1 Departments of Pediatrics and Physiology, University of Turku, FIN-20520 Turku, Finland and 2 Division of Endocrinology and Metabolism, Department of Internal Medicine, General Clinical Research Center and Center for Biomathematical Technology, University of Virginia Health Sciences Center, Charlottesville, Virginia 22908, USA
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Abstract |
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Key words: bioassays/hormone physiology/LH isoforms/oligosaccharide heterogeneity
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Introduction |
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The most common method for determining LH bioactivity is in-vitro bioassay of plasma or pituitary extracts, based on testosterone secretion by mouse or rat interstitial (Leydig) cells (Dufau et al., 1976b; Lucky et al., 1979
; Robertson et al., 1979
; Marut et al., 1981
; Huhtaniemi et al., 1996
). By comparing LH concentrations obtained by biological and immunological [such as radioimmunoassay (RIA), immunoradiometric (IRMA) or immunofluorometric assay (IFMA)] techniques in a given sample, one can also estimate the apparent bioactive to immunoreactive (B/I) LH ratio. This derived parameter has been used as an indirect index of the relative biopotency of LH, at least in relation to any one immunological estimate. The latter is an important consideration, inasmuch as large non-uniformities can exist among immunoassay technologies (Chappel, 1990
; Jaakkola et al., 1990
; Demers, 1991
; Phillips and Wide, 1994
; Clark et al., 1997
). Subject to these constraints, and within any given validated assay system, a changing B/I ratio should mirror variations in the relative in-vitro biopotency of LH.
In-vitro biopotency may diverge several-fold from in-vivo LH biopotency, especially when LH's metabolic clearance is altered by the post-translational addition of key residues, such as sulphate, sialic acid and/or galactosamine (Dufau and Veldhuis, 1987; Veldhuis et al., 1989; Burgon et al., 1996
). Few studies have simultaneously appraised in-vitro and in-vivo bioactivities of human LH as a function of variable isoform subtypes. One study utilized in-vitro Leydig cell responsiveness and in-vivo testosterone secretion in the rodent to compare the lutrophic effects of a wide gradation of human LH isotypes differing prominently by sialic acid and/or sulphate charge (Sardanons et al., 1987
; Burgon et al., 1996
). The degree of sialylation and sulphation strongly determined the whole-animal LH half-life, and thus influence in-vivo bioactivity more remarkably than in-vitro biopotency. For evident ethical reasons, analogous homologous-species comparisons have not been achievable using human Leydig cells in vitro and purified pituitary or plasma human LH preparations for injection in humans in vivo. However, such studies in the hypophysectomized sheep revealed a relatively small range of in-vivo LH biopotencies, at least in this particular ruminant species. Larger kinetic and potency differences emerged in the rat (Dufau et al., 1976b
; Sairam and Fleshner, 1981
; Clarke et al., 1990
). The recent report of a human recombinant LH-receptor-based in-vitro bioassay of LH-stimulated alpha inhibin promoter-driven luciferase expression in HEK 293 cells and of a human granulosa-luteal cell in-vitro assay model could make homologous in-vitro bioassay of human LH more practicable (LH assay) (Huhtaniemi et al., 1993).
Envisioning the eventual availability of biosynthetically pure human LH isoforms, we further suggest that a novel strategy for in-vivo homologous bioassay of human LH kinetics and actions could consist of GnRH antagonist-induced down-regulation of endogenous LH secretion followed by selective LH isoform infusion. One could then directly monitor LH elimination on the one hand and testosterone output on the other. This paradigm should clarify more expressly the relative in-vivo biopotencies of various human LH isotypes acting on the human gonad. Such studies would need to appraise the impact of LH isoforms on both acute and prolonged gonadal steroidogenesis in both sexes. Later analyses should also evaluate differential effects of FSH isotypes on gametogenesis in men and women.
Based on the above technical caveats, the following review of the physiological implications of LH isotypes must be viewed as provisional. Indeed, definitive physiological experiments in our view remain necessary to clarify several issues in this arena. Here, we will focus briefly on the following selected contexts of evident LH-isoform regulation in the human: (i) the adult male, (ii) the female menstrual cycle, (iii) the menopause, (iv) polycystic ovary syndrome (PCOS), (v) puberty, and (vi) in relation to molecular variants of LH polypeptide structure.
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LH isoforms in the normal adult male |
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Quantitative changes in plasma LH B/I ratios do not necessarily denote unequal secretion of bio-enriched versus bio-depleted isoforms of LH, but could also reflect their unequal rates of in-vivo metabolic interconversion or removal (Ashwell and Harford, 1982; Dufau and Veldhuis, 1987
; Veldhuis et al., 1987a
,b
, 1989a
, Veldhuis et al., b
; Pavlou et al., 1990
; Burgonm et al., 1996). In this regard, women show a rise in the LH B/I ratio postmenopausally (Dufau et al., 1976a
; Reader et al., 1983
; Davis et al., 1987
; Urban et al., 1988a
,b
,c
, 1990
; Kolp et al., 1992
), whereas ratios decrease or remain unchanged in ageing men (Marrama et al., 1984
; Warner et al., 1985
; Urban et al., 1988; Mitchell et al., 1995
). In one study, mean plasma concentrations of bioactive LH and the calculated half-life of endogenous LH were similar in healthy older and young men (Urban et al., 1988a
). However, in tamoxifen-treated older men, the amplitude (but not the frequency) of bioactive LH pulses failed to rise equivalently, suggesting an impaired reserve capacity for maximal bioactive LH secretion (Urban et al., 1988a
; Veldhuis et al., 1989a
). An age-related distinction in bioactive LH secretory responsiveness was also evident in response to short-term antiandrogen administration (Veldhuis et al., 1989a
, 1992
, 1994
).
Men with chronic renal failure treated with hemodialysis manifest elevated serum immunoreactive LH (and subunit) concentrations, but reduced B/I ratios and impaired testosterone output (Talbot et al., 1990
). In addition, the distribution of plasma LH isoforms in uremic patients includes more basic moieties. Conversely, acidic LH species in azotemic men correlated with higher B/I LH ratios and normal serum testosterone concentrations (Mitchell et al., 1994
). This relationship would coincide with the predictably longer in-vivo half-life of highly sialylated (more acidic) LH, and hence its extended availability in the circulation to stimulate gonadal steroidogenesis (Veldhuis et al., 1989a
).
Taken together, the foregoing clinical pathophysiological data suggest that differential LH isoform production may play a role in men during ageing and influence gonadal function in end-stage renal failure. However, the precise significance of LH heterogeneity in the normal young adult male remains to be determined (Urban et al., 1988a,b
).
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LH isoforms during the female menstrual cycle |
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No apparent changes were reported in LH B/I ratios in response to bolus i.v. GnRH stimulation (using conventional RIA or IFMA) during the early follicular phase of the menstrual cycle in healthy women (Ding and Huhtaniemi, 1991). During the late follicular phase, the LH B/I ratio increased according to a conventional RIA, but remained unchanged when estimated by IFMA. Opiate-receptor antagonist administration in healthy normally cycling women induced an equivalent elevation in bioactive and immunoreactive LH concentrations with a consequently invariant LH B/I ratio (Ding and Huhtaniemi, 1991
). Replication of this experiment across the menstrual cycle in the same volunteer using an homologous in-vitro LH bioassay and a high-specificity LH immunoassay will be important to corroborate this physiological inference in the female.
We conclude that the precise importance of varying LH micro-heterogeneity within the normal human menstrual cycle is unknown. However, most studies demonstrate strong physiological variations in LH bioactivity and/or isoform distribution in relation to menstrual cycle stage and in the postmenopausal context (Robertson et al., 1979; Lucky et al., 1980a
; Marut et al., 1981
; Dufau et al., 1983
; Reader et al., 1983
; Veldhuis et al., 1984
; Reiter et al., 1987
; Fauser et al., 1992
; Imse et al., 1992
).
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LH isoforms in the menopause |
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In summary, LH bioactivity rises markedly in the ovariprival postmenopausal woman, and exhibits increased isoform acidity due to greater post-translational sialylation and/or sulphation. Oestrogen reverses these changes. The putatively more short-lived alkaline LH isotypes induced by oestrogen replacement might thus contribute to effective suppression of plasma LH concentrations by oestrogen in postmenopausal women by accelerating LH removal (Veldhuis et al., 1987a). How the route of oestrogen delivery might alter these responses is not established. Moreover, how oestrogen-induced changes in endogenous LH isoforms might impact LH-dependent ovarian stromal-cell androgen secretion in the post-climacteric female is not known.
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LH isoforms in PCOS |
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Available data allow the important consideration that the highly oestrogenic milieu in PCOS modulates LH bioactivity (Bogdanove et al., 1971; Lucky et al., 1979
; Tsatsoulis et al., 1990
; Matikainen et al., 1994
). Indeed, short-term oestradiol replacement in postmenopausal women facilitates exogenous GnRH-stimulated release of bioactive LH, by augmenting the so-called self-priming effects of recurrent GnRH drive of gonadotrophes. Thus, in PCOS patients, the ample availability of androgenic substrates for the aromatase enzyme could enhance LH isotype alkalinity indirectly via oestrogen- dependent effects on hypothalamic and/or pituitary sites. In this interpretation, basic LH isoforms in PCOS may represent another oestrogenic feature of this syndrome (Carmina and Lobo, 1999
; Ropelato et al., 1999
). Alternatively, Nagamani and co-workers have suggested that hyperinsulinemia may enhance the secretion of more bioactive LH isoforms in women with hyperthecosis and hyperandrogenism (Nagamani et al., 1999
).
We thus infer that in-vitro estimates of plasma LH bioactivity are elevated in girls and women with PCOS, reflecting the presence of more basic LH isotypes. Whether the molecular heterogeneity of circulating LH isospecies is due to relative hyperoestrogenism and/or hyperinsulinism in PCOS is not known. However, we speculate that the inferentially more rapid in-vivo clearance of basic LH molecules in PCOS offers some protection against the degree of LH elevation, which would otherwise exacerbate hyperandrogenism further.
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LH isoforms in puberty |
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The heterogeneity of gonadotrophin isoforms is appraised by chromatofocusing procedures, which quantify charge variations among molecules (Weise et al., 1983). Thereby, Wide and co-workers identified a shift toward more acidic isoforms of LH (and FSH) during the later stages of puberty in a small number of children with precocious puberty (Wide et al., 1996
). Less conspicuous qualitative changes emerged during female puberty. However, early normal male puberty was marked by a dramatic shift towards more acidic isoforms of both gonadotrophins (Phillips et al., 1997
). This sex difference in isoform estimates may allow for more ready detection of the initiation of puberty in boys. However, the underlying mechanisms regulating the changes in gonadotrophin charge distribution in puberty are unclear, and their precise in-vivo relevance (if any) to sexual maturation in girls and boys is unknown.
Haavisto and co-workers (Haavisto et al., 1990) reported that greater basal and GnRH-stimulated bioactive and immunofluorometric LH release predicted the diagnosis of idiopathic constitutional delay of puberty, while arguing against pathological hypogonadotropic hypogonadism. In other patients with idiopathic hypogonadotropic hypogonadism (IHH), pulsatile GnRH administration for 7 days elevated both bioactive and immunoreactive LH and FSH concentrations.
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Molecular LH variants |
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Recently, a more common genetic variant of the LH protein was discovered in Finland (Pettersson et al., 1992) and subsequently in Japan (Furui et al., 1994
). The genetic LH polymorphism is caused by two discrete point mutations in the LH ß subunit gene, which result in corresponding amino acid alterations: Trp 8
Arg and Ile 15
Thr. The latter substitution introduces an extra N-linked glycosylation signal for oligosaccharide attachment. The variant LH beta allele has a prevalence ranging from 0 to >50% in various ethnic populations (Pettersson et al., 1992
; Furui et al., 1994
; Huhtaniemi et al., 1999
). Variant LH shows increased bioactivity in-vitro, but a reduced half-life in-vivo (Jameson, 1996
). The promoter of the variant LH ß gene also is ~50% more active in transfected cell lines than the wild-type gene (Jiang et al., 1999
). The foregoing differences in LH bio-synthesis, in-vitro action and in-vivo kinetics in subjects who are homo- or heterozygous for the variant LH allele can be associated epidemiologically with delayed puberty (Raivio et al., 1996
), PCOS (Rajkhowa et al., 1995
; Tapanainen et al., 1999
) and subfertility (Ramanujam et al., 2000
). Two studies also reported a higher prevalence of menstrual irregularity in Japanese women homozygous for the variant LH ß allele (Furui et al., 1994
; Suganuma et al., 1995
). Finally, in one analysis, the above variant LH gene type was associated with relatively greater frailty in healthy older men (van den et al., 1999
).
Boys with normal pubertal onset but a variant (homo- or heterozygous) LH genotype have smaller testis volumes, shorter stature, slower linear growth rates, and lower serum IGFBP-3 levels than their peers (Raivio et al., 1996). However, in cohorts of boys with a delayed onset of puberty, the frequency of the variant LH ß allele does not differ from that in the reference population, indicating that existence of the variant LH genotype does not generally influence the timing of pubertal GnRH activation (Raivio et al., 1996
). Based on these studies in pubertal boys, we infer that the polymorphic LH variant may affect testis growth and the somatotropic GH-IGF-1-BP-3 axis in pubertal boys, but not the age of pubertal onset.
Obese patients with PCOS exhibit a lower frequency of variant LH molecules both in Finland and the Netherlands, with a similar trend in the United States (Tapanainen et al., 1999). If these observations are correct, then the variant LH gene type may in part protect obese women from developing symptomatic PCOS. In contrast, Rajkhowa and co-workers in the UK (Rajkhowa et al., 1995
) observed a higher frequency of variant LH isotypes in obese women with PCOS compared with eugonadal controls. Thus, the role of variant LH in PCOS remains to be established.
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Selected technical issues |
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Summary |
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Notes |
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References |
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