Ultrastructure of the male genital ducts of the clearnose skate Raja eglanteria

Tissues from the male genital ducts of six specimens of the clearnose skate Raja eglanteria, comprising the Leydig gland, upper and lower epididymis, ductus deferens and seminal vesicle, were fixed and embedded for ultrastructural examination. In the Leydig gland, two types of columnar cells were identified, one bearing microvilli, a basal nucleus and evidence of active secretion with plentiful endoplasmic reticulum and numerous secretory droplets, and the other pyriform with cilia, and swathes of cytofilaments emanating from prominent desmosomes. Occasional crystalloid intramitochondrial inclusions were seen in the first type, with a periodicity of 24 nm. The upper epididymis was composed of cuboidal cells with microvilli and cilia and irregular electron dense granules, some of which were basally situated and extremely large, often within cells resembling intraepithelial leucocytes; such cells were also seen in the stroma underlying the epithelium. The lower epididymis cells also bore microvilli and cilia and were heavily vacuolated with fatty inclusions as well as the granule-laden leucocytes seen previously. In the ductus deferens, cells had masses of long cilia with occasional microvilli; endoplasmic reticulum was well developed, forming complex arrays with sparse secretory droplets and basal mitochondria. In the seminal vesicle there were two cell types, the most common having long cilia and short microvilli and an occasional, paler cell with supranuclear accumulations of small, round mitochondria. These ultrastructural appearances have been related to cell glycosylation and functions including protein secretion, water absorption and waste removal, and illustrate how structure and function vary down the length of the genital tract.     

Mating behavior,egg deposition,incubation period,and hatching in the clearnose skate,Raja eglanteria

Synopsis Adult clearnose skates, Raja eglanteria, were captured during the winters of 1981 and 1983, and observed to mate in captivity. Mating and egg depositions take place on the central west coast of Florida from December through mid-May. During copulation the male holds the trailing edge of the female's right or left pectoral fin firmly in his mouth, swings his tail beneath hers and inserts one clasper into the distal end of her reproductive tract. Copulation may last one to four hours during which time sperm pass from the urogenital papilla of the male along the clasper groove to the female. Sperm move cranially to the upper portion of the shell gland where they are stored and remain viable for at least three months. The ovum is fertilized in the shell gland. The egg case bears a prominent projection or horn at each corner. The two posterior ones are shorter and bear tendrils which are covered with a sticky substance that insures attachment to the substrate when the egg is deposited. Fertilized eggs are laid in pairs at intervals ranging from 1 to 13 days (mean of 4.5 ± 2.2 days). As development proceeds within the egg case a plugged slit on the lateral side of each horn opens and permits seawater to wash the developing embryo. Incubation periods for eggs maintained between 20–22°C decrease in duration throughout the egg laying season, ranging from 94 days initially to 77 days for eggs laid later in the spring. At hatching, the anterior end of the egg case ruptures, and the skate emerges abruptly with its pectoral fins rolled dorsally.     

Glycosylation of the male genital ducts and spermatozeugmata formation in the clearnose skate Raja eglanteria

Genital ducts of three male Raja eglanteria were fixed and embedded in epoxy and methacrylate resin. Epoxy resin sections from the Leydig gland, upper and lower epididymis, ductus deferens and seminal vesicle were stained with 20 labelled lectins to examine their glycosylation. The Leydig gland consisted of columnar epithelial cells expressing N-linked glycans, N-acetyl galactosamine, glucosamine and lactosamine residues and sialic acid. Interspersed were ciliated cells of a different glycotype. The upper epididymis of cuboidal epithelium had a strongly glycosylated, ciliated apical surface and cytoplasmic granules that stained heavily with many lectins, with increased glycosylation compared to the Leydig gland. In the lower epididymis, tall, vacuolated cells showed some differences and a slight reduction in lectin staining. The ductus deferens contained two cell types and showed increased terminal N-acetyl galactosamine. The ciliated cuboidal epithelium of the seminal vesicle had marked differences from the ductus epithelium, with decreased N-acetyl galactosamine and lactosamine expression but increased subterminal N-acetyl lactosamine and galactosamine expression and sialylation. Spermatozoa were suspended in a glycosylated matrix and, in the seminal vesicle, were embedded in solid masses of matrix forming spermatozeugmata. These data show changes in glycan expression along the male genital tract, probably related to the nurture and maturation of the spermatozoa as they travel towards the seminal vesicle.     

Scanning electron microscopic observations on the inner ear of the skate,Raja ocellata

The inner ear of the skate, Raja ocellata, was examined by scanning electron microscopy. The otolithic membranes have a gelatinous component and an endogenous class of otoconia. Cupulae are reticulate in form. The morphology and polarization of sensory cell hair bundles are described for the various regions of the labyrinth, and are compared with published observations on other species. In the otolithic maculae, the more centrally located receptor cells generally have longer sterecolia than the peripheral cells. The hair bundles of the lacinia are similar to those of the central portion of the sacculus and differed from those of the rest of the utricular macula. Hair bundles in the peripheral regions of all maculae and cristae are similar. The polarization pattern of the utriculus is similar to that of teleosts, while that of the lagena is less clearly dichotomized. The receptor cells of most of the sacculus are oriented in a bivertical direction, with cells in the anterior portion, and a few in the posterior region, being aligned longitudinally. The significance of morphology and polarization with respect to the functions of the otolithic organs is discussed. The relationship of cell processes of the ampullary receptors to the cupula is briefly considered.     

Alterations in serum steroid concentrations in the clearnose skate, Raja eglanteria: correlations with season and reproductive status.

Serum steroid hormones in the peripheral circulation of the clearnose skate, Raja eglanteria, were measured at the time of capture and at various times throughout the year while the animals were maintained as a captive breeding population. These analyses demonstrate interesting correlations between changes in hormone concentrations and annual reproductive events. Animals were sampled once (78 females, 20 males) or multiple times (15 females). For both groups of females, 17beta-estradiol was detected throughout the year with significant elevations occurring during October and November when ovarian follicles begin to mature (as determined through necropsy examinations), and January and February when maximum mating activity is observed and egg laying begins. Testosterone and dihydrotestosterone concentrations were significantly elevated in females only during January and February. Testosterone elevations were synchronous with longer-term elevations in 17beta-estradiol in females sampled either once or repetitively. Testosterone concentrations in males were significantly elevated during times of maximum breeding activity compared to periods of sexual inactivity. Data from females sampled during five stages of the egg laying process, as defined by the position of palpable egg capsules within the reproductive tract, revealed that 17beta-estradiol was highest when egg capsules were forming in the nidamental gland (stage 2) or uterus (stage 3); testosterone and dihydrotestosterone were maximal when eggs were in the uterus (stage 3) or cloaca (stage 4); and progesterone was significantly elevated immediately after oviposition (stage 5), suggesting a possible role for progesterone in the regulation of sequential laying of egg pairs. J. Exp. Zool. 284:575-585, 1999.     

Gastro-intestinal handling of water and solutes in three species of elasmobranch fish,the white-spotted bamboo shark,Chiloscyllium plagiosum,little skate,Leucoraja erinacea and the clear nose skate Raja eglanteria

The present study reports aspects of GI tract physiology in the white-spotted bamboo shark, Chiloscyllium plagiosum, little skate, Leucoraja erinacea and the clear nose skate, Raja eglanteria. Plasma and stomach fluid osmolality and solute values were comparable between species, and stomach pH was low in all species (2.2 to 3.4) suggesting these elasmobranchs may maintain a consistently low stomach pH. Intestinal osmolality, pH and ion values were comparable between species, however, some differences in ion values were observed. In particular Ca²⁺ (19.67 ± 3.65 mM) and Mg²⁺ (43.99 ± 5.11 mM) were high in L. erinacea and Mg²⁺ was high (130.0 ± 39.8 mM) in C. palgiosum which may be an indication of drinking. Furthermore, intestinal fluid HCO₃^? values were low (8.19 ± 2.42 and 8.63 ± 1.48 mM) in both skates but very high in C. plagiosum (73.3 ± 16.3 mM) suggesting ingested seawater may be processed by species-specific mechanisms. Urea values from the intestine to the colon dropped precipitously in all species, with the greatest decrease seen in C. plagiosum (426.0 ± 8.1 to 0 mM). This led to the examination of the molecular expression of both a urea transporter and a Rhesus like ammonia transporter in the intestine, rectal gland and kidney in L. erinacea. Both these transporters were expressed in all tissues; however, expression levels of the Rhesus like ammonia transporter were orders of magnitude higher than the urea transporter in the same tissue. Intestinal flux rates of solutes in L. erinacea were, for the most part, in an inward direction with the notable exception of urea. Colon flux rates of solutes in L. erinacea were all in an outward direction, although absolute rates were considerably lower than the intestine, suggestive of a much tighter epithelia. Results are discussed in the context of the potential role of the GI tract in salt and water, and nitrogen, homeostasis in elasmobranchs.     

Effects of insulin-like growth factor-I, corticosterone, and 3,3', 5-tri-iodo-L-thyronine on glycosaminoglycan synthesis in vertebral cartilage of the clearnose skate, Raja eglanteria.

Effects of insulin-like growth factor-I (IGF-I), corticosterone, and triiodothyronine (T(3)) on in vitro growth of vertebral cartilage of the clearnose skate, Raja eglanteria, were investigated. Uptake of [(35)S]sulfate in cultured vertebrae was used to characterize glycosaminoglycan (GAG) synthesis and cartilage growth. IGF-I significantly enhanced cartilage growth when concentrations of 1.28 and 12.8 nM were present in the culture system. Corticosterone significantly inhibited vertebral GAG synthesis at concentrations of 1, 10, and 100 nM. This effect was markedly pronounced in cartilage exposed to 1 and 10 nM corticosterone, in which GAG synthesis was virtually ceased. In contrast, T(3) (0.75, 7.5, and 75.0 nM) had no significant effect on sulfate uptake. These data suggest that IGF-I and corticosteroids may play important roles in regulating skeletal growth of elasmobranchs, as they appear to do in other vertebrates. While T(3) does not appear to exert an immediate, direct effect on vertebral growth, it may still influence elasmobranch chondrogenesis over longer culture periods or indirectly through other regulatory pathways. Thus, further information is necessary to characterize the role of thyroid hormones in the skeletal growth of these fishes. The present study is the first in vitro investigation on the hormonal regulation of elasmobranch cartilage growth. As such, the methods described herein provide a useful technique for examining these physiological processes. J. Exp. Zool. 284:549-556, 1999.     

Nitric oxide production by nurse shark (Ginglymostoma cirratum) and clearnose skate (Raja eglanteria) peripheral blood leucocytes

Reactive nitrogen intermediates, such as nitric oxide (NO), are important immunomodulators in vertebrate immune systems, but have yet to be identified as mediators of host defence in any member of class Chondrichthyes, the cartilaginous fishes. In the present study, production of NO by nurse shark (Ginglymostoma cirratum) peripheral blood leucocytes (PBL) stimulated with bacterial cell wall lipopolysaccharide (LPS) was investigated. PBL were cultured for 24 to 96 h following stimulation with LPS at concentrations ranging from 0 to 25 microg ml(-1), in both serum-supplemented and serum-free culture conditions. Production of NO was measured indirectly using the Griess reaction, with maximal NO production occurring after 72 h using 10% FBS and 10 microg LPS ml(-1). Application of these culture conditions to PBL from another cartilaginous fish (clearnose skate, Raja eglanteria) resulted in a similar NO response. Addition of a specific inhibitor of inducible nitric oxide synthase (iNOS), L-N(6)-(1-iminoethyl)lysine (L-NIL), resulted in a significant decrease in the production of NO by PBL from both species.     

Flow cytometric DNA analysis of nurse shark, Ginglymostoma cirratum (Bonaterre) and clearnose skate, Raja eglanteria (Bosc) peripheral red blood cells

Flow cytometric DNA analysis was used to determine the DNA content of red blood cells from Ginglymostoma cirratum (Bonaterre) and Raja eglanteria (Bosc). The DNA content/nucleus was calculated to be 7.6 and 7.1 pg/nucleus, respectively. Peripheral red blood cells obtained both from G. cirratum and from R. eglanteria were demonstrated to be actively cell cycling. Percentages of red blood cells in the phases of the cell cycle ( G ₀/ G ₁, S , and G ₂/ M ) not only varied considerably between different fishes but also were quite variable in weekly samples obtained from individual animals.     

Preliminary observations on the effects of vector-averaged gravity on the embryonic and larval development of the gastropod mollusk, Ilyanassa obsoleta Stimpson

Fertilized eggs of Ilyanassa obsoleta Stimpson were collected immediately after their deposition in egg capsules. Unopened egg capsules then were affixed to glass slides, and incubated either statically (controls) or on a clinostat (experimentals). After incubation for 9-14 days, hatching occurred sooner and in a higher percentage of clinostated capsules than in controls. Embryos that hatched while undergoing clinostat incubation were abnormal in morphology, whereas other embryos present in non-hatched capsules in the same tubes appeared normal, as did embryos in the control tubes. Although the results are compatible with a conclusion that vector-averaged gravity in the experimental tubes caused the altered development, some other aspects of how the incubations were done may have contributed to the differences between the control and experimental results.     

The hepatic glutathione transferases of the male little skate, Raja erinacea

Five cytosolic glutathione transferases were isolated from the liver of the male little skate, Raja erinacea, a marine elasmobranch. They were designated E-1 through E-5 in order of their elution from a DEAE-cellulose column with a 0 to 100 mM KCl gradient in 0.01 M Tris (pH 8.0). Each eluted peak of glutathione transferase activity, after concentration, was applied to an affinity column prepared by reaction of epoxy-activated Sepharose 6B with glutathione (GSH). Elution of the various glutathione transferases from this column with GSH resulted in the further purification of each enzyme; the major glutathione transferase, E-4 and E-1, were purified to apparent homogeneity by this procedure. Skate glutathione transferase E-4 is dimeric and the subunits are either very similar or identical in molecular weight (about 26 000 daltons). Enzymes E-2 through E-5 were acidic proteins (pI less than 7.0) and had high specific glutathione transferase activity (0.3--12 mumol/min/mg protein) with benzo[a]pyrene 4,5-oxide (BPO) as substrate, whereas the other enzyme (E-1) had low activity (0.01 mumol/min/mg) with BPO and a basic pI (greater than 9.5). Bilirubin and hematin, non-substrate ligands, bound tightly to homogeneous E-4, with dissociation constants in the micromolar range.     

The reproductive cycle of the roundel skate Raja texana

The reproductive cycle of Raja texana was determined from 63 females (510-630 mm total length, L(T)) and 81 males (355-546 mm L(T)) collected from the Gulf of Mexico. The results, based on follicle size, mature spermatocysts and gonadosomatic index (I(G)) suggests year-round reproductive activity.     

Relaxin from an oviparous species, the skate (Raja erinacea)

An acid-acetone extract prepared from ovaries of the skate, Raja erinacea, contained a weakly crossreacting molecule when tested in a pig relaxin radioimmunoassay. The material was isolated and purified to homogeneity by ion exchange chromatography, molecular exclusion chromatography, and HPLC. Analytical tests proved the molecule to consist of two chains and to have a molecular weight of 7,500. Sequence analyses of the A and B chains yielded the following sequence: Glu-Glu-Lys-Met-Gly-Phe-Ala-Lys-Lys-Cys-Cys-Ala-Ile-Gly-Cys-Ser-Thr-Glu- Asp-Phe-Arg-Met-Val-Cys and Arg-Pro-Asn-Trp-Glu-Glu-Arg-Ser-Arg-Leu-Cys-Gly-Arg-Asp-Leu-Ile-Arg-Ala- Phe- Ile-Tyr-Leu-Cys-Gly-Gly-Thr-Arg-Trp-Thr-Arg-Leu-Pro-Asn-Phe-Gly-Asn-Tyr- Pro-Ile-Met respectively. Skate relaxin has 0.2% of the activity of B29 pig relaxin in the symphysis pubis assay and 0.5% in the mouse uterine muscle strip contraction inhibition assay.     

Effects of luminal osmolarity on gastric acid secretion in the little skate,Raja erinacea

Summary Isolated gastric mucosa of the skate shows marked changes in acid secretory rate (J _H ), electrical potential difference (PD), and transepithelial resistance (R) with changes in mucosal bathing solution composition and a constant serosal solution. Removal of the 350 mM urea usually present in the mucosal solution reduces acid secretory rate by 25%, while adding urea to 1 M has no significant effect. Complete removal of osmotic solutes (distilled water) inhibits secretion by 78%, isotonic urea (no salts) inhibits by 54%, while isotonic salts alone (no urea) gives control secretory rates. The changes in PD and R are consistent with acid secretory changes. Theory and experience with terrestrial organisms would not predict these changes. The most likely explanation is osmotic swelling and shrinking of the surface cells, and occlusion of the secretory tubules in the swollen condition. Since marine species never encounter hypo- or hyperosmotic conditions due to food ingestion, their surface cells may be water permeable, unlike the situation in terrestrial and fresh water animals.Abbreviations J _H acid secretory rate per square centimetre tissue area - OC oxyntic cell - PBC pit border cell - PD transepithelial electrical potential difference - R transepithelial electrical resistance per square centimetre tissue area - SEC Surface epithelial cell     

Immunoglobulin heavy chain gene organization and complexity in the skate, Raja erinacea

Immunoglobulin heavy chain genes from Raja erinacea have been isolated by cross hybridization with probes derived from the immunoglobulin genes of Heterodontus francisci (horned shark), a representative of a different elasmobranch order. Heavy chain variable (VH), diversity (DH) and joining (JH) segments are linked closely to constant region (CH) exons, as has been described in another elasmobranch. The nucleotide sequence homology of VH gene segments within Raja and between different elasmobranch species is high, suggesting that members of this phylogenetic subclass may share one VH family. The organization of immunoglobulin genes segments is diverse; both VD-J and VD-DJ joined genes have been detected in the genome of non-lymphoid cells. JH segment sequence diversity is high, in contrast to that seen in a related elasmobranch. These data suggest that the clustered V-D-J-C form of immunoglobulin heavy chain organization, including germline joined components, may occur in all subclasses of elasmobranchs. While variation in VH gene structure is limited, gene organization appears to be diverse.     

A primitive ATP receptor from the little skate Raja erinacea

P2Y ATP receptors are widely expressed in mammalian tissues and regulate a broad range of activities. Multiple subtypes of P2Y receptors have been identified and are distinguished both on a molecular basis and by pharmacologic substrate preference. Functional evidence suggests that hepatocytes from the little skate Raja erinacea express a primitive P2Y ATP receptor lacking pharmacologic selectivity, so we cloned and characterized this receptor. Skate hepatocyte cDNA was amplified with degenerate oligonucleotide probes designed to identify known P2Y subtypes. A single polymerase chain reaction product was found and used to screen a skate liver cDNA library. A 2314-base pair cDNA clone was generated that contained a 1074-base pair open reading frame encoding a 357-amino acid gene product with 61-64% similarity to P2Y(1) receptors and 21-37% similarity to other P2Y receptor subtypes. Pharmacology of the putative P2Y receptor was examined using the Xenopus oocyte expression system and revealed activation by a range of nucleotides. The receptor was expressed widely in skate tissue and was expressed to a similar extent in other primitive organisms. Phylogenetic analysis suggested that this receptor is closely related to a common ancestor of the P2Y subtypes found in mammals, avians, and amphibians. Thus, the skate liver P2Y receptor functions as a primitive P2Y ATP receptor with broad pharmacologic selectivity and is related to the evolutionary forerunner of P2Y(1) receptors of higher organisms. This novel receptor should provide an effective comparative model for P2Y receptor pharmacology and may improve our understanding of nucleotide specificity among the family of P2Y ATP receptors.     

Kenojeinin I, antimicrobial peptide isolated from the skin of the fermented skate, Raja kenojei

An antimicrobial peptide was purified from fermented skate skin extract using the solid-phase extraction and separation on HPLC reversed-phase chromatography. Amino acid sequence of the purified peptide (Peak A) having an antimicrobial activity revealed the presence of many cationic residues of the total 28 amino acids. Its molecular mass was found to be 3059 Da. This result was in excellent agreement with the theoretical molecular mass calculated from the amino acid sequence. The synthetic kenojeinin I had inhibitory effects on B. subtilis (MIC, 12 microg/ml), E. coli (28 microg/ml), and S. cerevisiae (12 microg/ml). These results indicate that fermented skate skin is potentially antimicrobial.