Regular monitoring on experimental animal management found the fluctuation of ART outcome, which showed a necessity to explore whether superovulation treatment is responsible for such unexpected outcome. This study was subsequently conducted to examine whether superovulation treatment can preserve u...
Regular monitoring on experimental animal management found the fluctuation of ART outcome, which showed a necessity to explore whether superovulation treatment is responsible for such unexpected outcome. This study was subsequently conducted to examine whether superovulation treatment can preserve ultrastructural integrity and developmental competence of oocytes following oocyte activation and embryo culture. A randomized study using mouse model was designed and in vitro development (experiment 1), ultrastructural morphology (experiment 2) and functional integrity of the oocytes (experiment 3) retrieved after PMSG/hCG injection (superovulation group) or not (natural ovulation; control group) were evaluated. In experiment 1, more oocytes were retrieved following superovulation than following natural ovulation, but natural ovulation yielded higher (p < 0.0563) maturation rate than superovulation. The capacity of mature oocytes to form pronucleus and to develop into blastocysts in vitro was similar. In experiment 2, a notable (p < 0.0186) increase in mitochondrial deformity, characterized by the formation of vacuolated mitochondria, was detected in the superovulation group. Multivesicular body formation was also increased, whereas early endosome formation was significantly decreased. No obvious changes in other microorganelles, however, were detected, which included the formation and distribution of mitochondria, cortical granules, microvilli, and smooth and rough endoplasmic reticulum. In experiment 3, significant decreases in mitochondrial activity, ATP production and dextran uptake were detected in the superovulation group. In conclusion, superovulation treatment may change both maturational status and functional and ultrastuctural integrity of oocytes. Superovulation effect on preimplantation development can be discussed.
Regular monitoring on experimental animal management found the fluctuation of ART outcome, which showed a necessity to explore whether superovulation treatment is responsible for such unexpected outcome. This study was subsequently conducted to examine whether superovulation treatment can preserve ultrastructural integrity and developmental competence of oocytes following oocyte activation and embryo culture. A randomized study using mouse model was designed and in vitro development (experiment 1), ultrastructural morphology (experiment 2) and functional integrity of the oocytes (experiment 3) retrieved after PMSG/hCG injection (superovulation group) or not (natural ovulation; control group) were evaluated. In experiment 1, more oocytes were retrieved following superovulation than following natural ovulation, but natural ovulation yielded higher (p < 0.0563) maturation rate than superovulation. The capacity of mature oocytes to form pronucleus and to develop into blastocysts in vitro was similar. In experiment 2, a notable (p < 0.0186) increase in mitochondrial deformity, characterized by the formation of vacuolated mitochondria, was detected in the superovulation group. Multivesicular body formation was also increased, whereas early endosome formation was significantly decreased. No obvious changes in other microorganelles, however, were detected, which included the formation and distribution of mitochondria, cortical granules, microvilli, and smooth and rough endoplasmic reticulum. In experiment 3, significant decreases in mitochondrial activity, ATP production and dextran uptake were detected in the superovulation group. In conclusion, superovulation treatment may change both maturational status and functional and ultrastuctural integrity of oocytes. Superovulation effect on preimplantation development can be discussed.
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제안 방법
A 10-point standard curve (0–10 pmol/tube) was constructed for every 20 oocytes, and ATP content was calculated using the formula from linear regression of the standard curve.
The experimental protocols were approved by the Institutional Animal Care and Use Committee (approval number SNU-091028-4). Additionally, experimental samples were managed appropriately, and quality control of the laboratory facility and equipment were conducted.
All experiments were replicated more than three times, and the data obtained were subjected to statistical analysis. Not all oocytes retrieved from each experiment were provided because the superovulation treatment yielded different number of oocytes retrieved.
Number of oocytes retrieved and developed to the metaphase II (mature), pronuclear, 2-cell, 4-cell, morula and blastocyst stages was monitored. In experiment 2, ultrastructural normality of oocytes were monitored with TEM, which includes mitochondria, cortical granules, multivesicular bodies, lipid droplets, microvilli, and the endoplasmic reticulum system. In experiment 3, ATP generation representing mitochondrial function and endocytosis in mature oocytes retrieved from superovulation or natural ovulation were evaluated by JC-1 staining and dextran uptake, respectively.
Oocytes that had been activated parthenogenetically or inseminated in vitro were then cultured constantly in 5 μl droplets of modified Chatot, Ziomek, and Bacister (CZB) medium for a further 120 h at 37℃ under an atmosphere of 5% CO2 in air. Pronucleus formation, cleavage, and development to the 4-cell, morula, and blastocyst stages were monitored under an IX70 inverted microscope (Olympus, Japan) at 6, 24, 48, 72 and 120 h after activation or fertilization.
As a matter of fact, no significant difference on developmental competence of oocytes between the natural cycle and the superovulation groups confirmed normality of mature oocytes retrieved after superovulation (Table 1). The results of these data can contribute not only to obtaining toxicological data, but also to improving ART efficiency by decreasing potential adverse effect of superovulation.
대상 데이터
Vaginal smears were performed to check the estrous cycle stage of the female donors. The flushing medium was M2 medium (Sigma-Aldrich, St Louis, MO). For ovarian hyperstimulation, 5 IU of PMSG (Folligon™; Intervet International, Boxmeer, the Netherlands) was injected intraperitoneally and ovulation was induced by intraperitoneal injection of 5 IU of hCG (Pregnyl™; Organon, Oss, the Netherlands) 48 h later (Jang et al.
Total 11 mice were euthanized for observing oocyte ultra-structure and, 3 and 8 mice were allotted into the superovulation and the natural cycle group, respectively. The number of oocytes examined was 110 for the superovulation and 109 for the natural cycle groups.
이론/모형
1 (SAS Institute, USA) was used to analyze the data. When a significant model effect was detected, comparisons among groups were subsequently conducted using the leastsquares or Duncan methods. A p-value of less than 0.
성능/효과
Dextran uptake was measured in 87 oocytes. As shown in Fig. 5, dextran uptake, measured as a marker of early endocytosis, was lower in the hyperstimulation group compared with the control group (65.14 vs. 5.32; p = 0.004), although there was an increase in the number of multivesicular bodies, indicating that late endocytosis was increased.
4) detected. The developments to the 2-cell, 4-cell, morula and blastocyst stages were 94 to 100%, 87 to 96%, 87 to 93% and 72 to 82%, respectively.
The results of this study demonstrated that compared with natural ovulation, the superovulation treatment with PMSG and hCG decreases the proportion of mature oocytes to retrieved oocytes. In some oocytes being superovulated, ultrastructural alterations in mitochondria and lysosomal complexes, characterized by increased numbers of vacuolated mitochondria and multivesicular bodies were observed.
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