Transient expression of progesterone receptor and cathepsin-l in human granulosa cells during the periovulatory period Víctor García, M.D., Ph.D., Paulina.

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Transient expression of progesterone receptor and cathepsin-l in human granulosa cells during the periovulatory period Víctor García, M.D., Ph.D., Paulina Kohen, B.S., Carola Maldonado, Ph.D., Walter Sierralta, Ph.D., Alex Muñoz, B.S., Claudio Villarroel, M.D., Jerome F. Strauss, M.D., Ph.D., Luigi Devoto, M.D. Fertility and Sterility Volume 97, Issue 3, Pages 707-713.e1 (March 2012) DOI: 10.1016/j.fertnstert.2011.12.039 Copyright © 2012 American Society for Reproductive Medicine Terms and Conditions

Figure 1 (A) Western blot of progesterone receptor (PR) isoform A (92 kd) and isoform B (116 kd) in human granulosa cells (GCs) collected from patients before and at the time of the luteinizing hormone (LH) surge and after human chorionic gonadotropin (hCG) administration in IVF stimulated cycles. The GCs collected at the time of LH surge showed the most abundant levels of PR-A isoforms. Molecular mass standard is indicated on the right; β-actin was used as loading control and MCF-7 cells as positive control. Data represent an individual patient. (B) Immunofluorescence of PR in human GCs obtained from patients before LH surge and after hCG administration: (a) PR isoforms not detected before LH surge. (b) Total PR (green) detected after hCG administration (white arrows). (c) PRB isoforms exhibiting a weak signal. Nucleis were staining with DAPI (blue). (d) Negative control. (e, f) MCF-7 cells and endometrial cells for PR-A/B and PR-B isoform used as a positive control, respectively. Images are representative of three experiments. Fertility and Sterility 2012 97, 707-713.e1DOI: (10.1016/j.fertnstert.2011.12.039) Copyright © 2012 American Society for Reproductive Medicine Terms and Conditions

Figure 2 Progesterone receptor (PR) messenger RNA (mRNA) expression by quantitative real-time polymerase chain reaction in granulosa luteal cells (GLCs). (A, B) Total PR-A/B and PR-B messenger RNA (mRNA) increased after 6 hours incubation with human chorionic gonadotropin (hCG) (10 IU/mL) or Forskolin (1 μM). (C, D) Protein kinase A (PKA) inhibitor (H89) abolished the stimulatory effect of hCG (10 IU/mL) and forskolin (1 μM) of PR expression in GLCs culture. The values were expressed as mean ± standard error of the mean (n = 6). ∗P<.05 compared with control and other experimental conditions. Fertility and Sterility 2012 97, 707-713.e1DOI: (10.1016/j.fertnstert.2011.12.039) Copyright © 2012 American Society for Reproductive Medicine Terms and Conditions

Figure 3 (A) Western blot and (B) densitometric analysis of the protein kinase A (PKA) catalytic subunit (40 kd) in human preovulatory granulosa cells (GCs) collected before the luteinizing hormone (LH) surge (lanes 1, 2, and 3) and after human chorionic gonadotropin (hGC) administration (lanes 4, 5, and 6). Total proteins (40 μg) were loaded in each lane. Lane 7: homogenized rat brain used as a positive control. Densitometric relative units (RU) are normalized with Ponceau staining as loading control. Values area ± standard error of the mean from three individual observations in each group (n = 6). ∗P<.05. Fertility and Sterility 2012 97, 707-713.e1DOI: (10.1016/j.fertnstert.2011.12.039) Copyright © 2012 American Society for Reproductive Medicine Terms and Conditions

Figure 4 (A) Immmunoblots of pro-cathepsin-L (43 kd) and cathepsin-L active (25 kd) in human preovulatory granulosa cells (GCs) collected before the luteinizing hormone (LH) surge and after human chorionic gonadotropin (hCG) administration in the IVF cycle. Densitometric analysis shows that cathepsin-L active is statistically significantly expressed in the post-hCG group (∗P<.05). (B) Effect of hCG (10 IU/mL) on cathepsin-L active and pro-cathepsin-L avoided when cells are incubated for 24 hours in the presence of antiprogestin RU486 (1 μM). Densitometric results are expressed as a fold of basal value for cathepsin-L active and pro-cathepsin-L in human granulosa luteal cells (GLCs). Values are expressed as mean ± standard error of the mean (n =5); ∗P<.05 compared with control and hCG + RU486 (mifepristone) + Dx (dexamethasone). (C–F) Immunolocalization of cathepsin-L in human GLCs in presence and absence of RU486. (C) GLCs negative control. (D) GLCs basal. (E) GLCs culture + hCG (10 IU/mL). (F) GLCs culture + hCG (10 IU/mL) and RU486 (1 μM). Fertility and Sterility 2012 97, 707-713.e1DOI: (10.1016/j.fertnstert.2011.12.039) Copyright © 2012 American Society for Reproductive Medicine Terms and Conditions