1 Departments of Biotechnology and 2 Physiology, University of Turku, Finland
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Abstract |
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Key words: bioassay/GnRH stimulation test/immunoassay/luteinizing hormone/variant
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Introduction |
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LH is a heterodimer consisting of two non-covalently linked subunits, and ß, which are expressed in the anterior pituitary. The
subunits of the different glycoprotein hormones [follicle-stimulating hormone, chorionic gonadotrophin (HCG) and thyroid-stimulating hormone] within the same species are nearly identical, and the differing ß subunits confer the hormone specificity. Both subunits are glycosylated, with two N-linked carbohydrate chains on the common
subunit and one on the LHß subunit (Gharib et al., 1990
).
A common genetic variant of LH (v-LH) due to two point mutations in the ß subunit gene, both altering the amino acid sequence, Trp8Arg and Ile15Thr (Furui et al., 1994; Okuda et al., 1994
; Nilsson et al., 1998
), was initially discovered by us as an immunologically anomalous form of LH (Pettersson and Söderholm, 1991
). These mutations change the recognition site for a specific monoclonal antibody recognizing the intact
/ß heterodimer, thus offering a convenient method for assessing v-LH status by a combination of two immunoassays, one detecting only wild type (wt-LH), the other both forms of LH. The ratio of LH concentrations measured with the two assays provides a reliable tool to compare concentrations of the two hormone forms. v-LH is suggested to have an extra carbohydrate chain at Asn13, similar to that exhibited by HCG (Talmadge et al., 1984
), because the amino acid change in codon 15 introduces a new glycosylation consensus sequence. Indeed, this has been demonstrated with recombinantly produced v-LH (Suganuma et al., 1996
). Furthermore, v-LH has elevated bioactivity in vitro but significantly shorter half-life in circulation compared to wt-LH (Haavisto et al., 1995
; Suganuma et al., 1996
). As the pulse frequency of v-LH is normal (Haavisto et al., 1995
), this results in an overall LH action that is more potent at the receptor site, but shorter in duration in vivo.
In a very recent report, we described eight point mutations in the promoter region of the v-LHß allele and their effect on basal activity and response to hormonal stimulation in cell line transfections using promoter/reporter fusion genes (Jiang et al., 1999). In comparison to wt-LH, the v-LHß promoter had significantly higher basal activity and differed in response to hormonal stimulation in an immortalized mouse pituitary cell line, LßT2, and in a human embryonic kidney cell line, HEK 293. These results on altered promoter function of the v-LHß allele provide evidence for differences in regulation of synthesis, and possibly of secretion, of the two LH forms (Jiang et al., 1999
).
In this study, we investigated the circulating levels of the two LH forms in men and women heterozygous for the v-LHß allele. In principle, about half of the circulating LH should be wt- and v-LH forms, but if the secretion or clearance rates of the two LH types are different after GnRH stimulation, a change should be observed in the wt/total-LH ratio. The subjects underwent a GnRH stimulation test, and the relative proportion of the two LH forms were monitored using a ratio of two LH immunoassays measuring either wt or wt + v-LH. A mouse Leydig cell in-vitro bioassay was performed to investigate the biological activity of the LH. It was expected that these measurements would reveal whether differences exist in vivo in the activity and responses to hormonal stimulation between the wt and v-LHß alleles.
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Materials and methods |
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GnRH stimulation test
The test was always started in the morning between 08000900 h. A catheter was inserted into a vein of the forearm and a 10 min sample was taken. The 0 min sample was taken immediately before an i.v. injection of 50 µg GnRH (Relefact®; Hoechst, Frankfurt am Main, Germany) through the indwelling cannula. Similar blood samples of 4 ml were drawn 10, 20, 40, 60, 120 and 180 min after the GnRH injection. The blood samples were allowed to clot and the tubes were centrifuged before the sera were separated and stored at 20°C until analysed.
Immunofluorometric assays (IFMA) of LH
The LH concentrations of all serum samples collected during the GnRH stimulation test were determined by two different IFMA. The total LH concentration was determined using LHspec (Wallac Oy, Turku, Finland) as this assay also recognizes v-LH (Pettersson et al., 1992). The other assay, I3/A2, recognizes only the wt form of LH (Pettersson and Söderholm, 1991
). A ratio of the two assays (wt-LHI3/A2 /total LHLHspec) was used to determine the LH status of these individuals. Our previous studies have shown that heterozygotes for v-LHß have a wt/total LH ratio between 0.2 and 0.9 as the I3/A2 assay recognizes only about half of the total immunoreactive LH in heterozygote serum (Nilsson et al., 1997
).
All female samples were also analysed for their oestradiol content by DELFIA® oestradiol kit (Wallac OY), and male samples for testosterone using the DELFIA® Testosterone kit (Wallac Oy). All immunoassay determinations were performed using a 1235 autoDELFIA® automatic immunoassay system (Wallac Oy).
In-vitro bioassay of LH
The mouse interstitial cell in-vitro bioassay was used for measuring bioactive LH in serum. The method was performed as originally described (van Damme et al., 1974) and later modified by us (Ding and Huhtaniemi, 1989
). Bioactive LH was determined from a mixture of the serum samples collected at 10 min and immediately before injection of GnRH (0 sample) and from serum taken 60 min after the stimulation. In short, interstitial cells of adult (2-month-old) NMRI mouse testis were dispersed in medium 199, pH 7.4, supplemented with 0.1% BSA, 0.1 mmol/l 3-isobutyl-1-methylxanthin (Sigma Chemical Co., St Louis, MO, USA), 20 mmol/l HEPES and 10 000 IU/l sodium heparin (Leiras, Turku, Finland). Two or three dilutions of the serum samples, 1:10 and 1:20 of 0 samples and 1:10, 1:30, 1:100 or 1:300 of 60 min samples, were made in medium 199 with 0.5% BSA, and were analysed in triplicate. Each assay tube contained 100 µl of interstitial cell suspension (30 00050 000 cells), 50 µl standard or serum dilution and 50 µl diluted LH-free serum. The tubes were incubated for 3 h at 34°C in an atmosphere of 5% CO2 and 95% O2. LH free serum was prepared by incubating male serum in anti-LH coated microtitre plates (Wallac Oy), and it was added to the assay tubes in order to make the serum concentration equal in each reaction. After stopping the reaction by heating for 5 min at 100°C the tubes were stored at 20°C. The biological response was measured as testosterone production and quantified by radioimmunoassay (Huhtaniemi et al., 1985
).
Statistics
The results of the assays were analysed by Statview computer program (Abacus Concepts Inc., Berkeley, CA, USA). One-way analysis of variance (ANOVA) for repeated measures was performed, and P < 0.05 was regarded as statistically significant.
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Results |
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The bioactivity to immunoreactivity (B/I) ratio of LH was calculated from the bioassay and LHspec results (Figure 3). It decreased in the female samples from 4.92 ± 0.38 to 3.67 ± 0.37 (P = 0.010) and in males from 4.86 ± 0.26 to 4.11 ± 0.24 (P = 0.012). According to these results, the proportion of immunoreactive LH increases significantly compared to bioactive LH after GnRH stimulation in female and male heterozygotes.
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Discussion |
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The increased wt/total LH immuno-ratio after GnRH stimulation, which was significant in males, indicated that the wt form of LH accumulates in serum of v-LHß heterozygotes. As v-LH is more rapidly cleared from the circulation due to its shorter half-life (Haavisto et al., 1995), the proportion of wt-LH would consequently increase in the serum of heterozygotes. This explanation of the findings is also supported by the trend of immediate decrease in immuno-ratio values of the females. This finding may indicate a higher activation of the v-LHß gene in women than men upon GnRH stimulation.
In clinics the GnRH stimulation test is usually performed during the early follicular phase and 100 µg of GnRH is given. We chose to perform the test in the late follicular phase and use a lower GnRH dose because a similar test has previously been performed by us on wild type and homozygous individuals and we wanted to make the test designs similar (Haavisto et al., 1995). In the earlier study the response of one female who had already entered the ovulatory phase was clearly distinguished. Although the responses of the subjects in this study were similar it cannot be excluded that the timing of the test contributes to the observed sex difference seen in the immuno-ratio response. GnRH in submaximal doses stimulates LH secretion from both the acutely releasable (1st) and the reserve (2nd) pools of pituitary gonadotrophs (Wang et al., 1976
; Hoff et al., 1977
). Both pools increase significantly during the follicular phase, while after ovulation the pools decrease towards the end of the cycle (Wang et al., 1976
). These results were later supported by studies during the luteal-follicular transition (Messinis et al., 1996
). GnRH stimulation resulted in a decreased LH increase in the late luteal phase before increasing at day 2. The LH response decreased again on days 3 and 4, and increased on days 5 and 7.
The first B/I ratio measurements of LH demonstrated increases after GnRH stimulation (Dufau et al., 1976; Lucky et al., 1980
), but this was later shown to be due to poor sensitivity of older radioimmunoassays. These overestimated low LH levels, hence automatically resulting in higher B/I ratios when the absolute LH levels increased. When more reliable and sensitive immunoassays were used in the B/I ratio measurements, no change in this parameter was found (Jaakkola et al., 1990
; Huhtaniemi et al., 1992
). The present data are the first to show a decrease in the B/I ratios after GnRH stimulation. The nature of the subjects studied can explain this. Since they were heterozygotes for the v-LHß allele, their LH in fact consists of two components, the long-half-life wt hormone and the short half-time v-LH. If the synthesis of both is stimulated roughly to a similar extent by GnRH, the secretion of both increases in parallel, but then the relative proportion of wt hormone increases as v-LH is eliminated faster. This should suppress the B/I ratio of LH, since v-LH has higher B/I ratio than wt hormone. This finding is in perfect concordance with the increased wt/total LH ratio, as measured with the two immunoassays. If LH is homogeneous as is the case with wt and v-LH homozygotes, no alterations in the B/I ratio after GnRH stimulation can be expected.
Recently we reported eight mutations in the v-LHß promoter rendering the transcriptional activity higher than that of the wt-LHß promoter in LßT2 cells, known to express the LHß subunit gene, and in HEK 293 cells (Jiang et al., 1999). Progesterone and pulsatile GnRH administration also increased the activity of the v-LHß promoter more than that of the wt-LHß promoter. These results indicated that the v-LHß promoter would be stronger than the wt-LHß promoter in cell culture systems, which provide a compensatory means to maintain sufficient levels of v-LH in circulation, despite the shorter half-time. The fact that the expected increase in the wt/total LH ratio did not occur in women may be caused by higher GnRH response of their v-LHß promoter.
The increased bioactivity of v-LH may not be of crucial significance, as can be expected from the wide distribution of v-LH in different populations, varying between zero and 52% in carrier frequency (Nilsson et al., 1997, 1998
). The homozygotes for v-LH in the Finnish population are apparently healthy, whereas other findings have connected this genotype with infertility (Suganuma et al., 1995
; Takahashi et al., 1998
). In healthy women, v-LH has been associated with elevated serum levels of oestradiol, testosterone and sex hormone binding globulin in the follicular phase of the menstrual cycle (Rajkhowa et al., 1995
). Pubertal boys have slower height development and smaller testicular sizes, resulting in a delayed progression of puberty (Raivio et al., 1996
). The v-LHß allele occurs also less frequently in obese women with polycystic ovarian syndrome (PCOS) than in control subjects (Tapanainen et al., 1999
). There is thus ample evidence that the action of LH in individuals homo- or heterozygous for the v-LHß allele differs from that of individuals with wt-LH.
In this study we found that the wt-form of LH accumulated in serum of individuals heterozygous for the v-LHß allele after a GnRH stimulation (significantly in males), which was shown by an increase of the wt/total LH immuno-ratio. We also reported a decrease of the B/I ratio 60 min after the GnRH injection due to the increase of wt-LH relative to v-LH. These results further elucidate the physiology of the common genetic variant of LH. This polymorphism is also important to remember when LH is measured by immunoassays as antibodies recognizing the intact LH heterodimer have a poor recognition for v-LH.
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Acknowledgments |
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Notes |
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References |
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Submitted on June 1, 2000; accepted on October 20, 2000.