MICROTUBULE-ASSOCIATED PROTEIN-2 IN THE RAT TESTIS - A NOVEL SITE OF EXPRESSION

The testis is one of the most abundant sources of microtubule networks . These networks include mitotic and meiotic spindles, the spermatid m anchette and axoneme, and the Sertoli cell cytoskeleton. Microtubules are composed of alpha- and beta-tubulin subunits that are polymerized and stabilized by a variety of microtubule-associated proteins (MAPs). One of these, MAP2, has been extensively characterized as a brain-spe cific protein with the capacity to bind tubulin, cAMP-dependent kinase , and calmodulin. MAP2 mRNA is processed into at least two variants en coding proteins designated MAP2a, MAP2b, and MAP2c. Of the 5.7 kb of c oding sequence in the 9-kb mRNA that encodes MAP2a and MAP2b, a deleti on of approximately 4 kb produces mRNA encoding MAP2c, which consists of only the N- and C-terminal regions of MAP2b. To determine whether M AP2 was present in the rat testis, microtubule preparations were isola ted from adult rat testis and brain by means of taxol-mediated polymer ization and analyzed by gel filtration, ELISA, and Western blotting us ing polyclonal and monoclonal antibodies reactive with MAP2. A 74-kDa protein corresponding to MAP2c was detected in the testis. These resul ts were confirmed by Northern blot analysis of total RNA from adult ra t brain and testis with cDNA probes that distinguish between the known MAP2 splice variants. The predominant mRNAs in testis of 6 kb and 2.5 -3.5 kb corresponded to MAP2c. A single 6-kb mRNA with the potential t o encode MAP2c was detected in enriched preparations of immature Serto li cells and adult Leydig cells, Round spermatids contained at least t wo MAP2 mRNAs between approximately 2.5 and 3.5 kb in size that displa yed a stage-specific pattern of expression. Immunohistochemistry showe d a MAP2-like protein in both somatic and germ cells, with a particula rly distinct localization within the cytoplasm of primary and secondar y spermatocytes at stage XIV of the seminiferous cycle during meiotic metaphase. In addition to cytoplasmic staining, a novel localization o f this protein was observed in the nucleus of many testicular cells.