251 DEVELOPMENT OF A TECHNIQUE FOR STAINING CHROMOSOME AND SPINDLE OF MI AND MII BABOON OOCYTES

Abstract

The objective of this study was to develop a technique for staining baboon oocytes in order to detect chromosome and spindle disorganizations in abnormal oocytes. Baboon oocytes were retrieved by laparoscopy at Southwest National Primate Center/San Antonio following ovulation induction. The metaphase-I (MI) and metaphase-II (MII) oocytes were placed in one-mL sterile plastic tubes containing 10% formaldehyde solution. The tubes were sent by regular mail from San Antonio to the Reproductive Sciences Laboratory at Texas A&M University/College Station. The duration of oocyte fixation was from two to three weeks. The technique of baboon oocyte staining was based on the modification of the mouse oocyte staining protocol. A total of nine oocytes were collected for staining. The age of the baboon was six years. The oocytes were washed four times each, 20–30 min at room temperature, with PBS containing 10 µL Triton, 0.2 g dry milk, 2 g bovine serum albumin, and 0.75 g of glycine. The oocytes were kept over night in a refrigerator. The oocytes were stained with primary antibody solution (Monoclonal Anti Beta-Tubulin, Sigma Co., St. Louis, MO, USA) for 60 min at 37°C. After staining, five washes were performed for 20–30 min each at room temperature (RT). The oocytes were stained with secondary antibody solution (Alexa fluor 488, Invitrogen Co., Carlsbad, CA, USA) for 60 min at 37°C. Three washes were carried out at the end of the second staining, 20–30 min each at RT. Final staining was performed with Vectashield^reg propidium iodide (PI; Vector Laboratories, Burlingame, CA, USA) on a barrier slide which was then covered with a cover slide. Each slide contained 2–3 stained oocytes. The barrier slides were kept in a refrigerator for at least 48–72 h before imaging the oocytes. Chromosomes were stained with PI (red) and micro-tubular spindles were stained with anti Beta-tubulin antibodies + Alexa 488 (green). Cumulus cells appeared as bright red dots. The chromosomes of the immature oocytes (MI) did not yet appear completely aligned at the metaphase plate. The mature oocytes (MII) had extruded the polar body and it was clearly visible. The chromosomes looked elegantly organized at the metaphase plate. One chromosome was found to be disorganized (not aligned at the methaphase plate) in some oocytes which may be due to the side effect of superovulation treatment, but the spindles looked organized. In conclusion, the technique is found to be suitable for study of chromosome and spindle disorganizations which may cause recurrent abortions, birth defects, and infertility in nonhuman and human primates.