Fig 1.
Treatment with nicotinamide (NAM) and Sirtinol affects meiotic maturation in porcine oocytes.
(A) In cumulus-oocyte complexes (COCs), the peripheral layer of the cumulus expanded to more than 5 layers in the control group, whereas it became progressively thinner in the NAM- and Sirtinol-treated COCs. Most oocytes in the control group extruded the first polar body (indicated by white arrow). Upon treatment with NAM and Sirtinol, a large proportion of oocytes failed to extrude polar bodies. Bar = 100 μm. (B) Rate of polar body extrusion in the control, NAM-, and Sirtinol-treated groups. Rate of polar body extrusion in treatment groups was significantly reduced comparing to control group. *, significant, p < 0.05.
Fig 2.
NAM and Sirtinol treatment disrupts actin cap formation.
(A) Actin signals in the oocytes were examined by immunofluorescence staining. Oocytes treated with NAM and Sirtinol exhibited profoundly decreased actin fluorescence intensity. Bar = 20 μm (B) Actin fluorescence intensity at the membrane in porcine oocytes. The signal intensity of ROI of actin was the entire area of the oocyte membrane. Compared to control oocytes, actin fluorescence intensity was significantly decreased in inhibitor-treated oocytes. *significant, p < 0.05.
Fig 3.
Effects of NAM and Sirtinol on cortical granule-free domain (CGFD) formation in porcine oocytes.
In the control group, cortical granules (CGs) were absent at the cortex near the site of polar body extrusion in MI oocyte. However, in the treatment group, cortical granules were distributed uniformly across the entire cortex. CGFD formation was failed to observation. The arrow indicates a CGFD. Green, CGs; blue, chromatin. Bar = 20 μm.
Fig 4.
Treatment with NAM and Sirtinol induces meiotic defects in porcine oocytes.
(A) Meiotic spindle morphology in second meiosis (MII) oocytes. Control oocytes had barrel-shaped spindles and well aligned chromosomes, whereas spindle defects were frequently observed in NAM and Sirtinol-treated oocytes. Moreover, chromosomes displayed misalignment in treatment oocytes. Green, Tubulin; blue, chromatin. Bar = 20μm (B) Quantification of spindle defects and chromosome misalignment in control and inhibitor-treated oocytes. The percentage of abnormal spindle morphology was significant higher in the NAM- and Sirtinol-treated oocytes than in the control oocytes. *, significant, p < 0.05.