1204 Elena Ruggeri et al.
modifications occur in gametes and in early embryos (Reik et al., 2001; Bromfield et al., 2007; Cantone & Fisher, 2013), and ART procedures and in vitro culture could cause epi- genetic disturbances that affect oocyte competence, preg- nancy, and offspring health (Bromfield et al., 2007; El Hajj & Haaf, 2013). In our study, no definitive differences in zygote development were noted with the limited numbers of IVO and IVM oocytes, although zygotes from IVM subjectively appeared to develop slower. However, oocyte maturation groups differed in that significantly more zygotes from IVM, than IVO, had abnormalities suggestive of developmental failure. One potential risk of maturation in vitro is that nuclear and cytoplasmic maturation are not synchronized and can result in collateral effects on embryonic develop- ment (Smitz et al., 2011; Sanfins et al., 2015). Transcriptional profiling after in vitro maturation of oocytes from cattle, women and mice has confirmed changes in genes and pathways, which could affect postfertilization events (Rinaudo&Schultz, 2004; Mamo et al., 2011; El Hajj&Haaf, 2013). The extent that maturation in vitro affected oocyte quality in this study could not be directly determined, pri- marily because of the inherent variability within the popu- lation of immature oocytes. After sperm injection, factors associated with the sperm
and oocyte are critical for oocyte activation, decondensation of the sperm chromatin, and initiation of embryo develop- ment (Choi et al., 2004; Galli et al., 2007). Alterations in these processes can result in abnormal development, and abnor- mal morphologies were observed in some potential zygotes. Sperm-transmitted DNA damage leads to diverse abnormal reproductive outcomes and paternal genome loss, and it can be caused by different sperm chromatin defects, including premature sperm condensation and ectopic polar body extrusion after ICSI (Schmiady et al., 1996; Marchetti & Wyrobek, 2005; Deng & Li, 2009; Marchetti et al., 2015). After aneuploidy, the most common cause of fertilization failure in human in vitro fertilization and ICSI is premature sperm condensation (Edirisinghe et al., 1997; Moghbelinejad et al., 2013). Proper meiotic resumption of the oocyte is required to avoid premature condensation of the sperm head, which leads toDNAdamage and aneuploidy in human oocytes (Manandhar & Toshimori, 2003). In our study, premature sperm condensation was observed only in IVM, as previously reported (Tremoleda et al., 2003). Another sperm-related abnormality, specific to IVM in our study, was sperm chromatin induced ectopic polar body extrusion, with a spindle forming around sperm chromatin and leading to failure of normal zygote development because of the loss of paternal chromatin (Deng & Li, 2009). Premature sperm chromatin condensation and sperm chromatin induced ectopic polar bodies were only observed in IVM, suggesting that progression to the pronuclei stage was disrupted during oocyte activation in IVM oocytes or alterations of sperm DNA specific packaging and protamine deficiency (Schmiady et al., 1996; Deng & Li, 2009). However, we can- not exclude the potential that intrinsic oocyte quality or the stallion affected these results, as these variables were
different for the two ICSI systems. Additionally, the two post insemination culture conditions differed between labora- tories where the experiments were performed. Both DMEM/ F12 and SOF media are used clinically with high success rates, but we acknowledge that diverse culture media, and therefore the environment during embryo development, could have affected the results observed in this study. Additional zygote abnormalities included multiple pro-
nuclei and multipolar spindle or presence of scattered maternal chromosomes and intact sperm head (chromo- some fragmentation). Multipolar spindles suggest failure in spindle assembly checkpoints, and they were observed in a single oocyte from IVO and IVM (Sluder et al., 1997; Courtois et al., 2012). Scattered chromosomes and a sperm head were also observed in two potential zygotes from IVO and two from IVM. Multiple pronuclei suggest the failure of extrusion of the second polar body, possibly due to poor oocyte quality or sperm chromatin defects (Rosenbusch, 2001). Overall, we observed more morphological abnorm- alities in zygotes from IVM than IVO. This could have been caused by alterations in normal oocyte maturation in vitro; however, the increased diversity of oocytes collected from equine ovaries is also likely to affect developmental potential. Although the clinical use and success of ICSI has dramati-
cally increased in the equine industry over the last decade, this is one of few studies that has focused on factors that impact zygote development. The lack of ongoing research is primarily due to our lack of knowledge regarding normal equine zygote forma- tion and the expense and difficulty in obtaining equine oocytes, making projects that require large numbers of oocytes imprac- tical and overly expensive. Often, the end points of projects studying equine ICSI are cleavage and blastocyst
rates.Although these end points are important, they do not assess if fertilization was normal or why fertilization failure might have occurred. Therefore, more research is needed in this area to increase our understanding of equine fertilization and embryo development and to optimize success of ICSI. In this study, we demonstrated that confocal microscopy could provide meaningful data when using relatively low numbers of sperm-injected oocytes. Although multiple factors were different in regard to IVO or IVM, the results demonstrated some of the benefits and draw- backs for oocyte collection and maturation methods that are currently being used for clinical and research ICSI programs. The timelines provided by this research will allow future investi- gators to pick optimal time point(s) for observations of equine zygotesafter ICSI of
IVOorIVM.Thisinformation canbeused for future investigations, especially as related to sperm and oocyte quality. The research demonstrates that confocal micro- scopy of individual zygotes can be a powerful tool in assessing factors impacting fertilization and zygote development in the horse, and it provides methodology that can be used by researchers to practically investigate treatment effects.
CONCLUSIONS
In conclusion, we used confocal microscopy to observe equine zygotes at timed intervals after ICSI of IVO or IVM.
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