Effects of Sperm Pre-Treatment on Chromosomal Integrity Following Intracytoplasmic Sperm Injection in Mice.

Abstract

In intracytoplasmic sperm injection (ICSI), spermatozoa are selected by operator using a vague indicator such as progressive motility and morphological normality. Generally, non-capacitated and/or acrosome intact spermatozoon is injected into an oocyte for initiation of fertilizing process. In many species, it is believed that injection of capacitated or acrosome reacted spermatozoa is not essential for initiation of fertilization following ICSI. However, it is unknown whether the non-physiological condition like non-capacitated and acrosome intact sperm injection may induce any harmful effects on ICSI embryos or not. Therefore, we evaluated the zygotic chromosomal integrity after micro-injection of in vitro capacitated or acrosome reacted spermatozoa using a mouse model. The cytogenetic effects of another widely used sperm pre-treatment such as dithiothreitol (DTT), disulfide bond reducing agent, were also examined. For oocytes collection, B6D2F1 female mice (7-12 weeks old) were superovulated by injection of PMSG and hCG 48 h apart. The oocytes were recovered from oviducts between 14 and 16 h after hCG injection followed by denuding of their cumulus cells. Spermatozoa were collected from the cauda epididymis of male B6D2F1 mice (7-12 weeks old). Before ICSI, collected spermatozoa were underwent four treatments: i) 1 mg/ml methyl-β-cyclodextrin (MBCD) for 90 min for capacitation, ii) 0.02% lysolecithin (LL) and iii) 0.02% Triton X-100 (TX) for 1 min for disruption of acrosome, and iv) 5 mM DTT for 10, 30 and 60 min for reduction of disulfide bonds. The treated spermatozoa were injected within 30 min after treatment. After 19 to 21 h of ICSI, the presumptive zygotes were fixed by gradual-fixation/air drying methods on glass slides. The chromosomes on slides were stained with 2% Giemsa in buffered saline (pH 6.8) for 10 min. The fertilization rates of MBCD, LL and TX groups were 97, 94 and 97%, respectively, and 59, 69 and 60% of zygotes for MBCD, LL and TX groups, respectively, had normal chromosome sets. In non-treated control group, 87% of zygotes had normal chromosome sets. Although there was no significant difference among treatment groups, the proportions of zygotes having normal chromosome sets were significantly lower in sperm treatment groups than that of non-treated control (P<0.05). The fertilization rates (92 to 98%) did not vary among different periods of DTT treatments. However, chromosomal integrity decreased in a time depending manner (10 and 30 min vs. 60 min: 63 and 56% vs. 17%; P<0.001). Furthermore, severe chromosomal aberration (10 or more aberrations per one karyoplate) was observed in higher proportion (28%) of zygotes treated with 60 min DTT than those of 10 min (0%) and 30 min (8%) of counterparts (P<0.05). When compared with non-treated control group, DTT treatment for any duration had harmful effects on chromosomal integrity of zygotes (non-treatment vs. 10 and 30 min: 13% vs. 37 and 36%; P<0.05, non-treatment vs. 60 min: 13% vs. 55%; P<0.001). These results clearly show that sperm pre-treatment prior ICSI is a detriment to their genetic safety, even if the duration of treatment is extremely short, 1 min exposure to LL or TX. Moreover, prolonged sperm treatment such as DTT for 60 min increases the genetic risk as indicated by severe aberrant in their chromosomes. In conclusion, chromosomal integrity of spermatozoa treated with any chemicals should be confirmed prior ICSI.