Knockout mouse technology has been used over the last decade to define the essential roles of ovarian-expressed genes and uncover genetic interactions. In particular, we have used this technology to study the function of multiple members of the transforming growth factor-beta superfamily including inhibins, activins, and growth differentiation factor 9 (GDF-9 or Gdf9). Knockout mice lacking GDF-9 are infertile due to a block in folliculogenesis at the primary follicle stage. In addition, recombinant GDF-9 regulates multiple cumulus granulosa cell functions in the periovulatory period including hyaluronic acid synthesis and cumulus expansion. We have also cloned an oocyte-specific homolog of GDF-9 from mice and humans, which is termed bone morphogenetic protein 15 (BMP-15 or Bmp15). To define the function of BMP-15 in mice, we generated embryonic stem cells and knockout mice, which have a null mutation in this X-linked gene. Male chimeric and Bmp15 null mice are normal and fertile. In contrast to Bmp15 null males and Gdf9 knockout females, Bmp15 null females (Bmp15(-/-)) are subfertile and usually have minimal ovarian histopathological defects, but demonstrate decreased ovulation and fertilization rates. To further decipher possible direct or indirect genetic interactions between GDF-9 and BMP-15, we have generated double mutant mice lacking one or both alleles of these related homologs. Double homozygote females (Bmp15(-/-)Gdf9(-/-)) display oocyte loss and cysts and resemble Gdf9(-/-) mutants. In contrast, Bmp15(-/-)Gdf9(+/-) female mice have more severe fertility defects than Bmp15(-/-) females, which appear to be due to abnormalities in ovarian folliculogenesis, cumulus cell physiology, and fertilization. Thus, the dosage of intact Bmp15 and Gdf9 alleles directly influences the destiny of the oocyte during folliculogenesis and in the periovulatory period. These studies have important implications for human fertility control and the maintenance of fertility and normal ovarian physiology.

Many studies of the molecular and biochemical aspects of mammalian fertilization have focused on the interaction of the spermatozoa with the zona pellucida (ZP). The zona pellucida, a unique extracellular matrix surrounding the mammalian oocyte, is formed during ovarian follicular development. Following ovulation of the mature ovum, the spermatozoa must bind to and penetrate this matrix before the fertilization process is completed and the male and female genetic information combine. Although numerous models for this interaction have been proposed, the complete process has yet to be elucidated. The precise mechanisms by which these interactions occur also vary markedly among different mammalian species, making it more difficult to establish a unified model. To a great extent, the study of the molecules involved in these interactions have been limited because small numbers of female gametes are available for these studies. The recent development of techniques to isolate large numbers of zonae pellucidae as well as advances in immunological and molecular biology techniques have permitted the detailed characterization of ZP proteins. Although there is a paucity of information on the post-translational modification and extracellular processing of these molecules which result in matrix formation, a number of properties have been elucidated allowing better correlation between the structure and function of different ZP proteins among species. This review reflects these studies in relation to protein nomenclature and the molecular complexity of ZP antigens.

The zona pellucida (ZP) is the extracellular matrix that plays important roles in sperm-egg interaction. The ZP is composed of three major glycoproteins that exhibit heterogeneity due to extensive post-translational modifications including glycosylation and sulfation. Because of these modifications the nomenclature of ZP proteins from different species based on electrophoretic mobilities has been confusing. As the cDNAs and genes encoding the different ZP proteins have been isolated and sequenced, it is now possible to relate these ZP proteins according to gene families. Using the mouse ZP nomenclature, the ZP proteins from different mammalian species can be classified into three protein families: ZP1, ZP2, and ZP3. Although some of the structural domains of the ZP proteins of different species are conserved within each family, they exhibit distinct biological properties. In the mouse it has been established that ZP3 is the primary sperm receptor while ZP2 has secondary sperm receptor properties. In the pig, however, ZP1 has been shown to have sperm receptor activity similar to that observed in the rabbit and nonhuman primates. It is of interest that the human ZP2 and ZP3 gene families are 60–70% conserved with respect to the mouse ZP amino acid sequence, while the mouse ZP1 is only 39% conserved with respect to human ZP1. Such differences in protein structure and glysosylation may explain the marked species differences in the biochemical, physicochemical and immunochemical properties of the ZP. Studies have now shown that the proteins of the ZP are expressed in a stage specific manner and that there is increasing evidence that ZP proteins are expressed by both granulosa cells and the oocyte and may play a role in granulosa cell differentiation.

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