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BOR - Papers in Press, published online ahead of print April 16, 2003.
Biol Reprod 2003, 10.1095/biolreprod.103.016915
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BIOLOGY OF REPRODUCTION 69, 379–389 (2003)
DOI: 10.1095/biolreprod.103.016915
© 2003 by the Society for the Study of Reproduction, Inc.

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Impact of Carbohydrate Heterogeneity in Function of Follicle-Stimulating Hormone: Studies Derived from in Vitro and in Vivo Models1

Alfredo Ulloa-Aguirre2,3, Carlos Timossi3, Jorgelina Barrios-de-Tomasi3,4, Angélica Maldonado3, and Penelope Nayudu4

Research Unit in Reproductive Medicine,3 Hospital de Ginecobstetricia Luis Castelazo Ayala, IMSS, México D.F Gamete Biology Laboratory,4 Department of Reproductive Biology, German Primate Centre, Göttingen, Germany


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 IN VITRO STUDIES
 COMPLEX CELL-TYPE ASSAYS
 IN VIVO STUDIES
 CONCLUDING REMARKS
 REFERENCES
 
Carbohydrates attached to the protein core of glycoprotein hormones influence a number of intracellular and extracellular processes. As with other members of the glycoprotein hormone family, FSH is produced and released as an array of isoforms that differ from each other in the structure of their oligosaccharide attachments. In this review, we discuss how carbohydrate heterogeneity can impact on FSH action in different in vitro and in vivo systems. We present evidence for diverse effects of distinct charge isoforms at the target cell level, including differential and unique effects on various end responses, and discuss how the use of multiple cell-type assays has allowed identification of some specific effects of FSH isoforms on different cell populations and follicle compartments as well as oocyte maturation. Finally, we discuss recent information on the ability of naturally occurring and laboratory manufactured FSH isoforms to evoke particular effects on granulosa cell function and ovarian follicular maturation in vivo. Such studies have provided evidence that the type(s) of FSH signal delivered may in fact regulate distinct biological outcomes irrespective or in addition to outcomes dictated solely by clearance rate differences.

anterior pituitary, follicle-stimulating hormone, follicular development, granulosa cells, pituitary hormones


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
IN VITRO STUDIES
 COMPLEX CELL-TYPE ASSAYS
 IN VIVO STUDIES
 CONCLUDING REMARKS
 REFERENCES
 
Ovulation of developmentally competent oocytes depends on the capacity of the ovaries to produce Graafian follicles in response to endocrine, paracrine, autocrine, and intracrine signals. Folliculogenesis and maturation of fertilizable oocytes is therefore the ultimate result of the coordinated expression of multiple genes controlling an array of functions including cell growth, cytodifferentiation, cell-cycle progression, and apoptosis [1]. The endocrine signals that trigger such effects are the pituitary gonadotropins. In particular, the presence of FSH and a normally functioning receptor are obligatory requisites for folliculogenesis. In fact, in experimental animals and humans, inactivating mutations in the FSH ß-subunit or the FSH receptor gene lead to absence of follicular maturation and profound ovarian failure [2, 3].

As with all members of the glycoprotein family of hormones, FSH is a heterodimer comprised of a common {alpha}-subunit noncovalently associated with a ß-subunit, the latter that is structurally unique in its peptide sequence, to each member of the family [4]. The primary sequence of the human FSH-{alpha} and ß-subunits each encodes two glycosylation sequons located at positions Asn52 and Asn78 in FSH-{alpha} and Asn7 and Asn24 in FSHß-subunit [4, 5]; thus, glycans on FSH are major structural components comprising in excess of 30% of the mass of the gonadotropin [6, 7]. In all glycoprotein hormones, posttranslationally added oligosaccharides play a key role in determining a number of functional properties of the hormone, including {alpha}/ß subunit assembly, intracellular sorting, and metabolic clearance [5, 8]. In addition, these structures are also important in determining receptor-binding affinity and capability of the hormone to activate its receptor and efficiently trigger intracellular signaling [5].

Carbohydrate chain composition and structure among these hormones are highly variable, a feature that depends on the genetic background of the cell in which the hormones are synthesized [9–13]. Moreover, a wide spectrum in glycosylation, and particularly in terminal sialylation and sulfation, is found within a particular glycoprotein hormone [5, 14–16] (Fig. 1). These variations constitute the chemical basis for isoform formation and the extensive charge heterogeneity seen with all glycoprotein hormones [9, 16, 17]. In human (h) FSH, carbohydrate variability includes: 1) degree of complexity in branching [9–11, 18]; 2) presence of hybrid- and high-mannose oligosaccharide types [9, 16, 18]; 3) content in bisecting GlcNAc moieties linked to the core mannose residue [9]; 4) variations in inner fucose and terminal sialic acid residues [9, 11, 16, 18]; and 5) presence of a nonglycosylated FSHß-subunit [19]. Heterogeneity of hFSH is due solely to the carbohydrates, in which nearly 95% of FSH is variably acidic owing to the variable proportion of negatively charged, terminally positioned sialic acid residues [9, 10]. This variability allows the separation of its different isoforms by charge-based procedures [16].



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  		FIG. 1. Some of the N-linked oligosaccharide structures present on ovine, bovine, and human pituitary FSH. Only the complete structures are shown. Many other glycans may be found that are incompletely processed variants of these structures, mainly lacking terminal residues such as sulfate, sialic acid, and fucose. Because CHO cells are not provided with the enzymatic machinery to sialylate glycoproteins in {alpha}2,6 position, recombinant human FSH produced by these particular cells exhibits a lower number of isomers than pituitary- and urine-extracted FSH; in addition, FSH from this cell source does not contain bisecting N-acetyl glucosamine moieties (structure c) and exhibits a limited amount of complex branched carbohydrate structures. Recombinant FSH produced by baculovirus-infected Hi5 insect cells, exhibits a net basic charge because its oligosaccharides are practically unsialylated [5, 11, 12]

It is currently accepted that FSH is synthesized and released in multiple molecular forms so that cells are exposed simultaneously to a mix of a heterogenous population of isoform molecules [5, 16, 17]. We also know with certainty that the relative abundance of intrapituitary and circulating isoforms correlates with the endocrine status of the individual so that a close relationship exists between the presence of a particular isoform pattern within the pituitary and in circulation and the functional state of the gonad [5, 14, 16, 20, 21]. Finally, there is also evidence that isoforms differ from each other not only in their posttranslationally determined carbohydrate composition but also in their ability to bind to target cell receptors, survive in circulation, and provoke a range of biological responses in vitro and in vivo [5, 12–14, 16, 22–26].

Despite the above-mentioned facts, precise information about the physiological significance of FSH heterogeneity at the cellular level has remained elusive. Among the reasons for this knowledge deficit are: 1) the difficulties in interpreting the results derived from in vitro FSH bioassays in which heterologous or homologous (mostly based on cloned receptors) cell assay systems are employed [27, 28]; 2) the unavailability of reliable quantitative methods to measure individual isoforms in blood, which has precluded a clear identification of potential relationships between a given follicular event and selective changes in concentration levels of a particular isoform [29]; and 3) the difficulty in obtaining highly purified isoforms (particularly the less sialylated isoforms, which are the less abundant and more rapidly cleared from the circulation) in quantities sufficient to test them either individually or in different combinations in in vivo systems. Nevertheless, the recent development of multiple end point and multiple cell-type in vitro assays as well as in vivo experimental paradigms has facilitated the unfolding of new insights into the biological effects of the glycosylation variants of FSH that may be potentially useful to unveil the physiological relevance of FSH heterogeneity [5, 26, 28, 30–35]. In this review, we summarize recent data that have emerged from different laboratories, including the authors' own, on the in vitro and in vivo effects of the naturally occurring hFSH isoforms and discuss some of the mechanisms subservient to their particular combination of biological effects, emphasizing their potential role as physiological regulators of follicular function.


   IN VITRO STUDIES
TOP
 ABSTRACT
 INTRODUCTION
 IN VITRO STUDIES
COMPLEX CELL-TYPE ASSAYS
 IN VIVO STUDIES
 CONCLUDING REMARKS
 REFERENCES
 
Single-Cell-Type Assays

The potency and biological effects of the alternatively glycosylated variants of hFSH have been analyzed in vitro employing different heterologous cell assay systems [15, 16, 19, 23, 27]. These studies have revealed that more sialylated isoforms are less potent in provoking androgen aromatization and tissue-type plasminogen activator (tPA) enzyme activity than their less sialylated counterparts [18, 25, 36], although it is not known whether this difference is due to variations in sialylation alone. More recently we analyzed the possibility that the charge variants of hFSH may exert differential and even unique effects on granulosa cell function by monitoring several intermediate and end-products, including cAMP, estrogens, and tPA as well as cytochrome P450 aromatase (P450arom), tPA, and {alpha}-inhibin subunit ({alpha}-inh) mRNA expression [32].

For these studies, hFSH charge isoforms were obtained through high-resolution chromatofocusing of anterior pituitary glycoprotein extracts [36]; this procedure allowed separation of ~20 charge isoforms with elution pH values ranging from >7.0 to <3.80, which were subsequently pooled according to charge into seven hFSH isoform ranges [32]. Cyclic AMP, estrogen, and tPA production by cultured rat granulosa cells exposed to increasing doses of less sialylated FSH isoforms (elution pH values 6.60 to 4.60) was greater than those stimulated with similar doses of more acidic/sialylated isoforms (pH value 4.76 to <3.80) (Fig. 2) [32]. Similar effects were observed on P450arom and tPA mRNA expression (Fig. 3a). In contrast, {alpha}-inh mRNA production progressively increased as the pH of the isoform declined (Fig. 3b). The relatively low production of {alpha}-inh mRNA by the less acidic isoforms was not due to their enhanced ability to stimulate androgen aromatization and estrogen production because addition of the potent estradiol receptor antagonist ICI 182,780 or omission of the aromatization substrate (androstenedione) in the incubations did not significantly modify the isoforms-induced {alpha}-inh mRNA production by cultured rat granulosa cells [32]. Furthermore, the addition of recombinant hFSH or anti-hFSH to granulosa cells cultured in the presence of isoforms with pHs 6.60–6.20 or <=3.80 either increased or decreased, respectively, {alpha}-inh mRNA production stimulated by these isoforms, thus ruling out the possibility that the differential effect of the FSH isoforms on {alpha}-inh mRNA production had been due to non-FSH factors that may unspecifically inhibit or stimulate the production of this particular mRNA [32].



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  		FIG. 2. The ability of increasing doses of human FSH isoforms with elution pH values 5.47–5.10 and 4.76–4.12 to stimulate estrogen production by rat granulosa cells in culture. The isoforms were obtained by high-resolution chromatofocusing (column dimensions 90 x 1.5 cm) of anterior pituitary glycoprotein extracts [36]. Concentrates from these isoforms (with pH values >7.10, 6.60–6.20, 5.47–5.10, 5.06–4.60, 4.76–4.12, 4.05–3.82, and <3.80) were further chromatographed in columns of monoclonal anti-LH-IgG immobilized in Sepharose 4B to remove the LH that coeluted during the chromatofocusing separation. The dose is expressed in terms of human FSH-I-3 (National Hormone and Pituitary Program, Torrance, CA) assuming that the immunological potency is equal for both preparations



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  		FIG. 3. Cytochrome P450arom (a) and {alpha}-inh subunit mRNA expression (b) in cells exposed to different human FSH isoforms (at a dose of 2.8 ng/culture well) for 48 h (P450arom mRNA) or 24 h ({alpha}-inh mRNA). Upper panels: representative Northern blot hybridization analysis of P450arom and {alpha}-inh mRNA production in response to FSH exposure. +, positive control (ovarian tissue from 18-day pregnant rats); - and C, negative controls (granulosa cells cultured in the absence of FSH). Lower panels: relative optical densities (2.6 kb P450arom or {alpha}-inh mRNA/18S rRNA optical density ratio) of the autoradiographs shown above. (Adapted from ref. 32, with permission from The Society for Endocrinology, UK)

Interestingly, in contrast to isoforms with pH 6.60 to <=3.80, which behaved as FSH agonists in this heterologous primary cell culture system, the least acidic isoform (pH >=7.10) exhibited dose-dependent inhibitory effects on FSH action (Fig. 4) [30, 32]. Although this particular isoform was able to partially potentiate the effects of recombinant FSH on cAMP production, it profoundly inhibited estrogen formation and tPA enzyme activity (Fig. 4, a and b) and efficiently blocked P450arom and tPA mRNA expression (Fig. 4c) [30, 32].



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  		FIG. 4. a) Changes in concentrations of estrogens released into the culture medium of granulosa cells culture in the presence of 1.4–2.1 ng/culture well human FSH isoform with pH >7.10, 2.7 ng/culture well rec/hFSH produced by CHO cells or 0.5 mM dibutyryl cAMP (DBcAMP). Inset: Total estrogens released into the culture medium of granulosa cells incubated in the presence of DBcAMP and a strongly acidic hFSH isoform (pH <3.80). b) Detection of tPA enzyme activity in conditioned media from granulosa cells exposed for 48 h to rec/hFSH (5.3 ng/well) and DB-cAMP (0.5 mM) in the presence or absence of a basically charged hFSH isoform (pH >7.10, 2.8 ng/well) or a highly sialylated isoform (pH <3.80, 1.4 ng/well). The location of tPA (Mr ~70 000) and high-molecular-weight (Mr ~50 000) urokinase-type plasminogen activator (uPA) activities are noted. C, control incubations with no hormones or DBcAMP added. Note the selective effect of the hFSH isoform with pH >7.10 on the inhibition of DBcAMP- and rec/hFSH-evoked tPA enzyme activity (adapted from ref. 30 with permission from Karger AG, Basel). c) Reverse transcription-polymerase chain reaction of cytochrome P450arom mRNA exposed to 2.8 ng/culture well rec/hFSH, hFSH isoform with pH >7.10 or rec/hFSH plus hFSH isoform with pH >7.10. Std, Molecular mass standards; C, control incubations in the absence of FSH. (Adapted from ref. 32, with permission from The Society for Endocrinology, UK)

The mechanisms subservient to this variety of responses to hFSH isoforms are not yet known. One candidate mechanism is the variability in receptor affinity. In this regard, we found competitive displacement curves of 125I-labeled hFSH by the hFSH isoforms in both rat granulosa cells and seminiferous tubule homogenates, with receptor-binding activities that decreased as the elution pH of the corresponding isoform declined [37]. Furthermore, in both heterologous cell receptor assays, a significant inverse relationship was detected between the median elution pH value of the isoforms and their corresponding dissociation constant (Kd)[37]. However, in homologous cell assay systems, not all isoform fractions differ in Kd (e.g., the less sialylated isoforms versus the more acidic-sialylated isoforms, as discussed below). Accordingly, a more complex model introduces the concept that alternative downstream pathways are triggered by particular glycosylation variants of the gonadotropin, independent of receptor-binding affinity. In this model, the same receptor or its membrane-expressed variants [38, 39] may evoke a spectrum of responses reflected in activation/inhibition of various intracellular signaling pathways [39, 40].

This raises the interesting possibility for isoform-specific, oligosaccharide-determined stimulation of different functions. A number of observations support this latter mechanism. First, in homologous, single-cell assay systems (i.e., human embryonic kidney-derived 293 cells, expressing a unique form of FSH receptor population), less sialylated isoforms more effectively triggered Gs-mediated intracellular signaling than more acidic/sialylated analogs. This difference occurred despite both having the same affinity for the cloned receptor [37]. In addition, fucose-enriched thyrotropin (TSH) activates both cAMP and inositol phosphate (Gq/11 protein dependent in the activated TSH receptor system) intracellular signaling pathways, whereas the fucose-depleted analog stimulates only the Gs-mediated pathway [41]. This result introduces the possibility that fucose residues may play a role in the selective stimulation of specific G proteins. Lastly, chemically deglycosylated pituitary hFSH or insect cell-expressed (high-mannose) recombinant hFSH appear to selectively activate G{alpha}i, whereas intact hFSH does not activate this particular G protein primarily [42]. Thus, it appears that FSH glycoforms may have variable capacities to evoke signal transduction and that in addition to their net effects on metabolic clearance rate and in vivo bioactivity of the hormone (see below), they may function as selective stimulators or inhibitors of particular pathways. The changing abundance of different isoforms exerting antagonistic or diverse effects throughout the cycle may be critical for a more precise regulation of the gonadal response to the gonadotropic stimulus in vivo. A particular example may be the significant increase in secretion of less acidic isoforms during puberty and the periovulatory period, which have been well documented [16, 27, 43–46].


   COMPLEX CELL-TYPE ASSAYS
TOP
 ABSTRACT
 INTRODUCTION
 IN VITRO STUDIES
 COMPLEX CELL-TYPE ASSAYS
IN VIVO STUDIES
 CONCLUDING REMARKS
 REFERENCES
 
The biological effects of the different hFSH isoforms have additionally been studied using heterologous multiple cell-type assay systems. Such systems have allowed identification of some specific effects of FSH isoforms on different cell populations and follicle compartments. Results from these studies have revealed important aspects concerning the divergent but complementary actions of FSH glycoforms on follicle development and function.

Cumulus-Oocyte Complex Assay

The effects of several hFSH glycoforms on cumulus cells (which represent a specific specialized form of granulosa cells), and cumulus-enclosed oocytes have been explored by Yding Andersen et al. in a series of carefully performed studies [31, 34, 35]. In this assay system, cumulus-oocyte complexes (COCs) from pregnant mare's serum/human gonadotropin-treated mice are isolated by manual dissection of large preovulatory follicles and immediately exposed to experimental conditions; FSH but not LH provokes oocyte maturation of COC, whereas naked oocytes are unaffected by FSH [47, 48]. Oocytes are prevented from spontaneous maturation by incubation with hypoxanthine, which blocks cAMP degradation [49]. Exposure to different FSH isoform admixtures during 24 h of culture revealed that at physiological doses (~6 IU/L) both less acidic/sialylated (pH 6.4–5.7) and midacidic (pH 5.6–5.0) isoform mixtures induced oocyte resumption of meiosis more effectively than the highly sialylated fractions (pH 4.7–3.8) [31]. In fact, COCs exposed to the more acidic isoform mixture required more than twice the physiological dose of FSH to allow half of the oocytes to undergo maturation.

In subsequent time-course studies, these investigators found that COCs from preovulatory follicles respond to different hFSH isoforms with a rapid and immediate production of cAMP; less and midacidic isoforms were almost twice as effective as the more acidic fractions to evoke cAMP production by COCs exposed either continuously or intermittently to the FSH isoforms [34, 35]. For less acidic and midacidic isoforms, maximal cAMP accumulation in COCs occurred rapidly, after 15–30 min of exposure, a period of time that is within the plasma half-life reported for these isoforms [15, 34]. Thus, it is conceivable that the consistently documented increase in less acidic glycoforms occurring during the late follicular and preovulatory phases of the menstrual cycle [43–46] may play a role on the coordinated function between the oocyte and companion somatic cells. In this setting, the absence of ß-subunit oligosaccharides in the less acidic isoforms [19] could conceivably facilitate delivery of these particular FSH molecules from the follicle capillaries to the interior of the large, preovulatory follicle, favoring their interaction with the cumulus cells and oocyte.

Intact Isolated Follicle Culture

By contrast to other cell culture assays, intact follicle culture is the only in vitro system that can simulate, with any degree of accuracy, the sequential events of in vivo follicle development, allowing the evaluation of short- and long-term responses to a given stimulus on several functional and developmental aspects of target cells (including the oocyte itself) in their in situ arrangements with each other [28, 33, 50]. This system employs late primary-early secondary-preantral intact follicles from juvenile (<=21 days) mice, whose structure is maintained and remains intact and unattached for the whole culture period (up to 5 days), developing into an antral follicle (Fig. 5). Follicles in this developmental stage do not require LH and are particularly sensitive to and dependent on FSH for their rapid growth [33].



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  		FIG. 5. The appearance of isolated mouse follicles after culture with pituitary hFSH isoforms with pH values 5.62–4.96, 5.31–4.63, and 4.69–3.75 individually at their antral threshold doses, compared with the appearance of a follicle cultured with a 1:1 mixture of hFSH isoforms with pH 5.62–4.96 and 4.69–3.75 and a follicle from a mouse at the proestrus day of the cycle. Note the dramatically improved clarity of the somatic cell organization as well as the more normal appearance of the oocyte in the follicle exposed to both hFSH glycoforms. Particularly noticeable are the changes in the definition of the follicle wall region and the cells in this region of the follicle. (Adapted from ref. 28, with permission from Reproductive Healthcare Ltd., Cambridge, UK)

This isolated follicle culture system has been employed by Nayudu et al. [28] and Vitt et al. [51] to dissect the effects of charge isoforms derived from Chinese hamster ovary (CHO) cells [recombinant (rec) hFSH whose isoelectric point (pI) is relatively narrow (5.3–3.5) [17]] and from human pituitaries. They found that the functional and developmental fate of follicles exposed to different rec/hFSH isoforms differed, depending on the isoform and dose to which they were exposed during the 5-day incubation period. At antral threshold doses (i.e., the dose at which antral development is triggered), the less acidic fraction (pI 5.6–5.0) induced a high percentage (>70%) of rapidly and continuously growing (>=40 µm/day) antral follicles [28, 51]. More specifically, follicles exposed to this particular isoform showed: 1) inner follicle growth during the first 24 h of culture; 2) increase in growth rate during Days 2 or 3 of the culture at the expense of an exaggerated rise in granulosa cell number at the late preantral stage; 3) early estradiol production (after only 2 days of exposure); and 4) attainment of a large end-size after 5 days of culture [51]. Interestingly, high (supraoptimal) doses of this isoform had marked negative effects on the somatic cells and the oocyte of antral follicles, including overproliferation of the tightly packed mural granulosa cells, absence of the more loosely packed antral granulosa cells, and signs of oocyte degeneration [28, 52]. Overall, the effects provoked by this isoform on isolated follicle development suggest that its primary role may be to promote the development of mid- and late preantral follicles in preparation for antral development.

Compared with the effects observed in response to the less acidic isoform, follicles treated with its more acidic counterpart (pI 4.6–3.6) required significantly higher threshold doses (50 ng/ml vs. 1.5–2.5 ng/ml for the less acidic fraction), exhibited slower growth rate and reduced efficiency to evoke antral formation, and showed delayed and reduced estradiol production [27, 46]. In addition, with acid FSH, a marked difference from the other isoforms, including a midacidic (pI 5.0–4.5) isoform, was that indications of reduction in oocyte quality were coincident with antral formation [28, 52]. Interestingly, unfractionated rec/hFSH incorporates features of all the three isoforms, with a threshold dose slightly higher than that of the less acidic isoform (2.5 ng/ml) and a dose tolerance (the concentration between the threshold dose and the maximal dose) encompassing nearly the entire range of the isoforms tested [28]. This suggests that the more extreme fractions (less acidic and acidic), which are only minor components of the complete range of rec/hFSH [16, 28], may have effects well in excess of their actual concentrations in the unfractionated preparation and that the pattern of follicle development can be influenced even by minor changes in the proportional balance of isoforms.

In agreement with the findings for isolated COCs described above, the timing and effectiveness of acquisition of full oocyte maturation (metaphase II) capacity during in vitro follicle culture also differed, depending on which of the two extreme charge rec/hFSH isoforms were used. With the less acidic fraction, the oocytes already achieved their highest maturation capacity (over 80%) by the third day of culture, regardless of antral status, a result that was consistent with the accelerated growth and differentiation of follicles exposed to this particular isoform [28, 52]. Thereafter the capacity for maturation reduced slightly with time. A more extreme relationship with time existed for the antral follicles induced by acid fraction. On the third day, the oocytes from the small proportion of follicles that were antral were as maturable as those treated with the less acidic fraction, but by the fifth day of culture, the capacity for maturation was drastically reduced [28, 52]. This contrasted with the situation for nonantral follicles, in which on the third day, the oocyte maturation capacity was very low and thereafter the capacity for maturation increased, reaching 80% after 5 days of culture.

The effects of rec/hFSH isoform dose and follicle culture duration on the capacity of oocytes to acquire embryonic development has been additionally evaluated [52]. At threshold doses, the major difference between the less acidic and acid FSH isoform fractions was the timing and effectiveness of acquisition of two-cell embryonic developmental capacity. With the less acidic isoform, the highest rate of two-cell development (~80%) occurred after 3 days of exposure and only at the threshold dose, whereas under exposure to the acidic isoform, this rate was lower (~60%), occurred later (after 5 days of culture), and was present at equivalent rates over a range of doses between 10 ng/ml and 100 ng/ml. For either isoform, at threshold doses or below, the capacity for two-cell embryo production was not influenced by antral status. However, above the threshold dose, exposure to the least acidic isoform resulted in an increased proportion of antral follicles associated with a progressive decrease in embryo production [28, 52]. The more acidic isoform was by comparison less bioactive and less sensitive to overdosing.

The results from these studies with rec/hFSH isoforms suggest that a balance among isoforms is required to prevent negative effects and achieve optimal follicle development and oocyte maturation. This possibility has been further investigated using naturally occurring hFSH charge isoforms. The authors' laboratories have carried out mixing experiments to examine the effects of pituitary-derived hFSH isoforms [28]. Three partially purified fractions (elution pH values 5.62–4.96, 5.31–4.63, and 4.69–3.75) within the pI range of the rec/hFSH isoforms were studied to allow the possibility of comparison between the two FSH sources. The most striking difference from previous studies employing rec/hFSH isoforms was that at threshold doses, all three isoforms individually induced follicle development that lacked clear organization, particularly in the region of the basal membrane and theca (Fig. 5). Additionally, although the granulosa cells had proliferated, they appeared not to be regionally differentiated. Furthermore, the cumulus oocyte complex appeared abnormal, and antral formation was poor and associated with metachromatic staining of the follicular fluid, suggesting an abnormal follicular fluid composition. The reason for the morphological differences evoked by the natural hFSH and the rec/hFSH charge isoforms is not yet clear.

Because the recombinant FSH fractions are more restricted in their pI range than the naturally produced isoforms, it may be postulated that the basis for the effect may lie in the differential composition of the oligosaccharide chains. In fact, naturally produced less acidic isoforms exhibit a greater affinity for concanavalin A and therefore have a greater component of mannose [17]. These neutral sugar differences may be a source of hidden variance within isoform fractions separated on charge-based differences [16]. Furthermore, recombinant FSH produced in CHO cells lacks bisecting N-acetyl glucosamine residues, the role of which is uncertain in determining the biochemical properties of the gonadotropin. Minor differences in effects between naturally occurring glycoforms and rec/hFSH isoforms produced by human cell lines (e.g., human embryonic kidney-derived 293 cells) is what should be expected considering that the pH distribution of the latter is quite similar to that exhibited by their pituitary hFSH counterparts [13, 16].

The effects of antral threshold doses of the two more extreme pI isoforms (pH 5.62–4.96 and 4.69–3.75) combined at a 1:1 ratio were next examined. This produced a remarkable effect, restoring the apparently normal follicle organization as well as improving growth rate (Fig. 5) [28]. The basal membrane region and associated cells, both granulosa and theca, were clearly and more appropriately organized, regional differentiation of the granulosa cell layers was evident, and the cumulus oocyte complex improved. This organizational change strongly suggests that the mixture provides a more balanced stimulus for normal cell communication and differentiation. Thus, it is possible that a balance among variant gonadotropin forms, perhaps shifting over the progressive growth of the follicle, may be critical in determining the developmental pattern and ultimately the fate of the follicle and the oocyte in vivo. Further investigations are underway to clarify this possibility.


   IN VIVO STUDIES
TOP
 ABSTRACT
 INTRODUCTION
 IN VITRO STUDIES
 COMPLEX CELL-TYPE ASSAYS
 IN VIVO STUDIES
CONCLUDING REMARKS
 REFERENCES
 
There is compelling evidence showing that oligosaccharide residues in glycoprotein hormones play a pivotal role in determining the plasma half-life and consequently the in vivo bioactivity of the secreted hormone [5, 16, 53–58]. As mentioned previously, nearly 95% of hFSH is acidic owing to the negatively charged, terminally positioned sialic acid residues; this sugar content and particularly the number of exposed galactose residues play an essential role in determining the survival of the gonadotropin in the circulation [53, 54, 57]. Exposure of terminal galactose residues on FSH oligosaccharides dramatically increases their rate of clearance from plasma through a mechanism involving hepatocyte receptors for the asialo galactose-terminated complex molecules [53]. In addition, the presence of oligosaccharides bearing terminal mannose or N-acetylglucosamine also accelerates clearance of the molecule by specific receptors present in hepatic endothelial and Kupffer cells [56]. As a consequence, heavily sialylated FSH glycoforms circulate for longer times than their less acidic/sialylated counterparts [8, 15, 16, 22] (Fig. 6); this differential clearance among the various isoforms is what ultimately determines the plasma half-life of the circulating FSH isoform mix. In fact, it has been documented that the increased release of less sialylated FSH isoforms occurring during the preovulatory phase of the human menstrual cycle [43–46] correlates with a significant reduction in the plasma half-life of FSH secreted during this cycle phase [45].



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  		FIG. 6. The effects of hFSH isoforms with pH values 5.62–4.96 (I), 5.31–4.63 (II), 5.08–4.27 (III), and <3.75 (V), and rec/hFSH (F) administration in proliferation of 50–199 µm (excluding primordial follicles) and 200–400 µm preantral and antral follicles granulosa cells from hypophysectomized rats. Immature (21 days old) rats were hypophysectomized, and 6 h later they received a single s.c. injection (at doses equivalent to 8 IU rec/hFSH as assessed by an FSH bioassay procedure [36]) of one isoform, rec/hFSH, or vehicle (saline). Five, 11, or 17 h post injection, rats were treated with [3H]-thymidine (2.5 µCi/g body weight) and killed 1 h later. Ovaries were dissected and processed for histological and autoradiographic examination. Every 10th section from each ovary was examined (18 total sections examined/ovary) and the number of labeled granulosa cells was recorded by two independent investigators. Only follicles showing both oocyte and nucleolus were considered. The hFSH isoforms were obtained by preparative chromatofocusing (column dimensions 50 x 1 cm) and affinity chromatography [31]. Different letters above bars indicate the existence of significant (P < 0.05) differences between the treatment groups within the same period of time. The inset shows the plasma disappearance curves of FSH present in isoforms I, II, III, and V. Values in parentheses indicate the elution pH value of each isoform. (Adapted from ref. 25, with permission from Elsevier Science Ireland Ltd)

A possibility existed that the enhanced capability of the less acidic hFSH variants to evoke a biological effect at the target cell level (see above) may effectively compensate for the drawback imposed by their relatively shorter plasma half-life. This has been experimentally explored employing end points more specific than those traditionally used to assess the biological potency of this particular gonadotropin in vivo (i.e., the increase in ovarian weight provoked by repeated administration of the hormone) [23, 24]. In the first study, we analyzed the acute induction of tPA enzyme activity and mRNA expression in ovaries from phenobarbital-blocked proestrus rats treated with comparable doses of either CHO cell-produced rec/hFSH (less acidic; biological to immunological ratio 2.47 ± 0.21, plasma clearance rate 0.34 ± 0.09 ml/min) or highly purified urinary hFSH (more acidic; biological to immunological ratio 1.99 ± 0.03, plasma clearance rate 0.14 ± 0.01 ml/min) [59]. Although both preparations exhibited similar potencies, maximal tPA enzyme activity and mRNA responses occurred earlier after administration of the less acidic hFSH compound [59]. This finding is consistent with the observed differences in timing between less and more acidic hFSH isoforms to trigger and evoke maximal estradiol production by cultured follicles [28, 51].

In a more recent study [25], we analyzed the effects of pituitary hFSH isoform fractions (elution pH values 5.62–4.96, 5.31–4.63, 5.08–4.27, and <3.75) devoid of LH activity on granulosa cell proliferation in immature hypophysectomized rats. The effects of isoform treatment on granulosa cell proliferation in preantral, early antral, and antral follicles measuring 50–199 µm (excluding primordial follicles) and 200–240 µm in largest diameter, are shown in Fig. 6. Less acidic isoforms (pH 5.62–4.27) were equally or even more efficient than the more acidic counterpart in maintaining granulosa cell proliferation immediately after hypophysectomy. The effect of the less acidic isoform fraction (pH 5.62–4.96) was more pronounced on preantral follicles measuring 50–199 µm in diameter, in which the rate of cell proliferation after 6 and 12 h of exposure to this shorter-lived, less sialylated isoform was higher than that exhibited by follicles stimulated with the more acidic isoform fraction (pH <3.75) [25]. These observations are important from the physiological point of view, particularly considering that a number of FSH variants (including the less sialylated glycoforms) identified within the pituitary are also released to the circulation [43–46] and that the FSH receptor requires only nanomolar concentrations of the ligand to become activated. Even a brief exposure of the target cell to short-lived but highly potent gonadotropin forms may be sufficient to evoke a biological response. Thus, factors other than plasma half-life (including receptor-binding affinity and capability of the ligand to activate its receptor and efficiently trigger intracellular signaling) play an important role in determining the net in vivo effects of a given FSH variant.

As mentioned above, a more detailed analysis of the in vivo effects of naturally occurring hFSH variants has been hampered by the difficulty in obtaining highly purified pituitary FSH isoforms (particularly the less sialylated isoforms) in large quantities. To overcome this limitation, West et al. [26] produced a less acidic mix of isoforms by partial neuraminidase digestion of a highly purified ovine FSH preparation (primarily conformed by a predominantly acidic mix of isoforms) and administered equivalent molar concentrations of either mix, at 2-h intervals during the first 24 h and then hourly during an additional 24-h period, to GnRH antagonist-, LH-treated female prepuberal lambs. In this particular experimental paradigm, the acidic mix more efficiently stimulated estradiol production and growth of estrogenic follicles than the less acidic mix. The serum immunoreactive FSH levels in the group treated with the acidic mix increased progressively from the time of administration, whereas the levels in the group treated with the less acidic preparation remained unchanged throughout the course of treatment, illustrating the effects of the differential rates in clearance of each isoform mix on the circulating levels and in vivo bioactivity of the gonadotropin.

Although these interesting observations may be potentially useful for the design of therapeutic strategies oriented to obtain better control of ovarian hyperstimulation, the results are, however, more difficult to extrapolate to what actually occurs in physiological puberty and ovulatory cycles, conditions in which the FSH signal is far more complex: 1) enzymatically derived, laboratory-manufactured, less acidic isoforms are not true replicates of their naturally occurring counterparts, whose processing within the gonadotroph and in circulation involves more than omission or removal of sialic acid residues [5, 9]; 2) during puberty and the transition from early to late follicular phase, a progressive increase in less acidic isoforms occurs so that the gonad receives a gonadotropin signal containing both less acidic and acidic isoforms [43–46, 60–62]; and 3) the progressive shift from more acidic to less acidic, shorter-lived FSH glycoforms that occurs during the mid- to late follicular phase of the cycle [39–42] is tightly controlled by the increasing concentrations of estradiol [5, 21, 45, 63, 64], an ovarian-gonadotroph dialogue interrupted during GnRH antagonist treatment. In this regard, we have found that estradiol selectively regulates the expression levels of pituitary {alpha}2,3-sialyltransferase mRNA in vivo [64, 65]. In the rat, the lowest expression levels of this enzyme mRNA are found during the morning of the proestrus and estrus days and the highest in the morning of diestrus 1 (Fig. 7); thereafter the levels progressively decrease during the afternoon and evening of diestrus 1 and the morning of diestrus 2 [64]. Furthermore, early administration of a potent estradiol receptor antagonist on diestrus 1 completely reverted the time-dependent decrease in {alpha}2,3-sialyltransferase mRNA levels, whereas estradiol benzoate administered on the estrus day abolished the physiological increase in the enzyme mRNA levels [64, 65]. Thus, regulation of the structure and biological properties of FSH by ovarian products apparently represents an important feedback mechanism by which the target cell regulates the duration and intensity of the trophic signal released from the gonadotroph.



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  		FIG. 7. Changes in 2,3-sialyltransferase (ST3N) mRNA expression throughout the rat estrous cycle as disclosed by in situ hybridization (left) and Northern blot hybridization analyses (right bottom). Anterior pituitary glands were obtained on the morning (1000 h) of the estrous (E), diestrus 1 (D1), diestrus 2 (D2), and proestrus (P) days (n = 5 rats/cycle day). In situ hybridization (ISH) was performed employing a 346-bp digoxigenin-labeled cDNA probe encoding for a nonconserved sequence of the catalytic domain of the enzyme. Hybridization was detected by adding an antidigoxigenin antibody conjugated to alkaline phosphatase, followed by color development with the chromogenic substrate nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate. Slides were slightly counterstained with nuclear fast red. Specific localization of the ST3N mRNA in gonadotrophs was performed by immunohistochemistry of sections adjacent to the ISH sections employing an anti-FSH antiserum (not shown). Ten sections per gland were examined by three independent investigators. The ST3N mRNA signal increased by 160% and 108% in sections from D1 and D2 animals, respectively. Omission of digoxigenin-labeled antisense cDNA probe or use of digoxigenin-labeled sense cDNA probe, yielded no significant labeling. *, P < 0.05 vs. E, D2, and P; **, P < 0.05 vs. E and P. Original magnification: x400


   CONCLUDING REMARKS
TOP
 ABSTRACT
 INTRODUCTION
 IN VITRO STUDIES
 COMPLEX CELL-TYPE ASSAYS
 IN VIVO STUDIES
 CONCLUDING REMARKS
REFERENCES
 
FSH-mediated signaling is a complex process involving variant receptor populations [38, 39]; a variety of intracellular signaling pathways [40, 42, 66, 67]; and a number of polypeptide growth and differentiation factors, steroids, and other extracellular substances that modulate gonadotropin action [68]. In this review we presented experimental evidence that the molecular variants of FSH may exert a variety of effects, adding more complexity to the already intricate mechanisms whereby this gonadotropin regulates gonadal function. An unanswered question is how a variety of isoforms presumptively coupled to the same intracellular effector(s) may evoke such a variety of divergent responses. In this review we described some potential mechanisms that may account for these pleiotropic effects. One attractive possibility is that, depending on its particular oligosaccharide structure, FSH may provoke different hormone-receptor complex conformations enabling the receptor to activate alternate effectors and signaling cascades. Alternatively, glycoforms may bind differentially to variant receptor populations, in turn coupled to alternate signal transduction pathways [69, 70]. Whatever the mechanism, the biological features of the FSH glycoforms described herein indicate that normal follicle development and oocyte maturation require an appropriate balance among variant gonadotropic glycoforms. They also suggest that the changing abundance of different isoforms exerting antagonistic or diverse effects throughout the cycle and on different follicle stages may thereby orchestrate a symphony of shifting effects that serve to refine the regulation of the ovarian response to the gonadotropic stimulus.


   FOOTNOTES
 
1 Supported by grants from the Consejo Nacional de Ciencia y Tecnología (CONACyT; grant 38056M and 38074M) to A.U.-A. and C.T., and from Serono, Organon, Zebet (1328-134 bgvv) and BMBF (grant 01 GB 9601/2) to P.N. J.B.T. is a postgraduate student supported by the CONACyT and Fondo para el Fomento de la Investigación (FOFOI)-Instituto Mexicano del Seguro Social, Mexico. Back

2 Correspondence: Alfredo Ulloa-Aguirre, P.O. Box 99-065, Unidad Independencia, IMSS, C.P. 10101, México D.F. FAX: 52 55 5616 2278; aulloaa{at}servidor.unam.mx Back

Received: 5 March 2003.

First decision: 2 April 2003.

Accepted: 10 April 2003.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 IN VITRO STUDIES
 COMPLEX CELL-TYPE ASSAYS
 IN VIVO STUDIES
 CONCLUDING REMARKS
 REFERENCES
 

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