Fig 2: Relative mRNA expression of calcineurin isoforms in mouse oocytes (A) and granulosa cells (B).The relative mRNA levels represent the amount of mRNA expression normalised to Actb. The comparison of calcineurin mRNA expression was made between each oocytes’ stage and oocytes in one stage, so was in granulosa cells. The statistical difference between two different lowercase letters represents P < 0.05, such as “a” and “b,” “a” and “c,” or “b” and “c” represents P < 0.05.

Fig 3: Expression and localization of CLTC in metaphase oocytes treated with either taxol or nocodazole. (A) Immunoblotting analysis of CLTC from MI-stage oocytes after treatment with taxol or nocodazole. Beta-actin was used as a loading control. Noco, nocodazole. (B) Immunoblotting analysis of CLTC from MII-stage oocytes after treatment with either taxol or nocodazole. Beta-actin was used as a loading control. Noco, nocodazole. (C) Subcellular localizations of CLTC in MI-stage oocytes after treatment with either taxol or nocodazole were detected by immunofluorescence staining, respectively. Slides were examined using a confocal microscope. Red, CLTC; green, TUBB; blue, DNA. Scale bar = 20 µm. (D) Subcellular localizations of CLTC in MII-stage oocytes after treatment with either taxol or nocodazole were detected by immunofluorescence staining, respectively. Slides were examined using a confocal microscope. White arrows indicate polar bodies. Red, CLTC; green, TUBB; blue, DNA. Scale bar = 20 µm. CLTC, clathrin heavy chain 1; MI, metaphase I; MII, metaphase II.

Fig 4: MII-stage knockdown of CLTC impairs embryonic development. (A) Scheme for sample collections for immunoblotting analyses after MO injections into MII-stage oocytes. (B) CLTC-expression immunoblotting after CLTC-MO or control-MO injections into MII-stage sheep oocytes. Beta-actin was used as a loading control. (C) Band intensities (representing CLTC expression) were assessed using gray-level analyses after CLTC-MO or control-MO injections in MII-stage sheep oocytes. Data are presented as mean ± SD from three independent experiments. P < 0.05 was considered statistically significant. (D) The CLTC, spindles, and DNA in two-cell embryos from the control-MO and CLTC-MO groups were stained with anti-CLTC (red), anti-tubulin (green), and DAPI (blue), respectively. Scale bar = 20 µm. White arrows indicate the other blastomere nucleus. (E) MII-stage sheep oocytes were injected with either CLTC-MO or control-MO, and then the rate of early embryo developmental was measured. Data are presented as mean ± SD from eight independent repeats, and at least 12 MII oocytes were included for parthenogenetic activation in each group for each repeat. P < 0.05 was considered statistically significant. (F) Representative images of early embryos after CLTC-MO or control-MO injections. Scale bar = 100 µm. MII, metaphase II; CLTC, clathrin heavy chain 1; MO, morpholino; DAPI, 4',6-diamidino-2-phenylindole.

Fig 5: CLTC knockdown in sheep oocytes induces spindle defects and misaligned chromosomes. (A) Immunoblotting of CLTC expression after either CLTC-MO or control-MO injections. Beta-actin was used as a loading control. (B) Band intensities (representing CLTC expression) were assessed using gray-level analyses after CLTC-MO or control-MO injections. (C) The CLTC, spindles, and DNA in MII-stage oocytes (control-MO and CLTC-MO groups) were stained with anti-CLTC (red), anti-tubulin (green), and DAPI (blue), respectively. Scale bar = 20 µm. (D) The percentage of oocytes demonstrating PB1 extrusion in the control-MO and CLTC-MO groups. (E) Percentages of oocytes with abnormal spindles in the control-MO and CLTC-MO groups. (F) Immunofluorescence staining of CLTC expression after CLTC-MO or control-MO injections. Green, TUBB; blue, DNA. Scale bar = 20 µm. (G) Percentages of oocytes with chromosome misalignments in the control-MO and CLTC-MO groups. Data are presented as mean ± SD from at least three independent experiments. P < 0.05 was considered statistically significant. CLTC, clathrin heavy chain 1; MO, morpholino; PB1, first polar body; DAPI, 4',6-diamidino-2-phenylindole.