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After separation by means of preparative isoelectrofocusing, the isohormones of a Chinese hamster ovary (CHO)-derived recombinant follicle stimulating hormone (rFSH, Puregon) were characterized with respect to structural and functional features. A carbohydrate analysis revealed that rFSH isohormones with a low isoelectric point (pl) have a high sialic acid/galactose ratio and are rich in tri- and tetra-antennary N-linked carbohydrate chains in comparison with the high pl isohormones. The relative basic isohormones exhibit receptor binding activity and intrinsic bioactivity 2-3-fold higher than the relative acidic isohormones. However, due to their lower clearance rate these acidic isohormones displayed a 20-fold higher in-vivo bioactivity in the rat. A comparison of the isohormone profile of rFSH and urinary FSH (Metrodin) revealed that rFSH contains about 2-fold more basic isohormones with pl > or = 4.7 and 2-fold less acidic isohormones with pl < 4.1. In-vitro studies showed that the receptor binding affinity and intrinsic bioactivity of both FSH preparations are similar. Also the in-vivo efficacy and the pharmacokinetic behaviour of rFSH and urinary FSH in the rat were similar, which is not surprising since both preparations were compared in terms of in-vivo bioactivity calibrated in the rat Steelman-Pohley assay. However, in dogs the bioavailability of rFSH was lower than that of urinary FSH, which is in agreement with the higher percentage of relative basic isohormones in rFSH. This suggests that the pharmacokinetic behaviour of FSH in rats and dogs is different, which is supported by the much longer elimination half-life of rFSH and urinary FSH in dogs (27.9 and 30.4 h respectively) compared with rats (11.4 and 10.4 h respectively) for rFSH and urinary FSH respectively. The observed differences in pharmacokinetic behaviour in dogs and rats indicate that the rat Steelman-Pohley assay might not be a valid model for the prediction of the FSH bioactivity in species other than rat.

The N-linked carbohydrate chains of porcine zona pellucida glycoproteins were released by digestion with peptide-N4-(N-acetyl-~-glucosaminyl)asparagine amidase F and subsequently separated from the 0-glycoprotein by gel-permeation chromatography on Bio-Gel P-100. The 0-linked carbohydrate chains were released from the 0-glycoprotein by alkaline borohydride treatment. Fractionation of the extremely heterogeneous mixture of 0-linked oligosaccharide alditols was achieved by a combination of chromatographic techniques comprising gel-permeation chromatography on Bio-Gel P-4 and P-6, anion-exchange FPLC on Mono Q, and high-pH anion-exchange chromatography on CarboPac PA-1. The primary structures of 32 0-glycans were determined by one-and twodimensional 'H-NMR spectroscopy. The major part of the analyzed compounds contain a combination of the structural elements Gal~l-4GlcNAc/?l-3Gal/?l-3GalNAc-ol, GalP1 -4(6S04_)GlcNAc, and a2-3-linked Neu5Gc or NeuSAc. This series of compounds has the following structure,In addition, smaller compounds were identified in which the Gal/?l-3GalNAc-ol core is substituted by NeuSGc/Ac a2-6-linked to GalNAc-ol and/or NeuSGc/Ac a2-3-linked to Gal. Furthermore, oligosaccharides were obtained in which the distribution of 6-0-sulfated GlcNAc residues differs from that in the above-mentioned general structure, and a small portion of the oligosaccharides has the GlcNAc/31-3GalNAc-o1 core structure. Analysis of the endo-P-galactosidase digests of pools of N-and 0-glycans indicated that the two types of oligosaccharides contain qualitatively similar poly(N-acetyllactosamine) chains. In the case of the N-linked carbohydrate chains, multiple branching of the core structures occurs, resulting in an even larger heterogeneity than observed for the 0-linked carbohydrate chains.The zona pellucida (ZP) is the glycoprotein matrix surrounding the mammalian oocyte during the final stages of its development. It mediates several critical steps in the fertilization process, including the species-specific binding of spermatozoa to the ovum, induction of the acrosome reaction, prevention of polyspermy and the physical protection of the Abbreviations. FID, free induction decay ; HOHAHA, homonuclear Hartmann-Hahn ; HPAEC, high-pH anion-exchange chromatography; MLEV, composite pulse devised by M. Levitt; NAc, Nacetyl ; NGc, N-glycolyl ; NeuSAc, N-acetylneuraminic acid ; NeuSGc, N-glycolylneuraminic acid; NeuSGc/Ac, mixture of N-glycolylneuraminic acid and N-acetylneuraminic acid; PAD, pulsed amperometric detection; PNGase-F, peptide-N4-(N-acetyl-~-glucosaminy1)asparagine amidase F; pZP, porcine zona pellucida; Sia, sialic acid ; WEFT, water-eliminated Fourier transform; ZP, zona pellucida.Enzymes. Peptide-N4-(N-acetyl-~-glucosaminyl)asparagine amidase F (EC 3.5.1.52); endo-p-galactosidase (EC 3.2.1.103).growing embryo during the preinplantation stages of development [l, 21. As shown for different species, the ZP consists of families of glycoproteins differing in molecular mass and containing both N-and 0-linked ca...

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