The degradation of maternal proteins is one of the most important events during early development, and it is presumed to be essential for embryonic genome activation (EGA), but the precise mechanism is still not known. It is thought that a large proportion of the degradation of maternal proteins is mediated by the ubiquitin-proteolytic system. In this study we focused on the expression of the Skp1-Cullin1-F-box (SCF) complex, a modular RING-type E3 ubiquitin-ligase, during bovine preimplantation development. The complex consists of three invariable components--Cul1, Skp1, Rbx1 and F-box protein, which determines the substrate specificity. The protein level and mRNA expression of all three invariable members were determined. Cul1 and Skp1 mRNA synthesis was activated at early embryonic stages, at the 4c and early 8c stage, respectively, which suggests that these transcripts are necessary for preparing the embryo for EGA. CUL1 protein level increased from MII to the morula stage, with a significant difference between MII and L8c, and between MII and the morula. The CUL1 protein was localized primarily to nuclei and to a lesser extent to the cytoplasm, with a lower signal in the inner cell mass (ICM) compared to the trophectoderm (TE) at the blastocyst stage. The level of SKP1 protein significantly increased from MII oocytes to 4c embryos, but then significantly decreased again. The localization of the SKP1 protein was analysed throughout the cell and similarly to CUL1 at the blastocyst stage, the staining was less intensive in the ICM. There were no statistical differences in RBX1 protein level and localization. The active SCF-complex, which is determined by the interaction of Cul1 and Skp1, was found throughout the whole embryo during preimplantation development, but there was a difference at the blastocyst stage, which exhibits a much stronger signal in the TE than in the ICM. These results suggest that all these genes could play an important role during preimplantation development. This paper reveals comprehensive expression profile, the basic but important knowledge necessary for further studying.

Department of Veterinary Sciences Czech University of Life Sciences Prague Prague Czech Republic

Faculty of Science Charles University Prague Prague Czech Republic

Laboratory of Developmental Biology Institute of Animal Physiology and Genetics Academy of Science of Czech Republic v v i Libechov Czech Republic

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Yokoi H, Natsuyama S, Iwai M, Noda Y, Mori T, Mori KJ, et al. Non-radioisotopic quantitative RT-PCR to detect changes in mRNA levels during early mouse embryo development. Biochem Biophys Res Commun. 1993;195:769–75. PubMed

Svarcova O, Laurincik J, Avery B, Mlyncek M, Niemann H, Maddox-Hyttel P. Nucleolar development and allocation of key nucleolar proteins require de novo transcription in bovine embryos. Mol Reprod Dev. 2007;74:1428–35. PubMed

Ohsugi M, Zheng P, Baibakov B, Li L, Dean J. Maternally Derived FILIA-MATER Complex Localizes Asymmetrically in Cleavage-Stage Mouse Embryos. Development. 2008;135:259–69. PubMed

Toralová T, Benešová V, Kepková KV, Vodička P, Šušor A, Kaňka J. Bovine preimplantation embryos with silenced nucleophosmin mRNA are able to develop until the blastocyst stage. Reprod Camb Engl. 2012;144:349–59. PubMed

Suzumori N, Burns KH, Yan W, Matzuk MM. RFPL4 interacts with oocyte proteins of the ubiquitin-proteasome degradation pathway. Proc Natl Acad Sci U S A. 2003;100:550–5. PubMed PMC

DeRenzo C, Seydoux G. A clean start: degradation of maternal proteins at the oocyte-to-embryo transition. Trends Cell Biol. 2004;14:420–6. PubMed

Mtango NR, Latham KE. Ubiquitin proteasome pathway gene expression varies in rhesus monkey oocytes and embryos of different developmental potential. Physiol Genomics. 2007;31:1–14. PubMed

Verlhac M-H, Terret M-E, Pintard L. Control of the oocyte-to-embryo transition by the ubiquitin-proteolytic system in mouse and C. elegans. Curr Opin Cell Biol. 2010;22:758–63. 10.1016/j.ceb.2010.09.003 PubMed DOI

Glickman MH, Ciechanover A. The ubiquitin-proteasome proteolytic pathway: destruction for the sake of construction. Physiol Rev. 2002;82:373–428. PubMed

Soucy TA, Smith PG, Milhollen MA, Berger AJ, Gavin JM, Adhikari S, et al. An inhibitor of NEDD8-activating enzyme as a new approach to treat cancer. Nature. 2009;458:732–6. 10.1038/nature07884 PubMed DOI

Bai C, Sen P, Hofmann K, Ma L, Goebl M, Harper JW, et al. SKP1 connects cell cycle regulators to the ubiquitin proteolysis machinery through a novel motif, the F-box. Cell.1996;86:263–74. PubMed

Skowyra D, Craig KL, Tyers M, Elledge SJ, Harper JW. F-box proteins are receptors that recruit phosphorylated substrates to the SCF ubiquitin-ligase complex. Cell. 1997;91:209–19. PubMed

Zheng J, Yang X, Harrell JM, Ryzhikov S, Shim EH, Lykke-Andersen K, et al. CAND1 binds to unneddylated CUL1 and regulates the formation of SCF ubiquitin E3 ligase complex. Mol Cell. 2002;10:1519–26. PubMed

Latres E, Chiarle R, Schulman BA, Pavletich NP, Pellicer A, Inghirami G, et al. Role of the F-box protein Skp2 in lymphomagenesis. Proc Natl Acad Sci U S A. 2001;98:2515–20. PubMed PMC

Li H-H, Kedar V, Zhang C, McDonough H, Arya R, Wang D-Z, et al. Atrogin-1/muscle atrophy F-box inhibits calcineurin-dependent cardiac hypertrophy by participating in an SCF ubiquitin ligase complex. J Clin Invest. 2004;11:1058–71. PubMed PMC

Liu J, Furukawa M, Matsumoto T, Xiong Y. NEDD8 modification of CUL1 dissociates p120(CAND1), an inhibitor of CUL1-SKP1 binding and SCF ligases. Mol Cell. 2002;10:1511–8. PubMed

Zheng N, Schulman BA, Song L, Miller JJ, Jeffrey PD, Wang P, et al. Structure of the Cul1-Rbx1-Skp1-F boxSkp2 SCF ubiquitin ligase complex. Nature. 2002;416:703–9. PubMed

Pennetier S, Uzbekova S, Guyader-Joly C, Humblot P, Mermillod P, Dalbiès-Tran R. Genes preferentially expressed in bovine oocytes revealed by subtractive and suppressive hybridization. Biol Reprod. 2005;73:713–20. PubMed

Skaar JR, Pagano M. Control of cell growth by the SCF and APC/C ubiquitin ligases. Curr Opin Cell Biol. 2009;21:816–24. 10.1016/j.ceb.2009.08.004 PubMed DOI PMC

Silverman JS, Skaar JR, Pagano M. SCF ubiquitin ligases in the maintenance of genome stability. Trends Biochem Sci. 2012;37:66–73. 10.1016/j.tibs.2011.10.004 PubMed DOI PMC

Dealy MJ, Nguyen KV, Lo J, Gstaiger M, Krek W, Elson D, et al. Loss of Cul1 results in early embryonic lethality and dysregulation of cyclin E. Nat Genet. 1999;23:245–8. PubMed

Wang Y, Penfold S, Tang X, Hattori N, Riley P, Harper JW, et al. Deletion of the Cul1 gene in mice causes arrest in early embryogenesis and accumulation of cyclin E. Curr Biol CB. 1999;9:1191–4. PubMed

Hwang K-C, Cui X-S, Park S-P, Shin M-R, Park S-Y, Kim E-Y, et al. Identification of differentially regulated genes in bovine blastocysts using an annealing control primer system. Mol Reprod Dev. 2004;69:43–51. PubMed

Lee YL, Lee KF, Xu JS, Kwok KL, Luk JM, Lee WM, et al. Embryotrophic factor-3 from human oviductal cells affects the messenger RNA expression of mouse blastocyst. Biol Reprod. 2003;68:375–82. PubMed

Zhang Q, Chen Q, Lu X, Zhou Z, Zhang H, Lin H-Y, et al. CUL1 promotes trophoblast cell invasion at the maternal-fetal interface. Cell Death Dis. 2013;4:e502 10.1038/cddis.2013.1 PubMed DOI PMC

Kepkova KV, Vodicka P, Toralova T, Lopatarova M, Cech S, Dolezel R, et al. Transcriptomic analysis of in vivo and in vitro produced bovine embryos revealed a developmental change in cullin 1 expression during maternal-to-embryonic transition. Theriogenology. 2011;75:1582–95. 10.1016/j.theriogenology.2010.12.019 PubMed DOI

Galan JM, Wiederkehr A, Seol JH, Haguenauer-Tsapis R, Deshaies RJ, Riezman H, et al. Skp1p and the F-box protein Rcy1p form a non-SCF complex involved in recycling of the SNARE Snc1p in yeast. Mol Cell Biol. 2001;21:3105–17. PubMed PMC

Mandel SA, Fishman-Jacob T, Youdim MBH. Targeting SKP1, an ubiquitin E3 ligase component found decreased in sporadic Parkinson’s disease. Neurodegener Dis. 2012;10:220–3. 10.1159/000333223 PubMed DOI

Rhodes SL, Fitzmaurice AG, Cockburn M, Bronstein JM, Sinsheimer JS, Ritz B. Pesticides that inhibit the ubiquitin-proteasome system: effect measure modification by genetic variation in SKP1 in Parkinson׳s disease. Environ Res. 2013;126:1–8. 10.1016/j.envres.2013.08.001 PubMed DOI PMC

Piva R, Liu J, Chiarle R, Podda A, Pagano M, Inghirami G. In vivo interference with Skp1 function leads to genetic instability and neoplastic transformation. Mol Cell Biol. 2002;22:8375–87. PubMed PMC

Ohta T, Michel JJ, Xiong Y. Association with cullin partners protects ROC proteins from proteasome-dependent degradation. Oncogene. 1999;18:6758–66. PubMed

Ohta T, Michel JJ, Schottelius AJ, Xiong Y. ROC1, a homolog of APC11, represents a family of cullin partners with an associated ubiquitin ligase activity. Mol Cell. 1999;3:535–41. PubMed

Morimoto M, Nishida T, Nagayama Y, Yasuda H. Nedd8-modification of Cul1 is promoted by Roc1 as a Nedd8-E3 ligase and regulates its stability. Biochem Biophys Res Commun. 2003;301:392–8. PubMed

Sasagawa Y, Urano T, Kohara Y, Takahashi H, Higashitani A. Caenorhabditis elegans RBX1 is essential for meiosis, mitotic chromosomal condensation and segregation, and cytokinesis. Genes Cells Devoted Mol Cell Mech. 2003;8:857–72. PubMed

Moore R, Boyd L. Analysis of RING finger genes required for embryogenesis in C. elegans. Genes N Y N 2000. 2004;38:1–12. PubMed

Tan M, Davis SW, Saunders TL, Zhu Y, Sun Y. RBX1/ROC1 disruption results in early embryonic lethality due to proliferation failure, partially rescued by simultaneous loss of p27. Proc Natl Acad Sci U S A. 2009;106:6203–8. 10.1073/pnas.0812425106 PubMed DOI PMC

Jia L, Bickel JS, Wu J, Morgan MA, Li H, Yang J, et al. RBX1 (RING box protein 1) E3 ubiquitin ligase is required for genomic integrity by modulating DNA replication licensing proteins. J Biol Chem. 2011;286:3379–86. 10.1074/jbc.M110.188425 PubMed DOI PMC

Gutierrez GJ, Vögtlin A, Castro A, Ferby I, Salvagiotto G, Ronai Z, et al. Meiotic regulation of the CDK activator RINGO/Speedy by ubiquitin-proteasome-mediated processing and degradation. Nat Cell Biol. 2006;8:1084–94. PubMed

Marangos P, Verschuren EW, Chen R, Jackson PK, Carroll J. Prophase I arrest and progression to metaphase I in mouse oocytes are controlled by Emi1-dependent regulation of APC(Cdh1). J Cell Biol. 2007;176:65–75. PubMed PMC

Zhou L, Yang Y, Zhang J, Guo X, Bi Y, Li X, et al. The role of RING box protein 1 in mouse oocyte meiotic maturation. PloS One. 2013;8:e68964 10.1371/journal.pone.0068964 PubMed DOI PMC

Jia L, Soengas MS, Sun Y. ROC1/RBX1 E3 ubiquitin ligase silencing suppresses tumor cell growth via sequential induction of G2-M arrest, apoptosis, and senescence. Cancer Res. 2009;69:4974–82. 10.1158/0008-5472.CAN-08-4671 PubMed DOI PMC

Roovers EF, Rosenkranz D, Mahdipour M, Han C-T, He N, Chuva de Sousa Lopes SM, et al. Piwi proteins and piRNAs in mammalian oocytes and early embryos. Cell Rep. 2015;10:2069–82. 10.1016/j.celrep.2015.02.062 PubMed DOI

Pavlok A, Lucas-Hahn A, Niemann H. Fertilization and developmental competence of bovine oocytes derived from different categories of antral follicles. Mol Reprod Dev. 1992;31:63–7. PubMed

Kanka J, Kepková K, Nemcová L. Gene expression during minor genome activation in preimplantation bovine development. Theriogenology. 2009;72(4):572–83. 10.1016/j.theriogenology.2009.04.014 PubMed DOI

Muzzopappa M, Wappner P. Multiple roles of the F-box protein Slimb in Drosophila egg chamber development. Dev Camb Engl. 2005;132:2561–71. PubMed

Doronkin S, Djagaeva I, Beckendorf SK. The COP9 signalosome promotes degradation of Cyclin E during early Drosophila oogenesis. Dev Cell. 2003;4:699–710. PubMed

Ohlmeyer JT, Schüpbach T. Encore facilitates SCF-Ubiquitin-proteasome-dependent proteolysis during Drosophila oogenesis. Dev Camb Engl. 2003;130:6339–49. PubMed

Shcherbata HR, Althauser C, Findley SD, Ruohola-Baker H. The mitotic-to-endocycle switch in Drosophila follicle cells is executed by Notch-dependent regulation of G1/S, G2/M and M/G1 cell-cycle transitions. Dev Camb Engl. 2004;131:3169–81. PubMed

Bader M, Arama E, Steller H. A novel F-box protein is required for caspase activation during cellular remodeling in Drosophila. Dev Camb Engl. 2010;137:1679–88. PubMed PMC

Guardavaccaro D, Kudo Y, Boulaire J, Barchi M, Busino L, Donzelli M, et al. Control of meiotic and mitotic progression by the F box protein beta-Trcp1 in vivo. Dev Cell. 2003;4:799–812. PubMed

Toralová T, Susor A, Nemcová L, Kepková K, Kanka J. Silencing CENPF in bovine preimplantation embryo induces arrest at 8-cell stage. Reprod Camb Engl. 2009;138:783–91. PubMed

Falco G, Lee S-L, Stanghellini I, Bassey UC, Hamatani T, Ko MSH. Zscan4: a novel gene expressed exclusively in late 2-cell embryos and embryonic stem cells. Dev Biol. 2007;307:539–50. PubMed PMC

Salilew-Wondim D, Hölker M, Rings F, Phatsara C, Mohammadi-Sangcheshmeh A, Tholen E, et al. Depletion of BIRC6 leads to retarded bovine early embryonic development and blastocyst formation in vitro. Reprod Fertil Dev. 2010;22:564–79. 10.1071/RD09112 PubMed DOI

Graf A, Krebs S, Heininen-Brown M, Zakhartchenko V, Blum H, Wolf E. Genome activation in bovine embryos: review of the literature and new insights from RNA sequencing experiments. Anim Reprod Sci. 2014;149:46–58. 10.1016/j.anireprosci.2014.05.016 PubMed DOI

Filippov V, Filippova M, Sehnal F, Gill SS. Temporal and spatial expression of the cell-cycle regulator cul-1 in Drosophila and its stimulation by radiation-induced apoptosis. J Exp Biol. 2000;203:2747–56. PubMed

Davis W, Schultz RM. Developmental change in TATA-box utilization during preimplantation mouse development. Dev Biol. 2000;218:275–83. PubMed

Karabinova P, Kubelka M, Susor A. Proteasomal degradation of ubiquitinated proteins in oocyte meiosis and fertilization in mammals. Cell Tissue Res. 2011;346:1–9. 10.1007/s00441-011-1235-1 PubMed DOI

Sutovsky P, Motlik J, Neuber E, Pavlok A, Schatten G, Palecek J, et al. Accumulation of the proteolytic marker peptide ubiquitin in the trophoblast of mammalian blastocysts. Cloning Stem Cells. 2001;3:157–61. PubMed

Liu J, Furukawa M, Matsumoto T, Xiong Y. NEDD8 modification of CUL1 dissociates p120(CAND1), an inhibitor of CUL1-SKP1 binding and SCF ligases. Mol Cell. 2002;10:1511–8. PubMed

Zhang H, Kobayashi R, Galaktionov K, Beach D. pl9skp1 and p45skp2 are essential elements of the cyclin A-CDK2 S phase kinase. Cell. 1995;82:915–25. PubMed

Kaplan KB, Hyman AA, Sorger PK. Regulating the yeast kinetochore by ubiquitin-dependent degradation and Skp1p-mediated phosphorylation. Cell. 1997;91:491–500. PubMed

Piva R, Liu J, Chiarle R, Podda A, Pagano M, Inghirami G. In vivo interference with Skp1 function leads to genetic instability and neoplastic transformation. Mol Cell Biol. 2002;22:8375–87. PubMed PMC

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