Lapatinib inhibits meiotic maturation of porcine oocyte-cumulus complexes cultured in vitro in gonadotropin-supplemented medium Eva Nagyova, D.V.M.,

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Lapatinib inhibits meiotic maturation of porcine oocyte-cumulus complexes cultured in vitro in gonadotropin-supplemented medium Eva Nagyova, D.V.M., Ph.D., Lucie Nemcova, Ph.D., Alzbeta Mlynarcikova, Ph.D., Sona Scsukova, Ph.D., Jaroslav Kalous, Ph.D. Fertility and Sterility Volume 99, Issue 6, Pages 1739-1748 (May 2013) DOI: 10.1016/j.fertnstert.2012.12.040 Copyright © 2013 American Society for Reproductive Medicine Terms and Conditions

Figure 1 (A) Effect of lapatinib (10 μM; Lap10) on FSH/LH-stimulated cumulus expansion assessed after 24 or 44 hours. Oocyte-cumulus complexes (OCC) cultured in fetal bovine serum (FBS) represent control groups. (B) Effect of lapatinib (1, 10 μM; Lap1, Lap10) on expression of the cumulus expansion-related transcripts: TNFAIP6 and PTGS2 messenger RNA after 4 hours of FSH/LH stimulation. Relative abundance of the respective messenger RNA is expressed in arbitrary units as fold-strength increase of specific gene/ACTB ratio more than the level found in unstimulated OCC (FBS). Different superscripts above columns indicate significant differences (P<.01). (C) Effect of lapatinib (1, 10 μM; Lap1, Lap10) on FSH/LH-stimulated synthesis (total) and retention of hyaluronan (HA) by OCC after 24 hours. Data represent the mean ± SEM from at least five replicates for each treatment, prepared in duplicate (at least 50 OCC per treatment). Columns with different letters are significantly different (P<.01). (D) Effect of lapatinib (1–100 μM; Lap1, Lap10) on FSH/LH-stimulated secretion of P by OCC after 44 hours. Values represent the mean ± SEM obtained from triplicates of two independent experiments. The absolute value corresponding to control (FBS) was 10.1 ± 1.1 ng/mL. Bars with different superscripts indicate significant differences (P<.05). Fertility and Sterility 2013 99, 1739-1748DOI: (10.1016/j.fertnstert.2012.12.040) Copyright © 2013 American Society for Reproductive Medicine Terms and Conditions

Figure 2 Tumor necrosis factor (TNF) alpha-induced protein 6 (TNFAIP6; green) detection in oocyte-cumulus complexes after 24 hours of stimulation with FSH/LH in the absence/presence of lapatinib (Lap; 10 μM). The oocyte-cumulus complexes were incubated with rabbit–anti-human TNFAIP6 and with fluorescent secondary probe AF488 goat–anti-rabbit IgG. Nuclear DNA was counterstained with DAPI (blue). Transmitted light images were obtained with the use of a differential interference contrast (DIC). Fertility and Sterility 2013 99, 1739-1748DOI: (10.1016/j.fertnstert.2012.12.040) Copyright © 2013 American Society for Reproductive Medicine Terms and Conditions

Figure 3 Hyaluronan (HA; green) detection in oocyte-cumulus complexes after 24 hours of stimulation with FSH/LH in the absence/presence of lapatinib (Lap; 10 μM). The oocyte-cumulus complexes were labeled with biotinylated HA-binding protein (HABP) and with fluorescent secondary probe AF488 streptavidin conjugate (green). Nuclear DNA was counterstained with DAPI (blue). Transmitted light images were obtained with the use of a differential interference contrast (DIC). Fertility and Sterility 2013 99, 1739-1748DOI: (10.1016/j.fertnstert.2012.12.040) Copyright © 2013 American Society for Reproductive Medicine Terms and Conditions