Yuriko Hasegawa

Isolation and properties of androgenic gland hormone from the terrestrial isopod, Armadillidium vulgare

Abstract The androgenic gland hormone (AGH) is known to control sex differentiation in crustaceans. AGH was purified from isolated androgenic glands (AGs) of the male isopod Armadillidium vulgare by three steps of reverse-phase high performance liquid chromatography (RP-HPLC) and its chemical properties were examined. The purified AGH-active fraction showed masculinizing activity when 38 pg of this preparation was injected into a young female of the same species. Only 160 ng of the material was obtainable from 2000 animals at about an 11% rate of recovery. The elution of AGH activity by molecular sieve HPLC indicated that molecular weight of AGH was 11,000 ∼ 13,000. AGH was inactivated by treatment with trypsin or by reductive carboxymethylation. The AGH activity was not affected by heat treatment at 100°C for 3 min. These results indicated that AGH was a heat-stable protein with disulfide bond(s).

Molecular Cloning and Expression Analysis of cDNAs Encoding Androgenic Gland Hormone Precursors from Two Porcellionidae Species, Porcellio scaber and P. dilatatus

Abstract Male sexual characteristics in Crustacea are induced by androgenic gland hormone (AGH), which is produced by the male-specific androgenic gland. Recently, AGH in the terrestrial isopod Armadillidium vulgare was characterized and its cDNA cloned, the first example in which the structure of AGH was elucidated. We report here the molecular cloning of cDNAs encoding AGH precursors from two additional terrestrial isopods, Porcellio scaber and P. dilatatus. cDNA fragments encoding Porcellio scaber AGH (Pos-AGH) and P. dilatatus AGH (Pod-AGH) were amplified by RT-PCR using degenerate oligonucleotide primers designed based on the amino acid sequence of A. vulgare AGH (Arv-AGH). Subsequently, full length cDNAs were obtained by 5′- and 3′-RACE. Both AGH precursors consisted of a signal peptide, B chain, C peptide and A chain, and exhibited the same organization as that of Arv-AGH. The amino acid sequences of the A and B chains, which comprise mature AGH peptide, were highly conserved among the three species, while that of the C peptide showed only low sequence similarity. In Northern blot analysis, each cDNA fragment used as a probe specifically hybridized with a single band (0.75 kb) in mRNA extracted from whole male reproductive organs. In analysis of the tissue-specific gene expression of these two AGHs by RT-PCR, it was revealed that both AGH transcripts were detected only in cDNA synthesized using total RNA from the testis carrying the androgenic glands, but not in that from testis only, seminal vesicle, vas deferens, or hepatopancreas.

PURIFICATION AND PROPERTIES OF ANDROGENIC GLAND HORMONE FROM THE TERRESTRIAL ISOPOD ARMADILLIDIUM VULGARE

Abstract The androgenic gland hormone (AGH) is known to control sex differentiation in crustaceans. AGH was purified from isolated androgenic glands (AGs) of the male isopod Armadillidium vulgare by three steps of reverse-phase high performance liquid chromatography (RP-HPLC) and its chemical properties were examined. The purified AGH-active fraction showed masculinizing activity when 38 pg of this preparation was injected into a young female of the same species. Only 160 ng of the material was obtainable from 2000 animals at about an 11% rate of recovery. The elution of AGH activity by molecular sieve HPLC indicated that molecular weight of AGH was 11,000 ∼ 13,000. AGH was inactivated by treatment with trypsin or by reductive carboxymethylation. The AGH activity was not affected by heat treatment at 100°C for 3 min. These results indicated that AGH was a heat-stable protein with disulfide bond(s).

Preparation of an active recombinant peptide of crustacean androgenic gland hormone

In crustaceans, male sexual characteristics are induced by a hormone referred to as androgenic gland hormone. We have recently cloned a candidate cDNA in the terrestrial isopod Armadillidium vulgare. In order to prove that this cDNA encodes the hormone, recombinant single-chain precursor molecules consisting of B chain, C peptide and A chain were produced using both baculovirus and bacterial expression systems. Neither recombinant precursors showed activity. Digestion of only the precursor carrying a glycan moiety with lysyl endopeptidase gave a heterodimeric peptide with hormonal activity by removing a part of C peptide. These results indicate that the cDNA encodes the hormone.

Masculinization of females of the isopod crustacean, Armadillidium vulgare, following injections of an active extract of the androgenic gland

Injections of 64 U of an active extract of the androgenic gland into young females of Armadillidium vulgare induced masculinization of the external sexual characteristics and transformation of the internal female reproductive organs into testes, seminal vesicles, and vasa deferentia. Following injections of 10-U doses, the internal organs were hardly affected in 8 of 10 females although the external characteristics underwent masculinization.

Correct Disulfide Pairing Is Required for the Biological Activity of Crustacean Androgenic Gland Hormone (AGH): Synthetic Studies of AGH

Androgenic gland hormone (AGH) of the woodlouse, Armadillidium vulgare, is a heterodimeric glycopeptide. In this study, we synthesized AGH with a homogeneous N-linked glycan using the expressed protein ligation method. Unexpectedly, disulfide bridge arrangement of a semisynthetic peptide differed from that of a recombinant peptide prepared in a baculovirus expression system, and the semisynthetic peptide showed no biological activity in vivo. To confirm that the loss of biological activity resulted from disulfide bond isomerization, AGH with a GlcNAc moiety was chemically synthesized by the selective disulfide formation. This synthetic AGH showed biological activity in vivo. These results indicate that the native conformation of AGH is not the most thermodynamically stable form, and correct disulfide linkages are important for conferring AGH activity.

Immunological identification of crustacean androgenic gland hormone, a glycopeptide.

Androgenic gland hormone (AGH) is known to be responsible for sex differentiation in crustaceans. The amino acid sequence of AGH-active fraction purified from androgenic glands of the terrestrial isopod Armadillidium vulgare was determined by immunoprecipitation employing three types of antibodies raised against differing parts of the amino acid sequence deduced from the putative AGH cDNA sequence. As all antibodies adsorbed AGH activity, it was confirmed that the sequence examined was that of AGH. The affinity of AGH to certain lectins indicated that AGH possesses a carbohydrate moiety, which is in agreement with the observation that AGH possesses an N-glycosylation consensus sequence.

Alterations in the integument of Armadillidium vulgare masculinized by implantation of androgenic glands

Summary Alterations in the integument of masculinized females of Armadillidium vulgare caused by the implantation of androgenic glands from normal males involve both pigment cells and epidermal cells with respect to the intracellular structure and the ommochrome content. Epidermal cells beneath the cuticle of masculinized females are thicker than those of normal females, having a number of small secretory granules and well developed rough endoplasmic reticulum in the cytoplasm. As a result of epidermal cell thickening, pigment cells are more widely separated from the cuticle in masculinized females than in normal females. The ommochrome content in the integument of masculinized females, which was initially the same as that of normal females, increased with each postoperative stage and coincided with that of male integument by the fourth postoperative stage. The observations suggest that the slate grey color characteristic of the male body, shown during masculinization of females after the implantation of ...

Novel structures in secreting the androgenic gland hormone.

Abstract The secretory granules in the androgenic gland of the terrestrial isopod Armadillidium vulgare, which have been indistinct for long time because of vulnerable structures, were revealed by using the rapid-freezing and freeze-substitution method. The fine struture of the androgenic gland is conspicuous by the distribution of numerous particular organelles in the cytoplasm consisting of the endoplasmic reticulum and the Golgi complex, and by having a number of highly organized structures developed between the androgenic gland cells. The structures connect to the intercellular space, which is seen as intercellular canaliculi for exporting the androgenic gland hormone. The plasma membranes near the particular structure of the intercellular canaliculi in the androgenic gland are often specialized to form cellular junctions. The secretory granules including the electron-dense materials, which are supposed to be peptides of androgenic gland hormone, are distributed beside the particular structure of the intercellular canaliculi. Some of the granules are seen to fuse with the plasma membranes. This observation suggests that, in the Armadillidium vulgare, the secretory granules containing androgenic gland hormone are transferred to the extracellular space through the intercellular canaliculi particularly developed for exporting the peptide hormone. This is the first evidence to show the secretory mechanism of the androgenic gland hormone in the Isopoda.

An Immunoassay for the Androgenic Gland Hormone of the Terrestrial Isopod, Armadillidium vulgare

Summary Antiserum was raised in a rabbit against the purified androgenic gland hormone (AGH) of Armadillidium vulgare. The specificity of the antiserum was confirmed by immunoblotting and by absorption of the biological activity of AGH. The immunological activity measured by ELISA correlated well with the biological activity of AGH. It is therefore possible to measure AGH by immunoassay. The immunoassay, which is time-saving and avoids the handling problems of the bioassay was used to measure seasonal variation of AGH content in male reproductive organs. Lack of immunological crossreactivity between preparations from different isopod species supported former results on biological species-specificity.