Cyritestin is a membrane-anchored sperm protein belonging to the ADAM (f1.gif" BORDER="0"> f2.gif" BORDER="0">isintegrin and f1.gif" BORDER="0"> f3.gif" BORDER="0">etalloprotease) family of proteins, which are proposed to be involved in cell-cell adhesion through binding to integrin receptors. Several lines of evidence support a role of cyritestin and other members of this protein family in the fusion of sperm and the egg plasma membrane. In an effort to elucidate the physiological function of cyritestin, we have disrupted its locus by homologous recombination. Male homozygous null mutants are infertile, even though spermatogenesis, mating, and migration of sperm from the uterus into the oviduct are normal. In vitro experiments showed that infertility is due to the inability of the cyritestin-deficient sperm to bind to the zona pellucida. However, after removal of the zona pellucida, sperm-egg membrane fusion monitored by the presence of pronuclei and generation of 2- and 4-cell embryos did not reveal any differences from the wild-type situation. These results demonstrate that cyritestin is crucial in the fertilization process at the level of the sperm-zona pellucida interaction.

The pelota gene of Drosophila melanogaster encodes a protein that was found to be included in cell cycle regulation. Mutations were found to result in spermatogenic arrest, female sterility and disturbances in the patterning of the eye. Here we describe the cloning of the human pelota cDNA (PELO) that encodes a 385-amino-acid protein. Southern blot and fluorescence in situ hybridization analyses revealed that PELO is present as a single copy gene in the human genome and is localized on chromosome 5q11.2. Northern blot analysis revealed the presence of a 1.6-kb transcript in all tissues studied and an additional 2.0-kb transcript in testis.

The pelota gene of Drosophila melanogaster encodes a protein which is included in cell cycle regulation. Mutations were found to result in spermatogenic arrest, female sterility and disturbances in the patterning of the eye. We have recently isolated and characterized cDNA clones coding for the human pelota gene (PELO). Here we describe the cloning of the murine pelota cDNA and gene (Pelo) that encodes a 385-amino-acid protein. The exon-intron structure of the gene, which contains three exons, was determined. Comparison of the mouse amino acid sequences with the human and Drosophila sequences revealed an overall high identity (96% and 70%, respectively). Northern blot analysis detected a 1.7-kb transcript in all tissues studied. Southern blot analyses revealed that the pelota gene is present as a single copy in the mouse genome. The mouse pelota gene (Pelo) was mapped to the distal end of chromosome 13, in a region that is homologous with a segment of human chromosome 5q11 containing the orthologous human gene. Cloning of the mouse gene is an important step to study the function of the pelota gene in mammals and to create a mouse model for this evolutionarily conserved gene.

Germ cell cyritestin is a membrane-anchored protein belonging to the ADAM family of proteins. Sequencing of eight human cyritestin cDNA clones revealed that they are identical at their 5' and 3' ends but differ from each other in the length of an internal deletion, suggesting that the human cyritestin mRNA is alternatively spliced. Internal deletions that are present in some cDNA isoforms do not cause a frameshift in the C-terminal coding region. Analysis of the predicted amino acid sequences demonstrated that the human cyritestin is a polymorphic protein that could include membrane-anchored and soluble forms. Southern blot analysis and characterization of human cyritestin genomic fragments revealed that the human genome contains two copies of the cyritestin gene instead of one as in the mouse. The human CYRN1 and CYRN2 genes were assigned to the region p12-21 of chromosome 8 and q12 of chromosome 16, respectively. Northern blot and RT-PCR analyses revealed that both human genes are expressed in the testis. Amino acid sequence comparisons between cyritestin and other members of the metalloprotease-disintegrin family of proteins suggested that human and mouse cyritestin and monkey tMDCI are homologous molecules.

In humans, male and female partners contribute more or less equally to the infertility problem. In approximately 20% of infertile couples, the concurrence of male and female factors is suggested to be responsible for infertility. Neither of these factors are known nor is there a model system to prove this assumption. We present such a model system in the mouse, in which the lack of acrosin in the male and modifications of the zona pellucida (ZP) in the female result in a significant reduction of the fertilization rate in vitro. We generated mice carrying a deletion in the proline-rich region (PRR) of the proacrosin gene, resulting in the absence of proacrosin in the homozygous PRR(-/-) male mouse. Under normal conditions, sperm from the proacrosin-deficient mice are still capable of ZP penetration and fertilization. In this study, modifications of the ZP of oocytes after superovulation were achieved by treatment with dimethylsulphoxide or aroclor-1254 or by in-vitro ageing. It is known that under these conditions, a time-dependent hardening of the ZP occurs. The rates of fertilization in vitro of treated and aged oocytes using sperm from PRR(-/-) mice were found to be significantly reduced when compared with those reached with wild-type sperm. The relevance of the acrosin status and ZP condition for fertilization success were further substantiated by the finding that the fertilization rate with PRR(-/-) sperm is affected by the thickness of the ZP. Our results demonstrate that the lack of acrosin in sperm in combination with modifications to the ZP can affect fertility and can be an experimental model for the study of unexplained infertility in human couples in which both male- and female-derived factors are suggested to be the underlying causes.