Shrimp thrombospondin (TSP): presence of O-ß1,4 N-acetylglucosamine polymers and its function in TSP chain association in egg extracellular matrix.

We characterized the existence of O-ß(1,4)-GlcNAc polymers (ß1,4GNP) that were anchored on the O-linked glycosylation sites of shrimp thrombospondin (pmTSP-II). There were five putative ß1,4GNP linkages on the epithelial growth factor-like domain of pmTSP-II. Antibody against O-ß-GlcNAc (CTD110.6) was used to prove the existence of linear and complex ß1,4GNP. The antibody well reacted with linear chito-triose, -tetraose and -pentaose conjugated with phosphatidylethanolamine lipid. The immunoreactivity could also be detected with a complex ß1,4GNP within pmTSP-II (at MW > 250 kDa). Upon denaturing the protein with SDS-PAGE buffer, the size of pmTSP-II was shifted to be 250 kDa, approximately 2.5 folds larger than the deduced molecular mass of pmTSP-II (110 kDa), suggesting additional association of pmTSP-II apart from its known disulfide bridging. This was confirmed by chitinase digestion on pmTSP-II protein leading to the subsequent smaller protein bands at 110-170 kDa in time- and concentration-dependent manners. These bands well reacted with CTD110.6 antibody and disappeared after extensive chitinase hydrolysis. Together, we believe that ß1,4GNP on pmTSP-II serve the function in an inter-chain association to provide structural architecture of egg extracellular matrix, a novel function of pmTSP-II in reproductive biology.

N-linked mannose glycoconjugates on shrimp thrombospondin, pmTSP-II, and their involvement in the sperm acrosome reaction.

Glycoconjugates in egg extracellular matrices are known to serve several functions in reproductive processes. Here, the presence of N-linked mannose (Man) glycoconjugates on shrimp thrombospondin ( pmTSP-II) and their physiological functions were investigated in the black tiger shrimp Penaeus monodon. A molecular analysis of pmTSP-II demonstrated anchorage sites for N-linked glycans in both the chitin-binding and TSP3 domains. The presence of Man residues was verified by concanavalin A lectin histochemistry on the purified fraction of pmTSP-II (250 kDa with protease inhibitor). The function of the Man glycoconjugates was evident by the Con A interference with the pmTSP-II-induced acrosome reaction (AR) as well as by the ability to recover the induction of the AR by the inclusion of Mans in the treatment mixture. In addition, the recombinant proteins of the three signature pmTSP-II domains expressed in E. coli (lacking glycosylation) and mannosidase-treated pmTSP-II showed a minimal ability to initiate the AR response. Together, these results provide evidence of the pivotal role that Man-linked pmTSP-II plays in modulating the shrimp sperm AR, a novel role for a TSP family protein in shrimp reproductive biology.

Neuronal classification and distribution in the central nervous system of the female mud crab, Scylla olivacea.

The mud crab, Scylla olivacea, is one of the most economically valuable marine species in Southeast Asian countries. However, commercial cultivation is disadvantaged by reduced reproductive capacity in captivity. Therefore, an understanding of the general and detailed anatomy of central nervous system (CNS) is required before investigating the distribution and functions of neurotransmitters, neurohormones, and other biomolecules, involved with reproduction. We found that the anatomical structure of the brain is similar to other crabs. However, the ventral nerve cord (VNC) is unlike other caridian and dendrobrachiate decapods, as the subesophageal (SEG), thoracic and abdominal ganglia are fused, due to the reduction of abdominal segments and the tail. Neurons in clusters within the CNS varied in sizes, and we found that there were five distinct size classes (i.e., very small globuli, small, medium, large, and giant). Clusters in the brain and SEG contained mainly very small globuli and small-sized neurons, whereas, the VNC contained small-, medium-, large-, and giant-sized neurons. We postulate that the different sized neurons are involved in different functions.

Characterization of the thrombospondin (TSP)-II gene in Penaeus monodon and a novel role of TSP-like proteins in an induction of shrimp sperm acrosome reaction.

We have recently shown that water-soluble materials from the egg extracellular cortical rods (wsCRs) exert the ability to induce the sperm acrosome reaction in Penaeus monodon. In this study, we further demonstrated that the thrombospondin protein family (TSP) existed in wsCRs, and that their mRNA transcripts were detected in developing oocytes as early as stage I. Full sequence analysis revealed that our pmTSP sequence was considerably different from the recently reported pmTSP in the 5' nonconserved region and in many TSP signature domains, hence, the name pmTSP-II was given to our variant. The transcripts of pmTSP-II were detected only in early developing oocytes (stage-I and -II) while TSP-like proteins were detected in all developing oocytes, particularly at the outer rim of cortical rods situated in the extracellular crypts of the mature, stage-IV oocytes. In addition, wsCRs contained anti-TSP-reactive proteins, suggesting that TSP-like proteins are dissolved in and are part of the egg water during spawning. The functional importance of TSP-like proteins was evident by the interference of a wsCR-induced acrosome reaction response with anti-TSP in a concentration-dependent manner. In summary, we found that pmTSP-II transcripts were present in the developing oocytes and pmTSP-II protein accumulated in cortical rods, which are partly secreted and thus solubilized to produce dissolved TSP-like proteins that participate in induction of the sperm acrosome reaction-a novel reproductive role for TSP protein family.

Effect of progesterone administration on the distribution of oviductal carbohydrates in Rana tigrina.

Our aim has been to determine whether carbohydrate distribution in the oviducts of progesterone-treated animals is comparable with that of seasonal breeders in Rana tigrina. Like many other anurans, R. tigrina oviduct exhibits a short straight portion (pars recta, pr) at the beginning followed by a long, highly coiled portion (pars convoluta, pc). Histologically, the oviduct of this species revealed some unique features, one of which was intense toluidine blue staining, specifically in the upper mucosal glands of pc4. Based on lectin reactivities in the epithelial cells and mucosal glands, patterns of lectin staining in the seasonal breeders were classified into seven types: R1-R3 (for pr) and C1-C4 (for pc). Typically, some lectins reacted selectively either with ciliated cells (concanavalin A) or non-cialiated cells (Ricinus communis agglutinin I and wheatgerm agglutinin); however, Bandeiraea simplicifolia agglutinin I reacted with both cell types. These staining patterns were different in the progesterone-treated animals. Differences in glycan distribution in the oviductal secretions were revealed by lectin blotting. Compared with the seasonal breeders, an enhanced staining of some lectins was noted in the hormone-treated animals: either an increased staining intensity of existing protein bands or additional staining of new protein bands. Inversely, the staining of wheatgerm agglutinin was markedly diminished in the hormone-treated animals, suggesting the inhibitory effect of progesterone on oviductal glycan distribution. Whether alteration in glycan distribution upon progesterone treatment affects the physiological properties of the released jelly substances remains to be addressed.