Iron oxide nanoparticles modulate heat shock proteins and organ specific markers expression in mice male accessory organs.

With increased industrial utilization of iron oxide nanoparticles (Fe2O3-NPs), concerns on adverse reproductive health effects following exposure have been immensely raised. In the present study, the effects of Fe2O3-NPs exposure in the seminal vesicle and prostate gland were studied in mice. Mice were exposed to two different doses (25 and 50 mg/kg) of Fe2O3-NPs along with the control and analyzed the expressions of heat shock proteins (HSP60, HSP70 and HSP90) and organ specific markers (Caltrin, PSP94, and SSLP1). Fe2O3-NPs decreased food consumption, water intake, and organo-somatic index in mice with elevated iron levels in serum, urine, fecal matter, seminal vesicle and prostate gland. FTIR spectra revealed alterations in the functional groups of biomolecules on Fe2O3-NPs treatment. These changes are accompanied by increased lactate dehydrogenase levels with decreased total protein and fructose levels. The investigation of oxidative stress biomarkers demonstrated a significant increase in reactive oxygen species, nitric oxide, lipid peroxidation, protein carbonyl content and glutathione peroxidase with a concomitant decrement in the glutathione and ascorbic acid in the male accessory organs which confirmed the induction of oxidative stress. An increase in NADPH-oxidase-4 with a decrease in glutathione-S-transferase was observed in the seminal vesicle and prostate gland of the treated groups. An alteration in HSP60, HSP70, HSP90, Caltrin, PSP94, and SSLP1 expression was also observed. Moreover, accumulation of Fe2O3-NPs brought pathological changes in the seminal vesicle and prostate gland of treated mice. These findings provide evidence that Fe2O3-NPs could be an environmental risk factor for reproductive disease.

The expression of ß-microseminoprotein but not CRISP3 is reduced in ovarian cancer and correlates to survival.

BACKGROUND: ß-Microseminoprotein (MSMB) is an abundant protein in seminal plasma. Most of it is present as a free protein but a small part is bound to cysteine-rich secretory protein 3 (CRISP3) as a non-covalent complex. Even though their physiological function is unknown, both MSMB and CRISP3 have been ascribed roles in prostate carcinogenesis. Thus, several recent experimental studies indicate a tumor-suppressor role for MSMB. The present study was undertaken in order to evaluate, for the first time, the expression of MSMB and CRISP3 in ovaries and in ovarian tumors and to determine if their expression might indicate a role in ovarian tumor development. MATERIALS AND METHODS: Biopsies from prospectively collected samples from ovaries and benign, borderline and invasive ovarian tumors were analyzed for expression of MSMB and CRISP3 by immunohistochemistry. In patients with ovarian cancer the expression was compared to survival. RESULTS: Both MSMB and CRISP3 were strongly stained in ovarian epithelial cells and weakly stained in the stroma. In ovarian blood vessels, CRISP3 exhibited strong to medium staining, while MSMB was only weakly expressed. In benign and borderline tumors the staining pattern was similar to the one observed in the ovaries. In invasive neoplasms, the expression of MSMB in the tumor cells was significantly reduced. In univariate analysis, decreased expression of MSMB correlated to reduced survival. No correlation was found with stage, the strongest prognostic indicator for ovarian cancer, which supports an independent role of MSMB in ovarian carcinogenesis. For CRISP3, a staining pattern comparable to that for MSMB was observed in all groups, except the fact that decreased expression was not observed in invasive tumor cells. CONCLUSION: MSMB and CRISP3 were widely distributed in ovaries and in ovarian tumors; the expression of MSMB fits well with a tumor-suppressor function in ovarian carcinogenesis.

Reproductive tissue expression and sperm localization of porcine beta-microseminoprotein.

Beta-microseminoprotein (MSP) is a predominant protein of human seminal plasma and originates from prostate secretions. MSP from boar seminal plasma has been sequenced and shows only 50%-52% homology with that of human. Porcine MSP is synthesized by the post-natal prostate gland and is identical with the sperm motility inhibitor. Although MSP is a protein characteristic of the prostate gland, we have established the presence of its mRNA transcript not only in boar prostate but also in other reproductive organ tissues. In extracts of all these organs, specific polyclonal antiMSP antibody recognizes a 12-kDa protein band identified by mass spectrometry as MSP. Immunofluorescence (IMF) has revealed the occurrence of MSP in the epithelial tissue of the prostate, epididymis, seminal vesicles and Cowper's glands. MSP has been localized on epididymal spermatozoa in the acrosomal region and on the flagellum of ejaculated spermatozoa. The absence of MSP on the surface of capacitated spermatozoa together with the antibody detection of MSP in the sperm acidic extract after in vitro capacitation indicates its acrosomal origin. Additionally, MSP has been localized by IMF in the sperm acrosome in capacitated spermatozoa with a permeabilized plasma membrane and by electron microscopy in ejaculated spermatozoa. The function of MSP in seminal plasma and spermatozoa is not fully understood. Nevertheless, the secretion of porcine MSP by various reproductive organs indicates its multiple roles in the reproductive process. For the first time in mammalian species, MSP has been localized in various physiological stages of sperm.

A functional polymorphism in MSMB gene promoter is associated with prostate cancer risk and serum MSMB expression.

BACKGROUND: To explore the reported association of SNP marker rs10993994 with prostate cancer identified by two independent in two genome-wide association studies (GWAS) further, we performed a case-control study in southern Chinese Han population. Consequently, we detected the serum levels of MSMB expression with different genotypes in the cases and controls to characterize the functional consequences of rs10993994. MATERIALS AND METHODS: Two hundred fifty-one prostate cancer and 258 control subjects were included in the cancer association study and 90 serum samples were used to test the expression of the MSMB by Enzyme-linked immunosorbent assay (ELISA). RESULTS: We found that the T allele displayed an increased prevalence of prostate cancer compared with the C allele (OR = 1.30, 95% CI = 1.01-1.67, P = 0.040). Moreover, the prostate cancer patients carrying CT/TT genotype had significantly decreased serum MSMB levels compared to those with CC genotype (16.32 +/- 3.98 microg/L vs. 19.33 +/- 4.27 microg/L, P = 0.022). CONCLUSIONS: rs10993994 in MSMB promoter affects serum MSMB expression, contributes to the genetic predisposition to prostate cancer in southern Chinese Han population.

Expression pattern of serine protease inhibitor kazal type 3 (Spink3) during mouse embryonic development.

Recent evidence shows that the serine protease inhibitor Kazal type 3 (Spink3) has more diverse functions than expected. To gain insight into its function, we analyzed the spatiotemporal expression profile of Spink3, using in situ hybridization (ISH) and a Spink3+/lacZ knock-in mouse, in which lacZ was inserted into the Spink3 locus. Spink3lacZ expression was first observed in the foregut, midgut, hindgut and the forebrain/midbrain junction region at 9.5 days post coitus (dpc). In the pancreas, Spink3 mRNA was detected at 11.5 dpc, before formation of the typical shape of the exocrine structure of the pancreas. Acinar cell expression was clearly identified by 13.5 dpc. After differentiation of the intestinal tract, Spink3lacZ expression was observed in the large intestine at 11.5 dpc, followed by expression in the small intestine at 13.5 dpc, before appearance of intestinal digestive enzymes. Spink3 mRNA and Spink3lacZ activity were also detected in other tissues, including the mesonephric tubules and the urogenital ridge at 11.5 dpc, the genital swelling at 13.5 dpc, the ductus epididymis at 17.5 dpc, and the seminal vesicle at 8 weeks. These data suggest that Spink3 may play important roles in proliferation and/or differentiation of various cell types during development.

Increased intratumoral expression of prostate secretory protein of 94 amino acids predicts for worse disease recurrence and progression after radical prostatectomy in patients with prostate cancer.

OBJECTIVES: To examine the impact of prostate secretory protein of 94 amino acids (PSP94) expression within the primary tumor on disease recurrence and progression using radical prostatectomy specimens from patients with adenocarcinoma of the prostate. METHODS: PSP94 immunohistochemistry was performed on 59 radical prostatectomy specimens. The degree of PSP94 expression was reported as either present (group 1) or absent (group 2). Clinical data, including survival outcome measures, were correlated with PSP94 expression. RESULTS: The time to disease progression for group 1 was shorter compared with that for group 2 (P = 0.042). Disease-free survival was also less in group 1 than in group 2 (P = 0.033). Multivariate analyses demonstrated that PSP94 expression was an independent prognostic factor for a shorter interval to disease progression (P = 0.046) and disease-free survival (P = 0.049). CONCLUSIONS: PSP94 expression in radical prostatectomy tumor specimens appears to be associated with worsened survival outcomes and may provide additional prognostic information.

Differential expression of the trypsin inhibitor SPINK3 mRNA and the mouse ortholog of secretory granule protein ZG-16p mRNA in the mouse pancreas after repetitive injury.

A mouse model using repetitive acinar cell injury caused by supraphysiologic doses of cerulein to induce the characteristic fibrosis and loss of acinar cell mass found in human chronic pancreatitis was employed to identify early changes in gene expression. A gene array was used to detect changes in 18,000 expressed sequence tags; changes in specific transcripts were confirmed by RNase protection assays. These methods identified SPINK3, the mouse homologue of human and rat protease inhibitor genes, as being highly expressed in the pancreas and induced after pancreatic injury. Because SPINK3 may be an important serine protease inhibitor, its up-regulation may reflect an important endogenous cytoprotective mechanism in preventing further injury. The up-regulation of SPINK3 was specific; the mouse homologue of the zymogen-processing protein ZG-16p was also highly expressed in the pancreas but sharply down-regulated early in the course of injury. These findings suggest that the pancreatic acinar cell may respond to injury with a program of self-preservation and loss of normal function.

Transcriptional silencing of zinc finger protein 185 identified by expression profiling is associated with prostate cancer progression.

We profiled the expression of genes in benign and untreated human prostate cancer tissues using oligonucleotide microarrays. We report here 50 genes with distinct expression patterns in metastatic and confined tumors (Gleason score 6 and 9; lymph node invasive and noninvasive). Validation of expression profiles of 6 genes by quantitative PCR revealed a strong inverse correlation in the expression of zinc finger protein 185 (ZNF185), bullous pemphigoid antigen gene (BPAG1), and prostate secretory protein (PSP94) with progression of prostate cancer. Treatment of prostate cancer cell lines with 5-aza-2'-deoxycytidine (5-Aza-CdR), an inhibitor of DNA methylation, restored ZNF185 expression levels. Moreover, methylation-specific PCR confirmed methylation of the 5'CpG islands of the ZNF185 gene in all of the metastatic tissues and 44% of the localized tumor tissues, as well as in the prostate cancer cell lines tested. Thus, transcriptional silencing of ZNF185 by methylation in prostate tumor tissues implicates the ZNF185 gene in prostate tumorigenesis.

Mouse PSP94 expression is prostate tissue-specific as demonstrated by a comparison of multiple antibodies against recombinant proteins.

Prostate tissue-specific gene expression is crucial for driving potentially therapeutic genes to target specifically to the prostate. Prostate secretory protein of 94 amino acids (PSP94), also known as beta-MSP (microseminoprotein), is one of the three most abundant secretory proteins of the prostate gland, and is generally considered to be prostate tissue-specific. We have previously demonstrated that the expression of the rat PSP94 gene is strictly prostate tissue-specific by an antibody against a recombinant rat PSP94. In order to study prostate targeting utilizing the PSP94 gene in a mouse pre-clinical experimental model, we need to establish antibodies against mouse PSP94 to confirm if it is prostate tissue-specific as well. In this study, firstly we raised a polyclonal antibody against a recombinant glutathione-S-transferase- (GST-) mouse mature form of PSP94. However, it showed very poor immunoreactivity against prostate tissue PSP94 as tested in Western blotting experiments. Neither antibodies against rat PSP94 nor mouse PSP94 showed significant cross-reactivity. Thus a second antibody was established against a recombinant mouse mature PSP94 containing N-terminal polyhistidines, and stronger immunoreactivity against mouse prostate tissue PSP94 was identified in Western blotting experiments. Both of these antibodies showed immunohistochemical reactivity, while the latter showed stronger reactivity in IHC when tested with different fixatives. By studying tissue distribution, we demonstrated that, as with rat PSP94, mouse PSP94 is strictly prostate tissue-specific in experiments of both Western blotting and immunohistochemistry (IHC). This conclusion was also derived from a comparison among antibodies against human, rat, and mouse PSP94, showing very different immunoreactivities in Western blotting and IHC. Finally, a competitive assay between different species was performed. We demonstrated that antibodies against PSP94 from different species (human, primate, rodents) have poor cross-reactivities. These observations also indicate that the PSP94 gene is a rapidly evolving gene in all species. Results from this study have led to the possibility of utilizing PSP94 as a targeting agent specifically to the prostate in a mouse experimental model.