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.     

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.     

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 selenate on the development of the embryonic skate, Raja eglanteria

Morphogenesis of the clearnose skate, Raja eglanteria, was not significantly inhibited as a result of 7 days of exposure to 1-2 mM selenate in the sea water during Days 59-69 of embryonic development (hatching would normally have occurred at 82 +/- 4 days of incubation). Although corneal transparency appeared normal in the eye, preliminary measurements of the thickness of Bowman's layer of the cornea suggested that it was significantly thinner in the corneas of embryos exposed to 1-2 mM selenate. Selenate is an ion reported to inhibit sulfation of glycosaminoglycans in connective tissue.     

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.     

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.     

Ultrastructure of sperm storage and male genital ducts in a male holocephalan, the elephant fish, Callorhynchus milii.

In chondrichthyes, the process of spermatogenesis produces a spermatocyst composed of Sertoli cells and their cohort of associated spermatozoa linearly arrayed and embedded in the apical end of the Sertoli cell. The extratesticular ducts consist of paired epididymis, ductus deferens, isthmus, and seminal vesicles. In transit through the ducts, spermatozoa undergo modification by secretions of the extratesticular ducts and associated glands, i.e., Leydig gland. In mature animals, the anterior portion of the mesonephros is specialized as the Leydig gland that connects to both the epididymis and ductus deferens and elaborates seminal fluid and matrix that contribute to the spermatophore or spermatozeugmata, depending on the species. Leydig gland epithelium is simple columnar with secretory and ciliated cells. Secretory cells have periodic acid-Schiff positive (PAS+) apical secretory granules. In the holocephalan elephant fish, Callorhynchus milii, sperm and Sertoli cell fragments enter the first major extratesticular duct, the epididymis. In the epididymis, spermatozoa are initially present as individual sperm but soon begin to laterally associate so that they are aligned head-to-head. The epididymis is a highly convoluted tubule with a small bore lumen and an epithelium consisting of scant ciliated and relatively more secretory cells. Secretory activity of both the Leydig gland and epididymis contribute to the nascent spermatophores, which begin as gel-like aggregations of secretory product in which sperm are embedded. Fully formed spermatophores occur in the ductus. The simple columnar epithelium has both ciliated and secretory cells. The spermatophore is regionalized into a PAS+ and Alcian-blue-positive (AB+) cortex and a distinctively PAS+, and less AB+ medulla. Laterally aligned sperm occupy the medulla and are surrounded by a clear zone separate from the spermatophore matrix. Grossly, the seminal vesicles are characterized by spiral partitions of the epithelium that project into the lumen, much like a spiral staircase. Each partition is staggered with respect to adjacent partitions while the aperture is eccentric. The generally nonsecretory epithelium of the seminal vesicle is simple columnar with both microvillar and ciliated cells.     

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.     

In vivo exposure of clearnose skates, Raja eglanteria, to ionising X-radiation: acute effects on the peripheral blood, spleen, and epigonal and Leydig organs

The effects of ionising radiation on the peripheral blood, spleen, and epigonal and Leydig organs of cartilaginous fishes were investigated using juvenile clearnose skates, Raja eglanteria. Skates (N = 80) were sacrificed 12 days after exposure to 0-75 Gy of X-radiation, and morphometrics (body mass, disc width, total length), mass of spleens and epigonal organs, and peripheral blood leucocyte (PBL) counts were compared to controls using ANOVA. Spleen and epigonal organ mass and PBL counts declined logarithmically as a function of radiation dose. To assess recovery from X-radiation, skates (N = 40) were exposed to 0, 9 or 18 Gy and sacrificed when moribund or on days 10, 20, 30 and 40 post-irradiation. Partial recovery of Leydig organ and splenic red pulp was evident after 40 days in skates exposed to 9 Gy, but no indication of recovery was apparent at higher doses. Median lethal dose by 30 days (LD50/30) was calculated to be 9-18 Gy, similar to that determined for other fishes.     

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.     

Identification of vitellogenin in the little skate (Raja erinacea).

1. Vitellogenin was isolated from mature female skates by selective precipitation with MgCl2/EDTA followed by chromatography on DEAE-cellulose columns. 2. A single monomer of approximately 205 kDa was identified on 6.0% SDS-PAGE gels. 3. In addition, isolation of yolk proteins with ammonium sulfate yielded proteins of 94 and 38 kDa (putative phosvitins) and putative lipovitellins of ca 105, 91 and 67 kDa. 4. In vivo phosphate incorporation in female and male skates implanted with estradiol indicated that vitellogenin was phosphorylated. 5. Total protein phosphate incorporation was significantly higher in females than male skates. 6. In male skates treated with estradiol, phosphate incorporation increased from 2 days after implantation to a maximum at approximately 11 days after implantation. 7. Determination of the rate of disappearance of 32P-labeled protein suggests a half-life of ca 200 hr in normal female skate plasma.     

Ultrastructure of genital system ducts of Diphyllobothrium latum (Cestoda:Pseudophyllidae): the ducts of the female reproductive system

The components of the female reproductive system of Diphyllobothrium latum, including ovary, ovicapt, oviduct, vitelline ducts, vitelline reservoir, vagina, seminal receptacle, ootype with unicellular Mehlis's gland, ootype-uterine duct and uterus were observed with the electron microscope. The epithelium of the female reproductive system ducts consists of a nucleate syncytial layer. Structural differences in apical surface of the ducts, the number of nuclei and organoids in syncytial layer as well as the number of underlaid muscles were revealed. The regional differentiations of the uterus wall were registered. The middle and distal region of uterus was covered with microtriches. The filamentous microtriches were observed in apical surface of vagina. The epithelium of seminal receptacle and distal region of uterus were underlaided by the powerful muscle layers. The fertilization canal was revealed. It was shown that the formation of egg shells implemented by the deposit of vitelline globules in their surface in the ootype-uterine duct. Structural and functional differences of different parts of female apparatus in various groups of cestodes are conditioned by species biology.     

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.     

scyllo-inositol and myo-inositol levels in tissues of the skate Raja erinacea.

1. Many organs of the skate Raja erinacea have been found to contain scyllo-inositol levels that are much higher than myo-inositol, the opposite to that found in mammals. 2. Both inositols were found in all skate organs studied. 3. myo-Inosose-2 was found to accompany the inositols in many of these organs.     

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.     

Neuropharmacological analysis of synaptic transmission in the Lorenzinian ampulla of the skate Raja clavata

Summary Dissected ampullae of Lorenzini of the skate (Raja clavata) were studied with the aim of determining the synaptic transmitter between electroreceptor cell and afferent fibre. Resting activity and stimulus-evoked activity in response to electrical pulses were recorded in single afferent units at constant perfusion with normal and test solutions containing different putative neurotransmitters. Presynaptic transmitter release was blocked by Mg²⁺ (up to 50 mM) to investigate the effects of the test substances upon the postsynaptic membrane. l-Glutamate (l-GLU) and l-aspartate (l-ASP), both at concentrations between 10^-7 and 10^-3 M, enlarged strongly resting and stimulus-evoked discharge frequency in the afferent fibre. If transmission was blocked by high Mg²⁺, resting discharge frequency could be restored by l-GLU or l-ASP. The glutamate agonists quisqualate (10^-8–10⁵ M) and N-methyl-D-aspartate (10^-5–10^-3 M) enlarged spontaneous activity in the afferent fiber. The same was found for kainic acid (10^-9–10^-5 M). Taurine at concentrations between 10^-5 and 10^-3 M caused a concentration-dependent decrease in afferent activity. The same was found for gammaaminobutyric acid (GABA; 10^-5–10^-4 M), and for the catecholamines adrenaline and noradrenaline, both in concentrations between 10^-5 and 10^-3 M. Serotonine (10^-5–10^-3 M) and dopamine (10^-5-10^-3 M) had no effect on resting or evoked activity in the Lorenzinian ampulla afferents. Acetylcholine (ACh; 10^-4 M) enlarged discharge frequency in those units with initial rates lower than 22–25 Hz, but diminished discharge frequency in fibres with initial activity higher than 25 Hz. When synaptic transmission was blocked by high Mg²⁺ solution, perfusion with additional ACh did not restore resting activity in the afferent fibre. The results suggest that the most probable transmitter in the afferent synapse of the ampullae of Lorenzini is l-GLU or l-ASP, or a substance of similar nature.Abbreviations ACh acetylcholine - GABA gamma aminobutyric acid - KA kainic acid - l-ASP l-aspartate - l-GLU l-glutamate - NMDA N-methyl-D-aspartate - Q quisqualate - n.s. normal solution     

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.     

Structure and functions of the genital ducts of the male Port Jackson shark,Heterodontus portusjacksoni

Synopsis The genital ducts ofHeterodontus portusjacksoni consist of the sperm carrying ducts (the rete testis, ductuli efferentes, and initial and terminal segments of the ductus epididymidis) and the Leydig glands (anterior opisthonephros). The ducts are lined by a ciliated epithelium which maintains a barrier to the transport of solute between blood and the lumen of the duct. Spermatozoa, Sertoli cell bodies, Sertoli cell cytoplasts and cellular debris are released from spermatocysts into the longitudinal canal of the rete testis. However, only the Sertoli cell cytoplasts persist throughout the sperm ducts. The epithelia lining the initial segment of the ductus epididymidis and secretory tubules of the Leydig glands are specialized for protein secretion and (particularly the Leydig glands) must be the main source of luminal protein in the ductus epididymidis. The epithelium lining the terminal segment of the ductus epididymidis also secretes protein, reabsorbs fluid and sodium, and may carry out heterophagic digestion. Spermatozoa develop the capacity for motility in the extratesticular sperm ducts, but do not undergo structural changes. However, they form spherical bundles in the terminal segment of the ductus epididymidis. It is suggested that the reduction in ratio of sodium:potassium from 48:8 in the ductuli efferentes to 3:4 in the distal end of the terminal segment of the ductus epididymidis may favour sperm survival.