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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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Differential Expression of Genes: RESULTS(8)

Similarly, the relative expression of Psmbl after DNB exposure was the same in the semiquantitative and RT-RT methods, except for the increased expression shown by the semiquantitative method in the cauda epididymis. Similar to our semiquantitative studies, the RT-RT analysis of Psmbl showed a significantly reduced expression in the THP-treated animals when compared with the control animals (Supplemental Table 4B, line 1). While semiquantitative PCR did not show a significant difference among treatment days (Supplemental Table 4B, line 2), RT-RT showed that the mean RQ value of 42 days was significantly lower than in the 18- and 30-day animals, while the 18- and 30-day groups were not significantly different from each other.

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Differential Expression of Genes: RESULTS(7)

RESULTS(7)

On the basis of the compounded relative gene expression in all animals, wherein each gene was compared against Actb, the highest induction of expression of the UPP genes was observed in the caput epididymis and testis, followed by the corpus epididymis in both trials (Fig. 6, A and B, and Supplemental Fig. 2, A and B; available online at www.biolreprod.org). Overall, the lowest levels of UPP gene expression relative to actin gene expression were noted in the cauda epididymis (Fig. 6, A and B).

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Differential Expression of Genes: RESULTS(6)

There was a significant reduction in the expression of Ube2d3 after DNB treatment (Supplemental Table 3A).

In segment analysis (Fig. 5 and Supplemental Table 3, A and B; available online at www.biolreprod.org), there was a significantly (P < 0.01) reduced expression of all three constitutive proteasomal subunits in the corpus epididymis after THP treatment. Also, Psmb8 showed a significant reduction in the testis and caput epididymis and a significant increase in the cauda epididymis.

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Differential Expression of Genes: RESULTS(5)

RESULTS(5)

To evaluate the effect of DNB on mature sperm, flow cytometric analysis was performed in sperm samples from the control and DNB-exposed rats (Fig. 4, A-D). Each line represented peaks of a 5000-measured-cell histogram. The exposure to 6 mg/kg DNB significantly reduced the levels of ubiquitin in the sperm sample, causing the shift of the histogram peak to the left, toward low-fluorescent cells (Fig. 4, A and B). This was not caused by sperm fragmentation, as seen after THP exposure. Compared to the control rats, the average ubiquitin median levels (Fig. 4C) were increased slightly in the 2-mg/kg DNB group (P = 0.46) and significantly (P = 0.04) in the 6-mg/kg group.

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Differential Expression of Genes: RESULTS(4)

Many spermatozoa of a THP-exposed rat on Day 18 were still within marker M2; however, the peak was wider and shifted to the right toward the higher ubiquitin level (Fig. 3B), where a distinct second peak of highly fluorescent, high-ubiquitin cells was noted within marker M3.

On Day 30 (Fig. 3C) and Day 42 (Fig. 3D), the THP-exposed rat sperm samples displayed an increased amount of sperm fragments with lesser relative fluorescence and a reduced contribution of normal spermatozoa within marker M2.

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Differential Expression of Genes: RESULTS(3)

RESULTS(3)

Flow Cytometry

Samples for flow cytometric sperm evaluation were processed with monoclonal anti-ubiquitin antibody KM691, causing intense fluorescence of the surface (no permeabiliza-tion was used during processing) of the defective spermatozoa from both the control and the THP- and DNB-exposed rats (see Fig. 4D for an example). It was expected that a toxic exposure would increase the surface ubiquitination of defective cauda epididymal spermatozoa because of a previously established association between sperm abnormalities and sperm surface ubiquitination.

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Differential Expression of Genes: RESULTS(2)

Two such animals had anomalies in the testis, and two had anomalies in the epididymis. Only one of the eight rats given 2 mg/kg DNB and one of the seven control rats showed similar anomalies. The most frequent pathologies observed in the DNB-exposed rats (Fig. 1, J-O) were the degeneration and loss of spermatocytes and round spermatids, sometimes multifocally distributed, which affected multiple stages. Formation of multinucleated spermatids was also observed (Fig. 1M).

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