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Changes In the Isoforms of Luteinizing Hormone and Follicle-Stimulating Hormone during Puberty In Normal Children

Department of Clinical Chemistry (D.J.P., K.E., L.W.), University Hospital, Uppsala; The International Pediatric Growth Research Center, Department of Pediatrics (K.A.-W.), University of Göteborg, Göteborg, Sweden

Address all correspondence and requests for reprints to: Dr. Leif Wide, Department of Clinical Chemistry, University Hospital, S-751 85 Uppsala, Sweden. E-mail: Leif.Wide{at}clinchem.uas.se

	Abstract

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Concentrations of LH and FSH are known to increase during normal pubertal development, but changes in the isoforms of the gonadotropins at this time have not been investigated in depth. We examined the median charge of serum LH and FSH using agarose suspension electrophoresis in 81 normal children at pubertal stages I–V. In pubertal girls there were no significant (P > 0.05) differences in the median charge of LH, but there was a small (P = 0.05) shift to more acidic FSH isoforms between pubertal stages I and IV. In boys there was a significant (P < 0.01) shift to more acidic isoforms for both LH and FSH by pubertal stage II. Further changes were not found later in puberty. Except for LH at pubertal stage I, where the median charge was similar (P > 0.05) for both sexes, the median charge was more basic (P < 0.001) for both LH and FSH in girls compared with boys at all five pubertal stages. The degree of charge heterogeneity of FSH, estimated as the peak width at half the peak height, was significantly (P < 0.01) larger at pubertal stage I than at pubertal stages III-V in both boys and girls. The charge heterogeneity of LH was similar for all pubertal stages in both sexes. In conclusion, there were few qualitative changes in the gonadotropins during normal female puberty, whereas in the male there was a dramatic shift to more acidic isoforms of LH and FSH early in puberty. This information may assist our understanding of normal and pathological processes during puberty and may be of clinical relevance in detecting the initiation of puberty in boys.

	Introduction

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DURING PUBERTAL development of normal children, peripheral concentrations of LH and FSH increase sequentially from low values that are characteristic of prepuberty to concentrations in young adults that may be many-fold greater. In addition, a number of studies have suggested that there are qualitative changes in LH and FSH during puberty, i.e. a change in the isoform distribution. For the most part these changes have been inferred by examining the bioactive to immunoreactive (B/I) ratio of gonadotropins in serum. Several studies examining LH reported that there was an increase in the circulating B/I ratio for LH during pubertal development (1, 2, 3, 4, 5, 6). However, a recent study has suggested that this trend may reflect artifacts introduced when the in vitro bioassay and/or the immunoassay are used at the limit of their sensitivity (7). In the latter study using a more sensitive in vitro bioassay and immunoassay, no changes in the B/I ratio for LH were detected during pubertal development. Studies examining B/I ratios of FSH during puberty have generally recorded a decrease in the pituitary (8) or serum B/I ratio (5, 6, 9).

Another measure of changes in the isoforms of LH and FSH is to use a separation technique such as agarose suspension electrophoresis, which measures the charge, mainly of the terminal carbohydrate groups, on the gonadotropin molecules. Using this approach, it was found that the isoforms of FSH in the pituitary were more acidic in adult men compared with young boys (8). Furthermore, we have noted a shift to more acidic forms of LH and FSH in children during the later stages of puberty compared with earlier stages in puberty (10). However, the number of subjects at each pubertal stage was small, and all the children were undergoing precocious or early puberty, which may not be indicative of the normal pubertal environment.

In this study, we sought to clarify the pattern of LH and FSH heterogeneity in normal children undergoing pubertal development by examining the median charge and the degree of charge heterogeneity of the two gonadotropins using agarose suspension electrophoresis.

	Materials and Methods

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Subjects

All blood samples were obtained with the informed consent of the children and their parents. The samples were obtained from normal children of various pubertal stages through the Department of Pediatrics, University of Göteborg. The numbers of children at each stage and their median age are given in Table 1. All subjects were undergoing normal maturation; of the total of 81 children evaluated for this study, 7 girls and 4 boys were short-for-age but otherwise healthy and normal and without growth hormone insufficiency. Pubertal stage was scored according to Tanner (11) for pubic hair and breast development and according to Prader (12) for testicular volume. When adrenarche and gonadarche differed, the pubertal stage was rated according to gonadarchal development, i.e. breast development in girls and testicular volume in boys. All the serum samples were collected at 0200 or 0400 h and kept at -20 C until analyzed.

Measurement of LH and FSH in serum samples and in fractions eluted after agarose suspension electrophoresis was carried out using time-resolved sandwich fluoroimmunoassays, as previously described (13, 14). The assay kits were used as recommended by the manufacturer (Delfia hLH Spec and Delfia hFSH kits, Wallac Oy, Turku, Finland), with the exception that standards or samples (25 µL) were assayed in the presence of 0.5 µL/well normal mouse serum. Gonadotropin concentrations were expressed as international units per liter, with the reference standards being the World Health Organization Second International Standard for pituitary LH for immunoassay (80/552) and the Second International Reference Preparation of Pituitary FSH/LH (78/549) for LH and FSH, respectively. The detection limits for LH and FSH in serum were 20 and 35 mIU/L, respectively, and the intraassay coefficients of variation (CV) for both assays were less than 2% at levels between 3–250 IU/L and about 10% at 0.3 IU/L. The interassay CV was less than 3% at 4–18 IU/L for both FSH and LH.

For the detection of LH and FSH in eluted fractions following electrophoresis, aliquots of 250 µL were taken from each fraction and incubated overnight at 4 C with the capture antibody and in the presence of 1 µL/well normal mouse serum. Standards supplied with the kit were diluted in the buffer used in the electrophoresis. The detection limits for LH and FSH in column elutes were 2 and 3 mIU/L, respectively, and the intraassay CV was less than 3% at levels between 0.1 and 10 IU/L.

Agarose suspension electrophoresis

All serum samples were analyzed with the electrophoretic technique as previously described (15, 16), using a suspension of 0.10% agarose (Agarose IEF, Pharmacia, Uppsala, Sweden) in 75 mmol/L sodium veronal buffer (pH 8.6). Each serum sample (0.3–1 mL) was run on a 1.3 x 67 cm column for 20 h at 50 mA. The technique gives a nonsieving separation of isoforms with migration velocities, similar to those in free solution, and differences in mobility of the isoforms are due to differences in electric charge. The FSH or LH activities were measured in 20–25 eluted fractions and the areas under the hormone peaks calculated. In almost every case the shape of each hormone activity gave an extremely close approximation to a normal distribution. The median electrophoretic mobility (median charge) of the hormone activity was calculated as the mobility at the 50-percentile of the area and was expressed in relation to the mobility of human serum albumin, given as albumin mobility units (AMU). The degree of charge heterogeneity was estimated as the width, expressed in AMU, of the elution profiles of the gonadotropic activities at half the peak height.

The mobility of the human serum albumin was estimated to be 12 cm²/s/V towards the anode. There is no detectable adsorption to the agarose suspension and the recovery is close to 100% of both LH and FSH in the column eluates (17). For reference, purified FSH preparations with pI values of around 4.1, 4.4, 5.1, and 5.5 had median mobilities of 600, 520, 440, and 410 mAMU respectively (18). The between-assay CV of median electrophoretic mobility was less than 1% (15).

Data analyses

Before analysis, all serum concentrations were log-transformed to correct for heterogeneity of variance; AMU values from electrophoreses were analyzed untransformed. Statistical comparisons between groups were made using two-tailed, unpaired Student’s t-test.

	Results

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In the girls, the serum concentrations of LH and FSH increased from pubertal stage I to III, with no further change during Stages IV–V. In the boys there was a similar pattern of the LH concentrations with increases until pubertal stage III and thereafter, no further change. The FSH concentrations did not increase until pubertal stage III, after which the concentration plateaued (Table 1 and Fig. 1).

View larger version (19K): [in this window] [in a new window]   Figure 1. Relationship between serum concentration and median mobility of LH and FSH (mean ± SE) in girls () and boys (•) at pubertal stages I–V. The number of children in each group is given in Table 1. , cathode; , anode. *, P 0.05; **, P < 0.01 compared with pubertal stage I.

In terms of sex differences in the charge of the gonadotropins, the girls had significantly (P < 0.001) more basic isoforms of both LH and FSH at all stages compared with the boys of the corresponding stage, with one exception. This was before puberty at pubertal stage I, when the mean median mobility of LH was similar (P > 0.05) in both sexes. Thereafter, the LH isoforms in the males became more acidic, whereas in the females there was essentially no change in the charge of the LH isoforms with pubertal development.

The degree of charge heterogeneity of FSH estimated as the peak width at half the peak height was significantly (P < 0.01) larger at pubertal stage I than at pubertal stages II–V for boys and at pubertal stages III-V for girls (Table 1). The charge heterogeneity of LH was similar for all pubertal stages in both boys and girls.

	Discussion

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This study demonstrates that there are remarkable differences in the gonadotropin isoforms between pubertal girls and boys, and that changes in the isoforms are confined specifically to early puberty. In the girls, the charge of the LH isoforms was unchanged with pubertal stage, whereas for FSH there was a minor shift to more acidic forms by pubertal stage IV. The median charge of pituitary FSH isoforms is similar in prepubertal girls and adult women (8). Therefore, in the human pubertal female, the major change in the gonadotropins appears to be an increase in concentrations, from the lower levels in childhood.

Treatment of postmenopausal women with estradiol causes a shift to more basic isoforms of LH and FSH (16). Despite increasing concentrations of estrogen during puberty in girls (19), there was no detectable shift to more basic isoforms of the gonadotropins. This may indicate that during pubertal development the pituitary in females becomes less sensitive to the feedback effects of estrogen, resulting in increasing gonadotropin concentrations but also a reduced effectiveness of estrogen in inducing a shift to more basic isoforms.

In pubertal boys, there was a shift to the production of more acidic isoforms of both LH and FSH with, however, no further change detected after pubertal stage III. Of note was that for FSH there was a profound shift to more acidic forms between pubertal stage I and II without any significant change in the mean FSH concentration. This finding may indicate that the mechanisms controlling changes in the isoform distribution and changes in concentration are separated. We have noted similar examples in previous studies where the endocrine environment has been experimentally manipulated using exogenous GnRH (20), GnRH agonists (10), or treatment with progestagens (21).

The change in the serum isoform distribution in pubertal boys is consistent with previous studies examining the median charge of pituitary FSH (8). In that study, the mean median charge of pituitary FSH in young prepubertal boys was more basic than that in adult men. The present data indicate that this shift between prepuberty and adulthood occurs mainly around pubertal stage II.

The observed gender difference in charge of FSH, with more acidic forms in males than in females, is in agreement with previous observations in prepubertal children (8), pubertal children (10, 20), and adults of reproductive age (22, 23). In pubertal children a gender difference has also been found for LH with more acidic isoforms in boys than in girls (10, 20). On the other hand, the pituitary isoforms of LH were found to have a similar charge in young men and women (23). The current finding that prepubertal males have a similar charge on the LH isoforms in serum to prepubertal females warrants further investigation.

The degree of charge heterogeneity of LH was similar at all pubertal stages in both boys and girls. For serum FSH, however, the peaks were significantly wider before puberty (pubertal stage I) than at pubertal stages III–V in both boys and girls. It has previously been shown that the FSH peaks, but not the LH peaks, were wider when more basic isoforms appeared after an acute challange with GnRH or after treatment with an GnRH agonist (triptorelin) in pubertal children (10, 20).

Previous studies examining changes in the bioactivity of the gonadotropins during human puberty have used B/I ratios, reporting an increased B/I ratio across puberty (1, 2, 3, 4, 5, 6), or no change in the B/I ratio (7). The interpretation of these findings is difficult, particularly as we found no change in the isoforms in the girls and a shift only between pubertal stage I and II in boys. It is unclear if the above reports are biased because of artifacts induced by insensitive assays as discussed by Huhtaniemi (7), or indeed what properties of the LH isoforms the in vitro bioassays detect.

The underlying mechanisms regulating the changes in gonadotropin isoforms during puberty is unclear. The differences in charge observed for the isoforms probably reflect variations in the N-linked carbohydrate structures, three in LH and four in FSH, with different numbers of negatively charged terminal sialic acid residues and/or sulfate (SO₃^-) groups (24). The changes in charge, particularly in the boys, are significant over a relatively short time period. It would therefore appear unlikely that androgens or inhibin are responsible for this pattern, as these regulatory factors increase gradually over the entire pubertal process (19, 25, 26). Furthermore, castration of adult men does not affect the charge of serum FSH isoforms (22), administration of testosterone had no effect on the isoforms of serum FSH in men with Klinefelter’s syndrome (27), and normal men administered supraphysiological levels of testosterone had more basic isoforms of serum FSH (28). In pubertal boys to whom testosterone was administered, no changes in serum bioactive or immunoreactive FSH were observed (29).

The activity of hypothalamic GnRH increases during puberty, initially during nighttime pulsatile secretory episodes (19). The nighttime levels of LH and FSH are known to rise during puberty relative to daytime concentrations (19, 30). Our earlier studies suggest that increased stimulation by GnRH is not responsible for the shift to more acidic isoforms during puberty, as exogenous administration of GnRH (20) or GnRH agonists (10) to pubertal children results in the secretion of more basic isoforms of both LH and FSH. Therefore, the factors that initiate the production of more acidic isoforms during puberty in boys remain to be determined.

In summary, we report major differences between girls and boys in the patterns of LH and FSH isoform distribution during pubertal development. In pubertal girls there is little change in the charge of LH and a small shift to more acidic FSH isoforms, whereas in boys there is a shift to more acidic gonadotropin isoforms early in puberty. These findings illustrate that there are structural changes in the gonadotropins in pubertal children, which may assist our understanding of the normal and pathological processes during puberty. These observations may also be of clinical importance in detecting the initiation of puberty in boys.

	Acknowledgments

 
We thank Mr. Christer Bengtsson for excellent technical assistance, Catharina Jansson and the staff at the ward 34T of Göteborg Children’s Hospital, and Professor Timo Lövgren and Dr. Kim Pettersson of Wallac Oy (Turku, Finland) for providing the Delfia kits used in this study.

	Footnotes

 
¹ Present address: Institute of Reproduction and Development, Monash University, Clayton, Victoria 3168, Australia.

Received January 30, 1997.

Revised May 20, 1997.

Accepted June 16, 1997.

	References

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