Activation of the embryonic genome in mice begins late in one-cell zygote and is fully underway by the two-cell cleavage stage [38
]. The silencing of nuclear transcription occurring between meiotic maturation in oocytes and activation of the embryonic genome implies critical roles for preexisting stores of proteins and transcripts [39
]. Through knockout and knockdown strategies, individual maternal proteins have been demonstrated as essential for cleavage stage development in mice. In the present study, we identified many new and unknown maternal proteins in mice by constructing an MII oocyte proteome. In-depth analysis of these maternal proteins will assist us in screening for a proportion of great interest.
Interestingly, many maternal transcripts deposited in mammalian oocytes are not polyadenylated and therefore not translated into proteins [40
]. Independent confirmation of the protein expression of maternal genes is therefore necessary. This was also an important reason for us to construct the oocyte proteome. As a case in point, NALP14, NALP5 (MATER), and NALP4f were included in our subset of abundantly expressed maternal proteins. These three proteins belong to the multifunctional NACHT nucleoside triphosphatase (NTPase) family. NALP14 and NALP5 were previously reported as maternal effect proteins and play significant roles in mouse preimplantation embryo development [7
]. NALP4f was represented by 14 unique peptides in our present study and a previous analysis demonstrated that NALP4f
was an oocyte-specific gene [42
]. Our research has independently confirmed the high protein expression level of NALP4f in mature oocytes. Assuming that NALP4f has similar roles to NALP14 and NALP5, it is highly likely that NALP4f is an important factor necessary for normal embryogenesis and is a good candidate to be a maternal effect protein. In addition, we identified NALP2, NALP4b, and NALP9b in our oocyte proteome. Although the precise functions of NACHT NTPase family members remain to be determined, we speculate that these members play significant roles in early embryo development based on their homology to NALP14 and NALP5.A distinguishing characteristic of maternal effect proteins identified to date is that the majority of them have an abundant mRNA expression in oocytes and many are expressed only in oocytes [7
]. This fact led us to filter maternal products in our protein list by analyzing their corresponding mRNA expression patterns. As a result, 76 maternal proteins with high mRNA expression levels in oocytes and fertilized eggs were selected out. Of these proteins, we discovered that 9 previously described maternal effect proteins (MATER, STELLA, DNMT1, ZAR1, NPM2, PADI6, TLE6, TCL1, FILIA) were enriched in this subset. These maternal effect proteins have been reported to be absolutely necessary for oogenesis, fertilization or early embryo development. Indeed, apart from these well-known examples, the majority of proteins in this subset have not been previously studied or reported in oocytes. We suggest that these proteins are excellent candidates as maternal effect proteins.
A group of proteins belonging to the T-cell leukemia/lymphoma 1 (TCL1) protein family was of particular interest because their corresponding genes had dramatically similar mRNA expression patterns. Figure demonstrates that TCL1, TCLB1, TCLB2
are almost oocyte-specific genes. TCL1
was initially identified as a gene involved in recurrent chromosomal translocation in human prolymphocytic leukemia (T-PLL) and overexpression of TCL1
played a causative role in T cell leukemias of humans and mice [43
]. However, in TCL1
-deficient mice, a female fertility defect was observed. TCL1
-deficient females display normal oogenesis and rates of oocyte maturation/ovulation and fertilization, but the lack of maternally derived TCL1 impairs the embryo's ability to undergo normal cleavage and develop to the morula stage, especially under in vitro
culture conditions [44
]. The TCL1
loss-of-function phenotype indicates that maternal protein TCL1 plays a significant role in early embryo development. Unfortunately the functions of TCLB1 and TCLB2 have not yet been investigated and we can speculate that the two proteins may play similar or complementary roles in embryogenesis.
mRNA expression patterns of three maternal proteins. GNF SymAtlas database analysis shows that TCL1, TCLB1 and TCLB2 are almost solely expressed in the mouse oocyte and fertilized egg.
Domain composition analysis is an effective way to predict the functions of proteins identified in a proteomics analysis. Among 76 proteins singled out because their corresponding genes are highly expressed in oocytes, 6 proteins (FBXL10, FBXW14, FBXW16, FBXW19, EG382106, E330009P21Rik) contained an F-box domain, which was first described as a sequence motif in cyclin-F that interacts with the SKP1 protein. Different F-box proteins, as substrate-specific adaptor subunits of the Skp1-Cullin1-F-box (SCF) complexes, recruit particular substrates for ubiquitination via specific protein-protein interaction domains. Coincidentally, three core protein subunits (SKP1, RBX1, CUL1) of the SCF complex were all definitively identified in our proteome. As one of the major classes of ubiquitin ligases, the SCF complex plays a central role in cell-cycle regulation [34
]. In early stages of embryo development, degradation of maternal proteins is crucial for the oocyte-to-embryo transition [45
]. Our results suggest that the maternal SCF complex probably exists in oocytes and may be important for the oocyte-to-embryo transition by recruiting specific substrates for degradation.
Pluripotent stem cells are of considerable current interest as they can proliferate indefinitely in vitro
and give rise to many adult cell types, serving as a potentially unlimited source for tissue replacement in regenerative medicine. Recently, Takahashi et al. demonstrated that pluripotent stem cells can be induced from mouse fibroblasts by retroviral introduction of Oct3/4, Sox2, c-Myc and Klf4 [46
], indicating that the combination of these four factors can induce reprogramming of somatic cells to a pluripotent state. However, the use of retrovirus-transduced oncogenes represents a serious barrier to the eventual use of reprogrammed cells for therapeutic application because of tumor formation by c-myc reactivation [47
]. Therefore it is necessary to discover factors responsible for reprogramming that would be safer for therapeutic use. We compared the maternal proteins in our oocyte proteome with a recently published mouse ES cell proteome and identified an overlap of 371 proteins. In addition to some pluripotency markers, this group included many uncharacterized proteins, some of which may be good candidates for studying the mechanism of reprogramming. A good example is translationally-controlled tumor protein (Tpt1), which facilitates the first step of somatic cell reprogramming [48
]. Recent studies on Tpt1 demonstrate that this protein activates transcription of oct4
in transplanted somatic nuclei [49
]. We believe that further analysis of these candidate proteins at the functional level will uncover novel proteins that are essential for reprogramming and indirectly promote the application of therapeutic cloning.