FSH Glycosylation Research Articles

Determining the impact of FSH glycosylation variants on the pre-antral follicle transcriptome in the ageing ovary

Searchable abstracts of presentations at key conferences in endocrinology ISSN 1470-3947 (print) | ISSN 1479-6848 (online)

FSH glycosylation variants differentially modulate FSHR trafficking

FSH glycosylation variants differentially modulate FSHR trafficking

P–661 Comparative assessment of the structural features of human follicle-stimulating hormone in products from multiple markets

Abstract Study question The aim of the study is to explore the structural differences occurring in recombinant human follicle-stimulating hormone alfa (r-hFSH- α), originator and its biosimilars, from various countries. Summary answer When compared with r-hFSH-α originator (Gonal-f), its biosimilars presented structural differences, namely Primapur showed a significant different glycosylation profile. What is known already FSH is part of cystine knot growth factor superfamily and plays a central role in reproduction, as FSH stimulates follicular development and estrogen synthesis. R-hFSH- α is commonly used in assisted reproductive technologies to achieve multifollicular development. At the present r-hFSH- α biosimilars are available in Europe and other regions. R-hFSH-α is a complex glycoprotein, that possesses several structural features critical for its efficacy and safety1–2. Glycosylation profile is one of the most impactful attributes of the molecule defining a moiety of FSH isoforms with impact on its biological net effect3. Efficacy and safety of r-hFSH- α are strictly correlated with glycoforms’ composition3–8. Study design, size, duration At least two different batches of each r-hFSH- α originator and its biosimilars have been included in the study. Participants/materials, setting, methods The structural features of products from six different marketed r-hFSH α (Gonal-f, Primapur, Folisurge Intas, Corneumon, Jin Sai Heng, Follitrope LG) have been investigated with a variety of analytical techniques in order to evaluate the presence of molecular differences, which could have a severe impact on the efficacy and safety of the product. The attributes which have been investigated in-depth include primary, secondary and tertiary structure as well as post-translational modifications (PTMs), including glycosylation and contaminants. Main results and the role of chance All r-hFSH- α biosimilars analyzed presented differences compared to the originator. We firstly investigated Primapur and found significant differences regarding multiple structural attributes, particularly in the glycosylation profile. Gonal-f exhibited lower glycan branching, expressed by an A-index* of 2.5, while Primapur showed an A-index of 2.4. Furthermore, Primapur showed a lower level of sialylation in comparison with Gonal-f, as measured by their respective S-index* of 1.8 and 2.1. FSH glycosylation exhibits both macroheterogeneity and microheterogeneity, impacting both FSH protein’s half-life and affinity with the follicle-stimulating hormone receptor (FSHR). Antennarity, representing FSH microheterogeneity, influence r-hFSH-α activity since it has been shown that bulky and extended glycans may take longer to fit into the FSHR cavity compared to less sterically hindering glycans, resulting in a delayed response 7,8.Additionaly, sialylation has been shown in-vivo to correlate with plasma half-life and effect on granulosa cells proliferation 1,2,3. The slower clearance of highly sialylated r-hFSH has been shown to lead to a higher in-vivo activity, despite the lower in-vitro bioactivity 1,2,3. *A-index and S-index express respectively a measure of the number of antennae and sialic acid per glycan. Final values are generated from many relative abundances normalized to 100, highlighting the significance of small numerical differences. Limitations, reasons for caution More batches should be tested for each product. The authors are presenting full characterization of only one of the biosimilars since the rest of the products are under characterization. Wider implications of the findings: r-hFSH-α originator and its biosimilar showed differences in terms of glycosylation profile that is well known as the major protein characteristic impacting FSH activity as extensively demonstrated in in-vivo and in-vitro models. This structural difference could have impact also on product efficacy and safety. Trial registration number ‘not applicable’

New Human Follitropin Preparations: How Glycan Structural Differences May Affect Biochemical and Biological Function and Clinical Effect.

It is well accepted that pituitary follitropin is secreted into the circulation as a mixture of variants, which differ not in primary structure but rather at the level of glycosylation. These glycosidic forms vary in the number of glycosylation sites filled, complexity of glycosidic chains, and sialylation and sulfation. It is generally agreed that high sialylation, 2,3 sialic acid capping of terminal N-acetyl galactosamine or galactose leads to longer circulating half-life, by blocking binding of asialoglycoprotein receptor (ASGPR) in the liver. In contrast, 2,6 sialic acid found in humans does not prevent recognition of galactose and N-acetyl galactosamine by ASGPR. Few studies on clinical outcomes comparing differences in sialylation of follitropin found in commercially available preparations are available. Thus, there is a clear need for a consortium of open data to address this unmet need. Recently, FSH glycosylation, primarily on the β-subunit, which varies as women age, has emerged as a key modifier of follitropin action, with profound biological effects in vivo in animal models. To date, limited information of recombinant follitropin hormone preparations is available. Thus, most of the studies with FSH that is well characterized biochemically have been done in vitro, with engineered non gonadal host cells bearing recombinant receptors or in animal models. Since limited studies in human granulosa cells are available, a question is whether structural differences in glycosylation in commercially available follitropin affects biological function and clinical effect in humans. The presence of fucose, for example, has not been studied greatly even though, in the case of antibody therapy it has been shown to have a large effect on antibody targeting. This review on glycosidic variability of follitropin from the biochemical/structural point of view reflects on this question and presents an assessment in the context of available published data. If clinical differences are to be expected or not, the readers will have a better understanding of the evidence for and limitations of such expectations.

Deciphering the effects of testosterone on FSH glycosylation variant-dependent ERK/MAPK activation

Deciphering the effects of testosterone on FSH glycosylation variant-dependent ERK/MAPK activation

Follicle-Stimulating Hormone Glycobiology.

FSH glycosylation varies in two functionally important aspects: microheterogeneity, resulting from oligosaccharide structure variation, and macroheterogeneity, arising from partial FSHβ subunit glycosylation. Although advances in mass spectrometry permit extensive characterization of FSH glycan populations, microheterogeneity remains difficult to illustrate, and comparisons between different studies are challenging because no standard format exists for rendering oligosaccharide structures. FSH microheterogeneity is illustrated using a consistent glycan diagram format to illustrate the large array of structures associated with one hormone. This is extended to commercially available recombinant FSH preparations, which exhibit greatly reduced microheterogeneity at three of four glycosylation sites. Macroheterogeneity is demonstrated by electrophoretic mobility shifts due to the absence of FSHβ glycans that can be assessed by Western blotting of immunopurified FSH. Initially, macroheterogeneity was hoped to matter more than microheterogeneity. However, it now appears that both forms of carbohydrate heterogeneity have to be taken into consideration. FSH glycosylation can reduce its apparent affinity for its cognate receptor by delaying initial interaction with the receptor and limiting access to all of the available binding sites. This is followed by impaired cellular signaling responses that may be related to reduced receptor occupancy or biased signaling. To resolve these alternatives, well-characterized FSH glycoform preparations are necessary.

Hormonal Regulation of Follicle-Stimulating Hormone Glycosylation in Males

The Follicle-Stimulating Hormone plays an important role in the regulation of gametogenesis. It is synthesized and secreted as a family of glycoforms with differing oligosaccharide structure, biological action, and half-life. The presence of these oligosaccharides is absolutely necessary for the full expression of hormone bioactivity at the level of the target cell. The endocrine milieu modulates the glycosylation of this hormone. During male sexual development a progressive increase in FSH sialylation and in the proportion of glycoforms bearing complex oligosaccharides are the main features in this physiological condition. In late puberty, FSH oligosaccharides are largely processed in the medial- and trans-Golgi cisternae of the gonadotrope and remain without changes throughout adult life. In experimental models, the absence of gonads severely affects FSH sialylation; androgen administration is able to restore the characteristics observed under physiological conditions. The expression of ST6 beta-galactoside alpha-2,6-sialyltransferase 1 is hormonally regulated in the male rat; it decreases after short periods of castration but increases markedly at longer periods of androgen deprivation. Although ST3 beta-galactoside alpha-2,3-sialyltransferase 3 is expressed in the male rat pituitary it is not influenced by changes in the endocrine milieu. The oligosaccharide structure of FSH has an impact on the Sertoli cell endocrine activity. In more advanced stages of Sertoli cell maturation, both sialylation and complexity of the oligosaccharides are involved in the regulation of inhibin B production; moreover, FSH glycoforms bearing incomplete oligosaccharides may enhance the stimulatory effect exerted by gonadal growth factors. In this review, we discuss available information on variation of FSH glycosylation and its hormonal regulation under different physiological and experimental conditions, as well as the effect on Sertoli cell endocrine activity.

Macro- and Micro-heterogeneity in Pituitary and Urinary Follicle-Stimulating Hormone Glycosylation.

FSH glycosylation macroheterogeneity in pituitary and urinary hFSH samples was evaluated by Western blotting. Microheterogeneity in two highly purified urinary and pituitary hFSH preparations was evaluated by nano-electrospray mass spectrometry of peptide-N-glycanase-released oligosaccharides. An age-related loss of hypo-glycosylated hFSH in individual female pituitaries was indicated by progressively reduced abundance of hFSH21 relative to hFSH24. Urinary hFSH was evaluated as a potentially non-invasive indicator of glycoform abundance, as the only way for pituitary FSH to reach the urine is through the blood. Both highly purified and crude postmenopausal urinary hFSH preparations possessed the same amount of hFSH21 as postmenopausal pituitary gland FSH. Considerable microheterogeneity was encountered in both pituitary and urinary hFSH glycan populations, as 84 pituitary hFSH glycan ions were observed as compared with 68 urinary hFSH glycans. The biggest quantitative differences between the two populations were reduced abundance of bisecting GlcNAc-containing and fucosylated glycans, along with sulfated glycans in the urinary hFSH glycan population. The relative abundance of sialic acid and glycan antenna did not rationalize the retarded electrophoretic mobilities of the urinary hFSHβ21- and α-subunit bands relative to the corresponding pituitary hFSH bands, as the most abundant glycans in the former possessed only 2 more branches and the same sialic content as in the latter. Site-specific glycosylation information will probably be necessary.

Comparison of hFSH Glycosylation by Electrospray Ionization Mass Spectrometry.

FSH is a highly heterogenous glycoprotein hormone, which possesses 4 N-glycosylation sites, each decorated with a family of glycans. Glycosylation heterogeneity is usually evaluated by fractionation methods that separate on the basis of negative charge. From the pattern of isoform isoelectric points, differences in glycosylation have been inferred. For example, because different isoform patterns were encountered when pituitary extract, serum samples, and urine samples were fractionated by chromatofocusing then evaluated by FSH RIA, glycosylation of FSH from all three sources was assumed to differ. Recent advances in mass spectrometry now permit direct comparison of FSH glycans. In the present study, we compared FSH glycosylation in human FSH preparations derived from pituitary glands, postmenopausal urine, and recombinant hFSH expressed in the rat GH3 cell line. The glycan populations released from reduced, carboxymethylated hFSH preparations obtained from the pituitary and from postmenopausal urine were virtually identical. The major glycans were identical, the major differences occurred in relatively minor glycan populations, including a 35% decrease in sulfated glycans, 27% decrease in glycans with a bisecting GlcNAc residue, and a 55% decrease in fucosylated glycans. The great similarity in pituitary and urinary hFSH glycan populations is a highly significant finding as it the absence of major changes in glycan popultations between pituitary and urinary FSH means that serum FSH glycans must also be the same. Therefore, examination of urinary hFSH glycosylation is directly relevant to serum hFSH glycosylation. We also compared glycosylation of tetra-glycosylated hFSH and two di-glycosylated hFSH preparations. The di-glycosylated hFSH derived from FSH preparations possessed a glycan population that was highly similar to that from tetra-glycosylated hFSH. Glycans from both glycoform preparations were very similar to pituitary hFSH, except the sulfated glycan populations were greater in the glycoform preparations. Glycosylation of di-glycosylated hFSH isolated from hLH preparations was strikingly different from all other hFSH preparations. The glycans were largely high mannose and in combinantion with their abundance, it could be argued that these represent an intracellular form. Glycosylation of recombinant hFSH revealed the absence of tetra-antennary glycans, which comprised 15% of pituitary hFSH glycans. Moreover, recombinant hFSH triantennary glycans possessed a third branch linked beta1-6 to the alpha1-6 mannose residue, which is due to the action of GlcNAc transferase V. In pituitary this branch is linked beta1-4 to the alpha1-3 mannose residue, as a consequence of GlcNAc transferase IV. This suggested the absence of GlcNAc transferase IV in rat GH3 cells. However, RT PCR found evidence for expression of two isoforms of this enzyme, while failing to detect GlcNAc transferase V. In order to make a more native-like recombinant hFSH, GH3 cells will have to be engineered to express the more human pituitary-like glycosyltransferase population. Supported by NIH grant P01 AG029531. (poster)

Hormonal regulation of pituitary FSH sialylation in male rats

Sialic acid content in FSH is modulated by GnRH and sexual steroids. Galβ1,3GlcNAcα2,3-sialyltransferase (ST3Gal III) and Galβ1,4GlcNAcα2,6-sialyltransferase (ST6Gal I) incorporate sialic acid residues into FSH oligosaccharides. The aim of the present study was to assess pituitary FSH molecular microheterogeneity and ST3Gal III/ST6Gal I expression during sexual development and after castration in male rats. Preparative isoelectric focusing and lectin chromatography were used to isolate FSH glycosylation variants according to charge and complexity of their oligosaccharides; RT-PCR and immunohistochemistry were employed to analyse sialyltransferase expression. Sexual development was associated with a progressive shift towards more acidic/sialylated FSH glycoforms concomitantly with an increment in ST6Gal I gene and protein expression. After castration, a transient decrease followed by a marked increase in ST6Gal I expression were observed. Less acidic/sialylated FSH glycoforms bearing incomplete oligosaccharides increased after castration, despite high ST6Gal I expression. ST3Gal III expression remained unchanged in all the experimental conditions examined. These results show that the synthesis of FSH isoforms possessing α2,6-linked sialic acid is hormonally regulated in male rats.

All-or-none N-glycosylation in primate follicle-stimulating hormone β-subunits

Human FSH exists as two major glycoforms designated, tetra-glycosylated and di-glycosylated hFSH. The former possesses both α- and β-subunit carbohydrates while the latter possesses only α-subunit carbohydrate. Western blotting differentiated the glycosylated, 24,000 Mr hFSHβ band from the non-glycosylated 21,000 Mr FSHβ band. Postmenopausal urinary hFSH preparations possessed 75–95% 24,000 Mr hFSHβ, while pituitary hFSH immunopurified from 21- to 43-year-old females and 21–43-year-old males possessed only 35–40% 24,000 Mr hFSHβ. The pituitary hFSH from a postmenopausal woman on estrogen replacement was 75% 21,000 Mr hFSHβ. Other immunopurified postmenopausal pituitary hFSH preparations possessed 50–60% 21,000 Mr hFSHβ. Gel filtration removed predominantly 21,000 Mr free hFSHβ and reduced its abundance to 13–22% in postmenopausal pituitary hFSH heterodimer preparations. A major regulatory mechanism for FSH glycosylation involves control of β-subunit N-glycosylation, possibly by inhibition of oligosaccharyl transferase. Two primate species exhibited the same all-or-none pattern of pituitary FSHβ glycosylation.

Basal and gonadotropin-releasing hormone-releasable serum follicle-stimulating hormone charge isoform distribution and in vitro biological-to-immunological ratio in male puberty.

Follicle-stimulating hormone is synthesized and secreted as a mixture of heterogeneous isoforms that differ from each other in carbohydrate structure, biological potency, and plasma half-life. The relative abundance of the FSH isoforms will depend on the endocrine status of the donor at the time of sample collection. In the present study, we attempted to define the impact of the changing endocrine milieu characteristic of male puberty on the charge heterogeneity and plasma half-life of the serum FSH isoforms released under endogenous and exogenous GnRH drives, and examined whether such a varying hormone milieu modifies the capability of the circulating hormone to trigger intracellular signal transduction at the human FSH receptor level. Forty healthy male subjects at Tanner stages (Ts) 1 to 5 were sampled at 10 min intervals for 10 h. Serum from successive samples collected across 2-4 h intervals containing FSH released under basal, low-dose (10 microg), and high-dose (90 microg) exogenous GnRH-stimulated conditions was subjected to preparative chromatofocusing and tested for bioactivity employing a homologous cell in vitro bioassay system. Deconvolution analysis was applied to estimate the apparent endogenous FSH plasma half-life in samples obtained after administration of low-dose exogenous GnRH. Under all conditions studied, serum FSH charge isoforms were distributed along a pH range of 7.0 to less than 3.0. Comparisons across the different Tanner stages revealed a significant and selective increase in the ratio of FSH isoforms with elution pH values <4.50 relative to those with values >/=4.50 at Ts-2. At Ts-3, this ratio returned to that present at Ts-1, to decline thereafter during the ensuing pubertal stages. Serum bioactive FSH concentrations progressively increased (from 3.72 +/- 1.3 to 16.2 +/- 5.3 IU/L) throughout puberty, and in all conditions bioactive FSH concentrations exceeded those detected by a specific radioimmunoassay. The biological to immunological (B:I) FSH ratio at baseline was significantly (p < 0.05) lower at Ts-1 and Ts-2 (1.33 +/- 0.30 and 1.62 +/- 0.34, respectively) than at more advanced stages of pubertal development (2.28 +/- 0.20, 2.96 +/- 0.38, and 2.77 +/- 0.63 at Ts-3-, 4-, and -5, respectively) Similar differences were detected in samples containing FSH molecules released after low- and high-dose GnRH administration. The apparent endogenous FSH half-life of the deconvolved GnRH-induced FSH pulses was similar in the five study groups. These results demonstrate that the transition from infancy to sexual maturity in men is accompanied by qualitative changes in the circulating FSH isoform mixture. Although the changes in FSH glycosylation occurring throughout puberty are not of sufficient magnitude to alter the survival of the gonadotropin in circulation, they allow preferential secretion of bioactive FSH. The enrichment of the circulating mix of FSH isoforms with highly bioactive variants throughout spontaneous puberty may potentially favor the development of spermatogenesis and acquisition of reproductive competence.

Human FSH isoforms: carbohydrate complexity as determinant of in-vitro bioactivity

Differences in sialic acid content of the hormone have been considered the main determinant of FSH polymorphism. The aim of the present study was to investigate the effect of variations in the oligosaccharide structure of the intrapituitary human FSH (hFSH) glycosylation variants on their intrinsic biological activity. FSH charge isoforms obtained after chromatofocusing were further separated by lectin affinity chromatography [Concanavalin A (ConA), Wheat germ agglutinin (WGA), Lentil lectin (LcH)]. Isolated isoforms were separately tested for in-vitro bioactivity in a rat Sertoli cell aromatization bioassay. Our results show that: (1) FSH microheterogeneity is due not only to variations in the sialic acid content of the hormone but also to differences in the internal structure of the carbohydrate chains, and (2) variations in the sialic acid content as well as differences in the complexity of the glycans determine the full biological expression of FSH glycosylation variants.

Does the gonadotrophic axis play a role in the pathogenesis of Sertoli-cell-only syndrome?

Infertile men with Sertoli-cell-only syndrome (SCO) have highly elevated serum FSH immunoactivity related to the degree of histological damage. The activity that serum FSH exerts at the target site depends on its glycosylation pattern and FSH receptor (FSHR) function. Either could be impaired, leading to failure of spermatogenesis. The aim of the present investigation was to study bioactivity and the glycosylation pattern of serum FSH and the occurrence of mutations in the FSH receptor in infertile patients with SCO compared to normal men. Blood was taken from 19 patients with bilateral testicular focal or complete SCO and eight normozoospermic controls. FSH bioactivity in serum was measured using an in-vitro FSH bioassay based on recombinant rat FSHR. The glycosylation pattern of serum FSH was determined by concanavalin A chromatography. Inhibin B was determined in serum using a recently available assay. Genomic DNA extracted from blood lymphocytes was amplified by PCR using primers specific for the FSHR and screened by single-stranded conformation polymorphism gel electrophoresis. Men with SCO showed significantly higher FSH in-vitro bioactivity (34.9 +/- 5.0 IU/l) than controls (9.6 +/- 0.8 IU/l: p < 0.01), as well as significantly elevated FSH immunoactivity (14.9 +/- 1.7 IU/l) compared to controls (3.1 +/- 0.5; p < 0.01). Immunoactivity of serum FSH was correlated with in-vitro bioactivity (r = 0.9; p < 0.001) and was related to the degree of testicular damage (proportion of SCO-tubules) (ANOVA: p < 0.001) and total testicular volume (r = -0.76; p < 0.01). An inverse relationship between serum FSH and inhibin B levels (r = -0.93; p < 0.001) was found. In the serum of SCO patients a slight increase in less glycosylated FSH isoforms was found (6.7 +/- 0.6% versus 3.6 +/- 0.3%; p < 0.05). No mutations of the FSHR were observed in SCO patients. We conclude that the spermatogenic failure observed in infertile patients with SCO histology and elevated FSH serum levels can be explained neither by a change in FSH bioactivity nor by mutations in the FSHR. The slight change in the FSH glycosylation pattern is probably related to higher hormonal secretion rates in SCO patients. The inverse relationship between serum FSH and inhibin B points to an intact endocrine testicular-pituitary circuit responsible for the compensatory increase of FSH in SCO.

Differential Regulation of Serum Immunoreactive Luteinizing Hormone and Bioactive Follicle-Stimulating Hormone by Testosterone in Early Pubertal Boys*

The microheterogeneity and bioassayable activity of serum FSH (B-FSH) can be regulated by exogenous GnRH in boys with idiopathic hypogonadotropic hypogonadism and by estrogen in a women with gonadal dysgenesis, presumably via hormonally mediated changes in the degree of FSH glycosylation. To test the hypothesis that testosterone (T) regulates the circulating forms of B-FSH, we raised the serum T levels of early pubertal boys to adult levels. In this model, high dose T inhibits the pubertal nocturnal augmentation of LH secretion, apparently through decreased GnRH secretion. This model allowed us to test a second hypothesis, that B-FSH is a sensitive indicator of hypothalamic GnRH release. The boys were studied on two consecutive weekends, during which they received either saline (S) or T infusions. Beginning at noon on the study day, after an overnight acclimatization, the boys received either S or T at 33% or 100% of the adult male production rate. Blood was sampled from 2000-0800 at 10-min intervals for immunoactive LH and FSH (I-FSH) and for B-FSH, as determined by the in vitro Sertoli cell aromatase induction assay, and at 30-min intervals for T. Gonadotropin levels were analyzed as mean hourly or 3-h concentrations and as pulse profiles by two established objective peak detection programs, Cluster and Detect. During S treatment, mean LH increased after the onset of sleep (P = 0.0006) and, after plateauing for several hours, declined to baseline in the early morning hours. Mean levels of B-FSH were also minimally (but significantly) increased after the onset of sleep (P = 0.046) and paralleled the decline noted for LH. Mean levels of I-FSH did not demonstrate a diurnal rhythm. The effect of T was gonadotropin specific. High dose T abolished the nocturnal elevation in mean LH concentrations, but had no effect on the nocturnal elevation of B-FSH (P less than 0.05) or on I-FSH levels. The LH pulse frequency was greatest from 2300-0450 h, during S treatment (P = 0.016). The pulse frequency of B-FSH was also minimally increased after the onset of sleep (P = 0.045). The T infusion abolished the nocturnal increase in LH pulse frequency, without an effect on B-FSH pulse frequency. B-FSH pulse frequency exceeded LH pulse frequency during S treatment (8.0 +/- 0.7 pulses/12 h vs. 5.5 +/- 0.4), and B-FSH pulses persisted throughout the night. The pulse amplitudes of LH and B-FSH were not affected by T.(ABSTRACT TRUNCATED AT 400 WORDS)