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.
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.
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.
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.
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