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Ovigerous females of the estuarine crab Chasmagnathus granulatus were exposed to copper (0.01 and 1 mg/L), zinc (0.05, 1, and 10 mg/L), or lead (0.01 and 1 mg/L) during early, late, or whole embryonic development. None of the assayed heavy metals produced a significant mortality of females, neither a decrease in the number of hatched larvae nor a decrease in the egg incubation time, but several morphological abnormalities were detected in hatched larvae. The abnormalities were classified in three categories: eye, body pigmentary, and body morphological abnormalities. Those larvae with eye and body pigmentary abnormalities, particularly those involving retinal pigments and chromatophores, showed the highest incidence by exposure to the assayed metals. In addition, embryos were more susceptible to copper and zinc during the late period of development, whereas the effect of lead was greater during the early period of embryogenesis. Some teratogenic effects observed in C. granulatus embryos exposed to heavy metals, particularly the hypertrophy and hypopigmentation of eyes observed in the laboratory at a lead concentration as low as that reported for the natural environment, could be considered as sensitive biomarkers for this kind of pollutant.

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Objective: The aim of the present study is to assess the correlation between the presence, quantity and size of nuclear vacuoles and DNA damage and chromatin status in sperm samples of men who underwent to assisted reproduction technology. Methods: Forty six males who underwent to assisted reproductive technology (ART) were considered. According to their latest semen analysis (<3 months), were grouped into: (A) strict morphology index ≤4% (26) and (B) strict morphology index ≥14% (20). Motile sperm were selected by density gradient, and MSOME study was conducted to assess the number and size of nuclear vacuoles. DNA fragmentation (TUNEL) and DNA strand status (acridine orange) were assessed over the selected spermatozoa accordingly to their vacuole pattern. Results: In group A, sperm without vacuoles (1°) have similar levels of DNA fragmentation (TUNEL) in compare to the rest of observed patterns (2°-6°). Regarding to AO, spermatozoa with large or several vacuoles that cover more than 30-50% of the nuclear surface are AO+, but not necessarily TUNEL positive. The first three patterns of vacuoles patterns had lower levels of AO in compare to grades 4° and 6°. In group B, those sperm with one or more vacuoles greater than 30%-50% (4° and 6°), had a significant increase in TUNEL values, in relation to group 1°-3°. Considering AO, it was found that the 4° and 6° pattern had a significantly elevated level of this marker, as same of group A (P <0.05). Conclusions: There is no relationship between the greater number and size of sperm vacuoles with high levels of DNA fragmentation in patients with severe teratozoospermia (Kruger <4%). Conversely, this relationship is evident in normal semen samples (normal morphology. Sperm selection by IMSI technique, to select non-fragmented sperm in patients with Kruger <4%, is not necessarily secured when non-vacuolated sperm is selected.

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