A method to quantitate maternal transcripts localized in sea urchin egg cortex by RT-qPCR with accurate normalization

The sea urchin egg cortex is a peripheral region of eggs consisting of cell membrane and adjacent cytoplasm, which contains actin and tubulin cytoskeleton, cortical granules and some proteins required for early development. Method for isolation of cortices from sea urchin eggs and early embryos has been developed in 70s of 20th Century. Since that time this method has been reliable tool to study protein localization and cytoskeletal organization in cortex of unfertilized eggs and embryos during first cleavages. This study is an estimation of reliability of RT-qPCR to analyze levels of maternal transcripts that are localized in egg cortex. Firstly, we selected seven potential reference genes, 28S, Cycb , Ebr1 , GAPDH , Hmg1 , Smtnl1 and Ubb , which transcripts are maternally deposited in sea urchin eggs. The candidate reference genes were ranked by five different algorithms (BestKeeper, CV, ΔCt, geNorm and NormFinder) upon calculated level stability in both eggs and isolated cortices. Our results show that gene ranking differs in total RNA and mRNA samples, though Ubb is most suitable reference gene in both cases. To validate feasibility of comparative analysis of eggs and isolated egg cortices by RT-qPCR, we selected Daglb-2 as a gene of interest, which transcripts potentially localized in cortex, and found increased level of Daglb -2 in egg cortices. This suggests that proposed RNA isolation method with subsequent quantitative RT-qPCR analysis can be used to approve cortical association of transcripts in sea urchin eggs.

Integrins on eggs: focal adhesion kinase is activated at fertilization, forms a complex with integrins, and is necessary for cortex formation and cell cycle initiation

We investigate the proposal that integrins and focal adhesion kinase (FAK) form a complex that has structural and signaling functions in eggs. FAK protein is present in eggs and is phosphorylated at fertilization. pY397FAK localizes to the membrane 30 min after fertilization, which correlates with the expression of βC integrins and egg cortex development. The βC integrin and pY397FAK coimmunoprecipitate from egg cortex lysates. PF573 228 and Y11, inhibitors of FAK, interfere with pronuclear fusion and reduce the abundance of pY397FAK and cortical actin without affecting microvillar actin. Cyclin E normally accumulates in the nucleus 15 min after fertilization, then returns to background levels. PF573 228– or Y11-treated eggs accumulate cyclin E in the nucleus; however, levels remain high. In addition, PF573 228 interferes with the accumulation of pERK1/2 in the nucleus and in eggs initiating mitosis. Injection of eggs with a fusion protein consisting of the focal adhesion–targeting domain of FAK fused to green fluorescent protein interferes with cortex formation and produces abnormal nuclei. These data indicate that an integrin–FAK adhesion complex forms at the egg surface that functions in formation of actin arrays in the egg cortex and provides signaling inputs for cell cycle initiation.

Kinesin-1 mediates translocation of the meiotic spindle to the oocyte cortex through KCA-1, a novel cargo adapter

In animals, female meiotic spindles are attached to the egg cortex in a perpendicular orientation at anaphase to allow the selective disposal of three haploid chromosome sets into polar bodies. We have identified a complex of interacting Caenorhabditis elegans proteins that are involved in the earliest step in asymmetric positioning of anastral meiotic spindles, translocation to the cortex. This complex is composed of the kinesin-1 heavy chain orthologue, UNC-116, the kinesin light chain orthologues, KLC-1 and -2, and a novel cargo adaptor, KCA-1. Depletion of any of these subunits by RNA interference resulted in meiosis I metaphase spindles that remained stationary at a position several micrometers from the cell cortex during the time when wild-type spindles translocated to the cortex. After this prolonged stationary period, unc-116(RNAi) spindles moved to the cortex through a partially redundant mechanism that is dependent on the anaphase-promoting complex. This study thus reveals two sequential mechanisms for translocating anastral spindles to the oocyte cortex.