Recombinant rat follicle-stimulating hormone; production by Chinese hamster ovary cells, purification and functional characterization

Hakola, K; Boogaart, P Van der; Mulders, John W. M.; Leeuw, R. de; Schoonen, W.G.E.J.; Heyst, J Van; Swolfs, A; Casteren, J Van; Huhtaniemi, Ilpo; Kloosterboer, Helenius J.

doi:10.1016/s0303-7207(96)03990-1

Cited by 34 publications

(19 citation statements)

References 30 publications

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“…With regard to LH, the lower bioactivity of rat LH was coupled with faster T Y value in the circulation of this hormone, as compared with the respective human hormone. In contrast, whereas rat recFSH had lower bioactivity (Hakola et al 1997a) and receptor affinity than the cognate human hormone, the T Y of the rat hormone was longer than that of the human hormone. In accordance, the elimination half-times of human recFSH were previously reported to be about 5·7 h in rats, but about 30 h in dogs and humans (de Leeuw et al 1996), which makes human FSH a suboptimal choice to study the physiology of FSH action in rats.…”

Section: Discussionmentioning

confidence: 82%

“…The highly purified recombinant (rec) forms of rat LH (95% purity, 1100 IU/mg by in vitro bioassay, in relation to human recLH; Hakola et al 1997b) and FSH (98% purity, 8820 IU/mg by in vivo bioassay, Hakola et al 1997a) and of human FSH (Org 32489, >95% purity, 11 500 IU/mg by bioassay; batch 65), were donated by Organon (Organon International BV, Oss, The Netherlands). Highly purified human recLH (99·9% purity, 15 900 IU/ mg by in vivo bioassay, batch C31) was a generous gift of Ares-Serono (Geneva, Switzerland), and highly purified urinary hCG (NIH CR 121, 11 500 IU/mg) was a generous gift of NICDH (NIH, Bethesda, MD, USA).…”

Section: Hormone Preparationsmentioning

confidence: 99%

“…We have produced, purified and partially characterized rat recombinant (rec) FSH (Hakola et al 1997a) and rat recLH (Hakola et al 1997b), in order to study further the physiology of gonadotropin functions in the rat, the most common animal model in reproduction research. The advantages of the rec gonadotropins are that they can be produced and purified in large quantity, as is needed, for instance, for in vivo tests.…”

Section: Introductionmentioning

confidence: 99%

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Recombinant forms of rat and human luteinizing hormone and follicle-stimulating hormone; comparison of functions in vitro and in vivo

Hakola

Haavisto²,

Pierroz³

et al. 1998

Journal of Endocrinology

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We have previously described the preparation, purification and partial characterization of recombinant (rec) forms of rat luteinizing hormone (LH) and follicle-stimulating hormone (FSH). In the present study, the special functional features of these hormones were studied further, in vitro and in vivo, and compared with human recLH and recFSH, as well as with human urinary choriongonadotropin (hCG) and rat pituitary LH (NIDDK-RP3). In radioreceptor assay, the affinity of hCG binding to rat testis membranes was 5-fold higher than that of human recLH and 100-fold higher than that of rat recLH. In in vitro bioassay, using dispersed adult mouse interstitial cells or a mouse Leydig tumor cell line (BLT-1), hCG and human recLH were 10-to 20-fold more potent than rat recLH. Correspondingly, rat pituitary LH was about 10-fold less potent than rat recLH, and evoked a maximum testosterone response that was about half of that elicited by the other LH/CG preparations. Rat recFSH was about 10-fold less potent than human recFSH in stimulating cAMP production of a mouse Sertoli cell line (MSC-1) expressing the recombinant rat FSH receptor.The circulating half-times (T Y ) of rat and human rec hormones were assessed after i.v. injections into adult male rats rendered gonadotropin-deficient by treatment with a gonadotropin-releasing hormone antagonist. A novel immunometric assay was used for the rat FSH measurements. In the one-component model the T Y values of rat and human recLH were 18·2 1·9 min (n=7) and 44·6 3·1 min (n=7) respectively and those of rat and human recFSH were 88·4 10·7 min (n=6) and 55·0 4·2 min (n=6) respectively; the two-component models revealed similar differences between the rec hormone preparations. Collectively, rat recLH was eliminated significantly faster from the circulation than human recLH (P<0·0001). In contrast, the elimination of rat recFSH was significantly slower than that of human recFSH (P=0·02).In conclusion, rat recFSH and rat recLH display lower biopotencies per unit mass than the respective human hormones in vitro, and also in vivo for LH. This is paralleled by shorter T Y of rat recLH than the respective human hormone in the circulation, whereas human recFSH has a shorter T Y than human FSH. The special functional features of the rat rec gonadotropins emphasize the use of these preparations on studies of gonadotropin function in the rat, an important animal model for reproductive physiology.

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Section: Discussionmentioning

confidence: 82%

Section: Hormone Preparationsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Recombinant forms of rat and human luteinizing hormone and follicle-stimulating hormone; comparison of functions in vitro and in vivo

Hakola

Haavisto²,

Pierroz³

et al. 1998

Journal of Endocrinology

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“…Biologically active recombinant FSH has been produced previously using transformed mammalian and insect cell lines and transgenic mice (Keene et al 1989, Greenberg et al 1991, Mountford et al 1994, Arey et al 1997, Hakola et al 1997). The CHO cell-derived recombinant human FSH has been used successfully in infertility treatment (Devroey et al 1993).…”

Section: Discussionmentioning

confidence: 99%

Expression and secretion of a biologically active glycoprotein hormone, ovine follicle stimulating hormone, by Pichia pastoris

Fidler¹,

Lun²,

Young³

et al. 1998

Journal of Molecular Endocrinology

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The methylotrophic yeast, Pichia pastoris, has been used to co-express recombinant genes formed by fusion of the mating factor-alpha (MF ) leader and ovine follicle stimulating hormone (oFSH) and subunit coding sequences. Pichia strains carrying single copies of the two fusion genes secreted recombinant oFSH (roFSH) to concentrations of approximately 51·0 ng/ml and 17·5 ng/ml, measured by RIA or in vitro bioassay respectively, whereas a strain with two copies of the and one copy of the subunit fusion genes secreted roFSH to concentrations of 61 ng/ml (RIA) and 22 ng/ml (bioassay).It appears that the Pichia-derived roFSH had about one-third the in vitro bioactivity of native oFSH or, alternatively, only one-third of the roFSH is bioactive. Measurements of secreted roFSH and subunit concentrations indicated less than 10% of and 25-33% of subunits were stably dimerized. The receptor binding properties of the roFSH resemble those of native oFSH. In summary this paper reports the production, by P. pastoris, of a heterodimeric glycoprotein hormone (roFSH) that has in vitro biological activity.

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“…Placenta-derived material, such as PMSG, has the disadvantage of a very long biological half-life, which necessitates its neutralization by the injection of specific antibodies (Bouters et al 1983). Furthermore, stimulation of the target animal should preferably be with a homologous hormone preparation, as indicated by Hakola et al (1997). Finally, all currently used FSH preparations do contain some LH activity, which is considered responsible, at least in part, for the observed large variation in the results of superovulation (Kelly et al 1997).…”

Section: Introductionmentioning

confidence: 99%

High-level expression of biologically active recombinant bovine follicle stimulating hormone in a baculovirus system

Wiel¹,

Rijn²,

Meloen³

et al. 1998

Journal of Molecular Endocrinology

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Superovulation treatment of cows can benefit from the application of very pure recombinant bovine FSH (rbFSH), which is produced in nonmammalian cells. rbFSH is completely free of LH, and therefore can possibly reduce the variability in the results of superovulation. Furthermore, it does not contain brain-tissue-derived proteins and, when produced under serum-free conditions, it is free of other mammalian substances or potentially infectious material. We have produced rbFSH in insect cells, with the ultimate aim of inducing superovulation in cattle. Sf21 insect cells were coinfected with two recombinant baculoviruses, containing the cDNAs of bovine FSH alpha- and beta-subunits respectively. High levels of production of bioactive rbFSH were obtained after cloning cDNA that contained a major part of the 3' untranslated region of the bFSH beta gene. Maximum production of rbFSH 1-5 micrograms/ml (as measured by immunoassay) was obtained 70-90 h after infection. The recombinant material was highly potent in two in vitro bioassays, giving biological activities of 13 IU/ml (Y1 cell rounding assay), 22 IU/ml (Y1 cell cAMP assay), and 23 IU/ml (bovine oocyte maturation inhibition assay), and had a lower but significant activity of 6 IU/ml in the rat Sertoli cell assay. rbFSH was purified by immunoaffinity chromatography, using a monoclonal antibody directed against the human FSH beta-subunit. The purified heterodimer appeared to be homogeneous by SDS-PAGE, whereas the free beta-subunit appeared as a doublet, possibly indicating differently glycosylated forms. Intact heterodimer and both subunits were further identified by western blot analysis, and showed apparent molecular masses of 20 kDa (alpha-subunit), 23 kDa (beta-subunit) and 32.5 kDa (heterodimer). This insect-cell-produced rbFSH did not bind to wheat germ agglutinin, thus indicating that glycosidic side-chains may not contain terminal sialic acid. The relevance of a large 3' untranslated region in bFSH beta cDNA to the level of production of rbFSH, and the possible implications of the pattern of glycosylation for the biological activity of the recombinant hormone are discussed.

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Recombinant rat follicle-stimulating hormone; production by Chinese hamster ovary cells, purification and functional characterization

Cited by 34 publications

References 30 publications

Recombinant forms of rat and human luteinizing hormone and follicle-stimulating hormone; comparison of functions in vitro and in vivo

Recombinant forms of rat and human luteinizing hormone and follicle-stimulating hormone; comparison of functions in vitro and in vivo

Expression and secretion of a biologically active glycoprotein hormone, ovine follicle stimulating hormone, by Pichia pastoris

High-level expression of biologically active recombinant bovine follicle stimulating hormone in a baculovirus system

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