1 Departments of GynecologieObstétrique, CHU Clémenceau, Caen, 2 Endocrinologie, CHU Saint-Antoine, Paris and 3 BiochimieIRBA, Université de Caen, 14032 Caen Cédex, France
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: hypophysectomy/inhibitory factors/LH bioactivity/plasma
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Collection of plasma samples for LH bioassay
Control samples were obtained from healthy fertile men (mean age: 35.8 ± 3.5 years; n = 6), postmenopausal women (mean age: 53.6 ± 3.3 years; n = 14) and women on day 7 of their follicular phase (mean age: 32.3 ± 3.8 years; n = 10). Samples were also obtained from four hypophysectomized women (4161 years), who had not received hormonal treatment; only plasma samples with undetectable concentrations of I-LH were used and these were defined as LH-free.
LH bioactivity was also determined in plasma samples from young women (mean age: 31.6 ± 1.2 years; n = 16) during the GnRH test. None of them had hyperprolactinaemia, signs of ovarian dystrophy, polycystic ovarian disease (PCOD) or hyperthyroidism. All I-LH plasma values were in the normal range and plasma samples with a high LH/follicle-stimulating hormone (FSH) ratio (>1.5) were excluded from the study. A GnRH test was performed on day 7 of the follicular phase because LH concentration is quite stable during this early part of the follicular phase (days 37). Blood samples were collected following a single i.v. GnRH injection (100 µg; StimuLH, Roussel-Uclaf, Paris) at 0 (T0), 15 (T15), 30 (T30), 60 (T60) and 90 (T90) min. All blood samples were collected in EDTA, centrifuged at 2500 g for 10 min at 4°C and stored as aliquots at 20°C until further use.
Treatment of plasma samples
Plasma aliquots obtained from control and hypophysectomized women were submitted to one of the following treatments:
All treated samples were stored at 4°C, their I-LH values were measured and they were then subjected to in-vitro hLH bioassay.
In-vitro LH bioassay
The testes from Sprague Dawley rats (34 days old) were removed, decapsulated and then disrupted gently with scissors before incubation at 32°C for 10 min in a solution collagenase-dispase (0.05%), soybean trypsin inhibitor (0.005%) and deoxyribonuclease I (0.001%) in Ham's F12-DME medium. After sedimentation (twice) and filtration through nylon gauze (30 Mesh), the testicular cells were centrifuged and the pellet washed with fresh medium. From the final pellet, viability and 3ß-hydroxysteroid deshydrogenase activity (3ß-HSD) were performed (Papadopoulos et al., 1985). Incubations (106 testicular cells) were realized in duplicate at 32°C, under an air-CO2 (5%) atmosphere in Ham's F12-DME medium supplemented with fatty acid free BSA (2.5 g/l), sodium heparin (1 IU/l) and MIX (0.125 mM) (Denis et al., 1994
). Diluted human plasma was added at four increasing concentrations to the Leydig cell incubation medium and the rate of testosterone synthesis was determined in the same assay. Biological value of LH was then calculated from two plasma dilutions (Revol et al., 1997
). Results were expressed in percentages compared to two reference values X and Z [testosterone productions expressed in ng/106 Leydig cells/4h, X: in absence of LH and Z: after incubation with a saturating dose (1 IU/l) of human LH standard NBSB 80/552, which led to maximum stimulation of the Leydig cells in terms of testosterone output].
Hormone assays
After incubations, media were collected by centrifugation at 2000 g for 10 min at 4°C and testosterone measured by radioimmunoassay (Papadopoulos et al., 1985). Intra-and interassay coefficients of variation were 3 and 6% respectively and the sensitivity was 4 pg/tube. Plasma LH was measured by an immunoradiometric assay (IRMA) technique using 125I-hLH Coatria kit (bioMérieux); intra- and interassay coefficients of variation were 2.6 and 5.3%. Results were expressed in IU/l in terms of 1st IRP 68/40 (the normal LH range of values was 3.510 IU/l for men, 2.511 IU/l for women in follicular phase and 1865 IU/l for postmenopausal women). The FSH concentration was evaluated by an 125I-FSH Coatria kit from bioMérieux (normal range: 1.58.0 IU/l for men, 213 IU/l for women in follicular phase; 25145 IU/l for postmenopausal women).
Statistical analysis
Data are expressed as means ± SEM. Student's t-test was used to compare mean values obtained from at least three different experiments, performed in duplicate; a P value < 0.05 was considered to be significant. Correlation coefficients were determined by the method of least squares.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Bioactive LH concentrations in normal ovulatory women
The effects of a single GnRH injection on B-LH and I-LH concentrations are shown in Figure 1. Two groups were determined according to the intensity of the release of the plasma gonadotrophins: group I (50 < FSH < 150% and 150 < LH < 300%, n = 9) and group II (FSH > 150% and LH > 3 00%, n = 7). The basal I-LH values were not significantly different in the two groups (5.8 ± 0.8 IU/l in group I versus 4.4 ± 0.8 IU/l in group II). The B-LH concentrations at T0 were significantly lower in group II than in group I (12.1 ± 2.2 IU/l versus 21.4 ± 3.2 IU/l; P < 0.05). According to their respective control values and taking into account that the Tm for groups I and II were not significantly different from each other, there was a greater increase in B-LH in group II (6.7-fold) compared to group I (2.9-fold) between T0 and Tm respectively. A close correlation between I-LH and B-LH values in group I (r = 0.96) and in group II (r = 0.98) was noted. Within the two groups, the profile of the B/ILH ratios according to the sampling time following GnRH administration did not change. Group I was characterized by a significantly higher B/I LH ratio at T0 (P < 0.001), T15, T30, T60 and T90 when compared to group II (P < 0.01). The B/I LH ratio remained stable during the GnRH test, although a slight decrease was observed at T15 (12 versus 16%; group I versus group II).
|
|
|
|
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The variations of LH bioactivity in normally cycling women might be explained by the existence of either structural alterations in the LH molecule (variants) or variations in glycosylation (isoforms). Recently, polymorphic variants resulting from single substitutions of Trp8/Arg and Ile15/Thr, inserting a new potential glycosylation site into the LHß-chain, have been isolated from the serum of women (Suganuma et al., 1996). Functional differences of the LH variant from wild-type LH have been observed such as a higher B/ILH ratio, a shorter half-life in peripheral circulation and an increased bioactivity (Haavisto et al., 1995
). The frequency of LH variants seems to differ widely in healthy women from various populations: 28% in Finland, 15% in UK and 7.5% in North American Hispanics (Haavisto et al., 1995
; Rajhkowa et al., 1995). The consequences for fertility of these LH variants in healthy women are still being discussed (Furui et al., 1994
; Haavisto et al., 1995
).
The qualitative modification of LH bioactivity could be also explained by the existence of numerous isoforms with a wide range of biological potencies in vitro and different half-lives in vivo (Stanton et al., 1996). The isoform profile can be modified according to physiopathological situations: change of relative proportions of plasma HCG isoforms at the 13th week of pregnancy (Wide et al., 1994
), enrichment of basic LH isoforms in PCOD patients (Ding and Huhtaniemi, 1991
), appearance of more basic isoforms in pubertal children during chronic GnRH agonist therapy (Wide et al., 1996
).
For plasmas with low or nil I-LH values, the significance of the concentration of bioactive LH is more difficult to evaluate, because of the proportion of plasma volume added to the in-vitro bioassay. It is well known that plasma contains numerous factors which interfere with Leydig cell steroidogenesis (Papadopoulos et al., 1990). Discrepancies between the parallelism of male serum and standard LH curves have been observed for LH determination in a mouse in-vitro bioassay: the addition of serum exceeding 5% of the total volume assay results in a lower testosterone response (Lichtenberg and Pahnke, 1976
; Rajalakshmi et al., 1979
). However, the above observation has not been shown when male serum is used with a rat Leydig cell bioassay (Dufau et al., 1976
). It has been reported that factors present in the serum of infertile stallions (Whitcomb et al., 1991
) and in testicular and peripheral blood of rams, either healthy, hypophysectomized or castrated (Papadopoulos et al., 1990
), inhibit the binding of LH to its receptor in vitro. Indeed, we have shown that plasma collected from hypophysectomized women leads to a decrease of both basal and hLH-induced testosterone production. The concentration of inhibitory factors differs between samples, whereas a complete inhibition of testosterone output has been observed in the presence of 20% (v/v) plasma. Together with other data, this demonstrates that inhibitory factors are present in samples of plasma obtained from hypophysectomized men (Lichtenberg et al., 1982
; Boujrad et al., 1991
), young women using oral contraception (Lichtenberg et al., 1982
), and healthy men and postmenopausal women whose serum was treated with a ß-LH antibody (Ding and Huhtaniemi, 1989
). The presence of inhibitory factors was also observed when plasma from normal ovulatory women were treated with an antibody directed against the whole LH molecule, but not when treated with antibodies against either
- or ß-LH subunits, suggesting the existence of LH agonist or antagonist molecules in normal I-LH plasma. Our data are different from those published by Ding and Huhtaniemi (1989) showing an interference of postmenopausal plasma with the LH bioassay which may be explained by the age and the hormonal status of the patients studied.
The inhibitory effect on steroidogenesis was not modified when LH-free plasma was treated with activated charcoal although it was increased after diethylether extraction which may unmask other inhibitory factors. Only treatment by 12% PEG partially restored the testosterone production through precipitation of inhibitory factors. These factors were also eliminated by heating at 50°C for 15 min (Lichtenberg and Pahnke, 1976), 60°C for 15 min (Ding and Huhtaniemi, 1989
) or 50°C for 30 min (data not shown). The effects of blood sample treatments, particularly 12% PEG or diethylether, gave different values for the I-LH concentration. The consequences of these treatments on the testosterone production were similar in LH free and low I-LH plasmas (decrease with diethylether and partial restoration with 12% PEG) suggesting that inhibitory factor activities induce similar effects whatever their origin but differ from those in normal ovulatory women. The nature of these inhibitory factors remains unknown: the diverse effects of diethyl ether and PEG suggest that lipids are not likely to be involved, although plasma cholesterol and triglycerides are decreased by these two treatments (data not shown). Perhaps these inhibitory factors should be compared to the truncated LH molecules able to bind or mask LH receptors.
In conclusion, this data demonstrates that the value of bioactive LH determined by assay represents probably a balance between the bioactive LH molecule and inhibitory(s) factor(s) present in the plasma matrix. In plasmas with low I-LH concentrations (< 1 IU/l), the determination of LH bioactivity is strongly related to the plasma volume added to the bioassay and, consequently, to the proportion of inhibitory factors present. Nevertheless, the measurement of LH bioactivity remains useful, especially in physiopathological situations associated with an increased or normal immunoreactive gonadotrophin concentration.
![]() |
Acknowledgments |
---|
![]() |
Notes |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Denis, I., Drosdowsky, M.A. and Carreau, S. (1994) In vitro effects of various albumins on Leydig cell testosterone production in immature rat. Horm. Metab. Res., 26, 5556.[ISI][Medline]
Ding, Y.Q. and Huhtaniemi, I. (1989) Human serum LH inhibitor(s): behavior and contribution to in vitro bioassay of LH using dispersed mouse Leydig cells. Acta Endocrinol., 121, 4654.[Medline]
Ding, Y.Q., Ranta, T., Nikkanen, V. et al. (1991) Discordant concentrations of serum bioactive LH in man as measured in different in vitro bioassay systems using rat and mouse interstitial cells and human granulosaluteal cells. J. Endocrinol., 128, 131137.[Abstract]
Ding, Y.Q. and Huhtaniemi, I. (1991) Preponderance of basic isoforms of serum luteinizing hormone (LH) is associated with the high bio/immuno ratio of LH in healthy women and in women with polycystic ovarian disease. Hum. Reprod., 6, 346350.[Abstract]
Dufau, M.L., Mendelson, C.R. and Catt, K.J. (1974) A highly sensitive in vitro bioassay for luteinizing hormone and chorionic gonadotropin: testosterone production by dispersed Leydig cells. J. Clin. Endocrinol. Metab., 39, 610613.[ISI][Medline]
Dufau, M.L., Beitins, I.Z., McArthur, J.W. et al. (1976) Effects of luteinizing hormone releasing hormone (LHRH) upon bioactive and immunoreactive serum LH concentrations in normal subjects. J. Clin. Endocrinol. Metab., 43, 658667.[Abstract]
Fauser, B.C.J.M., Pache, T.D., Lamberts, S.W.J. et al. (1991) Serum bioactive and immuno reactive luteinizing hormone and follicle-stimulating-hormone concentrations in women with cycle abnormalities, with or without polycystic ovarian disease. J. Clin. Endocrinol. Metab., 73, 811817.[Abstract]
Furui, K., Suganuma, N., Tsukahara, S. et al. (1994) Identification of two point mutations in the gene coding luteinizing hormone (LH) ß-subunit, associated with immunologically anomalous LH variants. J. Clin. Endocrinol. Metab., 78, 107113.[Abstract]
Haavisto, A.M., Pettersson, K., Bergendahl, M. et al. (1995) Occurrence and biological properties of a common genetic variant of luteinizing hormone. J. Clin. Endocrinol. Metab., 80, 12571263.[Abstract]
Imse, V., Holzapfel, G., Hinney, B. et al. (1992) Comparison of luteinizing hormone pulsatility in the serum of women suffering from polycystic ovarian disease using a bioassay and five different immunoassays. J. Clin. Endocrinol. Metab., 74, 10531061.[Abstract]
Jaakkola, T., Ding, Y.Q., Kellokumpu-Lehtinen, P. et al. (1990) The ratios of serum bioactive/immunoreactive luteinizing hormone and follicle-stimulating hormone in various clinical conditions with increased and decreased gonadotropin secretion: reevaluation by a highly sensitive immunometric assay. J. Clin. Endocrinol. Metab., 70, 14961505.[Abstract]
Lichtenberg, V. and Pahnke, V.G. (1976) Measurement of biologically active LH/hCG by in vitro testosterone production assay and problems when applied to serum. Acta Endocrinol. Suppl., 202, 5457.
Lichtenberg, V., Pahnke, V.G., Graesslin, D. et al. (1982) Biological and immunological potencies of lutropin (LH) in human serum: comparative studies using different standard preparations. Horm. Metab. Res., 14, 3945.[ISI][Medline]
Lobo, R.A., Kletzky, O.A., Campeau, J.D. et al. (1983) Elevated bioactive luteinizing hormone in women with the polycystic ovary syndrome. Fertil. Steril., 39, 674678.[ISI][Medline]
Papadopoulos, V., Carreau, S. and Drosdowsky, M.A. (1985) Effects of phorbol ester and phospholipase C on LH-stimulated steroidogenesis in purified rat Leydig cells. FEBS Lett., 188, 312316.[ISI][Medline]
Papadopoulos, V., Kamtchouing, P., Boujrad, N. et al. (1990) Evidence of LH-inhibiting activity in ovine peripheral and testicular blood. Acta Endocrinol., 123, 345352.[ISI][Medline]
Rajalakshmi, M., Robertson, D.M., Choi, S.K. et al. (1979) Biologically active luteinizing hormone (LH) in plasma. III. Validation of the in vitro bioassay when applied to male plasma and the possible role of steroidal precursors. Acta Endocrinol., 90, 585598.[ISI][Medline]
Rajkhowa, M., Talbot, J.A., Jones, P.W. et al. (1995) Prevalence of an immunological LH ß-subunit variant in a UK population of healthy women and women with polycystic ovary syndrome. Clin. Endocrinol., 43, 297303.[ISI][Medline]
Revol, A., Carreau, S., Castanier, M. et al. (1997) Mesure de l'activité biologique de l'hLH: étude multicentrique. Ann. Biol. Clin., 55, 123128.[ISI]
Stanton, P.G., Burgon, P.G., Hearn, M.T.W. et al. (1996) Structural and functional characterisation of hFSH and hLH isoforms. Mol. Cell. Endocrinol., 125, 133141.[ISI][Medline]
Suganuma, N., Furui, K., Kikkawa, F. et al. (1996) Effects of the mutations (Trp8 Arg) and (Ile15
Thr) in human luteinizing hormone (LH) ß-subunit on LH bioactivity in vitro and in vivo. Endocrinology, 137, 831838.[Abstract]
Van Damme, M.P., Robertson, D.M. and Diczfalusy, E. (1974) An improved in vitro bioassay method for measuring luteinizing hormone activity using mouse Leydig cells preparations. Acta Endocrinol., 77, 655671.[ISI][Medline]
Veldhuis, J.D. and Dufau, M.L. (1993) Steroidal regulation of biologically active luteinizing hormone secretion in men and women. Hum. Reprod., 8 (suppl. 2), 8496.[Abstract]
Whitcomb, R.W., Schneyer, A.L., Roser, J.F. et al. (1991) Circulating antagonist of luteinizing hormone in association with infertility in stallions. Endocrinology, 128, 24972502.[Abstract]
Wide, L., Lee, J.Y. and Rasmussen, C. (1994) A change in the isoforms of human chorionic gonadotropin occurs around the 13th week of gestation. J. Clin. Endocrinol. Metab., 78, 14191423.[Abstract]
Wide, L., Albertsson-Wikland, K. and Phillips, D.J. (1996) More basic isoforms of serum gonadotropins during gonadotropin-releasing hormone agonist therapy in pubertal children. J. Clin. Endocrinol. Metab., 81, 216221.[Abstract]
Zaidi, A.A., Qazi, M.H. and Diczfaluzy, E. (1982) Molecular composition of human luteinizing hormone: biological and immunological properties of highly purified preparations after electrofocusing. J. Endocrinol., 94, 2936.[ISI][Medline]
Submitted on May 11, 1998; accepted on November 17, 1998.