Boris Baibakov

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.

HLA-G inhibits the functions of murine dendritic cells via the PIR-B immune inhibitory receptor

Human histocompatibility leukocyte antigen (HLA)‐G is an MHC class Ib molecule that has been proposed to regulate immune responses during pregnancy. One of the possible mechanisms of that modulation is based on its interaction with immunoglobulin‐like transcript (ILT) receptors. In this study, we show that HLA‐G modifies the function of murine dendritic cells via interactions with the paired immunoglobulin‐like inhibitory receptor, a homologue of the human inhibitory receptor ILT4. Triggering of the immune inhibitory receptors resulted in prolongation of allogeneic graft survival. This demonstration forms the basis for exploring and exploiting HLA‐G molecules and immune inhibitory receptors in the development of a new therapeutic strategy to improve allogeneic graft survival.

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.

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.

A single domain of the ZP2 zona pellucida protein mediates gamete recognition in mice and humans

The ZP251–149 domain is necessary for human and mouse gamete recognition on the surface of the zona pellucida and for mouse fertility.

Human sperm bind to the N-terminal domain of ZP2 in humanized zonae pellucidae in transgenic mice

Use of transgenic mouse eggs expressing human zona pellucida proteins identifies an N-terminal domain of ZP2 that is essential for human sperm–egg binding.

FIGLA, a Basic Helix-Loop-Helix Transcription Factor, Balances Sexually Dimorphic Gene Expression in Postnatal Oocytes

ABSTRACT Maintenance of sex-specific germ cells requires balanced activation and repression of genetic hierarchies to ensure gender-appropriate development in mammals. Figla (factor in the germ line, alpha) encodes a germ cell-specific basic helix-loop-helix transcription factor first identified as an activator of oocyte genes. In comparing the ovarian proteome of normal and Figla null newborn mice, 18 testis-specific or -enhanced proteins were identified that were more abundant in Figla null ovaries than in normal ovaries. Transgenic mice, ectopically expressing Figla in male germ cells, downregulated a subset of these genes and demonstrated age-related sterility associated with impaired meiosis and germ cell apoptosis. Testis-associated genes, including Tdrd1, Tdrd6, and Tdrd7, were suppressed in the transgenic males with a corresponding disruption of the sperm chromatoid body and mislocalization of MVH and MILI proteins, previously implicated in posttranscriptional processing of RNA. These data demonstrate that physiological expression of Figla plays a critical dual role in activation of oocyte-associated genes and repression of sperm-associated genes during normal postnatal oogenesis.

A polycation causes migration of negatively charged phospholipids from the inner to outer leaflet of the liposomal membrane

Aggregation of the negatively charged liposomes caused by the addition of the linear polycation, poly‐N‐ethyl‐4‐vinylpyridine bromide, was studied. At the point of maximal size and zero electrophoretic mobility of aggregates, the concentration of positive charges brought in by the adsorbed polycation was found to be equal to the total concentration of negative charges both on the outer and inner surface of the lipid bilayer. Since polycation saturation of the liposomal negative charges was found to occur without disruption of the membrane, it was concluded that the polycation induced migration of negatively charged phospholipid molecules from the inner to outer leaflet of the bilayer.

ZP2 and ZP3 Traffic Independently within Oocytes prior to Assembly into the Extracellular Zona Pellucida

ABSTRACT The extracellular zona pellucida surrounds mammalian eggs and mediates taxon-specific sperm-egg recognition at fertilization. In mice, the zona pellucida is composed of three glycoproteins, but the presence of ZP2 and ZP3 is sufficient to form a biologically functional structure. Each zona pellucida glycoprotein is synthesized in growing oocytes and traffics through the endomembrane system to the cell surface, where it is released from a transmembrane domain and assembled into the insoluble zona pellucida matrix. ZP2 and ZP3 colocalize in the endoplasmic reticulum and in 1- to 5-μm post-Golgi structures comprising multivesicular aggregates (MVA), but a coimmunoprecipitation assay does not detect physical interactions. In addition, ZP2 traffics normally in growing oocytes in the absence of ZP3 or if ZP3 has been mutated to prevent incorporation into the zona pellucida matrix, complementing earlier studies indicating the independence of ZP3 secretion in Zp2 null mice. N glycosylation has been implicated in correct protein folding and intracellular trafficking of secreted proteins. Although ZP3 contain five N-glycans, enhanced green fluorescent protein-tagged ZP3 lacking N glycosylation sites is present in MVA and is incorporated into the zona pellucida matrix of transgenic mice. Thus, ZP2 secretion is seemingly unaffected by ZP3 lacking N-glycans. Taken together, these observations indicate that ZP2 and ZP3 traffic independently through the oocyte prior to assembly into the zona pellucida.

PtdIns(3,4,5)P3 is constitutively synthesized and required for spindle translocation during meiosis in mouse oocytes.

Summary Prior to ovulation, mammalian oocytes complete their first meiotic division and arrest at metaphase II. During this marked asymmetric cell division, the meiotic spindle moves dramatically from the center of the oocyte to the cortex to facilitate segregation of half of its chromosomal content into the diminutive first polar body. Recent investigations have documented crucial roles for filamentous actin (F-actin) in meiotic spindle translocation. However, the identity of the upstream regulators responsible for these carefully orchestrated movements has remained elusive. Utilizing fluorescently tagged probes and time-lapse confocal microscopy, we document that phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P3] is constitutively synthesized with spatial and temporal dynamics similar to that of F-actin and Formin 2 (Fmn2). Blockage of PtdIns(3,4,5)P3 synthesis by LY294002, a specific inhibitor of phosphoinositide 3-kinase (PI3K), disrupts cytoplasmic F-actin organization and meiotic spindle migration to the cortex. F-actin nucleator Fmn2 and Rho GTPase Cdc42 play roles in mediating the effect of PtdIns(3,4,5)P3 on F-actin assembly. Moreover, the spatial and temporal dynamics of PtdIns(3,4,5)P3 is impaired by depletion of MATER or Filia, two oocyte proteins encoded by maternal effect genes. Thus, PtdIns(3,4,5)P3 is synthesized during meiotic maturation and acts upstream of Cdc42 and Fmn2, but downstream of MATER/Filia proteins to regulate the F-actin organization and spindle translocation to the cortex during mouse oocyte meiosis.