The thrombospondins (TSPs) are a family of five proteins that are involved in the tissue remodeling that is associated with embryonic development, wound healing, synaptogenesis, and neoplasia. These proteins mediate the interaction of normal and neoplastic cells with the extracellular matrix and surrounding tissue. In the tumor microenvironment, TSP-1 has been shown to suppress tumor growth by inhibiting angiogenesis and by activating transforming growth factor β. TSP-1 inhibits angiogenesis through direct effects on endothelial cell migration and survival, and through effects on vascular endothelial cell growth factor bioavailability. In addition, TSP-1 may affect tumor cell function through interaction with cell surface receptors and regulation of extracellular proteases. Whereas the role of TSP-1 in the tumor microenvironment is the best characterized, the other TSPs may have similar functions. (Part of a Multi-author Review)

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Thrombospondins: from structure to therapeutics

The transferrin family spans both vertebrates and invertebrates. It includes serum transferrin, ovotransferrin, lactoferrin, melanotransferrin, inhibitor of carbonic anhydrase, saxiphilin, the major yolk protein in sea urchins, the crayfish protein, pacifastin, and a protein from green algae. Most (but not all) contain two domains of around 340 residues, thought to have evolved from an ancient duplication event. For serum transferrin, ovotransferrin and lactoferrin each of the duplicated lobes binds one atom of Fe (III) and one carbonate anion. With a few notable exceptions each iron atom is coordinated to four conserved amino acid residues: an aspartic acid, two tyrosines, and a histidine, while anion binding is associated with an arginine and a threonine in close proximity. These six residues in each lobe were examined for their evolutionary conservation in the homologous N- and C-lobes of 82 complete transferrin sequences from 61 different species. Of the ligands in the N-lobe, the histidine ligand shows the most variability in sequence. Also, of note, four of the twelve insect transferrins have glutamic acid substituted for aspartic acid in the N-lobe (as seen in the bacterial ferric binding proteins). In addition, there is a wide spread substitution of lysine for the anion binding arginine in the N-lobe in many organisms including all of the fish, the sea squirt and many of the unusual family members i.e., saxiphilin and the green alga protein. It is hoped that this short analysis will provide the impetus to establish the true function of some of the TF family members that clearly lack the ability to bind iron in one or both lobes and additionally clarify the evolutionary history of this important family of proteins.

Evolution of the transferrin family: conservation of residues associated with iron and anion binding.

Thrombospondins are large secreted, multi-modular, calcium-binding glycoproteins that have complex roles in mediating cellular processes. Determination of high-resolution structures of thrombospondins has revealed unique and interesting protein motifs. Here, we review this progress and discuss implications for function. By combining structures of modules from thrombospondins and related extracellular proteins it is now possible to prepare an overall model of the structure of thrombospondin-1 and thrombospondin-2 and discern features of other thrombospondins. (Part of a multi-author Review)

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Structures of thrombospondins.

Crustaceans produce complex yolk proteins to meet the substrate and energy requirements of embryonic development. Early electron microscopic investigations point to a biphasic yolk synthesis, first within the ovary, followed by heterosynthesis at extra-ovarian sites. Recent advances in molecular techniques have enhanced our understanding of the genetic control of yolk synthesis in crustaceans. Amino acid sequencing of crustacean vitellogenin (Vg) has enabled the elucidation of the cDNA sequence associated with it, the identification of genes, and the examination of their expression patterns in different tissues. Yolk processing in crustaeans involves cleavage of the pro-Vg at consensus sites by subtilisin-like endoproteases within the hepatopancreas, hemolymph and oocytes. The structural elucidation of crustacean yolk proteins, as well as the comparison of amino acid sequences of vitellogenins from various crustacean species, has revealed molecular phylogenetic relationships not only among them but also with other large lipid transfer lipoproteins of disparate function. The combinatorial effects of eyestalk neuropeptides and a variety of trophic hormones achieve the hormonal coordination of molting and reproduction. Biogenic amines secreted by the central nervous system may also play an integrative role by stimulating neuropeptide secretion.

Mechanisms and control of vitellogenesis in crustaceans

Amino acids are vital for all living organisms. During early fish ontogeny, they are important fuel molecules, signalling factors and major substrates for the synthesis of a wide range of bioactive molecules and proteins. Because the majority of fish eggs are cleidoic, i.e. closed free-living systems following ovulation and activation, early development of fish depends on the maternal provision of amino acids during oogenesis. While more than 600 proteins have been identified in the growing oocytes of fish, the major vehicles for supplying amino acids to the growing oocyte before ovulation are the vitellogenins, of which many genes and multiple forms are known. Here we review the importance of amino acids for the intermediary metabolism of fish embryos and larvae, where amino acids have been shown to be the preferred catabolic substrate. Subsequently, we address the specialization of the lysosomal pathway involved in the uptake and degradation of yolk proteins. This latter pathway is specifically modified in the germline to facilitate the long-term storage of egg yolk proteins. In marine teleosts, the degradative pathway may be activated before fertilization during oocyte maturation to release free amino acids for oocyte hydration and the acquisition of egg buoyancy. In other species, including freshwater fish, a more latent activation of acid hydrolases occurs after fertilization during the four phases of yolk resorption. The developmental contributions of the yolk syncytial layer, vitelline circulation and liver are essential components of the amino acid supply during fish ontogeny.

Requirement for amino acids in ontogeny of fish

To elucidate the molecular mechanism of oocyte maturation in the kuruma prawn (Marsupenaeus japonicus), subtractive suppression hybridization (SSH) was initially used to identify novel up-regulated genes during the final stages of oocyte maturation, followed by evaluation of the differential expression profile by macroarray and quantitative real-time RT-PCR analyses. The cathepsin C (dipeptidyl peptidase I) gene was thus found to exhibit a significantly higher expression around the onset of cortical rod (CR) formation (early CR stage, appearance of round CRs), progress to a higher mRNA level until the middle CR stage (elongation of CRs), then rapidly revert to a low expression level at the late CR stage (occurrence of germinal vesicle breakdown, GVBD), as also observed at the non-CR stage (previtellogenesis and vitellogenesis). In situ hybridization analyses revealed that the sites of the expression of cathepsin C transcripts in the ovary were distributed in both oocyte and follicle cells, particularly at the early CR stage. A full-length cDNA sequence of this stage-specific gene was subsequently determined by rapid amplification of the cDNA 3' and 5' ends (3' and 5' RACE). The deduced amino acid sequence of the 230-residue mature peptide shared 67-70% identity to the known cathepsin C in mammals. Western blot analysis showed that expression of procathepsin C protein was exclusively at CR stages. The storage site of procathepsin C protein was localized in CRs as revealed by immunohistochemical analysis. This is the first report on the full-length cDNA sequence of cathepsin C and a demonstration of its involvement in the final stages of oocyte maturation in crustacean species.

Cathepsin C transcripts are differentially expressed in the final stages of oocyte maturation in kuruma prawn Marsupenaeus japonicus

In penaeid shrimps, vitellogenin (VTG), the precursor of vitellin, is synthesized in the ovary and hepatopancreas and accumulated in oocytes during ovarian development. In the present study, VTG gene expression levels and hemolymph VTG levels were determined throughout ovarian development in female kuruma prawn, Marsupenaeus japonicus. Hemolymph VTG levels and VTG mRNA levels in the ovary and hepatopancreas were high during vitellogenesis, remained high until final maturation, and then decreased after oviposition. This profile suggests that VTG synthesis activity increases during vitellogenesis and decreases after oviposition. Absence of a significant increase in ovary size in final maturation suggests cessation of yolk accumulation and low activity of VTG synthesis in spite of high VTG mRNA levels. VTG mRNA levels in ovary and hepatopancreas were both highly correlated during vitellogenesis. Thus, their contribution to yolk accumulation seems to be similar. In contrast, VTG mRNA levels in the hepatopancreas increased more slowly at the start of vitellogenesis and declined more sharply after oviposition than in the ovary. This suggests a difference in the regulation of VTG synthesis between the ovary and the hepatopancreas.

Vitellogenin gene expression and hemolymph vitellogenin during vitellogenesis, final maturation, and oviposition in female kuruma prawn, Marsupenaeus japonicus

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.

https://bioone.org/journals/biology-of-reproduction/volume-70/issue-6/biolreprod.103.025379/Molecular-Cloning-and-Ovarian-Expression-Profiles-of-Thrombospondina-Major-Component/10.1095/biolreprod.103.025379.pdf

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

In penaeid shrimp species, ovarian development is characterized by the accumulation of a major yolk protein (vitellin) and the formation of cortical rods in the oocytes. The process is considered to be under the control of a neuroendocrine organ in the eyestalk (the X-organ sinus gland complex). In the present study, the synthesis of vitellogenin (VTG, precursor of vitellin) and two kinds of cortical rod proteins (cortical rod protein, CRP; thrombospondin, MjTSP) was induced by bilateral eyestalk ablation (removal of the X-organ sinus gland complex) in immature female kuruma prawn, Marsupenaeus japonicus, and the synthesis process was monitored over a 7-day period after the ablation. The ovarian weight and hemolymph VTG levels increased in the ablated females. The VTG mRNA levels in the ovary increased concomitantly with vitellin accumulation in the ovary after eyestalk ablation. On the other hand, the CRP and MjTSP protein levels in the ovary increased after eyestalk ablation, whereas the CRP and MjTSP mRNA levels in the ovary did not change concomitantly. The results suggest that the regulatory mechanism of gene expression by eyestalk hormones is different between VTG (transcriptional control) and CRP-MjTSP (translational control).

Expression of vitellogenin and cortical rod proteins during induced ovarian development by eyestalk ablation in the kuruma prawn, Marsupenaeus japonicus

Only a few biochemical and molecular studies on yolk proteins (vitellins) have been carried out in mollusks, mainly in bivalves, while information on prosobranch vitellogenesis is still limited. In this study, we cloned a full-length cDNA encoding vitellogenin (Vg) in the Pacific abalone Haliotis discus hannai. The complete Vg cDNA consists of 7753 nucleotides with a long open reading frame encoding 2391 amino acid residues. The deduced primary structure contains the N-terminal amino acid sequences of the 95 kDa and 150 kDa subunits of vitellin of the abalone and shows similarities to Vgs of other mollusk, fish, nematode and coral species. In common with bivalve Vgs, the abalone Vg gene was expressed only in the ovary. In situ hybridization analysis further localized Vg mRNA to the follicle cells in the ovary. We conclude that the follicle cells are the site of Vg synthesis in H. discus hannai.

Keisuke Yamano

Papers

Cathepsin C transcripts are differentially expressed in the final stages of oocyte maturation in kuruma prawn Marsupenaeus japonicus

Vitellogenin gene expression and hemolymph vitellogenin during vitellogenesis, final maturation, and oviposition in female kuruma prawn, Marsupenaeus japonicus

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

Expression of vitellogenin and cortical rod proteins during induced ovarian development by eyestalk ablation in the kuruma prawn, Marsupenaeus japonicus

Ovarian follicle cells are the site of vitellogenin synthesis in the Pacific abalone Haliotis discus hannai.