Aquaculture Europe 2021

October 4 - 7, 2021

Funchal, Madeira

Add To Calendar 05/10/2021 16:10:0005/10/2021 16:30:00Europe/LisbonAquaculture Europe 2021MOLECULAR MECHANISMS INVOLVED IN ATLANTIC HALIBUT Hippoglossus hippoglossus EGG QUALITYCongress AuditoriumThe European Aquaculture Societywebmaster@aquaeas.orgfalseDD/MM/YYYYaaVZHLXMfzTRLzDrHmAi181982

MOLECULAR MECHANISMS INVOLVED IN ATLANTIC HALIBUT Hippoglossus hippoglossus EGG QUALITY

O . Yilmaz1* , A. Mangor Jensen1, T. Harboe1 , M . Møgster1, R . Mangor Jensen1, O . Mjaavatten2, E . Birkeland2 , E. Spriet3, L. Sandven3 , T . Furmanek4, F.S. Berven2 and B. Norberg1

 

1 Institute of Marine Research, Austevoll Research Station, 5392, Storebo Norway

2 The Proteomics Facility of the University of Bergen (PROBE), 5009 Bergen, Norway

3 The Molecular Imaging Center (MIC), University of Bergen, 5009, Bergen, Norway 

4 Institute of Marine Research, P.O. Box 1870, Nordnes, NO-5817, Bergen, Norway.

email: ozlem.yilmaz@hi.no

 



Introduction

Egg quality has a powerful influence on reproductive success. It remains a serious problem of largely variable cause(s) in human reproductive medicine (Keefe et al., 2015) and livestock production (Bobe and Labbé, 2010). Recent research in teleosts has focused on differential abundance of maternal mRNA and protein stockpiles deposited in the egg for clues to the origin of the problem (Cheung et al., 2019; Ma et al., 2019; Sullivan et al., 2015). Earlier studies revealed several impaired mechanisms involved in early development related cellular processes in zebrafish (Yilmaz et al., 2021, 2017). The evolutionary conserved stereotypical procedure of cellular events led us to investigate whether these findings are common in marine species of aquaculture interest. An unsteady supply of high quality eggs and fry in Atlantic halibut (Hippoglossus hippoglossus), makes this species a perfect candidate to study egg quality related mechanisms. The objective of our study was to analyze proteomic profiles in good (GQ) versus poor quality (BQ) eggs, to identify proteins that can serve as egg quality markers, and to discover molecular mechanisms determining egg quality.

Material and methods

Eggs from a total of 25 spawns were collected in 2019-2021 reproductive seasons. For each egg batch, aliquots were snap frozen at 1 hour post fertilization (1hpf) for subsequent analyses, while 100 ml was incubated for egg quality assessment, based on survival rates prior to hatching at ≃ 12 dpf. Proteomic profiling between GQ and BQ eggs was performed via tandem mass tags labeling (TMT) based liquid chromatography tandem mass spectrometry (LC-MS/MS). Obtained spectra was searched against an in-house built proteome database originated from Atlantic halibut egg transcriptome. Differentially abundant proteins (DAPs) were determined based on p values resolved from Student’s t-test (p < 0.05) followed by Benjamini Hochberg correction for multiple testing (p < 0.05). Functional annotation of DAPs was performed using the GO, KEGG, and UNIPROT databases. Overrepresentation analyses were done via GESTALT using human proteome as reference database. DAPs were additionally tested for protein-protein interaction networks using the STRING Network search tool. Eight DAPs were selected as candidate markers based on fold differences in abundance and were subjected to parallel reaction monitoring (PRM) based targeted LC-MS/MS. Relative abundance of gene expression for 21 DAPs were also determined via TaqMan based quantitative PCR. Eggs from GQ and BQ spawners were also processed for transmission electron microscopic (TEM) evaluations at 1hpf for analysis of differences in mitochondrial number and structure.

Results

A total of 115 proteins were differentially abundant between GQ and BQ eggs with 64 being down-regulated and 51 being up-regulated in BQ eggs compared to GQ eggs. Frequency distribution of DAPs showed clear differences in proteomic profiles between GQ and BQ eggs (p < 0.05). Accordingly, GQ eggs seem to contain significantly higher number of proteins related to protein folding, while BQ eggs seem to be enriched with proteins related to transcription, protein degradation and synthesis inhibition, and mitochondrial biogenesis. Overrepresentation-test-based enrichment analysis (p < 0.05) revealed significant biological processes, molecular functions and cellular components which are in close agreement with frequency distribution analysis findings. STRING network analysis of proteins down-regulated in BQ eggs revealed a network of three subclusters of proteins related to cytoskeletal regulation and energy homeostasis, protein homeostasis, and fatty acid degradation. Proteins which were up-regulated in BQ eggs formed a network made of two major subclusters of proteins mainly related to mitochondrial biogenesis, energy and protein homeostasis.    

PRM based LC-MS/MS results revealed five out of the eight candidate marker proteins (MT-ND5, DHRS9, GATD3a, FBP1, UQCRFS1) to be significantly different in abundance between GQ and BQ eggs. Accordingly, MT-ND5, DHRS9 and GATD3a are down-regulated while FBP1 and UQCRFS1 are up-regulated in BQ eggs. UQCRFS1 and FBP1 were additionally found to be part of the STRING network cluster which was formed by BQ up-regulated proteins. Relative abundance of expressions of 7 out of the 21 tested genes exhibited an increase with the same tendency pattern (p > 0.05) and 6 genes exhibited a converse tendency pattern (p > 0.05) to protein abundance, while 8 genes exhibit a similar increasing tendency pattern to protein abundance and significant differences between GQ and BQ eggs (p < 0.05). These genes (cyc1, fh, gcn1, ghitm, uqcrb, uqcrfs1, fbp1a, and atp5f1a) were also found within the STRING network cluster revealed to be significant in BQ up-regulated proteins. The overall findings were additionally supported by TEM observations which revealed clear signs of differences in mitochondrial number and structure between GQ and BQ eggs.

Conclusions

Frequency distribution, enrichment and protein-protein interaction analyses of DAPs between GQ and BQ A. halibut eggs revealed matching results pointing to impairments in mitochondrial structure and functions and protein folding related activities. Additional findings based on TEM imaging fortifies the hypothesis concerning the impairments in mitochondrial structure and functions. The overall findings were found to be corresponding to previous findings in zebrafish and thus there seem to be high similarities in the pattern of impaired molecular mechanisms in BQ eggs of zebrafish, a well-established biomedical model, and Atlantic halibut, a representative of marine flatfishes with aquaculture interest. We expect discoveries of such mechanisms in poor quality eggs to spur development of practical strategies to determine and eliminate the potential causes leading to egg quality problems in Atlantic halibut and other farmed fishes, thereby contributing significantly to development of effective strategies for improving breeding practices and sustainable growth of Norwegian and global aquaculture.

References

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Cheung, C.T., Nguyen, T., Le Cam, A., Patinote, A., Journot, L., Reynes, C., Bobe, J., 2019. What makes a bad egg? Egg transcriptome reveals dysregulation of translational machinery and novel fertility genes important for fertilization. BMC Genomics 20, 584. https://doi.org/10.1186/s12864-019-5930-8

Keefe, D., Kumar, M., Kalmbach, K., 2015. Oocyte competency is the key to embryo potential. Fertil. Steril. 103, 317–322. https://doi.org/10.1016/j.fertnstert.2014.12.115

Ma, H., Martin, K., Dixon, D., Hernandez, A.G., Weber, G.M., 2019. Transcriptome analysis of egg viability in rainbow trout, Oncorhynchus mykiss. BMC Genomics 20, 319. https://doi.org/10.1186/s12864-019-5690-5

Sullivan, C.V., Chapman, R.W., Reading, B.J., Anderson, P.E., 2015. Transcriptomics of mRNA and egg quality in farmed fish: Some recent developments and future directions. Gen. Comp. Endocrinol. 221, 23–30. https://doi.org/10.1016/j.ygcen.2015.02.012

Yilmaz, O., Patinote, A., Com, E., Pineau, C., Bobe, J., 2021. 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. BMC Genomics 22, 308. https://doi.org/10.1186/s12864-021-07606-1

Yilmaz, O., Patinote, A., Nguyen, T.V., Com, E., Lavigne, R., Pineau, C., Sullivan, C.V., Bobe, J., 2017. Scrambled eggs: Proteomic portraits and novel biomarkers of egg quality in zebrafish (Danio rerio). PLOS ONE 12, e0188084. https://doi.org/10.1371/journal.pone.0188084