The neglected part of early embryonic development: maternal protein degradation

Abstract

The degradation of maternally provided molecules is a very important process during early embryogenesis. However, the vast majority of studies deals with mRNA degradation and protein degradation is only a very little explored process yet. The aim of this article was to summarize current knowledge about the protein degradation during embryogenesis of mammals. In addition to resuming of known data concerning mammalian embryogenesis, we tried to fill the gaps in knowledge by comparison with facts known about protein degradation in early embryos of non-mammalian species. Maternal protein degradation seems to be driven by very strict rules in terms of specificity and timing. The degradation of some maternal proteins is certainly necessary for the normal course of embryonic genome activation (EGA) and several concrete proteins that need to be degraded before major EGA have been already found. Nevertheless, the most important period seems to take place even before preimplantation development—during oocyte maturation. The defects arisen during this period seems to be later irreparable.

Introduction

The early embryonic development is initially driven by maternally inherited mRNAs, proteins and other molecules (such as NTPs). As development proceeds, the stored stocks are degraded and replaced by embryonally synthesized molecules. There are plenty of articles dealing with maternal RNA processing during early embryonic development (summarized in [1, 2]). The degradation of maternal mRNAs is a gradual process, which peaks around the major wave of embryonic genome activation (EGA) and results in the degradation of the vast majority of maternal mRNAs at this time [3]. On the other hand, the way of maternally inherited protein degradation is not well described, especially in mammals. Protein degradation is relatively well described during oocyte maturation and fertilization ([4–6] and others) and several proteins such as securin and cyclin B1 are known to be degraded during oocyte maturation [5, 7]. However there is much fewer studies dealing with protein degradation during early embryogenesis.

During the pre-EGA stages of preimplantation development, the embryos are transcriptionally silent. This indicates that some of the inherited mRNA must be unusually stable [8], so that it persists even until the EGA stage. Therefore, embryonic life regulation is driven mostly at the post-transcriptional level with a high impact on the balance of protein synthesis/storage/degradation.

Due to the strict post-transcriptional regulation of stored maternal mRNA, mRNA and protein expression often do not correlate with each other (Stat1, G10 in Xenopus or Nanog in rabbit) [9, 10]. The onset of transcription of a concrete gene often coincides with a decrease in protein level as a result of its maternal mRNA degradation [9]. The levels of other proteins increase during the mid-blastula transition (MBT; an equivalent of EGA in mammals) in Xenopus [9]. Surprisingly, this increase is often caused not only by translation from embryonic but also from maternal mRNA. The maternal mRNA might be translationally silent, and its activation can be postponed to later stages of the development (e.g. fibronectin in Xenopus) [2, 11–13]. Besides maternal mRNAs and proteins, oocyte also contains metabolites necessary for several embryonic divisions. For example, maternal supply of deoxyribonucleotides (dNTPs) is important for increasing DNA content during the fast embryonic cell cycles in Drosophila [14]. Almost one-third of required dNTPs are of maternal origin, the rest must be synthesized de novo. The shift from using of maternally deposited to newly synthesized dNTPs is an important step leading to maternal-to-zygotic transition [15]. For successful EGA and histone modification, the presence of enzymes of tricarboxylic acid cycle localized to the nucleus is important in mouse embryos [16].

The preimplantation development of mammals is in many ways very specific and differs from the early embryogenesis of non-mammalian species [17]. The meiotic division of the oocyte is only completed after fertilization, and the cleavage of the early embryo is really slow in comparison to other animals (Fig. 1). In the time it takes mammalian embryos to go from fertilization to implantation, some non-mammalian species have already formed most of their organ primordia. As far as the developmental stage is concerned, the EGA occurs very early in mammalian embryos—the major wave occurs at the 2-cell to 16-cell stage for mammalian species [18–20]. Nevertheless, with respect to the time since fertilization, EGA occurs significantly later in mammals (compare Figs. 2, 3). At the 8-cell stage (at the same developmental stage for all mammalian species) the mammalian embryo undergoes a process called compaction (Figs. 2, 3). During this process, blastomeres that used to loosely attach to each other start to express adhesive molecules such as E cadherin, and the embryo becomes a compact structure. Asymmetry appears after the 8-cell stage with the formation of polar and apolar cells [21, 22], but can still be altered when needed [23, 24]. The cells are still able to adapt to a new environment (reviewed in [25]). The definitive formation of two distinct cell lineages comes at the blastocyst stage with the formation of the inner cell mass (ICM) and trophectoderm (TE). Afterward the blastocyst hatches, which is taken to be the final step of preimplantation development.

Fig. 1.

Comparison of development of selected mammalian and non-mammalian species in timeline. The figure shows progression of development during time after fertilization in Xenopus, Drosophila, mouse, bovine and human embryos. While the fast developing species, Xenopus and Drosophila, undergoes the first divisions in rapid manner and during several hours are able to develop to the gastrula stage, mammalian embryos divide slowly, approximately every 12–24 h and reach the blastocyst stage after a few days [17]. hpf hours post fertilization, hped hours post egg deposition, EGA embryonic genome activation, MBT mid-blastula transition

Fig. 2.

Processes during early development of selected mammalian and non-mammalian species in timeline. The figure shows important events during the early development of Xenopus, Drosophila, mouse, bovine and human embryos compared to each other in timeline. These important processes occur very soon after fertilization in Xenopus and Drosophila. Similar processed occur later in mammalian embryos, but these embryos are in a lower developmental stages that non-mammalian species at this time. Green shading density illustrate the maternal to zygotic transition of developmental control. The white arrow marks minor EGA (mouse, bovine, human) or minor MBT (Xenopus, Drosophila). The green arrow marks major EGA (mouse, bovine, human) or major MBT (Xenopus, Drosophila). hpf hours post fertilization, hped hours post egg deposition, EGA embryonic genome activation, MBT mid-blastula transition

Fig. 3.

Processes during early development of selected mammalian and non-mammalian species according to the cell cycle. The figure shows important events occurring during the early development of Xenopus, Drosophila, mouse, bovine and human embryos compared to each other during the cell cycle progression, focused specially on maternal protein level changes. Although some processes occur very quickly after fertilization in Drosophila and Xenopus, these embryos are in higher developmental stages than embryos of mammals, where the similar processes occur in earlier developmental stages actually. Green shading density illustrate the maternal to zygotic transition of developmental control. The white arrow marks minor EGA (mouse, bovine, human) or minor MBT (Xenopus, Drosophila). The green arrow marks major EGA (mouse, bovine, human) or major MBT (Xenopus, Drosophila). hpf hours post fertilization, hped hours post egg deposition, EGA embryonic genome activation, MBT mid-blastula transition

In this review, we summarize current knowledge on protein degradation during preimplantation development in mammals. Since a much larger dataset is available on the early embryogenesis of non-mammalian species, we also use this information and try to find a way to transfer this knowledge from non-mammalian to mammalian species.

Protein degradation pathways involved in preimplantation development

The degradation pathways of maternal proteins during early embryogenesis are to a large extent unknown. The most attention is paid to the ubiquitin–proteasome system (UPS) and autophagy, nevertheless other minor pathways are involved as well. For example in Xenopus, miRNAs are involved in protein degradation during early embryogenesis [26].

In particular UPS is a highly branched pathway comprising of a lot of different enzymes, many of them involved during preimplantation development. Results to date suggest that maternal protein degradation in embryos is not a mass process, but the degradation of each protein is instead controlled separately. Although the hypotheses and experiments can be largely based on the knowledge gained in somatic cells, there are clearly many specifics in the usage of an enzyme in early embryos for the degradation of a particular protein, its timing or localization.

Ubiquitin–proteasome system

UPS is a highly specific degradation pathway that primarily deals with the degradation of endogenous proteins. The proteins targeted for degradation are marked using the small protein ubiquitin that is reversibly linked by a covalent bond to targeted protein [27]. The proteins are polyubiquinated, which directs them to be degraded by the 26S proteasome. The ubiquitination of proteins is performed by three enzyme complexes: E1—ubiquitin activating enzyme; E2—ubiquitin-conjugating enzyme and E3—ubiquitin ligase [27]. While there is only one type of E1 enzyme and a little over 40 types of E2 enzymes, there are more than 500 types of E3 ligases [28]. The plurality of E3 ligases enables the polyubiquitination of different protein targets. The ubiquitinated proteins are rapidly degraded by the proteasome system.

The protein degradation using UPS regulates many cellular functions, including H2B methylation and chromatin remodelling [29, 30], cell cycle progression, the continuous running of transcription [31] and also its termination [32], without which the cell division does not occur. Moreover ubiquitination without the activation of UPS is also involved in DNA repair, autophagy, transcription and vesicle processing [33]. It is thought that a great deal of maternal protein degradation is mediated by the ubiquitin-proteolytic system [34–37] and the activity of UPS reflects the embryo quality [35]. Thus the role of the ubiqutin-proteasome pathway (UPS) in preimplantation development seems to be undeniable. The involvement of UPS in oocyte maturation and early preimplantation development has been shown by mass spectrometry (MS) [38–40]. MS is an ideal method for finding candidate proteins to be degraded during normal development. It seems that in a certain moment, degradation of only a few proteins is needed. This results were shown by Yang et al. [38] who identified just one protein, which is degraded by examined enzyme at specific time, between thousands of potential proteins analysed using MS. Thus, in spite of high material requirements, MS provide valuable insights into protein processing during embryogenesis.

The amount of proteasomes is mainly regulated at the transcriptional level of its subunits (reviewed in [41]). A decrease in proteasomal activity results in an increased mRNA expression of proteasomal genes and consequently in de novo proteasome formation [42]. However, transcription is inactive during the early stages of preimplantation development, and so the expression of proteasomal genes is not the way proteasome activity is regulated in embryos.

In mice, the polysomal maternal mRNA of genes responsible for ubiquitination is equally present in matured oocytes and one-cell embryos [43]. The embryonic expression of genes involved in protein ubiquitination is activated at the 8-cell stage in bovines; at the same time, the genes required for translation initiation and ribosome biogenesis are activated [44]. The major wave of embryonic genome activation takes place at the late 8-cell stage in cows. When experiments were focused on the expression of SCF complex members (Skp1-Cul-F box complex, one of the most common E3 ligases), it was found that the mRNA transcription of Cullin1 and Skp1 starts already at the early 8-cell (i.e. before major EGA) and of Rbx1 at the late 8-cell stage (EGA stage) [45]. The level of proteins related to UPS sharply increases in mouse zygotes [39], the Cullin1 protein level is the highest at the late 8-cell and morula stage, the level of SKP1 is the highest at the 4-cell stage [45]. This shows that the maternal mRNA of genes involved in UPS-based protein degradation is stored and translated until the major wave of EGA, and the expression of embryonic mRNA of these genes is initiated even before the major EGA, so that there is no gap in their protein synthesis. Interestingly, there are large interspecies differences in the expression of UPS genes mRNAs, as was shown by Mtango and Latham [35] after a comparison of mouse and rhesus monkey oocytes and embryos, and also after comparison with our results [45]. It would be very interesting to compare the expression of these genes at the protein level.

Polyubiquinated proteins are accumulated in the embryo around the major EGA—i.e. in mice from the 2-cell stage with rapid degradation at the 4-cell stage [46]. This degradation is consistent with the increase in the chymotrypsin-like activity of the proteasome at the 2-cell stage in mice [46]. However, it has been found that SCF complex activity does not significantly change during preimplantation development [45]. In preimplantation embryos, proteasomes and ubiquinated proteins are preferentially localized to the nucleus [47–49], active SCF complex (Cullin1 and Skp1 complex interaction) and Cullin 1 are mainly localized to the cytoplasm [45, 50]. At the blastocyst stage, ubiquitination is mainly localized to the trophectoderm. Sutovsky et al. [51] found that in bovine and murine blastocysts the diffuse cytoplasmic labelling of ubiquitin was present in both the TE and ICM, however in the TE, large granules of ubiquitin were accumulated, whilst these granules were not present in the ICM. Similarly in our laboratory, almost no SCF complex activity in the inner cell mass and high SCF complex activity in the TE was found [45]. On the other hand, the murine deubiquitinase Dub-2 is required for the development of the ICM and hatching of the blastocyst [52], and the localization of proteasomes seems to be the same in ICM and TE (nevertheless, this was observed using ivf-discarded triploid blastocysts; [47]). This indicates that UPS—mediated protein degradation is needed throughout the whole of preimplantation development from oocyte maturation to blastocyst formation. However, the two parts of the UPS—ubiquitination and proteasomal degradation—do not always correlate with each other, and the regulation of protein degradation is driven mainly by the proteasomal activity level rather than the level of ubiquitination in preimplantation embryos.

Moreover, the interaction of a protein with E3-ubiquitin ligase does not necessarily mean its degradation. For example, RECQL4 (RecQ like helicase 4) is ubiquinated by DDB1-CUL4A (DNA damage-binding protein 1-cullin 4A) E3 ubiquitin ligase, which triggers RECQL4 to mediate the repair of double-strand breaks in DNA instead of RECQL4 being degraded [53]. Further, it is associated with the ubiquitin ligases UBR1/UBR2, nevertheless this interaction does not even cause the ubiquitination of RECQL4 [54].

Deubiquitinating enzymes

Deubiquitinating enzymes (DUBs) are thiol proteases that remove the bound ubiquitin from its substrates (proteins, polyubiquitin chains) and thus reverses ubiquitination and enables the renewal of free monoubiquitin ([55, 56] reviewed in [57]). In this way, DUBs not only control (prevent) the substrate degradation, but also recycling of the ubiquitins for further use. It has been shown that silencing or inhibition of a DUB causes affected preimplantation development [58, 59] and others. The deterioration of their preimplantation development is likely mainly caused by the lack of free monoubiquitin (reviewed in [60]). The silencing of DUB USP36 causes decreased mRNA translation in murine morulas. USP36 is a nucleolar protein that is necessary for ribosome biogenesis and RNA processing [59]. To the best of our knowledge, there are no data on USP36 silencing in other mammalian species, however such an effect will likely occur earlier in development, according to the stage at which nucleoli are formed (e.g. at the 8-cell stage in cattle). DUBs certainly play an important role during fertilization in anti-polyspermy defence, specifically the Ubiquitin C-terminal hydrolases (UCHLs) [58]. UCHLs are the most important subgroup of DUBs. Further, there are four other families of DUBs: ubiquitin-specific proteases (USPs), ovarian-tumour domain (OTU DUBs), Machado–Joseph domain (MJD DUBs) and a Jab1/MPN metalloenzyme (JAMM) zinc-dependent metalloprotease (reviewed in [60]), whose role during preimplantation development has however not been elucidated yet.

UCHLs

As for oocytes and early embryos, UCHL1 and 3 are the most important members of the UCHL family. UCHL 1 and 3 are necessary for normal oocyte maturation and fertilization and the mechanism of their action seems to be highly conserved (reviewed in [60]). UCHL1 is highly expressed in porcine and bovine oocytes and is localized to the oocyte cortex [58, 61, 62]. This is likely because of its need for cortical granule maturation as part of the later polyspermy block [58]. It is further thought to regulate the level of maternal protein Mater [63]. UCHL3 is localized to the spindle and is probably involved in correct cumulus expansion [60]. Both UCHL1 and 3 are involved in correct polar body extrusion, which ensures leaving enough cytoplasm for the growing oocyte [60, 64]. The incorrect expression of UCHLs during oogenesis and fertilization causes abnormal embryo development, most defects are seen especially during morula compaction and blastocyst formation [64]. A potential role in early embryogenesis may also be played by UCHL2 (mostly referred to as BRCA1-associated protein 1; BAP1), which is needed for the removal of ubiquitin from histone H2B [65]. However, its role during preimplantation embryo development has not been studied at all yet.

Ring finger protein 114 (RNF114)

RNF114 is an E3 ubiquitin ligase that is predominantly expressed in oocytes and early embryos, and it is one of the most abundant proteins during the late stage of oocyte maturation [40, 66]. Recently, Yang et al. [38] found that RNF114 plays a crucial role in murine preimplantation development through the degradation of TAB1 (TGF-beta activated kinase 1 (MAP3K7) binding protein 1). Embryos with silenced RNF114 arrest at the two-cell stage (i.e. the stage of major EGA), which is likely caused by a defect in NFkB pathway activation during major EGA after TAB1 accumulation [38]. Interestingly, the authors studied more than 9000 proteins, and TAB1 was shown to be the only target of RNF114, whose degradation is necessary for normal preimplantation development [38]. This suggests that the degradation of individual proteins rather than all maternal proteins is important for the initiation of EGA.

specific proteasome assembly chaperone (ZPAC)

ZPAC is a protein that is specifically expressed in murine gonads, germ cells and early embryos, and not expressed in other somatic cells [46, 67]. This is probably because of an increased demand for protein degradation. The expression of ZPAC-like protein in other species has not been found to date [46]. The expression of ZPAC is increased at major EGA and is involved in the degradation of maternal proteins, as it interacts with and stabilizes proteasome assembly chaperone UMP1 (ubiquitin-mediated-proteolysis 1) and promotes 20S proteasome biogenesis [46]. The expression of these proteins is interconnected in the above-mentioned tissues and cells, as the silencing of one of them results in depleting the other [46]. ZPAC replaces PAC proteins that work as proteasome chaperones in somatic cells, probably because of the high abundance of β-precursors of the proteasome in quickly developing cells such as embryos [42, 46, 68]. Its protein level is highest from 12 to 36 h post fertilization (hpf; peak at 24 hpf) and is almost undetectable after the 8-cell stage in mice. Its mRNA becomes undetectable as soon as 36 hpf [46]. The UMP1 protein remains highly expressed until 48 hpf, but its level sharply decreases thereafter and is also almost undetectable from the 8-cell stage onwards [46]. This is a very early degradation compared to other maternal factors such as MATER that are present until the blastocyst stage. Both of these proteins are degraded in proteasomes, have a short life-time and are degraded and de novo synthesized even during early embryogenesis [46]. The majority of ZPAC knock-down and Ump1-knockdown embryos arrested between the 1- and 2-cell stage, i.e. just before the initiation of major EGA in mice because of the accumulation of β-subunits of the 20S proteasome [46, 67]. Both of these inhibitions cause a decrease in proteasomal activity and an accumulation of polyubiquinated proteins in the treated embryos [46]. ZPAC is also needed for normal spermatogenesis in mice [67, 69].

SCF complex

An SCF complex is an E3 ubiquitin ligase that is formed of Cullin1, SKP1, RBX1 and an F box protein. The F box determines the specificity of the complex to its substrate. There are also other types of similar ligases based on Cullin 2, 3, 4A, 4B, 5, 7 and 9 that are called Cullin 2–9 based CRL (Cullin-RING ubiquitin ligases) complexes, or often Cullin 2–9 based SCF complexes [70, 71]. Of these, CRL4 is known to be involved in mammalian preimplantation development [72–76].

SCF complexes are probably involved to a large extent in the protein degradation of maternal proteins, since there are a large number of SCF complexes in mouse oocytes and zygotes [39, 40], it is active throughout preimplantation development, and the embryonic expression of its invariant members is activated no later than the major EGA stage [45]. Moreover, the inhibition of SCF ligase activity causes an increase in total protein level [77]. It is certain that an SCF complex is responsible for the degradation of CDC6 (cell division cycle 6) before GVBD (germinal vesicle breakdown), CPEB (cytoplasmic polyadenylation element-binding protein) during oocyte maturation ([78, 79], CDC25A [M-phase inducer phosphatase 1]) in Xenopus around the major wave of MBT or DRF1/DFB4B (DBF4 zinc finger B) before the major MBT in Xenopus [80–84]. CRL4^DCAF1 is responsible for protein phosphatase 2A degradation that is necessary for successful GVBD during murine oocyte maturation [85]. The processing of CDC6 and DRF1/DFB4B proteins is discussed in detail in the Specific protein degradation chapter. CRL4A is responsible for degradation of SUV39H1 (Suppressor Of Variegation 3–9 Homolog 1) and CDT1 (Chromatin Licensing and DNA Replication Factor 1) during early murine development and its malfunction during murine oocyte maturation causes early embryonic arrest and female infertility [72–76]. Normal function of CRL4 is involved in control of correct expression of cell fate genes like Cdx2, Nanog or Oct4 [72, 74].

Other UPS—related proteins involved in preimplantation development

In addition to the above-mentioned genes, several other proteins related to UPS were found to be necessary for normal preimplantation development. These are mainly E3 ubiquitin ligases (RNF20, MDM2 and Anaphase promoting complex/Cyclosome (APC/C) and the ubiquitin conjugation enzyme UBE2A/HR6A. RNF20 is responsible for H2B monoubiqutination. After the silencing of Rnf20, the ubiquitin ligase of H2B, the majority of embryos arrest at the morula stage and only one third of them develop to the blastocyst stage [86]. This arrest is however not related to protein degradation defects, but to the decrease in H2B monoubiquitination level. MDM2 causes the degradation of P53, and its knockout causes embryonic lethality due to P53 accumulation [87–89]. Nevertheless, this arrest occurs during the postimplantation period of development around E5.5 (embryonic day; [88, 89]). Anaphase promoting complex/cyclosome (APC/C) prevents securin degradation in mice oocytes and zygotes and causes arrest at the metaphase phase of the cell cycle [90, 91]. APC/C is known to be responsible for cyclin B and securin degradation, which is needed for the cell cycle transition to anaphase [92]. UBE2A/HR6A seems to play the most important role during preimplantation development as the embryos of a UBE2A/HR6A-deficient mother arrest at the 2-cell stage or earlier in mice [93], however the reason for this arrest is unknown. The early arrest of embryos shows that Ube2a/HR6A belongs to the Maternal effect genes group, like the Mater or Filia gene.

Autophagy

Autophagy is, after UPS, the second most known pathway for degrading proteins, but also other molecules like lipids or small organelles present in the cytoplasm, and it is involved in the degradation of maternal mRNA during early embryogenesis [94–96]. Besides the degradation of unnecessary protein, protein degradation by autophagy is also needed for the recycling of amino acids that are necessary for de novo protein synthesis [97]. Autophagy degrades its substrates using lysosomes. It starts with the engulfment of part of the cytoplasm into an autophagosome. The autophagosome then fuses with the lysosome, in which proteins are broken down into amino acids. The regulation of autophagy is driven mainly by mTOR and ATG proteins (reviewed in [98, 99]). Murine Atg5−/− embryos arrest during the preimplantation phase of development [97], Beclin1 (ortholog of Arg6)−/− embryos arrest after implantation at E7.5 [100] and the inhibition of autophagy causes a significant decrease in blastocyst number in the treated group [101]. Autophagy is also involved in the regulation of pluripotency-associated proteins OCT4, NANOG and SOX2 [101, 102], which together suggest that autophagy plays a crucial role during early embryogenesis. The genes involved in the autophagy pathway are conserved from yeast to humans (reviewed in [103]). The autophagy pathway needs to be started shortly after fertilization, and in mice is active until the late 1-cell stage [97]. Interestingly, this fertilization-induced activation of autophagy is not dependent on mTORC1 [104] (mTOR is a member of two complexes, mTORC1 and mTORC2 [105]). During the middle 2-cell stage it becomes reactivated and is active until the 8-cell stage. After that, the autophagy activity decreases to the basal level and is only reactivated after birth [103]. Nevertheless, autophagy is clearly also important during the 8-cell to implantation period of development [106–108].

A higher rate of autophagy seems to correlate with a higher embryo quality. It was shown that mouse embryos with higher autophagic activity at 4-cell are of higher quality [109], LC3 (autophagosome marker)-induced autophagy can improve porcine oocyte quality [110], the induction of autophagy in bovine preimplantation embryos increases their developmental competence and on the other hand the inhibition of autophagy leads to a decrease in developmental competence [111]. Autophagy further improves the blastocyst survival rate during implantation [108]. On the other hand, a higher autophagy of oocytes and embryos can result from a poor maturation or developmental condition and is known as pro-death autophagy [107, 112–116]. Autophagy (not only) during preimplantation development is driven by poly(ADP-ribosyl)ation (PARylation) and PARylation inhibition leads to a decreased autophagic degradation of ubiquinated aggregates, and consequently to a decreased developmental competence of porcine and murine preimplantation embryos, especially at the blastocyst stage [106, 107]. Both the inhibition and induction of autophagy during the earliest stages of preimplantation development (from 1- to 2-cell stage or longer) decreases the number of blastomeres and increases apoptosis in blastocysts in mice. Nevertheless, the induction of autophagy does not influence the number of embryos reaching the blastocyst stage, in contrast to the inhibition of autophagy [101]. This is probably caused by the overexpression of Cdx2 (caudal type homeobox 2), Nanog and Sox2 mRNA in the treated blastocysts. This was true after both the induction and inhibition of autophagy, yet the increase was more significant after the induction of apoptosis, and in addition Pou5fl (Oct3/4) was also overexpressed [101]. However, the induction/inhibition of autophagy after reaching the 2-cell stage does not affect the development of embryos [101]. The normal operation of autophagy during the division from the 1-cell to the 2-cell stage thus seems to be the most important period of preimplantation development for the production of a normal blastocyst. Surprisingly in pigs, the number of embryos reaching the blastocyst stage was not affected after the inhibition of autophagy from the 1-cell to morula stage, but the number significantly decreased after inhibition from the morula to the blastocyst stage [107]. The authors suggest that only UPS based degradation and not autophagy may be involved in the maternal protein degradation in early stages of porcine preimplantation development [107, 117]. Nevertheless, the development of pig embryos arising from oocytes with an affected autophagy pathway significantly decreases the number and quality of blastocysts [116].

The autophagic activity in embryos declines with maternal age [109] and the level of autophagy in 2–4-cell-stage cloned porcine embryos influences major EGA initiation [96]. Interestingly, the activity of autophagy is dependent on the origin of the embryos. The highest activity of autophagy in porcine cloned embryos was found at the 2-cell stage, however the same was true for 4-cell-stage parthenogenetically activated embryos and 1-cell-stage IVFderived embryos [96, 107]. This is also true for the relocalization of the LC3 autophagosome marker from nuclei to the cytoplasm that coincides with a decrease in autophagic activity [96, 107].

Further, autophagy is interconnected with ubiquitination, as it degrades protein complexes that were imperfectly degraded using UPS or in organelle degradation [103]. How ubiquitination is further processed depends on the type of ubiquitin (Ub) chain binding. Chains of 4 or more Ubs on lysine 48 results in processing by UPS, the binding of Ubs on lysine 63 (K63) results in autophagy, the endocytosis of membrane proteins or DNA repair [33, 118, 119]. The timing of K48 and K63 ubiquitination in the peri-fertilization time of C. elegans is different. While UPS-related ubiquitination is present especially before the MII stage, K63 ubiquitination starts from the MII stage onwards and is connected not only to autophagy but also endocytosis [119, 120]. During the degradation of damaged mitochondria, autophagy collaborates with the E3 ubiquitin ligases Parkin and MUL1, which ubiquitinates the mitochondrial membrane protein. In this way, autophagy becomes selective, as the ubiquitination targets the marked mitochondria for lysosomal degradation [121]. The selective degradation is otherwise typical, especially for starvation (reviewed in [60]). Ubiquitination and autophagy also collaborate during paternal mitophagy after fertilization [121–123]. The selective degradation of ubiquitinated proteins is mediated through protein P62, which is a receptor for ubiquitinated proteins for autophagy (reviewed in [124]).

In conclusion, autophagy and ubiquitination clearly intersect with each other to correctly degrade/store maternal proteins during preimplantation development. The activity of both these pathways reflects the quality of the embryo [35, 103]. Similarly to UPS-related pathways, autophagy (autophagosomes) is mainly localized to the trophectoderm at the blastocyst stage [45, 51, 108, 125]. However, autophagy during preimplantation development is to a large extent related to the degradation of targets other than proteins (maternal mRNA, lipids, epigenetic changes etc.) [95, 96].

Endocytosis

Endocytosis is involved in plasma membrane proteins degradation. The selected proteins are first loaded into an intraluminal vesicle and then form multivesicle bodies that fuse with lysosomes [33, 126, 127]. It may be triggered by monoubiquitination or K63 ubiquitination (which also triggers autophagy) [33, 127]. In C. elegans, endocytosis is involved in the selective degradation of plasma membrane proteins after fertilization (e.g. caveolin 1, receptor mediated endycytosis 2 RME-2, chitin synthase 1 CHS-1, EGG-1) [128–132]. In mammals, the role of endocytosis in maternal plasma membrane protein degradation has not been demonstrated to date, but its role seems to be undeniable, as it is one of the earliest activated pathways in the preimplantation embryo [133].

Ornithine decarboxylase

Ornithine decarboxylase (ODC1) is a crucially important enzyme for the synthesis of polyamines [134]. Its inhibition causes implantation defects and entry of the murine blastocyst into an embryonic diapause [135]. ODC1 expression is also important during peri-implantation stages in species that do not normally undergo embryonic diapause [136]. The ODC1−/− offspring of ODC1 ± parents are able to implant, but die soon thereafter. This is likely related to the fact that ODC1 expression is needed for ICM expansion [137]. The enzyme is unusual in its specific way of targeting for proteasomal degradation. Instead of being marked with ubiquitin, ODC1 interacts with ornithine decarboxylase antizyme (OAZ), which directs it to be degraded by the 26S proteasome. An intact COOH-terminal part of the protein is needed for the degradation (reviewed in [138]). It seems that ODC1 can be also degraded in an OAZ/COOH-terminal/ubiquitin-independent manner by the 20S proteasome, when not bound to NAD(P)H quinone oxidoreductase (reviewed in [138]). In Xenopus, the maternal protein is stable and is not degraded before MBT [139]. No such data are available for mammalian preimplantation embryos.

Timing of protein degradation during early embryogenesis

The first extensive protein degradation occurs even before fertilization, and enables the resumption of meiosis and transition to mitosis. This is when the degradation occurs of proteins like cytoplasmic polyadenylation element-binding protein 1 (CPEB1; involved in translational control) in Xenopus, bovine and mouse oocytes [140–143], ELAVL2 in mouse oocytes (translational control) [6], or MEI1 and 2 (microtubule severing complex) in C. elegans (reviewed in [144]). CPEB is degraded with E3 ubiquitin ligase SCF^β−TrCP [80]. MEI1 and 2 are degraded by Cul3-based ubiquitin ligase [145, 146]. The Skp1-Cullin1-Fbox (SCF) complex ligases are suggested to be involved in protein degradation during bovine oocyte maturation and preimplantation development [45, 77, 125], since after the inhibition of SCF-ligases activity during oocyte maturation, an increase in total protein level occurs (see chapter SCF ligases). Interestingly, this inhibition largely influences further preimplantation development even after the transfer of the MII oocytes to standard culture medium for fertilization [77]. The quality of the embryo likely depends on the correct course of protein degradation during oocyte maturation [35, 77]. UPS is also involved in cumulus cell expansion, as the inhibition of UCHL3 or SCF ligases during oocyte maturation prevents cumulus cell expansion [60, 77]. The embryo arising from an oocyte with a defective process of protein storage/degradation may not be able to overcome this handicap, even if its protein processing is appropriate during preimplantation development [35]. Interestingly, the inhibition of UPS using the proteasome inhibitor MG132 in oocytes leads to an increased quality of cloned mammalian embryos [147–152]. The treatment prevents the degradation of cyclin B, maintains MPF activity and cause premature chromosome condensation, which enables a better availability of remodelling and reprogramming of the somatic nucleus and thus better reorganization of chromatin [150, 153]. Furthermore, it likely prevents the inappropriate degradation of proteins essential for initiation of EGA in cloned embryos and regulates the expression of genes involved in histone acetylation [153].

The next extensive protein degradation comes after fertilization of the oocyte. This early degradation depends on both maternally derived UPS and autophagy [49, 97, 104]. After the inhibition of proteasomal activity, polyubiquinated proteins become accumulated after fertilization [154]. UPS is likely involved in the proper reprogramming of the differentiated oocyte into a totipotent zygote after fertilization and its dysfunction is likely the reason for the deteriorated development of somatic cell nuclear transfer embryos [154]. The mouse zygote is enriched in proteins involved in the ubiquitin–proteasome pathway in comparison to MII oocytes, and the amount of proteins in zygotes is decreased compared to MII [39]. In murine MII oocytes, proteins related to ubiquitination and protein degradation amount to 9% [155]. This points to the rapid degradation of maternal proteins after fertilization in mice mentioned in the Ubiquitin proteasome pathway chapter. Moreover, a large set of F-box proteins is present in murine MII and zygotes, which suggests the involvement of an SCF complex in maternal protein degradation in mice [39]. Nevertheless, F-box proteins are involved in many other processes such as epigenetic regulation or reprogramming and its overexpression during the perifertilization period is likely also connected to these UPS-unrelated functions [39]. These results point to the involvement of UPS in post-fertilization protein degradation.

Further degradation is not thoroughly described, and is thought to not be so massive. Instead individual proteins are degraded at specific time points. We suppose that there is a smaller wave of maternal protein degradation before/at the major EGA stage, similarly to such degradation in Xenopus (discussed in the Specific protein degradation chapter). The degradation of maternal proteins is the easiest to explore in genes that are only expressed from maternally derived reserves. For example, the maternal protein MATER (NLRP5) is stored until the blastocyst stage and hatched blastocyst stage, when it is degraded in mice and bovines, respectively [156, 157]. Gao et al. [158] detected a decrease in the protein amount of maternal factors MATER (NLRP5), Floped or TLE6 even at the 8-cell stage. We suppose that the discrepancy in results is caused by the different method used, as Gao et al. used mass spectrometry (MS) and Pennetier and Ohsugi and their colleagues used western blot analysis [156–158]. Murine ZPAC and UMP1 are among the earliest degraded maternal proteins. Their degradation occurs at the 8-cell stage [46] and is discussed in the ZPAC chapter. The degradation of TAB1 occurs even earlier (2-cell stage). This degradation is necessary for the normal course of major EGA [38].

Table 1.

Proteins degraded in the time of EGA

Protein	Function	Organism	References
TAB1	Regulator of MAP3K7/TAK1, NFKB, MAPK14/p38alpha	Mouse	[38]
CDC25A	Induces progression of mitosis; tyrosine protein phosphatase	Xenopus, Drosophila	[81, 82, 185]
CDC6	Controls progression of DNA replication	Xenopus	[9]
Treslin	Regulates DNA replication	Xenopus	[9]
RECQL4	DNA dependent ATPase	Xenopus	[9]
TOPBP1	Involved in DNA replication	Xenopus	[9]
DRF1/DBF4B	Plays crucial role DNA replication control, Cdc7 subunit	Xenopus	[168]

The degradation of these proteins in necessary for normal course of EGA

Specific protein degradation

Even though there is a large decrease in protein number from MII stage oocytes to zygotes in mouse oocytes [39], not all proteins are degraded before the initiation of EGA. Some of the proteins are stored throughout the whole preimplantation development [157, 159, 160]; reviewed in [161]. In Xenopus, a stable amount of maternally stored proteins is stored long after the MBT, until beating heart [162].

Maternal proteins often form complexes that persist to preimplantation development and are then involved in the regulation of embryogenesis even after major EGA (reviewed in [161]). These are mainly functional complexes of proteins such as Zona pellucida sperm-binding proteins (ZPs) or SCMC (subcortical maternal complex; formed of MATER, FLOPED, TLE6, Filia, PADI6) and likely other proteins [163]. The expression of these genes is exclusively maternal, and mRNAs of its members are degraded at EGA. However, the proteins are stored until the blastocyst stage (reviewed in [164]). Another case of protein modification is the temporary masking of the protein CENPE during oocyte maturation, and it is possible that other proteins are also modified or form complexes to enable their storage to preimplantation development [165]; reviewed in [161]. Some cell cycle regulating proteins (like stem-loop binding protein or centromeric protein F) that are expressed in a cell-cycle dependent manner are expressed constantly in oocytes and early embryos [166, 167].

On the other hand, some maternal proteins need to be degraded, so that the embryo can take control over their development. The list of proteins that needs to be degraded before the major wave of EGA/MBT is shown in Table 1. Inmouse, the degradation of the TAB1 protein is needed for the normal course of major EGA [38]. Degradation of SUV39H1 by Cullin Ring Ligase 4A (CRL4A) is necessary for normal preimplantation development [74]. SUV39H1 is degraded by CRL4A^DCAF13. The SUV39H1-enriched embryos develop normally to 8-cell but are not able to compact and form blastocyst [74]. CRL4 is necessary also for CDT1 degradation. The CDT1 enriched murine embryos arrest already at 1–2-cell, i. e. before the major EGA. Nevertheless, this degradation seems to be cell cycle specific rather than EGA-specific. Moreover, several other unidentified substrates seems to be degraded by CRL4 enzymes during preimplantation development too [74, 76]. Even though it seems that in general maternal protein degradation is not needed for embryonic genome activation, Shin et al. [5] have shown that the transient inhibition of the ubiquitin proteasome pathway in murine preimplantation embryos using MG132 delays embryonic genome activation. This delay occurs regardless of whether the embryo is arrested or developing. This suggests that the degradation of maternal protein is highly specific and the degradation of one is just as important as keeping the others.

In Xenopus, five proteins that need to be degraded at major EGA were identified: TOPBP1 (a human ortholog of Xenopus Cut5), RECQ4, Treslin, DRF1 (Dumbbell-forming 4; a homolog of mammalian DBF4B) and XCDC6 (cell division control protein; [9, 168]). All these genes are involved in replication control. DRF1/DBF4B, being part of CDC7, is essential for the activation of the replication origin complex that the others are part of. CDC6 is one of the first proteins loaded into the complex. TOPBP1/CUT5, RECQ4 (or RECQL4 in mammals) and Treslin are part of the replication pre-initiation complex and are loaded to the replication origin in the second step (reviewed in [169]). Thus, the need for degradation of these proteins is likely connected to the lengthening of the cell cycle after the MBT and less a need for replication initiation. Whether the degradation of these genes is needed for the normal course of major EGA in mammals is to the best of our knowledge unknown. Certainly, these genes are important for mammalian preimplantation development as well. The knockouts are mostly lethal at peri-implantation stages in mammals, however the development of lower animals is enabled far beyond the MBT stage [170–177]. The only exception is Drf1/DBF4B. Drf1−/− mice are viable, only develop lymphopenia [178] and mutant cell cultures show affected cytoskeletal remodelling [179]. A complete knockout of Drf1/DBF4B was found as a rare sequence variant in humans [180] which further confirms that embryonic development is possible without Drf1/DBF4B expression even though it participates in the manifestation of disease. This similarity of null phenotypes in these genes suggests that the initiation of embryonic protein synthesis is as important as the degradation of the maternal protein. The differences between mammalian and non-mammalian species are likely due to the difference in developmental speed and must be taken into account when data are transferred between different model organisms. Taken together, it is clear that all the above-mentioned genes are needed for the normal preimplantation development of mammals, however there is no evidence on whether the degradation of maternal protein before major EGA is as necessary as in the Xenopus embryo. Certainly, these genes together form a group of proteins that play a similar role during the earliest phases of development and are processed in a similar manner. The necessity of their degradation is further supported by the overexpression experiments. Embryos overexpressing XCdc6 exhibit a high level of apoptosis [9]. Embryos that combined the overexpression of Cut5, RecQ4, Treslin and Drf1 do not lengthen cell cycles (dependently on protein level) and arrest at stage 17 (i.e. at 17th division) [168]. After the overexpression of Drf1/DBF4B, the viability of the Xenopus embryos decreases after stage 11 [83]. Interestingly, DBF4 overexpression in pre-MBT embryos can suppress the activation of Chk1, but the overexpression is not lethal, in contrast to Drf1 overexpression [83]. Thus, it would be extremely interesting to study the whole group thoroughly and complexly during mammalian preimplantation development.

In Xenopus, two of the above-mentioned proteins—DRF1/DBF4B and CDC6—have two distinct variants expressed from two different genes with a switch between those variants around the major MBT. DRF1/DBF4B is replaced by DBF4 before the major wave of MBT and cannot be detected afterwards (Figs. 2, 3). Consequently DRF1/DBF4B is present mainly in embryonic cells and DBF4 is found mainly in adult cells [181, 182]. With Cdc6, the Xcdc6A, and Xcdc6B variants were found. Only Xcdc6A is functional at early embryonic stages (Xcdc6B is present in early stages, however not functional). It switches around the major MBT stage from XCdcd6A to XCdcd6B and Xcdc6A is undetectable after stage 12 (Fig. 3) and only Xcdc6B is present in somatic cells [183]. Interestingly, Xcdc6A is not expressed in a cell-cycle dependent manner like the somatic Xcdc6B is [183]. Tikhmyanova and Coleman [183], however, suppose that the CDCD6A/B isoforms may be restricted to Xenopus since they did not find the isoforms in humans, fruit flies, puffer fish and worms in the available genomic databases, and their regulation significantly differs in different organisms [183]. In mammals, a similar switch was found in bovine embryos in Cullin 1, where the maternal variant of Cullin 1 mRNA switches to the embryonic (adult) Cullin 1 just before the major wave of EGA [45, 50]. The time of protein variant switch is unfortunately unknown. In Xenopus embryos, DRF1/DBF4B is degraded by SCF^β−TRCP (Skp1–Cullin 1–Fbox complex where β-TRCP represents the F-box protein) at the major MBT. This ensures a DRF1/DBF4B shortage and consequently a lengthening of cell cycles [83]. Thus, we suppose that the switch between DBF4 and Cullin 1 variants might be interconnected and it would be really interesting to find out whether there is a similar switch between DRF1/DBF4B and DBF4 in mammals and study the link between the maternal to embryonic Cullin 1 switch and DRF1/DBF4B degradation. The way that XCDC6 degrade at the major wave of MBT is to the best of our knowledge unknown. However, the degradation of CDC6 before GVBD is UPS-dependent and is mediated by SCF^FBXW7 [84]. (The decrease in CDC6 protein level in prophase arrest is caused by a decrease in its translation and is UPS independent.)

Similarly to the above-described proteins involved in replication control, CDC25A is degraded in Xenopus embryos around the minor MBT by SCF^β−TRCP (Figs. 2, 3) [81, 82]. CDC25A is a mitotic progression inducer and is involved in the resumption of meiosis during mouse oocyte maturation [184]. In Drosophila, a steep decrease in CDC25A level in cycles 10–13 causes cell cycle lengthening [185]. This is partly caused by CHK1 inhibition, which in Xenopus mediates the degradation of DRF1 by SCF^β−TRCP [83]. In Drosophila, the inhibition of CHK1 is sufficient for the prevention of cell cycle lengthening, which is not true for Xenopus embryos [83, 186].

The necessity of UPS for EGA was also found in mammalian species, as the deactivation of UPS or its part delays the initiation of both the minor and major wave of EGA [5, 38, 46, 67, 77, 154]. However, specific proteins that need to be degraded have not yet been found in mammals. Higuchi et al. [154] suppose that the delay is caused by the delayed recruitment of Pol II into the embryo pronuclei. According to their results, the delayed initiation of DNA replication does not seem to cause the delay in major EGA initiation [154]. Together with the results of Collart et al. [168], Sun et al. [9], Farrell et al. [185] and others, this suggests that for the normal course of EGA and further development, the replication rate decrease around major EGA and consequent slowing of development speed is especially important.

Concluding remarks

In this review, we summarized current knowledge of the way proteins are processed during preimplantation development. The degradation of maternal proteins is slower than the degradation of maternal RNAs with a large impact on specificity, timing and likely also localization of the degradation. It seems that the most important period for normal preimplantation development in terms of protein degradation occurs already during oocyte maturation. Normal maternal protein degradation during proper preimplantation development is definitely important as well, however without correct protein processing during oocyte maturation, the embryos are not able to develop normally, even under standard conditions. Both autophagy and UPS are necessary for normal preimplantation development. However, the extent to which each of these pathways takes part in maternal protein degradation has not been determined yet.

Since there is limiting data available concerning mammals, the data were also based on a comparison with non-mammalian species and the timing of individual events related to maternal protein degradation in several species was compared. For this purpose, the preparation of a theoretical model that will facilitate the transfer of knowledge from one model organism to the others will be highly beneficial. For a proper understanding of protein processing during preimplantation development, it will be necessary to concentrate especially on the time period around the major wave of EGA. This period is not well described in mammals, and based on the results obtained with lower organisms, it seems to be to a large extent driven by protein degradation. During this period, it is likely that no general or extensive degradation will occur (such as during oocyte maturation or fertilization). We suppose there is a specific degradation of some groups of proteins or even of individual proteins. Identifying such proteins will however be a very challenging and time-consuming work, when we take into account the identification by Yang et al. [38] of just one protein whose degradation is necessary for the normal course of major EGA.

Compliance with ethical standards

Conflict of interest

The authors declare no competing interests.