The Effects of Subacute Oral Exposure to
Vinclozolin on
Sperm Integrity in Post-pubertal Swine
Amanda Estabrooks1*, Tim
J. Evans2,
Eric M. Walters2,5,
Crystal Kinnison2,
Preston R. Buff3,
Fred Williams III2,
Thomas J. Fangman4,
Venkataseshu K. Ganjam3 and John K. Critser2,5
1Atlantic
Veterinary College, University of Prince Edward Island, Departments of 2Veterinary Pathobiology, 3Biomedical Science and 4Veterinary Medicine and Surgery,
College of Veterinary Medicine, 5National Swine Resource and Research Center,
University of Missouri-Columbia
•There
is increasing awareness of the potential adverse effects of
endocrine-disrupting chemicals (EDCs) on male reproduction.
•The
ability to assess the impact of these compounds on male fertility has been
hindered by the limitations of available animal models and the relative
insensitivity of traditional research end points.
•The
anatomical, physiological and genetic similarities between humans and swine
make the porcine model an attractive, non-rodent alternative to accurately
define EDC exposures which are potentially harmful to humans.
•Flow
cytometric evaluations of sperm integrity using different fluorescent dyes to
assess sperm viability, acrosomal status, mitochondrial function and sperm
chromatin structure, are potentially more sensitive indicators of toxic insult
to the male reproductive tract than traditional sperm parameters and
conception rates.
Overall Research Objective
Background
Further development and refinement of a
porcine model for human risk assessment involving EDC exposures
Preliminary Results
Conclusions
Using the known antiandrogen, vinclozolin
(VCZ), as a model EDC, we hypothesized that VCZ will alter selected indices of
sperm integrity in a manner dependent on VCZ dosage.
Specific Aims
•Demonstrate
the effects of different dosages of VCZ on sperm integrity of post-pubertal
swine.
•Optimization
of flow cytometric methods for the evaluation of porcine sperm.
•Generate
preliminary data for the design of future experiments evaluating the effects
of various EDCs on male reproductive morphology and function in swine.
Experimental Design
•A total of
thirty-two post-pubertal (12 to 36 month-old) boars were divided into four
replicates (n=8) for arrival on April 15, May 15, June 15 and September 1,
respectively.
• The eight boars in each replicate were
divided into four treatment groups (n=2) housed in separate buildings and
dosed orally with either 0, 0.25, 5 or 100 mg/kg BW of VCZ for 15 days (day
0-14).
•Boars were
sacrificed on Day 15 of the study, and terminal sperm samples from all
thirty-two boars were collected from the cauda epididymis, cryopreserved and
stored in liquid nitrogen (≈-196°C) prior to flow
cytometric analyses.
•Samples of sperm
will be stained with fluorescent dyes and evaluated using a flow cytometer to
assess sperm plasma membrane permeability (viability), acrosomal status,
mitochondrial function and sperm chromatin structure.
•Flow cytometric
data will be analyzed to determine percentages of viable cells with normal
acrosomes, functional mitochondria and chromatin structure.
•Statistical
analysis of the effects of VCZ exposure on sperm integrity in post-pubertal
swine will be analyzed as a Latin square design for repeated and non-repeated
variables, using PROC Mixed in SAS (SAS; Statistical Analysis Systems, Release
9.1, Cary, NC).
Research Impact
This
research will contribute to the design of future experiments and to the
further development of a porcine model to accurately define EDC exposures which are
potentially harmful to humans, as well as other non-rodent animals.
Acknowledgements
Experimental Hypothesis
VCZ-induced
Effects on Distribution of Cellular Events
Figure 4: A.
Distribution of cellular events in cryopreserved sperm from a control
boar. B. Distribution of cellular events in
cryopreserved sperm from a VCZ-treated
boar showing three populations of cellular events similar to those observed in
flash-frozen sperm stained for ubiquitin. C. The mean percentage of live sperm
with an intact acrosome was significantly higher in the lowest VCZ treatment
groups than in the boars treated with 5 or 100 mg of VCZ/kg BW (* denotes P < 0.05) .
Flow Cytometric Method Development
VCZ-induced
Morphologic Effects in Post-pubertal Boars
Figure 1: Sections of PAS-stained testes from boars
treated with 0, 0.25, 5 and 100 mg VCZ/kg BW are shown in A, B, C and D,
respectively (200X magnification). The effects of VCZ on testicular
interstitial cell density in this study are shown in E (** and *** denote P < 0.05 and P < 0.001 with
respect to pair-wise comparisons between treatment groups). and were
reflective of the observed Leydig (interstitial) cell hyperplasia. The effects of VCZ
on sperm ubiquitination in these boars are shown in F, G and H, respectively.
Materials and Methods for Flow Cytometry
Fluorescent
Staining of Sperm
Figure 3: A.
Sperm double labeled with Mitotracker® and Propidium Iodide (PI) demonstrating
high mitochondrial staining and viable (PI– head). B.
Sperm double labeled with Mitotracker® and PI showing nonviable (PI+
head) with very low mitochondrial staining.
C. Sperm double labeled with PI and Mitotracker® demonstrating a
nonviable sperm (PI+ head) and no mitochondrial staining. D. Boar sperm with damaged acrosome stained
with Alexa-488-PNA illustrating damaged acrosomes. E. Sperm stained with
Acridine Orange indicating chromatin damage (red and yellow) and intact
chromatin (green).
•Flow
cytometric evaluations of sperm integrity have the potential to detect subtle
EDC-induced sperm defects.
•Flow
cytometric methods need to be optimized to account for sample preservation,
the excitation and emission wavelengths of fluorescent dyes and toxicant-induced changes in cellular events.
•The
boar shows promise as a comparative model for studying the effects of EDCs on
DNA sperm integrity in non-rodent mammals.
E
A
B
C
D
http://images.google.com/imgres?imgurl=http://www.vetmed.ucdavis.edu/apc/MeyersLab
D
http://uuhsc.utah.edu/andrology/photo_gallery.html
A
B
C
E
A
B
C
Flow
Cytometric Evaluation of Sperm Integrity
Figure 2: A. A flow
cytometer emits a laser beam of a selected wavelength to interact with a
fluorescent dye, which differentially stains some component of the sperm cell. The “excited” fluorescent dye emits light
of specific wavelength(s), which can be
collected and analyzed. The stains used in this research project are
Propidium Iodide (PI) for intact plasma membranes, Alexa 488-PNA for reacted
acrosomes, Mitotracker-633 Deep Red for mitochondrial function and Acridine
Orange for single stranded DNA. B. Flow cytometry was performed using a
Beckman-Dickinson FACS Scan.
A
B
F
H
G