Cross-linking of ZP2 and ZP3 by transglutaminase is required for the formation of the outer layer of fertilization envelope of carp egg.

A transglutaminase (TGase) cDNA was cloned from carp ovary. It was highly homologous to zebrafish TGase. Immunoblot and enzymatical assay showed that TGase was present on the chorion and in the cytoplasm of carp eggs. Addition of TGase inhibitor, cadaverine or ethylene diaminetetracetic acid (EDTA) to the cortical reaction medium impaired the formation of the outer layer of fertilization envelope (FE(o)), the adhesive structure of carp egg. Fibroin-like substance (FLS), cystatin, cathepsin-like substance (CLS), and FEO-1 were the components of FE(o), wherein the majority of the former three were conjugated to form macromolecules of 90-205 kDa while the latter one was present in monomer of 22 kDa. Cadaverine interfered slightly the discharge of FLS conjugates out of the perivitelline space (PVS) but affected profoundly the recruitment of FLS conjugates to FE, whereas EDTA completely inhibited both the release and the recruitment of FLS conjugates to FE. Both EDTA and cadaverine did not inhibit the discharge of FEO-1 out of PVS but could inhibit the recruitment of FEO-1 to FE. The mechanism was studied. ZP2 and ZP3, the major constituents of inner layer of FE, were cross-linked during cortical reaction, which rendered FE hardened. In the presence of EDTA, the cross-linking of ZP2 and ZP3 were inhibited, thus FE remained soft. The PVS of an egg with a hardened FE was less expanded than an egg with a soft FE. It was assumed that a less expanded PVS would generate a higher fluid pressure than a more expanded PVS did. Therefore, the transportation of the macromolecules such as the FLS-cystatin-CLS conjugates out of PVS was facilitated in control and cadaverine-treated eggs whose FE were hardened but was blocked in EDTA-treated eggs whose FE were unhardened. On the other hand, the transportation of small molecules such as FEO-1 out of FE was not restrained, so they were discharged out of the PVS of the control and TGase inhibitor-treated eggs. In addition, TGase activity was also required for the recruitment of FLS conjugates to FE.

Fibroin-like substance is a major component of the outer layer of fertilization envelope via which carp egg adheres to the substratum.

Seven cDNA encoding silkworm fibroin homologues were cloned from a carp ovarian cDNA library. The encoded proteins are denoted as carp ovarian fibroin-like substances (FLS). FLS contain a repetitive domain consisting of tandem repeats of dipeptide of Gly-X, where X may be any amino acid. Each FLS has its own unique repeating sequence, such as GQGAGQGS, GQGMGQGM, GRGQGEGHGS, and GFGFGQGS, indicating a family of FLS genes exists in carp. FLS is exclusively expressed in oocytes and is stored in cortical granules. During cortical reaction, FLS is exocytosed to perivitelline space and then gradually added to the outer layer of the fertilization envelope (FEo). The FLS of fertilization envelope is conjugated with cystatin and cathepsin-like substance (CLS) and appears in multiple bands of molecular weights ranging from 40 to 205 kDa. After fertilization or artificial activation, carp eggs adhere firmly to the substratum via FEo. FLS is a major component of FEo. The presence of transglutaminase inhibitor, cadaverine or ethylene diaminetetraacetic acid, in the cortical reaction medium can impair or block the recruitment of FLS and other substances to FEo. As a consequence, FEo is not formed or is greatly reduced, resulting in a great reduction of egg adhesion.