Simy Weil

Temporal Silencing of an Androgenic Gland-Specific Insulin-Like Gene Affecting Phenotypical Gender Differences and Spermatogenesis

Androgenic glands (AGs) of the freshwater prawn Macrobrachium rosenbergii were subjected to endocrine manipulation, causing them to hypertrophy. Transcripts from these glands were used in the construction of an AG cDNA subtractive library. Screening of the library revealed an AG-specific gene, termed the M. rosenbergii insulin-like AG (Mr-IAG) gene. The cDNA of this gene was then cloned and fully sequenced. The cysteine backbone of the predicted mature Mr-IAG peptide (B and A chains) showed high similarity to that of other crustacean AG-specific insulin-like peptides. In vivo silencing of the gene, by injecting the prawns with Mr-IAG double-stranded RNA, temporarily prevented the regeneration of male secondary sexual characteristics, accompanied by a lag in molt and a reduction in growth parameters, which are typically higher in males of the species. In terms of reproductive parameters, silencing of Mr-IAG led to the arrest of testicular spermatogenesis and of spermatophore development in the terminal ampullae of the sperm duct, accompanied by hypertrophy and hyperplasia of the AGs. This study constitutes the first report of the silencing of a gene expressed specifically in the AG, which caused a transient adverse effect on male phenotypical gender differences and spermatogenesis.

Stabilization of amorphous calcium carbonate by phosphate rich organic matrix proteins and by single phosphoamino acids

Stable amorphous calcium carbonate (ACC) is a unique material produced naturally exclusively as a biomineral. It was demonstrated that proteins extracted from biogenic stable ACC induce and stabilize synthetic ACC in vitro. Polyphosphate molecules were similarly shown to induce amorphous calcium carbonate formation in vitro. Accordingly, we tested the hypothesis that biogenic ACC induction and stabilization is mediated by the phosphorylated residues of phosphoproteins. We show that extracellular organic matrix extracted from gastroliths of the red claw crayfish Cherax quadricarinatus induce stable ACC formation in vitro. The proteinaceous fraction of this organic matrix is highly phosphorylated and is incorporated into the ACC mineral phase during precipitation. We have identified the major phosphoproteins of the organic matrix and showed that they have high calcium binding capacity. Based on the above, in vitro precipitation experiments with single phosphoamino acids were performed, indicating that phosphoserine or phosphothreonine alone can induce the formation of highly stable ACC. The results indicate that phosphoproteins may play a major role in the control of ACC formation and stabilization and that their phosphoamino acid moieties are key components in this process.

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.

Pore properties and pharmacological features of the P2X receptor channel in airway ciliated cells

Airway ciliated cells express an ATP‐gated P2X receptor channel of unknown subunit composition (P2Xcilia) which is modulated by Na+ and by long exposures to ATP. P2Xcilia was investigated by recording currents from freshly dissociated rabbit airway ciliated cells with the patch‐clamp technique in the whole‐cell configuration. During the initial continuous exposure to extracellular ATP, P2Xcilia currents gradually increase in magnitude (priming), yet the permeability to N‐methyl‐d‐glucamine (NMDG) does not change, indicating that priming does not arise from a progressive change in pore diameter. Na+, which readily permeates P2Xcilia receptor channels, was found to inhibit the channel extracellular to the electric field. The rank order of permeability to various monovalent cations is: Li+, Na+, K+, Rb+, Cs+, NMDG+ and TEA+, with a relative permeability of 1.35, 1.0, 0.99, 0.91, 0.79, 0.19 and 0.10, respectively. The rank order for the alkali cations follows an Eisenman series XI for a high‐strength field site. Ca2+ has been estimated to be 7‐fold more permeant than Na+. The rise in [Ca2+]i in ciliated cells, induced by the activation of P2Xcilia, is largely inhibited by either Brilliant Blue G or KN‐62, indicating that P2X7 may be a part of P2Xcilia. P2Xcilia is augmented by Zn2+ and by ivermectin, and P2X4 receptor protein is detected by immunolabelling at the basal half of the cilia, strongly suggesting that P2X4 is a component of P2Xcilia receptor channels. Taken together, these results suggest that P2Xcilia is either assembled from P2X4 and P2X7 subunits, or formed from modified P2X4 subunits.

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 sexual shift induced by silencing of a single insulin-like gene in crayfish: ovarian upregulation and testicular degeneration.

In sequential hermaphrodites, intersexuality occurs naturally, usually as a transition state during sexual re-differentiation processes. In crustaceans, male sexual differentiation is controlled by the male-specific androgenic gland (AG). An AG-specific insulin-like gene, previously identified in the red-claw crayfish Cherax quadricarinatus (designated Cq-IAG), was found in this study to be the prominent transcript in an AG cDNA subtractive library. In C. quadricarinatus, sexual plasticity is exhibited by intersex individuals in the form of an active male reproductive system and male secondary sex characters, along with a constantly arrested ovary. This intersexuality was exploited to follow changes caused by single gene silencing, accomplished via dsRNA injection. Cq-IAG silencing induced dramatic sex-related alterations, including male feature feminization, a reduction in sperm production, extensive testicular degeneration, expression of the vitellogenin gene, and accumulation of yolk proteins in the developing oocytes. Upon silencing of the gene, AG cells hypertrophied, possibly to compensate for low hormone levels, as reflected in the poor production of the insulin-like hormone (and revealed by immunohistochemistry). These results demonstrate both the functionality of Cq-IAG as an androgenic hormone-encoding gene and the dependence of male gonad viability on the Cq-IAG product. This study is the first to provide evidence that silencing an insulin-like gene in intersex C. quadricarinatus feminizes male-related phenotypes. These findings, moreover, contribute to the understanding of the regulation of sexual shifts, whether naturally occurring in sequential hermaphrodites or abnormally induced by endocrine disruptors found in the environment, and offer insight into an unusual gender-related link to the evolution of insulins.

Androgenic gland implantation promotes growth and inhibits vitellogenesis in Cherax quadricarinatus females held in individual compartments

Summary Androgenic glands (AGs) were implanted into young female red claw crayfish, Cherax quadricarinatus, with the aim of investigating the role played by the AG in the balance between growth and reproduction under conditions of minimal social interaction (individual compartments). The growth rate of the females with AG implants was significantly higher than that of the control non-implanted females (0.11 ± 0.03 g/day vs. 0.08 ± 0.02 g/day). This difference was attributed to the larger molt increments and slightly shorter molt intervals of the females with implants vs. the control females. At the end of the experiment (538 days), the mean weight of the implanted females was significantly higher than that of the control females (64.58 ± 18.24 g vs. 51.07 ± 12.71 g, respectively), a lead of 26.4% for the implanted females that started 91 days after implantation and became significant at 153 days after implantation. By that time, 55.5% of the implanted females had developed typical male secondary characters, such as the red patch on the propodus. The shift of energy from female reproduction to growth was further demonstrated by the level of expression of the vitellogenin gene in the hepatopancreas: gene expression was high in control females but lower by several orders of magnitude in the AG-implanted females, as shown by real time RT-PCR relative quantification. Confirmation of these findings was provided by an ELISA test, which showed that the level of vitellogenic cross-reactive protein in the hemolymph of AG-implanted females was significantly lower than that in intact females. The significant growth promotion in AG-implanted females was clearly not due to social interaction. It may be attributed to a direct growth factor—like the effect of androgens in vertebrates—in combination with an indirect effect, through the shift of energetic investment from reproduction and vitellogenesis to growth. Since the AG implant had a more marked effect on molt increment than on molt interval, it seems likely that the AG acts as a growth promoter rather than as a molt enhancer.

Timing Sexual Differentiation: Full Functional Sex Reversal Achieved Through Silencing of a Single Insulin-Like Gene in the Prawn, Macrobrachium rosenbergii

ABSTRACT In Crustacea, an early evolutionary group (∼50 000 species) inhabiting most ecological niches, sex differentiation is regulated by a male-specific androgenic gland (AG). The identification of AG-specific insulin-like factors (IAGs) and genomic sex markers offers an opportunity for a deeper understanding of the sexual differentiation mechanism in crustaceans and other arthropods. Here, we report, to our knowledge, the first full and functional sex reversal of male freshwater prawns (Macrobrachium rosenbergii) through the silencing of a single IAG-encoding gene. These “neofemales” produced all-male progeny, as proven by sex-specific genomic markers. This finding offers an insight regarding the biology and evolution of sex differentiation regulation, with a novel perspective for the evolution of insulin-like peptides. Our results demonstrate how temporal intervention with a key regulating gene induces a determinative, extreme phenotypic shift. Our results also carry tremendous ecological and commercial implications. Invasive and pest crustacean species represent genuine concerns worldwide without an apparent solution. Such efforts might, therefore, benefit from sexual manipulations, as has been successfully realized with other arthropods. Commercially, such manipulation would be significant in sexually dimorphic cultured species, allowing the use of nonbreeding, monosex populations while dramatically increasing yield and possibly minimizing the invasion of exotic cultured species into the environment.

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.

Search for hepatopancreatic ecdysteroid-responsive genes during the crayfish molt cycle: from a single gene to multigenicity

SUMMARY The expression of the vitellogenin gene of the red-claw crayfish Cherax quadricarinatus (CqVg) was previously demonstrated in male crayfish during an endocrinologically induced molt cycle. The hypothesis that this expression is under the direct control of ecdysteroids was tested in this study both in vivo and in vitro. Unlike vitellogenin of insects, CqVg was not found to be ecdysteroid-responsive. Thus, a multigenic approach was employed for the identification of other hepatopancreatic ecdysteroid-responsive genes by a cDNA microarray. For the purposes of this study, a multi-parametric molt-staging technique, based on X-ray detection of gastrolith growth, was developed. To identify ecdysteroid-responsive genes during premolt, the molt cycle was induced by two manipulations, 20-hydroxyecdysone administration and X-organ–sinus gland complex removal; both resulted in significant elevation of ecdysteroids. Two clusters of affected genes (129 and 122 genes, respectively) were revealed by the microarray. It is suggested that only genes belonging to similarly responsive (up- or downregulated) gene clusters in both manipulations (102 genes) could be considered putative ecdysteroid-responsive genes. Some of these ecdysteroid-responsive genes showed homology to genes controlling chitin metabolism, proteases and other cellular activities, while 56.8% were unknown. The majority of the genes were downregulated, presumably by an energetic shift of the hepatopancreas prior to ecdysis. The effect of 20-hydroxyecdysone on representative genes from this group was confirmed in vitro using a hepatopancreas tissue culture. This approach for ecdysteroid-responsive gene identification could also be implemented in other tissues for the elucidation of ecdysteroid-specific signaling pathways during the crustacean molt cycle.