The sperm centrosome is an essential organelle playing a key role just after penetration into the oocyte. It serves to organise the sperm aster, which is required for syngamy and formation of the first mitotic spindle. It is also associated with acquisition of motility during epididymal transit. Previously, we demonstrated that testicular spermatozoa exhibit reduced developmental potential after oocyte injection due to the presence of an immature centrosome [Comizzoli et al. 2006 Biol. Reprod. 75, 252–260]. Centrosome and flagellum maturation naturally occur during epididymal transit where secreted proteins impart changes on the sperm to acquire its functional properties. The objective of this study was to better understand centrosome and flagellum maturation and identify key proteins that could be used to artificially mature testicular spermatozoa. Specifically, we focused our effort on cenexin, a protein that has been reported to aid in maturation of the flagellum and somatic cell centrosome. Epididymides were dissected from adult cat testes (>1 yr old). Spermatozoa were then extracted from the different regions (caput, corpus, cauda, and vas deferens) by slicing with a scalpel blade in phosphate buffered saline at 37°C and processed separately. Control samples were also collected from the rete testis. After recording sperm motility and forward progressive movement (FPM, from 0 = immotile to 5 = fast and straight), cells were fixed in 4% paraformaldehyde and immunostained with anti-cenexin antibodies labelled with a fluorescent probe. The proportion of cells with cenexin at the location of the centrosome and the intensity of immunofluorescence were quantified (n = 8 and 4 testes, respectively). The same methods were followed for detection of cenexin in the tail portion (n = 4 testes). Statistical analyses were conducted using repeated-measures and treatments were further compared using either a protected Tukey's or F-test for orthogonal contrasts. The proportion of sperm with cenexin localised at the centrosome progressively increased along the tract with the lowest percentage of stained cells in the testis and highest proportion in the cauda (45 v. 81%, T28 = 4.65, P < 0.0001). Among the labelled sperm, the intensity of immunofluorescence also significantly increased from the testis to vas deferens (4.33 v. 8.57 mean grey value; T12 = 3.29, P < 0.0065). Both motility and FPM increased from the testis to cauda segment (0 v. 93%, F4,15 = 13.53, P < 0.0001 and 0 v. 3.8 FPM, F4,15 = 26.67, P < 0.0001); however, the proportion with cenexin in the tail (range, 20 to 36%) as well as the labelling intensity (range, 3.14 to 5.26 mean grey value) did not change (P > 0.05) along the tract. These results clearly indicate that cenexin may be associated with centrosome but not flagellum maturation. Epididymal epithelial cells and luminal fluid from each segment are being examined to better understand the source of cenexin secretion and its incorporation into spermatozoa. Results from these studies will aid in further understanding the physiology of sperm maturation during epididymal transit and increase male fertility preservation options.
Free Amino Acids and Protein in Rete Testis Fluid, Vas Deferens Plasma, Accesory Sex Gland Fluid, and Seminal Plasma of the Conscious Bull