Role of cytosolic malate dehydrogenase in oocyte maturation and embryo development  Se-Jin Yoon, Ph.D., Deog-Bon Koo, Ph.D., Jung Sun Park, M.Sc., Kyung-Hee Choi, Ph.D., Yong-Mahn Han, Ph.D., Kyung-Ah Lee, Ph.D.  Fertility and Sterility  Volume 86, Issue 4, Pages 1129-1136 (October 2006) DOI: 10.1016/j.fertnstert.2006.02.105 Copyright © 2006 American Society for Reproductive Medicine Terms and Conditions

FIGURE 1 Northern blot analysis of mouse Mor2 mRNA in various tissue types. Total RNA was extracted from multiple mouse tissues (20 μg per lane), as indicated by 18S and 28S. Yoon. Role of Mor2 in mouse oocyte and embryo. Fertil Steril 2006. Fertility and Sterility 2006 86, 1129-1136DOI: (10.1016/j.fertnstert.2006.02.105) Copyright © 2006 American Society for Reproductive Medicine Terms and Conditions

FIGURE 2 Alignment of amino acid sequences of cytosolic malate dehydrogenase of mouse, human, and porcine. Different residues are boxed, and the numbers left indicate the amino acid position in each sequence. The GenBank accession numbers for mouse, human, and porcine are NM_008618, NM_005917, and NM_213878, respectively. Yoon. Role of Mor2 in mouse oocyte and embryo. Fertil Steril 2006. Fertility and Sterility 2006 86, 1129-1136DOI: (10.1016/j.fertnstert.2006.02.105) Copyright © 2006 American Society for Reproductive Medicine Terms and Conditions

FIGURE 3 Alignment of amino acid sequences of mouse malate dehydrogenase isozymes. Identical residues (73 amino acids) are boxed, and the numbers left indicate the amino acid position in each sequence. The GenBank accession numbers for Mor1 and Mor2 are NM_008617 and NM_008618, respectively. Yoon. Role of Mor2 in mouse oocyte and embryo. Fertil Steril 2006. Fertility and Sterility 2006 86, 1129-1136DOI: (10.1016/j.fertnstert.2006.02.105) Copyright © 2006 American Society for Reproductive Medicine Terms and Conditions

FIGURE 4 Quantitative real-time PCR analysis of cytosolic (Mor2) and mitochondrial (Mor1) malate dehydrogenase isozymes. Transcripts isolated from oocytes and various embryonic stages were reverse-transcribed. For the PCR reaction, one oocyte- or one embryo-equivalent cDNA was used as template. (A) Levels of mRNA were estimated from CT values, and the mRNA ratio (AU) was calculated against that of GV oocytes. Experiments were repeated at least three times, and data were expressed as mean ± SEM. (B) After real-time PCR analysis, the PCR products were electrophoresed on 1.5% agarose gels with 18S as an internal control. 2C = two-cell; 4C = four-cell; 8C = eight-cell; MO = morular; BL = blastocyst embryo. Yoon. Role of Mor2 in mouse oocyte and embryo. Fertil Steril 2006. Fertility and Sterility 2006 86, 1129-1136DOI: (10.1016/j.fertnstert.2006.02.105) Copyright © 2006 American Society for Reproductive Medicine Terms and Conditions

FIGURE 5 Specific suppression of Mor2 mRNA expression by Mor2 RNAi. Injection of Mor2 dsRNA into GV oocytes markedly inhibited Mor2 RNA expression. The mRNA levels in a single oocyte were determined by RT-PCR analysis. Expression of untargeted Mor1 was not altered by microinjection of Mor2 dsRNA, suggesting the Mor2-specific silencing of the present data. The expression of 18S is shown as a loading control. Buffer = buffer injection; dsMor2 = Mor2 dsRNA injection. Yoon. Role of Mor2 in mouse oocyte and embryo. Fertil Steril 2006. Fertility and Sterility 2006 86, 1129-1136DOI: (10.1016/j.fertnstert.2006.02.105) Copyright © 2006 American Society for Reproductive Medicine Terms and Conditions