Jurrien Dean

Mater , a maternal effect gene required for early embryonic development in mice

Maternal effect genes produce mRNA or proteins that accumulate in the egg during oogenesis. We show here that Mater, a mouse oocyte protein dependent on the maternal genome, is essential for embryonic development beyond the two-cell stage. Females lacking the maternal effect gene Mater are sterile. Null males are fertile.

Maternal control of early mouse development

The hiatus between oocyte and embryonic gene transcription dictates a role for stored maternal factors in early mammalian development. Encoded by maternal-effect genes, these factors accumulate during oogenesis and enable the activation of the embryonic genome, the subsequent cleavage stages of embryogenesis and the initial establishment of embryonic cell lineages. Recent studies in mice have yielded new findings on the role of maternally provided proteins and multi-component complexes in preimplantation development. Nevertheless, significant gaps remain in our mechanistic understanding of the networks that regulate early mammalian embryogenesis, which provide an impetus and opportunities for future investigations.

Expression of Cre recombinase in mouse oocytes: a means to study maternal effect genes.

., 1998). Thesematernally derived transcripts are responsible for thecompletion of meiosis, initiation of mitosis, activation ofthe embryonic genome, and completion of the transfor-mation of the highly differentiated oocyte into a totipo-tent embryonic cell. To study the effect of specific ma-ternal transcripts on these processes, we have sought toestablish a Cre-

Autoimmune disease of the ovary induced by a ZP3 peptide from the mouse zona pellucida.

We describe a novel experimental system in mice for the study of ovarian autoimmune disease, a condition encountered in women with premature ovarian failure. The ovarian autoimmune disease is induced in B6AF1 mice by a 15-amino acid peptide (Cys-Ser-Asn-Ser-Ser-Ser-Ser-Gln-Phe-Gln-Ile-His-Gly-Pro-Arg) from mouse ZP3, the sperm-binding component of the zona pellucida that surrounds growing and mature oocytes. Whereas the peptide induces both T cell and antibody responses, adoptive transfer of CD4+ T cell lines derived from affected animals causes oophoritis without observable antibodies to the zona pellucida peptide. The primacy of the T cell response in the pathogenesis of disease is further substantiated by defining oophoritogenic peptides as small as eight amino acids (Asn-Ser-Ser-Ser-Ser-Gln-Phe-Gln) that do not elicit an antibody response to the full-length ZP3 peptide. The identification of a well characterized peptide as a causative agent of autoimmune oophoritis should facilitate understanding of the pathogenesis of this T cell-mediated autoimmune disease. Because the proteins of the zona pellucida are conserved among mammals (the mouse and human ZP3 proteins are 67% identical), this murine model may lead to better understanding of the pathogenesis of human autoimmune oophoritis.

A subcortical maternal complex essential for preimplantation mouse embryogenesis.

We have identified a subcortical maternal complex (SCMC) that assembles during oocyte growth and is essential for zygotes to progress beyond the first embryonic cell divisions. At least four maternally encoded proteins contribute to this MDa complex: FLOPED, MATER, and TLE6 interact with each other while Filia binds independently to MATER. Although the transcripts encoding these proteins are degraded during meiotic maturation and ovulation, the SCMC proteins persist in the early embryo. The SCMC, located in the subcortex of eggs, is excluded from regions of cell-cell contact in the cleavage-stage embryo and segregates to the outer cells of the morulae and blastocyst. Floped(tm/tm) and/or Mater(tm/tm) eggs lack the SCMC but can be fertilized. However, these embryos do not progress beyond cleavage stage development and female mice are sterile. The proteins are conserved in humans, and similar maternal effect mutations may result in recurrent embryonic loss.

Oocyte-specific expression and developmental regulation of ZP3, the sperm receptor of the mouse zona pellucida.

The mouse zona pellucida is composed of three sulfated glycoproteins, encoded by the oocyte genome, that have important biological functions in preimplantation development. One of the zona gene products, ZP3, functions as the sperm receptor at fertilization. Our present data demonstrate that the ZP3 gene is transcribed in oocytes where its expression is developmentally regulated. Resting primordial oocytes do not express ZP3 mRNA, but these transcripts rapidly accumulate in growing oocytes so that they represent 0.1-0.2% of the total poly(A+) RNA. As oocytes complete their growth and undergo meiotic maturation, the abundance of ZP3 transcripts falls off dramatically; ovulated eggs contain less than 15% of peak levels. The oocyte-specific accumulation of ZP3 transcripts serves as an attractive system for further studies of factors that modulate developmentally regulated genes during mammalian oogenesis.

Structural Characterization of Native Mouse Zona Pellucida Proteins Using Mass Spectrometry

The zona pellucida is an extracellular matrix consisting of three glycoproteins that surrounds mammalian eggs and mediates fertilization. The primary structures of mouse ZP1, ZP2, and ZP3 have been deduced from cDNA. Each has a predicted signal peptide and a transmembrane domain from which an ectodomain must be released. All three zona proteins undergo extensive co- and post-translational modifications important for secretion and assembly of the zona matrix. In this report, native zonae pellucidae were isolated and structural features of individual zona proteins within the mixture were determined by high resolution electrospray mass spectrometry. Complete coverage of the primary structure of native ZP3, 96% of ZP2, and 56% of ZP1, the least abundant zona protein, was obtained. Partial disulfide bond assignments were made for each zona protein, and the size of the processed, native protein was determined. The N termini of ZP1 and ZP3, but not ZP2, were blocked by cyclization of glutamine to pyroglutamate. The C termini of ZP1, ZP2, and ZP3 lie upstream of a dibasic motif, which is part of, but distinct from, a proprotein convertase cleavage site. The zona proteins are highly glycosylated and 4/4 potential N-linkage sites on ZP1, 6/6 on ZP2, and 5/6 on ZP3 are occupied. Potential O-linked carbohydrate sites are more ubiquitous, but less utilized.

Gamete recognition in mice depends on the cleavage status of an egg's zona pellucida protein.

Getting Gametes Together Despite decades of research, the molecular basis of sperm-egg recognition in mammals remains unresolved. Models in which a glycan ligand in the zona pellucida (ZP) surrounding ovulated eggs binds to a sperm surface receptor have been widely embraced. A more recent model proposes that the cleavage status of a ZP protein, ZP2, renders the structure of the zona matrix either permissive or nonpermissive for sperm binding. Gahlay et al. (p. 216) tested predictions of each model by replacing endogenous zona proteins with either a mutant form of ZP2 that could not be cleaved or of ZP3 that lacked O glycan attachment sites. Sperm-egg recognition depended on the cleavage status of ZP2 rather than on glycan ligands released following fertilization. The cleavage status of the egg protein ZP2 regulates the recognition of sperm and prevents adherence of sperm to the two-cell embryo. At fertilization, mouse sperm bind to the zona pellucida (which consists of glycoproteins ZP1, ZP2, and ZP3) that surrounds eggs. A ZP2 cleavage model of gamete recognition requires intact ZP2, and a glycan release model postulates that zona glycans are ligands for sperm. These two models were tested by replacing endogenous protein with ZP2 that cannot be cleaved (Zp2Mut) or with ZP3 lacking implicated O glycans (Zp3Mut). Sperm bound to two-cell Zp2Mut embryos despite fertilization and cortical granule exocytosis. Contrary to prediction, sperm fertilized Zp3Mut eggs. Sperm at the surface of the zona pellucida remained acrosome-intact for more than 2 hours and were displaced by additional sperm. These data indicate that sperm-egg recognition depends on the cleavage status of ZP2 and that binding at the surface of the zona is not sufficient to induce sperm acrosome exocytosis.

Monoclonal antibodies to the murine zona pellucida protein with sperm receptor activity: Effects on fertilization and early development

During development and maturation, mammalian oocytes are surrounded by the zona pellucida which in the mouse is comprised of three sulfated glycoproteins, ZP-1, ZP-2, and ZP-3. Previously, monoclonal antibodies to ZP-2 have been isolated. The isolation and characterization of monoclonal antibodies specific for ZP-3, the zona protein with sperm receptor activity are now reported. Following passive immunization, these monoclonal antibodies localize to the intraovarian zonae pellucidae and their presence precludes both in vivo and in vitro fertilization of subsequently ovulated eggs. Monoclonal antibodies specific for either ZP-2 or ZP-3 also completely block in vitro fertilization at relatively low concentration ranging from 0.4 to 75 micrograms/ml. The contraceptive effect requires the presence of the zona and appears to inhibit the penetration of the zona pellucida by sperm rather than by blocking the sperm binding site. Neither antibody interferes with in vitro development from the two-cell to the blastocyst stage or with subsequent hatching from the enveloping zona pellucida.

Ovastacin, a cortical granule protease, cleaves ZP2 in the zona pellucida to prevent polyspermy

After fertilization, the metalloendoprotease ovastacin is released by cortical granule exocytosis and cleaves the zona pellucida glycoprotein ZP2, an essential step to block sperm binding to an already fertilized egg.