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Accumulation of the mitochondrial form of the sulphydryl oxidase Erv1p/Alrp during the early stages of spermatogenesis

Monika Klissenbauer¹, Silke Winters², Uwe A. O. Heinlein² and Thomas Lisowsky^1,*

¹ Institut für Botanik, Heinrich-Heine-Universität Düsseldorf, Universitätsstraße, D-40225 Düsseldorf, Germany
² Institut für Genetik, Heinrich-Heine-Universität Düsseldorf, Universitätsstraße, D-40225 Düsseldorf, Germany

View larger version (63K): [in a new window]   Fig. 1. Expression of Alrp in rat organs and blood samples. Samples (20 µg) of total protein extract were separated in non-reducing 4 % to 12 % gradient gels. The respective western blot was probed with an antibody specific for mammalian Alrp. Proteins were analysed with (+) and without (-) reducing agent (20 mmol l-1 dithiothreitol) in the sample buffer. The molecular masses (MW) of marker proteins are listed in kDa. Three forms of Alrp with molecular masses of 23, 21 and 15 kDa were identified. Under non-reducing conditions, all three forms of Alrp exist as dimers. S, spleen; K, kidney; H, heart, L, lung; C, cerebellum; B, brain; Li, liver; F, fat tissue; M, muscle, T, testis; LB, liver blood; vB, venous blood; aB, arterial blood. DTT, dithiothreitol.

View larger version (37K): [in a new window]   Fig. 2. Comparison of rat Alrp expression with that of actin, mitochondrial subunit Vb of cytochrome oxidase (CoxVb) and mitochondrial cytochrome c (Cyt c). Samples (20 µg) of protein were separated in a non-reducing 4 % to 12 % gradient gel. The respective western blot was probed with antibodies specific for mammalian Alrp, actin, CoxVb and Cyt c as indicated. Proteins were analysed with (+) and without (-) reducing agent (20 mmol l-1 dithiothreitol) in the sample buffer. The sample from muscle demonstrates dimer formation. The molecular masses (MW) of marker proteins are listed in kDa. Li, liver; K, kidney; H, heart; M, muscle, S, spleen; L, lung; B, brain; F, fatty tissue; LB, liver blood; vB, venous blood; aB, arterial blood; T, testis. DTT, dithiothreitol.

View larger version (33K): [in a new window]   Fig. 3. Expression of Alrp in mice from postnatal days 13-29. Testis samples from two animals were prepared for each listed day. Samples (20 µg) of protein were separated in a 4 % to 12 % gradient gel. The respective western blots were probed with antibodies specific for mammalian Alrp, mouse cyritestin and actin. Alrp is detectable at the earliest stages of spermatogenesis examined. The largest amounts of Alrp are observed at days 13, 19, 25 and 29. In contrast, cyritestin is expressed during the later stages of spermatogenesis. The molecular masses (MW) of marker proteins and of cyritestin (110kDa) and actin (42kDa) are listed.

View larger version (56K): [in a new window]   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.

View larger version (156K): [in a new window]   Fig. 5. Alrp immunodetection with alkaline-phosphatase-conjugated secondary antibody. (a) Sagittal section through seminiferous tubule with immunopositive (brown colour) spermatogonia along the basal lamina. (b) Lateral section through the spermatogonial layer of the seminiferous epithelium. Scale bar, 50 µm.

View larger version (153K): [in a new window]   Fig. 6. Confocal laser scanning microscope image of double-immunofluorescence assay using anti-Alrp (green) and anti-CoxVb (mitochondrial subunit Vb of cytochrome oxidase) (red). The highest levels of Alrp are observed in spermatogonia and elongated sperm cells, whereas the highest levels of CoxVb are found in other cells. The yellow colour therefore indicates that co-localisation of Alrp and CoxVp inside mitochondria occurs only when approximately equal amounts of the two proteins are present. Scale bar, 50 µm.