In Crustacea, reproductive function and mechanisms regulating vitellogenesis have not been fully elucidated. This is due in great part to a lack of information concerning the biochemical nature of the vitellogenin molecule, the hemolymph precursor of yolk protein, vitellin, as well as the functional expression of the vitellogenin-encoding gene. We have therefore cloned a cDNA encoding vitellogenin in the kuruma prawn, Penaeus japonicus based on the N-terminal amino acid sequence of the 91 kDa subunit of vitellin. The open reading frame of this cDNA encoded 2,587 amino acid residues. This is the first investigation reporting a full-length cDNA and its corresponding amino acid sequence for vitellogenin in any crustacean species.Northern blot analysis and in situ hybridization have revealed that mRNA encoding vitellogenin was expressed in both the follicle cells in the ovary and the parenchymal cells in the hepatopancreas. In nonvitellogenic females, vitellogenin mRNA levels were negligible in both the ovary and hepatopancreas, but in vitellogenic females, levels were dramatically increased in both tissues. In the ovary, highest levels were observed during the early exogenous vitellogenic stage, and thereafter rapidly decreased, whereas in the hepatopancreas, high levels were maintained until the onset of the late vitellogenic stage. Differing profiles of vitellogenin mRNA levels in the ovary and hepatopancreas suggest that the contribution of these tissues to vitellogenin synthesis harbor separate and complementary roles during vitellogenesis.

A cDNA encoding vitellogenin (Vg) in the giant freshwater prawn, Macrobrachium rosenbergii, was cloned based on the cDNA sequence of vitellin (Vn) fragments A-N and B-42 determined previously, and its amino acid sequence deduced. The open reading frame (ORF) encoded 2,537 amino acid residues and its deduced amino acid sequence possessed three consensus cleavage sites, R-X-R-R, similar to those reported in Vgs of insects. The deduced primary structure of Vg in M. rosenbergii was seen to be similar to that of Penaeus japonicus, especially in the N-terminal region. It is therefore likely that Vgs in crustacean species including prawns and other related decapods exhibit a similar structural pattern. Based on the deduced primary structure of Vg and analysis of the various Vg and Vn subunits found in the hemolymph and ovary during ovarian maturation, we demonstrated the post-translational processing of Vg in M. rosenbergii. This is the first time that Vg processing has been clearly demonstrated in a crustacean species. Vg, after being synthesized in the hepatopancreas, is considered to be cleaved by a subtilisin-like endoprotease to form two subunits, A and proB, which are then released into the hemolymph. In the hemolymph, proB is possibly cleaved by a processing enzyme of unknown identity to give rise to subunits B and C/D. The three processed subunits A, B, and C/D are sequestered by the ovary to give rise to three yolk proteins, Macr-VnA, VnB, and VnC/D.

This study describes the morphological and histological characteristics of the male reproductive system of the orange mud crab, Scylla olivacea (Herbst, 1796). Three maturation stages were determined on the basis of the vas deferens diameter, the gonad colour and the gonadosomatic index (GSI). Significant differences were observed in the GSI value as the crabs matured. All germ cells were present in the histological sections of the testes, and these decrease significantly in size as they progress from spermatogonia, spermatocytes, and spermatids to spermatozoa. Spermatophores are found in the anterior vas deferens (AVD) and median vas deferens (MVD) of all maturation stages but not in the posterior vas deferens (PVD), with a significantly smaller size in immature and maturing specimens. Thus, the classification of S. olivacea gonads into various maturation stages based solely on histological characteristics (i.e., the presence of spermatophores) is impossible. Therefore, the combination of both microscopic and macroscopic features is essential for determining the gonad maturation stages of male S. olivacea.

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SUMMARY: As a first step in understanding the mechanism of vitellogenesis in the kuruma prawn Penaeus japonicus, biochemical characteristics of vitellin (Vt) were investigated. Vitellin was purified from vitellogenic ovary by gel filtration and ion‐exchange chromatography, and an anti‐Vt antiserum was raised in rabbits. The molecular weight of the purified Vt was estimated to be 530 kDa by native‐polyacrylamide gel electrophoresis and gel filtration. Vitellin was composed of three polypeptide subunits of 91, 128, and 186 kDa in sodium dodecylsulfate–polyacrylamide gel electrophoresis. The 91 kDa subunit was further purified by reversed‐phase high‐performance liquid chromatography. A protein sequencer was used to analyze the amino‐terminal amino acid sequence of the 91 kDa subunit; residues up to position 29 were identified, except for two residues at positions 10 and 14. Positive immunohistochemical reaction against the anti‐Vt antiserum was observed in the cytoplasm of oocytes at endogenous and exogenous vitellogenesis stages.

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The dynamics of vitellogenin mRNA expression during ovarian maturation in Macrobrachium rosenbergii were examined by measuring hemolymph vitellogenin (Vg) levels and Vg mRNA expression in the hepatopancreas and ovary at differing reproductive stages in both intact and eyestalk ablated animals. Vg mRNA was quantified using real-time RT-PCR and hemolymph Vg was measured by enzyme immunoassay. In intact animals, Vg mRNA levels in the hepatopancreas and hemolymph Vg levels showed a gradual increase during the molt cycle concomitant with increasing gonadosomatic index (GSI), with Vg levels decreasing prior to ecdysis although GSI continued to increase. Eyestalk ablation was seen to accelerate Vg synthesis as well as ovarian maturation, although it did not alter the overall pattern of Vg expression. Vg mRNA expression was negligible in the ovary of both intact and eyestalk ablated animals, confirming that the hepatopancreas is the principal site of Vg synthesis in M. rosenbergii with the ovary being only a minor contributor. This study has shown that Vg synthesis is correlated to ovarian maturation and the molt cycle in M. rosenbergii.