Conservation of mammalian secondary sperm receptor genes enables the promoter of the human gene to function in mouse oocytes

Abstract

The human zona pellucida is an extracellular sheath composed of three major proteins (ZP1, ZP2, and ZP3) which surround the ovulated egg and mediate the initial interactions with sperm. Although fertilization is relatively species-specific and human sperm will not bind to mouse zona, there is a high degree of conservation between the coding regions of human ZP3 and mouse Zp-3 (the primary sperm receptor) genes. We now report the characterization of the human ZP2 gene and demonstrate that the sequences of its coding regions are 70% identical with those of the mouse Zp-2 (the secondary sperm receptor) gene. In addition, the first 300 bp of the 5' flanking regions of human ZP2 and mouse Zp-2 are highly conserved. This region of 5' flanking DNA contains a previously described 12-bp DNA sequence (element IV) that forms an oocyte-specific DNA-protein complex important for mouse Zp-2 and Zp-3 promoter activity. Human element IV forms a DNA-protein complex in gel mobility shift assays when incubated with human or mouse ovarian extracts. The formation of this complex is inhibited with molar excess of either human or mouse element IV sequences and is not present in extracts of testes, uterus, spleen, lung, or kidney. The human promoter region (0.3 kbp), coupled to a luciferase reporter gene and microinjected into the nuclei of 50-microns-diameter mouse oocytes, results in reporter gene activity at a level comparable to that of the homologous mouse promoter. Clustered point mutations in element IV in either the mouse or the human sequence dramatically decrease reporter gene activity. These results indicate that the similarity between mouse Zp-2 and human ZP2 genes enables the human promoter to utilize the heterologous transcription machinery in mouse oocytes. The observed transcription may involve the recognition of promoter sequences in element IV by conserved transcription factor(s).