Gene expression profiling during early folliculogenesis in the mouse ovary  Shalmali J. Dharma, M.Sc., Deepak N. Modi, Ph.D., Tarala D. Nandedkar, Ph.D.  Fertility and Sterility  Volume 91, Issue 5, Pages 2025-2036 (May 2009) DOI: 10.1016/j.fertnstert.2008.02.088 Copyright © 2009 American Society for Reproductive Medicine Terms and Conditions

Figure 1 Semiquantitative RT-PCR analysis of the selected genes from the array. The mRNA samples from the mouse ovaries on day 2 and day 4 were amplified by gene-specific primers (Table 1). The PCR products were resolved on 2% agarose gel. The signal intensities were normalized with a housekeeping gene, 18S, for comparison. Each bar represents mean ± SEM for three individual experiments. IOD = integrated optical density. Fertility and Sterility 2009 91, 2025-2036DOI: (10.1016/j.fertnstert.2008.02.088) Copyright © 2009 American Society for Reproductive Medicine Terms and Conditions

Figure 2 Immunohistochemical identification of C-KIT (a–c), AMH (d–f), GDF-9 (g–i), and ERβ (j–l) in day-2 and day-4 mouse ovaries. C-KIT protein expression (arrowhead) is detected in the oocytes of primordial follicles (a) and primary follicles (b). Anti-müllerian hormone protein expression is detected in granulosa cells of the primary follicles of day-4 ovaries (e) and absent in primordial follicles. Arrowheads (e) point to immunopositive granulosa cells. No staining is observed in day-2 ovaries (d). Growth differentiation factor 9 protein expression in the oocytes of primary follicles (h) and absence in primordial follicles of day-4 ovaries. No immunostaining is seen in day-2 ovaries (g). Estrogen receptor β immunolocalization is seen in oocytes and granulosa cells of primary follicles (k) but absent in primordial follicles in day-4 ovaries. No staining is observed in day-2 ovaries (j). The representatives of day-4 ovary are shown as negative controls (c, f, i, l) (without primary antibody) immunohistochemical control for C-KIT, AMH, GDF-9 and ERβ respectively, is included. Original magnification, ×400. Fertility and Sterility 2009 91, 2025-2036DOI: (10.1016/j.fertnstert.2008.02.088) Copyright © 2009 American Society for Reproductive Medicine Terms and Conditions

Figure 3 Immunohistochemical localization of PCNA (a–c) and TUNEL detection (d–f) in day-2 and day-4 mouse ovaries. Proliferating cell nuclear antigen staining is seen in the interstitial/stromal cells in day-2 ovaries containing primordial follicles (a). Distinct nuclear staining is observed in the granulosa cells of the primary follicles in day-4 ovaries (b). The negative control (c) is clear, without any staining. TUNEL positivity was not observed in day-2 (d) and day-4 (e) ovaries. The arrow shows TUNEL-positive granulosa cells in day-22 mouse ovarian section (f) (positive control). Original magnification, ×400. Fertility and Sterility 2009 91, 2025-2036DOI: (10.1016/j.fertnstert.2008.02.088) Copyright © 2009 American Society for Reproductive Medicine Terms and Conditions

Figure 4 Immunohistochemical localization of PCNA (left) and AMH (right) in paraffin sections of cultured day-4 neonatal mouse ovaries treated with estrogen (b, e), estrogen plus ICI (c, f), or medium (a, d). Proliferating cell nuclear antigen staining is present in granulosa cells of primary follicles of the estrogen-treated group (b) (arrows). Proliferating cell nuclear antigen immunoreactivity is weak (a) in the medium control group and absent in the ICI-treated group (c). Anti-müllerian hormone staining is absent in all three groups (d–f). Inset in f shows positive control of AMH in day-4 ovaries, where granulosa cells exhibiting staining. Original magnification, ×400. Fertility and Sterility 2009 91, 2025-2036DOI: (10.1016/j.fertnstert.2008.02.088) Copyright © 2009 American Society for Reproductive Medicine Terms and Conditions