UBE2 showed a distinct perinuclear focus in the principal cells of the control epididymis, reminiscent of endoplasmic reticulum localization pattern (Fig. 9B). Some of the principal cells showed a nuclear localization of the ubiquitinated protein species recognized by antibody MK12-3 (Fig. 9B). The endoplasmic reticulum-like localization pattern was absent from the DNB epididymis (Fig. 9B’), while the nuclear accumulation of anti-ubiquitin immunoreactive proteins was more pronounced (Fig. 9B0, insert). UCHL1 showed similar patterns in both the control (Fig. 9C) and DNP-exposed epididymis (Fig. 9C0).
In both types of tissue, a diffuse labeling was present in the cytosol of the principal cells, while a concentrated labeling that coincided with that of the ubiquitinated proteins was seen in the clear cells. A similar distribution pattern was followed by the proteasomes in the principal and clear cells of the control (PSMA/PMSB; Fig. 9D) and DNB-exposed (Fig. 9D0) epididymis. In this example, proteasomes were identified by a polyclonal antibody that recognized multiple 20S proteasomal a and p subunits. Noteworthy was the accumulation of ubiquitin and/or ubiquitinated substrates on the apical surface of the epididymal epithelium, recognized by anti-ubiquitin antibody KM691. Negative control slides did not show any of the patterns described above for the testis or epididymis in the control or DNB-exposed animals.