321 CYTOSKELETAL ALTERATIONS OF EQUINE OOCYTES THAT FAILED TO CLEAVE AFTER INTRACYTOPLASMIC SPERM INJECTION: EVALUATION OF MATERNAL AND CELL AGING

Abstract

Intracytoplasmic sperm injection (ICSI) is used for assisted fertilization of equine oocytes. However, not all oocytes cleave after ICSI. Maternal aging deleteriously affects fertility in mares and women, with reduced oocyte quality and success of assisted reproductive technologies. In the oocyte, senescence and cell-programmed death begins after maturation; the extent that maternal age affects these events is unknown. We hypothesised that formation of α/β tubulin asters and f-actin bubbles are associated with aging of the oocyte in vitro and/or aging of the oocyte in vivo, in aged donors. In Exp 1, oocytes were collected from ovaries obtained from an abattoir and matured for 28 h and selected for polar body extrusion (0 h). At 0, 24, and 48 h, oocytes (n = 38 total) were fixed in MTSB-XF and transferred into wash solution with 1% BSA and 0.1% Triton X-100 in PBS for immunostaining. For experiment 2, oocytes were collected from preovulatory follicles of mares (9–25 yr) in a clinical ICSI program and injected with sperm from various stallions after extrusion of a polar body. Between 24 to 51 h after ICSI, uncleaved oocytes (n = 52, single cell without evidence of fragmentation or indentation of the oolemma) were fixed. All oocytes were incubated with α/β tubulin and human-anti-centromere antibody-CREST/ACA (1 : 100 each). Following primary incubation, oocytes were washed and incubated with Alexa 488, Alexa 647, Alexa 561-phalloidin, and Hoechst 33258. Images and Z-stacks were acquired on an Olympus IX81 spinning disk confocal microscope. Morphometric and intensity analyses of images were performed using SlideBook software (Denver, CO). Student's t-test, Fisher's exact test, and chi-square analyses were used for statistical comparisons. After aging in vitro (experiment 1), the number of oocytes with tubulin multiasters increased (P < 0.001; 9% at 0 h, 14% at 24 h, 85% at 48 h); however, actin bubbling was observed in only 5/38 (13%) oocytes, with no effect of incubation time. In experiment 2, tubulin multiasters were present in 62% of oocytes that failed to cleave. More multiasters were observed per oocyte from mares ≤13 yr than ≥20 yr (P = 0.03) and fixed at 24 to 28 h than 44 to 51 h (P = 0.04). Actin bubbles were observed in 71% of oocytes that failed to cleave after ICSI, with more actin bubbles in oocytes from mares ≥20 yr than ≤13 yr (P = 0.01) and fixed 44 to 51 h versus 24 to 28 h after ICSI (P = 0.05). The sum intensity and area of the actin bubbles were higher in oocytes fixed at 44 to 51 h than 24 to 28 h (P = 0.01 and P = 0.04). The area occupied by the actin bubbles was larger (P = 0.05) in oocytes from mares ≥20 yr than ≤13 yr. This study demonstrates actin bubbles and tubulin asters are involved in oocyte aging and cytoskeleton remodelling with or without fertilization. Although actin structures were associated with donor age and hours after ICSI, they were not present in unfertilized oocytes aged in vitro. Multiaster formation was associated with cell senescence in oocytes aged in vitro. Although not previously reported for the equine oocyte, multiaster formation appeared to be an initial fertilization event within the oocyte associated with attempted zygote development.