Abstract
Many bivalves have an unusual mechanism of mitochondrial DNA (mtDNA) inheritance called doubly uniparental inheritance (DUI) in which distinctly different genomes are inherited through the female (F genome) and male (M genome) lineages. In fertilized eggs that will develop into male embryos, the sperm mitochondria remain in an aggregation, which is believed to be delivered to the primordial germ cells and passed to the next generation through the sperm. In fertilized eggs that will develop into female embryos, the sperm mitochondria are dispersed throughout the developing embryo and make little if any contribution to the next generation. The frequency of embryos with the aggregated or dispersed mitochondrial type varies among females. Previous models of DUI have predicted that maternal nuclear factors cause molecular differences among unfertilized eggs from females producing embryos with predominantly dispersed or aggregated mitochondria. We test this hypothesis using females of each of the two types from a natural population. We have found small, yet detectable, differences of the predicted type at the proteome level. We also provide evidence that eggs of females giving the dispersed pattern have consistently lower expression for different proteasome subunits than eggs of females giving the aggregated pattern. These results, combined with those of an earlier study in which we used hatchery lines of Mytilus, and with a transcriptomic study in a clam that has the DUI system of mtDNA transmission, reinforce the hypothesis that the ubiquitin-proteasome system plays a key role in the mechanism of DUI and sex determination in bivalves. We also report that eggs of females giving the dispersed pattern have higher expression for arginine kinase and enolase, enzymes involved in energy production, whereas ferritin, which is involved in iron homeostasis, has lower expression. We discuss these results in the context of genetic models for DUI and suggest experimental methods for further understanding the role of these proteins in DUI.
Highlights
From the ‡Institute of Life Science, College of Medicine, Swansea University, Swansea SA28PP, Wales UK; §Department of Biochemistry, Genetics and Immunology, Faculty of Biology, University of Vigo, 36310, Vigo, Spain; ¶Bedford Institute of Oceanography, Ocean and Ecosystem Science Division, Department of Fisheries and Oceans, Dartmouth, NS, Canada B2Y 4A2; ʈDepartment of Biology, University of Crete, Iraklion 71409, Crete, Greece
This phenomenon, called doubly uniparental inheritance of mitochondrial DNA (mtDNA) (DUI),1 suggests a connection between mtDNA inheritance and sex determination
Since the 1990s, DUI has been under intensive investigation, but the molecular mechanism for DUI and how it relates to sex determination, is still to be elucidated
Summary
Mussel Collection, Spawning, and Fertilization—Mussels were collected in Lameque New Brunswick and grown out at Mahone Bay, Nova Scotia, Canada (44.44° North 64.38° West). Experimental Design—From each of 40 female mussels successfully spawned, a sample of eggs was fertilized and 16 embryos were examined and classified according to whether they exhibited the dispersed or aggregated mitochondrial DNA pattern. There were 12 female mussels employed in the experiment (three types x four biological replicates), each providing a pool of unfertilized eggs from which a protein extract was obtained and analyzed separately using 2-DE electrophoresis. For five mussels (one of the Femalebiased type, two of the Male-biased type, and two from the Mixed type) the 2-DE electrophoresis was repeated on the extracted protein to provide technical replicates to allow estimation of experimental variation associated with the technique. Protein Extraction and 2-DE Electrophoresis—The experimental procedures for proteomics are given in full as supplemental experimental procedures These followed closely those used previously for mussels [47]. Use of the full sequences from the Mytilus EST and RNA-seq for the Blastp search provide more information for searching than the shorter peptide sequences matching the spectra
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