Technical Reference #3189

3189.

Potential Role for MATER in Cytoplasmic Lattice Formation in Murine Oocytes Boram Kim; Rui Kan; Lynne Anguish; Lawrence M. Nelson; Scott A. Coonrod, Cornell University, PLoS ONE, 5(3189), (2010)
Link To Paper

Abstract:
Background: Mater and Padi6 are maternal effect genes that are first expressed during oocyte growth and are required for embryonic development beyond the two-cell stage in the mouse. We have recently found that PADI6 localizes to and is required for the formation of abundant fibrillar Triton X-100 (Triton) insoluble structures termed the oocyte cytoplasmic lattices (CPLs). Given their similar expression profiles and mutant mouse phenotypes we have been testing the hypothesis that MATER also plays a role in CPL formation and/or function. Methodology/Findings: Herein we show that PADI6 and MATER co-localize throughout the oocyte cytoplasm following Triton extraction suggesting that MATER co-localizes with PADI6 at the CPLs. Additionally the solubility of PADI6 was dramatically increased in Matertm/tm oocytes following Triton extraction suggesting that MATER is involved in CPL nucleation. This prediction is supported by transmission electron microscopic analysis of Mater+/+ and Matertm/tm germinal vesicle stage oocytes which illustrated that volume fraction of CPLs was reduced by 90% in Matertm/tm oocytes compared to Mater+/+ oocytes. Conclusions: Taken together these results suggest that similar to PADI6 MATER is also required for CPL formation. Given that PADI6 and MATER are essential for female fertility these results not only strengthen the hypothesis that the lattices play a critical role in mediating events during the oocyte-to-embryo transition but also increase our understanding of the molecular nature of the CPLs.

Materials & Methods:
Confocal microscopy Immunostaining and Triton extraction procedures for oocytes have been described elsewhere (Yurttas et al. 2008). Oocytes and embryos were fixed in 4% paraformaldedyde (PFA) (EM Sciences) for 30 minutes. For extraction oocytes were incubated in extraction buffer containing 0.1M KCl 20 mM MgCl2 3 mM EGTA 20 mM HEPES (pH 6.8) 0.1% Triton X-100 and 16 Complete Protease Inhibitor Cocktail (Roche) for 10 minutes and rinsed in PBS quickly and fixed. Oocytes were permeabilized with 0.5% Triton X-100 in PBS for 30 minutes washed and incubated with rabbit anti-MATER (1:1000) (Tong et al. 2004) or guinea pig anti-PADI6 (1:1000) (Wright et al. 2003) in IF buffer (1% BSA 0.5% Normal Goat Serum in PBS) for 1 hour followed by another 1 h incubation with the appropriate Alexa Fluor-conjugated (1:450) secondary antibody (Molecular Probes) in IF buffer. Oocytes were mounted on slides with Slowfade Gold antifade agent (Molecular Probes) and imaged using an LSM 510 laser scanning confocal microscope (Carl Zeiss). Confocal microscope settings for comparison of MATER expression between Mater+/+ and Matertm/tm oocytes were identical. For Nile red staining oocytes were quickly washed 5 times in M2 medium 3 times in MEM alpha medium (Invitrogen) and then incubated with Nile red (Sigma 5 mg/1 ml) in MEM alpha medium for 5 minutes. Oocytes were briefly washed in PBS/PVA and PBS attached to MatTek dishes (MatTek Corporation) which were then filled with MEM alpha. All procedures were carried out at room temperature.

Microscopic Technique
Confocal microscopy

Cell Type(s)
oocytes