Vascular endothelial growth factor A and its two receptors in human preantral follicles from fetuses, girls, and women  Ronit Abir, Ph.D., Asangla Ao, Ph.D., Xiao Yun Zhang, M.Sc., Roni Garor, M.Sc., Shmuel Nitke, M.D., Benjamin Fisch, M.D., Ph.D.  Fertility and Sterility  Volume 93, Issue 7, Pages 2337-2347 (May 2010) DOI: 10.1016/j.fertnstert.2009.01.111 Copyright © 2010 American Society for Reproductive Medicine Terms and Conditions

Figure 1 Vascular endothelial growth factor (VEGF) A isoforms: primer design, and representative reverse transcriptase polymerase chain reaction (RT-PCR) gel illustrating VEGF-A121, VEGF-A165, and VEGF-A189 gene expression. (A) Schematic diagram showing VEGF-A isoforms and primer design. (B) Representative RT-PCR gel illustrating VEGF-A gene expression in fetal and adult ovaries: a) VEGF-A121 (283 bp); b) VEGF-A165 (214 bp); c) beta-actin (274 bp); d) VEGF-A189 (248 bp); lane 1: 100-bp ladder; lane 2: ovary from a 28-year-old woman; lane 3: ovary from a 21-year-old woman; lane 4: ovary from a 37-year-old woman; lane 5 and lane 7 in d: ovary from 35-gestational-week-old (GW) fetus; lane 6: ovary from 25-GW normal fetus; lane 7: pooled ovarian tissue from two 25-GW fetuses (VEGF-A189 was undetectable in this sample); lane 8 and lane 5 in d: pooled ovarian tissue from three 22–23-GW fetuses; lane 9 and lane 8 in d: RT− control. Fertility and Sterility 2010 93, 2337-2347DOI: (10.1016/j.fertnstert.2009.01.111) Copyright © 2010 American Society for Reproductive Medicine Terms and Conditions

Figure 2 Immunohistochemistry (IMH) photographs of vascular endothelial growth factor (VEGF) A expression. (A) VEGF-A protein staining. IMH staining of human ovary from 31 gestational-week-old (GW) fetus. Note the primordial follicles, with the strong red-brown staining indicating VEGF-A protein expression in the oocytes (full cytoplasmic without nuclear staining) and in a portion of the granulosa cells (GCs) and the stroma cells. Original magnification ×400. (B) VEGF-A protein staining. IMH staining of human ovary from 18-year-old girl. Note the primordial and primary follicles, with medium staining indicating VEGF-A expression in the oocytes (full cytoplasmic with nuclear staining) and in a portion of the GCs and the stroma cells. Original magnification ×400. (C) Negative control for VEGF-A. Section of human ovary from 27-GW fetus. Note the primordial and primary follicles, the overall blue staining, and the lack of red-brown staining. Original magnification ×400. (D) Negative control for VEGF-A. Section of the same human ovary as in Figure 2B. Note the primordial and primary follicles, the overall blue staining, and the lack of red-brown staining. Original magnification ×400. Fertility and Sterility 2010 93, 2337-2347DOI: (10.1016/j.fertnstert.2009.01.111) Copyright © 2010 American Society for Reproductive Medicine Terms and Conditions

Figure 3 Photographs of vascular endothelial growth factor receptor 1 (VEGFR1) expression. (A) VEGFR1 protein staining. Immunohistochemical (IMH) staining of the same human ovary as in Figure 2C. Note the primordial and primary follicles, with the medium staining indicating VEGFR1 expression in the oocytes (full cytoplasmic with partial as well as lack of nuclear staining) and in a portion of the granulosa cells (GCs) and stroma cells. Original magnification ×400. (B) VEGFR1 mRNA staining. In situ hybridization (ISH) staining of human ovary from 31-year old woman. Note the primary follicle, with medium-weak staining indicating VEGFR1 expression in the oocytes (partial cytoplasmic with nuclear staining) and in a portion of the GCs and stroma cells. Original magnification ×400. (C) Negative control of VEGFR1 IMH staining. Section of the same human ovary as in Figure 3A. Note the primordial and primary follicles, the overall blue staining, and the lack of red-brown staining. Original magnification ×400. (D) Negative control of VEGFR1 ISH staining. Section of the same human ovary as in Figure 3B. Note the primary follicle, the overall blue staining, and the lack of red-brown staining. Original magnification ×400. Fertility and Sterility 2010 93, 2337-2347DOI: (10.1016/j.fertnstert.2009.01.111) Copyright © 2010 American Society for Reproductive Medicine Terms and Conditions

Figure 4 Photographs of vascular endothelial growth factor receptor 2 (VEGFR2) expression and positive control for beta-actin. (A) VEGFR2 protein staining. Section of human ovary from a 5-year girl. Note the large secondary follicle with its weak oocyte staining (full cytoplasmic staining) and expression in a portion of the granulosa cells (GCs) and stroma cells. Original magnification ×400. (B) VEGFR2 mRNA staining. Section of human ovary from 33-gestational-week-old fetus. Note the primordial and primary follicles with the staining in the oocytes (full cytoplasmic with nuclear staining) and in a portion of the GCs and stroma cells. Original magnification ×400. (C) Negative control of VEGFR2 immunohistochemical expression. Section of the same ovary as in Figure 4A. Note the primary-secondary follicle, the overall blue staining, and the lack of red-brown staining. Original magnification ×400. (D) Negative control of VEGFR2 mRNA staining. Section of the same human ovary as in Figure 4 (B). Note the primordial and primary follicles, the overall blue staining, and the lack of red-brown staining. Original magnification ×400. (E) In situ hybridization positive control of beta-actin expression. Section of the same ovary as in Figure 4B. Note the primordial and primary follicles, with the medium-strong red-brown staining indicating expression in the oocytes (full cytoplasmic with nuclear staining) and in the GCs and stroma cells. Original magnification ×400. Fertility and Sterility 2010 93, 2337-2347DOI: (10.1016/j.fertnstert.2009.01.111) Copyright © 2010 American Society for Reproductive Medicine Terms and Conditions