No abstract

A line of transgenic mice that carries an insertional mutation in a gene essential for spermatogenesis is described. Males homozygous for the transgenic insert are sterile, while female homozygotes and both male and female heterozygotes exhibit normal fertility. Developing spermatids in homozygous males form prominent abnormal multinucleated syncytia (symplasts) and do not complete maturation. In addition, abnormal cytoplasmic vacuolation is commonly seen in Sertoli cells. One flank of the transgenic integration site within the genome has been cloned and used to show linkage between homozygosity for the transgene and the mutant phe-

Studies of the precise steps of spermiogenesis at which dramatic changes occur in the nuclear proteins have been limited by the inability to obtain sufficient quantities of these cells in narrowly defined developmental stages, especially those between steps 1 and 12. This limitation can now be overcome by vitamin A-induced synchronization of rat testes into a few stages of the seminiferous epithelial cycle. Cell suspensions from stage-synchronized rat testes were separated by centrifugal elutriation, and selected fractions were further purified on Percoll gradients. Fractions enriched in spermatids in steps 1-3, 7-8, 9-10, and 11-12 were obtained. Analysis of the basic nucleoproteins from these cells by PAGE revealed the following changes. Between steps 3 and 7, histone (H) 2A variants, H2A.1, H2A.2, and TH2A, became post-translationally modified; and during steps 9-11, H1t became modified. H4, which was monoacetylated in steps 1-3, showed maximal levels of hyperacetylation in steps 11-12. The histones were the major basic nuclear proteins in spermatids through step 12. The low levels of transition proteins 1 and 2 observed in a fraction enriched in steps 11-12 could be largely accounted for by contamination from step 13-15 spermatids. All results were consistent with those obtained from normal, unsynchronized rats. The technique of vitamin A synchronization is therefore useful in more precisely defining biochemical changes during spermiogenesis.

Using a variation of a previously published method for manipulating vitamin A levels, we obtained synchronized rat testes and determined the frequency of stages of the seminiferous epithelium in each rat. In this study, we have demonstrated a method for quantitative analysis of the synchrony. The degree of synchronization was expressed as a fraction of the cycle of the seminiferous epithelium, and thus in terms not influenced by the different durations of the stages of this cycle. The median stage about which the tubules were synchronized was calculated. This method may be used to compare the effects of different synchronizing treatments, which may be subtle, and to study various aspects of spermatogenesis in the synchronized testes. For example, the duration of the cycle of the seminiferous epithelium in synchronized testes is estimated to be 12.5 days.