Isam Khalaila

Sexual differentiation in decapod crustaceans: role of the androgenic gland

Summary In male crustaceans—unlike vertebrates—the endocrine and gametogenic functions are clearly separated into distinct organs, the androgenic gland and the testis, respectively. The androgenic gland is thought to be the exclusive source of hormone responsible for sex-differentiation and sexual characteristics in crustaceans. Information on this unique crustacean organ is revised with respect to its structure, secretion and role in the regulation of the expression of sexual characteristics, and intersexuality. Several decapod models are presented for research on sexual differentation in higher crustaceans.

A gastrolith protein serving a dual role in the formation of an amorphous mineral containing extracellular matrix

Despite the proclamation of Lowenstam and Weiner that crustaceans are the “champions of mineral mobilization and deposition of the animal kingdom,” relatively few proteins from the two main calcification sites in these animals, i.e., the exoskeleton and the transient calcium storage organs, have been identified, sequenced, and their roles elucidated. Here, a 65-kDa protein (GAP 65) from the gastrolith of the crayfish, Cherax quadricarinatus, is fully characterized and its function in the mineralization of amorphous calcium carbonate (ACC) of the extracellular matrix is demonstrated. GAP 65 is a negatively charged glycoprotein that possesses three predicted domains: a chitin-binding domain 2, a low-density lipoprotein receptor class A domain, and a polysaccharide deacetylase domain. Expression of GAP 65 was localized to columnar epithelial cells of the gastrolith disk during premolt. In vivo administration of GAP 65 dsRNA resulted in a significant reduction of GAP 65 transcript levels in the gastrolith disk. Such gene silencing also caused dramatic structural and morphological deformities in the chitinous-ACC extracellular matrix structure. ACC deposited in these gastroliths appeared to be sparsely packed with large elongated cavities compared with the normal gastrolith, where ACC is densely compacted. ACC spherules deposited in these gastroliths are significantly larger than normal. GAP 65, moreover, inhibited calcium carbonate crystallization in vitro and stabilized synthetic ACC. Thus, GAP 65 is the first protein shown to have dual function, involved both in extracellular matrix formation and in mineral deposition during biomineralization.

The eyestalk-androgenic gland-testis endocrine axis in the crayfish Cherax quadricarinatus

In decapod crustaceans, a number of neurohormones regulating a variety of physiological processes, including reproduction, are to be found in the X-organ-sinus gland complex of the eyestalk. Bilateral eyestalk ablation was thus performed in mature males of the Australian red claw crayfish Cherax quadricarinatus with the aim of studying the role of eyestalk-borne hormones on spermatogenic activity in the testis and on the androgenic gland (AG). The latter gland controls the differentiation and functioning of male sexual characteristics in crustaceans. Eyestalk ablation caused hypertrophy of the AG, as indicated by an increase in gland weight (3.9 +/- 0.44 mg vs < 0.1mg in intact males) and by overexpression of AG polypeptides. In the testes of eyestalk-ablated males, empty spermatogenic lobules were common, while lobules containing primary spermatocytes were infrequent. These findings were reflected in decreased amounts of DNA in these testes and a consequent increase in the relative weights of the sperm ducts. Since it was found that eyestalk ablation affected both the AG and the reproductive system, in vitro experiments were conducted to study the direct effects of the sinus gland on the AG and testes and of the AG on the testes. Sinus gland extracts inhibited by 30% the incorporation of radiolabeled amino acids into AG polypeptides and almost totally inhibited the secretion of radiolabeled AG polypeptides into the culture medium. However, sinus gland extracts had no significant effects on testicular tissue. On the other hand, AG extracts affected the in vitro phosphorylation of a testicular polypeptide (of 28 kDa), in a time- and dose-dependent manner, suggesting a direct effect of AG-borne hormones on the testes. The above findings, together with the evidence for direct inhibition by the sinus gland on the AG, suggest an endocrine axis-like relationship between the sinus gland, the AG, and the male reproductive system in decapod crustaceans.

A mass spectrometric and molecular modelling study of cisplatin binding to transferrin

A combination of mass spectrometry, UV/Vis spectroscopy and molecular modelling techniques have been used to characterise the interaction of cisplatin with human serum transferrin (Tf). Mass spectrometry indicates that cisplatin binds to the hydroxy functional group of threonine 457, which is located in the iron(III)‐binding site on the C‐terminal lobe of the protein. UV/Vis spectroscopy confirms the stoichiometry of binding and shows that cisplatin and iron(III) binding are competitive. The binding of cisplatin has been modelled by using molecular dynamic simulations and the results suggest that cisplatin can occupy part of both the iron(III)‐ and carbonate‐binding sites in the C‐terminal lobe of the protein. Combined, the studies suggest that cisplatin binding sterically restricts iron(III) binding to the C‐terminal lobe binding site, whereas the N‐terminal lobe binding site appears to be unaffected by the cisplatin interaction, possibly allowing the iron(III)‐induced conformational change necessary for binding to a Tf receptor.

Intersex Red Claw Crayfish, Cherax quadricarinatus (von Martens): Functional Males with Pre-vitellogenic Ovaries

Intersex individuals, possessing both male and female genital openings, were assessed in two groups-7 and 19 months old-of Australian red claw crayfish (Cherax quadricarinatus). All intersex individuals investigated were functional males, as suggested by their malelike morphology and the presence of testes, sperm ducts, androgenic glands, and viable spermatozoa. When an ovary was present in an intersex individual from either group, the gonadosomatic index, the diameter of the oocytes, and the ovarian cytosolic polypeptide profile were similar to those of immature, pre-vitellogenic females. We conclude that intersexuality in C. quadricarinatus does not indicate a case of protandric sequential hermaphroditism, as previously suggested. The case of intersexuality described here presents a unique model for the study of the role of the androgenic gland in the regulation of sex differentiation in crustaceans.

The Crustacean Androgen: A Hormone in an Isopod and Androgenic Activity in Decapods1

Abstract The androgenic gland has been described in a variety of crustacean species—isopods, amphipods and decapods. It has been shown to play a role in the regulation of male differentiation and in the inhibition of female differentiation. Upon its application for endocrine manipulation, it inhibits female characteristics. Recently, the androgenic hormone from the isopod Armadillidium vulgare was purified and characterized on the basis of a morphological bioassay. The hormone is a glycosylated protein composed of two peptide chains connected each to the other by two disulfide bridges. The pro-hormone consists of the same two chains connected by a third peptide in a complex that resembles the insulin super family hormones. The study of the androgenic gland in decapods lags behind that in the isopods, and a decapod androgenic hormone has yet to be identified. In this review, five decapod species are described as models, in which the androgenic gland exerts morphological, anatomical, physiological and behavioral effects. These models could serve as the basis of possible bioassays for the study of the structure and mode of action of the androgenic hormone in decapod crustaceans.

Endocrine Balance Between Male and Female Components of the Reproductive System in Intersex Cherax quadricarinatus (Decapoda: Parastacidae)

Intersex individuals of the crayfish Cherax quadricarinatus are functional males that also possess arrested ovaries. To study the role of eyestalk vs. androgenic gland factors in regulating the functional balance between the different components of the intersex reproductive system, andrectomy and/or unilateral destalking were performed. Andrectomized intersex speci- mens had atrophied testes and sperm ducts and showed a reduction in the number of testicular spermatogenic lobules. Large oocytes developed in their ovarian lobes, and the Gonadosomatic Index reached 1.60 ± 0.36 compared with 0.21 ± 0.03 for the control. In the polypeptide profiles of the ovarian lobes from andrectomized individuals, the 177-, 150-, and 106-kDa polypeptides pre- dominated, resembling the profile of the secondary vitellogenic ovary. The andrectomized indi- viduals neither lost their male external characteristics, such as the red patch on the propodus, nor developed ovigerous setae on their male-like pleopods. Unilateral eyestalk ablation did not cause significant differences in the male or the female components of the reproductive system compared with the control group. The maturation of the permanently arrested ovary and the arrest of the testis in andrectomized intersex individuals illustrated the central role of the androgenic gland in maintaining the endocrine balance in intersex C. quadricarinatus. J. Exp. Zool. 283:286n294, 1999.

O-Linked β-N-Acetylglucosaminylation (O-GlcNAcylation) in Primary and Metastatic Colorectal Cancer Clones and Effect of N-Acetyl-β-d-glucosaminidase Silencing on Cell Phenotype and Transcriptome

Background: O-GlcNAcylation regulates cellular processes such as transcription, signal transduction, metabolism, and cell cycle. Results: O-GlcNAcylation is elevated in CRC metastatic clone. OGA silencing resulted in the acquisition of a fibroblast-like morphology, growth retardation, and alteration of gene expression. Conclusion: O-GlcNAcylation affects gene transcription, metabolism, and proliferation. Significance: This research supports O-GlcNAcylation involvement in various aspects of tumor cell physiology. O-Linked β-N-acetylglucosamine (O-GlcNAc) glycosylation is a regulatory post-translational modification occurring on the serine or threonine residues of nucleocytoplasmic proteins. O-GlcNAcylation is dynamically regulated by O-GlcNAc transferase and O-GlcNAcase (OGA), which are responsible for O-GlcNAc addition and removal, respectively. Although O-GlcNAcylation was found to play a significant role in several pathologies such as type II diabetes and neurodegenerative diseases, the role of O-GlcNAcylation in the etiology and progression of cancer remains vague. Here, we followed O-GlcNAcylation and its catalytic machinery in metastatic clones of human colorectal cancer and the effect of OGA knockdown on cellular phenotype and on the transcriptome. The colorectal cancer SW620 metastatic clone exhibited increased O-GlcNAcylation and decreased OGA expression compared with its primary clone, SW480. O-GlcNAcylation elevation in SW620 cells, through RNA interference of OGA, resulted in phenotypic alterations that included acquisition of a fibroblast-like morphology, which coincides with epithelial metastatic progression and growth retardation. Microarray analysis revealed that OGA silencing altered the expression of about 1300 genes, mostly involved in cell movement and growth, and specifically affected metabolic pathways of lipids and carbohydrates. These findings support the involvement of O-GlcNAcylation in various aspects of tumor cell physiology and suggest that this modification may serve as a link between metabolic changes and cancer.

Identification and Quantification of Protein Glycosylation

Glycosylation is one of the most abundant posttranslation modifications of proteins, and accumulating evidence indicate that the vast majority of proteins in eukaryotes are glycosylated. Glycosylation plays a role in protein folding, interaction, stability, and mobility, as well as in signal transduction. Thus, by regulating protein activity, glycosylation is involved in the normal functioning of the cell and in the development of diseases. Indeed, in the past few decades there has been a growing realization of the importance of protein glycosylation, as aberrant glycosylation has been implicated in metabolic, neurodegenerative, and neoplastic diseases. Thus, the identification and quantification of protein-borne oligosaccharides have become increasingly important both in the basic sciences of biochemistry and glycobiology and in the applicative sciences, particularly biomedicine and biotechnology. Here, we review the state-of-the-art methodologies for the identification and quantification of oligosaccharides, specifically N- and O-glycosylated proteins.

On intersexuality in the crayfish Cherax quadricarinatus: an inducible sexual plasticity model

Summary Sexual differentiation is a plastic process. The plasticity may be manifested during embryo-genesis, when one set of primordial reproductive ducts develops while the other degenerates. In adults, many normal (e.g., sequential hermaphroditism) and abnormal (e.g., endocrine disorders or exposure to endocrine disrupters such as estrogenic pollutants) cases are known in which sexual plasticity may be expressed as various degrees of feminization. In crustaceans, the androgenic gland (AG) regulates the development of male characteristics; its absence results in feminization, often including the onset of vitellogenesis. A unique model of intersexuality was found in the crayfish Cherax quadricarinatus, in which some degree of natural sexual plasticity is observed. Two to 14% of the population are intersex individuals, having both male and female genital openings. Intersex specimens always function as males but may also contain an ovary in a permanently arrested, pre-vitellogenic state. This sexual plasticity model was recently characterized and investigated with respect to the role of the AG and the onset of vitellogenesis. Removal of the AG in intersex individuals induced the reproductive system to shift from a permanently active male state to a female state. This shift included changes in morphology, cessation of spermatogenesis and onset of secondary vitellogenesis manifested by a change in the ovarian protein profile, translocation of protein kinase C (PKC) in the ovary and appearance of secondary vitellogenic high-density lipoprotein (HDL) in the hemolymph. The vitellogenin gene was found to be induced in the hepatopancreas of AG ablated intersex individuals suggesting that the AG represses transcription of this gene in intact intersex individuals. The experimentally inducible sex shift in the crayfish provides a unique and controlled model system for the study of sexual differentiation and plasticity at the physiological and molecular levels. The findings presented here also illustrate the central role of the AG in the regulation of sexual differentiation in sexually plastic as well as gonochoristic crustacean species.