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From the Sperm Biology Laboratory, Department of Anatomy, Physiology, and Cell Biology, School of Veterinary Medicine, University of California, Davis, California.
| Correspondence to: Stuart A. Meyers, Sperm Biology Laboratory, Department 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{at}ucdavis.edu). |
| Received for publication October 16, 2002; accepted for publication February 7, 2003. |
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Abstract |
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Key words: Nonhuman primate, sperm, osmotic stress, motility, mitochondrial membrane potential
There have been attempts at freezing nonhuman primate sperm over the past 30 years, but reliable information 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 progress in developing a cryopreservation protocol that preserves sperm motility in all semen samples.
Attempts to cryopreserve rhesus spermatozoa have been developed on the basis of empirical approaches (Leverage 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 established cryopreservation diluents (Hammerstedt et al, 1990). Cryopreservation requires the exposure of spermatozoa to extreme variations in temperature and osmolality. When a solution is cooled below the freezing point, pure water crystallizes out as ice, so that the solutes dissolved 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 recognized 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 deleterious events and help cells to survive the process of cryopreservation, cryoprotective agents (CPAs) must be present during cooling and warming (Karow, 1969). During the addition of CPAs, cells gradually shrink as water 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 biophysics of nonhuman primate sperm.
There are primary biophysical properties with particular significance to cryopreservation, such as isosmotic cell volume (Viso), which is the volume of the cell in osmotic equilibrium within an isosmotic solution; the osmotically inactive fraction of cell volume (Vb); the hydraulic conductivity (Lp), which reflects the membrane permeability to water; and the activation energy for Lp (Ea), which is the temperature dependence of Lp. There are also crucial 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 (Garner and Thomas, 1999).
Electronic particle counters have been used to determine 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 (Willoughby 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 reproducible 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 determine the osmotic behavior and the osmotic tolerance limits of rhesus monkey spermatozoa. For the first objective, an electronic particle counter able to detect cell volume changes over anisotonic conditions was used. For the second objective, computer-assisted sperm analysis (CASA) and flow cytometry were used to analyze motility, viability, and MMP.
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Materials and Methods |
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Animals and Semen Sample Collection
Semen samples were obtained by electroejaculation from male rhesus macaques
(Macaca mulatta) (n = 7) as described elsewhere
(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 saline (DPBS). After 15 to 30 minutes, the
coagulum was removed, 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.
Media
The media used were isotonic (300 ± 5 mOsmol
kg–1) and anisotonic DPBS. Anisotonic solutions
were prepared by diluting isotonic DPBS (hypotonic solutions; 75 and 150
mOsmol kg–1) and by diluting 10-strength DPBS
(hypertonic solutions; 450, 600, and 900 mOsmol
kg–1) with reagent-grade water for experiments in
which the mean cell volume was determined. To achieve the final osmolalities
of 75, 150, 450, 600, and 900 mOsmol kg–1 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 kg–1, 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 5100 C; Wescor
Inc, Logan, Utah) that was calibrated against 100, 285, and 900 mOsmol
kg–1 standards for accuracy within ±5
mOsmol kg–1.
Experimental Design
Experiment 1. Osmotic Behavior of Rhesus Monkey
Spermatozoa—
A Coulter Counter (Z2 model; Coulter Corp, Miami, Fla) with a standard
50-µm aperture tube was used. Sperm mean cell volume (MCV) was calibrated
using spherical polystyrene latex beads of 3 different diameters (3, 5, and 10
µm, CC Size Standard 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 acquired using specific software (Accucomp;
Coulter Corp, Miami, Fla). Twenty microliters of sperm suspension adjusted to
20 x 106 cells mL–1 was placed in
20 mL of isotonic or anisotonic DPBS (final concentration 20 000 cells
mL–1). After 10 minutes, a histogram displaying
particle count versus volume (cell volume distribution) was recorded, as well
as different statistical parameters (mean, mode, median, and standard
deviation of volume). To observe whether sperm cells subjected to anisotonic
conditions could recover the isotonic volume, 20 µL of sperm suspension of
a solution of 20 x 106 cells mL–1
were placed in 2 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 in 300 mOsmol kg–1 DPBS were used as
control samples.
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:
Experiment 2. Osmotic Tolerance Of Rhesus Monkey Spermatozoa— Evaluation of sperm motility: For motility experiments, 50 µL of the sperm suspension (150 x 106 ml–1) was placed in 200 µL isotonic DPBS (300 mOsmol kg–1) or a range of anisotonic DPBS solutions to get a final concentration of 75, 150, 450, 600, and 900 mOsmol kg–1 and incubated at 22°C for 10 minutes (30 x 106 mL–1) prior to motility analysis. Sperm suspensions were then returned to near-isotonic conditions by adding 1.25 mL of isotonic DPBS (5 x 106 mL–1), 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 (CEROS, version 10.9i; Hamilton Thorne Biosciences, Inc, Beverly, Mass). The settings used were: frame rate 60 Hz, frames acquired 30, minimum contrast 30, minimum cell size 4, threshold straightness 80, medium average path velocity (VAP) cutoff 25, low VAP cutoff 5, low straight line velocity cutoff 10, nonmotile head size 12, nonmotile head intensity 80, static size limits 0.5 to 2.5, static intensity limits 0.55 to 1.4, and static elongation limits 0 to 80.
Evaluation of mitochondrial function: For MMP experiments, 200 µL of the sperm suspension (150 x 106 mL–1) was added to 800 µL of isotonic or anisotonic DPBS, for a final sperm concentration of 30 x 106 mL–1, and incubated at room temperature (22°C) for 10 minutes. Then, each 1-mL sample was divided into 2 500-µL samples. To the first set of tubes in each treatment, a final concentration of 2 µM JC-1 was added for 10 minutes, and the samples were evaluated using a flow cytometer, as described below. In the second set of tubes, sperm were diluted with 1.5 mL of isotonic DPBS (7.5 x 106 mL–1) for 10 minutes and then incubated for another 10 minutes with 2 µM 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 viability studies. The only difference was that propidium iodide (5 µM) was used instead of JC-1.
Flow cytometry: Information on 10 000 spermatozoa from each sperm sample used in the evaluation of mitochondrial function and determination of viability studies were collected using a FACS analyzer flow cytometer (FACSCalibur; Becton-Dickinson Immunocytometry Systems, San Jose, Calif), and generated data were examined using CellQuest software (version 3.3; Becton-Dickinson Immunocytometry Systems, San Jose, Calif).
Statistical analysis: Data were analyzed using one-way analysis of variance (ANOVA) with Minitab statistical software (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 normalized to control values prior to analysis. Data are presented as means ± standard error (SE).
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Results |
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The osmotic response of monkey spermatozoa was determined over the range 75 to 900 mOsmol at 22°C. Data are presented as a Boyle van't Hoff plot (volume vs 1/osmolality) and depicts a linear response (r2 = 0.99) and a Vb of 77.2% (Figure 2).
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Experiment 2
Evaluation of Sperm Motility—
Normalized total sperm motility was significantly lower (P <
.05) in 75, 600, and 900 mOsmol kg–1 anisotonic
conditions compared with controls (Figure
3). However, statistically significant differences in sperm
motility were not observed (P > .05) when incubated in 150 and 450
mOsmol kg–1 and compared with
controls.
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When sperm were returned to isosmolal conditions, only sperm previously incubated in 900 mOsmol kg–1 DPBS were not able to recover control levels of motility (P < .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 appropriate gating parameters for monkey sperm determined during preliminary experiments, the cell population (10 000 events) was divided into either high membrane potential (red/green fluorescence) or low membrane potential (green fluorescence), according to natural partitioning. The percentages of sperm with high and low MMPs subjected to different anisotonic conditions are shown in Figure 4A. Spermatozoa subjected to hypertonic conditions (600 and 900 mOsmol kg–1) showed a significant decrease (P = .004) of the percentage of population with high MMP and, consequently, a significant increase (P = .01) of the percentage of population with low MMP compared with control samples. However, when the intensity of ``red'' and ``green'' (high and low MMP, respectively) was quantified, an unexpected increase (P < .02) of individual cell intensity was observed in spermatozoa exposed to hypotonic conditions (Figure 4B). Sperm suspensions that returned to isotonic conditions after being exposed for 10 minutes to anisotonic solutions showed no significant differences in percentage of population with high and low MMP (Figure 4C) or cell intensity (Figure 4D) compared with control samples.
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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 incubated previously in 900 mOsmol kg–1 solution showed a significant decrease (P < .05) of viability demonstrated by the increased number of stained cells with propidium iodide.
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Discussion |
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Osmotic Behavior of Rhesus Monkey Spermatozoa
The isotonic cell volume of rhesus monkey spermatozoa determined using an
electronic particle counter was 36.8 ± 0.5 µm3 at
22°C with 77.2% of the total cell volume being osmotically inactive
(Vb) (both solids and bound water). The
determination of the osmotic response and Boyle van't Hoff relationship of a
cell type helps characterize 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 of 75 to 900 mOsmol
kg–1 (r2 = .99). When a cell
behaves as an ideal osmometer, the volume of the osmotically 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 – Vb) indicates
the amount of 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 22.8% of the total cell volume in isotonic
conditions is osmotically active, which approximates 8.4 µm3 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%, Willoughby et al, 1996;
boar 32.6%, Gilmore et al,
1996; and horse 29.3%, Pommer
et al, 2002). During the freezing process, water must leave the
cells by osmosis to prevent intracellular 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 macaque spermatozoa, and also on
the membrane hydraulic conductivity (Lp)
(Gao et al, 1995). Hydraulic
conductivity values at different temperatures in the presence or absence 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 hypertonic conditions (75–900 mOsmol kg–1), which suggests that the values for exosmotic and endosmotic hydraulic flows were similar in the tested osmolality range.
Rhesus monkey sperm cell volume has not been previously reported; therefore, we cannot compare our results to other studies. However, using measures obtained by automated sperm morphometric analysis (Gago et al, 1999) and scanning electron microscopy (Martin et al, 1975), the estimated macaque sperm volume is approximately 40 to 45 µm3. As has been described in mouse (Willoughby et al, 1996), human (Laufer et al, 1977; Gilmore 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.
Osmotic Tolerance of Rhesus Monkey Spermatozoa
The present study demonstrates that a high percentage of macaque sperm
(>80%) maintain viability or membrane integrity in the osmotic range tested
(75–900 mOsmol kg–1). Only sperm subjected
for 10 minutes to 900 mOsmol kg–1 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, 1997),
feline (Pukazhenthi et al,
2000) and, recently, in equine
(Pommer et al, 2002)
spermatozoa. Several hypotheses have been suggested for this finding
(Gao et al, 1993; Willoughby et al, 1996;
Pukazhenthi et al, 2000). The
first hypothesis is that spermatozoa respond as red blood cells do, which
undergo a process called posthypertonic hemolysis (spermolysis in this case),
which consists 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
microscopy that sperm subjected to hypertonic conditions display exvaginations
of the head membrane, resulting in a wrinkled surface
(Hammerstedt et al, 1990;
Gao et al, 1993). If this
wrinkled surface results in a fusion of contiguous membranes under the
influence of hypertonic stress, as has been described in other cell types
(Homann, 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 mOsmol kg–1 resulted in an almost 40% decrease in motility, and samples exposed to the extreme hypertonicity tested in the present study (900 mOsmol kg–1) 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; Songansen 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 conditions than membrane integrity. However, it appeared that, in macaque sperm, in contrast to other species, the shrinking process was more detrimental than the swelling process in terms of motility in the osmotic range tested. Sperm progressive motility is of primary importance, because it is required for sperm to reach the site of fertilization and penetrate into the oocyte. Therefore, viable but nonmotile sperm are not useful for AI and IVF purposes. These observations suggest that, during the cryopreservation 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.
Progressive motility is dependent on proper mitochondrial 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, hyposmotic and hyperosmotic, affect MMP differently. Hyperosmotic conditions induced a decrease in the percentage of spermatozoa with high MMP, but there are 2 observations 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 600 and 900 mOsmol kg–1 had a similar percentage of cells with high MMP (approximately 41%); however, the percentage of motile cells was significantly and disproportionately lower (30% and 9%, respectively). The second 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 incubated in 900 mOsmol kg–1 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 and Pi that makes ATP. However, a high MMP does not indicate concentration of ATP produced, which, ultimately, 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.
Hypotonic solutions did not affect cell proportion with high and low MMP; however, individual cell fluorescence intensity increased unexpectedly in hypotonic conditions (Figure 5B). As in many other species and other cell types, macaque sperm experience a volume expansion that can reach 1.68 times the isotonic volume under hypotonic conditions (75 mOsmol kg–1) and still maintain more than 84% viability (membrane integrity). Folded membrane structures or storage of excess surface membrane in isotonicity has not been reported for sperm cells (Hammerstedt et al, 1990); therefore, an increase in cell volume could be accompanied with membrane stretching processes, because sperm have a fixed membrane surface. As was discussed elsewhere (Mazur, 1990), cell membranes are incapable of stretching more than a minute amount. Membrane stretch due to volume expansion could likely provoke sublethal membrane leakiness, as has been discussed elsewhere (Noiles et al, 1993), that could increase the uploading of JC-1 in the mitochondria, inducing a higher individual cell fluorescence intensity.
In summary, the present study has determined several important biophysical characteristics of macaque spermatozoa that can be applied to improve current cryopreservation protocols. The results showed that 1) macaque spermatozoa exhibited a linear osmotic response in the range of 75–900 mOsmol kg–1; 2) macaque spermatozoa in isotonic media had a mean cell volume of 36.8 ± 0.5 µm3 at 22°C as measured with an electronic particle counter, 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 hypotonic conditions; 5) MMP did not explain the lack of sperm motility observed in our study; and 6) although most spermatozoa were able to recover initial volume after osmotic stress, they were not able to recover initial motility. This information, combined with current investigations of macaque sperm membrane hydraulic conductivity (Lp) at various temperatures, in the presence or absence of CPA, will be used to limit osmotic stress during cryopreservation.
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Acknowledgments |
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Y. Agca, J. Liu, S. Mullen, J. Johnson-Ward, K. Gould, A. Chan, and J. Critser Chimpanzee (Pan troglodytes) Spermatozoa Osmotic Tolerance and Cryoprotectant Permeability Characteristics J Androl, July 1, 2005; 26(4): 470 - 477. [Abstract] [Full Text] [PDF]
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