Project description:The zona pellucida plays a crucial role in the process of fertilization to early embryonic development, including cellular arrangement and communication between blastomeres. However, little is known regarding the role of the zona pellucida in pre- and post-implantation embryonic development associated with gene expression. We investigated the effects of the zona pellucida on the development and gene expression of pre- and post-implantation embryos. After the zona pellucida was removed at the 2-cell stage, compaction occurred earlier. In addition, the expression of differentiation-related genes in the inner cell mass (ICM) (Oct4, Sox21) and trophectoderm (TE) (Cdx2, Eomes, and Tfap2c) was significantly altered in both zona free (ZF) morula and blastocyst compared to zona intact embryos. After embryo transfer, the implantation rate and number of live fetuses were lower in ZF embryos than in control embryos, whereas the fetal weight at birth was not different. However, placental weight was significantly increased in ZF embryos. RNA-seq analysis of the placenta showed that a total of 473 differentially expressed genes that significantly influenced the biological process. The present study suggests that zona pellucida removal at the 2-cell stage not only disturbs the expression pattern of ICM/TE-related genes, but affects the post-implantation development of mouse embryos. Overall, this study provides a deeper insight into the role of the zona pellucida during early mouse embryonic development and the viability of post-implantation development. It also provides a valuable basis for future research related to the production of ZF embryos in mammals and the developmental properties of blastomeres.
Project description:Background<br>Vitellogenin is a well established biomarker for estrogenic exposure in fish. However, effects on gonadal differentiation at concentrations of estrogen not sufficient to give rise to a measurable vitellogenin response suggest that more sensitive biomarkers would be useful. Induction of zona pellucida genes may be more sensitive but their specificities are not as clear. The objective of this study was to find additional sensitive and robust candidate biomarkers of estrogenic exposure.<br>Results<br>Hepatic mRNA expression profiles were characterized in juvenile rainbow trout exposed to a measured concentration of 0.87 and 10ng ethinylestradiol/L using a salmonid cDNA microarray. The higher concentration was used to guide the subsequent identification of generally more subtle responses at the low concentration not sufficient to induce vitellogenin. A meta-analysis was performed with data from the present study and three similar microarray studies using different fish species and platforms. Within the generated list of presumably robust responses, several well-known estrogen-regulated genes were identified. Two genes, confirmed by quantitative RT-PCR (qPCR), fulfilled both the criteria of high sensitivity and robustness; the induction of the genes encoding zona pellucida protein 3 and a nucleoside diphosphate kinase (nm23).<br>Conclusions<br>The cross-species, cross-platform meta-analysis correctly identified several robust responses. This adds confidence to our approach used for identifying candidate biomarkers. Specifically, we propose that analyses of nm23 together with zona pellucida genes may increase the possibilities to detect an exposure to low levels of estrogenic compounds in fish.<br>
Project description:Purpose: Identifying the biological pathways affected by the deletion of the zona pellucida binding protein 2 in mouse lungs at different time points Method: RNA profiles of wild type C57BL6/J mice and Zpbp2 homozygous knock-out lungs were generated using next generation sequencing Illumina HiSeq4000 PE100
Project description:Purpose: Identifying the biological pathways affected by the deletion of the zona pellucida binding protein 2 in mouse lungs at different time points Method: RNA profiles of wild type C57BL6/J mice and Zpbp2 homozygous knock-out lungs were generated using next generation sequencing Illumina HiSeq2000 SR50
2019-08-24 | GSE136232 | GEO
Project description:Functional analysis of zona pellucida domain protein Dusky in Tribolium castaneum
Project description:The metalloproteinase ovastacin is released by the mammalian oocyte upon fertilization and cleaves zona pellucida protein 2, a component of the surrounding extracellular matrix, at a distinct cleavage site. This limited proteolysis hardens the zona pellucida, abolishes sperm binding and thereby regulates fertility. Therefore, this process has to be tightly controlled by the plasma protein fetuin-B, an endogenous competitive inhibitor. At present, little is known about how the cleavage characteristics of ovastacin differ from closely related proteases. Physiological implications of ovastacin beyond ZP2 cleavage are still obscure. In this study, we employed N-terminal amine isotopic labeling of substrates (N-TAILS) contained in the secretome of mouse embryonic fibroblasts to elucidate the substrate specificity and the precise cleavage site specificity. Furthermore, we were able to unravel the physicochemical properties governing enzyme-substrate interactions. Eventually, we identified several potential physiological substrates with significance for mammalian fertilization. These results suggest that ovastacin might affect sperm interaction beyond zona pellucida hardening
Project description:Despite passing routine laboratory tests for semen quality, bulls used in artificial insemination (AI) can exhibit a significant variation in fertility. During fertility analysis, a subfertile bull with a low pregnancy rate of 10% was identified. To fully characterize the phenotype, a range of in vitro, in vivo and molecular assays were carried out. Additionally, knockout mice were generated to investigate the function of the identified mutated gene. Sperm from the subfertile bull exhibited reduced motility and severely reduced caffeine-induced hyperactivation compared to control bulls of proven fertility. Ability to penetrate the zona pellucida, cleavage rate, cleavage kinetics, and blastocyst yield after IVF or AI were significantly lower than in control bulls. Whole-genome sequencing and RNA sequencing of testis revealed a critical mutation in AK9 that affect splicing, shaping the majority of AK9 transcripts that leads to a premature termination codon and a severely truncated protein. Transgenic mice deficient in AK9 were generated, resulting in the production of immotile sperm that were unable to fertilize the oocyte. These sperm exhibited abnormalities, including a low ATP concentration. RNA-seq analysis of testis revealed differential gene expression of components of the axoneme and sperm flagella, as well as steroid metabolic processes. Sperm ultrastructural analysis showed a higher percentage of sperm with abnormal flagella. The infertility produced by AK9 mutant bull and in AK9 deficient mice indicates the essential metabolic role of AK9 in sperm motility and/or hyperactivation, which in turn affects sperm binding and penetration of the zona pellucida.
Project description:CaV3.2 (encoded by Cacna1h gene) is necessary for the generation of calcium oscillations by maintaining voltage oscillations in zona glomerulosa cells of mice. However, Cacna1h knockout mice have normal plasma aldosterone levels, suggesting additional calcium entry pathways. We used RNA-seq to investigate such pathways in the murine zona glomerulosa. We want to investigate whether other calcium channels or transporters were upregulated to compensate for the loss of CaV3.2.