Mass Spectrometry Analysis of Rainbow Trout (Oncorhynchus mykiss) Outer Arm Dynein (OAD) Components. Expression and Localization of their mRNA During the Spermatogenesis Stages.

Abstract

Dynein-ATPases are macromolecular complexes involved in microtubule-based sliding during the beating cycle of the axoneme of cilia and flagella; this class of enzyme is also involved in cytoplasmic transports and chromosome movement. As a general feature, outer arm dyneins form large complexes of more than 1.2 MDa, which can be extracted by high salt treatment of isolated axonemes. These complexes are composed by one or two subunits (depending upon the species) containing one, two or three high molecular weight chains (>500 kDa), each exhibiting an ATPase activity. Each particle also contains intermediate and low molecular weight polypeptides. Analysis of the outer arm dynein from trout sperm flagella has shown that this vertebrate axonemal dynein was very similar in its structure, properties and behaviour to the sea urchin outer arm dynein. It is composed by two heavy chains (HC1 and HC2), four or five intermediate chains (ICs) and at least six light chains (LCs). We have used one and two dimensional gel electrophoresis coupled to tandem mass spectrometry (LC-MS-MS) to identify the different subunits composing the trout sperm axonemal OAD. Using the AGENAE trout transcripts database, we have obtained positive identification for the 5 ICs (for examples; IC1 and IC3 showed sequence similarity to IC1 from human, rat and dog; IC2 to bos, dog and ciona IC2; IC4 with thioredoxin domains containing protein; IC5 was identified mainly as tubulin) and the 6 LCs (for examples: LC2 showed sequence similarity with LC3 which was itself similar to the t-complex testis expressed-1 protein from mouse; LC4 to LC4 of several species; LC5 and LC6 were similar to the dynein cytoplasmic light chain 2 from human) and partial sequence information were obtained for alpha and beta HCs. Some of these proteins corresponded to cDNA probes spotted on the AGENAE trout microarray membrane (GPL3650). This array was used to analyze their expression profile during the course of the spermatogenesis. For example IC4 transcript increased strongly during gonadal maturation stages III-IV corresponding to the time of spermatocytes formation. This was further confirmed by in situ hybridization where the transcripts of IC4 have been found exclusively in spermatocytes I and II. Our results suggest that the transcription of the genes encoding axonemal components occurs early in spermatocytes much before the axoneme formation that takes place in spermatids. These first results on vertebrate axonemal OAD confirmed that a strong conservation of dynein intermediate and light chains exists from protists to vertebrates, suggesting strong selective pressure in relation with very specific function involved in the beating and/or flagellar waveform formation. This is also of importance since cilia and flagella have been involved in several important diseases and infertility in human.