Differential Expression of Genes Encoding Constitutive and Inducible 20S Proteasomal Core Subunits in the Testis and Epididymis of Theophylline- or 1,3-Dinitrobenzene-Exposed Rats1

Abstract

Theophylline (THP) and 1,3-dinitrobenzene (DNB) are thought to induce infertility by incapacitating the nurturing Sertoli cells and causing germ cell apoptosis in the testicular seminiferous epithelium, respectively. We hypothesized that THP and DNB exposure would alter the expression of the genes within the ubiquitin-proteasome pathway (UPP), implicated in spermatogenesis and epididymal sperm quality control. Rats were fed 0 or 8000 ppm of THP and necropsied on Days 18, 30, and 42 or administered 0, 2, or 6 mg/kg DNB via oral gavage and necropsied on Day 7. Tissues were collected from the testis and the caput, corpus, and cauda regions of the epididymis for transcriptional profiling by semiquantitative RT-PCR, real-time RT-PCR, and histopathology. Target UPP genes included those encoding for constitutive the 20S proteasomal core subunits Psmb1 (beta1), Psmb2 (beta2), and Psmb5 (beta5); the inducible 20S core subunits Psmb9 (LMP2), Psmb8 (LMP7), and Psmb10 (LMP10); and Ube1 (ubiquitin-activating enzyme E1), Ube2d3 (ubiquitin-conjugating enzyme E2), and Uchl1 (ubiquitin C-terminal hydrolase PGP9.5). Spermatozoa were collected from the cauda epididymis for analysis by light microscopy and flow cytometric evaluation of sperm surface ubiquitin. These data show that reprotoxic exposure alters the tissue-specific expression of UPP genes in the testis and epididymis, which may contribute to the aberrant spermatogenesis and epididymal processing of both normal and defective spermatozoa. Transcriptional profiling and flow cytometric analysis of the UPP thus captures the prodromal effects of reproductive toxicity not captured by conventional histology and functional cytology. Complementing seminal analysis with these measures may be useful in screening drug-induced toxicity or environmental infertility.

A comparative study of protein carbonylation and mitochondrial dysfunction using the neurotoxicants 1,3-dinitrobenzene, 3-nitropropionic acid, and 3-chloropropanediol

“…Another important consideration regarding the results of this study that was initially discussed in previous work is how relatively few proteins were identified as targets of oxidative carbonylation when compared to the overall number of proteins present in a DI TNC1 cell’s proteome; an observation made apparent by simply comparing an immunoblot with its corresponding 2D gel. This consistent finding might be explained by one of multiple reasons that include how only the most oxidationsensitive proteins are carbonylated at the toxicant concentrations used, that carbonylation stimulates proteasomal degradation of oxidized proteins, or that protein carbonyl formation may be dependent on molecular weight given that the majority of the carbonylated proteins identified in the current study were distributed among a mass range of 50–70 kDa (Floor and Wetzel 1998; Ceccarini et al, 2007; Tengowski et al, 2007). A recent study surveying current methods of detecting carbonylated proteins using immunoblotting discussed how derivatizing proteins with DNPH after electrophoresis resulted in the detection of more carbonyl-modified proteins than if one were to derivatize proteins prior to electrophoresis (Linares et al, 2011).…”

Sperm proteasome and fertilization

“…Proteasomes are tethered to acrosomal membranes and the acrosome surface (Morales et al 2004, Sutovsky et al 2004, Yi et al 2009b, most likely inserted there during acrosomal biogenesis in the spermatid (Mochida et al 2000). We have detected various proteasomal subunits and components of the UPS in the acrosomal granule and cap of rat spermatids (Tengowski et al 2007) but have only incomplete data from livestock species. A possible candidate for proteasome anchoring molecule in the acrosomal membranes is immunophilin FKBP38, shown to tether proteasomes to mitochondrial membranes (Nakagawa et al 2007).…”

Análise de citocinas pela RT-PCR em pacientes com rinite alérgica

“…The standard coefficient was then acquired from dividing the β-Actin value from each patient by the average of the 30 patients. This dosage was called relative because we measured the control expression in relation to the standard test [4][5][6][7] . In the statistical analysis, among the measures of interest, we used parametric and non-parametric tests, with statistical significance level fixed at 5% and a confidence interval at a minimum of 80% and a maximum of 95%.…”