The role of cilostazol, a phosphodiesterase 3 inhibitor, on oocyte maturation and subsequent pregnancy in mice

Abstract

It is important to identify effective contraceptive drugs that cause minimal disruption to physiological processes. Phosphodiesterase 3 (PDE3) inhibitors suppress meiosis in oocytes by decreasing the level of cAMP and blocking the extrusion of the first polar body. In this study, we tested the PDE3 inhibitor, cilostazol, as a potential contraceptive agent. The effects of cilostazol treatment in vitro and in vivo on the suppression of oocyte maturation in a mouse model were investigated. The results indicated that treatment with increasing concentrations of cilostazol led to a dose-dependent arrest in meiosis progression. The effective in vitro concentration was 1 µM and was 300 mg/kg in vivo. The effect of cilostazol was reversible. After removal of the drug, meiosis resumed and mouse oocytes matured in vitro, and showed normal chromosome alignment and spindle organization. After fertilization using an ICSI method, the oocytes showed normal morphology, fertilization rate, embryo cleavage, blastocyst formation, and number of viable pups when compared with controls. The offspring showed similar body weight and fertility. In vivo, the mice became infertile if the drug was injected sequentially, and became pregnant following discontinuation of cilostazol. More importantly, no side effects of cilostazol were observed in treated female mice as demonstrated by blood pressure and heart rate monitoring. It is concluded that cilostazol, a drug routinely used for intermittent claudication, can effectively inhibit oocyte maturation in vitro and in vivo, does not affect the developmental potential of oocytes following drug removal and has few side effects in female mice treated with this drug. These findings suggest that cilostazol may be a potential new contraceptive agent that may facilitate an efficacy and safety study of this drug.

Figure 1. Developmental stages of mouse oocytes after treatment of COCs and DOs with cilostazol and ORG9935 at different concentrations for 24 h.

As the concentration increased, the percentage of oocytes showing meiotic arrest increased (P<0.05). (A) Cilostazol and (B) ORG9935. Orange dot line represents COCs at GV stage, orange hyphen line represents COCs at GVBD stage, and orange straight line represents COCs at MII stage. Blue DOs dot line represents COCs at GV stage, blue hyphen line represents DOs at GVBD stage, and blue straight line represents DOs at MII stage.

Figure 2. The distribution and organization of microtubules and spindles in IVM oocytes treated and not treated with cilostazol.

(A) Representative normal spindle and chromosome alignment in the treatment group. (B) Representative normal spindle and chromosome alignment in the non-treatment group. Original magnification ×400.

Figure 3. Measurements of body weight and fertility in offspring from oocytes treated and not treated with cilostazol.

(A) body weight of female mice, (B) body weight of male mice, (C) fertility of female mice, (D) Fertility of male mice. The value of the litter size is not a percentage, but numerical data. In image (A) and (B), Black hyphen line with diamond represents mice conceived from female mice treated with cilostazol before, and black straight line with square represents mice conceived from female mice without cilostazol treatment. In image (C) and (D), Black solid rectangle represents cilostazol treatment group, and black hollow rectangle represents control group without cilostazol treatment.

Figure 4. Effects of in vivo treatment with different concentrations of cilostazol and ORG9935 on the GVBD ratio of oocytes in mice.

Orange straight line represents cilostazole treatment group, and blue straight line represents ORG9935 treatment group.

Figure 5. Effects of cilostazol administration on the fertility of female mice.

(A) treated with cilostazol, (B) treatment with cilostazol stopped. The value of the litter size is not a percentage, but numerical data. In image (A), Black solid rectangle represents cilostazol treatment group, and black hollow rectangle represents control group without cilostazol treatment. In image (B), Black solid rectangle represents cilostazol treatment group, but stopped treatment at present, and black hollow rectangle represents control group without cilostazol treatment.