ABSTRACT
A new quantitative evaluation technique, termed the fragmented testis method, has been developed for the detection of testis-ova in genotypic male fish using the medaka (Oryzias latipes). The routine traditional histological method for detection of testis-ova in male fish exposed to estrogens or suspected endocrine-disrupting chemicals has several disadvantages, including possible oversight of testis-ova due to limited sampling of selected tissue sections. The method we have developed here allows for the accurate determination of the developmental stages and the number and the size of testis-ova in a whole testis. Each testis was removed from the fish specimen, fixed with 10% buffered formalin solution, and then divided into small fragments on a glass slide with a dissecting needle or scalpel and aciform forceps in glycerin solution containing a small amount of methylene blue or toluidine blue. If present, all developing testis-ova of various sizes in fragmented testicular tissues were clearly stained and were observable under a dissecting microscope. Testis-ova occurred in controls were ascertained, while spermatozoa were also distinguishable using this method. This proved to be a convenient and cost-effective method for quantitatively evaluating testis-ova appearance in fish, and it may help to clarify the mechanism of testis-ova formation and the biological significance of testis-ova in future studies of endocrine disruption.
Subject(s)
Endocrine Disruptors/toxicity , Evaluation Studies as Topic , Ovum/drug effects , Testis/drug effects , Water Pollutants, Chemical/toxicity , Animals , Embryonic Development/drug effects , Female , Male , Microscopy , Oocytes/cytology , Oocytes/drug effects , Oryzias/abnormalities , Oryzias/embryology , Oryzias/metabolism , Ovum/cytology , Ovum/growth & development , Spermatozoa/cytology , Spermatozoa/drug effects , Testis/cytology , Testis/embryologyABSTRACT
Concentrations of vitellogenin (VTG) in different developmental stages: embryo (on the day of fertilization); yolk-sac larva (on the day of hatching); 2, 4, 6 and 8 weeks posthatching; were determined in medaka (Oryzias latipes) S-rR strain. Both sexes were bred separately from 1 week after hatching and fed only Artemia nauplii to avoid contamination by xenoestrogen from females and feed. Whole bodies of ten test fish/group were homogenized individually and the supernatants were quantified. Quantification of VTG was performed by an enzyme-linked immunosorbent assay (ELISA). In most males, VTG levels were less than 1 microg/g body weight and no fluctuation was observed throughout the developmental stages. In 2-week-old females, five had no detectable VTG, and the others had 5.18+/-2.37 microg/g of VTG. The 4-week-old females had 16.3+/-12.0 microg/g VTG, and the concentrations increased with maturity to 5.54+/-3.09 mg/g in 6-week-old and 8.99+/-3.10 mg/g in 8-week-old specimens. These results show that the concentrations of VTG in males are routinely close to the detection limit independent of the developmental stages in an environment with low contamination by xenoestrogen derived from artificial feed and females. Females have detectable VTG levels even in the juvenile phase, and the level increases markedly with maturation.
