Fig. 4. Gradient separation of testis cells and expression analysis for Alrp, cyritestin and mitochondrial subunit Vb of cytochrome oxidase (CoxVb). Samples (20 µg) of protein were separated in 4 % to 12 % gradient gels, and western blots were probed with the listed antibodies. Expression levels of Alrp are clearly distinct from those of testis-specific cyristestin. Alrp is found predominantly in spermatogonia and early spermatocytes. The 110 kDa form of cyritestin accumulates in the later stages of spermatogenesis, with the typical processing into the 55 kDa fragment during the final maturation steps of sperm cells. In contrast, mitochondrial CoxVb is present in comparable amounts in nearly all samples. T, total testis extract; numbers 3-80 represent the first number of the pooled fractions collected from the gradient. Pooled fractions contained predominantly one type of differentiating sperm cell as listed: 3, 3-14/pachytene spermatocytes; 15, 15-24/type A spermatogonia; 25, 25-35/leptotene—zygotene spermatocytes; 36, 36-50/Sertoli cells; 51, 51-56/type B spermatogonia; 57, 57-69/secondary spermatocytes; 70, 71-79/early spermatids; 80, 81-91/late spermatids. St, isolated spermatids from seminiferous tubules; Sp, mature sperm cells from epididymis.