Sculpting the Transcriptome During the Oocyte-to-Embryo Transition in Mouse. Svoboda P et al. (2015) In mouse, the oocyte-to-embryo transition entails converting a highly differentiated oocyte to totipotent blastomeres. This transition is driven by degradation of maternal mRNAs, which results in loss of oocyte identity, and reprogramming of gene expression during the course of zygotic gene activation, which occurs primarily during the two-cell stage and confers blastomere totipotency. Full-grown oocytes are transcriptionally quiescent and mRNAs are remarkably stable in oocytes due to the RNA-binding protein MSY2, which stabilizes mRNAs, and low activity of the 5' and 3' RNA degradation machinery. Oocyte maturation initiates a transition from mRNA stability to instability due to phosphorylation of MSY2, which makes mRNAs more susceptible to the RNA degradation machinery, and recruitment of dormant maternal mRNAs that encode for critical components of the 5' and 3' RNA degradation machinery. Small RNAs (miRNA, siRNA, and piRNA) play little, if any, role in mRNA degradation that occurs during maturation. Many mRNAs are totally degraded but a substantial fraction is only partially degraded, their degradation completed by the end of the two-cell stage. Genome activation initiates during the one-cell stage, is promiscuous, low level, and genome wide (and includes both inter- and intragenic regions) and produces transcripts that are inefficiently spliced and polyadenylated. The major wave of genome activation in two-cell embryos involves expression of thousands of new genes. This unique pattern of gene expression is the product of maternal mRNAs recruited during maturation that encode for transcription factors and chromatin remodelers, as well as dramatic changes in chromatin structure due to incorporation of histone variants and modified histones.//////////////////
A cytoplasmic marker for diplotene oocytes.
Gu W, et al reported that mammalian male and female germ cells express a germ
cell-specific Y-Box protein, MSY2.
MSY2 is
a member of the Y box multigene family of proteins; it contains the cold shock
domain that is highly conserved among all Y box proteins and four basic/aromatic
islands that are closely related to the other known germline Y box proteins from
Xenopus, FRGY2, and goldfish, GFYP2. Msy2 undergoes alternative splicing to
yield alternate N-terminal regions upstream of the cold shock domain. Although
MSY2 is a member of a large family of nucleic acid-binding proteins, Southern
blotting detects only a limited number of genomic DNA fragments, suggesting that
Msy2 is a single copy gene. By Northern blotting and immunoblotting, MSY2
appears to be a germ cell-specific protein in the testis. Analysis of Msy2 mRNA
expression in prepubertal and adult mouse testes, and in isolated populations of
germ cells, reveals maximal expression in postmeiotic round spermatids, a cell
type with abundant amounts of stored messenger ribonucleoproteins.
General function
Cell death/survival, Metabolism, RNA processing
Comment
Cellular localization
Cytoplasmic
Comment
Ovarian function
Oogenesis, Oocyte maturation
Comment
Absence of MSY2 in Mouse Oocytes Perturbs Oocyte Growth and Maturation, RNA Stability, and the Transcriptome. Medvedev S et al. Messenger RNA (mRNA) is remarkably stable during oocyte growth thus enabling mRNAs to accumulate during the growth phase and thereby provide mRNAs that support early embryonic development. MSY2, a germ cell-specific RNA-binding protein, is implicated in regulating mRNA stability. MSY2 is essential for development because female Msy2(-/-) mice are infertile. We describe here the characterization of Msy2(-/-) oocytes. Mutant oocytes grow slower during the first wave of folliculogenesis and maturation to and arrest at metaphase II is severely compromised due to aberrant spindle formation and chromosome congression. Consistent with MSY2 conferring mRNA stability is that the amount of poly(A)-containing RNA is reduced by ~25% in mutant oocytes. Stability of an exogenous mRNA injected into mutant oocytes is lower than when compared to their wild-type counterparts, and moreover, expression of wild-type MSY2 in mutant oocytes increases mRNA stability, whereas injection of a mutant form of MSY2 not capable of binding RNA does not. Transcription quiescence that normally occurs during the course of oocyte growth is not observed in mutant oocytes and the transcriptome of mutant oocytes is markedly perturbed. These results, and those of previous studies, strongly implicate a central role of MSY2 in regulating mRNA stability.
CDC2A (CDK1)-mediated phosphorylation of MSY2 triggers maternal mRNA degradation during mouse oocyte maturation. Medvedev S et al. Degradation of maternal mRNA is thought to be essential to undergo the maternal-to-embryonic transition. Messenger RNA is extremely stable during oocyte growth in mouse and MSY2, an abundant germ cell-specific RNA-binding protein, likely serves as a mediator of global mRNA stability. Oocyte maturation, however, triggers an abrupt transition in which most mRNAs are significantly degraded. We report that CDC2A (CDK1)-mediated phosphorylation of MSY2 triggers this transition. Injecting Cdc2a mRNA, which activates CDC2A, overcomes milrinone-mediated inhibition of oocyte maturation, induces MSY2 phosphorylation and the maturation-associated degradation of mRNAs. Inhibiting CDC2A following its activation with roscovitine inhibits MSY2 phosphorylation and prevents mRNA degradation. Expressing non-phosphorylatable dominant-negative forms of MSY2 inhibits the maturation-associated decrease in mRNAs, whereas expressing constitutively active forms induces mRNA degradation in the absence of maturation and phosphorylation of endogenous MSY2. A positive-feedback loop of CDK1-mediated phosphorylation of MSY2 that leads to degradation of Msy2 mRNA that in turn leads to a decrease in MSY2 protein may ensure that the transition is irreversible.
Yu J, et al 2003 reported the requirement for RNA-binding activity of MSY2 for cytoplasmic localization and retention in mouse oocytes.
MSY2, a mouse germ cell-specific Y-box protein, is implicated in the global regulation of the stability and/or translation of maternal mRNAs in the mouse oocyte. The authors report here that in the oocyte approximately 75% of MSY2 protein is associated with a Triton-insoluble preparation, whereas in either male germ cells or when exogenously expressed in transfected somatic cells almost all MSY2 is soluble. This retention in the oocyte, which is unlikely mediated either by microfilaments or by microtubules, markedly decreases beyond the two-cell stage of development. By microinjecting mutant MSY2-EGFP chimeric mRNAs into mouse oocytes and then assaying the expressed protein's localization by laser-scanning confocal microscopy, the authors find that an intact cold-shock domain (CSD), containing two RNA-binding motifs, is required to localize MSY2 to the oocyte cytoplasm. In addition, an additional basic/aromatic amino acid island (B/A), which can also interact with RNA, in the C-terminal tail domain is necessary to retain MSY2 following Triton permeabilization. Intact mRNA appeared required for this retention, since RNase A treatment of Triton-permeabilized oocytes or microinjection of RNase A into the oocyte released essentially all of the endogenous MSY2 protein. Furthermore, there is a positive correlation between the ability of the mutant MSY2-EGFP protein to remain associated with the Triton-insoluble preparations and its increased affinity for RNA, as determined by RNA electrophoretic mobility shift assays. These results suggest that binding of intact maternal mRNA by MSY2 is required for its cytoplasmic retention.
Expression regulated by
Comment
Ovarian localization
Oocyte
Comment
In the ovary,
MSY2 is present exclusively in diplotene-stage and mature oocytes Gu W, et al . MSY2 is
maternally inherited in the one-cell-stage embryo but is not detected in the late
two-cell-stage embryo. This loss of MSY2 is coincident with the bulk
degradation of maternal mRNAs in the two-cell embryo.
1
Junying Yu et al 2001 reported the expression of MSY2 in Mouse Oocytes and
Preimplantation Embryos.
Translational control plays a central role during oocyte maturation and early embryogenesis, as these processes occur
in the absence of transcription. MSY2, a member of a multifunctional Y-box protein family, is implicated in repressing
the translation of paternal mRNAs. Northern blot analysis indicates that MSY2 expression is highly restricted and essentially confined to the
oocyte in the female mouse. MSY2 transcript and protein, as assessed by reverse transcription-polymerase chain
reaction and immunoblotting, respectively, are expressed in growing oocytes, metaphase II-arrested eggs, and 1-cell
embryos, but then are degraded by the late 2-cell stage; no expression is detectable in the blastocysts. During oocyte
maturation, MSY2 is phosphorylated and following fertilization it is dephosphorylated. Quantification of the mass
amount of MSY2 reveals that it represents 2% of the total protein in the fully grown oocyte, i.e., it is a very abundant
protein. Both endogenous MSY2 and MSY2-enhanced green fluorescent protein (EGFP), which is synthesized
following microinjection of an mRNA encoding MSY2-EGFP, are primarily localized in the cytoplasm, and about 75%
of the MSY2 remains associated with oocyte cytoskeletal preparations. Results of these studies are consistent with the
proposal that MSY2 functions by stabilizing and/or repressing the translation of maternal mRNAs.
Follicle stages
Primary, Secondary, Antral, Preovulatory
Comment
Loss of SCP1, a Synaptonemal Complex Protein, Contributes to the Initiation of Follicular Assembly in the Developing Rat Ovary Paredes A, et al .
In the rat ovary, germ and somatic cells become organized into primordial follicles 48-72 h after birth. Although several genes have been implicated in the control of early follicular growth, less is known about the factors involved in the formation of primordial follicles. Using the method of differential display (DD) of messenger RNAs, we found several genes differentially expressed at the time of follicular assembly. One of them encodes Synaptonemal Complex Protein-1 (SCP1), a core component of the protein complex that maintains recombining chromosomes together during prophase I of the first meiotic division in germ cells. This association, evident during the pachytene stage, ends when chromosomal desynapsis begins in the diplotene stage at the end of prophase I. Oocytes become arrested in the diplotene/dictate stage before becoming enclosed into primordial follicles, suggesting that oocytes must complete meiotic prophase I before becoming competent to direct follicle assembly. We now show that attainment of the diplotene stage results in follicular formation. In developing rat ovaries, SCP1 mRNA expression is confined to oocytes and decreases precipitously within 24 h after birth, preceding the organization of primordial follicles. The premature loss of SCP1, achieved via treatment with an antisense oligodeoxynucleotide targeting SCP1 mRNA, resulted in more oocytes reaching the diplotene stage, as evidenced by a decrease in the number of oocytes containing germ cell nuclear antigen-1 (GCNA1, a nuclear protein whose expression ceases in diplotene), and an increase in the number of oocytes expressing MSY2 (a cytoplasmic Y box protein expressed in oocytes that have become arrested in diplotene). SCP1-deficient ovaries exhibited and increased number of newly formed follicles, suggesting that completion of meiotic prophase I endows oocytes with the capability of orchestrating follicular assembly.
Phenotypes
Mutations
2 mutations
Species: mouse
Mutation name: None
type: None fertility: subfertile Comment:Yu J, et al reported that transgenic RNAi-mediated reduction of MSY2 in mouse oocytes results in reduced fertility.
MSY2 is implicated in regulating the stability and translation of maternal mRNAs during mouse oogenesis. We report here that by driving the expression of a transgene encoding an Msy2 hairpin dsRNA in growing oocytes using the oocyte-specific Zp3 promoter, the amount of MSY2 protein was reduced by at least 60% in fully grown oocytes. The decrease appeared specific because no decrease was observed in either non-targeted mRNAs or proteins. Fertility of transgenic females was severely reduced. Although transgenic eggs could be inseminated, the eggs did not exhibit the normal series of oscillations in intracellular Ca(2+), resume meiosis, undergo cortical granule exocytosis, or ZP2 cleavage to ZP2(f). Transgenic oocytes also displayed a higher incidence of both the non-surrounded nucleolus chromatin morphology, and abnormal meiotic spindle formation was observed following oocyte maturation. Transgenic oocytes contained less total mRNA (approximately 75-80% that of non-transgenic oocytes) and displayed a reduced level of protein synthesis. Moreover, several of the maturation-associated changes in protein synthesis failed to occur in the transgenic oocytes. These results support a role for MSY2 in stabilizing maternal mRNAs in growing oocytes, a process essential to generate meiotically and developmentally competent oocytes.
Species: mouse
Mutation name: None
type: null mutation fertility: infertile - ovarian defect Comment: Absence of the DNA-/RNA-binding protein MSY2 results in male and female infertility Yang J, et al .
MSY2, a germ-cell-specific member of the Y-box family of DNA-/RNA-binding proteins, is proposed to function as a coactivator of transcription in the nucleus and to stabilize and store maternal and paternal mRNAs in the cytoplasm. In mice lacking Msy2, a normal Mendelian ratio is observed after matings between heterozygotes with equal numbers of phenotypically normal but sterile male and female homozygotes (Msy2(-/-)). Spermatogenesis is disrupted in postmeiotic null germ cells with many misshapen and multinucleated spermatids, and no spermatozoa are detected in the epididymis. Apoptosis is increased in the testes of homozygotes, and real-time RT-PCR assays reveal large reductions in the mRNA levels of postmeiotic male germ cell mRNAs and smaller reductions of meiotic germ cell transcripts. In females, there is no apparent decrease in either the number of follicles or their morphology in ovaries obtained from 2- and 8-day-old Msy2(-/-) mice. In contrast, follicle number and progression are reduced in 21-day-old Msy2(-/-) ovaries. In adult Msy2(-/-) females, oocyte loss increases, anovulation is observed, and multiple oocyte and follicle defects are seen. Thus, Msy2 represents one of a small number of germ-cell-specific genes whose deletion leads to the disruption of both spermatogenesis and oogenesis.