Variation of prostaglandin E2 concentrations in ovaries and its effects on ovarian maturation and oocyte proliferation in the giant fresh water prawn, Macrobrachium rosenbergii.

Prostaglandins (PGs) are important bioactive mediators for many physiological functions. In some decapod crustaceans, prostaglandin E2 (PGE2) has been detected in reproductive organs, and may play a role in the control of ovarian maturation. However, in the freshwater prawn, Macrobrachium rosenbergii, the presences of PGE2 and key enzymes for PGE2 biosynthesis, as well as its effects on ovarian maturation have not yet been investigated. In this study we reported the presence of PGE2, cyclooxygenase1 (COX1) and prostaglandin E synthase (PGES) in the ovarian tissues of M. rosenbergii, using immunohistochemistry. Intense immunoreactivities of PGE2 (PGE2-ir), COX1 (Cox1-ir) and PGES (PGES-ir) were detected in previtellogenic oocytes (Oc1 and Oc2), while the immunoreactivities were absent in the late vitellogenic oocytes (Oc4). This finding supports the hypothesis that the PGE2 biosynthesis occurs in the ovary of this prawn. To ascertain this finding we used LC-MS/MS to quantitate PGE2 concentrations during ovarian developmental cycle. The levels of PGE2 were significantly higher in the early ovarian stages (St I and II) than in the late stages (St III and IV). Moreover, we found that administration of PGE2 stimulated the ovarian maturation in this species by shortening the length of the ovarian cycle, increasing ovarian-somatic index, oocyte proliferation, and vitellogenin (Vg) level in the hemolymph.

Cloning of the crustacean hyperglycemic hormone and evidence for molt-inhibiting hormone within the central nervous system of the blue crab Portunus pelagicus.

The crustacean X-organ-sinus gland (XO-SG) complex controls molt-inhibiting hormone (MIH) production, although extra expression sites for MIH have been postulated. Therefore, to explore the expression of MIH and distinguish between the crustacean hyperglycemic hormone (CHH) superfamily, and MIH immunoreactive sites (ir) in the central nervous system (CNS), we cloned a CHH gene sequence for the crab Portunus pelagicus (Ppel-CHH), and compared it with crab CHH-type I and II peptides. Employing multiple sequence alignments and phylogenic analysis, the mature Ppel-CHH peptide exhibited residues common to both CHH-type I and II peptides, and a high degree of identity to the type-I group, but little homology between Ppel-CHH and Ppel-MIH (a type II peptide). This sequence identification then allowed for the use of MIH antisera to further confirm the identity and existence of a MIH-ir 9kDa protein in all neural organs tested by Western blotting, and through immunohistochemistry, MIH-ir in the XO, optic nerve, neuronal cluster 17 of the supraesophageal ganglion, the ventral nerve cord, and cell cluster 22 of the thoracic ganglion. The presence of MIH protein within such a diversity of sites in the CNS, and external to the XO-SG, raises new questions concerning the established mode of MIH action.

Evaluation of formaldehyde exposure among gross dissection after modified embalming solution and health assessment.

Mainly embalming fixative contains formaldehyde which is classified as a carcinogen. People who work with cadavers have been at higher risk of cancer after formaldehyde exposure. We have formulated a less-formalin fixative (contained 3.6% formaldehyde,23.8% ethanol, 15% glycerin, and 0.2% phenol in the water) for preserving cadavers. Therefore, the objective of the present study was to evaluate the level of atmospheric formaldehyde indoors and the breathing exposure of medical students during dissection classes. We also analyzed the pulmonary parameters and effects of formaldehyde. The levels of atmospheric formaldehyde indoors and personal breathing exposure were sampled during anatomy dissection classes (musculoskeletal system, respiratory system, and abdominopelvic organ system) using sorbent tubes with air sampling pumps. Samples were then analyzed using Gas Chromatography with Flame Ionization Detector (GC-FID). The mean level of formaldehyde indoor air among the three classes was 0.518 ± 0.156 ppm whereas the formaldehyde level in the personal breathing zone was 0.956±0.408 ppm, which exceeded the recommended exposure standards of international agencies, including NIOSH agency and PEL of Thailand legislation. The laboratory had high humidity, high room temperature, and poor air ventilation. There was a significant difference in FVC, FEV1, and PEF (p < 0.05) between the sexes of students. Comparison pulmonary parameters between students and instructors showed that all parameters of the pulmonary function test had no significant differences. General fatigue and burnings of eyes and nose associated with strong odor were the most common symptoms reported during the dissection classes. The modified embalming fixative was used less formalin with ethanol-glycerin mixture, and it was suitable for the study of medical students, with few side effects of respiratory problems. However, the modified exhaust ventilation with local table-exhaust ventilation and heating-ventilation-air conditioning system performance were urgent issues for reducing levels of formaldehyde indoor air in the dissection room.

Changes in the levels of serotonin and dopamine in the central nervous system and ovary, and their possible roles in the ovarian development in the giant freshwater prawn, Macrobrachium rosenbergii.

Serotonin or 5-hydroxytryptamine (5-HT) and dopamine (DA) are the two key neurotransmitters that control gonadal development in decapod crustaceans. This study investigated changes in the levels of 5-HT and DA in the CNS and ovary during different phases of the ovarian cycle of the freshwater prawn, Macrobrachium rosenbergii. The levels of 5-HT and DA were quantified by using High Performance Liquid Chromatography with electrochemical detection (HPLC-ECD). Moreover, changes of vitellogenin (Vg) concentrations in the hemolymph after treatment with 5-HT and DA (at doses of 2.5 x 10(-6) and 2.5 x 10(-7)mol per prawn) were also examined. 5-HT exhibited a gradual increase in concentration in the brain and thoracic ganglia from ovarian stage I (0.12+/-0.01 nmol/mg, 0.22+/-0.01 nmol/mg, respectively) to reach a maximum (0.66+/-0.03 nmol/mg, 1.48+/-0.03 nmol/mg, respectively) at ovarian stage IV. In contrast, DA in the brain and thoracic ganglia showed the highest concentrations at ovarian stage II (0.20+/-0.01 nmol/mg, 1.27+/-0.06 nmol/mg, respectively) and then decreased to the lowest concentrations (0.06+/-0.01 nmol/mg, 0.28+/-0.04 nmol/mg, respectively) at ovarian stage IV. The ovarian concentration of 5-HT was 0.53+/-0.11 nmol/mg at ovarian stage I and gradually increased to 1.63+/-0.16 nmol/mg at ovarian stage IV. In contrast, the concentration of DA was highest at ovarian stage I (29.05+/-1.31 nmol/mg), and lowest at the ovarian stage IV (11.43+/-0.74 nmol/mg). Injecting 5-HT into prawns significantly increased Vg concentration in the hemolymph at ovarian stage IV compared to control groups, and injecting DA into prawns had the opposite effect. The inverse relationship between 5-HT and DA levels in neural ganglia and ovaries, and their opposing effects on hemolymph Vg levels suggest that these two transmitters play opposite regulatory roles in controlling ovarian maturation and oocyte development in this species.

Production and characterization of a monoclonal antibody against recombinant glutathione S-transferase (GST) of Fasciola gigantica.

A monoclonal antibody (MoAb) against a recombinant glutathione S-transferase (rGST) of F. gigantica was produced in BALB/c mice. Reactivity and specificity of this monoclonal antibody was assessed by ELISA and immunoblotting. Six stable clones, namely 3A3, 3B2, 3C6, 4A6, 4B1 and 4D6 were obtained, All these MoAb reacted with rGST and native GST at a molecular weight of 28 kDa and found to be IgG1, kappa-light chain isotypes. These MoAb cross-reacted with Schistosoma mansoni and Schistosoma japonicum antigens at molecular weights of 28 and 26 kDa, respectively, but no cross-reactions were detected with antigens of Eurytrema and Paramphistomum spp. The localization of GST in metacercaria, 7-week-old juvenile and adult F. gigantica was performed by immunofluorescence technique, using MoAb as well as polyclonal antibody (PoAb) to the native protein as probes. In general, all clones of MoAb gave similar results and the pattern was quite similar to staining by PoAb. The fluorescence was intense, which implied the presence of a high concentration of GST in the parenchymal tissue in all stages of the parasite. However, the parenchymal cells were not evenly stained which implied the existence of subpopulations of this cell type with regard to GST production and storage. In addition, in adult and juvenile stages a moderate fluorescence was present in the basal layer of the tegument, while light fluorescence was observed in the caecal epithelium, cells in the ovary, testis and vitelline gland of the adult. In the metacercaria stage, in addition to parenchymal tissue, the tegument and tegumental cells were stained relatively more intense with MoAb and PoAb than in other stages.

Ultrastructure of differentiating oocytes and vitellogenesis in the giant freshwater prawn, Macrobrachium rosenbergii (De Man).

The ultrastructure of oogenesis in Macrobrachium rosenbergii, with reference to vitellogenesis, has not been reported. We used light and electron microscopy, as well as vitellin (Vn) purification and antibody production, to study the temporal and spatial production of Vn in the ovary by immunofluorescence. Histologically, the ovary is subdivided into cone-shaped ovarian pouches with a central core containing layers of oogonia. These divide to produce oocytes that migrate outwardly and differentiate into mature oocytes. During the course of differentiation, oocytes undergo modifications, including the rearrangement of nuclear chromatin, the accumulation of ribosomes, rough endoplasmic reticulum (RER), and lipid, and the formation of secretory and yolk granules, resulting in four stages. Ultrastructurally, early previtellogenic oocytes (Oc(1)) are characterized by the accumulation of new ribosomal aggregates, translocated from the nucleus. Late previtellogenic oocytes (Oc(2)) show nuclear heterochromatin with a "clock face" pattern, the presence of RER, and three types of secretory granules. Follicular cells occupy the intercellular spaces and surround the Oc(2). Early vitellogenic oocytes (Oc(3)) are larger, with nuclei containing predominantly decondensed euchromatin, and cytoplasm with yolk and secretory granules, and few lipid droplets. Late vitellogenic oocytes (Oc(4)) are characterized by completely euchromatic nuclei, an indistinct plasma membrane, yolk platelets and secretory granules, and abundant lipid. Vitellogenin (Vg) in ovaries of M. rosenbergii consist of two main bands at MW 90 and 102 kDa. Our data indicates that Vn is present, and probably synthesized in Oc(3) and Oc(4), but there may be some undetected exogenous Vg production.

Bilateral eyestalk ablation of the blue swimmer crab, Portunus pelagicus, produces hypertrophy of the androgenic gland and an increase of cells producing insulin-like androgenic gland hormone.

The androgenic glands (AG) of male decapod crustaceans produce insulin-like androgenic gland (IAG) hormone that controls male sex differentiation, growth and behavior. Functions of the AG are inhibited by gonad-inhibiting hormone originating from X-organ-sinus gland complex in the eyestalk. The AG, and its interaction with the eyestalk, had not been studied in the blue swimmer crab, Portunus pelagicus, so we investigated the AG structure, and then changes of the AG and IAG-producing cells following eyestalk ablation. The AG of P. pelagicus is a small endrocrine organ ensheathed in a connective tissue and attached to the distal part of spermatic duct and ejaculatory bulb. The gland is composed of several lobules, each containing two major cell types. Type I cells are located near the periphery of each lobule, and distinguished as small globular cells of 5-7 µm in diameter, with nuclei containing mostly heterochromatin. Type II cells are 13-15 µm in diameter, with nuclei containing mostly euchromatin and prominent nucleoli. Both cell types were immunoreactive with anti-IAG. Following bilateral eyestalk ablation, the AG underwent hypertrophy, and at day 8 had increased approximately 3-fold in size. The percentage of type I cells had increased more than twice compared with controls, while type II cells showed a corresponding decrease.

Spermatogenesis in the blue swimming crab, Portunus pelagicus, and evidence for histones in mature sperm nuclei.

Spermatogenesis in the blue swimming crab, Portunus pelagicus, is described by light and electron microscopy. The testis is composed of anterior (AT) and posterior (PT) lobes, that are partitioned into lobules by connective tissue trabecula, and further divided into zones (germinal, transformation and evacuation), each with various stages of cellular differentiation. The vas deferens is classified into three distinct regions: anterior (AVD), median (MVD), and posterior (PVD), on the presence of spermatophores and two secretions, termed substance I and II. Based on the degree and patterns of heterochromatin, spermatogenesis is classified into 13 stages: two spermatogonia (SgA and SgB), six primary spermatocytes (leptotene, zygotene, pachytene, diplotene, diakinesis, and metaphase), a secondary spermatocyte (SSc), three spermatids (St 1-3), and a mature spermatozoon. Spermatid stages are differentiated by chromatin decondensation and the formation of an acrosomal complex, which is unique to brachyurans. Mature spermatozoa are aflagellated, and have a nuclear projection and a spherical acrosome. AUT-PAGE and Western blots show that, during chromatin decondensation, there is a reduction of most histones, with only small amounts of H2B and H3 remaining in mature spermatozoa.