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. 2014 Apr 24;60(2):128-35.
doi: 10.1262/jrd.2013-098. Epub 2014 Feb 4.

The association of mitochondrial potential and copy number with pig oocyte maturation and developmental potential

Seul-Ki Lee  1 , Ming-Hui Zhao, Jung-Woo Kwon, Ying-Hua Li, Zi-Li Lin, Yong-Xun Jin, Nam-Hyung Kim, Xiang-Shun Cui
Affiliations

The association of mitochondrial potential and copy number with pig oocyte maturation and developmental potential

Seul-Ki Lee et al. J Reprod Dev. .

Abstract

ATP is critical for oocyte maturation, fertilization, and subsequent embryo development. Both mitochondrial membrane potential and copy number expand during oocyte maturation. In order to differentiate the roles of mitochondrial metabolic activity and mtDNA copy number during oocyte maturation, we used two inhibitors, FCCP (carbonyl cyanide p-(tri-fluromethoxy)phenyl-hydrazone) and ddC (2'3-dideoxycytidine), to deplete the mitochondrial membrane potential (Δφm) and mitochondrial copy number, respectively. FCCP (2000 nM) reduced ATP production by affecting mitochondrial Δφm, decreased the mRNA expression of Bmp15 (bone morphogenetic protein 15), and shortened the poly(A) tails of Bmp15, Gdf9 (growth differentiation factor 9), and Cyclin B1 transcripts. FCCP (200 and 2000 nM) also affected p34(cdc2) kinase activity. By contrast, ddC did not alter ATP production. Instead, ddC significantly decreased mtDNA copy number (P < 0.05). FCCP (200 and 2000 nM) also decreased extrusion of the first polar body, whereas ddC at all concentrations did not affect the ability of immature oocytes to reach metaphase II. Both FCCP (200 and 2000 nM) and ddC (200 and 2000 µM) reduced parthenogenetic blastocyst formation compared with untreated oocytes. However, these inhibitors did not affect total cell number and apoptosis. These findings suggest that mitochondrial metabolic activity is critical for oocyte maturation and that both mitochondrial metabolic activity and replication contribute to the developmental competence of porcine oocytes.

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Figures

Fig. 1.
Fig. 1.
Mitochondrial membrane potential (A, B), relative mtDNA copy number (C), and relative ATP content (D) in FCCP- or ddC-treated MII oocytes. Membrane potential was calculated as a ratio of the red florescence, which corresponded to activated mitochondria (J-aggregates) (A), to the green fluorescence, which corresponded to less-activated mitochondria (J-monomers, data not shown). Data corresponding to the control was arbitrarily set at 1. Ctrl, control; F, FCCP; D, ddC. Values represent means ± SEM from at least three separate experiments. *, P < 0.05, compared with the control.
Fig. 2.
Fig. 2.
Percentage of maturation in FCCP- or ddC-treated MII oocytes. The number of oocytes examined in each treatment group is shown in parentheses. Ctrl, control; F, FCCP; D, ddC. Values represent means ± SEM from at least five separate experiments. *P < 0.05 compared with the control, **P < 0.01 compared with the control.
Fig. 3.
Fig. 3.
Relative mRNA expression of maternal genes (Bmp15, Gdf9, cyclin B1, and c-mos) (A) and poly(A) tail length (B) in FCCP- or ddC-treated MII oocytes. Porcine Gapdh mRNA expression was used as an internal control, and the control group was set at 1. Ctrl, control; F, FCCP; D, ddC. Lane 1, control; lanes 2 and 3, oocytes treated with 200 or 2000 nM FCCP, respectively; lanes 4 and 5, oocytes treated with 200 or 2000 µM ddC, respectively. Arrows point to the initial length of the poly(A) tail. Values represent means ± SEM from three separate experiments. *P < 0.05, compared with the control.
Fig. 4.
Fig. 4.
Relative p34cdc2 kinase (A), p-ERK1/2 activity (B) and relative density of p-ERK1/2 activity (C) in FCCP- or ddC-treated MII oocytes. The upper panel in (B) is an immunoblot showing the levels of p-ERK1 and p-ERK2. The lower panel in (B) is a histogram summarizing immunoblotting results. Ctrl, control; F, FCCP; D, ddC, p, phospho. Values represent means ± SEM from three separate experiments. *P < 0.05, compared with the control.
Fig. 5.
Fig. 5.
Effects of FCCP and ddC on the developmental competence of oocytes. Percentage of cleavage (A), blastocyst formation (B), total cell number (C), and apoptosis (D) in blastocysts. Ctrl, control; F, FCCP; D, ddC. The number of oocytes examined in each treatment group is shown in parentheses. Values represent means ± SEM from at least four separate experiments. *P < 0.05, compared with the control.
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