Potential role for MATER in cytoplasmic lattice formation in murine oocytes

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 Mater(tm/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 Mater(tm/tm) germinal vesicle stage oocytes which illustrated that volume fraction of CPLs was reduced by 90% in Mater(tm/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.

Figure 1. Co-localization of MATER and PADI6 in non-extracted and Triton X-100 extracted GV oocytes and early embryos.

(A) Confocal microscopic images show non-extracted GV-stage oocytes, 2-cell, and 4-cell embryos after fixation, permeabilization and incubation with anti-MATER (green) and anti-PADI6 (red) antibodies. (B) GV oocytes extracted with 0.1% Triton X-100 and stained with MATER (green) and PADI6 (red) antibodies. Merged images highlight MATER and PADI6 co-localization. Scatter plot indicates a degree of co-localization of MATER and PADI6. Region A shows pixels with high MATER (green) intensities, region B shows pixels with high PADI6 (red) intensities, and region C shows pixels with both high MATER (green) and PADI6 (red) intensities. Mander's overlap coefficient: 0.97, Pearson's correlation coefficient: 0.7. (C) CD1 mouse oocyte lysates were chromatographed by FPLC. Eluted fractions (1 ml) were analysed by immunoblotting with antibodies to PADI6 and MATER. Densitometry was used to generate a graph and the values in fraction 11 were set at a relative intensity of 100%. Elution of each protein standard is indicated by arrow.

Figure 2. PADI6 Triton X-100 solubility is increased in Mater^tm/tm GV-stage oocytes.

(A) Confocal analysis shows Mater^+/+ and Mater^tm/tm GV-stage oocytes prior to, and following, extraction with 0.1% Triton X-100. Oocytes were incubated with PADI6 antibodies (red). (B) Western blotting shows expression of MATER and PADI6 protein in Mater^+/+ and Mater^tm/tm GV-stage oocytes prior to, and following, 0.1% Triton X-100 extraction. Isolated oocytes were either extracted or not extracted with Triton, and then evaluated by Western blotting using either anti-MATER, anti-PADI6, or anti-β actin antibodies.

Figure 3. EM analysis reveals that the volume fraction of CPLs is reduced in Mater^tm/tm oocytes.

(A) Representative TEM image (x11,500) of the ultrastructure of Mater^+/+ GV oocytes. A high magnification (x26,500) image of the lattice structure is shown in the inset. (B) Representative TEM image (x11,500) showing the ultrastructure of Mater^tm/tm GV oocytes. Arrows in A and B indicate CPLs.

Figure 4. Mater^tm/tm oocytes display elevated levels of lipid droplets.

(A) Low magnification (x1,700) TEM images of Mater^+/+ and Mater^tm/tm GV-stage oocytes. Oocytes were prepared for TEM as above. White arrows point to LDs. (B) Confocal images of GV-stage live Mater^+/+ and Mater^tm/tm oocytes following Nile red staining. DIC images show morphology of LDs in oocytes. Close up images (Right panel) highlight LD aggregates. Arrows indicate LDs. (C) Quantitation of the volume fractions of CPLs, LDs, mitochondria, and ER in Mater^+/+ and Mater^tm/tm GV-stage oocytes. Mean ± SEM is indicated. P value is <0.004 for CPLs, <0.04 for LD, >0.05 for mitochondria, and ER.