F.M. Uckun et al. / Reproductive Toxicology 16 (2002) 57–64
59
analyses was conducted using a Hewlett Packard GC/mass
spectrometer Model 6890 (Wilmington, DE) equipped with
a mass ion detector and Chem Station software. The tem-
perature of the oven was steadily increased from 70°C to
ing arsenicals, and sperm motility was evaluated as de-
scribed above.
In experiments designed to examine the kinetics of
sperm immobilization by arsanilate complexes, 1-ml ali-
6
2
50°C and the carrier gas was helium.
quots of a highly motile fraction of sperm (15 ϫ 10 ) or a
1
:1 dilution of liquefied semen (62 Ϯ 7% progressive mo-
2
.3. Computer-assisted sperm analysis (CASA)
Fresh human semen was obtained from 9 healthy volun-
tility) in BWW medium were mixed with 2.5 l of freshly
prepared stock (100 mM) solutions of sperm-immobilizing
arsanilates (S-370, PHI-370, and PHI-380) to yield a final
concentration of 250 M. A corresponding volume of
DMSO (0.25%) was added to control tubes. Following
addition of the test compounds, 5-l aliquots were imme-
diately transferred at 1 min intervals to prewarmed 20-m
Microcell chambers and the time (in min) required to com-
pletely immobilize sperm was recorded using CASA. Three
separate experiments were performed to assess the kinetics
of the sperm immobilizing activity of arsenicals.
teers participating in the donor semen program at the Parker
Hughes Institute. All donor specimens were obtained after
informed consent and in compliance with the guidelines of
the Parker Hughes Institute Institutional Review Board. The
effect of pentavalent arsenicals on human sperm motility
and kinematics was tested using semen and corresponding
swim-up fractions. To evaluate the effect of arsenicals on a
highly motile fraction of sperm, we first subjected liquefied
normospermic semen to discontinuous (90–45%) gradient
centrifugation using Enhance-S-Plus (Conception Technol-
ogies, San Diego, CA) cell isolation medium as previously
described [9,10]. The resulting pellet was washed twice and
resuspended in Biggers, Whitten, and Whittingam’s me-
dium (BWW) supplemented with 3% bovine serum albumin
In experiments designed to assess reversibility of sperm
6
immobilization, motile sperm (Ͼ10 ϫ 10 /mL) prepared
from 3-donor semen were resuspended in 1 ml aliquots of
BWW-0.3% BSA in the presence and absence of 250 M
PHI-370. A corresponding volume (0.25%) of DMSO was
added to control tubes containing assay medium. Following
(BSA) (BWW-3% BSA).
1
5 min incubation at 37°C, duplicate aliquots of control and
Two-milliliter aliquots of sperm suspension were centri-
test sperm suspensions were used for sperm motility assess-
ment using CASA. The remaining sperm suspension was
washed by addition of fresh assay medium and centrifuga-
tion (500 g for 5 min). The supernatants were discarded, and
the pellets were resuspended in 1 ml each of BWW-0.3%
BSA medium (without PHI-370 or DMSO). Following 60
min incubation at 37°C, duplicate aliquots were reassessed
for sperm motion parameters by CASA. The results were
compared to the sperm motion parameters of similarly pro-
cessed sperm suspensions of motile sperm suspended in
medium lacking PHI-370.
fuged (500 g, 5 min) and the tubes were incubated at a 45°
angle for 60 min at 37°C in a 5% CO atmosphere. The
2
supernatant containing primarily motile sperm was recov-
ered in BWW medium containing 25 mM HEPES and 0.3%
BSA (BWW-0.3% BSA), and resuspended in the same
medium. The effect of increasing concentrations (1.9 to 500
M) of 12 phenyl arsenicals and incubation time on sperm
head centroid-derived sperm motility parameters was deter-
mined using a Hamilton-Thorne Integrated Visual Optical
System (IVOS) version 10.9i instrument (Hamilton–Thorne
Research Inc., Danvers, MA) as described previously
The kinematic parameters determined for each sperm
sample included numbers of motile (MOT) and progres-
sively (PRG) motile sperm, curvilinear velocity (VCL, a
measure of the total distance traveled by a given sperm
during the acquisition divided by the time elapsed), average
path velocity (VAP, the spatially averaged path that elimi-
nates the wobble of the sperm head), straight line velocity
[
9–12]. This computer system measures several kinematic
parameters of sperm head motion.
To evaluate the sperm immobilizing activity of 3 amino-
phenyl arsenicals (S-370, S379, and S-381) and their 9
N-substituted derivatives (PHI-273, PHI-370, PHI-371,
PHI-378, PHI-379, PHI-380, PHI-381, PHI-385, and PHI-
3
86), highly motile (84 Ϯ 6% progressive motility) frac-
7
(VSL, the straight-line distance from beginning to end of
tions of sperm (10 /mL), prepared from pooled donor sperm
track divided by time taken), beat cross frequency (BCF,
frequency of sperm head crossing sperm average path), the
amplitude of lateral head displacement (ALH, the mean
width of sperm head oscillation), and the derivatives,
straightness (STR ϭ VSL divided by VAP ϫ 100), linearity
(LIN ϭ VSL divided by VCL ϫ 100, departure of sperm
track from a straight line). Data from each individual cell
track were recorded and analyzed. At least 200 sperm were
analyzed for each aliquot sampled. Nonlinear regression
analysis was used to find the EC50 and t1/2 values (i.e.
concentration and time required for 50% sperm motility
loss) from the concentration-effect and time course curves,
specimens (n ϭ 7) were incubated in 1 ml BWW-0.3% BSA
(
pH 7.4) containing serial two-fold dilutions of the test
compound (500 M to 1.9 M). After 3 h of incubation at
7°C, 5-l aliquots of sperm were transferred to two 20-m
3
Microcell chambers (Conception Technologies), and sperm
motility was assessed by CASA. Sperm motility in sham-
treated control suspensions of motile sperm (i.e. similarly
processed sperm suspended in 1 ml of the corresponding
medium containing 0.5% DMSO in the absence of arseni-
cals) was determined for comparison. Aliquots (0.25 ml;
6
Ͼ30 ϫ 10 sperm/mL) of semen specimens were also
treated with increasing concentrations of sperm immobiliz-