Proteolytic Activity of the 26S Proteasome Is Required for the Meiotic Resumption, Germinal Vesicle Breakdown, and Cumulus Expansion of Porcine Cumulus-Oocyte Complexes Matured In Vitro
Biol Reprod Yi et al.
78: 115
Supplemental Data
Files in this Data Supplement:
[Supplemental Figure 1] -
"Inhibition of cumulus expansion in vitro by MG132. A) Immature porcine oocytes surrounded by a compact mass of cumulus cells before IVM. B) Expanded COCs cultured in FSH/LH-supplemented medium for 22 h. C) COCs cultured in the presence of 10 µM MG132 in IVM medium for 44 h. Original magnification ×20."
[Supplemental Figure 3] -
"Immunostaining of RAC1 GTPase (red) in unstimulated (0h; left column) and gonadotropin-stimulated (44h; right column) porcine COCs. Filamenouts actin (F-actin/actin microfilaments; green) was counterstained with phalloidin-Alexa 488 and DNA (blue) with DAPI. Overlaps (Comb.) are shown among epifluorescence channels (conventional epifluorescence microscopy) and with the perfocal differential-interference contrast (DIC) images. Original magnification ×260."
[Supplemental Figure 2] -
"MG132 treatment can be used to prolong the period of in vitro maturation (IVM), yielding satisfactory (>50%) numbers of mature metaphase II oocytes after 88 h of IVM. COCs were cultured in the presence of 10 µM MG132 for 44 h in TCM199 medium. Subsequently, oocytes were cultured in IVM medium with hormones for 22 h and for additional 22 h without hormones. Experiments were repeated five times. Values are expressed as the mean percentage ± SEM. The numbers of cultured oocytes are indicated in parentheses. Different superscripts a, b, and c denote a significant difference at P < 0.05."
[Supplemental Figure 4] -
"Negative control for immunolabeling of 26S proteasomes (red) and nuclear pore complex proteins (NPC; green) in porcine oocytes. A) Double labeling of proteasomes and NPC in a GV-stage oocyte. B) Double labeling of proteasomes and NPC in an oocyte undergoing germinal vesicle breakdown (GVBD). C) Double-negative labeling in an oocyte undergoing GVBD. Images were collected by a conventional epifluorescence microscope. Original magnification ×450."
