Chromosomal DNA was isolated from rapidly dividing cells of Xenopus laevis embryos at blastulation, at gastrulation, and at the beginning of hatching. Few, if any, replication forks were seen by electron microscopy in DNA isolated at any stage of embryogenesis. Instead, unbranched DNA, which appeared to be single-stranded, was abundant at all stages. The percentage of chromosomal DNA that was single-stranded was quantitated by electron microscopy and by monitoring the release of acid-soluble radioactivity during digestion of labeled chromosomal DNA with nucleases specific for single-stranded DNA. The amount of single-stranded DNA was inversely correlated with the length of S phase during embryogenesis. We postulate that chromosomal DNA replication in X. Ievis embryos takes place by a mechanism in which strand separation is uncoupled from DNA synthesis.

Amplification of sequences by the polymerase chain reaction (PCR) has become a powerful tool in the study of gene expression. The technique is, in fact, so powerful that it may detect 'leaky transcription'. Thus, it is now important to be able to quantify the transcripts that are amplified to determine whether or not they represent legitimate transcription of target genes. In this paper, we describe a one-step amplification reaction coupled to solution hybridization/RNase protection that is capable of quantitating specific transcripts in total RNA from one to ten preimplantation mouse embryos and is generally applicable to any cloned mRNA sequence.

The testis-determining factor in the mouse is encoded by the Sry gene on the Y chromosome. Transcripts of this gene have been shown previously to be present in the genital ridge at the beginning of gonadal differentiation (11.5 days post coitum) and in adult testis. In this study, RNA transcripts of the Sry gene are also detected in male blastocyst-stage embryos (3.5 days post coitum) at approximately 40-100 copies per cell, long before overt sex differentiation. These results indicate that preimplantation mouse embryos have sexually dimorphic gene expression at least with respect to Sry transcripts. In addition, at least some of the Sry RNA transcripts in blastocysts are circular, as has been reported for Sry transcripts from adult testis. The appearance of Sry transcripts in blastocysts at this level raises the possibility that sex determination begins earlier during embryonic development than previously thought.

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