Evaluation of diagnostic utility, safety considerations, and effect on fertility of transvaginal ultrasound-guided ovarian biopsy in mares.

Ultrasound-guided biopsies of corpora lutea have been previously used for research purposes in the mare and cow. However, the health effects and fertility after transvaginal luteal biopsies (TVLB) or transvaginal ovarian biopsies (TVOB) obtained for diagnostic purposes in cases of suspected ovarian tumors have not been previously evaluated in the horse. The aim of this study was to determine the effects on health and fertility of TVLB and TVOB in mares; 53 mares were included in the study (11 control non-biopsied mares, 37 TVLB mares biopsied on one or more of the following Days 8, 10, 12, 15, 21, and 5 TVOB mares with ovarian abnormalities), resulting in a total of 108 TVLB and TVOB cycles and 183 procedures. Mares were divided into Groups 1 to 3 by the number of TVLB per estrous cycle (mare in Group 1 had 1 TVLB procedure, mares in Group 2 had 2 TVLB procedures, and mares in Group 3 had 3 TVLB procedures). Group 4 comprised TVOB mare cycles with ovarian abnormalities (n = 5). Mares were examined to determine day of ovulation (Day 0) and the presence of a corpora lutea using transrectal ultrasonography. Mares were sedated, and an ultrasound-guided transvaginal biopsy was performed of luteal or ovarian tissue. Health effects of TVLB or TVOB were assessed by daily rectal temperatures, appetite, and general demeanor for 72 hours post-procedure, and 3 mares were examined at necropsy. Fertility was not significantly different in control and TVLB Groups 1 to 3 (P = 0.7648) and in the first or subsequent cycles where the ovulation was from an ovary that had a previous TVLB (P = 0.7147). A TVLB on Day 8 post-ovulation may induce an early return to estrus. In conclusion, the TVLB or TVOB procedure had no effect on health and fertility in this study if the procedure was correctly performed with good technique. Because of the low number of cycles (n = 37), the fertility data should be interpreted with caution. The TVOB may be applied in the diagnosis of mares with ovarian abnormalities, and no adverse health effects were associated with TVOB of mares with granulosa theca cell tumor.

Changes in mitochondrial gene and protein expression in grass shrimp, Palaemonetes pugio, exposed to chronic hypoxia.

Spatial and temporal increases of hypoxia in estuaries are of major environmental concern. Since mitochondria consume most of the oxygen in the cell, we examined the potential role of mitochondrial gene and protein expression in adaptation to chronic hypoxia in the grass shrimp Palaemonetes pugio. Grass shrimp were exposed to DO levels slightly above and below the critical pO(2), 1.8 mg/L, for P. pugio, and hypoxia-induced alterations in gene expression were screened using custom cDNA macroarrays. Mitochondrial gene expression was not affected by exposure to moderate hypoxia (2.5mg/L DO). However, chronic exposure to severe hypoxia (1.5mg/L DO) for 7 days resulted in an increase of transcription of genes present in the mitochondrial genome (including 16S rRNA and Ccox 1), together with up-regulation of genes involved in Fe/heme metabolism. This pattern was completely reversed by day 14, when a significant down-regulation of these genes was observed. Separating mitochondrial proteins in two dimensions by IEF and reverse phase chromatography, followed by LC/MS/MS of differentially expressed proteins, showed cytochrome c oxidase subunit 2, encoded by Ccox 2, was down-regulated after 12d exposure to severe hypoxia. It appears therefore that decreases in mitochondrial Ccox gene transcription result in decreased mitochondrial Ccox protein synthesis. These results suggest that mitochondrial genes and proteins show promise as molecular indicators of exposure to hypoxia.

Molecular and developmental effects of exposure to pyrene in the early life-stages of Cyprinodon variegatus.

Polycyclic aromatic hydrocarbons (PAHs) have been connected to developmental toxicity in the early life-stages of many species by their ability to bind to the aryl hydrocarbon receptor (AHR), which dimerizes with ARNT (AHR nuclear translocator) to induce transcription of genes such as CYP1A1. ARNT also dimerizes with HIF (hypoxia-inducible factor alpha) to induce transcription of genes such as VEGF (vascular endothelial growth factor), suggesting that PAHs may interfere with transcription of VEGF by competing for ARNT. Herein, we address the molecular and developmental effects of exposures to the weak AHR agonist pyrene on the early life-stages of the sheepshead minnow Cyprinodon variegatus. Embryos were exposed under flow-through conditions to 0, 20, 60, or 150 ppb pyrene up to 432 hours post-fertilization (hpf). RNA was extracted at 5 time points (12, 24, 48, 96, and 432 hpf) and changes in CYP1A1 and VEGF expression were assessed by real-time RT-PCR. Since few genes have been documented for the sheepshead minnow, we first cloned and sequenced CYP1A1, VEGF and internal standard 18S rRNA. Pyrene significantly induced the AHR-regulated gene, CYP1A1, in a time- and dose-dependent manner, while pyrene failed to alter the HIF-regulated gene, VEGF. However, VEGF was found to change during various stages of normal development in this study. Although a normal hatch time (5 dpf) was observed for all treatments, pyrene-treated embryos showed dose-dependent abnormalities such as severe dorsal body curvature, mild pericardial and yolk-sac edema, and increased mortality. Taken together, these data indicate that embryonic exposure of sheepshead minnows to pyrene disrupts normal development and alters expression of an AHR/ARNT-regulated gene. In addition, embryonic exposure to pyrene failed to provide evidence of possible AHR-HIF pathway cross-talk since developmental expression of VEGF was unaltered.

Effects of long-term hypoxia on enzymes of carbohydrate metabolism in the Gulf killifish, Fundulus grandis.

The goal of the current study was to generate a comprehensive, multi-tissue perspective of the effects of chronic hypoxic exposure on carbohydrate metabolism in the Gulf killifish Fundulus grandis. Fish were held at approximately 1.3 mg l(-1) dissolved oxygen (approximately 3.6 kPa) for 4 weeks, after which maximal activities were measured for all glycolytic enzymes in four tissues (white skeletal muscle, liver, heart and brain), as well as for enzymes of glycogen metabolism (in muscle and liver) and gluconeogenesis (in liver). The specific activities of enzymes of glycolysis and glycogen metabolism were strongly suppressed by hypoxia in white skeletal muscle, which may reflect decreased energy demand in this tissue during chronic hypoxia. In contrast, several enzyme specific activities were higher in liver tissue after hypoxic exposure, suggesting increased capacity for carbohydrate metabolism. Hypoxic exposure affected fewer enzymes in heart and brain than in skeletal muscle and liver, and the changes were smaller in magnitude, perhaps due to preferential perfusion of heart and brain during hypoxia. The specific activities of some gluconeogenic enzymes increased in liver during long-term hypoxic exposure, which may be coupled to increased protein catabolism in skeletal muscle. These results demonstrate that when intact fish are subjected to prolonged hypoxia, enzyme activities respond in a tissue-specific fashion reflecting the balance of energetic demands, metabolic role and oxygen supply of particular tissues. Furthermore, within glycolysis, the effects of hypoxia varied among enzymes, rather than being uniformly distributed among pathway enzymes.

Brain aromatase in Japanese medaka (Oryzias latipes): Molecular characterization and role in xenoestrogen-induced sex reversal.

In female fish estrogen is required for the development of primary and secondary sex characteristics and is derived from the aromatization of androgens by aromatase. There are two isoforms of aromatase in several teleost species, brain and ovarian. The objective of this study was two-fold: clone and sequence the coding and promoter region of brain aromatase in medaka, and determine the effects of exposure to an environmental estrogen (o,p-DDT) on sex determination and brain aromatase transcription and activity. The brain aromatase coding sequence was obtained by reverse transcription polymerase chain reaction (RT-PCR) and PCR-based genomic DNA walking was used to clone the promoter of the brain aromatase gene. The promoter sequence revealed potential binding sites for the estrogen receptor and for transcription factors involved in primary neurogenesis and sex determination. Medaka fry were exposed to increasing o,p-DDT concentrations (0-5.5 microg/L) from days 1 to 15 after hatch and brain aromatase expression and activity were measured on days 5, 9, and 14. A complete male-to-female sex reversal occurred at 5.5 microg/L o,p-DDT and aromatase activity and expression data showed a significant five-fold increase at this concentration at day 14. This information suggests that brain aromatase is involved in the abnormal sexual differentiation of fish treated with xenoestrogens.

Effects of hypoxia on gene and protein expression in the blue crab, Callinectes sapidus.

Increases in hypoxic conditions are one of the major factors responsible for declines in estuarine habitat quality, yet to date there are no indicators for recognizing populations of estuarine organisms that are suffering from chronic hypoxic stress. Here we test the hypothesis that alterations in gene and protein expression of antioxidant enzymes and other stress-specific proteins can be used as molecular indicators of hypoxic stress. Blue crabs, Callinectes sapidus, were exposed to 2-3 ppm DO for 5 days. Gene expression was measured using macroarrays constructed from cDNA of 10 partial gene transcripts cloned from blue crab hepatopancreas. Significant (p< or =0.05) down-regulation of gene expression was found for MnSOD, hemocyanin, ribosomal S15 and L23. Subtractive hybridization using RNA from control and hypoxic hepatopancreas tissues also indicated down-regulation of hemocyanin transcription. In contrast, Western blotting showed a significant (p< or =0.05) increase of hemocyanin protein in the hepatopancreas and cross-linking of MnSOD proteins in hypoxia-exposed crabs. Thus, hypoxia-responsive cDNA arrays and Westerns may be useful diagnostic tools for monitoring effects of hypoxia in estuarine crustacea.

Reproductive effects of estrogenic and antiestrogenic chemicals on sheepshead minnows (Cyprinodon variegatus).

Environmental estrogens can activate genes of the reproductive system, such as vitellogenin (VTG), a precursor to egg yolk protein, by activating the estrogen receptor (ER), whereas antiestrogens can inhibit ER activation. Adult lab-reared male sheepshead minnows (Cyprinodon variegatus) were exposed to estrogenic 4-tert-octylphenol (OP) and females to antiestrogenic cadmium (Cd), and the effects on four potential indicators of impaired reproductive function were examined: VTG in F0 male blood as sign of feminization, F0 generation fecundity/fertility, embryonic development/egg hatching/survival rate of F1 generation fry, and F0 gonadal histology. Mean VTG in the control, 11.5, 33.6, and 61.1 microg/L OP male fish were 0, 10.7, 38.7, and 65.6 mg/ml postexposure and 0, 2.5, 19.4, and 30.0 mg/ml postreproduction. A significant inverse relationship between increasing VTG in male blood and reproductive success of mating groups involving these males was shown, with higher OP decreasing percent viable eggs (fertility) by approximately 60%. Histology showed increased testis anomalies and decreased spermatozoa with increasing OP exposure. No effects on F1 embryonic development, egg hatching, or fry survival rate were observed. A significant decline in percent viable egg production involving Cd-exposed females occurred only when mated with OP-exposed males, with no eggs produced by fish exposed to the highest OP and Cd concentration. A three-week field exposure near a wastewater treatment plant outfall showed no elevated VTG in male plasma, but significantly higher total egg production per female per collection day (approximately 45%) was observed at the site furthest from the outfall.