| BackgroundPhthalate esters (PAEs) is the most widespread environmental hormone pollutants in the world, which can be detected in air particulates, industrial waste water, rivers and soil, solid waste, food and drinking water, and also can be detected in adult urine, blood, amniotic fluid, and urine of newborn. Epidemiological studies have shown that PAEs was a recognized environmental endocrine disruptors (EEDs) which are closely related to reproductive health. If women exposured to PAEs during pregnancy, the babies would be suffering produced cryptorchidism, hypospadias, testicular cancer in adulthood, and risk of such low quality of semen significantly increased. Dibutylph -thalate (DBP) is one of commonly used PAEs, which is mainly as a plasticizer and additives of supplies that widespread in the environment. The male reproduction growth toxicity is the main toxic effect of DBP, also testicular atrophy, epididymal dysplasia and hypospadias are main performance of the rats exposed to DBP.Sertoli cells involve in spermatogenesis, which provide the necessary structural support, supplied nutrition, and regulation. Sertoli cell dysfunction can lead to disorders of spermatogenesis and germ cell loss. When male rats exposed to DBP, the Sertoli cell-related protein expressed abnormally and the Sertoli cell structure and function altered. This leads to seminiferous tubule changes in the structure and function, also results in spermatogenic dysfunction. At present, studies have shown that DBP can damage Sertoli cells, but its mechanism is very complicated, the exact injury mechanism is not clear. So far, DBP on Sertoli cell injury-specific markers had not yet discovered. Therefore, studying injury mechanisms and screening of the specific injury biomarkers of Sertoli cells which exposed DBP, it would provide evidence that monitor damage and develop effective preventive measures to injury mechanism of Sertoli cells which exposed DBP.ObjectiveTo analyse the differential protein expression profiling in Sertoli cells of rats which exposed to DBP by comparative proteomics method, to explore the damage mechanism of Sertoli cells which exposed to DBP at the protein levels, and try to find the specific protein biomarkers of DBP damagment to Sertoli cells.Methods1. Isolation, purification, culture and identification of rat Sertoli cells in vitro: Take 18 to 21 day-old male SD rat testis, culture primary Sertoli cells in vitro, identify cell viability by trypan blue, and identify cells purity by oil red O.2. Establishment of rat Sertoli cell model damaged by DBP: The different concentrations of DBP were added to primary cultures of rat Sertoli cells, and DMSO was used in control group.3. The rat Sertoli cell morphological changes were observed by light microscope and electron microscope.4. Proteomics method was used to screen different expressed proteins: to extract total protein of Sertoli cells, then analyze and identify the differences protein by two-dimensional electrophoresis (2-DE) and matrix-assisted laser desorption ionization time of flight tandem mass spectrometry (MALDI-TOF-MS/MS).5. Western blot method was used to validate the proteomics results.Results1. Morphological changes: Compared with the control group, the number of Sertoli cells reduced and the morphology of Sertoli cells changed under light microscope; Sertoli cell ultrastructure changed under electron microscopy.2.Compared with the control group, seven different expressed proteins were identified: Centromere protein -1, Myl6 protein, peroxiredoxin-5, cell division protein kinase 16 isoform b, Myl9 protein , profilin protein increased, and phosphatidylethanolamine-binding protein 1 decreased.4. Peroxiredoxin-5 and phosphatidylethanolamine-binding protein 1 were further validated by Western blot method. The western blot results were consistent with the two-dimensional electrophoresis results.Conclusions1. DBP could make Sertoli cells morphological change, leading to toxic effect on Sertoli cells.2. In this study, we successfully found seven different expressed proteins, which were centromere protein, peroxiredoxin-5, phosphatidylethanolamine-binding protein 1, profiling protein, and that related with male reproductive and developmental toxicity mechanisms. The damage mechanism may be: inhibit cell growth and proliferation by apoptosis; increased of germ cell apoptosis by abnormally expressing of vimentin and tubulin, cytoskeleton destruction.3. Centromere Protein may be the specific protein biomarkers of DBP damaged the Sertoli cells. |
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