The presence of the Y-chromosome, not the absence of the second X-chromosome, alters the mRNA levels stored in the fully grown XY mouse oocyte

Abstract

The oocytes of B6.Y(TIR) sex-reversed female mouse mature in culture but fail to develop after fertilization because of their cytoplasmic defects. To identify the defective components, we compared the gene expression profiles between the fully-grown oocytes of B6.Y(TIR) (XY) females and those of their XX littermates by cDNA microarray. 173 genes were found to be higher and 485 genes were lower in XY oocytes than in XX oocytes by at least 2-fold. We compared the transcript levels of selected genes by RT-PCR in XY and XX oocytes, as well as in XO oocytes missing paternal X-chromosomes. All genes tested showed comparable transcript levels between XX and XO oocytes, indicating that mRNA accumulation is well adjusted in XO oocytes. By contrast, in addition to Y-encoded genes, many genes showed significantly different transcript levels in XY oocytes. We speculate that the presence of the Y-chromosome, rather than the absence of the second X-chromosome, caused dramatic changes in the gene expression profile in the XY fully-grown oocyte.

Figure 1. RT-PCR detection of Y-encoded gene transcripts in XY and XX oocytes.

Agarose gel electrophoresis stained with ethidium bromide. High transcript levels of Eif2s3y, Ddx3y, and Ube1y1 and lower levels of Rbmy1 were visible in XY oocytes but not in XX oocytes. The transcript levels of Zfy1 was similar to those of Rbmy1 after the same amplification cycles, but increased, as shown here, by adding 4 more cycles. For each RT-PCR experiment, β-actin was included as an internal control. S, 100 bp ladders.

Figure 2. RT-PCR detection of X-encoded gene transcripts in XX, XO and XY oocytes.

A. Agarose gel electrophoresis stained with ethidium bromide. S, 100 bp ladders. B. Relative transcript levels. In each set of experiment, the transcript levels were normalized against the mean of two β-actin controls. Each column indicates the mean ± SEM (n = 6 for Xiap and n = 3 for others except for Usp9x in XO, n = 1). a and b above columns indicate statistical differences at P<0.05 by paired students t-test.

Figure 3. RT-PCR detection of additional X-encoded (in rectangular boxes) and autosomal gene transcripts in XX, XO and XY oocytes.

A. Agarose gel electrophoresis stained with ethidium bromide. S, 100 bp ladders. B and C. Relative transcript levels of. X-encoded (A) and autosomal (C) genes. In each set of experiment, the transcript levels were normalized against the mean of two β-actin controls. Each column indicates the mean ± SEM (n = 6 for Atrx and Xiap, n = 4 for Atp2g5 and Dub2, n = 3 for others). Different low case and capital letters above columns denote statistical differences at P<0.05 and 0.01, respectively, by one-way ANOVA.

Figure 4. RT-PCR detection of additional autosomal gene transcripts in XX, XO and XY oocytes.

A. Agarose gel electrophoresis stained with ethidium bromide. S, 100 bp ladders. B. Relative transcript levels of autosomal genes in XX, XO and XY oocytes. In each set of experiment, the transcript levels were normalized against the mean of two β-actin controls. Each column indicates the mean ± SEM (n = 3). Different low case and capital letters above columns denote statistical differences at P<0.05 and 0.01, respectively, by one-way ANOVA.

Figure 5. Relative transcript levels of Epas1 in XX, XO and XY oocytes collected from ovaries at 10 and 30 days after birth.

“Growing oocytes” were selected for their diameter between 40 and 50 µm. “Fully grown oocytes” were denuded from oocyte-cumulus complexes of antral follicles. The transcript levels were normalized against the mean of two β-actin controls. Each column indicates the mean ± SEM (n = 3). A and B above columns denote statistical differences at P<0.01 by paired students t-test.