134 THE DISTRIBUTION OF CYTOSKELETON AND CHROMATIN CONFIGURATIONS DURING EARLY EMBRYO DEVELOPMENT OF SWAMP BUFFALO (BUBALUS BUBALIS)

Abstract

Microtubules and actin microfilaments have been demonstratedto be actively involved with fertilization and early embryo development. The objective of this study was to examine the redistribution of cytoskeleton and chromatin configurations in swamp buffalo oocytes through the initial cleavage event after in vitro fertilization. Sperm penetration was analysed at 6 h post IVF in 63 oocytes (3 replicates), whereas the chronology embryonic development in terms of the redistribution of cell cytoskeleton and chromatin configurations was studied in a total of 462 oocytes (7 to 8 replicates) at 12, 18, 24, 30, and 48 h after IVF. The oocytes were matured in vitro for 22 h. Then, IVF was performed as described previously (Totey et al. 1993). After fertilization, presumptive zygotes and embryos were fixed at various times (6, 12, 18, 24, 30, and 48 h) to examine spermatozoa penetration, redistribution of the cytoskeleton (microtubules and actin filaments), and chromatin configurations using epifluorescent microscopy. Staining was undertaken with wheat germ agglutinin to visualize the zona pellucida, monoclonal-α-tubulin-TRIT C to show the microtubules, 488 phalloidin to identify microfilaments, and DAPI to label the chromatin. At 6 h after fertilization, sperm penetration was observed in 44.4% of examined oocytes. At 12 h post IVF, maternal chromosomes of fertilized oocytes progressed to the second meiotic division and formed the female pronucleus simultaneously with the decondensation of paternal chromosomes. A dense network of microtubules was observed radiating from the base of the decondensing sperm head (referred to as sperm aster) At 18 h post IVF, the sperm chromatins became the male pronucleus. Simultaneously, the sperm aster increased in size and filled the whole ooplasm. The syngamy of the male and female pronuclei was completed by 24 h post IVF, which was associated with a dense array of microtubules. Cell cleavage was observed by 30 h post IVF. This was apparently facilitated by a dense network of actin microfilaments that formed in the middle of the dividing embryo. These results indicated that microtubules and actin microfilaments undergo changes after fertilization consistent with a crucial role during fertilization in swamp buffalo. The centrosomal material was paternally inherited.

This work was supported by TRF-MAG (MRG-WII515S056) and CHE-TRF Senior Research Fund (RTA5080010).