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Differential Expression of Genes: DISCUSSION(6)

We have shown that defective spermatozoa become ubiquitinated in the caput epididymis, presumably by the enzymatic ubiquitination machinery residing within epididymal fluid. The percentage of defective spermatozoa is reduced after passage through the corpus epididymis. Since proteasomes have also been detected in the epididymal fluid, it is possible that defective spermatozoa are partially degraded intraluminally in the caput and corpus epididymis and that the resident clear cells of the corpus epididymis take up and degrade the ubiquitinated proteins released from moribund spermatozoa.

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Differential Expression of Genes: DISCUSSION(5)

DISCUSSION(5)

Alzheimer disease has been linked to the aberrant transcription of the ubiquitin-B (UB-B) gene, caused by a +1 frame-shift during transcription. This misreading results in the translation of the dysfunctional UB-B +1 protein with the elongated C-terminus not capable of ligation to a substrate protein. Consequently, the amyloid protein within neurons is not properly ubiquitinated and degraded by proteasomes, causing the formation of amyloid plaques in Alzheimer disease-affected brain tissue. Intriguingly, the UB-B+1 frame shift product has been found in the aging human epididymis.

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Differential Expression of Genes: DISCUSSION(4)

It could also be argued that the apocrine-secretory capabilities of the epididymal epithelium were impaired, resulting in a reduced content of UBE2 and other ubiquitin system enzymes in the epididymal fluid. We did not observe any major morphological changes in the epithelia, and the accumulation of presumed secretory ubiquitin on the apical secretory sites of the DNB epididymis appeared comparable to the control rats.

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Differential Expression of Genes: DISCUSSION(3)

DISCUSSION(3)

While this shortcoming could be compensated for by dual DNA ubiquitin flow cytometry, we found the current analysis, which was based on the distribution within markers M1-M3, sufficiently informative and reflective of THP exposure.

Because of the previously established association between sperm abnormalities and sperm surface ubiquitination, we expected that a toxic exposure to DNB in the absence of sperm fragmentation (as observed in THP rats) would increase the surface ubiquitination of defective cauda epididymal spermatozoa.

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Differential Expression of Genes: DISCUSSION(2)

Similarly, several components of the UPP showed a statistically significant expression change on Day 18 in expression analysis. On the basis of our studies of sperm ubiquitin in infertile men, we could expect an overall reduction in the percentage of presumably normal spermatozoa with background levels of surface ubiquitin (see Fig. 3, A and B) and predict an overall increase of the sperm ubiquitin levels in THP- and DNB-exposed rats. While we indeed observed a significant reduction in the normal sperm percentage after THP exposure, the overall sperm ubiquitin values were actually lower in the exposed animals than in the control animals on Days 30 and 42.

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Differential Expression of Genes: DISCUSSION(1)

DISCUSSION(1)

The present study demonstrates the reproductive toxic and spermatotoxic effects of THP and DNB by conventional means (histology and DIC microcopy) and by novel approaches, including flow cytometric analysis of isolated epididymal spermatozoa and transcriptional profiling of select gene products within the proteolytic UPP. Our goal was to determine if transcriptional analysis and flow cytometry could capture minor changes in the male reproductive system of rats exposed to reprotoxic chemicals. We were particularly interested in examining threshold toxic exposures at which a conventional histological examination did not reveal any pathology.

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Differential Expression of Genes: RESULTS(12)

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).

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Differential Expression of Genes: RESULTS(11)

RESULTS(11)

In the control testis, the distribution of PSMB9 showed a diffuse pattern in the nuclei and cytoplasm of all types of spermatogenic cells. Distinct accumulation was observed in the cytoplasmic lobe of elongated spermatids, in the residual bodies (Fig. 8D), and in the acrosomal cap of the round and elongated spermatids (Fig. 8D, insert). Acrosomal and residual body localization of PSMB9 (LMP2) was also seen in the DNB testis (Fig. 8D0, top insert), while there seemed to be an increased diffuse labeling in the cytoplasm and nuclei of the degenerated, single, and multinucleated spermatids.

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Differential Expression of Genes: RESULTS(10)

Similar to UBE1, UBE2 was localized in the residual body and cytoplasmic lobe (Fig. 8B) but also within the developing acrosome of round and elongating spermatids (Fig. 8B, top insert) and in the chromatin of the pachytene and dividing spermatocytes (Fig. 8B, bottom insert). In the DNB testis, UBE2 and ubiquitin showed a distinct accumulation in the multinucleated spermatids (Fig. 8B0). While the acrosomal localization was still observed in the spermatids, there was no labeling in the pachytene and dividing spermatocytes (Fig. 8B0, insert). UCHL1 was concentrated in the residual bodies and within the developing acrosomal caps of the round spermatids in the control testis (Fig. 8C).

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Differential Expression of Genes: RESULTS(9)

RESULTS(9)

The RT-RT PCR method confirmed that the expression of Ube2d3 was reduced by THP exposure in some of the examined compartments (Supplemental Table 4D). Similarly, the expression of Ube2d3 was reduced in the testis and cauda epididymis of the 2- and 6-mg/kg DNB-exposed animals (Fig. 7).

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