FLUORESCENT LOCALIZATION OF THIOLS AND DISULFIDES IN MARSUPIAL SPERMATOZOA BY BROMOBIMANE LABELING

The acrosome of marsupial spermatozoa is a robust structure which, unl ike its placental counterpart, resists disruption by detergent or free ze/thawing and does not undergo a calcium ionophore induced acrosome r eaction. In this study specific fluorescent thiol labels, bromobimanes , were used to detect reactive thiols in the intact marsupial spermato zoon and examine whether disulfides play a role in the stability of th e acrosome. Ejaculated brushtail possum (Trichosurus vulpecula) and ta mmar wallaby (Macropus eugenil) spermatozoa were washed by swim up and incubated with or without dithiothreitol (DTT) in order to reduce dis ulfides to reactive thiols. Spermatozoa were then washed by centrifuga tion and treated with monobromobimane (mBBr), a membrane-permeable bro mobimane, or with monobromotrimethylammoniobimane (qBBr), a membrane-i mpermeable bromobimane. Labelled spermatozoa were examined by fluoresc ence microscopy and sperm proteins (whole sperm proteins and basic nuc lear proteins) were analysed by gel electrophoresis. The membrane-perm eable agent mBBr lightly labelled the perimeter of the acrosome of non -DTT-treated possum and wallaby spermatozoa, indicating the presence o f peri-acrosomal thiol groups. After reduction of sperm disulfides by DTT, mBBr labelled the entire acrosome of both species. The membrane-i mpermeable agent qBBr did not label any part of the acrosome in non-DT T or DTT-treated wallaby or possum spermatozoa. Thiols and disulfides are thus associated with the marsupial acrosome. They are not found on the overlying plasma membrane but are either in the acrosomal membran es and/or matrix. The sperm midpiece and tail were labelled by mBBr, w ith increased fluorescence observed in DTT-treated spermatozoa. The nu cleus was not labelled in non-DTT or DTT-treated spermatozoa. Electrop horetic analysis confirmed the microscopic observations: Basic nuclear proteins (protamines) lacked thiols or disulfide groups. Based on the se findings, the stability of the marsupial acrosome may be due in par t to disulfide stabilization of the acrosomal membranes and/or acrosom al matrix. In common with placental mammals, thiol and disulfide conta ining proteins appear to play a role in the stability of sperm tail st ructures. It was confirmed that the fragile marsupial sperm nucleus la cked thiols and disulfides. (C) 1994 Wiley-Liss, Inc.