Table 1.
Primers used in this study.
Figure 1.
Expression of JMY in different tissues and during porcine oocytes meiotic maturation.
A, B: The relative expression of JMY in different tissues (A) and oocytes (B) were measured by quantitative real-time PCR. mRNA levels were normalized to those in heart. Values represent mean ± s.e.m. *p<0.05, n = 3. C: Protein levels of JMY in maturing oocytes measured by western blotting. Two hundreds oocytes were used per lane. D: Subcellular localization of JMY during porcine oocytes meiotic maturation as revealed by immunofluorescence staining. Red: JMY, Blue: chromatin.
Figure 2.
Knockdown of JMY during meiotic maturation.
A–D: GV oocytes injected with dsRNAs or control were cultured for 44 hours. Knockdown of JMY mRNA was determined by RT-PCR (A) and Western blotting (B). Subcellular localization (C) and quantitized fluorescence intensity (D) of JMY fluorescence of dsRNA or control injected oocytes measured at MI (20 h after culture) and MII (44 h after culture) stages respectively. E: Germinal vesicle breakdown (GVBD) and polar body extrusion (PBE) rates of JMY dsRNA induced oocytes. Red: JMY, Blue: chromatin. Values represent mean ± s.e.m. *p<0.05, n = 5.
Figure 3.
Actin, α-tubulin and Arp2 expression after knockdown of JMY in porcine oocytes.
A: Representative images of spindle defects in JMY knockdown oocytes at MI (20 h after culture) and MII (44 h after culture) stages are shown. Spindle was stained with anti-α-tubulin antibody (Green) and DNA was stained using Hoechest 33342 (Blue). B: Abnormal distribution of actin in control and dsRNA microinjection groups of oocytes at MI (20 h after culture) and MII (44 h after culture) stages. Actin(Red), DNA(Blue). Actin fluorescences were measured and quantitized (n = 6). Values represent mean ± s.e.m, *, p<0.05. C: Arp2 localization (left panel) and fluorescence intensity (right panel) in porcine oocytes at at MI (20 h after culture) and MII (44 h after culture) stages. Values represent mean ± s.e.m, *, p<0.05.
Figure 4.
JMY involved in DNA damage responses in maturing porcine oocyte.
A: Typical staining of γ-H2AX in porcine oocytes before and after the treatment with etoposide (25 mg/mL) at MI (A) and MII (B) stage. Red, γ-H2AX; blue, chromatin. B: JMY expression in porcine oocytes on 44 h after etoposide. Note that JMY is localized at nucleus in etoposide treated group (indicated in the arrow). Red, JMY; blue, chromatin. Values represent mean ± s.e.m, *p<0.05, n = 3. C–D: p53 expression affected by JMY knockdown and/or etoposide treatment. Porcine oocytes were treated with JMY dsRNA and/or etoposide (25 mg/mL) and expression levels of p53 were quantified by RT-PCR (C) or western blotting (D). GADPH expression was used as internal control.
Figure 5.
A working model for roles of JMY in porcine oocytes maturation and DSB pathway.
A: JMY is involved in oocyte maturation and asymmetric spindle migration, presumably via its actin nucleation promoting activity. B: Upon DNA damage, JMY have a tendency to nuclear accumulation, which is recruited to the promoters of p53 target genes and facilitates the p53 response.