Procedure for Bovine ICSI, not Sperm Freeze-drying, Impairs the Function of the Microtubule-organizing Center

Abstract

Abstract. This study was designed to investigate whether freeze-dried (FD) bull spermatozoa maintained the function of the microtubule-organizing center (MTOC) after rehydration and intracytoplasmic sperm injection (ICSI). In a preliminary attempt, the cleavage and blastocyst formation rates in FD-ICSI zygotes (36 and 1%, respectively) were found to be considerably lower than those in control ICSI zygotes (67 and 21%, respectively) or in IVF zygotes (78 and 43%, respectively). An alkaline comet assay indicated that the DNA fragmentation index (length of comet tail × % DNA liberated) was not significantly different between fresh and FD spermatozoa. In the main experiment, formation of sperm-asters in the FD-ICSI oocytes 7 h postinsemination occurred at a similar rate when compared with the control ICSI oocytes (41 vs. 49%). Among the oocytes exhibiting sperm aster formation, the extent of microtubule network assembly was comparable between the FD-ICSI and control ICSI groups. However, the MTOC of the ICSI oocytes was not as functional as that of IVF oocytes in terms of the aster formation rate (97%) and the fluorescent intensity of the microtubule network (2.0 folds). These results suggest that the freeze-drying process per se had no adverse effect on maintaining the MTOC function in bull spermatozoa. Key words: Bovine Intracytoplasmic sperm injection (ICSI), Freeze-drying, Microtubule-organizing center (MTOC), Sperm-aster (J. Reprod. Dev. 57: [428][429][430][431][432] 2011) centrosome is composed of a pair of centrioles surrounded by pericentriolar materials such as γ-tubulin, centrin and pericentrin. Since an interphase network of microtubules and the mitotic bipolar spindle are nucleated from the centrosome, the centrosome is considered to be the microtubule-organizing center (MTOC) [1]. During fertilization in most mammalian species, the centrosome brought into an oocyte by a spermatozoon plays a critical role in assembly of the microtubule network that brings both male and female pronuclei to the center of the newly formed zygote, as reported in humans [2], rhesus monkeys [3], rabbits [4], pigs [5] and cattle [6]. Interestingly, paternal inheritance of the MTOC does not occur in the mouse [7] and rat [8], and the microtubule network developed from multiple cytoplasmic asters, instead of a single sperm-aster, is involved in the migration of pronuclei [9].Freeze-drying has been proposed as an alternative method to preserve mammalian spermatozoa [10], although freeze-dried (FD) spermatozoa after rehydration lose their motility and application of intracytoplasmic sperm injection (ICSI) technique is necessary. Rodent spermatozoa can be stored practically at refrigeration temperatures [11][12][13]. However, in large domestic species, in vitro production of blastocysts derived from FD-ICSI is still considered to be a challenging endeavor [14][15][16]. We have recently reported that the freeze-drying protocol slightly reduced the ability of bull spermatozoa to induce calcium oscillations [17] and that it had no a...

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