Is there any physiological role for gonadotrophin oligosaccharide heterogeneity in humans?

I. Gondatrophins are synthesized and released in multiple molecular forms. A matter of fact

A. Ulloa-Aguirre,1, C. Timossi and J.P. Méndez

Research Unit in Reproductive Medicine and Developmental Biology, Instituto Mexicano del Seguro Social, Mexico City, Mexico


    Abstract
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 Abstract
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 The heterogeneity of...
 The isoforms of pituitary...
 Have the criteria to...
 Acknowledgements
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Carbohydrates attached to the protein core of all glycoprotein hormones play an essential role in the function of the molecule, influencing a number of intracellular and extracellular processes. As with other members of the glycoprotein hormone family, pituitary gonadotrophins are not produced as single or unique molecules but rather as arrays of isoforms that differ from each other mainly in the structure of their oligosaccharide attachments. In both experimental animals and in humans, the abundance of the different isoforms varies depending on the endocrine status of the donor present at the time of collection of the tissue or sample. Conditions characterized by an oestrogen-enriched hormonal milieu (eg. the preovulatory phase of the menstrual cycle) promote the formation and secretion of variants with relatively low sialic acid and/or sulphate content, whereas physiological deficiency of this sex steroid (as in the postmenopause) favours the production of highly sialylated, long-lived gonadotrophin variants. When tested individually, less sialylated isoforms exhibit higher receptor-binding and in-vitro biological activity but shorter plasma half-life than their more sialylated counterparts. Both the hormonal regulation and the functional differences among the naturally occurring isoforms strongly suggest that gonadotrophin heterogeneity represents a distinctly different mechanism through which the pituitary gland may regulate the intensity and duration of the gonadotrophic stimulus. Nevertheless, whereas the existence of the alternatively glycosylated variants of gonadotrophins in both the pituitary and in serum is currently without doubt, the physiological role of this phenomenon is still a controversial issue and a matter of debate.

Key words: glycoprotein hormones/gonadotrophin heterogeneity/oligosaccharides


    Introduction
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 Abstract
 Introduction
 The heterogeneity of...
 The isoforms of pituitary...
 Have the criteria to...
 Acknowledgements
 References
 
LH and FSH are the master anterior pituitary signals that regulate gonadal function. They belong to the glycoprotein family of hormones, which also includes thyrotrophin and chorionic gonadotrophin (CG). All members of this family are heterodimers, formed by a common {alpha}-subunit non-covalently associated with a ß-subunit, which is structurally unique in its peptide sequence to each member of the family (Bousfield et al., 1996Go). The subunits of all glycoprotein hormones contain various asparagine-linked oligosaccharide structures; as in other glycoproteins from multicellular eukaryotes, oligosaccharide structures on glycoprotein hormones are highly variable and play a key role in determining several functional properties of the hormone including metabolic clearance, regulation of potency, subunit assembly and intracellular sorting (Ulloa-Aguirre et al., 1999Go). The wide spectrum in glycosylation, particularly in terminal sialylation and sulphation, constitutes the chemical basis for isoform formation and the extensive charge heterogeneity seen with all these glycoprotein hormones (Baenziger and Green, 1988Go; Ulloa-Aguirre et al., 1995Go). Beginning with pioneering studies (Peckham et al., 1973Go; Bogdanove et al., 1974Go; Peckham and Knobil, 1976aGo,bGo), which, employing gel permeation chromatography, observed different elution patterns of gonadotrophins from pituitaries of monkeys and rats in different endocrine conditions, abundant evidence on the existence of gonadotrophin heterogeneity in the anterior pituitary and in serum has steadily accumulated during the last 25 years.


    The heterogeneity of gonadotrophins in the pituitary and in serum is thoroughly documented
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 Abstract
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 The heterogeneity of...
 The isoforms of pituitary...
 Have the criteria to...
 Acknowledgements
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Multiple charge isoforms of both gonadotrophins have been isolated from anterior pituitary extracts and serum of several animal species, including man (Ulloa-Aguirre et al., 1988Go, 1992Go, 1995Go; Wide, 1992Go; Cooke et al., 1996Go). Separation of human gonadotrophins by charge-based procedures has disclosed the existence of 20 or more charge isoforms of these hormones (Stanton et al., 1996Go; Zambrano et al., 1996Go). Although sialic acid and sulphate residues play a key role in determining the charge heterogeneity of LH and FSH (as well as other glycoprotein hormones), the origin of gonadotrophin polymorphism cannot be attributed fully to the variability in these negatively charged terminal residues, since a large number of different gonadotrophin glycoforms may have the same charge yet a different oligosaccharide composition (Ulloa-Aguirre et al., 1988Go; Creus et al., 1996Go).

That the target tissues within the gonads are exposed to a mixture of circulating gonadotrophin isoforms has also been abundantly documented (Ulloa-Aguirre et al., 1988Go, 1995Go). Several in-vivo and in-vitro studies in experimental animals and in humans have demonstrated that both LH and FSH are secreted in multiple forms. This indicates that detection of intracellular variants is not due to extraction of carbohydrate intermediates resulting from post-translational processing of the glycoproteins, but rather to synthesis of distinct final forms of the complex carbohydrate chains attached to the protein core of the gonadotrophin (Ulloa-Aguirrre et al., 1988, 1995). Further, evidence derived from studies in humans has indicated that the heterogeneous nature of circulating gonadotrophins is not the result of structural alterations during circulation, since the charge distribution of the basally circulating and gonadotrophin releasing hormone (GnRH)-releasable LH and FSH are essentially the same (Zambrano et al., 1995Go; Castro-Fernández et al., 2000).

The early studies mentioned above (Bogdanove et al., 1974Go; Peckham and Knobil, 1976aGo,bGo) also demonstrated that the apparent molecular size of the gonadotrophins could be altered as a result of castration or steroid hormone administration. Studies that followed these early reports, further documented the role of hypothalamic and gonadal factors in determining the changes in relative pH distribution of both pituitary and serum gonadotrophins in experimental animals and in humans (Chappel et al., 1983Go; Ulloa-Aguirre et al., 1988Go, 1995Go). Some of these studies demonstrated that at least in rodents and in humans, oestrogens strongly influence gonadotrophin glycosylation, thus raising the interesting possibility of an endocrine-governed mechanism for precise control of gonadotrophin heterogeneity and potency (Wide, 1985Go, 1989Go; Ulloa-Aguirre et al., 1995Go). In humans, significant variations in relative distribution of intrapituitary and circulating charge isoforms have been documented in several conditions including puberty (Phillips et al., 1997Go), the menstrual cycle (Padmanabhan et al., 1988Go; Wide and Bakos, 1993Go; Zambrano et al., 1995Go; Anobile et al., 1998Go) and senescence (Wide, 1985Go, 1989Go; Anobile et al., 1998Go), thus emphasizing the importance of the prevailing hormonal milieu in determining the type or types of gonadotrophins to be synthesized and secreted in a given time.

Two general concepts were derived from these studies: (i) Gonadotrophins are synthesized and secreted in multiple forms and (ii) Gonadal feedback on the pituitary not only regulates the quantity but also the quality of the gonadotrophin molecules to be secreted in a particular physiological time. May variations in gonadotrophin quality (due to alternative glycosylation) be translated into changes in potency? This obviously requires that the various glycoforms additionally differ from each other in functional properties.


    The isoforms of pituitary gonadotrophins exhibit distinct biological features
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 The heterogeneity of...
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 Have the criteria to...
 Acknowledgements
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It has been shown that more acidic/sialylated pituitary LH and FSH isoforms may exhibit lower receptor binding activity and in-vitro biological potency than the more basic or less acidic counterparts, when such functional parameters are expressed either as the receptor-binding activity/immunoactivity ratio (RRA/I) or the bioactivity/immunoactivity (B/I) ratio (Snyder et al., 1987Go; Ulloa-Aguirre et al., 1988Go, 1995Go; Zambrano et al., 1996Go, 1999Go). Further, less basic and neutral highly purified human LH isoforms exhibit higher in-vitro bioactivities per mass of protein (as determined by amino acid analysis) than the more acidic and basic variants (Stanton et al., 1996Go). In interpreting these observations, one should always have in mind that in-vitro bioassays may be potentially affected by a number of influences, which albeit unrelated to the ligand, may eventually alter the measured outcome. This may be the case, for example, for the heterologous cell assay systems (eg. rodent or bovine), which may differ functionally among species and thus extrapolation of results to human cells would be questionable. In addition, in naturally expressed cell systems the responses to a given gonadotrophin stimulus may be modulated by a number of endocrine, autocrine and paracrine factors, whose influence on the response measured in-vitro is difficult to control and thus it is not usually considered. On the other hand, the use of homologous assay systems bears the problem that the cells employed are not usually provided with all the intracellular machinery that may eventually be involved in a number of signalling pathways activated as the result of a particular ligand-receptor interaction (Gonzalez-Robayna et al., 2000Go). In this vein, measurement of one intermediate product (eg. cAMP) to assess the effect of a given agonist may not always correlate with its effects on the generation of a final product (eg. oestrogens) or in long-term trophic responses. The use of the relationships mentioned above to express the potency of the hormone at the target cell level has also raised some controversy due to the likelihood that gonadotrophin heterogeneity may alter immunoreactivities (Chappel, 1990Go; Tsatsoulis et al., 1991Go; Simoni et al., 1994Go; Stanton et al., 1996Go; Zambrano et al., 1996Go; Robertson et al., 1997Go; Dias, 2001Go). Nevertheless, the finding that the receptor-binding activity and in-vitro bioactivity of highly purified isoforms may vary over a fiveto eight-fold range when expressed on the basis of defined protein content (Stanton et al., 1996Go), further indicates that the reported variations in isoform bioactivity (as expressed by differences in B/I or RRA/I relationships) cannot be simply due to heterogenous immunoreactivity. Moreover, Yding Andersen and colleagues (1999) recently demonstrated that less acidic human FSH isoforms induced resumption of meiosis significantly more efficiently than acidic isoforms regardless of the method employed to determine the concentration of the isoforms tested (radioimmunoassay, radio-receptor assay and in-vitro bioassay) (Yding Andersen et al., 1999Go).

As expected, the more acidic/sialylated variants of the gonadotrophins exhibit the longest plasma half-life life (Wide, 1986Go; Ulloa-Aguirre et al., 1992Go). In addition, some studies have shown that the more acidic forms of FSH also exhibit the highest in-vivo bioactivity when assessed by the classical ovarian weight augmentation tests (Wide and Hobson, 1986Go; Mulders et al., 1997Go). Nevertheless, this assay, which is widely employed to assess the in-vivo effects of FSH preparations from diverse origin (ie. urinary or recombinant), has been also a matter of some controversy. In fact, recent studies employing more sensitive experimental approaches have shown that the in-vivo bioactivity of the naturally occurring pituitary isoforms of both gonadotrophins does not always correlate with their corresponding plasma half-life, thus suggesting that factors other than the metabolic clearance rate, may also influence the net in-vivo biological activity of the hormone. For example, when an acute effect (such as induction of tissue-type plasminogen activator enzyme activity and mRNA expression) was examined, less-sialylated, shorter-lived FSH molecules also exhibited a pronounced and rapid effect (Timossi et al., 1998bGo). In a more recent study, less-sialylated human FSH variants were equally or even more potent than heavily sialylated forms for inducing rat granulosa cell proliferation or preventing follicular atresia over a 6 to 12 h period (Barrios de Tomasi et al., 1999Go, 2001 Go). Likewise, pharmacokinetic studies on human pituitary LH have also suggested that the intrinsic differences in in-vivo bioactivities between its glycosylation variants may be primarily determined to the initial interaction of the hormone with the target cell, rather than to differences in metabolic clearance rate (Burgon et al., 1996Go). Evidence for the effectiveness of short-lived isoforms in-vivo has also been provided by experiments in male monkeys using desialylated and deglycosylated CG (Liu et al., 1989Go). Whether these effects of short-living glycoforms are relevant in physiological or pathological circumstances still remain to be investigated.

On the basis of a large number of studies, including those quoted above, the question of whether relative changes in the synthesis and secretion of the gonadotrophin glycoforms and thus in functional attributes of the mixture of circulating variants actually provide a means to regulate gonadal function, has been the subject of several experimental and clinical studies.


    Have the criteria to assign physiological and clinical importance to gonadotrophin polymorphism been fulfilled?
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 The heterogeneity of...
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Previously proposed criteria should be met in order to assign physiological and clinical importance to gonadotrophin heterogeneity (Padmanabhan et al., 1992Go; Ulloa-Aguirre et al., 1995Go). Some of these criteria include: (i) Isoforms identified in the pituitary should be secreted into the circulation and reach the target site; (ii) Circulating isoforms should be differentially regulated by the endocrine milieu; (iii) The changes in the distribution of pituitary and circulating isohormones during different physiological or clinical conditions should be of sufficient magnitude to alter significantly the net potency of the hormone or alternatively, the functional attributes, rather than potencies, should differ among the various isoforms.

Abundant evidence derived from clinical and experimental studies documents the first two criteria mentioned above and there is no doubt that multiple glycosylation variants of both pituitary gonadotrophins (as well as other glycoprotein hormones) are released from the pituitary gland, some of which may potentially reach the target tissue and interact with their cognate receptor. In addition, the existence of significant sexand age-related variations in the median charge of intrapituitay and circulating gonadotrophins has been abundantly documented; the forms of intrapituitary LH and/or FSH are more acidic/sialylated in males and in older individuals of both sexes (Wide, 1985Go, 1989Go) and circulating gonadotrophins from postmenopausal women are more sialylated than those exhibited by young women (Wide et al., 1992). Further, chronic administration of oestradiol efficiently counteracted the formation of more acidic isoforms of both gonadotrophins after menopause (Wide and Naessén, 1994Go; Wide et al., 1995Go). Several studies have also documented the occurrence of significant changes in charge isoform distribution of both serum LH and FSH throughout the menstrual cycle, with greater proportions of less acidic isoforms being released during midcycle than in the early-, mid-follicular and luteal phases (Padmanabhan et al., 1988Go; Wide and Bakos, 1993Go; Zambrano et al., 1995Go; Anobile et al., 1998Go). The finding that the distribution of circulating isoforms may vary in a significant manner in certain physiological conditions, indicates that gonadotrophin heterogeneity is regulated by hypothalamic inputs (GnRH) and/or by end products from the gonads. Further, the cellular mechanisms whereby gonadal inputs regulate glycosylation of pituitary gonadotrophins have been thoroughly explored (Dharmesh and Baenziger, 1993Go; Damián-Matsumura et al., 1999Go). For example, we have recently shown that the mRNA expression of the pituitary {alpha}-2,3-sialyltransferase (one of the enzymes responsible for the incorporation of terminal sialic acid residues on the oligosaccharide chains of the glycoprotein hormones) changes according to the time and day of the rat oestrous cycle and that the dynamics of these changes in the enzyme mRNA levels correlate with variations in serum oestradiol concentrations (Damián-Matsumura et al., 1999Go). As mentioned earlier, changes in the degree of terminal sulphation and sialylation may potentially alter the circulatory survival of the gonadotrophins and consequently their biological potency in vivo.

Despite new and more recent studies on this issue, evidence supporting the third criteria still remains highly suggestive, but not definitive. Changes in net biological activity of the mixture of circulating gonadotrophin isoforms expressed in terms of in-vitro B/I activity ratio as an index of variations in biological activity, albeit documented by some studies, does not invariably correlate with parallel changes in relative distribution of their corresponding isoforms or with the intrinsic in-vitro biopotency exhibited by the intrapituitary counterparts. Further, in some instances the results yielded by the heterologous or homologous bioassay systems used to quantitate biologically active gonadotrophins in serum have been rather contradictory (see above), thus emphasizing the technical limitations in accurately and unequivocally measuring changes in net biological activity (and immunoreactivity) of gonadotrophins. For example, significant shifts toward the release of less acidic (and presumptively more in-vitro biologically active) FSH and LH isoforms during the late follicular phase and/or mid-cycle of the human menstrual cycle has been consistently documented (Padmanabhan et al., 1988Go; Wide and Bakos, 1993Go; Zambrano et al., 1995Go; Anobile et al., 1998Go). However, whereas some studies employing heterologous systems have detected higher B/I ratios during the early follicular phase or mid-cycle, others have found constant ratios throughout the cycle (Dufau et al., 1976Go; Veldhuis et al., 1984Go; Suginami et al., 1985Go; Jia et al., 1986Go; Padmanabhan et al., 1988Go; Fauser and Hsueh, 1989Go; Fauser et al., 1989Go; Reddi et al., 1990Go). In contrast, studies using homologous assay systems that express the recombinant human FSH receptor have reported higher ratios during the late follicular phase and/or midto late luteal phase of the cycle (Guderman et al., 1994; Christin-Maitre et al., 1996Go). More recently, we have found that the in-vitro B/I ratio of FSH secreted in baseline and GnRH-stimulated conditions is higher during the mid-luteal phase than during the early follicular and mid-cycle phases, as disclosed by a homologous bioassay system (A.Ulloa-Aguirre et al., unpublished data). This observation rules out the possibility of overestimation of low hormone concentrations by our radioimmunoassay as the cause of decreased ratios during the early follicular phase [(Huhtaniemi et al., 1993Go); some other explanations for the apparent discrepancies between the results yielded by different immunoassays and in-vitro bioassays have been extensively discussed elsewhere (Simoni and Nieschlag, 1991Go; Tsatsoulis et al., 1991Go; Dahl and Stone, 1992Go; Jeffcoat, 1993Go; Lambert et al., 1998Go)]. As a corollary, this latter finding also implies that changes in terminal sialylation of FSH cannot be the sole explanation for the increase in in-vitro B/I ratio of the gonadotrophin, since there are not dramatic shifts in the charge-based microheterogeneity of serum FSH either after GnRH administration or during the luteal phase of the menstrual cycle (Zambrano et al., 1995Go).

In postmenopausal women, both bioactive serum LH concentrations (as measured by heterologous in-vitro assays) and the circulatory survival of the gonadotrophin are higher than in cycling women (Fauser et al., 1989Go; Sharpless et al., 1999Go); whereas the latter finding fits perfectly with the changes in charge of serum LH detected across the age, the former somehow contradicts the results showing decreased in-vitro B/I LH ratios of the more acidic/sialylated intrapituitary LH variants. This raises the question of whether intrapituitary and secreted variants are actually the same from the structural point of view. A similar situation occurs with serum FSH across the menstrual cycle, in which the increase in less acidic/sialylated isoforms at midcycle is temporarily accompanied by a marked decrease in the plasma half-life of the gonadotrophin (Zambrano et al., 1995Go). Despite the controversy regarding the real occurrence of changes in net biological potency of the circulating gonadotrophins during some physiological situations, it seems that variations in their circulatory survival, as a consequence of alternative glycosylation, may potentially influence the intensity and time of exposure of the ovary to the gonadotrophic stimulus in vivo. In this vein, identification of clear associations between particular patterns of circulating gonadotrophin isoforms and discrete physiological responses in vivo has been difficult to make because of the pleoiotropic actions of the hormones and the strong influence of autocrine factors on gonadal function. Nevertheless, some abnormal conditions in both men and women, in which the co-existence of changes in serum gonadotrophin charge isoform distribution and secondarily in the plasma half-life (and/or in in-vitro B/I ratio as well) of the hormone may potentially contribute to the impaired function of the reproductive axis have been reported (Ding and Huhtaniemi, 1991Go; Beitins et al., 1993Go; Mitchel et al., 1994; Ropelato et al., 1999Go; Castro-Fernández et al., 2000).

Finally, the possibility that the different gonadotrophin isoforms may exert distinctly different functions on the gonad has been documented by at least four studies (Dahl et al., 1988Go; Padmanabhan et al., 1993Go; Timossi et al., 1998aGo, 2000Go). Basically charged intrapituitary and circulating FSH isoforms with FSH antagonistic effects have been described (Dahl et al., 1988Go; Timossi et al., 1998aGo). More recently, we have found that the various intrapituitary FSH isoforms may exhibit differential effects depending on the end point measured; less acidic isoforms are more potent for stimulating cytochrome P450 aromatase and tissue-type plasminogen activator mRNA expression than the more acidic isoforms, whereas these latter analogs induce {alpha}-inhibin mRNA production with a higher potency than their less acidic counterparts (Timossi et al., 2000Go). Whether the circulating isoforms of this gonadotrophin may also display similar divergent functions at the target cell level remains to be investigated.

In conclusion, the glycosylation-based heterogeneity of pituitary and serum gonadotrophins has been exhaustively documented. A substantial number of experimentallyand clinically-derived observations strongly suggest that the phenomenon of gonadotrophin multiplicity may represent a fine-tuning mechanism through which the pituitary gland regulates gonadal function. Nevertheless, more studies are still necessary for a full understanding of the functional significance of this phenomenon. Undoubtedly, we need to refine our methods for measuring the biological activity of the hormones in vitro and in vivo as well as to develop immunoassay systems that may detect equally all glycosylation variants. Designing of monoclonal antibodies capable of identifying exclusively or at least preferentially a particular variant may also be desirable to help the monitoring of changes or alterations in the circulating concentrations of a given isoform during different physiological and pathological states and to allow the establishment of direct associations between particular isoform patterns and specific physiological responses. At the molecular level, it is necessary to prove experimentally the interesting hypothesis that the gonadotrophin isoforms may act through variant gonadotrophin receptor populations (Ulloa-Aguirre et al., 1995Go; Sairam et al., 1997Go; Babu et al., 2000Go), to corroborate the isoform-specific induction of different and divergent functions in vivo and to characterize the molecular mechanisms subservient to the unique biochemical features of the pituitary signals that govern gonadal function.


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 Abstract
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 The heterogeneity of...
 The isoforms of pituitary...
 Have the criteria to...
 Acknowledgements
 References
 
The Consejo Nacional de Ciencia y Tecnología (CONACyT) and the Fondo para Fomento de la Invetigación (FOFOI)-IMSS, México, have supported aspects of the authors' work in recent years.


    Notes
 
1 To whom correspondence should be addressed at: Research Unit in Reproductive Medicine, Instituto Mexicano del Seguro Social, Apdo. Postal 99–065, Unidad Independencia, Mexico 10101 D.F. E-mail: aulloaa{at}buzon.main.conacyt.mx Back


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