Reproductive and Developmental Alterations on Zebrafish (Danio rerio) Upon Long-Term Exposure of Di-ethyl phthalate (DEP), Di-isononyl phthalate (DINP) and Di (2-ethylhexyl) phthalate (DEHP).

Di (2-ethylhexyl) phthalate (DEHP), di-ethyl phthalate (DEP) and di-isononyl phthalate (DINP) are all endocrine disrupting chemicals (EDCs) for organisms. However, little research has been done on the effects of long-term EDC exposure. The present study found that the zebrafish barely grew during the 7 months of DINP exposure. The fecundity rate (%) of female spawning was lower in the DEHP treatment by 4 months compared to other exposure groups. Zebrafish treated with 12.5-25.0 ppm of DEP for 4 months presented no spawning. Gonadal-somatic index (GSI) levels significantly decreased, and there were more oocytes in the atresia and peri-nucleus stage compared to the control group. In addition, the hatching rate of embryos were 71.02%, 56.92%, and 21.70% for females treated with DINP, DEHP and DEP, respectively. There were also abnormal craniofacial chondrogenesis development on 72 hpf embryos upon females treated with the three EDCs. In conclusion, long term exposure of DEHP, DINP, and DEP did not only affect the reproductive capacity of female zebrafish, but the 3 plasticizers also influence craniofacial cartilage development of its offspring.

Effects of bisphenol A or diethyl phthalate on cartilage development and the swimming behavior of zebrafish (Danio rerio) through maternal exposure.

Waterborne bisphenol A (BPA) and diethyl phthalate (DEP) are endocrine disruptive chemicals that impact the reproductive system of fish. The present study checks the effectiveness of the reproductive capacity on zebrafish after BPA and DEP exposure, and consequently investigates its effect on their development and the swimming behavior of its offspring. The exposure of BPA and DEP to zebrafish reveals that the levels of ovarian 17ß-estradiol (E2) and relative mRNA expression (RRE) ratios (Treatment/Control) of hepatic vitellogenin (vtg1) could be induced and decreased. Liver RRE levels in estrogen receptors (ERs) are also affected. Among the ERs, esr2a significantly increased upon BPA exposure, and esr1 and esr2b decreased upon DEP exposure. In addition, the ceratohyal cartilage (CH) angle of larvae whose mothers were exposed to BPA (F-BPA) was significantly bigger, but the CH angle of larvae whose mothers were exposed to DEP (F-DEP) was significantly smaller than the control. The swimming performance of larvae from F-DEP was more compromised than the control, but the situation did not appear in the larvae from the F-BPA group. The success rate of larvae hatching from F-BPA and F-DEP was lower than control group. Moreover, the successful rate of female spawns was higher in the control group compared to the treatment groups exposed to BPA and DEP. We suggested that both maternal BPA and DEP disrupt E2 levels, and influence the CH development of larvae, resulting in a decrease in successful hatching. Only the swimming behavior of larvae from maternal DEP was disrupted.

Changes in vitellogenin and estrogen receptor expression and 17ß-estradiol concentration in male juvenile tilapia can be used to evaluate endocrine-disrupting chemicals.

The effects of endocrine disruption with respect to potential human toxicities have been extensively evaluated to date. However, the standard testing methods used have not always taken the most pertinent approach. In this study, we used juvenile male tilapia (Oreochromis niloticus) as an animal model to test for endocrine disruption by chemicals. We measured 17ß-estradiol (E2) concentrations, and the mRNA relative expression ratio (RER; treatment/control) of vitellogenin (vtg2) and estrogen receptors (ERs) to assess whether the effluent concentration of selected plasticizers disrupt E2 function in fish. We found that the vtg2 RER was significantly increased after exposure to 2.52 nM E2 for 5 days, 0.438 µM of bisphenol A (BPA) for 7 days, or 2.865 µM Cd2+ for 7 days. These data support vtg2 transcript level as a sensitive biomarker to evaluate contamination of water by endocrine disrupting chemicals (EDCs). However, vtg2 expression did not respond to fluctuations of E2 concentrations in the tilapia juveniles exposed to selected plasticizers. However, the RER of three types of ERs appeared to change dramatically upon exposure to plasticizers. ERα significantly increased, but ERß2 decreased with 3.6 µM DEP exposure. Both ERα and ERß2 decreased significantly after 1.44 µM DIBP exposure. We suggest that changes of vtg2 mRNA RER, E2 levels and ERs mRNA expression should be taken into consideration at the same time to determine if chemical contaminants in the water are endocrine disrupters.

Effectiveness of Oral Rinsing Solutions on Mucus, Odor, and Plaque in the Hospitalized Elderly in Taiwan.

This quasi-experimental and pretest-posttest study was conducted in Taiwan. The effectiveness of three types of oral rinsing solutions (normal saline, 0.2% chlorhexidine [CHX], and boiled water) was compared among 120 elderly patients (40 patients per group). Data on oral mucus, odor, and plaque were collected at admission on Day 1 (Time [T] 0), Day 4 (T1), Day 7 (T2), and Day 10 (T3). The results showed that the oral health condition in terms of mucus, plaque, and odor improved significantly over time. The effect for the oral condition did not differ significantly among the three groups, except for oral odor. At T2, the CHX group experienced higher oral odor than did the boiled water group (Solutions × Time interaction, F = 3.967, p = .002). Boiled water appears to be a safe and effective oral rinsing solution for hospitalized elderly patients.

The expression of VILL protein is hypoosmotic-dependent in the lamellar gill ionocytes of Otocephala teleost fish, Chanos chanos.

Milkfish, a species within the primitive teleost lineage Otocephala, can survive in water conditions ranging from hypo- to hyper-saline. This study explored the effects of environmental salinity on apical morphologies of ionocytes and the expression of villin homologs in the gills of milkfish acclimated to either seawater (SW) or fresh water (FW). Scanning electron microscopy revealed that the ionocytes in the gill filaments of SW and FW milkfish, respectively, cellular apical morphologies were hole-type and squint-type. The flat-type ionocytes were observed in the gill lamellae of FW milkfish. Furthermore, apical surfaces of some lamellar ionocytes exhibited microvilli. Villin 1 is a microvilli marker expressed in the epithelial cells of various vertebrates. In the phylogenetic tree of villin 1 homologs, primitive teleosts exhibit villin 1-like (VILL) and villin 1 proteins. Two mRNA sequences, villin 1 and VILL, were identified from the milkfish transcriptome by next generation sequencing. Low but constant expression of villin 1 (gene and protein) was observed in the gills for both SW and FW fish. VILL gene and protein expression levels in the gills were higher in FW fish, compared to SW fish. Double immunofluorescence staining demonstrated that VILL protein was present in some lamellar ionocytes of FW milkfish, but not in the filament ionocytes of either FW or SW milkfish. Taken together, these findings indicated that the VILL expression of ionocytes is hypoosmotic-dependent. The VILL might be involved in the formation of microvilli in the lamellar ionocytes for hyperosmoregulation of the milkfish.

Comparisons of two types of teleostean pseudobranchs, silver moony (Monodactylus argenteus) and tilapia (Oreochromis mossambicus), with salinity-dependent morphology and ion transporter expression.

There are essentially four different morphological types of pseudobranchs in teleosts, including lamellae-free, lamellae semi-free, covered, and embedded types. In the euryhaline silver moony (Monodactylus argenteus), the pseudobranch belongs to the lamellae semi-free type, which is characterized by one row of filaments on the opercular membrane and fusion on the buccal edge. The pseudobranchial epithelium of the moony contains two types of Na(+), K(+)-ATPase (NKA)-rich cells: chloride cells (CCs) and pseudobranch-type cells (PSCs). Our results revealed increased expression of NKA, the Na(+), K(+), 2Cl(-) cotransporter (NKCC), and the cystic fibrosis transmembrane conductance regulator (CFTR) for Cl(-) secretion and CCs profiles in the pseudobranchs of seawater (SW)-acclimated silver moonies, which indicates the potential role of pseudobranchs containing CCs in hypo-osmoregulation. In contrast, the pseudobranch of the Mozambique tilapia (Oreochromis mossambicus) belongs to the embedded type, which is covered by the connective tissues and only contains PSCs but not CCs. No sign of NKCC and CFTR-immunoreactivity (IR) was found in the pseudobranchs of SW and freshwater (FW) tilapia. However, higher NKA protein expression and larger sizes of NKA-IR PSCs were found in the pseudobranchs of FW-acclimated tilapia. Moreover, in the FW-acclimated moony, NKA-IR PSCs also exhibited higher numbers and larger sizes than in the SW individuals. Taken together, similar responses in low-salinity environments in different types of pseudobranchs indicated that the salinity-dependent morphologies of PSCs might be involved in critical functions for FW teleosts.

Profiles of thyrotropin, thyroid hormones, follicular cells and type I deiodinase gene expression during ontogenetic development of tilapia larvae and juveniles.

The aims of the present study are to determine whether triiodothyronine (T3) and/or thyroxine (T4) in tilapia larvae is gifted through the mother, and to investigate the change profiles of thyrotropin (TSH), thyroid follicular cells and type I deiodinase (D1) gene expression following larval development. T3 and T4 contents were measured using radioimmunoassay, thyrotropin was observed using immunocytochemistry, and the D1 gene was cloned and measured using real-time PCR. Results indicated that the ß-TSH-immunoreactive cells (thyrotropin ICC) signals were detected at 9 dph (i.e., 9 days of post-hatching). Thyroid follicular cells were observed first at 3 dph, while the T3 contents of the whole body gradually decreased before 11 dph. T4 contents were detected until 13 dph, with higher secretion during 19-21 dph. In addition, the T3 synthesis was not inhibited by thiourea (TU) before 13 dph, but the TU response in the larvae appeared after 13 dph. Type I deiodinase (D1: GenBank accession number KC591724) was found to contain 2444 bases and encoded 248 amino acids. The D1 mRNA expression began to increase at 13 dph, with a higher expression during 15-19 dph. These results suggested that the T3 contents were maternally derived before 13 dph. Both thyroid hormonal changes and some parameters related to thyroid hormone synthesis in ontogenetic tilapia are discussed.

Exploration of the mechanisms of protein quality control and osmoregulation in gills of Chromis viridis in response to reduced salinity.

Fish gills are the vital multifunctional organ in direct contact with external environment. Therefore, activation of the cytoprotective mechanisms to maintain branchial cell viability is important for fish upon stresses. Salinity is one of the major factors strongly affecting cellular and organismal functions. Reduction of ambient salinity may occur in coral reef and leads to osmotic stress for reef-associated stenohaline fish. However, the physiological responses to salinity stress in reef-associated fish were not examined substantially. With this regard, the physiological parameters and the responses of protein quality control (PQC) and osmoregulatory mechanisms in gills of seawater (SW; 33-35 ‰)- and brackish water (BW; 20 ‰)-acclimated blue-green damselfish (Chromis viridis) were explored. The results showed that the examined physiological parameters were maintained within certain physiological ranges in C. viridis acclimated to different salinities. In PQC mechanism, expression of heat-shock protein (HSP) 90, 70, and 60 elevated in response to BW acclimation while the levels of ubiquitin-conjugated proteins were similar between the two groups. Thus, it was presumed that upregulation of HSPs was sufficient to prevent the accumulation of aggregated proteins for maintaining the protein quality and viability of gill cells when C. viridis were acclimated to BW. Moreover, gill Na(+)/K(+)-ATPase expression and protein amounts of basolaterally located Na(+)/K(+)/2Cl(-) cotransporter were higher in SW fish than in BW fish. Taken together, this study showed that the cytoprotective and osmoregulatory mechanisms of blue-green damselfish were functionally activated and modulated to withstand the challenge of reduction in salinity for maintaining physiological homeostasis.

Anti-Mullerian hormone serum level as a predictive marker of ovarian function in Taiwanese women.

BACKGROUND: Anti-Mullerian hormone (AMH), which is secreted by preantral and small antral follicles, has been found to be a valuable marker of ovarian reserve. The purpose of this study was to determine age-related changes in AMH levels that occur in Taiwanese women and to determine whether measuring AMH is a highly sensitive and specific tool for diagnosing polycystic ovarian syndrome (PCOS) in Taiwanese women. METHODS: A group of 59 healthy, fertile, regularly cycling women, a second group of seven patients with premature ovarian failure or menopause, and a third group of 45 PCOS patients were enrolled. Serum AMH concentrations were measured using an enzyme-linked immunosorbent assay. RESULTS: AMH levels in healthy fertile women with regular menstrual cycles demonstrated an age-related decline, with a rapid drop between 30-40 years of age that was followed by a slow decrease after 40 years old. All patients with premature ovarian failure and menopause had undetectable AMH levels. AMH levels in PCOS patients were found to be significantly higher than those measured in healthy fertile controls. The sensitivity and specificity of AMH for detecting PCOS in patients aged 29-38 years were calculated to be 74% and 79%, respectively, using an AMH cut-off value of 3.5 ng/mL. CONCLUSION: Here, we provide data on Taiwanese women that demonstrate age-related decline in AMH levels and establish an AMH-based method for detecting PCOS, which may be used as reference data for future AMH studies on Taiwanese women.

Changes of glycogen metabolism in the gills and hepatic tissue of tilapia (Oreochromis mossambicus) during short-term Cd exposure.

The aim of the study was to test the hypothesis that the mechanism of glycogen metabolism has taken place in gills rather than in liver during Cd exposure. Male tilapia were exposed to 44.45 µM ambient Cd for 12h, and we found blood glucose significantly increased, however, lactate levels showed no significant changes. The glycogen phosphorylase (GP) activity increased immediately after 0.75 to 3h of Cd exposure in the gills, and after 1 to 6h in the liver, respectively. In addition, the glycogen level depleted faster in the gills than in the liver. Plasma cortisol level increased from 0.25 to 1h and recovered after 3h, while the glucagon did not significantly change during Cd exposure. Glucocorticoid receptor (GR) mRNA expression decreased after 0.75 h in the gills, while it significantly increased after 6h in the liver. Ca(2+), Na(+), Cl(-), and K(+) significantly decreased upon Cd exposure within 6h following Cd-induced toxic stress. We suggested that the cortisol is the spontaneous stimulation of glycogen metabolism in the gills, and it triggers a subsequent energy supply later in the liver. Taken together, the profile of glycogen metabolism between gills and liver during Cd-exposure stress provide good support to our hypothesis.

Elevated Na+/K+-ATPase responses and its potential role in triggering ion reabsorption in kidneys for homeostasis of marine euryhaline milkfish (Chanos chanos) when acclimated to hypotonic fresh water.

The milkfish (Chanos chanos) is an economic species in Southeast Asia. In Taiwan, the milkfish are commercially cultured in environments of various salinities. Na(+)/K(+)-ATPase (NKA) is a key enzyme for fish iono- and osmoregulation. When compared with gills, NKA and its potential role were less examined by different approaches in the other osmoregulatory organs (e.g., kidney) of euryhaline teleosts. The objective of this study was to investigate the correlation between osmoregulatory plasticity and renal NKA in this euryhaline species. Muscle water contents (MWC), plasma, and urine osmolality, kidney histology, as well as distribution, expression (mRNA and protein), and specific activity of renal NKA were examined in juvenile milkfish acclimated to fresh water (FW), seawater (SW 35 per thousand), and hypersaline water (HSW 60 per thousand) for at least two weeks before experiments. MWC showed no significant difference among all groups. Plasma osmolality was maintained within the range of physiological homeostasis in milkfish acclimated to different salinities, while, urine osmolality of FW-acclimated fish was evidently lower than SW- and HSW-acclimated individuals. The renal tubules were identified by staining with periodic acid Schiff's reagent and hematoxylin. Moreover, immunohistochemical staining showed that NKA was distributed in the epithelial cells of proximal tubules, distal tubules, and collecting tubules, but not in glomeruli, of milkfish exposed to different ambient salinities. The highest abundance of relative NKA alpha subunit mRNA was found in FW-acclimated milkfish rather than SW- and HSW-acclimated individuals. Furthermore, relative protein amounts of renal NKA alpha and beta subunits as well as NKA-specific activity were also found to be higher in the FW group than SW and the HSW groups. This study integrated diverse levels (i.e., histological distribution, gene, protein, and specific activity) of renal NKA expression and illustrated the potential role of NKA in triggering ion reabsorption in kidneys of the marine euryhaline milkfish when acclimated to a hypotonic FW environment.

Relative changes in the abundance of branchial Na(+)/K(+)-ATPase alpha-isoform-like proteins in marine euryhaline milkfish (Chanos chanos) acclimated to environments of different salinities.

Previous studies revealed that upon salinity challenge, milkfish (Chanos chanos), the euryhaline teleost, exhibited adaptive changes in branchial Na(+)/K(+)-ATPase (NKA) activity with different Na(+) and K(+) affinities. Since alteration of activity and ion-affinity may be influenced by changes in different isoforms of NKA alpha-subunit (i.e., the catalytic subunit), it is, thus, intriguing to compare the patterns of protein abundance of three major NKA alpha-isoform-like proteins (i.e., alpha1, alpha2, and alpha3) in the gills of euryhaline milkfish following salinity challenge. The protein abundance of three NKA alpha-isoform-like proteins in gills of milkfish reared in seawater (SW), fresh water (FW), as well as hypersaline water (HSW, 60 per thousand) were analyzed by immunoblotting. In the acclimation experiments, the SW group revealed significantly higher levels of NKA alpha1- and alpha3-like proteins than the FW or HSW group. Time-course experiments on milkfish that were transferred from SW to HSW revealed the abundance of branchial NKA alpha1-like and alpha3-like proteins decreased significantly after 96 and 12 hr, respectively, and no significant difference was found in NKA alpha2-like protein. Furthermore, when fish were transferred from SW to FW, the amounts of NKA alpha1- and alpha3-like proteins was significantly decreased after 96 hr. Taken together, acute and chronic changes in the abundance of branchial NKA alpha1- and alpha3-like proteins may fulfill the requirements of altering NKA activity with different Na(+) or K(+) affinity for euryhaline milkfish acclimated to environments of various salinities.

Differential expression of branchial Na+/K(+)-ATPase of two medaka species, Oryzias latipes and Oryzias dancena, with different salinity tolerances acclimated to fresh water, brackish water and seawater.

Previous studies on non-diadromous euryhaline teleosts introduced a hypothesis that the lowest level of gill Na(+)/K(+)-ATPase (NKA) activity occurs in the environments with salinity close to the primary natural habitats of the studied species. To provide more evidence of the hypothesis, two medaka species, Oryzias latipes and O. dancena, whose primary natural habitats are fresh water (FW) and brackish water (BW) environments, respectively, were compared from levels of mRNA to cells in this study. The plasma osmolalities of O. latipes and O. dancena were lowest in the FW individuals. The muscle water contents of O. latipes decreased with elevated external salinities, but were constant among FW-, BW-, and seawater (SW)-acclimated O. dancena. Expression of NKA, the primary driving force of ion transporters in gill ionocytes, revealed different patterns in the two Oryzias species. The highest NKA alpha-subunit mRNA abundances were found in the gills of the SW O. latipes and the FW O. dancena, respectively. The pattern of NKA activity and alpha-subunit protein abundance in the gills of O. latipes revealed that the FW group was the lowest, while the pattern in O. dancena revealed that the BW group was the lowest. Immunohistochemical staining showed similar profiles of NKA immunoreactive (NKIR) cell activities (NKIR cell numberxcell size) in the gills of these two species among FW, BW, and SW groups. Taken together, O. latipes exhibited better hyposmoregulatory ability, while O. dancena exhibited better hyperosmoregulatory ability. Our results corresponding to the hypothesis indicated that the lowest branchial NKA activities of these two medaka species were found in the environments with salinities similar to their natural habitats.

Characterization of two mosquito STATs, AaSTAT and CtSTAT. Differential regulation of tyrosine phosphorylation and DNA binding activity by lipopolysaccharide treatment and by Japanese encephalitis virus infection.

Two mosquito STATs, AaSTAT and CtSTAT, have been cloned from Aedes albopictus and Culex tritaeniorhynchus mosquitoes, respectively. These two STATs are more similar to those of Drosophila, Anopheles, and mammalian STAT5 in the DNA binding and Src homology 2 domains. The mRNA transcripts are expressed at all developmental stages, and the proteins are present predominantly at the pupal and adult stages in both mosquitoes. Stimulation with lipopolysaccharide resulted in an increase of tyrosine phosphorylation and DNA binding activity of AaSTAT and CtSTAT as well as an increase of luciferase activity of a reporter gene containing Drosophila STAT binding motif in mosquito C6/36 cells. After being infected with Japanese encephalitis virus, nuclear extracts of C6/36 cells revealed a decrease of tyrosine phosphorylation and DNA binding activity of AaSTAT which could be restored by sodium orthovanadate treatment. Taking all of the data together, this is the first report to clone and characterize two mosquito STATs with 81% identity and to demonstrate a different response of tyrosine phosphorylation and DNA binding of these two STATs by lipopolysaccharide treatment and by Japanese encephalitis virus infection.