Acquisition and loss of oocyte meiotic and developmental competence during in vitro antral follicle growth in mouse  Ingrid Segers, M.Sc., Tom Adriaenssens, M.Sc., Elife Ozturk, M.Sc., Johan Smitz, M.D.  Fertility and Sterility  Volume 93, Issue 8, Pages 2695-2700 (May 2010) DOI: 10.1016/j.fertnstert.2009.11.035 Copyright © 2010 American Society for Reproductive Medicine Terms and Conditions

Figure 1 Representative pictures of follicle development and corresponding nucleolar conformation in the oocyte during in vitro follicle growth. Follicles at the start of culture (110–130 μm) have intact oocyte-granulosa cell connections and adhering theca cells and are categorized as follicular (F). When granulosa cells outgrow the basal membrane, the follicle is termed diffuse (D). A follicle displaying a clear lumen around a delineated cumulus oocyte complex and a ring of mural granulosa cells at the outer boundaries of the follicle, is termed antral follicle (A). In the oocyte, three types of nucleolar conformation were seen. A nonsurrounded nucleolus (NSN) is characterized by a cloud of chromatin material occupying the entire nucleoplasm which contains some chromatin foci. In time, a partial rim of perinucleolar chromatin is formed: the intermediate stage (NSN/SN). The surrounded nucleolus (SN) is characterized by condensed chromatin surrounding the nucleolus and a nucleoplasm with some patches of chromatin. Fertility and Sterility 2010 93, 2695-2700DOI: (10.1016/j.fertnstert.2009.11.035) Copyright © 2010 American Society for Reproductive Medicine Terms and Conditions

Figure 2 Follicle development (A) and corresponding nucleolar conformation (B) of the oocyte during follicle culture. (A) Follicular stage (black bars), diffuse follicle stage (gray bars), and antral follicle stage (white bars). (B) Nonsurrounded nucleolar conformation (black bars), intermediate conformation (gray bars), and surrounded nucleolar conformation (white bars). Bars represent the mean percentages with SEM of follicles in each stage per plate in six culture plates. Fertility and Sterility 2010 93, 2695-2700DOI: (10.1016/j.fertnstert.2009.11.035) Copyright © 2010 American Society for Reproductive Medicine Terms and Conditions

Figure 3 Cleavage and blastocyst development after 8, 10, and 12 days of oocyte growth in culture and oocyte growth in vivo. Cleavage percentages represent the amount of normal 2-cell embryos out of the mucified cumulus-oocyte complexes used in the IVF procedure (black bars). Blastocyst development is calculated as the amount of blastocysts obtained out of the 2-cell embryos (gray bars). Bars represent mean percentages with SEM of embryos in each stage per two (day 10) or six (days 8 and 12 and in vivo) experiments. Statistical differences are indicated by different letters (analysis of variance + Tukey: P<.05). Fertility and Sterility 2010 93, 2695-2700DOI: (10.1016/j.fertnstert.2009.11.035) Copyright © 2010 American Society for Reproductive Medicine Terms and Conditions

Figure 4 Morphology of follicles cultured for 6 days and the nucleolar conformation of the corresponding oocytes in control conditions with 10 mIU/mL recombinant (r) FSH added from the start of culture or in the experimental condition with 10 mIU/mL r-FSH added only from day 4 of culture. Developmental stages are classified as follicular (black bars), diffuse (gray bars), and antral (white bars). Nucleolar conformation of oocytes is subdivided into nonsurrounded nucleolus (black bars), a transition stage (gray bars), and surrounded nucleolus (white bars). Bars represent the mean percentages with SEM in eight culture plates. Statistical significance is indicated by asterisks (t test: P<.05). Fertility and Sterility 2010 93, 2695-2700DOI: (10.1016/j.fertnstert.2009.11.035) Copyright © 2010 American Society for Reproductive Medicine Terms and Conditions