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

Feb 10, 2004Biology of reproduction

Thrombospondin, a key protein in egg cell structures, and its gene activity in the ovaries of mature penaeid shrimp

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Abstract

Three related forms of cortical rod proteins, designated mjTSP, are produced from a single gene in penaeid shrimp.

cDNA sequencing revealed identical sequences among three mjTSP protein forms, differing only by the loss of 246 and 369 base pairs.
The mjTSP proteins share similarities with extracellular matrix proteins in the thrombospondin/cartilage oligomeric protein family.
mjTSP transcripts were present at similar levels in immature, vitellogenic, and mature ovaries.
In situ hybridization showed that mjTSP transcripts localized primarily in previtellogenic oocytes across all developmental stages.
mjTSP proteins were absent in immature ovaries but became detectable during the early vitellogenic stage and maintained high levels in mature ovaries.

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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.

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

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