Using a newly developed polyclonal antibody against murine cyclin D3, we have found that protein levels of cyclin D3 were highly detectable only in thymus and testis in rats. Since testis offer unique opportunities to examine the cell cycle in vivo, we examined the temporal and spatial expression of cyclin D3 and the DNA synthesis indicator, proliferating cell nuclear antigen (PCNA), in the rat testis during development, The protein levels of cyclin D3 protein in testis from 7 days to 3 months old were almost constant and then decreased gradually thereafter. The protein levels of cyclin D1 and PCNA were high in the testis of 7- and 14-day-old rats and decreased during testicular development. In the seminiferous tubules of 7-day-old newborns, cyclin D3 was surprisingly located in cytoplasm of stem cells that had bigger nuclei than the nuclei of surrounding cells. Interestingly, cyclin D3 immunopositive cells did not immunostain with PCNA in nuclei. In the adult testis, anti-cyclin D3 antibody strongly stained the cytoplasm of early stage primary spermatocytes, lightly stained pachytene spermatocytes, but did not stain elongated spermatids. There was no detectable cyclin D3 in Sertoli cells, interstitial cells, or fibroblasts within seminiferous tubules, or in blood vessels within the interstitial matrix. The known cyclin D3 partner, cyclin dependent kinase 4, was located mainly in nuclei of spermatogonia and in early stage primary spermatocytes, Strong PCNA immunopositive staining was located in the nuclei of spermatogonia in adult testis. These results indicate that cyclin D3 is detectable in meiotically active male germ cells (PCNA-negative cells), but is conspicuously absent fi om mitotically active spermatogonia (PCNA-positive cells). Moreover, in contrast to in. vitro reports, cyclin D3 is not located in the nucleus, but rather in the cytoplasm of male germ cells in vivo. Taken together, the presence of cyclin D3 in spermatocytes and its location in the cytoplasm lead us to speculate that cyclin D3 may have functions in male germ cells other than mitosis. (C) 1997 Academic Press.