Relationship between the presence of endogenous nicks and sperm chromatin packaging in maturing and fertilizing mouse spermatozoa

Abstract

Mammalian spermiogenesis involves the replacement of histories by protamines, resulting in a highly compacted chromatin. Upon fertilization, the reverse process occurs. We have previously shown that the chromomycin A3 (CMA3) fluorochrome represents a useful tool for detecting protamine deficiency in spermatozoa. In this study we investigated CMA3 fluorochrome accessibility and the presence of endogenous nicks in maturing and fertilizing mouse sperm. Testicular sperm of stages 1-7 and 8-14 showed high positivity (> 96%) to CMA3, decreasing to 63% in stage 15-16 spermatids. In situ protamination of stage 15-16 spermatids saw an inhibition of CMA3 accessibility. Only 8% of the mature spermatozoa in the efferent ducts were CMA3-positive; this value decreased to 0% in the caput epididymidis. At fertilization, CMA3 fluorescence reappears in decondensing sperm. Fluorescein isothiocyanate (FITC) fluorescence, identifying endogenous nicks, was evident in 6% of stage 1-7 spermatids, increased to 22% in stage 8-14 spermatids, and disappeared in stage 15-16 spermatids. During fertilization, endogenous nicks were not observed in decondensing sperm. We propose that 1) the presence of nicks in mouse testicular spermatids suggests that DNA cutting and ligating occurs prior to completion of protamination and 2) the absence of nicks during fertilization indicates that decondensation is not simply the reversal of the initial chromatin packaging process.