The role of transcriptome and proteome in developmental competence of eggs of Sander lucioperca
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ABSTRACT: Molecular profiling of the eggs is an excellent approach aiming at understanding biological processes and mechanisms conditioning developmental competence in finfishes and, consequently, reproductive fitness. Despite many efforts, it is still unclear what is the specific role of transcriptome and proteome in determination of egg quality in Teleostei fishes. The aim of this study was to perform integrated transcriptomic-proteomic analysis of eggs of pikeperch – a commercially relevant freshwater fish species. Additionally, comparative analysis of transcriptome and proteome in eggs characterized by either high or low egg quality has been performed, in order to identify mechanisms leading to early embryonic lethality in pikeperch. Our study provides a novel insight into the understanding of the role of maternally-derived molecular cargo in finfishes. The data obtained sheds light on the importance of transcriptome in development of nervous system suggesting neurogenesis-related mRNAs as a very important, non-genetic inheritance factor. Proteomic analysis highlights specific role of proteins in the immune response in ovulated eggs. Integrated analysis brings attention to the galactose-specific lectin nattectin gene and protein as the frontline defense molecule for the egg and developing embryo. The molecular analysis of egg developmental competence emphasizes post-vitellogenic processes (final oocyte maturation and ovulation) as the ones potentially compromising transcriptomic profile, but not affecting proteomic cargo. It highlights the need for careful reconsideration of the commercial reproductive protocols as the very strong modulators of egg quality and their molecular profile. Considering the mechanisms driving these alterations as well as consequences stemming from the differential abundance of the transcripts are still to be explored, the candidate quality markers, first time provided for pikeperch along with the current study, are creating valuable resource for further studies.
Project description:Good quality or developmentally competent eggs result in high survival of progeny. Previous research has shed light on factors that determine egg quality, however, large gaps remain. Initial development of the embryo relies on maternally-inherited molecules, such as transcripts, deposited in the egg, thus, they would likely reflect egg quality. We performed microarray analysis on zebrafish fertilized eggs of different quality from unrelated, wildtype couples to obtain a global portrait of the egg transcriptome to determine its association with developmental competence and to identify new candidate maternal-effect genes. Fifteen of the most differentially expressed genes (DEGs) were validated by quantitative real-time PCR. Gene ontology analysis showed that enriched terms included ribosomes and translation. In addition, statistical modeling using partial least squares regression and genetics algorithm also demonstrated that gene signatures from the transcriptomic data can be used to predict reproductive success. Among the validated DEGs, otulina and slc29a1a were found to be increased in good quality eggs and to be predominantly localized in the ovaries. CRISPR/Cas9 knockout mutants of each gene revealed remarkable subfertility whereby the majority of their embryos were unfertilizable.Our novel findings suggested that even in varying quality of eggs due to heterogeneous causes from unrelated wildtype couples, gene signatures exist in the egg transcriptome, which can be used to predict developmental competence. Further, transcriptomic profiling revealed two new potential maternal-effect genes that have essential roles in vertebrate reproduction.
Project description:Humans and animals have problems producing eggs with high embryo developmental competence, but the causes of poor egg quality are usually unknown. This study delivered the first proteomic portraits of egg quality in zebrafish, a leading model for vertebrate development. Egg batches of good and poor quality, evidenced by embryo survival for 24 h, were used to create pooled or replicated sample sets subjected to different levels of fractionation before LC-MS/MS. Obtained spectra were searched against a custom zebrafish proteome database and detected proteins were annotated, categorized and quantified based on their normalized spectral counts. Manual and automated enrichment analyses were highly confirmative, showing that good and poor quality eggs have disparate proteomes. Proteins involved in protein synthesis, energy metabolism, and lipid metabolism, and certain vitellogenin products were strikingly underrepresented in poor quality eggs. Poor quality eggs also had significantly higher representation of proteins related to immune system and endosome/lysosome functioning, oncogenes, and apoptosis, as well as lectins and egg envelope proteins. Quantitative comparisons of highly abundant proteins revealed 9 candidate egg quality markers warranting further study. In conclusion, the zebrafish egg proteome appears to be linked to embryo developmental potential, a phenomenon that begs further investigation.
Project description:Humans and animals have problems producing eggs with high embryo developmental competence, but the causes of poor egg quality are usually unknown. This study delivered the first proteomic portraits of egg quality in zebrafish, a leading model for vertebrate development. Egg batches of good and poor quality, evidenced by embryo survival for 24 h, were used to create pooled or replicated sample sets subjected to different levels of fractionation before LC-MS/MS. Obtained spectra were searched against a custom zebrafish proteome database and detected proteins were annotated, categorized and quantified based on their normalized spectral counts. Manual and automated enrichment analyses were highly confirmative, showing that good and poor quality eggs have disparate proteomes. Proteins involved in protein synthesis, energy metabolism, and lipid metabolism, and certain vitellogenin products were strikingly underrepresented in poor quality eggs. Poor quality eggs also had significantly higher representation of proteins related to immune system and endosome/lysosome functioning, oncogenes, and apoptosis, as well as lectins and egg envelope proteins. Quantitative comparisons of highly abundant proteins revealed 9 candidate egg quality markers warranting further study. In conclusion, the zebrafish egg proteome appears to be linked to embryo developmental potential, a phenomenon that begs further investigation.
Project description:Egg quality is of pivotal importance in biomedicine, agriculture, ecology and environmental science because of its tremendous influence on reproductive success or failure in all animals. Despite species specific differences in physiological aspects of early development, the evolutionary conserved stereotypical procedure of cellular events, led us to investigate whether these findings are common with marine fish species of aquaculture interest. Atlantic halibut (Hippoglossus hippoglossus) is a highly prized species in global fish markets with increasing demand to its production. The objectives of this study were 1) to reveal the proteomic profiles of good versus poor quality halibut eggs, 2) to identify proteins that can serve as egg quality markers, and 3) to discover molecular mechanisms determining egg quality using a combination of quantitative proteomics workflows, tandem mass tags (TMT) labeling and parallel reaction monitoring (PRM).
Project description:The aim of the project was to identify differently expressed genes in eggs of European seabass (Dicentrarchus labrax) characterized by different quality. In this way it was expected to identify genes possibly being a molecular indicator of egg quality in this species, which was never studied to date. For the study microarray analysis of over 26 thousand genes in 16 egg batches was performed. Additionally, for each egg batch biological quality was determined, what allowed to compare the gene expression profile with overall egg quality (divided into two groups representing ‘high’ and ‘low’ egg quality). The analysis allowed to identify 39 differently expressed genes between the two groups representing ‘high’ and ‘low’ egg quality. From those genes, expression level of 7 were verified by real-time qPCR which confirmed significant difference in expression in 5 of them.
Project description:Sturgeon species, considered living fossils, exhibit unique reproductive characteristics, making it crucial to comprehend the molecular processes governing the formation and quality of their eggs. However, there is a notable lack of comprehensive data concerning the protein composition analysis of sturgeon eggs and ovarian fluid (OF) and their functional significance. To address this knowledge gap, this study aimed to conduct a comprehensive comparative proteomic analysis of Siberian sturgeon eggs and OF using liquid chromatography-mass spectrometry (LC-MS/MS). A total of 566 proteins were identified in eggs, while 617 proteins were identified in OF, with 772 proteins showing differential abundance. Among the differentially abundant proteins, 407 were more abundant in eggs, while 365 showed higher abundance in OF. Furthermore, we identified 288 proteins specific to eggs and 339 proteins specific to OF, along with the top ten most abundant proteins in each. Functional annotation analysis unveiled enriched metabolic pathways, such as oxidative phosphorylation and fatty acid metabolism, as well as protein ubiquitination and translation, in eggs. Conversely, ovarian fluid proteins primarily associated with immune system processes, including the complement and coagulation cascade, neutrophil and leukocyte-mediated immunity, cholesterol metabolism, and regulation of actin cytoskeleton. This study presents the first comprehensive characterization of the protein composition of sturgeon ovarian fluid and eggs, shedding light on their distinct functional roles. The findings not only advance our understanding of sturgeon reproduction but also shed light on egg-OF signaling and origin of the OF proteins. Moreover the identified proteins offer potential biomarkers for predicting egg quality contributing to the development of effective breeding strategies for sturgeon species.
Project description:We modeled profiles of ovary gene expression and their relationship to egg quality, evaluated as production of viable mid-blastula stage embryos, in striped bass (Morone saxatilis) using artificial neural networks and supervised machine learning. Collective changes in expression of a limited suite of genes (233) representing only 2% of the queried ovary transcriptome explained >90% of the eventual variance in embryo survival. Egg quality related to minor changes in expression (M-bM-^IM-$0.2-fold), with most gene transcripts making minor contribution (<1%) to the overall prediction of egg quality. Correlation analyses of this suite of candidate genes indicated that collective dysfunction of the ubiquitin-26S proteasome, COP9 signalosome, and subsequent control of the cell cycle engenders embryonic developmental incompetence in striped bass. Our results show that the transcriptomic signature evidencing this dysfunction is of, and therefore likely to influence, egg quality, a biologically complex trait that is crucial to reproductive fitness. Female striped bass were sorted into groups (N=8 each) producing M-bM-^@M-^Xhigh qualityM-bM-^@M-^Y or M-bM-^@M-^Xlow qualityM-bM-^@M-^Y eggs (spawns) based upon the percentage of eggs bearing viable 4 h embryos. Spawns with >50% of eggs producing 4 h embryos were considered to be of high quality and spawns with <30% of eggs producing 4 h embryos were considered to be of low quality.
Project description:Female Aedes aegypti mosquitoes impose a severe global public health burden as primary vectors of multiple viral and parasitic pathogens. Under optimal environmental conditions, Aedes aegypti females have access to human hosts that provide blood proteins for egg development, conspecific males that provide sperm for fertilization, and freshwater that serves as an egg-laying substrate suitable for offspring survival. As global temperatures rise, Aedes aegypti females are faced with climate challenges, like intense droughts and intermittent precipitation, which create unpredictable and suboptimal conditions for the egg-laying step of their reproductive cycle. Aedes aegypti mosquitoes nonetheless show remarkable reproductive resilience, but how they achieve this is unknown. Here we show that under drought-like conditions simulated in the laboratory, mated, blood-fed Aedes aegypti females carrying mature eggs retain them in their ovaries for extended periods, while maintaining the viability of these eggs until they can be deposited in freshwater. Using transcriptomic and proteomic profiling of Aedes aegypti ovaries, we identify two previously uncharacterized genes – here named tweedledee and tweedledum – that show ovary-enriched, temporally-restricted expression during egg retention. These genes are mosquito-specific, linked within a syntenic locus, and rapidly evolving under positive selection, raising the possibility that they serve an adaptive function. Using loss-of-function mutagenesis to disrupt both genes, we show that, tweedledee and tweedledum, which encode secreted proteins, are specifically required for extended retention of viable eggs, such as during intermittent precipitation or drought. These results highlight an elegant example of taxon-restricted genes at the heart of an important adaptation that equips Aedes aegypti females with “insurance” to, when contextually appropriate, flexibly extend their reproductive sequence without losing reproductive capacity, thus allowing this species to exploit diverse and unpredictable habitats.
Project description:Egg quality dictates fertility outcomes, and although there is a well-documented decline with advanced reproductive age, how it changes during puberty is less understood. Such knowledge is critical, since advances in Assisted Reproductive Technologies are enabling pre- and peri-pubertal patients to preserve fertility in the medical setting. Therefore, we investigated egg quality parameters in a mouse model of the pubertal transition or juvenescence (postnatal day; PND 11-40). Animal weight, vaginal opening, serum inhibin B levels, oocyte yield, oocyte diameter, and zona pellucida thickness increased with age. After PND 15, there was an age-associated ability of oocytes to resume meiosis and reach metaphase of meiosis II (MII) following in vitro maturation (IVM). However, eggs from the younger cohort (PND 16-20) had significantly more chromosome configuration abnormalities relative to the older cohorts and many were at telophase I instead of MII, indicative of a cell cycle delay. Oocytes from the youngest mouse cohorts originated from the smallest antral follicles with the fewest cumulus layers per oocyte, suggesting a more developmentally immature state. RNA Seq analysis of oocytes from mice at distinct ages revealed that the genes involved in cellular growth signaling pathways (PI3K, mTOR, and Hippo) were consistently repressed with meiotic competence, whereas genes involved in cellular communication were upregulated in oocytes with age. Taken together, these data demonstrate that gametes harvested during the pubertal transition have low meiotic maturation potential and derive from immature follicular origins.
Project description:The goal of this study was to use global gene expression as a diagnostic tool to compare hepatic gene expression patterns in both male and female FHM in streams with the lowest and highest reproductive success, and potentially identify a suite of mRNA transcripts indicative of reproduction in a population The goal of this study was to compare differences in hepatic mRNA expression between gender at high and low egg-producing streams, not differences between individual streams. A k-means cluster analysis was performed using eggs/pair/day on the original 17 streams to delineate 3 clusters: high, medium and low. From that analysis, FHM from 6 of the original 17-streams used in Crago et al. (2010) were chosen for the microarray experiment (Fig. 1, Table 1). In this study the experimental condition is reproductive success; High versus Low reproductive success. The streams grouped into High Reproductive Success were Oak Creek-2007 (2313 eggs), Point Creek (1277 eggs), Meeme Creek (1164 eggs) and Baird Creek (967 eggs). The streams grouped into Low Reproductive Success were: Ashwaubenon Creek (0 eggs), Devils Creek (541 eggs) and Oak Creek-2006 (642 eggs). Multiple regression analysis using the 22 sediment and water quality characteristics measured in the 6 streams with the highest (n = 4 and lowest (n = 3) streams demonstrated that there were no differences amongst the streams in regards to measure sediment and water variables. .5 One array was run for each gender from each stream. So that Males from Point Creek were pooled and run on one array, males from Ashwaubenon Creek were run on a separate array, and so forth. There were 14 arrays used in this study, 7 for males, 7 for females from individual rivers. So that Males from Point Creek were pooled and run on one array, males from Ashwaubenon Creek were run on a separate array, and so forth. In the case of Oak Creek, which was sampled in both years, there was a large difference in egg production between two years. Therefore separate arrays were run for Oak Creek 2006 and Oak Creek 2007. All streams chosen had overall survival rates of at least 80% through the 21-day sampling period, except Devils River. The survival rate for Devils River was at 100% until four days prior to the end of the experiment when six fish died or escaped.