Knock out of Specific Maternal Vitellogenins in Zebrafish (Danio Rerio) Evokes Vital Changes in Egg Proteomic Profiles that resemble the Phenotype of Poor Quality Eggs

Abstract

Background We previously reported the results of CRISPR/Cas9 knock-out (KO) of type-I and type-III vitellogenins (Vtgs) in zebrafish, which provided the first experimental evidence of essentiality and disparate functioning of specific types of Vtg at different times during vertebrate development. However, the lack of knowledge on specific contributions of different types of Vtg to molecular functions related to major developmental processes remained to be investigated. The present study employed liquid chromatography and tandem mass spectrometry (LC-MS/MS) to observe proteomic profiles of zebrafish eggs lacking three type-I Vtgs (Vtg 1, 4 and 5) simultaneously (vtg1-KO), or lacking type III Vtg (vtg3) only (vtg3-KO), as compared to those of wild type (Wt) eggs. Obtained spectra were searched against a zebrafish proteome database and identified proteins were quantified based on normalized spectral counts.Results The vtg-KO in zebrafish revealed impaired proteomes of 1 hour post fertilization (hpf) zebrafish eggs which were highly resembled the proteomic phenotype of poor quality eggs of the same developmental stage reported in our prior studies. Proteomic profiles of vtg-KO eggs and perturbations in abundances of hundreds of proteins revealed unique, noncompensable contributions of multiple Vtgs in protein homeostasis (synthesis and degradation) and in energy homeostasis even after zygotic genome activation. Increase in endoplasmic reticulum stress, Redox/Detox activities, glycolysis/gluconeogenesis, enrichment in cellular proliferation and human neurodegenerative disease related mechanisms in both vtg1- and vtg3-KO eggs point to severe molecular changes and consecutive dysfunctions in the abovementioned cellular activities. Distinctive increase in apoptosis and Parkinson disease pathways and decrease in lipid metabolism related activities in vtg3-KO eggs implies compelling roles of Vtg3, the least abundant form of Vtgs in vertebrate eggs, in mitochondrial activities. Several differentially abundant proteins representing the altered molecular mechanisms were unveiled to be considered as strong candidate markers to study details of these mechanisms during early embryonic development in zebrafish and potentially other vertebrates. Conclusions These findings indicate that the global egg proteome is subject to extensive modification depending on the presence or absence of specific Vtgs and that these modifications can have a major impact on developmental competence.

Inactivation of Exosc10 in the oocyte impairs oocyte development and maturation, leading to a depletion of the ovarian reserve in mice

“…Proteins (2 µg) were in-gel digested with 12.5 ng/μL sequencing grade modified trypsin (Promega, Charbonnières-les-Bains, France) after concentration on one band using a 4-12% Bis-Tris precast gel (NuPAGE ™ ) as previously described [15]. The resulting peptide mixtures were injected onto a TimsTOF Pro instrument (Bruker Daltonik GmbH, Bremen, Germany) coupled to a NanoElute HPLC System equipped with on a 75 µm × 250 mm IonOpticks Aurora 2 C18 column (Ion Opticks Pty Ltd, Fitzroy, Australia) and LC-MS/MS data were acquired using the PASEF method as previously described [16].…”