Expression of the insulin-like growth factor and insulin systems in the luteinizing macaque ovarian follicle Rebecca S. Brogan, Ph.D., Scott Mix, Muraly.

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Expression of the insulin-like growth factor and insulin systems in the luteinizing macaque ovarian follicle Rebecca S. Brogan, Ph.D., Scott Mix, Muraly Puttabyatappa, M.S., Catherine A. VandeVoort, Ph.D., Charles L. Chaffin, Ph.D. Fertility and Sterility Volume 93, Issue 5, Pages 1421-1429 (March 2010) DOI: 10.1016/j.fertnstert.2008.12.096 Copyright © 2010 American Society for Reproductive Medicine Terms and Conditions

Figure 1 Intrafollicular concentrations of IGF1, IGF2, and insulin during luteinization. IGF1, IGF2, and insulin concentrations were measured using specific RIAs before (0 hours) and 3, 6, 12, and 24 hours after an ovulatory hCG stimulus (n = 5, 5, 5, 4, 5). IGF1 and IGF2 samples were acidified before assay to free IGFs bound to IGFBPs. Fertility and Sterility 2010 93, 1421-1429DOI: (10.1016/j.fertnstert.2008.12.096) Copyright © 2010 American Society for Reproductive Medicine Terms and Conditions

Figure 2 Expression of IGF1R, IGF2R, and IR mRNAs during luteinization. Granulosa cells aspirated from rhesus monkeys undergoing controlled ovarian stimulation before (0 hours) and 3, 6, 12, and 24 hours after an ovulatory hCG bolus (n = 5, 5, 4, 3, 3). The mRNA expression of IGF1R (left panel), IGF2R (middle panel), and total IR (right panel) was determined by real-time RT-PCR. Data were normalized to RPL19 mRNA and expressed as relative units. Asterisk indicates significant differences (P<.05) from control (0 hours) samples. Fertility and Sterility 2010 93, 1421-1429DOI: (10.1016/j.fertnstert.2008.12.096) Copyright © 2010 American Society for Reproductive Medicine Terms and Conditions

Figure 3 Granulosa cell expression of IGFBPs during luteinization. Granulosa cells aspirated from rhesus monkeys undergoing controlled ovarian stimulation before (0 hours) and 3, 6, 12, and 24 hours after an ovulatory hCG bolus (n = 5, 5, 4, 3, 3). The mRNA expression of IGFBP2 (top left panel), IGFBP3 (top right), IGFBP4 (middle left), IGFBP5 (middle right), IGFBP6 (lower left), and PAPP-A (lower right) was determined by real-time RT-PCR. Data were normalized to RPL19 mRNA and expressed as relative units. Asterisk indicates significant differences (P<.05) from control (0 hours) samples. Fertility and Sterility 2010 93, 1421-1429DOI: (10.1016/j.fertnstert.2008.12.096) Copyright © 2010 American Society for Reproductive Medicine Terms and Conditions

Figure 4 Follicular fluid levels of IGFBPs and PAPP-A during luteinization. Follicular fluid was collected from rhesus monkeys undergoing controlled ovarian stimulation before (0 hours) and 3, 6, 12, and 24 hours after an ovulatory hCG bolus (n = 3, 3, 2, 3, 5). (A) Representative example of a Western blot showing the IGFBP2 bands during the time course of hCG treatment (top panel) normalized to the large proteins remaining in the gel post-transfer (bottom panel). (B). The follicular fluid protein profiles of IGFBP2–6 are shown in graphical form. The Western blots were completed, membranes and gels scanned into a PDF file, and IGFBP band density normalized to coomassie blue-stained protein using the program U-SCAN IT (Silk-Scientific Corporation). Asterisk indicates significant differences (P<.05) from control (0 hours) samples. The hatch mark (#) indicated significant differences between 3 and 6 hours of hCG treatment (IGFBP4). (C) follicular fluid levels of PAPP-A determined by ELISA. Fertility and Sterility 2010 93, 1421-1429DOI: (10.1016/j.fertnstert.2008.12.096) Copyright © 2010 American Society for Reproductive Medicine Terms and Conditions