cases in addition to biological activity against FSH, anti-LH activity is also induced. By systematic analysis of the antisera raised with FSH using systematic epitope scanning (PEPSCAN) we found differences between the FSH-specific and FSH-nonspecific sera. Only the FSH-specific antiserum contained antibodies that recognized amino acid sequence 37-55 on the beta-subunit in a linear manner. Because antibodies against this epitope have not been found in the cross-reactive sera this epitope forms a prime candidate for an anti-FSH contraceptive vaccine.     

Effects of profound suppression of luteinizing hormone during ovarian stimulation on follicular activity, oocyte and embryo function in cycles stimulated with purified follicle stimulating hormone

The effects of profound suppression of circulating luteinizing hormone (LH) during the follicular phase of in-vitro fertilization cycles were explored in normal women during treatment with a gonadotrophin-releasing hormone analogue and exogenous purified follicle stimulating hormone. Ovarian responses to treatment and the capacity of supernumerary embryos to undergo blastocyst formation were examined in groups of patients defined by the concentration of plasma LH in the mid-follicular phase. Concentrations < or = 0.5 IU/I diagnosed the group with profoundly suppressed LH (相似文献   

Rhythmicity of luteinizing hormone secretion expressed in vitro

In the present study we explored the possibility that the pituitary functions as an autonomous clock and is capable of generating rhythms of luteinizing hormone (LH) release independently of hypothalamic control. Pituitaries from estrous or diestrous day 1 female mice were perifused separately with Medium-199. Effluent samples were collected at 10-min intervals and assayed for LH levels. Fourier analysis and curve-fit analysis served to elucidate the presence of prominent periods whose significance was then determined by best-fit cosinor. The latter method was used to determine additional parameters for the significant rhythm. All perifused pituitaries exhibited LH release patterns that were composed of significantly long ultradian rhythms (approximately 16 and 8 h, p < 0.001). Continuous stimulation with gonadotropin-releasing hormone (GnRH) or estradiol did not alter the periods of the observed rhythms but affected other rhythm parameters. Gonadotropin-releasing hormone increased the mesor of the rhythm and estradiol increased the amplitude. The results indicate that pituitary gonadotropes are capable of producing rhythms of LH release for a long duration in vitro, in the absence of hypothalamic control. Both GnRH and estradiol affect different rhythm parameters but do not change the periods of these rhythms.     

Effects of thyrotropin, follicle stimulating hormone and luteinizing hormone on sIL-2R in vitro secretion from human peripheral blood mononuclear cells

The effect of thyroid stimulating hormone (TSH) or thyrotropin (0.06, 0.6, 6, and 60 microIU/ml), follicle stimulating hormone (FSH) and luteinizing hormone (0.1, 1, 10, and 100 mIU/ml) on soluble interleukin-2 receptor (sIL-2R) release in vitro from resting or phytohaemagglutinin (PHA) activated human peripheral blood mononuclear cells (PBMC) was evaluated. sIL-2R concentrations were measured in supernatants of cultured cells by quantitative sandwich enzyme immunoassay method (ELISA). TSH in a dilution of 0.6 microIU/ml and FSH in a concentration of 1 mIU/ml inhibited the secretion of sIL-2R only (p < 0.01) into supernatants from PHA activated PBMC cultures.     

Effect of profound suppression of luteinizing hormone during treatment with gonadotrophin-releasing hormone analogue and purified follicle stimulating hormone upon development of cryopreserved embryos

In response to previously published evidence from monkeys, this study examined the influence of the degree of luteinizing hormone (LH) suppression during the follicular phase of the stimulation cycle, upon cryopreserved embryo survival and development. The LH concentration of the mid-follicular phase was assessed in 250 in-vitro fertilization (IVF) cycles treated with gonadotrophin-releasing hormone analogue (GnRHa) and either purified follicle stimulating hormone (FSH) or human menopausal gonadotrophin (HMG), and was related to the performance of cryopreserved embryos in 351 subsequent embryo transfer cycles. Rates of embryo survival, embryo development rates, implantation rates, and pregnancy rates were examined with respect to the LH concentration recorded in the mid-follicular phase. In contrast to experimental evidence from other primates, there was no significant influence of the follicular phase LH concentration upon any of the parameters examined.     

Effect of growth hormone administration on circulating levels of luteinizing hormone, follicle stimulating hormone and testosterone in normal healthy men

A new area of growth hormone (GH) therapy in adults is the treatment of infertility. The aim of this study was to evaluate the effects of pharmacological GH administration on the secretion of pituitary and gonadal hormones in normal men. Eight healthy men, 23-32 years of age (mean 28.1 years), with a normal body mass index were studied in a double-blind, placebo-controlled crossover design. All participants had a normal semen analysis before entering the study. Each participant was treated with placebo and GH (12/IU/day, Norditropin; Novo Nordisk, Denmark) during two different 14-day periods, separated by a 6 week washout period. Administration of GH for 14 days resulted in a significant increase in serum insulin-like growth factor I (IGF-I; P < 0.01) but no changes occurred in IGF-I values during placebo treatment. The concentrations of follicle stimulating hormone and luteinizing hormone displayed no change during the two periods and did not differ between the GH treatment period and the placebo period. The concentration of testosterone was unchanged during the placebo/GH periods and there was no difference between the GH treatment period and the placebo period. We conclude that GH treatment for 14 days in normal healthy men does not affect gonadotrophin or testosterone patterns.     

Effect of endothelin-1 in man--impact on basal and stimulated concentrations of luteinizing hormone, follicle-stimulating hormone, thyrotropin, growth hormone, corticotropin, and prolactin

The effect of endothelin-1 on basal and stimulated serum (plasma) concentrations of luteinizing hormone (LH), follicle-stimulating hormone (FSH), thyrotropin (TSH), prolactin (PRL), growth hormone (GH), and corticotropin was investigated in healthy male volunteers (n = 5). Intravenous (IV) administration of endothelin-1 (5 ng/kg/min for 15 minutes, followed by 2.5 ng/kg/min for 105 minutes) induced an increase in basal plasma concentrations of corticotropin. Serum concentrations of PRL, TSH, LH, FSH, and GH remained unchanged. The increase in serum concentrations of these pituitary hormones induced by IV administration of LH-releasing hormone ([LH-RH] 100 micrograms), thyrotropin RH ([TRH] 400 micrograms), GH-RH (100 micrograms), and corticotropin-releasing factor ([CRF] 100 micrograms) was suppressed in regard to PRL (P < .01) and GH (P < .01) and enhanced in regard to corticotropin (P < .01). Stimulated serum concentrations of LH and FSH also tended to be higher following administration of endothelin-1 (P < .05), whereas the increase in serum concentrations of TSH remained unchanged. Thus, when administered in pharmacological doses, endothelin-1 influences pituitary hormone secretion in man.     

Effect of luteinizing hormone-releasing hormone on rat adrenocortical cells

The effects of luteinizing hormone-releasing hormone (LH-RH) on the function of rat adrenal cortex were investigated by using dispersed zona glomerulosa (capsular) and zona fasciculata-reticularis (inner) cells. LH-RH increased basal (but not adrenocorticotropic hormone (ACTH)-stimulated) corticosterone secretion of inner cells, without affecting either aldosterone or corticosterone production by capsular cells. LH-RH markedly raised basal (but not ACTH-enhanced) cyclic-AMP release by inner cells. The corticosterone secretagogue action of LH-RH was abolished by the protein kinase A inhibitor H-89. The conclusion is drawn that LH-RH specifically stimulates adrenal glucocorticoid secretion in rats through the activation of the adenylate cyclase signaling pathway.     

Increased biological activity due to basic isoforms in recombinant human follicle-stimulating hormone produced in a human cell line

FSH has four asparagine-linked oligosaccharides with variable sialic acid contents, so that FSH is not a single molecule, but a heterogeneous group of isoforms. These isoforms differ in their biological properties and their distribution changes in various physiological states, allowing the modulation of FSH activity. Recombinant human (h) FSH has been produced in Chinese hamster ovary cells and has an isoform profile similar to those of both pituitary FSH standard and purified urinary FSH. These FSH preparations, however, do not contain the full spectrum of FSH isoforms found in the circulation. Production of recombinant hFSH in a cell line with a different pattern of glycosylation could broaden its isoform profile and potentially alter its biological activity. Thus, we transfected human embryonal kidney cells (293) with the human alpha and FSH beta genes to produce recombinant hFSH (hFSH-293) and determined its biological activity in a rat granulosa cell bioassay. Although hFSH-293 was immunologically indistinguishable from pituitary FSH standard, its biological potency was 3- to 6-fold higher than those of two different pituitary FSH standards. To investigate this increased potency, we separated the isoforms of hFSH-293 by chromatofocusing and determined their biological potencies in the rat granulosa cell bioassay. The isoform profile of hFSH-293 demonstrated a greater number of basic isoforms than that of pituitary FSH standard. Several of these basic isoforms exhibited enhanced in vitro biological potency, accounting for the increased biological potency of hFSH-293. This pattern of high in vitro biological activity and more basic isoforms is analogous to the FSH circulating during GnRH stimulation, pubertal induction, and ovulation.     

Recombinant human luteinizing hormone: a partial physicochemical, biological and immunological characterization

The aim of this study was to partially characterize the glycoform composition of a recombinant human luteinizing hormone preparation (rhLH; Serono), an early version of the material (LHadi) which is currently being assessed for clinical application. Specifically, the charge (pl) and internal carbohydrate complexity of this rhLH was examined and compared with that of an alternative commercially available form of recombinant LH (Crystal Chem) and a pituitary International Reference Preparation (IRP). All preparations were separated by charge by chromatofocusing them on a pH gradient (7-4) using a 4 ml mono-P column is conjunction with a fast performance liquid chromatography system and by complexity of the oligosaccharide structures using concanavalin A (con-A) lectin affinity chromatography. LH in both the unfractionated and fractionated material was assessed by immunoradiometric assay (IRMA, I-LH) and by the in-vitro Leydig cell bioassay (B-LH). Both assays were calibrated against IRP 80/552. The in-vitro biopotency of the preparations was 18187 (Serono rhLH), 12063 (Crystal Chem rhLH) and 6658 (80/552) IU/mg; biological:immunological ratios were 1.14 (80/552), 1.90 (Crystal Chem rhLH) and 1.99 (Serono rhLH). However, similar qualitative data were obtained by both bioassay and immunoradiometric assay following fractionation, with the median pl of the bioactive LH in the preparations being 5.5 (24% > pH 6), 5.52 (18% > pH 6) and 4.97 (0% > pH 6) for the Serono, Crystal Chem and pituitary preparations respectively. Further all three contain < 1% of the complex carbohydrate structures and between 36-44% and 56-63% of the intermediate and simple forms of bioactive LH. In conclusion, the Serono recombinant LH preparation has a higher in-vitro bioactivity and is more basic than the other two preparations although the complexity of its carbohydrate moities appears to be similar.     

European collaborative study of LH assay: 3. relationship of immunological reactivity, biological activity and charge of human luteinizing hormone

This report describes the results of the third part of the collaborative study organized by a working group sponsored by the Community Bureau of Reference of the European Community Commission. The aim of the present work was to establish the link between immunoreactivity and biological activity of human LH, thus allowing to determine the antigenic domains of the molecule involved in the induction of the biological effect. The relationship between immunoreactivity and electric charge of hLH was also studied. This work allowed to further apprehend hLH isomorphism and its role in discrepancies observed among hLH assays and clinical status. It also made the feasibility of measuring biologically active isoforms by an immunological method to be assessed. The effect of 36 mAb with known epitopic specificity, was evaluated on both hLH binding to rat membrane receptor and hLH induced production of testosterone by porcine Leydig cells. All the epitopes located on the beta subunit were found to be strongly involved in the biological activity whereas 4/9 and 10/18 epitopes present on the alpha subunit or specific for the holomolecule respectively appeared weakly involved. Assaying biological hLH using immunological method would require that mAb specific for all the epitopes involved in the receptor activation be tested, and thus appears presently unsuitable for routine clinical evaluation. In the previous work some LH immunoassays were found to underestimate LH concentrations (J. Endocrinol. Invest 1994, 17: 397-406 and 407-416). The mAb used in liquid phase in these kits were found in the present work to be directed against the domains of LH weakly involved in the activation of the receptor and would suggest that bioactive LH would be misevaluated by these kits. The immunoreactivity of hLH isoforms separated by isoelectric focusing (IEF) in liquid phase was also determined. IEF allowed to separate three groups of hLH isoforms but none of them exhibited a specific discriminating pattern of immunoreactivity when they were tested against a panel of mAb. It suggests that, in our experimental conditions, the electric charge and the immunoreactivity of hLH were not closely linked.     

The unique exon 10 of the human luteinizing hormone receptor is necessary for expression of the receptor protein at the plasma membrane in the human luteinizing hormone receptor, but deleterious when inserted into the human follicle-stimulating hormone receptor

The LH receptor (LHR) is a member of the family of G protein-coupled seven-times plasma membrane transversing receptors. Its gene consists of 11 exons, the last one encoding the transmembrane and intracellular domains of the receptor. The FSHR, and its gene, resemble structurally those of the LHR, with the exception that the sequences corresponding to exon 10 in LHR are missing in FSHR, which is thus encoded by a total of ten exons. Our recent studies on the marmoset monkey testis LHR cDNA indicated that an 81 bp nucleotide sequence, encoding the complete exon 10 of the LHR gene in other mammalian species, is absent in this species without affecting the LHR function. To study further the role of the exon 10 encoded sequences of the LHR in the gonadotropin receptor function, a deletion of exon 10 from the human LHR (hLHdeltaexon10R), and a chimeric hFSHR with exon 10 from hLHR inserted (hFSHLHexon10R), were constructed in expression vectors. The results presented here demonstrate that 293 cells transfected with the hLHdeltaexon10R display a decrease in the proportion of the receptor binding at the cell surface, compared with cells transfected with wild-type hLHR. However, the cells expressing hLHdeltaexon10R showed similar high affinity binding of [125I]iodo-hCG as those transfected with wild-type hLHR, in either intact cells or their detergent extracts. In addition, cells expressing the hLHdeltaexon10R and wild-type hLHR displayed similar dose-response of cAMP production to hCG stimulation. Cells transfected with chimeric hFSHLHexon10R showed barely detectable [125I]iodo-FSH binding in intact cells compared with those transfected with wild-type hFSHR. The FSH binding detected in cellular detergent extracts displayed 10-fold lower binding activity than wild-type receptors, in spite of similar level of immunoreactive FSHR protein expression in the transfected cells. The hFSHLHexon10R had a modest 5-fold lower binding affinity for FSH as compared with wild-type hFSHR. In conclusion, the present study indicates that the sequences encoding exon 10 of the hLHR are essential for the LHR expression at the plasma membrane, but deleterious for function if inserted into the hFSHR.     

Effect of restraint stress on the preovulatory luteinizing hormone profile and ovulation in the rat

Plasma profiles of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) were measured during restraint stress on the day of pro-oestrus; these profiles were considered in relation to ovulation rate on the next day. Rats bearing a permanent jugular vein cannula were subjected to restraint, which was started 0, 1 or 2 h before the presumed onset of the LH surge and ended just before the beginning of the dark period. Exposure to restraint resulted in a suppression of the secretion of both gonadotrophins on the day of pro-oestrus. Suppression of the LH surge was virtually complete (plasma LH < or = 0.2 ng/ml) in 15 out of 32 stressed rats, and the ovaries of these rats contained graafian follicles with oocytes in germinal vesicle stage. In these rats, the LH surge did not occur 24 h later. In the remaining 17 rats, restraint resulted in a considerable suppression of the LH surge. Of these rats, five had an ovulation rate of 100% and four ovulated partially. In unruptured follicles of the latter, the oocyte had not resumed meiosis and the follicle wall was not luteinized. In the remaining eight rats with a reduced LH surge, ovulations had not occurred and graafian follicles were unaffected. The results of this study indicate that during pro-oestrus restraint stress suppresses and does not delay the release of preovulatory gonadotrophins. Partial suppression of LH by restraint does not result in induction of meiotic resumption without subsequent ovulation or in luteinized unruptured follicles.     

Purified urinary follicle stimulating hormone induces different hormone profiles compared with menotrophins, dependent upon the route of administration and endogenous luteinizing hormone activity

The effects of treatment of patients with gonadotrophin-releasing hormone analogue (GnRHa) combined with purified follicle stimulating hormone (FSH) for in-vitro fertilization (IVF) were investigated in detail to determine the influences of different administration routes and the degree of suppression of luteinizing hormone (LH). Responses to exogenous gonadotrophins were studied in infertile women (n = 60) with normal menstrual rhythm whose endogenous gonadotrophin activity was suppressed using a GnRHa in a long protocol. They were randomized to receive i.m. administration of human menopausal gonadotrophins (HMGim, Pergonal) or purified follicle stimulating hormone (FSH, Metrodin High Purity) administered either i.m. (MHPim) or s.c. (MHPsc). Responses were assessed by measuring plasma FSH, LH, oestradiol, testosterone and progesterone. After stimulation day 4, the MHPsc group showed significantly higher circulating concentrations of FSH than either the MHPim or HMGim group. However, the HMG group showed significantly higher oestradiol concentrations after stimulation day 5 than either MHP group. The differences in circulating oestradiol concentrations in the MHP-treated patients appeared to be strongly influenced by the mean circulating concentrations of LH in the follicular phase. The patients who showed mean follicular phase LH concentrations of < 1 IU/l showed longer follicular phases, lower circulating oestradiol and testosterone concentrations and also lower follicular fluid concentrations of oestradiol and testosterone, indicating a reduction in the normal follicular metabolism of progesterone to androgens and oestrogens under these conditions. This group of patients also showed longer follicular phases, which may have consequences for future clinical management.     

Plasma concentrations of prolactin, growth hormone, and luteinizing hormone in steers administered ergotamine or ergonovine

This research investigated whether ergot alkaloids associated with endophyte-infected tall fescue could alter plasma concentrations of pituitary hormones that regulate biological processes related to cattle performance. Seven Angus yearling steers received single i.v. injections of ergotamine tartrate, ergonovine maleate, or saline vehicle in a simple cross-over design. Each steer was given a different compound each week. Blood samples were collected at 15-min intervals for 45 min before and 240 min after treatments to assess plasma concentrations of prolactin, growth hormone, and LH. Respiratory rates were measured hourly to ascertain a systemic effect. Ambient temperature averaged 34 degrees C during data collection. Treatment x time was a significant source of variation for respiration rate and plasma concentrations of each hormone evaluated. Respiration rates were higher for ergonovine than for saline (P < .02) and ergotamine (P < .07) 30 min after treatment, but they were higher (P < .05) for ergotamine than for ergonovine and saline by 210 min after treatment. Both alkaloids transiently elevated (P < .01) plasma growth hormone concentrations compared with before alkaloid treatment and after saline treatment. Ergotamine reduced (P < .01) plasma concentrations of prolactin and LH throughout the 120-min period after treatment compared with concentrations before ergotamine treatment and after saline treatment. Ergonovine lowered (P < .01) prolactin concentrations for a shorter time than ergotamine and did not affect mean LH concentrations. Results indicated that ergot alkaloids implicated as contributing agents to fescue toxicosis can alter plasma concentrations of pituitary hormones important to cattle production.     

Levels of luteinizing hormone in semen of fertile and infertile men and possible significance of luteinizing hormone in sperm metabolism

Luteinizing hormone (LH) levels were measured in the seminal plasma of 68 fertile and infertile men. LH levels in the seminal plasma were severalfold higher than those normally found in serum and were significantly higher in oligospermic and normospermic samples than in azoospermic samples. However, no significant difference was observed in LH levels of oligospermic and normospermic men. The effects of LH on fructose utilization, glucose oxidation, and adenyl cyclase activity of spermatozoa were also examined. The results indicate a possible role of seminal plasma LH in sperm motility and metabolism.