Acknowledgments
We would like to thank Dr.
Zhongmin Lu and Seth Tomchik for use of histological equipment, confocal and
light microscopes and especially for technical and intellectual support. We thank Dr. Jim OÕReilly and the OÕLab for support. A special thanks to Alexandra Sterlin
for support and delivery of literature.
A hearty thanks to G-Rob Burgess for technical support. MM and LG would also like to extend thanks to the University of Miami
Department of Biology for travel support as well as the UM College of Arts and
Sciences Kriloff foundation for travel funding. LG is especially
grateful to SICB for student support for this meeting. This study was supported by the EPA
STAR-GRO fellowship number 91621401 for LG.
The brain illustration
for this poster was drawn by Mark Mandica. Please visit www. mandica.com for further information. You can contact MM at
mandica@bio.miami.edu or LG at ganser@bio.miami.edu. Visit us at the O'Lab website
www.bio.miami.edu/oreilly or www.bio.miami.edu/zlu.
Adult cane toads (Bufo marinus) display sexually dimorphic reproductive behaviors. Male toads call females to mate. Female toads respond by moving toward
the male`s call (phonotaxis) The goal of this study is to identify potential
sexually dimorphic areas of the brain of Bufo
marinus using histological
techniques.
During the amphibian larval period,
hormones help organize bipotential reproductive tissues in the brain and
gonads. It is during this critical
period of organization that certain man-made chemicals may disrupt the normal
endocrine cascade that governs the function and form of reproductive
tissues. These Òendocrine
disruptorsÓ, often found in agricultural chemicals and industrial wastes, have
been known to affect the morphology of the gonads as well as the mating
behaviors of exposed animals.
The putative endocrine disruptor,
atrazine, is liberally used on crops and residential grasses in South
Florida. It is the most commonly
used herbicide in the United States and is specifically used on South FloridaÕs
sugar cane crop (Gross et al. 2003), on recreational grasses and residential
lawns. A previous study (Hayes et al. 2002) concluded that atrazine feminized
the gonads of male clawed frogs (Xenopus). Cane toads (Bufo
marinus) are an invasive species in
Florida and flourish in developed and agricultural areas where herbicide use is
common. Cane toads collected from
agricultural areas in South Florida show evidence of abnormal gonad morphology,
while toads collected from reference areas appear to be normal (McCoy et al.
2002).
Male bufonids possess vestigial
ovarian tissue (BidderÕs organ) that may increase susceptibility to endocrine
disruption. We hypothesize that if
atrazine affects the morphology of the gonads it will also affect the areas of
the brain that govern mating behavior.
In this preliminary study, we compare neuroanatomical structures of
normal male and female Bufo marinus. We use
Nissl stain to identify common nuclei and to make qualitative comparisons of
nucleus size, cell body size, and cell density between males and females.
Cane toads were collected from
various sites in South Florida and euthanized for brain and gonad
analysis. Individuals without
obvious gonadal abnormalities were selected for this neuroanatomical study.
General anatomical observations were made on brain tissues stained with Cresyl
Violet stain for Nissl substance.
Brains were dissected from toads, fixed in 4% paraformaldehyde, and
embedded in gelatin with 30% sucrose.
Tissues were post-fixed in 30% sucrose with 4% paraformaldehyde. Serial frozen sections (50 µm) were
made on a sliding microtome.
Cresyl violet-stained tissues were viewed with a Nikon Eclipse E600
light microscope and analyzed using Neurolucida ¨ software. We qualitatively
compared potentially sexually dimorphic nuclei in the brains of male and female
toads. AVT is thought to
play a central role in mediating mating behavior in amphibians. Previous studies by Boyd et al. (1992)
and Gonzales and Smeets (1992) described the neuroanatomical locations of
arginine vasotocin immunoreactive (AVT-ir) cells and fibers in frogs and
salamanders. We based our identification of potentially dimorphic nuclei in the
brain of Bufo marinus on the results of these studies.
Previous studies by
Boyd et al. (1992) found sexual dimorphism in AVT-ir cell and fiber density in
the amygdala pars lateralis and habenula of the brain of Rana catesbeiana. We were unable to
notice any qualitative sexual dimorphism in cell body size, cell body density,
or nucleus size in these areas in Bufo
marinus. We did, however, note some subtle differences in cell body
density and cell body size in the glossopharyngeal motor nucleus. Cell bodies in the solitary tract seem
more organized in the male brain compared to the female brain.
Our preliminary
studies using Cresyl Violet stain for Nissl substance have helped us to
identify neuroanatomical structures and subtle sex differences in the cane toad
brain. It is clear that meticulous
study of previously identified sexually dimorphic areas of the anuran brain are
necessary. Future immuno-cytochemical studies to identify AVT-ir cells and
fibers paired with the identification of additional nuclei will help us to
identify potential sexual dimorphism in areas of the brain that govern mating
behavior.
These studies are a
necessary precursor to future experiments to determine the effects (if any) of
exposure to atrazine on areas of the brain that mediate mating behavior.
Boyd, SK, CJ Tyler, GJ De Vries.
1992. Sexual dimorphism in
the vasotocin system of the
bullfrog (Rana catesbeiana). J.
Comp. Neurol. 325:
313-325.
Gonzales, A and WJAJ Smeets. 1992.
Comparative analysis of
vasotocinergic and
mesotocinergic cells and fibers in the brain of two amphibians, the anuran Rana ridibunda and the
urodele Pleurodeles waltlii. J.
Comp. Neurol. 315:53-73.
Gross, TS, KA McCoy, M Sepulveda, JA Carr, JP Giesy, AJ Hosmer, RJ
Kendall, K Solomon, EE Smith, and G van der Kraak. 2003. Atrazine exposure and the occurrence of
reproductive abnormalities in field caught
Bufo marinus from South Florida.
McCoy, KA, M Sepulveda and TS Gross. 2002. Atrazine exposure and the occurrence of reproductive
abnormalities in field caught Bufo marinus from South Florida. In: Proceedings of the Society of
environmental Toxicology and
Chemistry, 23rd Annual Meeting.
Assessing Sex Differences in the Brain of the Cane
Toad, Bufo
marinus
Lisa R. Ganser and Mark L. Mandica. Department of Biology.
University of Miami. Coral
Gables, FL 33124
Results
