Single-step purification of equine chorionic gonadotrophin directly from plasma using affinity chromatography.

Equine chorionic gonadotrophin (eCG) is a hormone widely used in superovulation protocols because of its follicle-stimulating action, which increases reproductive efficiency in animals of productive interest. It contains 45% carbohydrate, 10% of which is N-acetylneuraminic acid (sialic acid). The eCG purification procedures from equine serum or plasma are mainly based on chromatographic methods. However, before these procedures, it is necessary to follow sample pre-conditioning steps, such as several precipitation stages and/or ultrafiltration/diafiltration processes. In this work, an efficient affinity chromatographic matrix for eCG purification directly from plasma was developed. The matrix consisted of chitosan mini-spheres with immobilized wheat germ agglutinin (WGA). The matrix allowed 98% adsorption of eCG directly from plasma without any pre-treatment with an overall yield of around 60%. The matrix chosen was able to maintain the efficient performance of the purification process for three consecutive cycles. Also, the process was scaled-up 500 times in volume and tested over seven consecutive cycles maintaining its chromatographic performance. The results presented here suggest the potential application of this matrix to one-step purification of eCG from plasma.

Gonadotropin preparations: past, present, and future perspectives.

This Educational Bulletin offers past, present and future perspectives on gonadotropin preparations.

Mammalian-like nonsialyl complex-type N-glycosylation of equine gonadotropins in Mimic insect cells.

Recombinant equine luteinizing hormone/chorionic gonadotropin (eLH/CG) was expressed in Mimic insect cells, that are commercial stably transformed Spodoptera frugiperda (Sf9) cells expressing five mammalian genes encoding glycosyltransferases involved in the synthesis of complex-type monosialylated N-glycans. We previously showed that it exhibited no in vivo bioactivity although expressing full in vitro bioactivity, and it was suspected that this was because of insufficient sialylation of eLH/CG N-glycans. Lectin binding analyses were performed with recombinant dimeric eLH/CG or its alpha subunit, secreted in the serum-containing supernatant of infected Sf9 and Mimic cells. Two types of specific lectin affinity assays (blot analyses and enzyme-linked immunosorbent assay) were used to compare the ability or inability of natural and recombinant gonadotropins to bind to various lectins. In natural equine chorionic gonadotropin (eCG), complex-type N-glycans terminating with both Siaalpha2,3Gal (based on Maackia amurensis agglutinin [MAA] binding) and Siaalpha2,6Gal (based on Sambucus nigra agglutinin [SNA] binding) were found, but in the alpha subunit dissociated from natural eCG, we only detected Siaalpha2-6Gal. In eLH/CG and its alpha subunit produced by Sf9 cells, N-glycans were found to be terminated by mannosyl residues (based on Galanthus nivalis agglutinin [GNA] binding), whereas those produced in Mimic cells were terminated by galactoses (based on binding to Ricinus communis agglutinin I [RCA I] , but not to SNA or MAA). This is in agreement with the fact that the nucleotide donor substrate of sialic acid is not naturally synthesized in insect cells. On the basis of binding to Arachis Hypogaea agglutinin [PNA], O-glycans exhibited the Galbeta1-3GalNAc structure in recombinant-free alpha and eLH/CG from both Sf9 and Mimic cell lines. Both N- and O-linked carbohydrate side chains synthesized in Mimic cells should thus be amenable to further acellular sialylation.

Extraction of equine chorionic gonadotrophin from pregnant mare plasma by direct adsorption on chromatographic media.

Equine chorionic gonadotrophin (eCG) is a hormone of practical value in veterinary medicine and animal production. Here we report a novel preparation procedure based on its direct adsorption onto anionic-exchange resins in a batch-wise mode. The active plasma is previously conditioned to reduce pH and ionic strength to required levels. After the adsorption stage, a 90% recovery of the initial eCG is achieved, with a concentration factor of about 50 and an enrichment factor around 500, with high preservation of biological activity. Further purification is carried out by cation-exchange column chromatography. The recovery for the whole process is higher than 70%, and the final potency of the preparation is close to 4000 IU/mg. The process is well suited for its application to the industrial scale.

A novel uterine protein that associates with the embryonic capsule in equids.

An apparently unique protein produced in large quantities by the endometrium of the mare which adheres to, or is incorporated into, the acellular capsule that surrounds the equine conceptus in early pregnancy, has been characterized and partially sequenced. It has a molecular mass of approximately 18 kDa on SDS-PAGE gels and is nonglycosylated as assessed by a sensitive carbohydrate detection kit. Comparison of its first 24 amino-terminal amino acids with all entries in the databases failed to show any significant identity with any other protein sequence. Secretion of the protein appears to be progesterone dependent, as its presence in uterine flushings correlates with peripheral serum progesterone profiles during the oestrous cycle and its secretion can be induced in anoestrous mares by administration of a synthetic progestagen. However, in pregnant mares, the protein disappears from the uterus after about day 20 (term = 320-340 days), despite the persistence of high serum concentrations of progesterone, indicating that additional mechanisms control its synthesis and secretion. The strong association of the protein with the glycoprotein capsule that surrounds the equine blastocyst suggests that it may be incorporated into the capsule as the capsule expands from day 11 after ovulation. Alternatively, or additionally, it may be involved in the transport of nutrients or other substances through the capsule, and may therefore play an important role in the maintenance of pregnancy.

Structure determination of the major N- and O-linked carbohydrate chains of the beta subunit from equine chorionic gonadotropin.

The carbohydrate moieties of equine chorionic gonadotropin alpha and beta subunits were released from the protein backbones by successive treatments with peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase F and alkaline borohydride and then fractionated by FPLC and HPLC. The major N- and O-linked glycans of the beta subunit were characterized by 500-MHz 1H-NMR spectroscopy, showing a remarkable structural heterogeneity for the N-glycosidically linked chains, comprising mono-, di-, tri- and tri'-antennary N-acetyllactosamine type of glycans, being partly alpha 1-6 fucosylated at the Asn-bound GlcNAc residue and having alpha 2-6 and alpha 2-3 linked N-acetyl- and N-acetyl-4-O-acetylneuraminic acid residues as sialic acid constituents. Significant differences in this respect were detected for the partially characterized glycans of the alpha subunit. The major part of the O-linked carbohydrate chains, occurring solely in the beta subunit, is formed by tri-, tetra-, penta- and hexa-saccharides. There are indications for the presence of oligo(N-acetyllactosamine) units in both the N- and O-linked glycans of the beta subunit.

Comparison of two reference preparations for horse chorionic gonadotrophin in four in-vivo and in-vitro assays.

A number of horse chorionic gonadotrophin (CG) preparations of different purities and from diverse sources have been compared in radioimmuno-, radioreceptor, in-vitro cell culture, and in-vivo assays. The relative activities of the great majority of the preparations tested were consistent in the 4 assay systems. Moreover, their relative activities in the 4 assays were consistent with those found for unfractionated plasmas. These preparations were therefore considered to represent the native form of hormone. The second International Reference Preparation (IRP2) was among the few preparations exhibiting discordant relative activities in the different assay systems. Its relative in-vivo activity was almost 50% lower than that found in the 3 other assays. This could be due to denaturation of the hormone during its preparation or to selection of isoform(s) not representative of the whole population of molecules. For standardization of horse CG preparations by in-vivo assay, IRP2 has proved to be a reliable standard. However, for the standardization of preparations by in-vitro methods, a standard giving consistent results in in-vivo and in-vitro assays must be used. The present report indicates that the NIH standard, but not IRP2, fulfils these requirements.

Pregnant mare serum gonadotropin. Rapid chromatographic procedures for the purification of intact hormone and isolation of subunits.

A method exploiting hydroxylapatite chromatography was developed to purify pregnant mare serum gonadotropin (PMSG or eCG) to high biological activity from partially purified commerical preparations. In addition, an alternative method utilizing chromatography on quaternary aminoethyl (QAE)-Sephadex and Sephadex G-200 is also presented. Both procedures are capable of producing, from commerical material with a potency of approximately 2,500 IU/mg, a product in excess of 12,000 IU/mg. If care is taken in the selection of fractions from the hydroxylapatite chromatography, essentially purified material may be obtained in a single step. The best fraction from the QAE-Sephadex and G-200 chromatography procedure contains a minor impurity. Pregnant mare serum gonadotropin subunits were purified by a single chromatographic step from the foregoing preparations utilizing 6 M guanidine hydrochloride for dissociation, followed by chromatography on Sephadex G-75. Analytical data, including amino acid composition, carbohydrate composition. NH2-terminal amino acid determinations, and electrophoretic behavior of the subunits in sodium dodecyl sulfate polyacrylamide gel electrophoresis are presented.

Purification and characterization of the gonadotropin secreted by cultured horse trophoblast cells.

Equine chorionic gonadotropin (eCG) secreted by horse trophoblast cells cultivated in vitro was isolated and its chemical, immunochemical, and biological characteristics were compared to those of the gonadotropin secreted in vivo and isolated from PMS. It was also compared with eCG isolated from the tissue of origin, the endometrial cups. The gonadotropin secreted in vitro had a smaller molecular size, contained appreciably less carbohydrate, and showed different amino-terminal residues from that secreted in vivo. In addition, there were significant differences in amino acid composition between eCG secreted in vivo and that secreted in vitro. The reactivity of eCG (isolated from the medium of cultured trophoblast cells) in homologous eCG (isolated from PMS) and equine LH and FSH RIAs suggested close antigenic similarities to eCG isolated from PMS. However, compared to serum-derived eCG, eCG secreted in vitro showed reduced activity in both LH (13%) and FSH (9-24%) bioassays and in LH and FSH radioreceptor assays (40-50%). The differences in bioassay potencies may be accounted for in part by the lower sialic acid content of the gonadotropin secreted in vitro. With respect to both bioactivity and chemical composition, the eCG secreted in vitro more closely resembled the eCG isolated from endometrial cups than that isolated from serum.