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Localisation of the hyaluronan receptor CD44 in porcine cumulus cells during in vivo and in vitro maturation

Published online by Cambridge University Press:  31 October 2002

Masaki Yokoo
Affiliation:
Department of Obstetrics and Gynaecology, Faculty of Veterinary Medicine, Swedish University of Agricultural Sciences (SLU), SE-750 07 Uppsala, Sweden Laboratory of Animal Reproduction, Graduate School of Agricultural Science, Tohoku University, Sendai 981-8555, Japan
Paisan Tienthai
Affiliation:
Department of Obstetrics and Gynaecology, Faculty of Veterinary Medicine, Swedish University of Agricultural Sciences (SLU), SE-750 07 Uppsala, Sweden Laboratory of Animal Reproduction, Graduate School of Agricultural Science, Tohoku University, Sendai 981-8555, Japan Division of Animal Science and Technology, Faculty of Agriculture, Okayama University, Okayama 700-8530, Japan
Naoko Kimura
Affiliation:
Department of Obstetrics and Gynaecology, Faculty of Veterinary Medicine, Swedish University of Agricultural Sciences (SLU), SE-750 07 Uppsala, Sweden Laboratory of Animal Reproduction, Graduate School of Agricultural Science, Tohoku University, Sendai 981-8555, Japan
Koji Niwa
Affiliation:
Division of Animal Science and Technology, Faculty of Agriculture, Okayama University, Okayama 700-8530, Japan
Eimei Sato
Affiliation:
Laboratory of Animal Reproduction, Graduate School of Agricultural Science, Tohoku University, Sendai 981-8555, Japan
Heriberto Rodriguez-Martinez
Affiliation:
Department of Obstetrics and Gynaecology, Faculty of Veterinary Medicine, Swedish University of Agricultural Sciences (SLU), SE-750 07 Uppsala, Sweden

Abstract

Polyspermy is fairly common during porcine in vitro fertilisation (IVF), perhaps due to incomplete in vitro oocyte maturation (IVM). Porcine cumulus cells (CCs) layered around the oocyte produce large amounts of extracellular hyaluronan (HA) when forming an expanding cell cloud during the last phase of oocyte maturation. The specific actions of HA are mediated via HA-binding proteins (HABPs), such as CD44, which act as receptors. In this study using immunocytochemistry and western blotting we investigated the localisation of CD44 in CCs obtained from in vivo-matured pig cumulus-oocyte complexes (COCs) and compared it with that in CCs from immature COCs and of COCs subjected to IVM and IVF procedures. Immunolabelling of CD44 was absent or very weak in CCs from immature COCs but strongly present on the surface of the CCs obtained from in vivo, displaying a similar localisation in the in vitro-matured COCs. In the latter, the labelling decreased but did not disappear in CCs 4 h after sperm co-incubation during IVF. Immunoblotting detected bands of between 73 and 88 kDa, corresponding to CD44, in the protein extract from in vivo CCs collected immediately prior to, or following spontaneous ovulation. The in vitro-matured CCs, however, presented bands ranging from 81 kDa to 88 kDa. Also, the bands found in the in vivo-matured CCs showed a larger variation of intensity and migration among animals than did the batches of in vitro-matured CCs. No CD44 band was detected on aliquots of the frozen-thawed boar spermatozoa used for IVF. The results clearly demonstrate that the specific HA receptor CD44 is present in expanding CCs of in vivo-matured pig COCs, in relation to increasing amounts of inter-CC HA. The subtle differences in molecular weight and migration ability observed between in vivo and in vitro samples may relate to differences in glycosylation and thus explain differences in HA-binding ability, of consequence for optimising in vitro culture conditions.

Type
Research Article
Information
Zygote , Volume 10 , Issue 4 , November 2002 , pp. 317 - 326
Copyright
2002 Cambridge University Press

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