102 REPEATED OVARIAN STIMULATIONS BY EXOGENOUS GONADOTROPIN COULD ALTER ATP CONTENT AND MITOCHONDRIAL DISTRIBUTION IN MOUSE OOCYTES

Abstract

Ovarian stimulation (OS) by exogenous gonadotropin enhances the availability of mammalian oocytes but compromises their developmental competence (Combelles and Albertini 2003 Biol. Reprod. 68, 812–821). Recently, several studies have reported mitochondrial function-related abnormalities in oocytes after single and repeated OSs. Because mitochondria can directly influence fertilization outcomes (El Shourbagy et al. 2006 Reproduction 131, 233–245), this study aimed to determine the relationship between mitochondria-related parameters and developmental competence on the basis of ATP content, mitochondrial DNA (mtDNA) copy number, mitochondrial distribution, and IVF results for oocytes after repeated OSs. Ovulated oocytes were recovered from ICR female mice treated with 7.5 IU of eCG and 5 IU of hCG at an interval of 48 h in 1 (control) to 3 stimulation cycles, which were performed at intervals of either 5 or 10 d (n = 15–25 in each treatment group). The ATP content in oocytes was determined using a luminometer and commercial kits (BacTiter-Glo; Promega, WI, USA; n = 15–29 in each treatment group). The mtDNA copy number in oocytes was quantified by performing absolute quantification with real-time PCR (n = 4–8 in each treatment group). Mitochondrial distribution in oocytes stained by rhodamine123 was observed under a confocal microscope (n = 12–26 in each treatment group). These analyses were performed only for morphologically normal oocytes. The data were analyzed by one-way ANOVA, followed by Fisher’s least significant difference, or by the chi-square test. Some mice did not ovulate in the third stimulation cycle for both intervals (5-d interval, 32%; 10-d interval, 80%). The mean numbers of ovulated oocytes gradually decreased with progression of the stimulation cycles. The ATP content of the oocytes significantly decreased both in the second and third stimulation cycles, performed with a 5-d interval (control, 1.038 ± 0.117 pmol; second cycle, 0.852 ± 0.189 pmol; third cycle, 0.932 ± 0.272 pmol). The mean mtDNA copy number in oocytes did not change significantly but varied widely in the third stimulation cycle (control, 146 000 ± 21 000; 5-d interval, 135 000 ± 35 000; 10-d interval: 148 000 ± 50 000; mean ± SD). The mitochondrial staining patterns were classified into homogeneous, aggregation, and perinuclear accumulation. The rates of aggregation and perinuclear accumulation increased after repeated stimulation cycles. The blastocyst rates did not significantly differ among the treatment groups after IVF (75–82%). Repeated OSs not only decreased the number of ovulated oocytes but also caused changes related with mitochondrial function, even in the morphologically normal ovulated oocytes. Translocation of active mitochondria, which are associated with energy production, has some functional correlation with successful pre-implantation development (Suzuki et al. 2006 J. Mamm. Ova. Res. 23, 128–134). Changes in mitochondrial distribution might compensate for the negative effect of the decrease in ATP content and low mtDNA copy number after repeated OSs to help reach the blastocyst stage.