Expression of the mouse testis-determining gene Sry in male preimplantation embryos.

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.

Effect on embryos of injection of phosphorothioate-modified oligonucleotides into pregnant mice.

Phosphorothioate-modified oligonucleotides were injected into pregnant female mice to assess the effect on developing embryos. Injections were carried out during two different time periods, one when embryos were in preimplantation stages of development (about 3.5 days of development) and the other after implantation, when both a fetus and placenta are present (from days 9.5 to 11.5 of development). Three different phosphorothioate-modified oligonucleotides were injected. One, which had a sequence not present in the mouse genome, was used to ask whether nonspecific toxic or teratogenic effects on embryos result from treatment of the mother. A second was complementary to the mRNA of the testis-determining factor gene Sry and was used to ask whether a specific developmental pathway (i.e., sex determination) could be disrupted in embryos in vivo. The third was the complement of the anti-Sry sequence. None of these oligonucleotides reduced the frequency of successful pregnancy after mating or the average litter size from that observed in controls animals. Furthermore, examination of 291 pups or fetuses from all oligonucleotide-injected pregnant females revealed no developmental defects regardless of which sequence was used. It is concluded that injection of phosphorothioate-modified oligonucleotides into pregnant females according to the protocols described here is not toxic or teratogenic to embryos in a nonspecific way. Also, anti-Sry oligonucleotides did not influence sex determination in embryos, although there are several possible explanations for this.

Mutually exclusive expression of the Strongylocentrotus purpuratus Spec1 gene and its Lytechinus pictus homologue in cells of hybrid embryos.

The expression of the Spec1 gene of Strongylocentrotus purpuratus and its Lytechinus pictus homologue LpS1 was analyzed in reciprocal hybrid embryos of these two species of sea urchin. While the time course of accumulation of Spec1 mRNA was nearly normal in hybrid embryo populations, the accumulation of LpS1 mRNA was not. This was particularly evident in plutei, where the level of LpS1 mRNA was less than 5% that in normal L. pictus plutei. In situ hybridization analysis of serial sections indicated that LpS1 mRNA was detectable in only about 2% of hybrid plutei in either cross, whereas Spec1 mRNA was present in nearly all hybrid plutei; expression of either homologue was appropriately restricted to the aboral ectoderm. In crosses of L. pictus eggs with S. purpuratus sperm (LpSp), about 1% of hybrid plutei expressed LpS1 RNA in most or all aboral ectoderm cells at normal levels, and did not express Spec1 RNA; in another 1% of the LpSp hybrid plutei the Spec1 and LpS1 transcripts were present at normal levels in complementary, non-overlapping patches of contiguous aboral ectoderm cells. In the reciprocal SpLp cross, each hybrid pluteus expressed either only the LpS1 gene (about 2%) or only the Spec1 gene throughout the aboral ectoderm. In SpLp hybrid gastrulae the level of LpS1 mRNA was less restricted; about 2% of the embryos contained only LpS1 RNA, and about half expressed only Spec1 transcripts, but in the remaining embryos Spec1 and LpS1 transcripts were coexpressed in the same aboral ectoderm cells.(ABSTRACT TRUNCATED AT 250 WORDS)

A simple method for quantifying specific mRNAs in small numbers of early mouse embryos.

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.

Replication forks are underrepresented in chromosomal DNA of Xenopus laevis embryos.

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. laevis embryos takes place by a mechanism in which strand separation is uncoupled from DNA synthesis.