298 DISRUPTION OF NUCLEAR MATURATION, APOPTOSIS AND CYTOSKELETAL CHANGES IN BOVINE OOCYTES EXPOSED TO HEAT SHOCK

Abstract

Heat shock (HS) can cause apoptosis and induce changes in cytoskeletal elements. A series of experiments were performed to determine whether physiologically relevant HS disrupts the progression of oocytes through meiosis, fertilization, and zygote formation, and causes corresponding changes in the cytoskeleton and apoptosis. Cumulus-oocyte complexes (COCs) were cultured at 38.5 (38C), 40 (40C), or 41°C (41C) for the first 12 h of maturation. Incubation during the last 10 h of maturation and 18 h post insemination (hpi) was at 38.5°C and 5% (v/v) CO₂ for both treatments. The CATMOD procedure of SAS (SAS Institute, Inc., Cary, NC, USA) was used to analyze the distribution of oocytes into various classes of nuclear maturation and the proportion of apoptotic oocytes. In Exp. 1, matured oocytes were fixed in 4% (w/v) paraformaldehyde, and either stained with Hoechst 33342 or labeled with TUNEL kit (Roche Diagnostics, Indianapolis, IN, USA). Pronuclei were classified as being either condensed or at metaphase I (MI), metaphase II (MII), anaphase I, or telophase I. HS affected (P < 0.001) the distribution of oocytes into stages of meiosis. The majority of 38C oocytes reached MII while 41C oocytes were mostly at MI. Both 40C and 41C increased the percentage of oocytes having TUNEL-positive nuclei (P < 0.001). In Exp. 2, matured oocytes were fixed and stained with Hoechst and markers for either filamentous actin (phalloidin) or microtubules (anti-bovine-α-tubulin labeled with Zenon). Microfilament localization was affected by stage of nuclear maturation and by HS. Actin microfilaments were more prominent in the cytoplasm of heat-shocked oocytes than for 38C oocytes. In addition, the intense ring of actin present under the plasma membrane was reduced for 41C oocytes and the transzonal actin processes present in 38C oocytes were absent in 41C oocytes. A subset of heat-shocked oocytes possessed misshapen MI spindles with disorganized microtubules and unaligned chromosomes. In Exp. 3, addition of 50 nM sphingosine 1-phosphate (S1P) to maturation medium blocked the effect of HS on progression through meiosis and apoptosis. There was a temperature × S1P interaction (P < 0.001) on distribution of oocytes into nuclear classes because S1P increased the proportion of 41C oocytes that were at MII. S1P also blocked the increase in percentage of TUNEL positive oocytes (temperature × treatment, P < 0.005). In Exp. 4, examination of the chromosomal organization for putative zygotes (18 hpi) revealed that HS affected (P < 0.001) their distribution into nuclear classes. The percentage of putative zygotes with a normal diploid pattern was 57% vs. 20% for 38C and 41C oocytes, respectively. In conclusion, HS during the first 12 h of maturation disrupts nuclear maturation, induces apoptosis, alters the cytoskeleteon, and reduces subsequent fertilization. These alterations are likely to be involved in the mechanism underlying heat shock induced disruption of oocyte competence and can be reduced by S1P.

This work received the following support: BARD FI-330-2002 and USDA Grant 2002-35203-12664.