Molecular Cloning and Ovarian Expression Profiles of Thrombospondin, a Major Component of Cortical Rods in Mature Oocytes of Penaeid Shrimp, Marsupenaeus japonicus1

Abstract

In penaeid shrimp, cortical rods (CRs) are formed in peripheral crypts of the oocyte after completion of yolk accumulation; subsequently the CRs are utilized as a source of jelly materials that surround fertilized eggs. In our previous study, of five major components, three CR proteins displayed quite similar immunological characteristics. In this study, cDNA sequences and developmental expression profiles at both transcriptional and protein levels were examined to elucidate the molecular characteristics of CR proteins and the process of CR formation. Sequencing cDNAs exhibited the presence of three related forms that have identical sequences except for the loss of 246 and 369 bp in medium and short forms, respectively, suggesting that a single gene generates three transcriptional variants corresponding to the three CR proteins. Their deduced amino acid sequences revealed similarities to those of extracellular matrix proteins in a thrombospondin (TSP) 3,4/cartilage oligomeric protein family, and thereby the CR proteins were designated mjTSP. Semiquantitative analysis by real-time polymerase chain reaction revealed the presence of mjTSP transcripts, at similar levels, in immature, vitellogenic, and mature ovaries. Furthermore, in situ hybridization localized the majority of transcripts in previtellogenic oocytes in ovaries at all developmental stages. By the Western blot, on the other hand, mjTSP proteins were undetectable in immature ovaries but became obvious at the early vitellogenic stage. The immunosignals were enhanced during vitellogenic stages and maintained a high intensity in mature ovaries. Thus, transcription, translation of mjTSP, and formation of the CR structure occurred at different stages of ovarian development.

Extracellular matrix retention of thrombospondin 1 is controlled by its conserved C-terminal region

“…Thus ECM retention appears likely to be an ancestral activity of TSPs. This is borne out by the recently reported functional role of the single TSP of Drosophila, D-TSP, in the ECM of muscle-tendon attachment sites and the localisation of a prawn TSP to specialised ECM structures -oocyte cortical rods (Yamano et al, 2004;Chanana et al, 2007;Subramanian et al, 2007).…”

“…Thus ECM retention appears likely to be an ancestral activity of TSPs. This is borne out by the recently reported functional role of the single TSP of Drosophila, D-TSP, in the ECM of muscle-tendon attachment sites and the localisation of a prawn TSP to specialised ECM structures -oocyte cortical rods (Yamano et al, 2004; Chanana et al, 2007;Subramanian et al, 2007).Studies of cell-attachment activities of TSP1 and TSP2 have relied on purified TSPs coated onto glass or plastic surfaces, where they probably form a uniform layer of individual trimers. Using classic procedures for isolation of insoluble ECM, we demonstrate that TSP1 and other TSPs are retained in ECM in the physical form of small, closely spaced puncta.…”

Reproductive Mechanisms in Crustacea Focusing on Selected Prawn Species: Vitellogenin Structure, Processing and Synthetic Control

“…japonicus (Kim et al 2004(Kim et al , 2005; and Mar. japonicus thrombospondin (MjTSP, 130, 140, and 150 kDa; Yamano et al 2003Yamano et al , 2004. Except for their molecular weights, SOP, CRP, and MjTSP show similar molecular structure in terms of repeated cysteinerich domains, and proteolytic sites for posttranslational modulation and glycosylation, as well as similar deduced amino acid sequences.…”

“…Except for their molecular weights, SOP, CRP, and MjTSP show similar molecular structure in terms of repeated cysteinerich domains, and proteolytic sites for posttranslational modulation and glycosylation, as well as similar deduced amino acid sequences. Yamano et al (2004) suggested that they are the products of a single gene or are encoded by orthologous genes.…”

Thrombospondins: from structure to therapeutics

“…There is one EGF-like repeat with the calcium-binding consensus sequence in THBS-1, two in vertebrate group B THBSs, and two in Drosophilia THBS. Shrimp (Marsupenaeus japonicus) THBS has at least six EGF-like repeats, with five repeats having a calcium-binding consensus sequence, except for an arginine at the position of the conserved Y/F [31]. It will be interesting to learn whether or not these repeats bind calcium.…”