Distribution of RNA binding protein MOEP19 in the oocyte cortex and early embryo indicates pre-patterning related to blastomere polarity and trophectoderm specification

Abstract

We report the cloning and characterization of MOEP19, a novel 19 kDa RNA binding protein that marks a defined cortical cytoplasmic domain in oocytes and provides evidence of mammalian oocyte polarity and a form of pre-patterning that persists in zygotes and early embryos through the morula stage. MOEP19 contains a eukaryotic type KH-domain, typical of the KH-domain type I superfamily of RNA binding proteins, and both recombinant and native MOEP19 bind polynucleotides. By immunofluorescence, MOEP19 protein was first detected in primary follicles throughout the ooplasm. As oocytes expanded in size during oogenesis, MOEP19 increased in concentration. MOEP19 localized in the ovulated egg and early zygote as a symmetrical spherical cortical domain underlying the oolemma, deep to the zone of cortical granules. MOEP19 remained restricted to a cortical cytoplasmic crescent in blastomeres of 2-, 4- and 8-cell embryos. The MOEP19 domain was absent in regions underlying cell contacts. In morulae, the MOEP19 domain was found at the apex of outer, polarized blastomeres but was undetectable in blastomeres of the inner cell mass. In early blastocysts, MOEP19 localized in both mural and polar trophectoderm and a subset of embryos showed inner cell mass localization. MOEP19 concentration dramatically declined in late blastocysts. When blastomeres of 4- to 8-cell stages were dissociated, the polarized MOEP19 domain assumed a symmetrically spherical localization, while overnight culture of dissociated blastomeres resulted in formation of re-aggregated embryos in which polarity of the MOEP19 domain was re-established at the blastomere apices. MOEP19 showed no evidence of translation in ovulated eggs, indicating that MOEP19 is a maternal effect gene. The persistence during early development of the MOEP19 cortical oocyte domain as a cortical crescent in blastomers suggests an intrinsic pre-patterning in the egg that is related to the apical-basolateral polarity of the embryo. Although the RNAs bound to MOEP19 are presently unknown, we predict that the MOEP19 domain directs RNAs essential for normal embryonic development to specific locations in the oocyte and early embryo.

Maternal control of early mouse development

“…This formulation is consistent with models in which cell fate is determined by cleavage pattern and by the positions of blastomeres (or by the molecular determinants in them) in the embryo (Tarkowski and Wroblewska, 1967;Johnson and McConnell, 2004;Zernicka-Goetz et al, 2009). The ability of the SCMC to redistribute during cleavage-stage embryogenesis (Ohsugi et al, 2008;Herr et al, 2008;Li et al, 2008a) may play an important role in the regulative nature of early mammalian development, in which ablation of individual blastomeres need not prevent normal development (Tarkowski, 1959;Tsunoda and McLaren, 1983). The observation that some inner cells become outer cells with trophectoderm descendents after incubation with morulae (Ziomek and Johnson, 1982) after immunosurgery (Ohsugi et al, 2008).…”

“…One could envision a role for the SCMC in sequestering macromolecules that eventually trigger, either directly or indirectly, a commitment of the outer cell progeny to becoming trophoblasts (Johnson and McConnell, 2004). The localization of Cdx2 transcripts in the periphery of outer cells (Jedrusik et al, 2008) and the observation that FLOPED binds to ribonucleotide homopolymers (Herr et al, 2008) are consistent with the complex having a role in sequestering RNA in outer cells to influence the subsequent pathways that direct cells into trophectoderm or ICM lineages.…”

“…The founding member of this complex is encoded by Mater (maternal antigen that embryos require; official symbol Nlrp5), which was originally characterized as an antigen associated with a mouse model of autoimmune oophoritis (see Glossary, Box 1) (Tong and Nelson, 1999). Additional members of the complex are encoded by Floped (factor located in oocytes permitting embryonic development; official symbol Ooep) (Pierre et al, 2007;Herr et al, 2008;Li et al, 2008a), Tle6 (transducin-like enhancer of split 6) (Li et al, 2008a) and Filia (RIKEN cDNA 2410004A20) (Ohsugi et al, 2008). The interactions of these proteins, which co-localize in the subcortex as seen by confocal microscopy, have been confirmed by co-expression of Myc-or HA-tagged recombinant proteins in heterologous cell lines and by co-immunoprecipitation (Ohsugi et al, 2008;Li et al, 2008a).…”

“…5B). The exclusion of the SCMC from cell-cell contact is reversed when blastomeres are separated (Ohsugi et al, 2008;Herr et al, 2008;Li et al, 2008a), which indicates that a dynamic equilibrium exists between a cytoplasmic pool of preassembled complex (or components of it) and the subcortical complex (Fig. 5).…”

The subcortical maternal complex: multiple functions for one biological structure?

“…The genes encoding the four SCMC components are expressed during oogenesis, and the transcripts are rapidly degraded after EGA; their proteins accumulate already in growing oocytes and persist in cleavage-stage embryos up to the blastocyst stage [10,13,16,17]. Mater, Floped, and Tle6 expression was observed in oocytes as early as the primary follicle stage [10,17,18].…”