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Differential Expression of Genes: DISCUSSION(9)

DISCUSSION(9)

While we found that both constitutive

p subunits and their inducible counterparts were expressed in the testis and epididymis, only the constitutional subunits showed significant expression changes in the tissues of the exposed animals. Products of two of the three examined UPP enzyme genes not directly associated with the proteasome, the Ubel and Ube2d3 genes, also showed significant changes overall or in some segments. This indicates that the screening of the ubiquitin system provides valuable information about the effect of toxic exposure on the male reproductive system.

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Differential Expression of Genes: DISCUSSION(8)

However, several other genes showed significant expression level changes in certain segments alone after either THP or DNB exposure. This indicates that THP and DNB cause significant changes in gene expression in specific compartments of the male reproductive system. For example, Psmb8 showed a P-value of 0.156 between the control and THP-treated animals when all four segments (i.e., the testis and the caput, corpus, and cauda epididymis) were analyzed together. However, according to the feed-segment interaction statistical analysis, the expression change of Psmb8 in the feed-segment interaction was statistically significant (P = 0.0047).

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Differential Expression of Genes: DISCUSSION(7)

DISCUSSION(7)

Inducible proteasomal core subunits PSMB9, PMSB8, and PSMB10 replace their constitutive counterparts PMSB5, PMSB1, and PMSB2 in the professional antigen-presenting cells but are also present in other cell types, such as eye lens cells and the sperm acrosome. The expression of inducible subunit Pmsb8 as measured by semiquantitative RT was significantly reduced within the testis and corpus epididymis of the THP-exposed rats, and yet overall, the changes in the expression of the other two inducible subunits were not significant. It is possible that the inducible subunit genes are less sensitive to THP.

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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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