Cortical Rod Research Articles

Molecular cloning and functional expression of the Penaeus monodon 5-HT receptor

Serotonin (5-HT) mediates a number of diverse physiological functions in crustaceans by interacting with various 5-HT receptor subtypes. A putative 5-HT receptor cloned from the ovary of the black tiger prawn ( Penaeus monodon) consisted of 2291 nucleotides, encoding a putative 5-HT 1Pem receptor protein of 591 amino acids. Transient expression of 5-HT 1Pem in HEK293 cells demonstrated a saturable [ 3H]-5-HT binding with a Kd of 10.43 ± 1.13 nM and B max of 1.53 ± 0.06 pmol/mg. The putative 5-HT 1Pem receptor is expressed in all tissues examined and is constitutively expressed in the ovary during ovarian maturation and spent phase. Polyclonal antibodies against the third intracellular loop (i3 loop) of the 5-HT receptor showed that the 5-HT 1Pem receptor protein was expressed in the trabeculae of ovarian stages 1 and 2 but on the cortical rod and surrounding the oocyte membrane of stages 3 and 4, suggesting that receptor localization plays a critical role in regulating ovarian maturation and spawning in penaeus shrimp.

Expression of vitellogenin and cortical rod proteins during induced ovarian development by eyestalk ablation in the kuruma prawn, Marsupenaeus japonicus

In penaeid shrimp species, ovarian development is characterized by the accumulation of a major yolk protein (vitellin) and the formation of cortical rods in the oocytes. The process is considered to be under the control of a neuroendocrine organ in the eyestalk (the X-organ sinus gland complex). In the present study, the synthesis of vitellogenin (VTG, precursor of vitellin) and two kinds of cortical rod proteins (cortical rod protein, CRP; thrombospondin, MjTSP) was induced by bilateral eyestalk ablation (removal of the X-organ sinus gland complex) in immature female kuruma prawn, Marsupenaeus japonicus, and the synthesis process was monitored over a 7-day period after the ablation. The ovarian weight and hemolymph VTG levels increased in the ablated females. The VTG mRNA levels in the ovary increased concomitantly with vitellin accumulation in the ovary after eyestalk ablation. On the other hand, the CRP and MjTSP protein levels in the ovary increased after eyestalk ablation, whereas the CRP and MjTSP mRNA levels in the ovary did not change concomitantly. The results suggest that the regulatory mechanism of gene expression by eyestalk hormones is different between VTG (transcriptional control) and CRP-MjTSP (translational control).

Identification and molecular characterization of a peritrophin-like protein from fleshy prawn ( Fenneropenaeus chinensis)

Peritrophin, one of the components of the peritrophic matrix, was first isolated from the intestine of insects. It is thought to protect insects from invasion of microorganisms and to stimulate digestion of food. Peritrophin-like proteins have also been found in crustaceans, as a component of the egg layer. In this study, one fragment of the peritrophin-like gene was obtained from fleshy prawn (Chinese shrimp) ( Fenneropenaeus chinensis) by panning the T7 phage display library constructed with the shrimp hemocyte cDNA. The total sequence of the peritrophin cDNA was cloned by modified SMART cDNA and LD-PCR methods. The full cDNA is 1048 bp and the deduced protein is composed of 274 amino acids, including 21 amino acid signal peptide, and four peritrophin A domains and the latter three forming three chitin-binding domains. Similarity analysis results showed that the peritrophin-like protein from F. chinensis has significant similarities with peritrophin-like and cortical rod proteins from other shrimp. It was inducing expression in hemocytes, heart, stomach, gut, and gills of the infected shrimp, and constitutive expression in the ovaries. No expression signal was detected in the hepatopancreas of either infected or noninfected shrimp. The recombinant peritrophin-like protein has the activity of binding Gram-negative bacteria and strong binding activity to chitin. Therefore, the bacteria and chitin binding activities of the peritrophin-like protein suggest that it may plays a role in immune defense and other physiological resposes.

Localization and Developmental Expression of mRNA for Cortical Rod Protein in Kuruma Prawn Marsupenaeus japonicus

The mature penaeid oocytes possess cortical rods that contain two related cortical rod proteins (CRP, 28.6 kDa and 30.5 kDa). In the present study, localization of CRP mRNA and gene expression profiles of CRP and vitellogenin (Vg) during ovarian development were examined in kuruma prawn, Marsupenaeus japonicus, an economically important species for shrimp and prawn farming. Northern blot analysis revealed that CRP mRNA was expressed in the ovary. In situ hybridization showed strong signals for CRP transcripts in the oocytes at early developmental stages in both immature and mature ovaries. Quantitative analysis by real-time polymerase chain reaction revealed that CRP mRNA levels were higher in the previtellogenic and endogenous (primary) vitellogenic stages than in more advanced stages. Unlike CRP mRNA, Vg mRNA levels were low in the ovary and hepatopancreas in previtellogenic females. When the ovary developed into the endogenous vitellogenic stage, ovarian Vg mRNA levels increased significantly, followed by rapid decrease in more advanced stages. The Vg mRNA levels in the hepatopancreas, on the other hand, tended to be high in the exogenous (secondary) vitellogenic and maturation stages, in which ovarian Vg mRNA levels were decreased. Our findings indicate that CRP mRNA is highly expressed before the onset of vitellogenesis, suggesting that the transcription, translation, and cortical-rod formation of CRP occur at different phases of oocyte development. The endogenous vitellogenic stage is a crucial stage for the initiation of CRP and Vg syntheses. The coincidence of these protein syntheses suggests that CRP and Vg syntheses are regulated by closely-related mechanisms.

Cathepsin C transcripts are differentially expressed in the final stages of oocyte maturation in kuruma prawn Marsupenaeus japonicus

To elucidate the molecular mechanism of oocyte maturation in the kuruma prawn ( Marsupenaeus japonicus), subtractive suppression hybridization (SSH) was initially used to identify novel up-regulated genes during the final stages of oocyte maturation, followed by evaluation of the differential expression profile by macroarray and quantitative real-time RT–PCR analyses. The cathepsin C (dipeptidyl peptidase I) gene was thus found to exhibit a significantly higher expression around the onset of cortical rod (CR) formation (early CR stage, appearance of round CRs), progress to a higher mRNA level until the middle CR stage (elongation of CRs), then rapidly revert to a low expression level at the late CR stage (occurrence of germinal vesicle breakdown, GVBD), as also observed at the non-CR stage (previtellogenesis and vitellogenesis). In situ hybridization analyses revealed that the sites of the expression of cathepsin C transcripts in the ovary were distributed in both oocyte and follicle cells, particularly at the early CR stage. A full-length cDNA sequence of this stage-specific gene was subsequently determined by rapid amplification of the cDNA 3′ and 5′ ends (3′ and 5′ RACE). The deduced amino acid sequence of the 230-residue mature peptide shared 67–70% identity to the known cathepsin C in mammals. Western blot analysis showed that expression of procathepsin C protein was exclusively at CR stages. The storage site of procathepsin C protein was localized in CRs as revealed by immunohistochemical analysis. This is the first report on the full-length cDNA sequence of cathepsin C and a demonstration of its involvement in the final stages of oocyte maturation in crustacean species.

Isolation and cDNA Cloning of Ovarian Cortical Rod Protein in Kuruma Prawn Marsupenaeus japonicus (Crustacea: Decapoda: Penaeidae)

Two cortical rod proteins having molecular weights of 28.6 kDa and 30.5 kDa were isolated from the mature ovary of Marsupenaeus japonicus using gel filtration and reversed-phase HPLC. Analysis of the N-terminal amino acid sequence of the 28.6 kDa molecule revealed that amino acid residues 1-21 corresponded to residues 9-29 of the 30.5 kDa molecule. Examination of homology using BLAST showed that 21 amino acids out of 29 residues of the 28.6 kDa molecule, and 14 out of 29 residues of the 30.5 kDa molecule were identical to that of the ovarian cortical rod proteins of Penaeus semisulcatus. Positive immunohistochemical reaction to antiserum raised against the 28.6 kDa protein was observed on cortical rods forming around the periphery of oocytes at the maturation stages. Western blotting analysis revealed that both the 28.6 kDa and 30.5 kDa molecules stained with the anti-28.6 kDa antiserum. Furthermore, the 28.6 kDa and 30.5 kDa proteins were both glycosylated, as evidenced by positive carbohydrate staining using Concanavalin A and production of positive PAS reaction. These results indicate that the cortical rods are comprised of the 28.6 kDa and 30.5 kDa molecules. We subsequently cloned two full-length cDNAs based on the N-terminal sequences of the 28.6 kDa and 30.5 kDa molecules. The open reading frame of 28.6 kDa and 30.5 kDa encoded 276 amino acid residues. Comparison analysis of the two cDNAs revealed that the location of the processing site and sequence of signal peptides differed, indicating that the two cDNAs are products of two separate genes and encode the 28.6 kDa molecule and 30.5 kDa molecule, respectively. Both proteins possessed one potential N-linked glycosylation site. It is considered that both molecules are components of the cortical rods, forming a jelly layer after fertilization.

Theoretical predictions for spatial covariance of the electroencephalographic signal during the anesthetic-induced phase transition: Increased correlation length and emergence of spatial self-organization.

In a recent series of papers, the authors have developed a stochastic theory to describe the electrical response of a spatially homogeneous cerebral cortex to infusion of a general anesthetic agent. We showed that by modeling the GABAergic (propofol-like) drug effect as a prolongation of the inhibitory postsynaptic impulse response, we obtain a prediction that there will be a hysteretically separated pair of first-order phase transitions in the population-average excitatory soma voltage, the first occurring at the point of induction of unconsciousness, and the second at the point of emergence from unconsciousness. In the present paper we generalize our earlier "zero-dimensional" homogeneous cortex to a one-dimensional (1D) line of cortical "mass," thus allowing for the possibility of spatial inhomogeneities in neural activity. Following the spirit of our earlier adiabatic ("slow membrane") philosophy, we impose a spatioadiabatic approximation that permits us to compute analytic expressions for changes in EEG (electroencephalographic) correlation length and EEG spatial covariance as a function of anesthetic effect. We establish that the correlation length of the EEG fluctuations is expected to increase at the approach to the transition points, and this finding is consistent with both the homogeneous-cortex prediction of increased correlation time ("critical slowing down") near transition, and the recent, comprehensive anesthetic study by John et al. [Conscious. Cogn. 10, 165 (2001)] reporting an increase in EEG coherence near the points of loss and recovery of consciousness. In addition, we find that if the long-range (corticocortical) excitatory-to-inhibitory connectivity in the 1D cortex is stronger than the long-range excitatory-to-excitatory connectivity, then the spatioadiabatic system can organize itself into large-amplitude spatial patterns ("dissipative structures") consisting of giant stationary quasiperiodic voltage fluctuations distributed along the cortical rod.

Ovarian development in relation to changes in the external genitalia in captive Penaeus monodon

Hatchery-reared Penaeus monodon postlarvae were cultured in ponds until they attained adult size. Thereafter, the shrimps were transferred to concrete broodstock tanks for maturation. During the culture period, the morphological changes of the ovary in relation to the development of the external genitalia and other quantitative parameters were examined. Based on histology, primordial germ cell and chromatin nucleolus in the early stages of ovarian development were reported in addition to the stages that have been described earlier on adult specimens, thus completing the description of ovarian development on P. monodon. Females with ovaries at the perinucleolus stage can be impregnated as long as the thelycum is structurally developed to receive the spermatophores. Females with cortical rod stage ovaries either spawned or resorbed their eggs before molting, indicating that the molting cycle overrides reproductive activity in first-maturing females.

The cytochemistry of oocytes of Chinese shrimpPenaeus orientalis

In the growth of oocytes ofPenaeus orientalis Kishinouye, five stages were distinguished. Histochemical tests showed the presence of DNA in the chromatin and nucleolus of the cell. The cytoplasm at the previtellogenetic stage and the nucleolus are rich in RNA and the proteins abounding with cysteine, tyrosine and tryptophan. The yolk consists mainly of proteins and phospholipids. The 1,2-glycol groups of carbohydrate occur in the cytoplasm at stage II, and aggregate mostly into cortical rods at stages IV and V. Neutral lipid droplets, and protein containing disulfides, appear in the cytoplasm at stages III and IV respectively. The proteins in the cortical rod differ from those in other components of the cell in the presence of cystine and absence of arginine.

Ovarian maturation stages of the wild giant tiger prawn, Penaeus monodon Fabricius

A qualitative and quantitative study of the ovarian maturation stages of wild-caught Penaeus monodon was conducted to refine the existing method of staging. For industrial purpose, measuring the ovarian width at the first abdominal region can minimize arbitrariness in staging. A width of 20 mm indicates readiness for spawning while reproductive performance is improved when ovarian width is 30 mm or more. Based on histology, the usual 6 to 8 stages of development can be reduced to four stages: previtellogenic, vitellogenic, cortical rod, and spent. The previtellogenic stage (P) is characterized by the predominance of oogonia and primary oocytes in the chromatin nucleolus and/or perinucleolus stage. The vitellogenic stage (V) is marked by the presence of yolky oocytes. The cortical rod stage (C) is distinguished by the appearance of yolky oocytes with spherical or rod-like bodies at the peripheral cytoplasm. The spent stage (S) can be distinguished by the presence of few oocytes with yolky substance and/or cortical rods, thicker layers of follicle cells, and few darkly-stained, irregularly shaped primary oocytes. Individuals classified as stages II+ and III+ showed similarity in size and shape of all lobes in the posterior thoracic region, and histologically, corresponded to the revised stages V and C, respectively. Histochemical staining shows that glycoproteins and lipids are absent in the primary oocytes and present in yolky oocytes. Glycoproteins but not lipids occur in the cortical rods. Analysis of variance showed significant differences in mean gonad weight and gonadosomatic index values but not in the mean body length and body weight values in stages P to C. Uniformity in the number, stage and composition of oocytes in the four regions of the ovary at each stage showed that stage of ovarian maturity in wild prawns can be represented by any region of the ovary. Differences in the oocyte size frequency and mean values of average and maximum oocyte diameter in the four stages showed that these are good indicators of stage of maturation in wild P. monodon.

MORPHOLOGY OF THE CORTICAL REACTION IN THE EGGS OFPENAEUS AZTECUS

The morphology of large cortical specializations (rods) located in the peripheral ooplasm is described in oocytes of the shrimp Penaeus aztecus and confirmed with P. setiferus. The rods, which are perpendicular to the oolemma, are club-shaped and approximately 39.9 µm long, with an apical diameter of 9.3 µm and a basal diameter of 4.6 µm. They are composed of numerous, tightly packed, fibrillar structures. Each cortical rod lies within a partially membrane-bound crypt and is separated from the external media by a thin investment coat. The investment coat lies directly adjacent to the oolemma and completely surrounds the egg. Upon exposure to sea water, the cortical rods begin to emerge from the crypts and appear to lift the investment coat off the egg surface. A corona is formed around the oocyte as the rods are expelled, but quickly dissipates. Extensive membrane vesiculation associated with cortical rod crypts is apparent at this time.

A Mg++ dependent cortical reaction in the eggs of penaeid shrimp

AbstractMature penaeid oocytes contain large cortical specializations (rods) in their cortex. These rods reside in membrane crypts and are isolated from the external environment by a thin investment coat which surrounds the oocyte. When oocytes contact seawater, at spawning, their cortical rods are expelled and form a striking investment layer which subsequently dissipates. During cortical rod dissipation a hatching membrane forms around the egg. Oocytes spawned into Mg++ free seawater do not exhibit cortical rod explusion. If oocytes spawned into Mg++ free seawater are returned to normal seawater cortical rod explusion proceeds, however the expelled rods do not dissipate.

Light and fine Structural Studies of Natural and Artificially Induced Egg Growth of Penaeid Shrimp1

ABSTRACTEgg differentiation in penaeids induced to undergo gonad maturation by eyestalk ablation has been examined with light and electron microscopes.Controls consisted of both wild brown and white shrimp (Penaeus aztecus and P. setiferus) caught in various stages of ovarian maturation. These animals were sacrificed and ovarian tissues were excised and fixed in a cold (4 C) aldehyde mixture, post‐fixed in osmic acid, dehydrated in acetone, and embedded in an epoxy resin. Experimental animals included laboratory‐reared, wild immature, and wild mature animals which had resorbed ovarian tissues. These animals were ablated with hot surgical clamps and sacrificed at various time intervals. Tissues were prepared as described for the controls.The development of ova from experimental animals was compared to control animals. In the stages early oocyte, cisternal phase, platelet phase, cortical rod phase, and mature oocyte, the experimental oocytes appeared the same as the control tissues. Though further studies (e.g., biochemical and zygote development) are needed, these preliminary fine structural studies are encouraging. While ablation, a mechanical process, may not be the technique of choice for maturing shrimp, it does indicate that manipulation of the endocrine system will be successful.