177 Collection of mouse immature oocytes using cilostazol, a phosphodiesterase 3A inhibitor treatment

Abstract

Administration of cilostazol in mice and swine has resulted in the ovulation of immature oocytes at the germinal vesicle (GV) or metaphase I (MI) stages. The present study aimed to define oocyte synchronised maturation, yield, health, and ease of collection from mice treated with cilostazol. The conventional method included mice primed with pregnant mare serum gonadotrophin (PMSG) and GV oocytes isolated from preovulatory follicles 48 h post-PMSG. Recovery of MI oocytes included the in vitro maturation of the isolated GV oocytes into the MI stage for 6 h or the superovulation of mice with PMSG and human chorionic gonadotrophin (hCG), 48 h apart, and the isolation of MI oocytes from preovulatory follicles 6 h post-hCG. The cilostazol method included the superovulation of mice, as described above, and oral treatment with 7.5 mg of cilostazol once (at the same time as hCG) or twice (at the same time as hCG plus 6 h post-hCG) to result in the ovulation of MI or GV oocytes, respectively. The cilostazol method resulted in immature oocytes that are uniform in size. For instance, the cilostazol method resulted in 98.0% (n = 110) of GV oocytes with a diameter range of 60-90 μm compared with only 49.5% (n = 118) of GV oocytes resulting from the conventional method (P < 0.0001). Similarly, 95.0% (n = 93) of MI oocytes obtained from the cilostazol method were synchronised within the diameter range of 50.1-70 μm compared with 60.0% (n = 89) of MI oocytes obtained from the conventional method (P < 0.0001). Cilostazol also resulted in immature oocytes with synchronised nuclear and cytoplasmic maturation. In this regard, the cilostazol method resulted in GV oocytes having higher levels of co-occurrence of peripheral cortical granules and surrounded nucleolus chromatin configuration compared with the conventional method (80.6% (n = 124) vs. 36.6% (n = 131), respectively; P < 0.0001). Similarly, the co-occurrence of normally organised spindles and chromosomes and peripheral cortical granules with free domains was observed more frequently in MI oocytes obtained from the cilostazol method than in those obtained from the conventional method (82.8% (n = 151) vs. 65.0% (n = 100), respectively; P = 0.001). The cilostazol method was more time and labour efficient (0.8 ± 0.2 vs. 3.2 ± 0.2 min; P < 0.001) and resulted in higher oocyte yields (22.7 ± 1.8 vs. 7.9 ± 0.7 oocytes; P < 0.0001) and normal morphology (94 ± 1.5% vs. 80.1 ± 3.3%; P = 0.02) than did the conventional method (P < 0.001). Finally, compared with the conventional method, the cilostazol method improved the blastocyst production rate of GV and MI oocytes from 39.6% (n = 188) to 65.0% (n = 169) and from 44.2% to 75.3%, respectively (P < 0.001). The presented method provides not only oocytes with uniform size and synchronised developmental maturation but also a technique of oocyte collection that is efficient and resourceful. It is possible that not all of the immature oocytes resulting from the conventional method are from preovulatory follicles, and they do not necessarily represent the cohort of oocytes that would develop adequately and consequently ovulate as opposed to the presented method.