Timed analysis of the nuclear maturation of oocytes in early preantral mouse follicle culture supplemented with recombinant gonadotropin  Rita G Cortvrindt, Yanxin Hu, M.D., Jun Liu, Johan E.J Smitz, M.D., Ph.D.  Fertility and Sterility  Volume 70, Issue 6, Pages 1114-1125 (December 1998) DOI: 10.1016/S0015-0282(98)00332-X

FIGURE 1 Schematic representation of follicle growth and differentiation during culture. (A), Early preantral follicle at day 1 of the culture. A single layer of theca cells is attached on the basement membrane. (B), Follicle cultured for approximately 2 days. Start of attachment of the follicle to the culture dish with the outgrowth of theca cells over the bottom of the dish. (C), Follicle cultured for 4–6 days. Breakthrough and proliferation of the granulosa cells over the outer part of the basal membrane and outgrowth of granulosa cells over the thecal monolayer. (D), Follicle cultured for 10 days. An antral cavity has been formed underneath the oocyte. Differentiation of the granulosa cell mass into a mural part and a cumulus part neighboring the oocyte. (E), In vitro ovulation of a mucified cumulus-oocyte complex by the addition of hCG and EGF. Fertility and Sterility 1998 70, 1114-1125DOI: (10.1016/S0015-0282(98)00332-X)

FIGURE 2 (A), Early preantral follicle immediately after mechanical dissection: note the intact apposition of oocyte and cuboidal granulosa cells, and the intact basal membrane with some attached theca cells (scale bar = 40 μm). (B), On day 1, a monolayer of cells has spread on the bottom of the dish, immobilizing the follicle (scale bar = 40 μm). (C), Translucent areas (onset of antral cavity formation) are visible surrounding the oocyte-cumulus complex in a day-8 follicle (scale bar = 240 μm). (D), A day-13 follicle showing a clear antral cavity surrounded by a granulosa cell wall. The oocyte-cumulus complex is located on top of the structure (scale bar = 240 μm). (E), The addition of hCG and EGF to the culture medium induced cumulus mucification and a mucified oocyte-cumulus complex is released from the basal structure of the follicle (scale bar = 240 μm). Fertility and Sterility 1998 70, 1114-1125DOI: (10.1016/S0015-0282(98)00332-X)

FIGURE 3 Mean oocyte diameter (±1 SD) on days 1, 6, 8, 12, 14, and 16 of in vitro culture. Open bars indicate FSH only, dotted bars indicate FSH plus 10 mIU/mL of LH, and hatched bars indicate FSH plus 100 mIU/mL of LH. Different letters above the days of in vitro culture mark significantly different diameters (P <.05; a<b<c). Within each culture day, oocyte diameters did not differ significantly. Fertility and Sterility 1998 70, 1114-1125DOI: (10.1016/S0015-0282(98)00332-X)

FIGURE 4 Percentage of spontaneous intrafollicular GVBD (open bars) and degeneration (hatched bars) as observed immediately after denudation of the corona-cumulus cells on days 6, 8, 10, 12, 14, and 16. Results shown are the mean ±SEM from the three gonadotropin conditions: (A), FSH; (B), FSH plus 10 mIU/mL of LH; and (C), FSH plus 100 mIU/mL of LH. The asterisk denotes significant (P < .01) increases from the preceding values for either spontaneous GVBD or degeneration. Fertility and Sterility 1998 70, 1114-1125DOI: (10.1016/S0015-0282(98)00332-X)

FIGURE 5 Percentages of GVBD (A) and MII (B) 24 hours after oocyte denudation from follicular cells at different times in culture with the three gonadotropin conditions (mean ±SEM). Open bars indicate FSH alone, dotted bars indicate FSH plus 10 mIU/mL of LH, and hatched bars indicate FSH plus 100 mIU/mL of LH. Fertility and Sterility 1998 70, 1114-1125DOI: (10.1016/S0015-0282(98)00332-X)

FIGURE 6 (A), Percentage of GVBD at 16 hours after the addition of hCG and EGF on different days (mean ±SEM). (B), Percentage of MII oocytes from the oocytes that underwent GVBD observed at 16 hours after the addition of hCG and EGF (mean ±SEM). Open bars indicate FSH alone, dotted bars indicate FSH plus 10 mIU/mL of LH, and hatched bars indicate FSH plus 100 mIU/mL of LH. The different letters above the columns indicate statistically significant (P <.05) differences. Fertility and Sterility 1998 70, 1114-1125DOI: (10.1016/S0015-0282(98)00332-X)

FIGURE 7 (A), Basal progesterone concentrations during culture. (B), Progesterone concentrations 16 hours after the addition of hCG and EGF. (C), Basal E2 concentrations. (D), Inhibin concentrations. Open bars indicate FSH alone, dotted bars indicate FSH plus 10 mIU/mL of LH, and hatched bars indicate FSH plus 100 mIU/mL of LH. The columns represent the mean ±SEM. Each column represents the average calculated from a variable number of measurements per condition, for which the minimums and maximums were as follows: minimum 4 measurements and maximum 21 measurements for E2, minimum 3 and maximum 6 for progesterone, and minimum 3 and maximum 8 for inhibin. The different letters over the columns indicate significantly different values. P = progesterone. Fertility and Sterility 1998 70, 1114-1125DOI: (10.1016/S0015-0282(98)00332-X)