Improvement of in vitro culture of mouse cumulus–oocyte complexes using PDE3- inhibitor followed by meiosis induction with epiregulin Sergio Romero, M.Sc.,

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Presentation transcript:

Improvement of in vitro culture of mouse cumulus–oocyte complexes using PDE3- inhibitor followed by meiosis induction with epiregulin Sergio Romero, M.Sc., Johan Smitz, M.D., Ph.D. Fertility and Sterility Volume 93, Issue 3, Pages 936-944 (February 2010) DOI: 10.1016/j.fertnstert.2008.10.016 Copyright © 2010 American Society for Reproductive Medicine Terms and Conditions

Figure 1 (A) Scheme of follicle culture and cumulus–oocyte complex (COC) subculture. After 8 days of culture, COCs were retrieved from the cultured follicles and subcultured for 4 more days. (B) Scheme for washing procedure applied to COCs subcultured with 1 μM PDE3-I. Fertility and Sterility 2010 93, 936-944DOI: (10.1016/j.fertnstert.2008.10.016) Copyright © 2010 American Society for Reproductive Medicine Terms and Conditions

Figure 2 Oocyte spontaneous meiotic resumption. Oocytes of follicles at 8 and 12 days of culture as well as subcultured cumulus–oocyte complexes (COCs) were separated from granulosa cells and placed in fresh medium. Spontaneous meiotic resumption was assessed 18 hours later. Results are expressed as mean ± standard deviation of the respective percentages. Different letters denote statistically significant differences. Fertility and Sterility 2010 93, 936-944DOI: (10.1016/j.fertnstert.2008.10.016) Copyright © 2010 American Society for Reproductive Medicine Terms and Conditions

Figure 3 (A) Cumulus–oocyte complex (COC) after retrieval from day-8 follicle culture. (B) COC after 4 days of subculture in control medium. (C) COC after 4 days of subculture in 10 nM PDE3-I. (D) Degenerated COC after 4 days of subculture. (cc) cumulus cells, (O) oocyte. Black bar = 100 μm; white bar = 200 μm. Fertility and Sterility 2010 93, 936-944DOI: (10.1016/j.fertnstert.2008.10.016) Copyright © 2010 American Society for Reproductive Medicine Terms and Conditions

Figure 4 Cumulus–oocyte complex (COC) survival after 4 days of COC subculture in different PDE3-I concentrations. Survival rate is expressed as percentage of total COCs plated at the start of the subculture. Results are from at least nine plates (5 to 10 COCs each) per treatment in three independent experiments (P<.05). Different letters denote statistically significant differences. Fertility and Sterility 2010 93, 936-944DOI: (10.1016/j.fertnstert.2008.10.016) Copyright © 2010 American Society for Reproductive Medicine Terms and Conditions

Figure 5 Oocyte meiotic resumption after 4 days of subculture in presence of PDE3-I, Org9935. Cumulus–oocyte complexes (COCs) were stimulated for ovulation (for 18 hours) with hCG/EGF. Results are expressed as mean ± SEM (P<.05) of the respective percentages, obtained from at least four plates (5 to 10 COCs each) per treatment in three independent experiments. Different letters denote statistically significant differences. Fertility and Sterility 2010 93, 936-944DOI: (10.1016/j.fertnstert.2008.10.016) Copyright © 2010 American Society for Reproductive Medicine Terms and Conditions

Figure 6 Oocyte meiotic resumption after stimulation with hCG, epiregulin, or combination of both. (A) After 4 days of subculture in PDE3-I free medium. Cumulus–oocyte complexes (COCs) were stimulated for ovulation with the defined stimuli. (B) Oocyte meiotic resumption in COCs retrieved from in vitro fully grown COCs. The COCs were subcultured in presence of 1 μM PDE-3-1, then retrieved and washed to remove the inhibitor and stimulated with same stimuli. Unstimulated COCs were used as control. All groups except follicle + hCG/EGF represent COCs. Follicles cultured for 12 days, where meiotic resumption is induced with hCG/EGF, are shown for reference. Results are expressed as mean + SEM of the respective percentages obtained from at least six plates (5 to 10 COCs each) per treatment in three independent experiments. Different letters denote statistically significant differences. Fertility and Sterility 2010 93, 936-944DOI: (10.1016/j.fertnstert.2008.10.016) Copyright © 2010 American Society for Reproductive Medicine Terms and Conditions