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The aim of the present study is to provide a morphological explanation of carbendazim (CBZ)-induced sloughing of germ cells that occurs in a stage-specific manner. Therefore, very early alterations in the seminiferous tubule epithelium were examined histologically in the rat testis after oral administration of CBZ (400mg/kg). Gaps between the elongated and round spermatids, the first indication of germ cell sloughing (pre-sloughing), were observed in stage late VI-early VII seminiferous tubules at 90-min post-treatment. Tubulin immunoreaction in the Sertoli cells was reduced in intensity in tubules with pre-sloughing. However, electron microscopy demonstrated that there were some intact microtubules in these cells. At 120 min, sloughing was seen in stage late VI-early VII and XIII-XIV. Tubulin immunoreaction in the Sertoli cells was greatly decreased in intensity in tubules where cell sloughing was observed. Electron microscopy showed that there were few microtubules in the body region of these cells. Stages II-V and mid-VII-VIII were exempt from the sloughing effect at 180 min. These changes in microtubules were not observed in Sertoli cells that did not exhibit sloughing characteristics, regardless of the post-treatment intervals. The present results suggest that stage specificity of sloughing is due to the stage-specific susceptibility of Sertoli cell microtubules to CBZ.

Microtubules are involved in many structural and functional changes that occur in Sertoli cells during the cycle of the seminiferous epithelium. However, few studies have addressed stage-specific changes in the distribution of microtubules that accompany the process of spermatogenesis. This study provides a stage-by-stage immunohistochemical evaluation of Sertoli cell microtubules in paraffin sections of whole rat testes using an antibody to tyrosinated alpha-tubulin. Microtubules that contain tyrosinated tubulin are considered to be less stable and are therefore expected to populate Sertoli cells undergoing dynamic changes during spermatogenesis. A quantitative method was developed to analyze the relative tyrosinated alpha-tubulin staining in different stages of the cycle. Immunostaining patterns of the stages were separated into five different groups. Stages VII-VIII had the least amount of tyrosinated alpha-tubulin, while stages IX-XI contained the greatest amount. The staining patterns were consistent with established structural changes in the seminiferous epithelium, such as the formation of ectoplasmic specializations, the presence of microtubule nucleation sites along the periphery of the apical cytoplasm, and the translocation of elongate spermatids from deep crypts in the Sertoli cell to the tubule lumen. These data should provide improved methods for the evaluation of microtubules in the study of Sertoli cell responses to environmental toxicants and testicular diseases.

To improve the study of epithelial function in rat ductuli efferentes (efferent ductules) and initial segment epididymis, we developed a primary cell culture system with modifications of the Klinefelter method (1992) . The cultured efferent ductal epithelium was grown to confluence and the cells maintained many of the organelles characteristic of these cells in vivo, including dense-staining granules, indented nuclei and apical cilia. Ciliary beat was observed for up to 10 days in culture. Cultured initial segment epithelial cells were elongated and characterized by apical branched microvilli. Electron microscopy revealed intact cell junctions, an endocytotic apparatus and lysosomal granules. Ultrastructurally, the initial segment epithelium contained a well developed Golgi apparatus. For both epithelia, cell characteristics were also confirmed by indirect immunofluorescent staining for cytokeratins 8, 18. Endocytotic activity was detected by the uptake of cationic ferritin at the apical surface and within vesicles. Estrogen receptor and clusterin mRNAs were expressed in the cultured epithelia and no difference was found in their expressions when cultured with or without 10 −9 17-β estradiol. Indirect immunofluorescent staining for clusterin further indicated that this protein was present in the cultures. In conclusion, these in vitro methods will be useful for the investigation of epithelial function in the head of the epididymis.

The effects of a microtubule poison, carbendazim, on rat spermiogenesis were examined for abnormalities of nuclei, acrosome and manchette in round and elongating spermatids in Stages VII-XII on days 7.5, 9.5, 10.0 and 10.5 post-treatment using routine electron microscopy. Spermatid nuclear abnormalities were observed in Stages IX-XI on day 9.5 and at greater post-treatment intervals. Nuclear abnormalities included nuclear distortions, various types of nuclear invaginations and abnormal positioning of the modified nuclear envelope. Acrosomal abnormalities were noted on day 7.5 and at greater intervals. Discontinuous, multiple granular and fragmentary acrosomes were observed in Stages VII-XI. In addition, spermatids with complete absence of acrosome (acrosome-deficient spermatids) were observed in Stages VII-X. Poorlyformed and absent ectoplasmic specializations were seen in the cytoplasm of Sertoli cells next to the acrosome-deficient spermatids. A major abnormality of the manchette was irregular positioning of the manchette microtubules in steps 9–11 spermatids on day 9.5 and at greater intervals, which resulted in nuclear invagination. The results indicate that carbendazim induces abnormalities in spermatid morphology that are common to those reported in testes treated with several chemical compounds and in testes of mutant animals.

Early morphological changes in the rat Sertoli cell induced by the fungicide carbendazim (methyl-2-benzimidazole carbamate; MBC), a metabolite of benomyl, were examined. Adult rats were treated with single doses of MBC (400 mg/kg) or vehicle and examined by light and electron microscopy at 3 hr post-treatment. Sloughing of elongating spermatid clusters was observed in all stages of spermatogenesis, except for Stages III-V. Cleavage occurred near the apical region of the seminiferous epithelium where cytoplasmic processes of the Sertoli cell surround the heads of elongating spermatids. The cleaved cytoplasm remained attached to the sloughed spermatids and ectoplasmic specializations remained undamaged. Intact microtubules were observed in the apical Sertoli cell cytoplasm (including sloughed tissues) but were decreased in the body region, where aggregates of mitochondria were found. Cytoplasm near the cleavage site exhibited rarefaction, which was associated with swollen cisternae of endoplasmic reticulum. It appears that the mechanism of germ cell sloughing induced by MBC treatment involves the disruption of microtubules in the body region of the Sertoli cell, the retraction of cytoplasmic organelles and the swelling of endoplasmic reticulum.