RELATIONSHIP BETWEEN THE PRESENCE OF ENDOGENOUS NICKS AND SPERM CHROMATIN PACKAGING IN MATURING AND FERTILIZING MOUSE SPERMATOZOA

Mammalian spermiogenesis involves the replacement of histones by prota mines, resulting in a highly compacted chromatin. Upon fertilization, the reverse process occurs. We have previously shown that the chromomy cin A(3) (CMA(3)) fluorochrome represents a useful tool for detecting protamine deficiency in spermatozoa. In this study we investigated CMA (3) 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 CMA(3), decreasing to 63% i n stage 15-16 spermatids. In situ protamination of stage 15-16 spermat ids saw an inhibition of CMA(3) accessibility. Only 8% of the mature s permatozoa in the efferent ducts were CMA(3)- positive; this value dec reased to 0% in the caput epididymidis. At fertilization, CMA(3) fluor escence reappears in decondensing sperm. Fluorescein isothiocyanate (F ITC) fluorescence, identifying endogenous nicks, was evident in 6% of stage 1-7 spermatids, increased to 22% in stage 8-14 spermatids, and d isappeared 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 cu tting and ligating occurs prior to completion of protamination and 2) the absence of nicks during fertilization indicates that decondensatio n is not simply the reversal of the initial chromatin packaging proces s.