1 Introduction Meri et al., 1990 Powell et al., 1997 Holt et al., 2001; Turnberg et al., 2003, 2004; Lin et al., 2004; Mead et al., 2004; Williams et al., 2004 Qian et al., 2000 Harris et al., 2003 Qin et al., 2003 Baalasubramanian et al., 2004 Qin et al., 2006 We, and others, are using CD59a knockout mice in disease models to explore roles of MAC based on our evidence that CD59a is the principal regulator of the MAC in most tissues. If CD59b is indeed widely distributed then the value of studies in CD59a knockouts is in question. It is therefore essential that we test the evidence. Here using multiple methods we revisit these published studies and undertake new analyses to explore the expression patterns of CD59a and CD59b at the mRNA and protein level. We conclude that expression of CD59b at the mRNA and protein level is essentially absent in all tissues other than testis. Low level expression on blood cells was confirmed and trace detection of mRNA in tissues was shown to be likely due to blood contamination. We further analysed expression of CD59b in testis and showed that expression coincided with onset of puberty and was restricted to spermatozoa and their immediate precursors. Ligation of CD59b on spermatozoa with monoclonal antibody markedly inhibited sperm motility, suggesting a specific role in reproductive function. 2 Materials and methods 2.1 Mice b cd59a Holt et al., 2001 2.2 Antibodies and reagents Harris et al., 2003 Baalasubramanian et al., 2004 2.3 Semi-quantitative RT-PCR Table 1 Qin et al. (2006) Table 1 2.4 Quantitative real-time PCR analysis Baalasubramanian et al., 2004 Baalasubramanian et al., 2004 Table 1 Qin et al. (2006) Table 1 2.5 Preparation of tissue lysates g 2.6 SDS-PAGE and Western blot analysis Lysates were mixed 1:1 with sample buffer for SDS-PAGE and separated under non-reducing conditions in 15% gels. Separated proteins were transferred onto nitrocellulose membranes (Schleicher & Schuell, London, UK), and membranes blocked with 5% (w/v) non-fat milk in PBS (PBS-M). Membranes were then probed with the primary mAb diluted in PBS-M, washed in PBS containing 0.1% Tween-20 (PBS-T), then probed with HRPO-conjugated donkey anti-rat Ig in PBS-M to detect the rat anti-CD59a or HRPO-conjugated donkey anti-mouse Ig (Jackson) to detect the mouse anti-CD59b. After further washing in PBS-T, bands were developed using ECL (Perbio Science UK Ltd.) and captured on autoradiographic film (Kodak Ltd., Hemel Hempstead, Hertfordshire, UK). 2.7 Spermatozoa preparation and analysis Mizuno et al., 2004, 2005 g g Mizuno et al., 2004 6 2.8 Functional inhibition assay of CD59a and CD59b in mouse sperm cd59a 2.9 Statistical analysis t P 3 Results 3.1 CD59b mRNA is highly expressed only in testis Baalasubramanian et al., 2004 Qin et al., 2006 Fig. 1 Fig. 1 Baalasubramanian et al., 2004 Qin et al. (2006) Table 1 Sommer and Tautz, 1989; Kwok et al., 1990; Christopherson et al., 1997; Löffert et al., 1998 Table 1 Baalasubramanian et al., 2004 Table 2 Table 1 Fig. 2 Table 2 Baalasubramanian et al., 2004 Fig. 2 Qin et al., 2006 Fig. 3 5 5 Fig. 3 Table 2 Baalasubramanian et al., 2004 5 Fig. 3 5 Baalasubramanian et al., 2004; Qin et al., 2006 Fig. 3 3.2 Expression of CD59b protein is testis restricted Baalasubramanian et al., 2004 Qin et al., 2004 Fig. 4 3.3 Expression of CD59b mRNA in testis coincides with puberty and plays a role in spermatozoal motility Baalasubramanian et al., 2004 Fig. 5 Fig. 5 Fig. 5 Baalasubramanian et al., 2004 Fig. 6 Fig. 6 Fig. 7 cd59a Qin et al., 2003 4 Discussion Baalasubramanian et al., 2004 Qin et al. (2006) in silico Figs. 3 and 4 Fig. 3 Qin et al. (2001) Fig. 5 Fig. 6 Fig. 7 cd59b Qin et al., 2005 5 Concluding remarks Baalasubramanian et al., 2004 Qin et al., 2006