Immunolocalization of alpha-tubulin, gamma-tubulin, and CENP-E in male rat and male mouse meiotic divisions: pathway of meiosis I spindle formation in mammalian spermatocytes.

Recent findings on cell division suggest that differences exist in spindle organization not only between mitotic and meiotic systems, but also between female and male meiosis. In mammals, this has been difficult to demonstrate due to lack of appropriate methods. By taking advantage of the strict organization and ordered kinetics of mammalian spermatogenesis, we harvested highly enriched populations of dividing mouse and rat spermatocytes using transillumination-assisted micro-dissection of seminiferous tubules. In the spermatocytes, we examined the localization and distribution of microtubules, centrosomes, and kinetochores at different phases of the first meiotic division using immunohistochemistry with antibodies against alpha-tubulin, gamma-tubulin, and CENP-E, respectively. Fluorescence and confocal microscope analysis of dividing spermatocytes provides evidence that the formation of the male mammalian meiosis I spindle differs from that of female meiosis and mitosis. A short (1-2 microns) bipolar aggregate of microtubules is nucleated by two adjacent centrosomes located next to the nucleus. After nuclear envelope breakdown, adjacent centrosomes and the short spindle become surrounded by the mass of paired meiotic chromosomes. At prometaphase the distance between the centrosomes increases resulting in elongation of the microtubule arrays and eventually formation of a full-length metaphase spindle (12-14 microns). Based on these results we suggest a model for spindle morphogenesis in mammalian spermatocytes.