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.
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).
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.
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.
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.
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.
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).
Histology and Semen Quality
No changes in the histology of testicular and epididymal tissues were observed in control rats at any time point (Fig. 1, A-C) or in the THP-exposed rats on Day 18 (data not shown; Supplemental Table is available online at www.biolreprod. org). Histological changes of seminiferous epithelia were noted in three of six exposed animals on Day 30 and in five of six rats on Day 42 (Supplemental Table 1). Sloughing of spermatids in the form of multinucleated bodies (Fig. 1, D-I) was the most frequent testicular anomaly and was consistent with the proposed mechanism of the THP action on Sertoli cells. This translated into the depletion of spermatogonia and spermatids from seminiferous epithelium and a reduction in the number of fully differentiated spermatozoa in the testis and epididymis.
Real-Time Quantitative PCR
Two-step RT-PCR, multiplex PCR, TaqMan probe-based chemistry, and the Comparative CT Method were used. TaqMan probes and primers (Table 2) were designed with Primer Express (Applied Biosystems). In all cases, the target amplicon spans an intron. A RealMasterMix Probe (Rox) was used as the real-time PCR reagent. On the basis of semiquantitative RT-PCR data from the present study and from a separate pilot study on the reproductive toxicity of DNB (unpublished results), two marker genes were selected: Ube2d3 and Psmbl.
The PCR products were resolved by electrophoresis on a 2% agarose gel stained poststain with ethidium bromide. Gels were photographed on top of an ultraviolet illuminator. The PCR products were confirmed as target sequences by sequencing with the Applied Biosystems 3730 DNA Analyzer (Applied Biosystems, Foster City, CA) by means of Applied Biosystems Prism BigDye Terminator cycle sequencing chemistry at the DNA core facility of the University of Missouri at Columbia.