Supplement of cilostamide in growth medium improves oocyte maturation and developmental competence of embryos derived from small antral follicles in pigs

- Effects of cAMP modulators on in vitro growth and maturation of oocytes derived from small antral follicles (SAFs) were examined.
- Cilostamide stimulated oocyte growth while inhibiting premature meiotic maturation by regulating intraoocyte cAMP.
- In vitro development after somatic cell nuclear transfer of SAF-derived oocytes was improved by cilostamide treatment.

This study was conducted to evaluate the effects of cyclic AMP (cAMP) modulator cilostamide (CIL) and forskolin (FSK) treatment during in vitro growth (IVG) on growth, maturation, and embryonic development of cumulus-oocyte complexes (COCs) derived from small antral follicles < 3 mm in diameter (SAFCOCs). SAFCOCs were untreated (control) or treated with 20 μM CIL and/or 50 μM FSK for 2 days for IVG. Next, IVG oocytes were cultured for maturation and then induced for parthenogenesis (PA) or used as recipient ooplasts for somatic cell nuclear transfer (SCNT). Nuclear maturation of oocytes was significantly lower in the control (49.6 ± 9.3%) group than in other groups (67.2 ± 5.0-79.8 ± 7.9%). The cumulus expansion score after IVG-IVM was significantly higher in the control and CIL group than in the FSK and CIL + FSK groups. CIL significantly increased mean diameter SAF-derived oocytes (120.0 ± 0.5 μm) compared to the control, FSK, and CIL + FSK (114.8 ± 0.5-116.7 ± 0.6 μm) and showed a comparable level of intracellular glutathione (GSH) contents (0.84 ± 0.07 pixels/oocyte) to medium antral follicle (MAF)-derived oocytes (1.00 ± 0.08 pixels/oocyte), but was higher than those of oocytes treated with FSK and CIL + FSK (0.29 ± 0.05 and 0.37 ± 0.05 pixels/oocyte, respectively). CIL treatment significantly increased blastocyst formation (55.1 ± 4.7%) after PA relative to the control (29.4 ± 6.4%), FSK (34.8 ± 7.1%), and CIL + FSK (41.1 ± 5.2%). A higher proportion of oocytes treated with CIL, FSK, and CIL + FSK (73.3 ± 1.7-82.8 ± 9.1%) remained at the germinal vesicle stage after IVG culture than control oocytes (40.0 ± 5.0%). Following SCNT, blastocyst formation of SAFCOCs treated with CIL (22.4 ± 6.3%) was higher than that of oocytes (0-10.4 ± 5.3%) in control, FSK, and CIL + FSK, but similar to that (25.3 ± 3.5%) of MAF-derived COCs not cultured for IVG. The cAMP level of SAFCOCs before IVG was 0.1 ± 0.03 fmol/oocyte. After 2 days of IVG culture, cAMP level was increased significantly by treatment with FSK and CIL + FSK (3.0 ± 0.57 and 12.1 ± 0.62 fmol/oocyte, respectively) relative to the control and CIL treatment (0.1 ± 0.03 and 0.3 ± 0.04 fmol/oocyte, respectively). Our results demonstrate that CIL treatment during IVG improves the low developmental competence of SAFCOCs to levels comparable to MAFCOCs by allowing oocyte growth while inhibiting premature meiotic maturation, probably via maintenance of cAMP concentrations at appropriate levels.