Sperm cell-free systems

It is interesting and important to note that several species, such as Schizosac-charomyces pombe, lack histone HI (Wood et al 2002) (see also section 2.4). The nucleosome-repeat length is slightly shorter in S. pombe than in human (Godde and Widom, 1992). The contribution of histone HI to the mitotic chromosome condensation has been examined with the use of a cell-free system of Xenopus eggs, in which the condensed sperm nuclei can be transformed into metaphase chromosomes. Even when histone HI is removed from the extract, the metaphase chromosomes can still be formed (Ohsumi et al, 1993). In addition, an elimination of all HI genes in Tetrahymena exerts no phenotypic effect (Shen et al, 1995). 

An advantage of cell-free systems is the potential to evaluate independently cytosolic and membrane vesicle (MV) contributions to nuclear development. Membrane-free cytosol is obtained after ultracentrifugation of crude lysates and MVs can be recovered from the pellets. Both cytosolic extracts and MVs can be stored frozen without detectable loss of envelope assembly activity. They can also be manipulated easily by chemical or enzymatic treatments. Such manipulations have enabled the identification of distinct steps of male pronuclear formation and of factors required for each of these steps, notably in Xenopus (Lohka and Masui, 1984 Wilson and Newport, 1988 Vigers and Lohka, 1 1 Boman et al., 1992) and the sea urchin (Cameron and Poccia, 1994 Collas and Poccia, 1995a,b Collas etal., 1995). Studies in the sea urchin and surf clam have indicated that decondensation of sperm chromatin in vitro meets several criteria established by microinjection of sperm nuclei into living eggs (Cothren and Poccia, 1993) and by electron microscopy observations of normal pronuclear formation in vivo (Longo and Anderson, 19( 1970). 

An interesting approach reported for obtaining purified male and female fractions on microdevices involves acoustic differential extraction (ADE) and exploits microacoustic transducer developments (see Chapter 44 by Laurell for details). This method involves the elution of biological material from the vaginal swab under mild lysis conditions, resulting in a mixture of sperm cells and epithelial cell lysate. The sample is then infused in the microdevice, and sperm cells trapped in a monolayer above an ultrasonic transducer while free DNA (from the lysed epithelial cells) flows through the system unretained. Flow of the epithelial cell lysate is directed to the outlet reservoir... 

A, Cell-Free Chromatin Remodeling Systems 13. Sperm Chromatin Preparation... 

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