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doi: 10.1242/10.1242/dev.00422

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Impaired cumulus mucification and female sterility in tumor necrosis factor-induced protein-6 deficient mice

¹ Section of Connective Tissue Biology, Department of Biomedical Engineering, The Cleveland Clinic Foundation, Cleveland, Ohio 44195, USA
² Section of Biochemistry and Molecular Biology, Departments of Orthopedic Surgery and Biochemistry, Rush University at Rush-Presbyterian-St. Luke's Medical Center, Chicago, Illinois 60612, USA
³ MRC, Immunochemistry Unit, Department of Biochemistry, University of Oxford, OX1 3QU Oxford, UK
⁴ Department of Public Health and Cell Biology, University of Rome `Tor Vergata', 00133 Rome, Italy

View larger version (55K): [in a new window]   Fig. 1. Generation of Tnfip6–/– mice. (A) Genomic organization of wild-type and recombinant alleles, and the targeting vector pPNT. The location of the probe used for Southern blot hybridization is shown underneath the wild-type allele. Black boxes represent exons. (B) Southern blot analysis of the genomic DNA samples (10 µg) derived from ES cell clones (wild type and clone no.135 with selected disruption of Tnfip6) or from mouse tails were digested with HindIII, separated in a 0.6% agarose gel, transferred to Nytran membrane and probed with a 2.2 kb 32P-labeled genomic DNA probe shown in A. (C) Genotyping results of Tnfip6–/–, Tnfip6+/– and wild-type (wt) littermates. PCR products of genomic DNAs using neomycin-specific primers (600 bp; lane 1); neomycin and intron 1-specific primers (441 bp; lane 2); exon 1 and intron 1-specific primers (365 bp without Neo gene and 2156 with Neo; lane 3). (D) RT-PCR results using total RNA samples from fibroblasts of Tnfip6–/–, Tnfip6+/– and wild-type mice without and with TNF stimulation (20 ng/ml; 24 hours) as indicated. (E) Media from the same fibroblast cultures used for RNA isolation were collected and TNFIP6 protein detected by Western blot analysis using affinity purified and biotinylated rabbit TSG-6-RC21 antibody (Bardos et al., 2001). The locations of TNFIP6 (36 kDa) and the previously described HC-TNFIP6 complex (125 kDa) (Mukhopadhyay et al., 2001) are marked.

View larger version (35K): [in a new window]   Fig. 2. Hematoxylin and eosin stained ovaries from sexually mature (12-week old) mice. Note the presence of corpus luteum (CL) in all three Tnfip6 genotypes. Scale bar: 500 µm.

View larger version (45K): [in a new window]   Fig. 3. Lack of fertilizability of oocytes from Tnfip6–/– females. Fertilizability was assessed by the ability of the oocytes to reach the two-cell stage using in vivo fertilization. Scale bar: 100 µm.

View larger version (119K): [in a new window]   Fig. 4. Deficient cumulus mucification in Tnfip6–/– females. (A-C) Hematoxylin stained preovulatory follicles in the three Tnfip6 genotypes 48 hours after the PMSG treatment (before hCG injection). (D-F) Hematoxylin stained preovulatory follicles 10 hours after induction of ovulation by hCG injection. (G-L) Localization of hyaluronan (green) in preovulatory follicles 10 hours after hCG injection. Low (G-I) and high (J-L) power magnifications are shown. White arrowheads in L point to cell surface hyaluronan. Nuclei are stained blue by DAPI. Scale bars: 100 µm (A-I) and 50 µm (J-L).

View larger version (87K): [in a new window]   Fig. 5. Morphology of ovulated COCs in mice with different Tnfip6 genotypes. Females of all three Tnfip6 genotypes are able to ovulate as indicated by the presence of oocytes within the oviduct (A-C). The dense cloud-like material (black arrowheads) around the oocytes in the oviducts of wild-type and Tnfip6+/– females are the expanded cumulus layer. Oocytes in the oviduct of the Tnfip6–/– females lack the same dense material (C, white arrowheads). This difference is clearly seen when isolated COCs are compared (D-F). COCs from wild-type and heterozygous females show normal morphology, while oocytes from the homozygotes do not have a cumulus layer. These latter oocytes are either completely nude (not shown) or they are associated with a dispersed network of granulosa/cumulus cells (F). Scale bars: 100 µm.

View larger version (122K): [in a new window]   Fig. 6. Impaired expansion of Tnfip6–/– COCs in vitro. (A-I) Compact COCs (48 hours after PMSG injection) from the three Tnfip6 genotypes were cultured in the absence (A-C) or in the presence of either 3 ng/ml EGF (D-F) or 1 mM dbcAMP (G-I). (J-K) Expansion of Tnfip6–/– COCs can be restored by supplementing recombinant TNFIP6 in the culture medium. Scale bars: 100 µm.

View larger version (43K): [in a new window]   Fig. 7. TNFIP6 is necessary for the transfer of heavy chains (HCs) from II and PI to hyaluronan. (A) Tnfip6–/– mice lack HCs covalently transferred to hyaluronan in their ovaries. Western blot of Streptomyces hyaluronidase-digested ovarian extracts are shown. Open arrowheads indicate dimers or clusters of HCs located close to each other on a single hyaluronan chain and resistant to hyaluronidase digestion. (B) TNFIP6 facilitates the covalent transfer of HCs from II and PI to hyaluronan in vitro. Note the formation of a high molecular mass hyaluronan-HC complex (HA-HC) in lane 3 in the presence of recTNFIP6, and the sensitivity of this band to hyaluronidase (h'ase) in lane 4. In both blots, serum with or without chondroitinase ABC (ch'ase) digestion was used as reference for II, PI and their HCs.

View larger version (23K): [in a new window]   Fig. 8. Schematic model for the proposed role of TNFIP6 in the stabilization of the cumulus extracellular matrix. For simplicity, only two hyaluronan chains are shown, but the proposed interactions can crosslink many of these chains creating a very stable hyaluronan meshwork. Asterisks indicate covalent interactions (ester bonds) between the heavy chains (HCs) and hyaluronan. All other protein-hyaluronan interactions are non-covalent. The presence of HC(1 or 2)-TNFIP6 complexes in the cumulus matrix was demonstrated in our previous study (Mukhopadhyay et al., 2001). Note that the exact structure of the HC-TNFIP6 has not yet been elucidated, therefore two hypothetical complex structures are shown. `?' indicates that no HC3-TNFIP6 complex has been detected yet. CS, chondroitin sulfate.