Coculture with cumulus cells improves maturation of mouse oocytes denuded of the cumulus oophorus: observations of nuclear and cytoplasmic events  Li Ge, Ph.D., Hong-Shu Sui, M.Sc., Guo-Cheng Lan, Ph.D., Na Liu, M.Sc., Jun-Zuo Wang, M.Sc., Jing-He Tan, Ph.D.  Fertility and Sterility  Volume 90, Issue 6, Pages 2376-2388 (December 2008) DOI: 10.1016/j.fertnstert.2007.10.054 Copyright © 2008 American Society for Reproductive Medicine Terms and Conditions

Figure 1 Nuclear progression of mouse oocytes during maturation under different conditions. Oocytes matured vivo (IVO), in vitro as COCs (COC), DOs cultured alone (DO), or cocultured on a CC monolayer (coDO) were recovered at 0, 3, 7, 11, and 15 hours after hCG injection or in vitro culture. The meiotic progression was divided into the GV, pMI, MI, A/TI, and MII stages under a phase-contrast microscope after orcein staining. Percentages of oocytes at different stages of nuclear maturation are compared across maturation conditions. Values without a common letter above the bars within the same column (the same stage of nuclear maturation at the same time point) differ (P<.05). Fertility and Sterility 2008 90, 2376-2388DOI: (10.1016/j.fertnstert.2007.10.054) Copyright © 2008 American Society for Reproductive Medicine Terms and Conditions

Figure 2 The mean time (hours) that an oocyte spent at each stage of nuclear progression during maturation in vivo (IVO), in vitro as COCs (COC), as DOs cultured alone (DO), or cocultured with CC monolayer (coDO). The calculation was done according to Sirard et al. (30) and was based on data collected at 0, 1, 2, 3, 4, 5, 7, 11, and 15 hours after hCG injection or in vitro culture. Fertility and Sterility 2008 90, 2376-2388DOI: (10.1016/j.fertnstert.2007.10.054) Copyright © 2008 American Society for Reproductive Medicine Terms and Conditions

Figure 3 Laser scanning confocal microscopic images (equatorial sections) of CGs (green) and chromatin (blue) in mouse oocytes at different stages of maturation. (A, B) Oocytes at the GV stage with stages 1 and 2 of CG distribution, respectively; (C) an oocyte of pMI nuclear state with a stage 2 CG distribution; (D and E) oocytes at the MI nuclear stage with stages 3 and 4 of CG distribution, respectively. (F) an oocyte at TI with a stage 5 CG distribution; (G) an MII oocyte with a stage 6 CG distribution; (H) an IVM MII oocyte collected at 11 hours of culture, with incomplete CG migration; (I) an IVM MII oocyte collected at 15 hours of culture showing a partial premature exocytosis of CGs. Fertility and Sterility 2008 90, 2376-2388DOI: (10.1016/j.fertnstert.2007.10.054) Copyright © 2008 American Society for Reproductive Medicine Terms and Conditions

Figure 4 Changes in CG distribution during maturation of mouse oocytes under different conditions. Percentages of oocytes at different stages of CG distribution (stages 1–6) are compared among oocytes matured in vivo (IVO), in vitro as COCs (COC), as DOs cultured alone (DO), or cocultured on a CC monolayer (coDO). Values without a common letter above the bars within the same column (the same stage of CG distribution at the same time point) differ (P<.05). Fertility and Sterility 2008 90, 2376-2388DOI: (10.1016/j.fertnstert.2007.10.054) Copyright © 2008 American Society for Reproductive Medicine Terms and Conditions

Figure 5 Laser scanning confocal microscopic images of mouse oocytes showing different patterns of mitochondrial distribution. (A) The symmetrical distribution at the GV stage; (B) partial congregation; (C) complete congregation; (D) an oocyte with blurred mitochondria, with a homogeneous distribution of fluorescence in the ooplasm. Fertility and Sterility 2008 90, 2376-2388DOI: (10.1016/j.fertnstert.2007.10.054) Copyright © 2008 American Society for Reproductive Medicine Terms and Conditions

Figure 6 Percentages of oocytes showing partially or completely congregated patterns of mitochondria distribution after maturation under different conditions. Percentages of oocytes with a partial or complete mitochondrial congregation were compared among the IVOs, COCs, DOs, and coDOs at both the MI and MII stages of maturation. Values without a common letter above their bars differ (P<.05) within patterns of mitochondrial distribution and stages of maturation. Fertility and Sterility 2008 90, 2376-2388DOI: (10.1016/j.fertnstert.2007.10.054) Copyright © 2008 American Society for Reproductive Medicine Terms and Conditions

Figure 7 Percentages of oocytes with the tine-pole (TinePole), even-pole (EvenPole), and barrel-shaped (Barrel) spindles after maturation under different conditions. Percentages of oocytes with different spindle forms were compared among the IVOs, COCs, DOs, and coDOs. Values above the bars without a common letter in their superscripts differ (P<.05) within spindle types. Fertility and Sterility 2008 90, 2376-2388DOI: (10.1016/j.fertnstert.2007.10.054) Copyright © 2008 American Society for Reproductive Medicine Terms and Conditions

Figure 8 Intracellular concentrations of GSH in oocytes matured under different conditions. The GSH level at different stages of maturation was compared among IVOs, COCs, DOs, and coDOs, with oocytes at the GV stage as controls. Means with different letters indicate statistically significant differences (P<.05). Fertility and Sterility 2008 90, 2376-2388DOI: (10.1016/j.fertnstert.2007.10.054) Copyright © 2008 American Society for Reproductive Medicine Terms and Conditions

Figure 9 Dynamic changes in MPF activities during oocyte maturation under different conditions. Oocytes at the late GV stage (lGV) were collected at 2 hours (IVOs and COCs), 1.5 hours (DOs), or 1.75 hours (coDOs) after hCG injection or culture; oocytes at the pMI stage were collected at 4 hours (IVOs and COCs), 3 hours (DOs), or 3.5 hours (coDOs); the MI oocytes were recovered at 7 hours (IVOs and COCs), 6 hours (DOs), or 6.5 hours (coDOs); oocytes at the A/TI stage were collected at 9.5 hours (IVOs and COCs), 8.5 hours (DOs), or 9 hours (coDOs); the early MII (eMII) oocytes were assayed at 11 hours (IVOs) or 12 hours (COCs, DOs, and coDOs); and the late MII (lMII) oocytes were collected at 12.5 hours after hCG (IVOs) or 14 hours of culture (COCs, DOs, and coDOs). The histone H1 activity values of newly ovulated oocytes were arbitrarily set as 100%, and the other values were expressed relative to this activity. The relative value of histone H1 activity at different stages of maturation was compared among the IVOs, COCs, DOs, and coDOs. Means without a common letter differ (P<.05) within maturation stages. Fertility and Sterility 2008 90, 2376-2388DOI: (10.1016/j.fertnstert.2007.10.054) Copyright © 2008 American Society for Reproductive Medicine Terms and Conditions