Inhibition of SCF complexes during bovine oocyte maturation and preimplantation development leads to delayed development of embryos

Kinterova Veronika2,3,4,, Kanka Jiri3, Petruskova Veronika3,5, Toralova Tereza3

Abstract

The mechanism of maternal protein degradation during preimplantation development has not been clarified yet. It is thought that a lot of maternal proteins are degraded by ubiquitinproteasome system. In this study, we focused on the role of the SCF (Skp1-Cullin-F-box) complexes during early bovine embryogenesis. We inhibited it using MLN4924, an inhibitor of SCF complex ligases controlled by neddylation. Oocytes maturated in MLN4924 could be fertilized, but we found no cumulus cells expansion and a high number of polyspermy after in vitro fertilization. We also found a statistically significant deterioration of development after MLN4924 treatment. After treatment with MLN4924 from the 4-cell to late 8-cell stage, we found a statistically significant delay in its development, some of the treated embryos were however able to reach the blastocyst stage later. We found reduced levels of mRNA of EGA markers PAPOLA and U2AF1A, which can be related to this developmental delay. The cultivation with MLN4924 caused a significant increase in protein levels in MLN4924-treated oocytes and embryos, no such change was found in cumulus cells. To detect the proteins affected by MLN4924 treatment, we performed a western blot analysis of selected proteins (SMAD4, Ribosomal protein S6, Centromeric protein E, P27, NFKB inhibitor alpha, RNA binding motif protein 19). No statistically significant increase in protein level was detected in either treated embryos or oocytes. In summary, our study shows that SCF ligases are necessary for the correct maturation of oocytes, cumulus cells expansion, fertilization and early preimplantation development of cattle.

Summary sentence: SCF complexes are involved in normal oocyte maturation, polysperm defence and correct course of preimplantation development

Key words: ubiquitin – proteasome system, MLN4924, oocyte, cumulus cells, early development, SCF complexes

Introduction

The early embryonic development of mammals is controlled by maternal mRNA and proteins synthesized during oogenesis. These reserves are used until embryonic genome activation (EGA), a process that occurs at a species-specific developmental stage (the late 8cell stage in cattle [1], 4-cell stage in pigs, 2-cell stage in mice [2]). After fertilization, maternal mRNA and proteins are gradually eliminated [3]. The degradation of maternal mRNA is a gradual process, which peaks around the major wave of EGA [4], but the mechanism underlying the elimination of maternal proteins remains unknown. Two main mechanisms are suggested to be involved in maternal protein degradation – autophagy and the ubiquitin-proteasome system (UPS). Although previous studies suggest the essential participation of autophagy in early embryogenesis [5], UPS seems to play a crucial role in maternal protein degradation during preimplantation development. It has been proved that UPS plays an important role in polar body emission, the formation of pronuclei, meiosis resumption and cell cycle proliferation [6]. The E3 ubiquitin ligase RNF114 is essential for the activation of the NF-KB pathway during EGA [7], and after silencing the E3 ubiquitin ligase Rnf20, the majority of embryos arrest at the morula stage [8]. UPS-based protein degradation is managed by ubiquitin covalently attached to the targeted proteins by the cooperation of three enzymatic complexes: E1 ubiquitin – activating enzyme, E2 ubiquitin – conjugating enzyme and E3 ubiquitin ligases. The E3 enzyme, which binds the substrate, mediates the interaction between ubiquitin and the substrate protein [9]. E3 enzymes are responsible for substrate specificity. One of the most abundant families of E3 enzymes are SCF complexes (Skp1-Cullin-F box, reviewed in [10]). This complex consists of three invariant members: Cullin (Cul), Rbx1, Skp1 and one of the F-box proteins, which determines the substrate specificity [11]. SCF ligases are assumed to mediate up to 20% of proteasome-dependent degradation [12]. The SCF complex is activated by neddylation, posttranslational modification conjugating the small ubiquitin-like protein NEDD8 to Cul. Deneddylation is caused by the CAND1 (Cullin-associated and neddylation dissociated 1) protein which binds to Cul and needs to be dissociated to reactivate the SCF complex [13].
According to our previous results, SCF complex activity is necessary throughout the whole of preimplantation development. Further, the early activation of Cul1 and Skp1 mRNA expression indicates its necessity for the preparation of embryos for EGA [14]. Therefore, we decided to explore the role of SCF complexes in preimplantation development in more detail. We show here that inhibiting the activity of SCF ligases leads to an increase in protein level and consequently influences both oocyte maturation (including cumulus expansion) and early embryo development.

Materials and Methods

IVF and embryo culture

Unless otherwise indicated, the chemicals were purchased from Sigma (SigmaAldrich, St. Louis, MO) and plastic from Nunclon (Nunc, Roskilde, Denmark). Bovine embryos were obtained after the in vitro maturation of oocytes and their subsequent in vitro fertilization (IVF) and in vitro culture. Briefly, abattoir-derived ovaries from cows and heifers were collected and transported in thermos containers in sterile saline at about 33 °C. The cattle had been slaughtered (Jatky Rosovice spol, s.r.o.; Slaughterhouse Rosovice) for the public edible meat. Those ovaries were discarded without any utilization, hence, an ethics statement in our paper was not required. The follicles with a diameter between 5 and 9 mm were dissected with fine scissors and then punctured. The cumulus-oocyte complexes (COC) were evaluated and selected according to the morphology of the cumulus and subjected to in vitro maturation in TCM 199 (Earle’s salt) supplemented with 20 mM sodium pyruvate, 50 U/ml penicillin, 50 µg/ml streptomycin, 10 % oestrus cow serum (ECS), serum gonadotropin and chorionic gonadotropin (P. G. 600, 15 U/ml; Intervet, Boxmeer, Holland) without a paraffin overlay in four-well dishes under a humidified atmosphere for 24 h at 39 °C with 5 % CO2.
For IVF, the COC were washed four times in phosphate-buffered saline (PBS) and once in Tyrode’s albumin lactate pyruvate (TALP) fertilization medium, and transferred in groups of up to 30 to four-well dishes containing 250 µl TALP per well. The TALP medium contained 1.5 mg/ml bovine serum albumin (BSA), 30 µg/ml heparin, 0.25 mM sodium pyruvate, 10 mM lactate and 20 µM penicillamine. One straw with frozen semen from one bull previously tested in the IVF system was thawed in a 40 °C water bath, diluted with 2 ml TALP and centrifuged at 3500 g for 10 min. The spermatozoa were layered under 5 x 1 ml TALP. The supernatant with the motile spermatozoa was isolated after 1 h of swim-up at 39 °C [15]. Spermatozoa were counted in a heamocytometer and diluted in the appropriate volume of TALP to give a concentration of 2 x 106 spermatozoa/ml. A 250 µl aliquot of this suspension was added to each fertilization well to obtain a final concentration of 1 x 106 spermatozoa/ml. Plates were incubated under a humidified atmosphere with 5 % CO2 – 5 % O2 – 90 % N2 for 20 h at 39 °C.
At approximately 20 hour post fertilization (hpf) presumed zygotes were denuded by gentle pipetting and transferred in groups up to 25 to 25 µl of Menezo B2 medium (Veterinary Research Institute, Brno, Czech Republic) supplemented with 10 % ECS and cultured in a humidified atmosphere 5 % CO2 – 5 % O2 – 90 % N2 under liquid paraffin (Origio, Malov, Denmark). Germinal vesicle (GV) oocytes were fixed immediately after isolation or further cultivated. The dishes were then examined at 24 h post isolation and 34, 44, 72, 96, 120, 156 and 180 hpf, and metaphase II (MII) oocytes and two-cell (2c), four-cell (4c), early eight-cell (e8c), late eight-cell (L8c), morula, blastocyst and hatched blastocyst were collected at each time point respectively.
Cumulus cells were denuded by gentle pipetting of oocytes, subsequently 2x washed by PBS, centrifuged and frozen from a group of 25 oocytes to each tube.

MLN4924 treatment

To determine the optimal dose of MLN4924 for our experiment, several concentrations of MLN4924 were used (in detail in Supplementary material section). To inhibit the SCF complex, MLN4924 was added to the culture medium at concentration of 1µM from 4c to L8c stage (44 – 96 hpf). After MLN4924 treatment, the embryos were washed thoroughly and subsequently cultivated in Menezo B2 medium. Part of L8c stage embryos after MLN4924 treatment was washed with PBS, frozen and stored at -80 °C. Control embryos without treatment were collected at the same time interval as their treated counterparts from the same fertilization/cultivation group.
In the case of inhibition of the SCF complex before IVF, oocytes were cultivated in MPM medium with MLN4924 immediately after COC isolation, cultivated for 24 h, then washed with MPM medium and frozen or thoroughly washed with fertilization medium and in vitro fertilized.

Quantification of mRNA expression

The embryos were washed using PBS in groups of five and store dry and deep-frozen at – 80 °C until used. Poly (A) + mRNA was extracted from the pools of 5 embryos using a Dynabeads mRNA DIRECT Micro Kit (Invitrogen Dynal AS, Eugene, OR) according to the manufacturer’s instruction.
The expression of mRNA was measured by quantitative RT-PCR and the reaction was performed using OneStep RT – PCR kit (Qiagen, Hilden, Germany) with real-time detection using SybrGreen fluorescent dye. The reaction composition was Qiagen OneStep RT – PCR buffer (1x), dNTP Mix (400 µM of each), forward and reverse primers (both 400 µM; Table 1), SybrGreen (1 : 50 000 of 1000x stock solution; Invitrogen), RNasin Ribonuclease Inhibitor (Promega; 0.2 µl), Qiagen OneStep Enzyme Mix (0.5 µM), and template RNA. Reaction conditions were as follow: RT at 50 °C for 30 min, initial activation at 95 °C for 15 min, cycling: denaturation at 94 °C for 15 s, annealing at 60 °C for 20 s (Cul1), at 58 °C for 20 s (U2 small nuclear RNA auxiliary factor 1, U2AF1), at 55 °C for 20 s (Eukaryotic translation initiation factor 1A, eiF1A), at 57 °C for 20 s (Poly(A) polymerase alpha, PAPOLA) and extension at 72 °C for 30 s. The final extension step was held for 10 min at 72 °C. The real-time RT-PCRs were run in duplicate, with all samples in the same reaction. The experiments were carried out in a RotorGene 3000 (Corbett Research, Morthlake, Australia). Fluorescence data were acquired at 3 °C below the melting temperature to distinguish the possible primer dimers. The qRT-PCR data were determined using serial dilutions of fibroblasts RNA and the standard curve was created using the take-off points. The take-off points were calculated by Internal RotorGene software (Corbett Research) and defined as the cycle at which the second-derivative curve is at 20 % of the maximum rate of fluorescence and indicates the transition to the exponential phase (RotorGene 3000 operation manual; Corbett Research). The starting amount of corresponding RNA in analyzed samples was determined by appointing the take-off points to the curve. Products were verified by melting analysis and gel electrophoresis on 1.5 % agarose gel with ethidium bromide staining.

Experiment were repeated at least 3 times.

Western blotting

Unless otherwise indicated, chemicals were purchased from Sigma. Embryos and oocytes (20 per extract) were lysed with 6 µl of Millipore H2O and 2.5 µl of 4 x lithium dodecyl sulfate, sample buffer NP 0007 and 1 µl reduction buffer NP 0004 (Novex, Thermo Fisher Scientific, Prague, Czech Republic), boiled for 5 min and subjected to 4 – 12 % SDS-PAGE. Protein were transferred from gels to an Immobilon P membrane (Millipore Biosciences, Billerica, MA) using a semidry blotting system (Whatman Biometra GmbH, Hoettingen, Germany) for 28 min at 5 mA/cm2. The blocking of the membranes were performed in 5 % non-fat milk in TBS-Tween (TBS-T, 20 mM Tris, pH 7.4, 137 mM NaCl and 0.5% Tween 20) for SMAD4 (Mothers against decapentaplegic homolog 4), Protein 27 (P27), RBM19 (Probable RNAbinding protein 19), zygote arrest 1 (ZAR1), alpha-tubulin, in 1% non-fat milk in TBS-T for Ribosomal protein S6 (RPS6) and Centromeric protein E (CENPE) and in 5 % BSA in TBS-T for NFKB inhibitor alpha (IKBA) for 1 h. Membranes were incubated overnight with the following primary antibodies: rabbit anti-CENPE (Cell Signaling Technology 14977, Leiden, Netherlands) 1 : 500, rabbit anti- IKBA (Cell Signaling Technology 9242S, Leiden, Netherlands) 1 : 1000, rabbit anti-P27 (Abcam ab32034, Cambridge, UK) 1 : 1000, rabbit anti-RBM19 (Abcam ab122515, Cambridge, UK) 1 : 250, mouse anti-RPS6 (Santa Cruz Biotechnology SC-74459, Santa Cruz, TX) 1 : 1000, rabbit anti-SMAD4 (Proteintech 10231I-AP, Manchester, UK) 1 : 7000, rabbit anti-ZAR1 (Bioss Antibodies bs-13549R, Woburn, Massachusetts) 1 : 300 and rabbit anti-alpha-tubulin (Abcam ab52866, Cambridge, UK) 1: 2000. After washing in TBS-T, the membranes were incubated for 1 h with secondary antibody Peroxidase Anti-Rabbit Donkey (711-035-152, Jackson Immuno Research, Suffolk, UK) or Peroxidase Anti-mouse Donkey (715-035-151, Jackson Immuno Research, Suffolk, UK) both in 1 : 7500 dilution in 1 % non-fat milk/TBS-T, 5 % non-fat milk/TBS-T or in 5 %/TBS-T 1h at room temperature. Proteins were visualized by ECL (Amersham, GE Healthcare life science), films were scanned using a GS-800 calibrated densitometer (BioRad) and quantified using Image J software (http://rsbweb.nih.gov/ij/).

Total protein amount measurement – Pierce BCA Protein Assay

Embryos were lysed by RIPA buffer (composed of: 150 mM NaCl, 5 mM EDTA pH=8, 50 mM Tris HCl pH=7.4, 0.5 % NP-40, 1 % sodium deoxycholate, 1 % Triton X-100, 0.1 % SDS). The protein amount was measured using Pierce BCA Protein Assay Kit (ThermoFisher Scientific, Prague, Czech Republic) according to manufacturer’s instruction [16]. Both standard dilution series and samples were prepared in duplicated and incubated for 1.5 h at 37 °C. Optical density was measured at 562 nm using Synergy HTX multimode reader (BioTek, Swindon, UK).

Polyspermy detection – immunofluorescence

Embryos were fixed in 4 % paraformaldehyde for 50 min at 4 °C. Fixed embryos were processed immediately or stored in PBS for up to 3 weeks at 4 °C. After washing in PBS, the embryos were incubated in 0.5 % (v/v) TritonX-100 for 15 min. All subsequent steps were done in PBS supplemented with 0.3 % (w/v) BSA and 0.05 % (w/v) saponin (PBS/BSA/sap). Embryos were blocked with 2 % normal goat serum (NGS; Millipore Biosciences; St. Charles, MO) for 1 h and incubated with mouse anti-lamin (Sigma-Aldrich SAB4200236, St. Louis, MO) 1 : 150 in PBS/BSA/sap overnight at 4 °C. After thorough washing, the embryos were incubated with goat anti-mouse conjugated with Alexa Fluor 594 1:350 (Invitrogen, Eugene, OR) in PBA/BSA/sap for 1 h at room temperature in the dark. After washing, the nuclei were stained and embryos were mounted in Vectashield HardSet Mounting Medium with DAPI (4′,6-Diamidine-2′-phenylindole dihydrochloride, Vector Laboratories, Peterborough, UK). Controls of immunostaining specificity were carried out by omitting the primary antibody or using another species-specific secondary antibody conjugate. The samples were examined using Leica TCS SP5 (Leica Microsystems AG, Wetzlar, Germany). The images were processed using Image J software.

Statistical analysis

The data were analyzed using SigmaStat 3.0 software (Jandel Scientific, San Rafael, CA). The data were tested for normality and then paired t-test, Mann-Whitney Rank Sum test or ttest were used. P ≤ 0.05 was considered statistically significant.

Results

Development of embryos after treatment with MLN4924

To investigate the role of SCF complexes during bovine preimplantation development, we used the selective inhibitor of NEDD8-activating enzyme (NAE) MLN4924. NAE is an activator of SCF ligases, and its inhibition prevents the formation of SCF complexes. We did not use the microinjection of cul1 dsRNA, since CUL1 protein is very stable during bovine preimplantation development. After the microinjection of Cul1 dsRNA into zygotes, the Cul1 mRNA was significantly decreased (to 25.31 % compared to the controls), nevertheless the protein level remained unchanged (S1 Fig. and S2 Fig.). At first, embryos were treated with several concentrations of MLN4924 to determine an optimal concentration for our experiment (S3 Fig.) and 1 µM MLN4924 was selected as the most appropriate concentration. The development of embryos treated with MLN4924 from the 4c to late 8c stage was significantly delayed. Only 26.6 ± 2 % (mean ± S.D.) of MLN4924-treated embryos were able to reach the morula stage compared to 57.2 ± 6.98 (p=0.025) of control embryos. At 168 hours post fertilization (hpf) 22.381 ± 10.852 % (mean±S.D) of control embryos reached the blastocyst stage (the number of 4-cell stage embryos was considered to be 100 %). However, only 3.889 ± 4.843 % of MLN4924-treated embryos reached the blastocyst stage at this time (p=0.01). On the other hand, some of the embryos treated with MLN4924 were able to reach the blastocyst stage later (Fig. 1). Under the microscope, we found no difference in blastocyst quality between them.
To determine whether the maternally inherited CUL1 protein is needed for fertilization and further preimplantation development, we treated the GV oocytes for 24 h with MLN4924. We have found no expansion of cumulus cells after the MLN4924 treatment of GV oocytes. The condition of cumulus cells remained the same during maturation. Nevertheless, it was possible to fertilize the oocytes. Cumulus cells closely surrounded embryos even after fertilization, in contrast to control embryos gradually losing the surrounding cumulus cells. (Fig. 2). After in vitro fertilization, the average ratio of normally fertilized embryos in MLN4924-matured oocytes was significantly reduced to 22.00 ± 16.28 % (mean ± S.D.) compared to 71.57 ± 14.39 % of normally fertilized embryos in the control group (p=0.014). In some MLN4924-treated oocytes, a high number of sperms were found attached to the zona pellucida, laying in the subzonal space or entering the oocyte. The proportion of unfertilized MLN4924-treated oocytes was significantly higher (26.2 % ± 14.70) compared to the control (11.91 % ± 14.88) (p<0.05). We observed a significantly higher rate of polysperm embryos in MLN4924-treated oocytes (51.798 ± 20.721 %) compared to the control group (16.52 ± 5.13 %; p=0.016) (Fig. 3A). Lamin staining was used for better visualization of pronuclei (Fig. 3B). The development of diploid MLN4924-treated embryos was significantly deteriorated from 8c onwards (8c – p=0.005; morula – p=0.015; blastocyst p<0.05) and only 8.67 % ± 4.59 (mean±S.D) of MLN4924-treated embryos, in compared to 20.99 % ± 7.69 (mean±S.D) of control embryos, developed to the blastocyst stage (the number of 2c-stage embryos was to be considered 100 %; Fig. 3C).

Changes in protein level after MLN4924 treatment

The treatment of embryos using 1μM MLN4924 from the GV stage to MII stage oocyte and from 4-cell stage to late 8-cell stage embryos caused a statistically significant increase in protein level (p<0.05 in both cases). It rose to 161.43 ± 122.28 (mean±S.D.) in MLN4924treated oocytes and to 150.17 % ± 30.71 (mean±S.D) in MLN4924-treated embryos compared to controls (Fig. 4Aand Fig. B). In contrast, the protein level was not changed in cumulus cells collected after MLN4924 treatment from GV- to MII stage oocytes (p>0.05) (Fig. 4C).
To determine which proteins were affected after the MLN4924 treatment of oocytes and embryos, we performed a western blot analysis of selected proteins. The proteins were selected as SCF complex substrates or based on their expression level during preimplantation development. SMAD4, RPS6, CENPE, P27, IKBA, ZAR1 and RBM19 were tested. Alpha-tubulin was used as the housekeeper gene. No statistically significant increase in protein level was detected in either treated embryos or oocytes (p>0.05). In embryos two bands after RBM19 staining were detected (130 and 150 kDa). The presence of the higher band is likely caused by phosphorylation or interaction with SUMO2. In MLN4924-treated oocytes, a statistically significant decrease was found in SMAD4 protein level compared to controls (p<0.001; 11.52 % ± 3.06; protein level in controls was considered 100 %, Fig. 5). No statistically significant difference was found in α-tubulin levels (p>0.05).

Delay in EGA initiation after MLN4924 treatment

We monitored the mRNA expression of three EGA markers: eukaryotic translation initiation factor 1A (eiF1A), U2 auxiliary splicing factor (U2AF1) and polyadenylate polymerase (PAPOLA) [17] after the MLN4924 treatment of embryos from 4c to L8c. The level of expression of U2AF1 and PAPOLA mRNA was significantly lower in MLN4924-treated embryos than in controls (U2AF1: p=0.003; 50.264 % ± 21.097; PAPOLA: p<0.001; 47.386 % ± 11.806; mean±S.D; the mean of mRNA expression level of controls was considered 100%). The level of eiF1A mRNA expression was not significantly decreased (p>0.05) (Fig. 6).

Discussion

The expression profiles of SCF complex members, the existence of two Cul1 variants, the activity of SCF complex during whole preimplantation development and the currently available data concerning protein degradation during early embryogenesis suggest the potential importance of SCF complexes during early embryonic development [14, 18–22]. To experimentally verify this hypothesis, we used the NAE inhibitor MLN4924. MLN4924 (also known as Pevonedistat) is an inhibitor of SCF ligases controlled by neddylation, primarily Cul1-based SCF ligases or, on a smaller scale, Cul2-, Cul3-, Cul4A/B- or Cul5- based SCFligases. It was developed as potent anti-cancer drug and to date, most studies with MLN4924 are focused on cancer cells [23]. The first option we considered for the silencing of a gene or connected enzyme in preimplantation embryos was the microinjection of dsRNA into zygotes. This method is well established in early mammalian embryos and frequently used [24–28]. However, the CUL1 protein is highly stable and remains present at a statistically unchanged level even after the silencing of Cul1 mRNA (S1 Fig. and S2 Fig.). Thus MLN4924 treatment was chosen instead.
At first we wanted to know whether oocytes matured with deactivated SCF complexes can be fertilized and establish viable embryos. The oocytes were matured in MLN4924 from the GV to MII stage, and even during this period we noticed a change in cumulus cells morphology. Whilst in controls the cumulus cells expanded and gradually dropped from the zygotes after fertilization, no such change was found in MLN4924-treated oocytes. The role of UPS in the formation of the expanded cumulus is still poorly understood. Yi et al. [29] have shown that after the treatment of GV oocytes with proteasome inhibitor MG132 for 22 h, no oocytes reached the MII stage. In comparison to other proteasome pathway inhibitors such as MG132, which completely block proteasome activity, MLN4924 specifically blocks the activation of SCF complexes. This is probably why almost 74 % of MLN4924-treated oocytes could be fertilized, even though only 40.3 % zygotes were diploid in comparison to 80 % of diploid controls. We assume that the higher polyspermy rate may be due to altered insufficient CG exocytosis. CG exocytosis after fertilization is involved in the ZP-mediated polyspermy blocking and its absence due to inhibition of deubiquinating enzyme (DUB) ubiquitin Cterminal hydrolase L1 (UCHL1) causes increased rates of polyspermy [30]. This suggests that the degradation of proteins by SCF ligases is necessary for the correct course of antipolyspermy defense, in which it likely cooperates with UCHL1 or maybe also other DUBs. Further, we showed that SCF ligases are an essential part of UPS that is involved in cumulus cell expansion. Yi et al. [29] showed that the expansion of cumulus cells is blocked and cumulus cells are firmly attached to oocytes even in the later stages of oocyte maturation in MG132-treated porcine oocytes. This suggests that SCF complexes play an important role in the expansion of cumulus cells. Interestingly, even though the cumulus cells are definitely influenced by the UPS inhibition, we found no change in the total protein level in cumulus cells after MLN4924 treatment compared to controls (Fig. 4C).
When we monitored the developmental competence of embryos arising from MLN4924treated oocytes, we found a statistically significant deterioration of development in comparison to controls. Interestingly, the most statistically significant difference was found at the 8c stage (p=0.005) and the strength of the significance decreased in further stages (morula p=0.016; blastocyst p=0.048). Similarly, after the MLN4924 treatment of embryos from 4c to L8c, we found a statistically significant difference between the number of treated embryos and controls at the morula stage (p=0.025) and blastocyst stage at 168 hpf (p=0.01). However, despite there being a clear difference at the blastocyst stage at 192 hpf, it was not statistically significant (p>0.05) (Fig. 1). This suggests that the degradation of proteins by SCF complexes during oocyte maturation and the early stages of embryogenesis was needed for normal preimplantation development, nevertheless some embryos after MLN4924 treatment are able to overcome this handicap and develop into blastocysts 24 hours later. We suppose that the embryos are temporarily arrested in their development at the 8c stage and resume their growth a few hours after the transfer of embryos to the pure culture medium. Since the deterioration of developmental competence is more distinct after the treatment of GV-MII oocytes than 4cL8c embryos, normal activity of the SCF complex is apparently more important during oocyte maturation than during the early stages of preimplantation development. The delay in the development of embryos may be related to reduced levels of mRNA of EGA markers PAPOLA and U2AF1A. The level of another EGA marker eiF1A was not significantly changed (Fig. 6). The genes involved in protein ubiquitination are activated at 8c in bovines [31], i.e. even before the major wave of EGA, and this also applies to two invariant members of the SCF complex, Cul1 and Rbx1 [14]. Hence embryonic UPS being responsible for protein degradation is probably necessary for the normal course of EGA. The involvement of proteolysis during EGA was discussed in Stitzel and Seydoux [32] and Liu et al. [33] and the delay in EGA initiation was also found in murine preimplantation embryos treated with MG132 independently of their developmental competence [6].Our results points to delayed EGA initiation and suggest the importance of protein degradation through the SCF complex. However since the initiation of eiF1A was not altered in our study in contrast to Shin et al. [6], it seems that for the normal course of EGA the cooperation of multiple parts of the UPS is necessary. The critical stage for embryos arising from MLN4924-treated oocytes is just the EGA stage, and the deterioration of development is also obvious in blastocysts. It is very interesting that in embryos that were treated with MLN4924 during the EGA period, the developmental deterioration stops being significant in the late blastocyst stage. This suggests that the preparation for EGA and post-EGA development is already going on during oocyte maturation and the early stages of embryogenesis.
Moreover, we have found a statistically significant increase in total protein level in both treated oocytes and embryos (Fig. 4A and Fig. B) in contrast to MLN4924-treated cumulus cells (Fig. 4C). This shows that as a consequence of SCF complex inhibition, some proteins cannot be degraded and are hoarded. We wanted to know which proteins are affected by the SCF complex inhibition. We tested several SCF complex substrates (SMAD4, RPS6, CENPE, IKBA, P27) [34–38] and other proteins based on their function or expression during preimplantation development (RBM19, ZAR1) [39–41] using western blot analysis. However, we did not find an increase in protein level in any of these proteins after the deactivation of SCF complexes (Fig. 5). An accumulation of P27 was found after the MLN4924 treatment of cell cultures [42, 43], and it is likely that the accumulation of some of the other proteins mentioned above would be found if it was tested in cell cultures in the same way as after SCF complex silencing [34, 44]. After the inhibition of E3 ligase RNF114 in murine preimplantation embryos, the accumulation of only one protein (TGF-beta activated kinase 1, TAB1) was found, even though the authors tested over 9000 proteins [7]. This suggests that the degradation of proteins during oocyte maturation and preimplantation development is subject to strict rules. In somatic cells, mRNAs and proteins can be degraded and synthesized again without limit. In preimplantation embryos, the maternal stores of mRNAs and proteins are stored at least until the EGA stage (late 8c in bovines) and thus those stores need to be conserved. Thus, we suppose that the SCF complexes might be involved in the degradation of the studied proteins even in preimplantation embryos, however the degradation takes place at a specific time point and/or specific location, similarly to the existence of translational hotspots during oocyte maturation [45]. It is known that the localization of proteins is to some extent driven by their degradation, as proteins are only degraded at a certain location [46]. Interestingly, we found a statistically significant decrease in SMAD4 protein level in MLN4924-treated MII stage oocytes in comparison to controls. We suppose that the decrease of SMAD4 protein amount may be related to atypical maturation of the treated oocytes and cumulus cells as the knockout of SMAD4 gene is connected to cumulus cells defects in mice [47].In conclusion, we have shown that SCF ligases are necessary for the normal maturation of oocytes, expansion of their cumulus cells and normal preimplantation development of embryos. Although we found an increase in protein level in both treated oocytes and embryos, we have not found a specific affected protein. To explore which proteins are degraded by SCF complexes during preimplantation development, we plan to subject the treated oocytes and embryos to mass spectrometry analysis. This is a big challenge, since bovine preimplantation embryos are quite a scarce material, and mass spectrometry is very demanding in terms of the amount of material. However, it is probably also the only way to find the SCF complex substrates in oocytes and preimplantation embryos, in which, as our results suggest, protein processing is strictly driven to avoid the degradation of essential maternal stores. This indicates that protein degradation by SCF complexes is an essential process in proper preimplantation development. Incomplete protein degradation and their subsequent higher content leads to developmental delay and to a decrease in the mRNA level of some EGA markers. This developmental delay persists until the early blastocyst stage. Nevertheless, at least some of these embryos are able to overcome this handicap and develop from a morula to a blastocyst about 24 hours later. Shin et al. [6], Stitzel and Seydoux [32] and Liu et al. [33] suggest that protein degradation is crucial for EGA, the initiation of which is delayed after UPS inhibition. Our results provide a new piece of the puzzle of the involvement of the UPS in EGA startup, and show the necessity of protein degradation by SCF ligases at this stage, even though other parts of the UPS must also be involved.

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