Salinity-dependent expression of calcium-sensing receptors in Atlantic salmon (Salmo salar) tissues.

Multiple reports suggest that calcium-sensing receptors (CaSRs) are involved in calcium homeostasis, osmoregulation, and/or salinity sensing in fish (Loretz 2008, Herberger and Loretz 2013). We have isolated three unique full-length CaSR cDNAs from Atlantic salmon (Salmo salar) kidney that share many features with other reported CaSRs. Using anti-CaSR antibodies and PCR primers specific for individual salmon CaSR transcripts we show expression in osmoregulatory, neuroendocrine and sensory tissues. Furthermore, CaSRs are expressed in different patterns in salmon tissues where mRNA and protein expression are modified by freshwater or seawater acclimation. For example, in seawater, CaSR mRNA and protein expression is increased significantly in kidney as compared to freshwater. Electrophysiological recordings of olfactory responses produced upon exposure of salmon olfactory epithelium to CaSR agonists suggest a role for CaSRs in chemoreception in this species consistent with other freshwater, anadromous, and marine species where similar olfactory responses to divalent and polyvalent cations have been reported. These data provide further support for a role of CaSR proteins in osmoregulatory and sensory functions in Atlantic salmon, an anadromous species that experiences a broad range of environmental salinities in its life history.

Polyvalent cation receptor proteins (CaRs) are salinity sensors in fish.

To determine whether calcium polyvalent cation-sensing receptors (CaRs) are salinity sensors in fish, we used a homology-based cloning strategy to isolate a 4.1-kb cDNA encoding a 1,027-aa dogfish shark (Squalus acanthias) kidney CaR. Expression studies in human embryonic kidney cells reveal that shark kidney senses combinations of Ca(2+), Mg(2+), and Na(+) ions at concentrations present in seawater and kidney tubules. Shark kidney is expressed in multiple shark osmoregulatory organs, including specific tubules of the kidney, rectal gland, stomach, intestine, olfactory lamellae, gill, and brain. Reverse transcriptase-PCR amplification using specific primers in two teleost fish, winter flounder (Pleuronectes americanus) and Atlantic salmon (Salmo salar), reveals a similar pattern of CaR tissue expression. Exposure of the lumen of winter flounder urinary bladder to the CaR agonists, Gd(3+) and neomycin, reversibly inhibit volume transport, which is important for euryhaline teleost survival in seawater. Within 24-72 hr after transfer of freshwater-adapted Atlantic salmon to seawater, there are increases in their plasma Ca(2+), Mg(2+), and Na(+) that likely serve as a signal for internal CaRs, i.e., brain, to sense alterations in salinity in the surrounding water. We conclude that CaRs act as salinity sensors in both teleost and elasmobranch fish. Their tissue expression patterns in fish provide insights into CaR functions in terrestrial animals including humans.

Stage-dependent accumulation of cadmium and induction of metallothionein-like binding activity in the testis of the Dogfish shark, Squalus acanthias.

Cadmium (Cd) is an established spermatotoxicant. Using the shark (Squalus acanthias) testis model, we investigated stage-related patterns of intratesticular Cd accumulation and effect. After a single injection of 109CdCl2, tracer was rapidly eliminated from plasma but accumulated and was retained in testis for at least 7 days. Intratesticular 109Cd was stage dependent, resulting in a 3- to 5-fold gradient: germinal zone (GZ) > premeiotic (PrM) > meiotic (M) > postmeiotic (PoM) stages. When measured as tissue:plasma ratios, the Cd-binding mechanism in GZ (71:1) was similar to that in liver (87:1) but lower than in kidney (381:1). The same intratesticular gradient was seen in untreated controls when tissue Cd levels were measured by atomic absorption spectroscopy, implying environmental exposure. A single CdCl2 injection (5 mg/kg i.v.) elevated testicular Cd > 160-fold in all stages but did not alter the direction or magnitude of the gradient. Intratesticular distribution of metallothionein-like Cd-binding protein was stage dependent (PrM = PoM > GZ = M), but the pattern differed from the Cd gradient. This binding component was Cd inducible in all but M stages, but induction did not alter the stage-dependent pattern of binding activity or Cd accumulation. Analysis of tissue subfractions after in vivo tracer injection indicated that the binding mechanism responsible for the intratesticular gradient is mainly cytosolic, but that a second less abundant component is associated with the nucleus. The functional significance of preferential Cd accumulation in GZ and PrM stages of spermatogenesis remains to be determined.

Negative feedback control of the spermatogenic progression by testicular oestrogen synthesis: insights from the shark testis model.

The organisation of the testis of the dogfish shark is technically advantageous for stage-by-stage analysis of spermatogenesis in vivo and in vitro. Prior studies using this model show that total oestrogen receptors (ER) are concentrated in regions where spermatocysts ("follicle-like" germ cell-Sertoli cell units) are in stem cell and spermatogonial stages: respectively, germinal zone (GZ) and premeiotic (PrM) regions. By contrast, key enzymes regulating oestrogen (E) concentrations (aromatase, 17 alpha-hydroxylase) are maximal in meiotic (M) and postmeiotic (PoM) regions, respectively, which are upstream in the intratesticular vascular pathway. To investigate the hypothesis that E is part of a signalling mechanism between stages of development, studies were undertaken to test direct effects of oestradiol-17 beta (E2) on processes in ER-rich regions. As measured by [3H]thymidine (-Tdr) incorporation. DNA synthesis in GZ and PrM regions was inhibited by E2 (0-1000 nM) in a dose-response fashion. The maximal response (30-40%) was significant, reproducible and observed within 72 hr of treatment. Insulin differentially affected DNA synthesis and the response to E2 in GZ in GZ and PrM regions. As measured by [3H]Tdr release after prelabelling spermatocysts of GZ regions, apoptosis progressively decreased with increasing concentrations of E2. At the maximal dose of E2 used, there was no effect on total protein synthesis or secretion in combined GZ/PrM cysts, indicating that effects on DNA synthesis and cell death were authentically physiological, not pharmacological, and consistent with a state of developmental arrest. These results support the hypothesis that E synthesised within the testis is part of a negative feedback regulatory mechanism whereby more mature stages regulate the developmental advance of less mature stages. A growth control mechanism of this type could explain the strict temporal, spatial and quantitative order of succeeding stages characteristic of normal spermatogenesis in all vertebrates. Further study is required to determine whether E signalling in this model is restricted to Sertoli cells or has a germ cell component.

The goldfish as a model for studying neuroestrogen synthesis, localization, and action in the brain and visual system.

Organizational and activational effects of estrogen (E) in the central nervous system (CNS) are exerted directly by circulating E and indirectly after aromatization of circulating androgen to E in the brain itself. Understanding an environmental chemical's ability to disrupt E-dependent neural processes, therefore, requires attention to both pathways. Because aromatase (Aro) is highly expressed in teleost brain, when compared to mammals and other vertebrates, fish are technically advantageous for localization and regulation studies and may also provide a model in which the functional consequences of brain-derived (neuro-)E synthesis are exaggerated. Recently, Aro was immunolocalized in cell bodies and fiber projections of second- and third-order neurons of the goldfish retina and in central visual processing areas. Authentic Aro enzyme activity was verified biochemically, suggesting a heretofore unrecognized role of sex steroids in the visual system. Initial studies show that in vivo treatment with aromatizable androgen or E increases calmodulin synthesis and calmodulin protein in retina and also affects retinal protein and DNA. Whether there are related changes in the processing of visual information that is essential for seasonal reproduction or in the generative and regenerative capacity of the goldfish visual system requires further investigation.