Missing and overexpressing proteins in domestic cat oocytes following vitrification and in vitro maturation as revealed by proteomic analysis

BACKGROUND: The domestic cat serves as an animal model for assisted reproductive studies of endangered felid species. To date, there are no data on the protein alterations following cryopreservation of oocytes in felid family. METHODS: Immature (germinal vesicle) domestic cat oocytes were vitrified in the vitrification solution containing 35% ethylene glycol, 20% DMSO and 0.5 mM sucrose. The vitrified-warmed oocytes were matured (metaphase II) in vitro and subjected to proteomic analysis using 1DE SDS-PAGE prefractionation combined with LC-MS/MS. RESULTS: A total of 1712 proteins were identified in in vitro matured oocytes. Of the 1712 proteins, 1454 proteins were found in both groups, whereas, 258 proteins were differentially expressed between control and vitrified-warmed groups. In vitrified-warmed oocytes, the missing proteins were membrane and nuclear proteins; whereas, apoptosis and DNA repair proteins were overrepresented. CONCLUSIONS: The identified missing and overexpressed proteins in vitrified-warmed oocytes represent potential markers of cryoinjuries and the developmental pathways of oocytes. The findings of differential expressed proteins may contribute to effective ways of proteome analysis of oocyte/embryo quality in order to assess safety of cryopreservation in felid species.

Impairment of CD4+CD25+ regulatory T cells in C4-deficient mice.

Objective: To investigate the association between deficiencies of early components in the classical complement pathway and the development of SLE. Methods: Forty inbred C57BL/6J mice and 40 knockout C4 complement gene (C4KO) mice, which included 10 mice in each age group (2, 4, 6, and 8 months) were used. The enumeration of CD4+CD25+ Tregs frequencies in bone marrow, spleen and peripheral blood from both normal and C4KO groups were performed by flow cytometry. The expression levels of Foxp3 and TGF-b in the same tested tissues were measured using real time PCR. The antinuclear antibodies (ANA) were semi-quantitatively measured using ELISA. Results: We report decreased frequencies of CD4+CD25+ Tregs and reduced expression levels of Foxp3 and TGF-β, which efficiently program the development and function of Tregs, in lymphoid tissues and peripheral blood of C4KO mice. In this study, C4KO mice have higher titers of ANA than those of normal mice. Higher frequencies of mice positive for ANA are also found in older mice.Conclusions: The deficiency of the C4 gene induces the decreased numbers of Tregs that further increase the production of ANA resulting in the development of an autoimmune disorder. The outcomes of our study help us to understand the association between the deficiency of C4 in the classical complement pathway and development of autoimmune disorder via the role of Tregs.

Serum antibodies and cytokines in C4-deficient mice and their responses to exercise.

Psychological stress is believed to be one of the predisposing factors for systemic lupus erythemato-sus (SLE), whereas physical stress such as exercise has never been reported to be related. We measured the cir-culating levels of antibodies (IgM, IgG, anti-dsDNA IgG), Th1 (IFN-γ), Th2 (IL-4, IL-6), and of pro-inflammatory (TNF-α, IL-1β) and anti-inflammatory (TGF-β) cytokines of C4-/- female mice at rest, after acute exercise and after exercise training, using an antibody-capture ELISA. Prior to the exercise, the C4-/- mice had higher levels of IgG and anti-dsDNA IgG but lower levels of IFN-γ, IL-1β, IL-6 and IL-4 than wild-type C57BL/6 (B6) mice. A single bout of exercise to exhaustion increased serum IgG, TNF-α, IL-1β and TGF-β in the B6 mice but only TGF-β in the C4-/- mice was increased. We conclude that exhaustive or moderate exercise has no effect on the levels of serum anti-bodies and cytokines and is thus unlikely to promote the onset of SLE.