Fertility and Sterility Localization and quantitative expression of the calcium-sensing receptor protein in human oocytes  Maria Elena Dell’Aquila, Ph.D., Teresa De Santis, B.Sc., Yoon Sung Cho, Ph.D., Stephan Joel Reshkin, Ph.D., Anna Maria Caroli, D.V.M., Filippo Maritato, O.T., Paolo Minoia, D.V.M., Valeria Casavola, Ph.D.  Fertility and Sterility  Volume 85, Pages 1240-1247 (April 2006) DOI: 10.1016/j.fertnstert.2005.11.033 Copyright © 2006 American Society for Reproductive Medicine Terms and Conditions

FIGURE 1 Identification of calcium sensing receptor (CaSR) in human oocytes (lane A) and cumuli oophori (lane B) by Western blot analysis. The protein equivalent of 100 oocytes and 50 cumuli oophori was loaded. The proteins were subjected to SDS-PAGE on a linear gradient running gel of 4%–12%. The CaSR protein was detected with a primary antibody against a 20–amino acid peptide sequence near the C-terminus of human CaSR. The doublet seen in cumulus cells corresponds to the nonglycosylated 120-kDa form and to a glycosylated 130-kDa form of CaSR. Dell’Aquila. Calcium-sensing receptor in human oocytes. Fertil Steril 2006. Fertility and Sterility 2006 85, 1240-1247DOI: (10.1016/j.fertnstert.2005.11.033) Copyright © 2006 American Society for Reproductive Medicine Terms and Conditions

FIGURE 2 Detection of the calcium sensing receptor (CaSR) protein expression in a human metaphase II (MII)-stage oocyte by immunofluorescence with a primary antibody against a 20–amino acid peptide sequence near the C-terminus of human CaSR. Confocal laser scanning microscopy. The optical section shows the oocyte (A) and corona radiata cells (B) with positive labeling for CaSR. Arrows indicate the oolemma (A) and protrusion of corona cells (B). (C) Negative control. Scale bar = 25 μm. Dell’Aquila. Calcium-sensing receptor in human oocytes. Fertil Steril 2006. Fertility and Sterility 2006 85, 1240-1247DOI: (10.1016/j.fertnstert.2005.11.033) Copyright © 2006 American Society for Reproductive Medicine Terms and Conditions

FIGURE 3 Calcium sensing receptor (CaSR) localization and distribution on 25 serial optical sections in a human metaphase I (MI)-stage oocyte. The sample is representative of confocal investigations performed on all examined oocytes. The section squared in red (plane 13) represents the equatorial plane that was used for quantitative analysis, as reported in Figure 4. Dell’Aquila. Calcium-sensing receptor in human oocytes. Fertil Steril 2006. Fertility and Sterility 2006 85, 1240-1247DOI: (10.1016/j.fertnstert.2005.11.033) Copyright © 2006 American Society for Reproductive Medicine Terms and Conditions

FIGURE 4 Quantitative investigation of calcium sensing receptor (CaSR) expression in human oocytes. The micrographs show the image obtained at the equatorial plane of oocytes rapresentative of the GV (A), metaphase I (MI) (B), and metaphase II (MII) (C) stages. In the corresponding right panels (A′–C′), the fluorescence intensity encountered along the programmed scan line was recorded and plotted against the coventional pixel unit scale (0–255). Each cell was measured 16 times, on the basis of different radii taken at random. Dell’Aquila. Calcium-sensing receptor in human oocytes. Fertil Steril 2006. Fertility and Sterility 2006 85, 1240-1247DOI: (10.1016/j.fertnstert.2005.11.033) Copyright © 2006 American Society for Reproductive Medicine Terms and Conditions