Equine Zygote Development After ICSI 1199
with maternal chromosomes and extruded first polar body (PB1); (2) anaphase to telophase transition including ana- phase/telophase shift of maternal chromosomes, condensed sperm chromatin, PB1, and second polar body (PB2) in the process of extrusion or fully extruded; (3) formation of the male pronuclei with maternal chromosomes, PB1 and PB2; (4) male and female pronuclei at distant positions; (5) male and female pronuclei in close apposition. Pronuclei (pre- sumed male and female) were identified by localization of DNA and a human anticentromere antibody (CREST poly- clonal antibody). Development of presumptive zygotes were considered
abnormal if the following images were observed: (1) pre- mature chromosome condensation of the sperm with male chromosomes flanked by a bipolar spindle; (2) multiple pronuclei; (3) sperm chromatin induced ectopic polar body; (4) multipolar spindles with incorrect separation of chromosomes and presence of multiple centrosomes; (5) scattered maternal chromosomes and intact sperm head.
Statistical Analysis
χ2 Analysis was used to determine overall effects across all time points, and Fisher’s exact test was used within each category for comparisons among time points if the overall difference was significant at p<0.05.
RESULTS
Presumptive Zygote Developmental Events and Pronuclei Assessment
A similar series of developmental events were observed during zygote development after ICSI of IVO or IVM (Fig. 1, Table 1). Because of the relatively low number of samples, differences in the number of potential zygotes at specific developmental stages were observed over time only for IVO with condensed sperm chromatin, maternal chromosomes and PB1 (p=0.02, Figs. 1a–1c), IVO for presence of distant pronuclei (p=0.06, Fig. 2b), and IVM for apposed pronuclei (p=0.008, Fig. 2a, Table 1). The number of zygotes with pronuclei (distant or apposed) was significantly elevated at 12 and 16 h when compared with earlier time points for IVM, with the numerically highest percentage of pronuclei observed at 12 h (80%). Although not different over time, the highest numerical percentages of pronuclei were observed at 6 (67%) and 8 h (80%) for IVO (Table 1). At the initial observation after ICSI, many zygotes had a
female spindle arrested at metaphase II with aligned and compact chromosomes, an extruded first polar body, and paternal DNA as identified by the presence of the sperm head and, in some samples, the tail (Figs. 1a, 1b). Kinetochores were associated with maternal chromosomes at the metaphase plate and in the extruded first polar body (Fig. 1c). This early phase of development was observed predominantly at 4 h after ICSI, including 80 and 38% of presumptive zygotes derived from IVO and IVM,
respectively. One zygote from each of the two groups had not progressed past this developmental stage at 6 h after ICSI, and two zygotes from each group appeared delayed or arrested in development and had not progressed from this stage by ≥8 h after ICSI. The anaphase to telophase transition was imaged as
maternal chromosomes approaching anaphase or extruding the second polar body during telophase, with the presence of a sperm head (Figs. 1d–1f). This stage was observed as early as 4 h after ICSI for IVO and IVM, with some presumptive zygotes arrested at this point in later hours. Decondensation of male chromatin with a microtubule
array around the sperm head nuclei, female chromosomes aligned at the metaphase plate, and PB1 with or without PB2 (Figs. 1g–1i) was only imaged in three oocytes, including IVO oocytes that appeared delayed in development at 12 h after ICSI. The presence of two pronuclei, presumed male and
female, were imaged as early as 6 h after ICSI (Fig. 2b). At 8 h for IVO and 12 h for IVM, 80% of zygotes had pronuclei. When the pronuclei were in different areas of the ooplasm (distant), they were surrounded by a complex tubulin net, which diffused throughout the developing zygote. When the two pronuclei were close, the tubulin net was concentrated and contracting at the site of apposition (Fig. 2a). The number of nucleolus precursor bodies (NPB) in both parental pronuclei varied from three to seven; NPB were imaged in all pronuclei when PB2 was not extruded and three pronuclei were present (Fig. 3b).
Abnormalities Observed in Presumptive Equine Zygotes After ICSI Failure of zygote development was observed for IVO and IVM and was associated with abnormal cytoskeletal and chromatin configurations (Figs. 3, 4). IVO had an incidence of abnormalities during zygote development of 3/28 (11%), with no significant differences for the various phenotypes over time (Table 2). Of the 44 potential zygotes from IVM, 13 (30%) had abnormal phenotypes, with the most common abnormalities being premature chromosome condensation and sperm chromatin induced ectopic polar body. Overall, potential zygotes from IVM had a higher number of abnor- mal phenotypes per total injected oocytes than IVO (p=0.04, Table 2).
DISCUSSION
Equine zygotes are difficult to obtain, and confocal micro- scopy provides a potentially efficient method to analyze the limited numbers of samples. The equine oocyte is a large cell with a high lipid content (Ambruosi et al., 2009). The ooplasm measures ~85 µmin diameter and is surrounded by the zona pellucida, a glycoprotein layer about 12-µmin thickness; the outer zona pellucida is ~117 µm in diameter (Altermatt et al., 2009). The size and properties of the equine oocyte affect immunostaining and confocal imaging, adding
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