Gene expression during the oocyte-to-embryo transition in mammals.

Abstract

The seminal question in modern developmental biology is the origins of new life arising from the unification of sperm and egg. The roots of this question begin from 19th to 20th century embryologists studying fertilization and embryogenesis. Although the revolution of molecular biology has yielded significant insight into the complexity of this process, the overall orchestration of genes, molecules, and cells is still not fully formed. Early mammalian development, specifically the oocyte-to-embryo transition, is essentially under "maternal command" from factors deposited in the cytoplasm during oocyte growth, independent of de novo transcription from the nascent embryo. Many of the advances in understanding this developmental period occurred in tandem with application of new methods and techniques from molecular biology, from protein electrophoresis to sequencing and assemblies of whole genomes. From this bed of knowledge, it appears that precise control of mRNA translation is a key regulator coordinating the molecular and cellular events occurring during oocyte-to-embryo transition. Notably, oocyte transcriptomes share, yet retain some uniqueness, common genetic motifs among all chordates. The common genetic motifs typically define fundamental processes critical for cellular maintenance, whereas the unique genetic features may be a source of variation and a substrate for sexual selection, genetic drift, or gene flow. One purpose for this complex interplay among genes, proteins, and cells may allow for evolution to transform and act upon the underlying processes, at molecular, structural and organismal levels, to increase diversity, which is the ultimate goal of sexual reproduction.

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Gene expression during the oocyte-to-embryo transition in
mammals
Alexei V Evsikov and Caralina Marín de Evsikova
The Jackson Laboratory, 600 Main Street, Bar Harbor, ME 04609
Abstract
The seminal question in modern developmental biology is the origins of new life arising from the
unification of sperm and egg. The roots of this question begin from 19th-20th century embryologists
studying fertilization and embryogenesis. Although the revolution of molecular biology has yielded
significant insight into the complexity of this process, the overall orchestration of genes, molecules,
and cells, is still not fully formed. Early mammalian development, specifically the oocyte-to-embryo
transition, is essentially under “maternal command” from factors deposited in the cytoplasm during
oocyte growth, independent of de novo transcription from the nascent embryo. Many of the advances
in understanding this developmental period occurred in tandem with application of new methods and
techniques from molecular biology, from protein electrophoresis to sequencing and assemblies of
whole genomes. From this bed of knowledge, it appears that precise control of mRNA translation is
a key regulator coordinating the molecular and cellular events occurring during oocyte-to-embryo
transition. Notably, oocyte transcriptomes share, yet retain some uniqueness, common genetic motifs
among all chordates. The common genetic motifs typically define fundamental processes critical for
cellular maintenance, whereas the unique genetic features may be a source of variation and a substrate
for sexual selection, genetic drift, or gene flow. One purpose for this complex interplay among genes,
proteins, and cells may allow for evolution to transform and act upon the underlying processes, at
molecular, structural and organismal levels, to increase diversity, which is the ultimate goal of sexual
reproduction.
Keywords
embryonic genome activation; gene duplication; maternal effect gene; translational control;
reproductive isolation
I am strongly inclined to suspect that the most frequent cause of variability may be
attributed to the male and female reproductive elements having been affected prior to
the act of conception.
Charles Darwin, The Origin of Species
I. Introduction
The origin of individual life, fertilization, along with its causal and consequential molecular
and cellular events, arguably remains one of the focal fields in modern developmental biology.
Indeed, the fundamental issue of cytoplasmic control of nuclear function in early
development (DiBerardino 1979; Gurdon and Woodland 1968) remains as essential as it was
in the times of Boveri, Spemann, Briggs and other founding fathers of modern embryology
Correspondence: The Jackson Laboratory, 600 Main Street, Bar Harbor, ME 04609, Dr. Alexei V. Evsikov, alexei.evsikov@jax.org, Dr.
Caralina Marín de Evsikova, caralina.evsikova@jax.org.
NIH Public Access
Author Manuscript
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Published in final edited form as:
Mol Reprod Dev. 2009 September ; 76(9): 805–818. doi:10.1002/mrd.21038.
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originating from the early 20th century. Setting aside the phenomenal discoveries of the recent
decades and dissection of critical components and pathways in the system, the whole ‘unified’
picture remains elusive.
Some of the major reasons for this setback in major conceptual advance are (i) the (unexpected)
complexity of a seemingly simple biological system, coupled with (ii) over-optimistic
expectations that seminal discoveries (e.g., mammalian cloning) will translate into new
paradigms, as well as (iii) the ungrounded shifts in the “community perception” of key
biological concepts (e.g., “ontogeny recapitulates phylogeny”) from the ever-changing
domains of theories to the firm grounds of axioms. In this review, we will focus mainly on one
aspect of the complex mechanisms governing early mammalian development, the regulation
of gene expression at the onset of oocyte maturation and into the early stages of development
known as the oocyte-to-embryo transition (OET). We will also discuss the (ii) and (iii) points,
as we believe that OET provides excellent, as well as enlightening examples on how dogmatism
impedes scientific advance.
II. Gene expression during OET: a historical perspective
In metazoans, maternal factors accumulated in the ooplasm during the growth period of the
egg are solely responsible for the control of maturation, fertilization and initial development
of the newly formed embryo. In most non-mammalian species studied, the first nuclear
divisions proceed rapidly and the ooplasm is programmed to progress through the first
morphogenetic events without the need for de novo transcription from the embryonic genome
(Davidson 1986). In contrast, the first cell cycles of a newly formed mammalian embryo are
quite prolonged, with no differentiation events occurring prior to activation of the embryonic
genome. For example, the developmental transition from oocyte maturation to embryonic
genome activation occupies 40–44 hours, or about 8% of Mus musculus prenatal development
(Braude et al. 1979; Howlett and Bolton 1985). The past decade of somatic cell nuclear transfer
experiments to generate genetic clones of different mammalian species underscores that the
oocyte cytoplasm contains factors pivotal for the remodeling of introduced nuclei. In mice,
nuclear reprogramming must be completed by the end of the second cell cycle, the time when
the M. musculus embryonic genome is completely activated, to produce viable embryos.
The studies of mammalian oocytes and embryos relied heavily, and evolved hand-in-hand with
development and implementation of milestone molecular techniques. Indeed, the invention of
SDS-PAGE electrophoresis by Laemmli (Laemmli 1970) spread quickly to study protein
expression patterns in the oocytes and embryos (Braude et al. 1979; Eppig and Eckhardt
1976; Golbus and Stein 1976; Howlett and Bolton 1985; Petzoldt and Hoppe 1980; Petzoldt
et al. 1981; Van Blerkom 1981; Van Blerkom and Brockway 1975; Van Blerkom and Manes
1974); further improvements in protein separation techniques such as 2D electrophoresis
(O’Farrell 1975) and principally, the introduction of qualitative and quantitative tools for 2D
electrophoregram analysis (Garrels 1983; Garrels 1989) had a similarly enthusiastic response
from the embryological community (Bensaude et al. 1983; Christians et al. 1995; Evsikov and
Solomko 1999; Howe and Solter 1979; Latham et al. 1991a; Latham et al. 1994; Latham et al.
1991b; Levinson et al. 1978; Richoux et al. 1991; Sanchez and Erickson 1985; Schultz and
Wassarman 1977b; Shi et al. 1994). However exciting, at the time these techniques carried a
major caveat: with an exception of a few known markers, the establishment of protein
identities was essentially impossible. For these and other reasons, the cDNA library technology
(Dworkin and Dawid 1980; Okayama and Berg 1982; Okayama and Berg 1983; Rougeon and
Mach 1976), once optimized for microgram amounts of harvested mRNA (Okayama and Berg
1982) quickly became the dominant technique to capture and study gene expression patterns
in oocytes and early embryos.
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While the first attempts to construct cDNA libraries of mammalian oocytes and embryos from
miniscule amounts of starting material were not very successful (Taylor and Piko 1987),
optimization of the techniques lead to the production of representative M. musculus cDNA
libraries (Rothstein et al. 1992) which are now the “gold standard” among high-quality
mammalian oocyte and early embryonic libraries (Evsikov et al. 2006). Application of
subtractive hybridization methods to these libraries (Sive and St John 1988) permitted
production of specialized cDNA libraries enriched in stage-specific transcripts (Rothstein et
al. 1993), and ultimately, isolation and characterization of genes differentially expressed during
early mammalian development (Hwang et al. 1996; Hwang et al. 1997; Hwang et al. 1999; Oh
et al. 1997). A modification of subtraction technique, known as suppression subtractive
hybridization, SSH (Diatchenko et al. 1996) has been widely used to construct ‘stage-specific’
cDNA libraries for pre-implantation embryos of several mammalian species, including Homo
sapiens (Bui et al. 2005; Leandri et al. 2009; Mohan et al. 2002; Morozov et al. 1999).
The next advance towards deciphering and enumeration of gene expression in oocytes and
embryos came with advances in automated DNA sequencing, which enabled implementation
of the large-scale Mouse Expressed Sequence Tag (EST) project and obtaining sequence
information for tens of thousands of randomly picked cDNA clones from oocyte and pre-
implantation embryo libraries (Marra et al. 1999). To date, this and similar projects (i.e.,
(Carninci et al. 2005; Kawai et al. 2001; Ko et al. 2000; Okazaki et al. 2002) provided sequence
information for approximately 210,000 ESTs from oocytes and pre-implantation embryos of
M. musculus alone. To facilitate the studies of changes in gene expression and move away from
time-consuming procedures of SSH and subtracted cDNA libraries, the microarray technology
developed in the 1990s – early 2000s (Fodor et al. 1993; Kargul et al. 2001; Lipshutz et al.
1999; Lockhart et al. 1996; VanBuren et al. 2002) proved very useful. Indeed, by now it is the
most popular technique to study the dynamic changes of gene expression during early
development (for recent reviews, see (Adjaye 2005; Aiba et al. 2006; Evans et al. 2008). At
the same time, expansion of the genome sequencing projects for other chordate model
organisms, together with production and representative sequencing of cDNA libraries from
eggs or ovaries for these species permitted to add a second – phylogenetic – dimension to the
studies of the oocyte cytoplasm’s role in early development (Evsikov et al. 2006; Evsikov and
Marin de Evsikova 2009). Finally, recent developments in sensitivity of the mass spectrometry
technology permitted establishment of peptide identities for individual spots on the 2D
electrophoregrams of minute amounts of cell lysates, making feasible and reinvigorating
research in the oocyte and pre-implantation embryo proteomics (Calvert et al. 2003; Coonrod
et al. 2004; Coonrod et al. 2002; Hao et al. 2002; Ma et al. 2008; Vitale et al. 2007).
III. Study of the transcriptome during OET
As noted above, vast amounts of EST sequence data for the oocytes and pre-implantation
embryos provided opportunity to establish identities of genes expressed during this critical
period of development. The first published studies of oocyte and embryo transcriptome
dynamics using EST analysis (Ko et al. 2000; Sasaki et al. 1998) had several caveats, such as
relatively small amounts of annotated sequences in GenBank, lack of Mus musculus genome
assembly, and relatively “primitive” data mining tools for unambiguous establishment of gene
identities. Expansion of GenBank sequence data via projects like Mammalian Gene Collection
(Gerhard et al. 2004; Strausberg et al. 2002; Strausberg et al. 1999) and RIKEN Mouse Gene
Encyclopaedia Project (Kawai et al. 2001; Okazaki et al. 2002), massive expansion of the
Mouse Genome Database effort to annotate all M. musculus genes (Blake et al. 2009; Nadeau
et al. 1995), as well as publication of publicly available M. musculus genome assembly
(Waterston et al. 2002) and development of genome navigation and mining tools such as
ENSEMBL (Hubbard et al. 2002) allowed to increase the rate of precise gene identification in
later studies. Indeed, in the “early post-genome era” gene identities could have been established
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for 71% EST sequences from the 2-cell stage M. musculus embryo cDNA library (Evsikov et
al. 2004), while just two years later, a methodologically identical analysis of the fully-grown
M. musculus oocyte cDNA library established identities of 91% ESTs (Evsikov et al. 2006).
One of the most critical issues affecting the quality of EST-based gene expression analysis is
the method used for cDNA library preparation. Due to low quantity of mRNA in mammalian
oocytes and pre-implantation embryos, linear amplification of cDNA by PCR is often used to
overcome this problem, especially in the studies of H. sapiens embryos (Adjaye et al. 1999;
Adjaye et al. 1997; Adjaye et al. 1998; Goto et al. 2002; Serafica et al. 2005). While this
technique is appropriate in the cases when the biological material is scarce, comparison of M.
musculus oocyte and embryonic cDNA libraries generated using the PCR amplification step
with those prepared by direct cloning reveals the superiority of the latter method in the
preservation of original diversity and relative abundance of transcripts (Evsikov et al. 2006).
A key feature of the M. musculus oocyte and early embryo transcriptomes is the under-
representation of “housekeeping” gene transcripts during OET. In fact, “normal” levels of
housekeeping gene expression in M. musculus are established only by the blastocyst stage, and
despite widespread unsubstantiated beliefs of the opposite, under-representation of
housekeeping transcripts are also observed in the oocytes of African clawed frog Xenopus
laevis as well (Evsikov et al. 2006). Arguably, this aspect of transcriptome reflects the
uniqueness of the oocyte as a “reprogramming machine” designed to create a totipotent embryo.
Among other intriguing characteristics of the M. musculus transcriptome dynamics during OET
is profuse expression of the oocyte-specific genes (Table 1), high representation of novel
“variants”, such as retrogenes and duplicated genes, and abundant usage of alternative
promoters such as Long Terminal Repeats (LTRs) of retrotransposons to drive gene expression
(Evsikov et al. 2004; Peaston et al. 2004). These enigmatic aspects of the oocyte biology are
discussed in the last sections of this review.
As noted above, microarray analysis of gene expression has recently become a method of
choice to study transcriptome dynamics during mammalian OET and pre-implantation
embryogenesis. Particularly, development of commercial high-density, easy-to-use and
relatively low-cost arrays like Affymetrix GeneChips opened the avenues not only to study
changes in gene expression patterns in normal M. musculus development (Wang et al. 2004),
but more importantly, in experimental systems such as oocytes and embryos harboring
mutations in candidate fertility or maternal effect genes (Hao et al. 2009; Tang et al. 2007;
Wan et al. 2008). Moreover, both commercial and “custom” arrays are now used in other
mammalian species as well. Indeed, because embryonic genome activation in Bos taurus
(bovine) and Oryctolagus cuniculus (rabbit) embryos occurs later than in the M. musculus,
these agriculturally important species may more closely model H. sapiens OET and
preimplantation development (Duranthon et al. 2008). For these reasons, studies of O.
cuniculus and B. taurus OET and preimplantation development (Kues et al. 2008; Marjani et
al. 2009; Vigneault et al. 2009), as well as the effects of in vitro maturation, fertilization and
culture on the transcriptomes of B. taurus oocytes and embryos (Katz-Jaffe et al. 2009; Smith
et al. 2009) are rapidly expanding. Moreover, microarrays have been used in studies aimed to
uncover the effects of somatic nuclei reprogramming in cloned B. taurus embryos (Beyhan et
al. 2007; Everts et al. 2008).
As any complex technological platform, microarray analysis has a number of caveats, and we
will talk about only those pertinent to the studies of oocytes and embryos. Firstly, contrary to
the common belief, microarrays by definition are not the tool for gene discovery, because
individual probes on the arrays represent known (or sometimes putative) transcripts. Only the
study of the transcriptome through direct sequence analysis, such as analysis of ESTs, is a valid
tool for mining for novel expressed loci or transcripts during OET (Peaston et al. 2004).
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Secondly, while microarrays are a perfect tool for validation and quantification of changes in
individual gene expression across biological samples, they are unsuitable to quantify the
transcript ratios among individual genes. Thirdly, due to the possibility of cross-hybridization,
microarray technology is not the best tool to study gene families that have greater than 95%
sequence identity among individual members and which are abundantly represented during
OET (Evsikov et al. 2004; Evsikov et al. 2006). However, the most important caveats arise
with experimental design and interpretation of microarray data. For example, mRNA content
of the 2-cell stage M. musculus embryo, 0.26 pg, is almost four times lower than 0.95 pg of
mRNA in the fully-grown oocyte (Piko and Clegg 1982) (Figure 1A). This reflects massive
degradation of maternal mRNAs during OET discussed in the next section. The designs of
microarray experiments usually employ equal amounts of mRNA from different biological
samples. However, this methodology is unacceptable for oocytes and early embryos. In a
putative experiment comparing the transcriptomes of fully-grown oocytes and 2-cell stage
embryos normalization to the amount of mRNA would result in false “overexpression” of stable
mRNAs (Figure 1B), and false “stability” of degrading mRNAs (Figure 1D). For this reason,
only normalization to the number of oocytes and embryos (Figures 1C, 1E) results in the correct
physiological representation of transcriptome dynamics (Su et al. 2007). Another important
consideration often overlooked in the microarray studies of OET is differential polyadenylation
of maternal transcripts. Indeed, common use of T7-oligo(dT) primer in the first step of sample
preparation results in complex and un-interpretable gene expression patterns, due to selective
bias for representation of mRNAs with longer poly(A) tails; however, usage of internally-
priming oligonucleotides such as Full Spectrum™ MultiStart Primers overcomes this obstacle
as well (Su et al. 2007).
IV. Regulatory mechanisms of OET
Commencing at oocyte maturation, and during the subsequent transcriptionally silent stages
of development, three major mechanisms contribute to the dynamic changes that occur in the
ooplasm: (i) the timely translation of stored maternal transcripts provides the cytoplasm with
new proteins, (ii) post-translational modification of existing and/or newly synthesized proteins
determines the exact timing of events during this period, and (iii) the machinery involved in
degradation of proteins and mRNAs removes the no longer needed molecules. Indeed, early
studies of specific changes in the patterns of synthesized proteins during oocyte maturation
(Schultz and Wassarman 1977a; Schultz and Wassarman 1977b), demonstrated the necessity
of protein synthesis for meiotic progression beyond premetaphase I (Schultz and Wassarman
1977a). Similarly, the adverse effects of protein phosphorylation inhibitors (Rime et al.
1989) and inhibitors of the major cellular protein-degrading organelle proteasome (Josefsberg
et al. 2000; Josefsberg et al. 2001; Solter et al. 2004) imply the global involvement of these
mechanisms from the onset of OET. Later studies, focused on the interplay among synthesis
and degradation of cyclin B and phosphorylation status of p34cdc2 to orchestrate the oscillations
in M-phase promoting factor (MPF) activity during oocyte maturation (Hampl and Eppig
1995) provided examples and understanding of particular molecular pathways affected by these
three global mechanisms. Here, we will specifically focus on the mechanisms of mRNA
translational recruitment and turnover during OET. Indeed, in the absence of de novo
transcription, timely recruitment of mRNA for translation, as well as differential message
stability, are the key governing mechanisms creating the dynamic changes in the molecular
environment of the cytoplasm (Oh et al. 2000).
During oocyte growth, many transcribed mRNAs are de-adenylated and stored in the ooplasm
for subsequent translation. During translational activation, their limited 3′ poly(A) tails
lengthen (Bachvarova 1992), a sign that active translation is occurring (Richter 1999). Half of
the poly(A) mRNA found in the fully-grown oocyte is de-adenylated, or degraded, during
maturation, and by the 2-cell stage, the embryo contains less than 30% original amount of
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adenylated mRNAs found in the egg (Piko and Clegg 1982). A large number of maternal
mRNAs degraded during the early phase of OET are ones with pivotal functions during oocyte
growth and most likely have been retained in the ooplasm due to some unknown mechanism
(s) for global inhibition of mRNA degradation (Evsikov et al. 2006). For example, mRNAs
for structural genes of zona pellucida (Zp1, Zp2 and Zp3) are highly abundant in fully-grown
oocytes (Evsikov et al. 2006) but are not translated (Bleil and Wassarman 1980) and after
maturation become virtually undetectable in ovulated, metaphase II (MII)-arrested oocytes.
However, stable maternal mRNAs that are translated during OET are regulated by interacting
with a number of RNA-binding regulatory proteins or other factors. Several seminal cis- and
transacting factors involved in these interactions are discussed below.
Polyadenylation of dormant mRNAs
In the oocytes of X. laevis, as well as M. musculus, mRNAs that are translated in a time-
dependent fashion contain certain “sequence signatures” in their 3′-untranslated regions
(UTRs) (Fox et al. 1989; Fox and Wickens 1990; McGrew et al. 1989; Vassalli et al. 1989).
These U-rich motifs, known as cytoplasmic polyadenylation elements, CPEs (consensus
sequence is UUUUUAU), are located from several to about 120 nucleotides upstream of the
nuclear polyadenylation signal AAUAAA in the M. musculus (Oh et al. 2000). The regulatory
CPE-binding protein (CPEB), and several proteins with which it interacts, have been
characterized in X. laevis and M. musculus (Hake and Richter 1994; Hodgman et al. 2001;
Mendez et al. 2000; Stebbins-Boaz et al. 1999). Essentially, CPEB in its non-phosphorylated
form binds to CPE and prevents, via interactions with other proteins, polyadenylation of mRNA
(Figure 2A). Phosphorylation of CPEB triggers bound mRNA polyadenylation and its
consequent translation (for recent reviews of this and related mechanisms, see (Richter and
Sonenberg 2005; Vardy and Orr-Weaver 2007). Similar regulation of mRNA recruitment for
translation is found in the synapto-dendritic compartment of neurons (Wells et al. 2000),
suggesting the existence of a common mechanism for rapid molecular changes in either
transcription-void systems, such as oocytes, or in cellular compartments located too far from
a nucleus to employ typical translational activation mechanisms to be effective, such as in
neurons. CPEB plays an essential role throughout oogenesis, since CPEB-knockout female
mice lack oocytes due to meiosis failure in germ cells at the pachytene stage (Tay and Richter
2001), while conditional depletion of Cpeb mRNA in growing follicles results in grossly
abnormal oocyte development (Racki and Richter 2006). The general nature of CPEB-
mediated mRNA silencing/re-activation is supported by the existence of homologous proteins
with similar functions across metazoans (Chang et al. 1999; Minshall et al. 1999). However,
other alternative models for CPEB-dependent mRNA repression in oocytes (e.g., Figure 2B),
as well as existence of multiple Cpeb paralogs in mammalian genomes (Cpeb1 – Cpeb4), three
of which are expressed in the oocytes, add a layer of complexity on CPEB-dependent
translational regulation.
Two additional motifs have been identified in the 3′UTRs of the messages stored in X. laevis
eggs, the “embryonic CPEs” (eCPEs; [U]10–18 or [C]10–18 tracts). mRNAs containing these
motifs are recruited for translation only after fertilization (Paillard et al. 2000; Simon et al.
1992; Wu et al. 1997). eCPEs may be located up to several hundred nucleotides upstream of
the poly(A) signal. Their known respective binding factors are X. laevis elrA, a member of the
highly conserved ELAV family of RNA binding proteins, whose likely counterparts in
mammalian oocytes are ELAVL1 and ELAVL2 proteins (Evsikov et al. 2004), and a 42 kDa
protein, a homolog of mammalian poly(rC)-binding protein 2 (PCBP2), also highly expressed
throughout OET in mammals (Evsikov et al. 2004; Evsikov et al. 2006). The high level of
sequence conservation of these proteins among vertebrates suggests that mechanisms of mRNA
recruitment for translation during OET are quite ancient and well conserved, despite significant
differences in the mode of reproduction (i.e. oviparity vs. viviparity).
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Factors affecting mRNA de-adenylation, stability and degradation
De-adenylation is a common mechanism for halting translation, and is often the first step in
the degradation of mRNA. At least three distinct pathways are known to be involved in mRNA
de-adenylation in oocytes and early embryos. Biochemical dissections of these pathways were
mostly studied using X. laevis oocytes. The first pathway, “default” de-adenylation in maturing
X. laevis oocytes, is relatively slow, and, as shown by microinjection of specific antibodies, is
achieved by de-adenylating nuclease PARN (Korner et al. 1998). The second mechanism for
“targeted” de-adenylation relies on the presence of AU-rich elements (AREs) in the 3′UTR of
certain mRNAs. AREs were originally identified as motifs responsible for destabilization and
rapid degradation of mRNAs in mammalian somatic cells (Chen and Shyu 1995). However,
in oocytes and early embryos these elements seem to be responsible for fast de-adenylation,
but not necessarily degradation, of mRNAs (Voeltz and Steitz 1998). AREs have been
classified into three types on the basis of their sequence. Type I AREs have 1–3 copies of an
AUUUA motif within a U-rich region; type II is characterized by at least two overlapping
copies of an UUAUUUA(U/A)(U/A) motif, also within a U-rich region; type III AREs contain
varying repeats of [U(A/G)]n stretches, again in the context of the U-rich region of the UTR.
Interestingly, in somatic cells ELAVL1, a potential eCPE binding protein (see above), binds
to mRNAs that contain type I/II AREs and protects them from degradation but not de-
adenylation (Fan and Steitz 1998; Peng et al. 1998). The list of proteins that have an effect on
the stability and dynamics of ARE-containing mRNAs in oocytes and early embryos is
constantly growing and include CUGBP1 (Paillard et al. 2002), ePAB (Voeltz et al. 2001),
CCR4b/CNOT6L (Morita et al. 2007) and C3H-4/ZFP36L2 (Belloc and Mendez 2008). Genes
encoding orthologs of these proteins are expressed in mammalian oocytes and early embryos
as well (Evsikov et al. 2004; Evsikov et al. 2006; Ramos et al. 2004; Seli et al. 2005). Moreover,
Cugbp1-null females are infertile (Kress et al. 2007), while M. musculus embryos from females
homozygous for truncated Zfp36l2 allele do not develop beyond the 2-cell stage (Ramos et al.
2004), pinpointing an important role for these proteins in the mammalian OET. A third
mechanism for mRNA de-adenylation, occurring after mid-blastula transition in X. laevis, is
α-amanitin sensitive and thus, depends on embryonic transcription (Audic et al. 2001; Audic
et al. 2002). Specifically, maternal mRNAs for cyclin A1 and cyclin B2 are de-adenylated and
degraded only after the initiation of zygotic transcription; interestingly, AREs present in the
3′UTRs regions of these mRNAs are dispensable for de-adenylation and thus other motifs in
the 3′UTRs regulate this mRNA destabilization. Overall, these data are consistent with the
observations of certain co-occurring sequence motifs found in the 3′UTRs of multiple stable
maternal mRNAs and suggest the “combinatorial code” allowing precise spatio-temporal
regulation of mRNAs during OET (Evsikov et al. 2006; Padmanabhan and Richter 2006; Pique
et al. 2008).
Antisense and small RNA regulation of maternal transcripts
Recently, it has been shown that maternal microRNAs (miRNAs) are essential for OET in
zebrafish, Danio rerio (Giraldez et al. 2006), and in M. musculus (Murchison et al. 2007; Tang
et al. 2007). In particular, D. rerio miR-430 miRNA is responsible for de-adenylation and
targeted degradation for a multitude of maternal messages (Giraldez et al. 2006). Another class
of regulatory RNAs involved in the regulation of gene expression and mRNA turnover in
oocytes are “small interfering RNAs” (siRNAs) (Tam et al. 2008; Watanabe et al. 2008). While
miRNAs are encoded by their own loci and usually target a number of different mRNAs, the
siRNAs are produced from long double-stranded RNAs (dsRNAs) and have more specific
targets. In oocytes, the sources for sense strand of dsRNAs are mRNAs of cellular genes, while
the antisense RNA strands are provided by retrogene inserts transcribed in “reverse”
orientation, or antisense transcripts of cellular genes, such as Suv39h1 antisense transcript;
ESTs for both types were described in the oocyte cDNA library (Evsikov et al. 2006). Key
proteins involved in miRNA and siRNA pathways during OET are DICER1 and AGO2/
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EIF2C2 (Tam et al. 2008; Watanabe et al. 2008); indeed, loss of Dicer1 in growing oocytes
results in female sterility linked to abnormal oogenesis (Murchison et al. 2007; Tang et al.
2007). For recent review of this bourgeoning field, see (Ghildiyal and Zamore 2009).
V. Phylogenetic aspects: conservation vs. uniqueness of oocyte
transcriptomes
It has been well recognized for some time that eutherian mammals are quite unique with respect
to early development, which does not rely, unlike most other metazoans, on antero-posterior
and dorso-ventral axis specification during oogenesis or after fertilization, and that blastomeres
of early mammalian embryos are highly regulative (Davidson 1990; Davidson 1991; Evsikov
et al. 1994; Hiiragi and Solter 2004; Motosugi et al. 2005; Motosugi et al. 2006). This and
other features of mammalian oocytes and early embryos, such as minute amounts of yolk, are
likely attributed to viviparity, as embryos receive nutrition directly from mothers upon
implantation. Indeed, eggs of placentotrophic viviparous reptiles are similar in morphology to
mammalian oocytes (Blackburn et al. 1984; Gomez and Ramirez-Pinilla 2004; Hernandez-
Franyutti et al. 2005). Despite this uniqueness, 80% of the genes expressed in the oocytes of
M. musculus are also transcribed in the eggs from phylogenetically distant chordates such as
X. laevis and sea squirt Ciona intestinalis (Evsikov et al. 2006). Similar results were reported
in comparative genomics studies of B. taurus, M. musculus and X. laevis oocyte transcriptomes
using custom multi-species or NIA 22K mouse cDNA microarrays (Vallee et al. 2008; Vallee
et al. 2006). Remarkably, the “most conserved” subset of the M. musculus oocyte transcriptome
overwhelmingly represents mRNAs stable throughout and thus required for the OET (Evsikov
et al. 2006). Additional support for our interpretation arises from recent microarray studies of
OET and preimplantation development in B. taurus (Kues et al. 2008). On the other hand,
expression of genes whose transcripts rapidly disappear at the onset of M. musculus OET is
the most divergent. Moreover, a substantial proportion of these genes underwent rapid
molecular diversifications such as gene duplications (Figure 3), exaptation of retrogenes, or
functional inactivation (Evsikov et al. 2004; Evsikov et al. 2006). Additionally, these “rapidly
evolving” genes within the M. musculus oocyte transcriptome tend to be oocyte-specific, i.e.
expressed only in oocytes, and often species-specific, i.e. present only in M. musculus genome.
A disproportionate number of maternal effect genes identified to date in M. musculus also
belong to this group (e.g., Mater/Nlrp5 (Tong et al. 2000), Oas1d (Yan et al. 2005), and others),
implying neofunctionalization as a strong evolutionary force behind duplications of oocyte-
specific genes. These data imply phylogenetic plasticity of the mechanisms mediating
oogenesis and may reflect, for example, the requirement for fast reproductive adaptation to a
new ecological niche during speciation. At the same time, evolution of OET per se rests upon
a stable molecular foundation of conserved gene interactions.
VI. Coda: oocytes as a playground of evolution
Analysis of oocyte transcriptomes gives hints about the processes underlying the cornerstones
of biological evolution, specifically gametic selection and reproductive isolation. For instance,
it has been established that genes associated with reproduction are subjected to strong selective
pressure (Swanson and Vacquier 2002; Swanson et al. 2001). A general explanation for this
phenomenon is that it serves as a primary mechanism attaining reproductive isolation during
speciation. For example, the zona pellucida genes, which encode sperm receptors required for
fertilization, are among the most rapidly evolving genes in mammals; in some instances, such
as M. musculus Zp4, the “fast-paced” molecular evolution ultimately lead to functional
elimination of the gene (Evsikov et al. 2006; Goudet et al. 2008). The rise of the oocyte-specific
gene families, such as Rfpl4 and Nlrp (Figure 3), may reflect the other side of the same process:
new genes arise by duplication, acquire a unique function, which facilitates reproductive
isolation, thereby providing a substrate for natural selection to act upon and result in speciation.
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Overall, this idea, which is obtaining more experimental and comparative genomics support
(Bikard et al. 2009; Clark et al. 2007; Evsikov et al. 2006; Evsikov and Marin de Evsikova
2009; Semon and Wolfe 2007), implies that when it comes to the role of oocyte in early
development, even subtle phylogenetic differences may play as big of a role as implied
“evolutionary conserved mechanisms” that have been governing the field of experimental
molecular embryology.
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