Toward using stable spermatozoal RNAs for prognostic assessment of male factor fertility  G. Charles Ostermeier, Ph.D., Robert J. Goodrich, B.S., Michael P. Diamond, M.D., David J. Dix, Ph.D., Stephen A. Krawetz, Ph.D.  Fertility and Sterility  Volume 83, Issue 6, Pages 1687-1694 (June 2005) DOI: 10.1016/j.fertnstert.2004.12.046 Copyright © 2005 American Society for Reproductive Medicine Terms and Conditions

FIGURE 1 Comparing two spermatozoal RNA populations of ideal quality. (A) Microarray distribution. Ribonucleic acid was purified from two ideal quality spermatozoal samples and radiolabeled cDNA probes were constructed. Expressed sequence tags (ESTs) labeled by probe 14001 are shown in blue whereas those labeled only by probe 14002 are shown in red. Where the two probes overlap, the ESTs are shown in purple. (B) Venn diagram summarizing microarray data. The two ideal RNA samples shared 132 ESTs, indicating 40 and 35 unique messages in samples 14001 and 14002, respectively. Ostermeier. Stable spermatozoal RNAs. Fertil Steril 2005. Fertility and Sterility 2005 83, 1687-1694DOI: (10.1016/j.fertnstert.2004.12.046) Copyright © 2005 American Society for Reproductive Medicine Terms and Conditions

FIGURE 2 Comparing two spermatozoal RNA populations from poor quality samples. (A) Microarray distribution. Ribonucleic acid was purified from two spermatozoal samples of poor quality then radiolabeled cDNA probes were constructed. Expressed sequence tags (ESTs) labeled by probe 14012 are shown in red whereas those labeled by probe 14013 are shown in green. Where the two probes overlap, the ESTs are shown in yellow. (B) Venn diagram summarizing microarray data. The two poor quality RNA samples shared 47 ESTs, indicating 28 and 19 unique messages in samples 14012 and 14013, respectively. Ostermeier. Stable spermatozoal RNAs. Fertil Steril 2005. Fertility and Sterility 2005 83, 1687-1694DOI: (10.1016/j.fertnstert.2004.12.046) Copyright © 2005 American Society for Reproductive Medicine Terms and Conditions

FIGURE 3 Comparing a medium quality RNA sample with ideal and poor quality populations. Ribonucleic acid was purified from two ideal (14001 and 14002), one medium (769), and two poor (14012 and 14013) quality spermatozoal samples; radiolabeled cDNA probes were constructed and used to interrogate the human tox 1.2 microarray (Clontech, Palo Alto, CA). (A) Venn diagram summarizing microarray data overlap. The two ideal samples shared 96 expressed sequence tags (ESTs) with the medium quality sample. This illustrates that all but two RNAs from sample 769 were in both of the ideal samples. (B) Venn diagram summarizing microarray data overlap. The two poor quality RNA samples shared 37 ESTs with the medium quality sample. This shows that just less than half of the RNAs (45 of 98) from sample 769 were not found in either of the poor quality samples. Ostermeier. Stable spermatozoal RNAs. Fertil Steril 2005. Fertility and Sterility 2005 83, 1687-1694DOI: (10.1016/j.fertnstert.2004.12.046) Copyright © 2005 American Society for Reproductive Medicine Terms and Conditions

FIGURE 4 Full-length transcripts exist among spermatozoal RNAs. Ribonucleic acid was isolated from spermatozoa subject to two bouts of freeze-thaw processes. Complementary DNA was created and then interrogated using intron spanning PCR primers (Table 2) designed specifically to amplify the 5′ and 3′ ends as well as the middle of the transcripts CTNNA2 (shown in A) and CYP2D6 (shown in B). For each transcript, the exons are shown as numbered boxes connected by lines representing the related intron. The specific exons interrogated are shown as gray boxes lettered A-F, which correspond to the labeled lanes in the gel electrophoresis images. In each gel image, lanes 1 and 9 show 100-bp ladders; lanes 2, 4, and 7, cDNA products; lanes 3, 6 and 8, water blanks; and lane 5, a genomic positive control. Ostermeier. Stable spermatozoal RNAs. Fertil Steril 2005. Fertility and Sterility 2005 83, 1687-1694DOI: (10.1016/j.fertnstert.2004.12.046) Copyright © 2005 American Society for Reproductive Medicine Terms and Conditions