Full text of DOI:10.1002/j.1939-4640.2003.tb02705.x

Journal of Andrology, Vol. 24, No. 4, July/August 2003
Copyright American Society of Andrology
᭧
Osmotic Tolerance Limits and Properties of Rhesus Monkey
Macaca mulatta) Spermatozoa
(
JOSEP RUTLLANT, ANGELA C. POMMER, AND STUART A. MEYERS
From the Sperm Biology Laboratory, Department of Anatomy, Physiology, and Cell Biology, School of Veterinary
Medicine, University of California, Davis, California.
ABSTRACT: Fundamental cryobiological characteristics of rhe-
sus spermatozoa must be determined for successful cryopres-
ervation techniques to be established. The main objectives of the
present study were to determine the osmotic behavior and os-
motic tolerance limits of rhesus macaque spermatozoa. Cell vol-
ume changes over anisotonic conditions were assessed using an
electronic particle counter and sperm motility was evaluated with
a computer-assisted sperm analysis system. Analysis of mem-
brane integrity and mitochondrial membrane potential was per-
formed using flow cytometry. Rhesus monkey spermatozoa be-
have as linear osmometers in the osmotic range tested (75–900
at 22ЊC, with 77.2% of the intracellular volume being osmotically
inactive. Results regarding sperm tolerance to osmotic stress
showed that sperm motility was more sensitive than membrane
integrity to deviations from isotonicity and, in addition, that rhesus
sperm motility and membrane integrity were more sensitive to
hypertonic than hypotonic conditions. Mitochondrial membrane
potential did not explain the lack of sperm motility observed under
anisosmolal conditions in our study. Although most spermatozoa
were able to recover initial volume after osmotic stress, they were
not able to recover initial motility.
Key words: Nonhuman primate, sperm, osmotic stress, motility,
mitochondrial membrane potential.
Ϫ1
2
mOsmol kg ), as shown by the Boyle van’t Hoff plot (r ϭ .99).
3
Rhesus spermatozoa have a mean cell volume of 36.8 Ϯ 0.5 ␮m
J Androl 2003;24:534–541
onhuman primates have been a relevant model for
the study of human disease because of the physio-
gress in developing a cryopreservation protocol that pre-
serves sperm motility in all semen samples.
N
logical and genetic similarities among members of the
primate order. The preservation of nonhuman primates as
important biomedical resources is critical, mainly for ge-
netically unique or altered individuals. There is also an
increasing need to preserve colonies of nonhuman pri-
mate species that are endangered because of human ac-
tivity such as habitat destruction and hunting. The devel-
opment of efficient nonhuman primate sperm cryopres-
ervation would aid in successful propagation of germ-
plasm through artificial insemination (AI) and in vitro
fertilization (IVF) programs.
Attempts to cryopreserve rhesus spermatozoa have
been developed on the basis of empirical approaches (Le-
verage et al, 1972; Sanchez-Partida et al, 2000), and, as
in other mammalian species, progress improving sperm
survival could not be achieved simply by modifying es-
tablished cryopreservation diluents (Hammerstedt et al,
1990). Cryopreservation requires the exposure of sper-
matozoa to extreme variations in temperature and osmo-
lality. When a solution is cooled below the freezing point,
pure water crystallizes out as ice, so that the solutes dis-
solved in the remaining liquid water fraction increases the
osmotic strength of the solution (Watson, 2000). In the
process of freezing cells in suspension, ice nucleation in
the extracellular space creates osmotic pressure changes
in the unfrozen fraction that affects the cells (Mazur,
1984; Watson and Duncan, 1988). It is generally recog-
nized that the duration of exposure to such events should
be minimized for optimal cell survival, implying that the
cooling rate should be rapid. However, the cooling rate
must be slow enough to allow water to leave the cells by
osmosis, preventing intracellular ice formation, which is
lethal (Mazur, 1984; Watson, 2000). To avoid these del-
eterious events and help cells to survive the process of
cryopreservation, cryoprotective agents (CPAs) must be
present during cooling and warming (Karow, 1969). Dur-
ing the addition of CPAs, cells gradually shrink as water
There have been attempts at freezing nonhuman pri-
mate sperm over the past 30 years, but reliable informa-
tion on the most appropriate methods is limited (Morrell
and Hodges, 1998). Cryopreserved sperm from some
nonhuman primates have been used for AI and IVF, but
macaque sperm, like human sperm, have highly variable
cryoprotection requirements depending on the individual
sperm donor. This biological variability has restricted pro-
Correspondence to: Stuart A. Meyers, Sperm Biology Laboratory, De-
partment of Anatomy, Physiology, and Cell Biology, School of Veterinary
Medicine, University of California, Davis, CA 95616 (phone: 530-752-
9511; fax: 530-752-7690; e-mail: smeyers@ucdavis.edu).
Received for publication October 16, 2002; accepted for publication
February 7, 2003.
534

Rutllant et al · Osmotic Tolerance and Properties of Rhesus Sperm
535
flows out of the cell to a hypertonic environment, then
cells return to normal volume as water and CPAs enter.
The reverse process occurs during CPA removal. These
osmotic events can severely damage the spermatozoa. A
more fundamental understanding of the biophysical and
biochemical characteristics that accompany the sperm
freezing and thawing processes is an essential prerequisite
to design successful cryopreservation protocols, as has
been described for other mammalian spermatozoa (Holt
and North, 1994). However, little information, if any, is
known regarding the fundamental cryobiology and bio-
physics of nonhuman primate sperm.
Animals and Semen Sample Collection
Semen samples were obtained by electroejaculation from male
rhesus macaques (Macaca mulatta) (n
ϭ 7) as described else-
where (Sarason et al, 1991). Animals were housed at California
National Primate Research Center and maintained according to
Institutional Animal Care and Use Committee protocols at the
University of California. Semen samples were collected into 15-
mL centrifuge tubes that containing 6 mL of HEPES-Biggers,
Whitten, and Whittingham or Dulbeccos’ phosphate-buffered sa-
line (DPBS). After 15 to 30 minutes, the coagulum was re-
moved, the semen was diluted with an additional 6 mL of the
same medium, and the sperm suspensions were evaluated for
motility and viability. After centrifugation, the sperm pellets
were resuspended, and samples were subsequently divided into
aliquots according to the experimental design.
There are primary biophysical properties with particu-
lar significance to cryopreservation, such as isosmotic cell
volume (V ), which is the volume of the cell in osmotic
iso
equilibrium within an isosmotic solution; the osmotically
Media
inactive fraction of cell volume (V ); the hydraulic con-
b
Ϫ1
The media used were isotonic (300
isotonic DPBS. Anisotonic solutions were prepared by diluting
isotonic DPBS (hypotonic solutions; 75 and 150 mOsmol kg )
and by diluting 10-strength DPBS (hypertonic solutions; 450,
Ϯ 5 mOsmol kg ) and an-
ductivity (L ), which reflects the membrane permeability
p
to water; and the activation energy for L (E ), which is
Ϫ1
p
a
the temperature dependence of L . There are also crucial
p
physiological properties that need to be preserved in order
to be able to fertilize that can be affected as a result of
osmotic stress, such as membrane integrity, motility, and
mitochondrial membrane potential (MMP). Progressive
motility is dependent on proper mitochondrial function,
since these organelles must produce energy in the form
of adenosine triphosphate (ATP) to power the flagellar
motion that propels the sperm to the site of fertilization
Ϫ1
6
00, and 900 mOsmol kg ) with reagent-grade water for ex-
periments in which the mean cell volume was determined. To
achieve the final osmolalities of 75, 150, 450, 600, and 900
Ϫ
1
mOsmol kg in the experiments in which the sperm motility,
MMP, and viability were assessed, an adjusted set of anisotonic
solutions was prepared (20, 115, 490, 675, and 1050 mOsmol
Ϫ1
kg , respectively), to avoid the dilution effect (1 :4) of the
sperm sample in the anisotonic media. Final osmolalities were
always confirmed using a vapor pressure osmometer (model
(Garner and Thomas, 1999).
5
100 C; Wescor Inc, Logan, Utah) that was calibrated against
Electronic particle counters have been used to deter-
Ϫ
1
1
00, 285, and 900 mOsmol kg standards for accuracy within
mine membrane permeability characteristics in the sperm
of several species—humans (Laufer et al, 1977; Gilmore
et al, 1995), pigs (Gilmore et al, 1996, 1998a), mice (Wil-
loughby et al, 1996), bulls (Petrunkina et al, 2001), and
horses (Pommer et al, 2002). The advantages of electronic
particle counters are that they allow rapid and reproduc-
ible collection and analysis of data, a variety of cell
shapes and sizes can be analyzed without any required
assumption regarding shape, and multiple cell populations
can be studied in a bulk sample (Acker et al, 1999).
The main objectives of the present study were to de-
termine the osmotic behavior and the osmotic tolerance
limits of rhesus monkey spermatozoa. For the first objec-
tive, an electronic particle counter able to detect cell vol-
ume changes over anisotonic conditions was used. For the
second objective, computer-assisted sperm analysis
Ϫ
1
Ϯ5 mOsmol kg .
Experimental Design
Experiment 1. Osmotic Behavior of Rhesus Monkey Spermato-
zoa—A Coulter Counter (Z2 model; Coulter Corp, Miami, Fla)
with a standard 50-
volume (MCV) was calibrated using spherical polystyrene latex
beads of 3 different diameters (3, 5, and 10 m, CC Size Stan-
dard 6602793, 6602794, and 6602796, respectively; Coulter
Corp, Miami, Fla) measured in each of the iso- and anisotonic
solutions used, as suggested by the manufacturer. The Coulter
counter was interfaced to a microcomputer, and data were ac-
quired using specific software (Accucomp; Coulter Corp, Miami,
␮m aperture tube was used. Sperm mean cell
␮
Fla). Twenty microliters of sperm suspension adjusted to 20
ϫ
6
Ϫ1
10 cells mL was placed in 20 mL of isotonic or anisotonic
Ϫ
1
DPBS (final concentration 20 000 cells mL ). After 10 minutes,
a histogram displaying particle count versus volume (cell volume
distribution) was recorded, as well as different statistical param-
eters (mean, mode, median, and standard deviation of volume).
To observe whether sperm cells subjected to anisotonic condi-
(CASA) and flow cytometry were used to analyze motil-
ity, viability, and MMP.
tions could recover the isotonic volume, 20
␮L of sperm sus-
Materials and Methods
6
Ϫ1
pension of a solution of 20
ϫ 10 cells mL were placed in 2
Chemicals and Reagents
Propidium iodide and JC-1 were purchased from Molecular
Probes (Eugene, Ore). All other chemicals were obtained from
Sigma Chemical Company (St Louis, Mo).
mL of anisotonic DPBS. After 10 minutes, 18 mL of isotonic
DPBS were added, and volume determinations were performed
after 5 more minutes (Gilmore et al, 1998a). Sperm incubated
Ϫ1
in 300 mOsmol kg DPBS were used as control samples.

536
Journal of Andrology · July/August 2003
To determine the inactive cell volume and estimate whether
monkey sperm membranes behave as linear osmometers, sperm
volumes recorded in iso- and anisotonicity conditions were fitted
to the Boyle van’t Hoff equation:
V
Miso
M
Vb
Vb
ϭ
1
Ϫ
ϩ
Viso
[
Viso
]
Viso
where V is the cell volume at the osmolality M, V is the cell
volume at isotonicity (M ), and V is the osmotically inactive
iso
iso
b
cell volume.
Experiment 2. Osmotic Tolerance Of Rhesus Monkey Sper-
matozoa—Evaluation of sperm motility: For motility experi-
6
Ϫ1
ments, 50
placed in 200
of anisotonic DPBS solutions to get a final concentration of 75,
␮
L of the sperm suspension (150
␮
ϫ 10 ml ) was
Figure 1. Sperm samples were incubated for 10 minutes at room tem-
perature in isotonic or anisotonic buffer. MCV was then determined using
a Beckman Coulter Counter (Z2). In separate tubes, after the initial 10-
minute incubation, samples were diluted with isotonic buffer and incu-
bated for 10 minutes before volume was determined. Superscript (a) de-
Ϫ
1
L isotonic DPBS (300 mOsmol kg ) or a range
Ϫ
1
1
1
50, 450, 600, and 900 mOsmol kg and incubated at 22
Њ
C for
6
Ϫ1
0 minutes (30
ϫ
10 mL ) prior to motility analysis. Sperm
Ϫ1
notes significance from control (300 mOsmol kg ) volume (n ϭ 5 mon-
suspensions were then returned to near-isotonic conditions by
keys).
6
Ϫ1
adding 1.25 mL of isotonic DPBS (5
ϫ 10 mL ), as described
by Gilmore et al (1998a). After 5 minutes of incubation, motility
was analyzed again. At least 200 cells were evaluated using
CASA for all experiments in which motility was determined
(
Minitab Inc, State College, Penn), with a level of significance
set at 5%. ANOVA was used to evaluate treatment differences
over a range of anisotonic treatments. End points included sperm
total motility, viability, MMP, and MCV. Data were also blocked
by individual male and pooled if no significant differences were
observed among males. In some experiments, data were nor-
malized to control values prior to analysis. Data are presented
(CEROS, version 10.9i; Hamilton Thorne Biosciences, Inc, Bev-
erly, Mass). The settings used were: frame rate 60 Hz, frames
acquired 30, minimum contrast 30, minimum cell size 4, thresh-
old straightness 80, medium average path velocity (VAP) cutoff
25, low VAP cutoff 5, low straight line velocity cutoff 10, non-
motile head size 12, nonmotile head intensity 80, static size lim-
its 0.5 to 2.5, static intensity limits 0.55 to 1.4, and static elon-
gation limits 0 to 80.
as means
Ϯ standard error (SE).
Evaluation of mitochondrial function: For MMP experiments,
6
Ϫ1
2
00
to 800
concentration of 30
perature (22 C) for 10 minutes. Then, each 1-mL sample was
divided into 2 500- L samples. To the first set of tubes in each
treatment, a final concentration of 2 M JC-1 was added for 10
␮
L of the sperm suspension (150
ϫ
10 mL ) was added
Results
␮
L of isotonic or anisotonic DPBS, for a final sperm
6
Ϫ
1
Experiment 1
The isotonic MCV (mean
matozoa was determined to be 36.8
measured with an electronic particle counter, with no sig-
nificant difference among males (P .958). The MCV
was different from the control volume (300 mOsmol kg )
ϫ
10 mL , and incubated at room tem-
Њ
Ϯ SE) of rhesus monkey sper-
␮
3
Ϯ 0.5 ␮m at 22ЊC
␮
minutes, and the samples were evaluated using a flow cytometer,
as described below. In the second set of tubes, sperm were di-
ϫ 10 mL ) for 10
minutes and then incubated for another 10 minutes with 2
JC-1 before being analyzed on the flow cytometer. Samples were
also visually assessed by epifluorescence microscopy by a single
observer and evaluated for the uptake of dye and fluorescence
pattern.
Determination of viability: The same protocol as described in
‘Evaluation of Mitochondrial Function’’ was used for the via-
ϭ
Ϫ
1
6
Ϫ1
luted with 1.5 mL of isotonic DPBS (7.5
Ϫ
1
in 75, 150, and 900 mOsmol kg anisotonic solutions (P
.05) but not statistically different in 450 and 600
␮
M
Ͻ
Ϫ
1
mOsmol kg . Spermatozoa in all treatments were able to
recover initial volume when returned to isotonic medium
(P
times initial volume when exposed to 75 mOsmol kg
DPBS and decreased to 0.83 times initial volume when
Ͼ .05) (Figure 1). MCV swelled approximately 1.68
Ϫ
1
‘
bility studies. The only difference was that propidium iodide (5
Ϫ
1
incubated in 900 mOsmol kg DPBS (Table).
The osmotic response of monkey spermatozoa was de-
termined over the range 75 to 900 mOsmol at 22ЊC. Data
␮
M) was used instead of JC-1.
Flow cytometry: Information on 10 000 spermatozoa from
each sperm sample used in the evaluation of mitochondrial func-
tion and determination of viability studies were collected using
a FACS analyzer flow cytometer (FACSCalibur; Becton-Dick-
inson Immunocytometry Systems, San Jose, Calif), and gener-
ated data were examined using CellQuest software (version 3.3;
Becton-Dickinson Immunocytometry Systems, San Jose, Calif).
Statistical analysis: Data were analyzed using one-way anal-
ysis of variance (ANOVA) with Minitab statistical software
are presented as a Boyle van’t Hoff plot (volume vs 1/
osmolality) and depicts a linear response (r2
ϭ 0.99) and
a V of 77.2% (Figure 2).
b
Experiment 2
Evaluation of Sperm Motility—Normalized total sperm
motility was significantly lower (P .05) in 75, 600, and
Ͻ

Rutllant et al · Osmotic Tolerance and Properties of Rhesus Sperm
537
Normalized monkey sperm cell volume, motility, and viability after
incubation in anisotonic solutions for 10 minutes and returning to
isotonic conditions and MMP in anisotonic DPBS
Normalized
Osmolality
mOsmol kg
High
Ϫ1
(
)
Volume
Motility (%) Viability (%) MMP (%)
7
5
1.68
1.32
1
0.92
0.88
0.83
63.76
89.88
100.00
79.76
30.59
8.94
84.92
95.67
100.00
98.78
86.62
80.36
81.77
79.49
76.93
62.48
41.78
41.15
1
3
4
6
9
50
00
50
00
00
Ϫ1
Figure 2. Boyle van’t Hoff plot (volume vs osmolality ) of monkey sper-
matozoa at 22ЊC. Sperm samples were exposed to different osmolalities:
Ϫ1
9
00, 600, 450, 300, 150, and 75 mOsmol kg DPBS. The y-intercept
Ϫ
1
9
00 mOsmol kg anisotonic conditions compared with
b
indicates that V , the osmotically inactive volume, is 77.2% of the isotonic
cell volume (n ϭ 5 monkeys).
controls (Figure 3). However, statistically significant dif-
ferences in sperm motility were not observed (P .05)
when incubated in 150 and 450 mOsmol kg and com-
pared with controls.
Ͼ
Ϫ
1
onstrated by the increased number of stained cells with
propidium iodide.
When sperm were returned to isosmolal conditions,
Ϫ
1
only sperm previously incubated in 900 mOsmol kg
DPBS were not able to recover control levels of motility
.05). A nonspecific decrease in motility was also
observed after dilution into isosmolal DPBS; however,
significant differences were not observed (P .27).
Evaluation of Mitochondrial Function—Using the ap-
propriate gating parameters for monkey sperm determined
during preliminary experiments, the cell population
(P Ͻ
Discussion
ϭ
Cells undergoing freezing are initially exposed to extra-
cellular ice crystallization from extracellular water that
results in hyperosmolal concentration of solutes (Watson,
1995, 2000). The cells respond to this insult by losing
(
10 000 events) was divided into either high membrane
water and shrinking in volume so that the solute concen-
tration between intra- and extracellular compartments can
equilibrate. Conversely, as cells are exposed to a hypo-
tonic extracellular environment, as in the case during
thawing, cell volume is increased by passive diffusion of
water. So the determination of the osmotic limits that
sperm cells can tolerate could be applied to minimize the
deleterious consequences associated with volume excur-
sion and therefore improve cryopreservation techniques.
potential (red/green fluorescence) or low membrane po-
tential (green fluorescence), according to natural parti-
tioning. The percentages of sperm with high and low
MMPs subjected to different anisotonic conditions are
shown in Figure 4A. Spermatozoa subjected to hypertonic
Ϫ
1
conditions (600 and 900 mOsmol kg ) showed a signif-
icant decrease (P .004) of the percentage of population
with high MMP and, consequently, a significant increase
.01) of the percentage of population with low MMP
ϭ
(P ϭ
compared with control samples. However, when the in-
tensity of ‘‘red’’ and ‘‘green’’ (high and low MMP, re-
spectively) was quantified, an unexpected increase (P
Ͻ
.02) of individual cell intensity was observed in sper-
matozoa exposed to hypotonic conditions (Figure 4B).
Sperm suspensions that returned to isotonic conditions af-
ter being exposed for 10 minutes to anisotonic solutions
showed no significant differences in percentage of pop-
ulation with high and low MMP (Figure 4C) or cell in-
tensity (Figure 4D) compared with control samples.
Sperm viability—The percentage of membrane-intact
spermatozoa after 10 minutes of incubation in anisotonic
solutions is shown in Figure 5. Membrane integrity was
apparently unaffected by exposure to anisotonic solutions,
but, after returning to isotonic conditions, spermatozoa
Figure 3. Monkey sperm samples were incubated for 10 minutes in iso-
tonic or anisotonic DPBS, and total motility was analyzed using a CASA
system. Samples were then diluted with adjusted DPBS, to bring solu-
tions near to isotonicity, incubated for 5 minutes, and motility analyzed
again. Superscript (a, b) denotes significant difference from control (300
Ϫ
1
incubated previously in 900 mOsmol kg solution
showed a significant decrease (P .05) of viability dem-
Ϫ1
Ͻ
mOsmol kg ) within each treatment group (n ϭ 7 monkeys).

538
Journal of Andrology · July/August 2003
Osmotic Behavior of Rhesus Monkey Spermatozoa
The isotonic cell volume of rhesus monkey spermatozoa
determined using an electronic particle counter was 36.8
3
Ϯ
0.5
␮
m at 22
ЊC with 77.2% of the total cell volume
being osmotically inactive (V ) (both solids and bound
b
water). The determination of the osmotic response and
Boyle van’t Hoff relationship of a cell type helps char-
acterize the response of a cell to osmotic stress that occurs
during the cryopreservation process. In the present study,
monkey sperm behaved as linear osmometers in the range
Ϫ
1
2
of 75 to 900 mOsmol kg (r
ϭ .99). When a cell be-
haves as an ideal osmometer, the volume of the osmoti-
cally available water contained in the cell is inversely
related to the osmolality of nonpermeable solutes in the
external medium (Mazur, 1984). Then, the osmotically
active cell volume (Viso
Ϫ V ) indicates the amount of
b
water in the cells that can be lost (unbound water) during
the osmotic stress occurred during cryopreservation or in
other physiological events. In monkey spermatozoa, only
2
2.8% of the total cell volume in isotonic conditions is
3
osmotically active, which approximates 8.4 ␮m of water.
Compared with spermatozoa from various species studied
thus far, 22.8% is the smallest active cell volume recorded
(
man 50%, Gilmore et al, 1995; murine 39.3%, Willough-
by et al, 1996; boar 32.6%, Gilmore et al, 1996; and horse
9.3%, Pommer et al, 2002). During the freezing process,
2
water must leave the cells by osmosis to prevent intra-
cellular ice formation, which is lethal. The duration for
water loss depends on the volume of water that has to be
released from the cell, which is a small amount for ma-
caque spermatozoa, and also on the membrane hydraulic
conductivity (L ) (Gao et al, 1995). Hydraulic conductiv-
p
ity values at different temperatures in the presence or ab-
sence of CPAs have not been determined for monkey
spermatozoa but are the basis for ongoing investigations
in our laboratory.
Rhesus monkey spermatozoa respond similarly to
sperm from other mammalian species exhibiting a linear
response to a given osmotic range (human, Gilmore et al,
1995; boar, Du et al, 1994; Gilmore et al, 1996; mouse,
Willoughby et al, 1996; impala, wart hog, elephant, and
lion, Gilmore et al, 1998b; horse, Pommer et al, 2002).
The observed linear osmotic behavior included hypo- and
←
of 2 ␮M JC-1 was added to each treatment for 10 minutes, and the
samples were read on the flow cytometer. (C) and (D) Samples previ-
ously subjected to anisotonicity conditions were diluted with 1.5 mL iso-
tonic DPBS for 10 minutes and then incubated for another 10 minutes
with 2 ␮M JC-1 before being analyzed on the flow cytometer. The graphs
show percentage of cells with high and low MMP (A, C) and normalized
fluorescence intensity for high and low MMP (B, D). Red-labeled cells
were considered to have high MMP, whereas those labeled green were
considered to have low MMP. Superscripts (a, b) denote significance
Figure 4. Sperm samples were incubated for 10 minutes at room tem-
perature in isotonic or anisotonic DPBS. (A) and (B) A final concentration
Ϫ1
from control (300 mOsmol kg ) values (n ϭ 3 monkeys).

Rutllant et al · Osmotic Tolerance and Properties of Rhesus Sperm
539
potheses have been suggested for this finding (Gao et al,
993; Willoughby et al, 1996; Pukazhenthi et al, 2000).
1
The first hypothesis is that spermatozoa respond as red
blood cells do, which undergo a process called posthy-
pertonic hemolysis (spermolysis in this case), which con-
sists of an overload of normally impermeable solutes due
to membrane leakage on hypertonic stress. Then, when
the cell returns to isotonic conditions, it swells to above
its normal isotonic volume and lyses (Lovelock, 1953;
Zade-Oppen, 1968). Another possible explanation is that
cell shrinkage induces membrane loss, so that, when they
return to isotonic conditions, the cells lyse in attempting
to return to their normal volume (Steponkus and Wiest,
1979). It has been observed by scanning electron micros-
copy that sperm subjected to hypertonic conditions dis-
play exvaginations of the head membrane, resulting in a
wrinkled surface (Hammerstedt et al, 1990; Gao et al,
Figure 5. Monkey sperm samples were incubated for 10 minutes in iso-
tonic or anisotonic DPBS, and duplicated samples were returned to iso-
tonic conditions. A final concentration of 5 ␮M propidium iodide was add-
ed to each treatment for 10 minutes, and the samples were analyzed
using a flow cytometer. Superscript (a) denotes significant difference
1993). If this wrinkled surface results in a fusion of con-
tiguous membranes under the influence of hypertonic
stress, as has been described in other cell types (Homann,
Ϫ1
from control (300 mOsmol kg ) within each treatment group (n ϭ 5
monkeys)
1998), an abrupt return to isosmolal conditions would
possibly result in cell lysis.
Although the percentage of cells showing adequate
membrane integrity was high through the osmotic ranges
tested here, sperm motility markedly declined in response
to deviations from isotonicity. Samples exposed to 75
Ϫ
1
hypertonic conditions (75–900 mOsmol kg ), which sug-
gests that the values for exosmotic and endosmotic hy-
draulic flows were similar in the tested osmolality range.
Rhesus monkey sperm cell volume has not been pre-
viously reported; therefore, we cannot compare our results
to other studies. However, using measures obtained by
automated sperm morphometric analysis (Gago et al,
Ϫ
1
mOsmol kg resulted in an almost 40% decrease in mo-
tility, and samples exposed to the extreme hypertonicity
Ϫ
1
tested in the present study (900 mOsmol kg ) resulted in
a decrease of more than 90% from the isotonic motility
(control). Similar to human (Gao et al, 1993; Curry and
Watson, 1994), mouse (Willoughby et al, 1996; Songan-
sen and Leibo, 1997), ram (Curry and Watson, 1994),
boar (Gilmore et al, 1998a), bovine (Liu and Foote,
1998), and equine (Pommer et al, 2002) sperm, macaque
sperm motility was more sensitive to anisotonic condi-
tions than membrane integrity. However, it appeared that,
in macaque sperm, in contrast to other species, the shrink-
ing process was more detrimental than the swelling pro-
cess in terms of motility in the osmotic range tested.
Sperm progressive motility is of primary importance, be-
cause it is required for sperm to reach the site of fertil-
ization and penetrate into the oocyte. Therefore, viable
but nonmotile sperm are not useful for AI and IVF pur-
poses. These observations suggest that, during the cryo-
preservation of macaque sperm, the freezing process
might be more detrimental than the thawing process. A
correlation between posthypertonic injury of human
sperm and the time of cell exposure to the hypertonic
environment before returning to isosmotic conditions has
been observed (Gao et al, 1993). The shorter the time,
the less cell injury, which indicates that cells can tolerate
severe shrinkage for a short time.
1
1
999) and scanning electron microscopy (Martin et al,
975), the estimated macaque sperm volume is approxi-
m . As has been described in mouse
Willoughby et al, 1996), human (Laufer et al, 1977; Gil-
more et al, 1995), and horse (Pommer et al, 2002) sperm,
the estimates obtained from electronic cell sizers are
slightly smaller than estimates obtained from microscopic
measurements.
3
mately 40 to 45
(
␮
Osmotic Tolerance of Rhesus Monkey Spermatozoa
The present study demonstrates that a high percentage of
Ͼ80%) maintain viability or membrane
integrity in the osmotic range tested (75–900 mOsmol
kg ). Only sperm subjected for 10 minutes to 900
mOsmol kg solution and returned to isotonicity showed
a significant decrease in viability (P .05) compared
with control spermatozoa (isotonic conditions), but even
then the viability was greater than 80%. The deleterious
effect on membrane integrity of the returning process to
isotonicity after being incubated in hypertonic conditions
has been described before in human (Gao et al, 1993),
mouse (Willoughby et al, 1996; Songansen and Leibo,
macaque sperm (
Ϫ1
Ϫ
1
Ͻ
1
997), feline (Pukazhenthi et al, 2000) and, recently, in
equine (Pommer et al, 2002) spermatozoa. Several hy-
Progressive motility is dependent on proper mitochon-

540
Journal of Andrology · July/August 2003
drial function, because mitochondria produce energy in
the form of ATP to power the flagellar motion that propels
the sperm (Garner and Thomas, 1999). Results from the
present study indicate that anisosmotic conditions, hypos-
motic and hyperosmotic, affect MMP differently. Hyper-
osmotic conditions induced a decrease in the percentage
of spermatozoa with high MMP, but there are 2 obser-
vations that indicate that sperm motility is affected by
factors other than MMP. The first is the lack of correlation
between the percentage of cells exhibiting high MMP and
the percentatge of motile cells. Spermatozoa subjected to
matozoa that can be applied to improve current cryopres-
ervation protocols. The results showed that 1) macaque
spermatozoa exhibited a linear osmotic response in the
Ϫ
1
range of 75–900 mOsmol kg ; 2) macaque spermatozoa
in isotonic media had a mean cell volume of 36.8 0.5
C as measured with an electronic particle coun-
Ϯ
3
␮
m at 22Њ
ter, with 77.2% being osmotically inactive; 3) motility
was more sensitive than membrane integrity to changes
in osmolality; 4) macaque sperm motility and membrane
integrity were more sensitive to hypertonic than hypoton-
ic conditions; 5) MMP did not explain the lack of sperm
motility observed in our study; and 6) although most sper-
matozoa were able to recover initial volume after osmotic
stress, they were not able to recover initial motility. This
information, combined with current investigations of ma-
Ϫ
1
6
00 and 900 mOsmol kg had a similar percentage of
cells with high MMP (approximately 41%); however, the
percentage of motile cells was significantly and dispro-
portionately lower (30% and 9%, respectively). The sec-
ond observation is that sperm suspensions that returned
to isotonic conditions after being exposed for 10 minutes
to anisotonic solutions showed no significant differences
in the percentage of the population with high and low
MMP compared with control samples, so a recovery of
MMP was observed. However, sperm previously incu-
caque sperm membrane hydraulic conductivity (L ) at var-
p
ious temperatures, in the presence or absence of CPA, will
be used to limit osmotic stress during cryopreservation.
Acknowledgments
Ϫ
1
bated in 900 mOsmol kg DPBS were not able to recover
control levels of motility. In the process of ATP synthesis,
a high MMP is needed to constitute the electrochemical
proton gradient that the enzyme ATP synthase will use to
drive the energetically unfavorable reaction between ADP
We gratefully acknowledge Dr Mary Delaney, Department of Animal
Science, College of Agriculture and Environmental Sciences, for techni-
cal assistance with the Z2 Coulter counter and Abigail Spinner, California
National Primate Research Center, for technical assistance with flow cy-
tometry.
and P that makes ATP. However, a high MMP does not
i
indicate concentration of ATP produced, which, ultimate-
ly, is the energy used by the flagellum. Perhaps JC-1 is a
good indicator of MMP, but it does not assess either the
final level of ATP or the lack of motility observed in our
study.
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