Follicular fluid (FF) accumulates in the antrum of the ovarian follicle and provides the microenvironment for oocyte development. FF plays an important role in follicle growth and oocyte maturation. The FF provides a unique window to investigate the processes occurring during buffalo follicular development. The observed low quality of buffalo oocytes may arise from the poor follicular microenvironment. Investigating proteins found in buffalo FF (BFF) should provide insight into follicular development processes and provide further understanding of intra-follicular maturation and oocytes quality. Here, a proteomic-based approach was used to analyze the proteome of BFF. SDS-PAGE separation combined with mass spectrometry was used to generate the proteomic dataset. In total, 363 proteins were identified and classified by Gene Ontology terms. The proteins were assigned to 153 pathways, including signaling pathways. To evaluate difference in proteins expressed between BFF with different follicle size (small, <4 mm; and large, >8 mm), a quantitative proteomic analysis based on multi-dimensional liquid chromatography pre-fractionation tandem Orbitrap mass spectrometry identification was performed. Eleven differentially expressed proteins (six downregulated and five upregulated in large BFF) were identified and assigned to a variety of functional processes, including serine protease inhibition, oxidation protection and the complement cascade system. Three differentially expressed proteins, Vimentin, Peroxiredoxin-1 and SERPIND1, were verified by Western blotting, consistent with the quantitative proteomics results. Our datasets offers new information about proteins present in BFF and should facilitate the development of new biomarkers. These differentially expressed proteins illuminate the size-dependent protein changes in follicle microenvironment.

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The phosphatidylinositol 3-kinase (PI3K)/protein kinase B (PKB/Akt) pathway plays an important role in regulating cell proliferation, metabolism, and survival. However, the distinct roles of Akt isoforms (Akt1, Akt2, and Akt3) in pluripotent stem cell maintenance are not fully defined. Using mouse embryonic stem cells (ESCs), we show that direct inhibition of Akt activity leads to ESC apoptosis. The Akt3, but not Akt1 or Akt2, activity specifically regulates this effect. Inhibiting Akt3 also leads to a cell cycle arrest at G1 phase. These regulatory roles of Akt3 are dependent on its kinase activity. Blocking the expression of Akt1 plus Akt2 in ESCs does not affect cell survival or proliferation, although blocking Akt1 aggravates the apoptotic effect induced by depletion of Akt3. We further show that blocking Akt3 in ESCs results in significant nuclear accumulation of p53, as well as the activation of its downstream targets, such as Mdm2, p21, and Fas. Inhibiting p53 and its downstream targets partially rescued the effects caused by Akt3-depletion. Our results revealed an Akt3 isoform-specific mechanism for ESC survival and proliferation involving the control of p53 activity.