Submitted August 14, 2007
Returned for revision October 10, 2007
Accepted January 29, 2008
Ovary
Synthesis of Tumor Necrosis Factor Alpha-Induced Protein 6 in Porcine Preovulatory Follicles: A Study with A38 Antibody
Eva Nagyova *,
Antonella Camaioni ,
Radek Prochazka ,
Anthony J Day ,
and
Antonietta Salustri
* To whom correspondence should be addressed. E-mail: nagyova{at}iapg.cas.cz.
Abstract
We have previously shown that the heavy chains (HC) of inter-alpha-trypsin inhibitor (IalphaI) become covalently linked to hyaluronan (HA) during in vivo and in vitro expansion of porcine oocyte-cumulus cell complexes (OCCs). We have now studied by immunoblotting the synthesis of tumor necrosis factor alpha induced protein 6 (TNFAIP6), which is essential for catalyzing this reaction in expanding mouse OCCs. Expanding OCCs were collected from preovulatory follicles of naturally cycling pigs and also after in vitro culture (24 or 42 h) in medium supplemented with FSH and pig serum. After isolation, OCCs were treated with Streptomyces hyaluronidase or Chondroitinase ABC. Matrix, cell pellet and total extracts were analyzed by Western blotting. A band of about 35-kDa and a doublet of about 120-kDa, corresponding to the molecular weight of the native and HC-linked form of TNFAIP6, respectively, were detected by a rabbit anti-human TNFAIP6 polyclonal antibody in matrix extracts of expanded cumuli. Moreover, we found, by using a cell-free assay that porcine follicular fluid collected from follicles at 24 h after hCG stimulation contains HC-HA coupling activity. This activity was abolished by the rat anti-human monoclonal antibody A38, which has an epitope within the Link module domain of TNFAIP6. These experiments suggest that free TNFAIP6 protein was present in follicular fluid aspirated from porcine follicles 24 h after hCG stimulation. In contrast to mouse, we show that the A38 monoclonal antibody does not affect in vitro cumulus expansion of porcine OCCs.
Key words:
Ovary •
Cumulus cells •
TNFAIP6 •
hyaluronan •
pig
