Expression of cell cycle regulatory genes during primordial-primary follicle transition in the mouse ovary  Chang-Eun Park, M.S., Kwang-Yul Cha, M.D., Kyungjin Kim, Ph.D., Kyung-Ah Lee, Ph.D.  Fertility and Sterility  Volume 83, Issue 2, Pages 410-418 (February 2005) DOI: 10.1016/j.fertnstert.2004.06.074 Copyright © 2005 American Society for Reproductive Medicine Terms and Conditions

FIGURE 1 Suppression subtractive hybridization subtraction efficiency. Subtraction efficiency was determined by analyzing the amount of G3PDH present in both the unsubtracted starting cDNA and the subtracted target cDNA through the use of increasing numbers of PCR cycles. MW stands for molecular weight marker. PCR was performed on subtracted (lanes 1–4) and unsubtracted (lanes 5–8) secondary PCR products with G3PDH primers in both forward (lanes 1–8) and reverse subtraction (lanes 9–16). Numbers above each lane indicate PCR cycles. Fertility and Sterility 2005 83, 410-418DOI: (10.1016/j.fertnstert.2004.06.074) Copyright © 2005 American Society for Reproductive Medicine Terms and Conditions

FIGURE 2 Northern blot analysis of the genes from the day-1 and day-5 subtracted cDNA library to confirm their differential expression between the two. GDF-8: growth differentiation factor-8; lats2: large tumor suppressor gene-2; Mater: maternal antigen that embryos require; MTi7: MT transposon-like element, clone-7; PTPRD: protein tyrosine phosphatase receptor type D; D1: day 1; D5: day 5. Fertility and Sterility 2005 83, 410-418DOI: (10.1016/j.fertnstert.2004.06.074) Copyright © 2005 American Society for Reproductive Medicine Terms and Conditions

FIGURE 3 Microphotographs depict the laser capture microdissection procedure for capturing each stage of follicles out of ovarian tissues. (A) Day-1 ovarian section before capturing. (B) Captured primordial follicles from the day-1 ovarian section. (C) Day-5 ovarian section before capturing. (D) Captured primary follicles from the day-5 ovarian section. Fertility and Sterility 2005 83, 410-418DOI: (10.1016/j.fertnstert.2004.06.074) Copyright © 2005 American Society for Reproductive Medicine Terms and Conditions

FIGURE 4 Real-time PCR analysis to confirm the differential expression with gene specific primers as listed in Table 1. (A) Amplification profile of septin2 and lats2 between primordial and primary follicles. PMF: primordial follicle; PRIF: primary follicle. (B) Melting curves for septin2 and lats2. (C) Amplification profile of G3PDH between primordial and primary follicles. (D) Melting curves for G3PDH. The mean CT values are as follows: septin2 (primordial: 13.28, primary: 15.42), lats2 (primordial: 19.70, primary: 21.14), and G3PDH (primordial: 12.13, primary: 12.00). Fertility and Sterility 2005 83, 410-418DOI: (10.1016/j.fertnstert.2004.06.074) Copyright © 2005 American Society for Reproductive Medicine Terms and Conditions

FIGURE 5 Differential expression of 11 genes related to cell-cycle regulation between primordial (blue bars) and primary (red bars) follicles. Expression amount was calculated from CT values, and the fold-difference of relative abundance of mRNA was calculated against that of primordial follicles. Experiments were repeated at least three times, and data are expressed as mean ± SEM. *Statistically significant, P<.05. Fertility and Sterility 2005 83, 410-418DOI: (10.1016/j.fertnstert.2004.06.074) Copyright © 2005 American Society for Reproductive Medicine Terms and Conditions