134 EVALUATION OF APOPTOSIS IN BOVINE EMBRYOS BY FLUORESCENT LABELING OF CASPASES-3 AND -7

Abstract

Reports indicate that apoptosis is more prevalent in in vitro-produced (IVP) embryos than in in vivo-derived (IVD) embryos. These studies most often utilize the TUNEL procedure to label fragmented DNA. DNA fragmentation, however, is not unique to apoptosis as cells undergoing necrosis also exhibit substantial DNA fragmentation. Also, improper tissue handling has been reported to induce enough DNA fragmentation for TUNEL-positive staining of nuclei. Thus, the objective of this study was to evaluate the presence of caspases-3 and -7 as indicators of apoptosis in bovine fresh IVD embryos (F-IVD; n = 32), frozen-thawed IVD embryos (FT-IVD; n = 32) and IVP embryos cultured in CR1aa (n = 64). Embryos (quality grades, QG = 1 to 4) were evaluated for apoptotic cells by using a fluorescence-based assay for the detection of active caspaces (Caspatag®, Caspase 3,7 In Situ Assay Kit, Chemicon International, Inc., Temecula, CA, USA). To validate this assay, positive controls (PC) were prepared by incubating IVP-embryos in cyclohexamide for 3 to 4 h prior to staining. After staining, embryos were placed on slides and the number of apoptotic cells/embryo were counted. Embryos in which individual fluorescent points were not apparent were categorized as having profuse staining patterns (PSP) and were assigned an arbitrary apoptotic cell value of 20. Data were analyzed using PROC GLM of SAS with a post hoc LSD test. More than 65% of all embryos displayed at least 1 fluorescent cell, regardless of treatment. PC embryos consistently exhibited PSP. Overall, culture conditions did not markedly affect the incidence of apoptosis. FT-IVD embryos had significantly more (P < 0.05) apoptotic cells (8.2 ± 2.9) than F-IVD (0.9 ± 0.3). However, when embryos exhibiting PSP were excluded, the statistical difference was no longer present (1.5 ± 0.9 vs. 0.9 ± 0.3, respectively). It is unclear why some embryos exhibit specific staining patterns while others exhibit PSP, but we hypothesize that PSP embryos are likely degenerate. Interestingly, different degrees of fluorescence were often present within an embryo. Often, small pinpoint areas of fluorescence were detected while other times the fluorescence appeared to consume the entire cytoplasm of the cell. The capase protein has been localized in the cytoplasm of the cell, so the different degrees of fluorescence may represent different degrees of apoptosis. There was no major effect of culture environment on apoptosis when embryos were categorized by QG. Furthermore, within a treatment, there was no significant difference in apoptosis across embryo QG. To our knowledge, this is the first report of apoptosis evaluated in relation to embryo QG. Based on these findings, apoptosis does not appear to be heavily involved in the reduced developmental potential of IVP bovine embryos. The authors would like to sincerely thank Dr. Neil Schrick for his generous donation of frozen in vivo-derived bovine embryos.