181 CYTOSKELETAL AND CHROMOSOMAL ORGANIZATION IN DEVELOPMENTALLY ARRESTED EQUINE ZYGOTES AFTER INTRACYTOPLASMIC SPERM INJECTION

Intracytoplasmic sperm injection (ICSI) has been developed as a clinical procedure in the equine industry. Not all sperm-injected oocytes develop into embryos; however, the causes of developmental failure after equine ICSI have not been studied. Our objective was to use confocal microscopy to evaluate sperm-injected equine oocytes from young and old mares that did not cleave in a clinical ICSI program, to study the hypothesis that cleavage failure is associated with cytoskeletal and chromosomal alterations and affected by mare age. Oocytes were collected from the dominant follicles of young (Y; 5–15 years old) and old (O; 20–24 years old) oestrous mares approximately 24 h after administration of deslorelin and hCG. At approximately 44 h after deslorelin and hCG administration, oocytes were injected using frozen-thawed sperm from various stallions. Injected oocytes (n = 15) that failed to cleave by 24 to 48 h were fixed in microtubule stabilization buffer extraction fixative (MTSB-XF) and stained for DNA, α/β tubulin, acetylated α-tubulin, and f-actin. Confocal z-stacks were obtained using a Zeiss LSM 5 microscope (Carl Zeiss Microscopy LLC, Thornwood, NY), and images were assessed for cytoskeletal structures and chromosome organisation using the Zeiss LSM image browser. Potential zygotes (Y: n = 6 and O: n = 9) were categorized for (1) 2 sets of chromosomes, (2) misaligned chromosomes, (3) oversized spindles (approximately twice the size of an MII spindle), and (4) actin bubbling. The number of zygotes in each category for Y and O mares were compared by Fisher's exact test. Some samples were included in more than 1 morphological category. Two separate sets of chromosomes were observed in 1 of 6 and 4 of 9 injected oocytes from Y and O mares, respectively (P = 0.58), with development stopping before fusion of male and female pronuclei. Misaligned chromosomes were present in 2 of 6 and 7 of 9 oocytes from Y and O mares, respectively (P = 0.14). Oversized spindles were observed in more Y than O potential zygotes (5 of 6 and 2 of 9, respectively, P = 0.04), representing fusion of male and female pronuclei before developmental arrest. Multiasters were associated with the oversized spindles in potential zygotes from Y (2 of 6) and O (2 of 9) mares (P = 1), with microtubule activity potentially associated with the first mitotic division. Structures associated with actin bubbling tended (P = 0.12) to be higher in uncleaved zygotes from Y than O mares (5 of 6 and 3 of 9, respectively). Of the 15 injected oocytes, 10 arrested after fusion of male and female pronuclei. We have previously observed a high incidence of chromosomal misalignment in metaphase II oocytes from old mares. In the small number of potential zygotes that were studied, chromosomal misalignment tended (P = 0.14) to be higher for old mares. Other observations, such as oversized spindles and actin bubbling, appeared to be more prevalent in the potential zygotes of Y than O mares; these observations could be associated with normal development or post-ovulatory aging of the oocyte. Additional work will focus on the roles of actin and microtubules in remodelling the cytoplasm and organizing the chromosomes after fertilization in the horse and on the role of maternal aging in fertilization failure.