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C and D show 8 μm of transverse cross-sections through the animals' right gonad stained with Mallory's trichrome stain. The yellow color in A and B is a result of fixation in Bouins' fixative. The ovary is distinguished by its greater length, lobed structure, and melanin granules.Although some specimens' ovaries lack pigment (especially atrazine-treated animals), testes never have melanin in this species.Each animal was weighed to the nearest 0.002 g on a Mettler AT 261 Delta Range balance and its total length was measured to the nearest 0.5 mm.Animals were anesthetized in 0.2% benzocaine (Sigma), assigned a unique identification number, fixed in Bouins' fixative, and preserved in 70% ethanol until further analysis.
We report results from two experiments that used frogs from two separate sources. 1 were from a long-term captive colony maintained at the University of California, Berkeley, whereas adults from Exp. In both experiments, three females and three males were injected with human choriogonadotropin (1,000 international units) 6 h before harvesting gametes.
Food levels were adjusted as the animals grew to maximize growth. 1, we exposed larvae to atrazine at nominal concentrations of 0.01, 0.1, 1.0, 10.0, and 25 parts per billion (ppb), whereas the second experiment used 0.1, 0.4, 0.8, 1.0, 25, and 200 ppb atrazine.
Concentrations were confirmed by two independent laboratories (PTRL West, Richmond, CA, and the Iowa Hygienic Laboratory, Univ. All stock solutions were made in ethanol (10 ml), mixed in 15-gallon containers, and dispensed into treatment tanks.
Experiments were carried out at 22°C with animals under a 12-h/12-h light/dark cycle (lights on at 6 a.m.).
Animals were exposed throughout the entire larval period, from hatching [Niewkwoop–Faber (NF) Stage 48 (16)] until complete tail reabsorption (NF Stage 66).
This disruption in steroidogenesis likely explains the demasculinization of the male larynx and the production of hermaphrodites.