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,
From the * University of Missouri Comparative
Medicine Center, College of Veterinary Medicine, Columbia, Missouri; and
Yerkes National Primate Research Center, Emory
University, Atlanta, Georgia.
| Correspondence to: John K. Critser, PhD, Comparative Medicine Center, College of Veterinary Medicine, University of Missouri, 1600 East Rollins, Room E-109, Columbia, MO 65211 (e-mail: critserj{at}missouri.edu). |
| Received for publication November 9, 2004; accepted for publication January 23, 2005. |
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Abstract |
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Key words: Cryopreservation, membrane permeability
In the context of conservation biology, significant numbers of nonhuman primate (NHP) species are currently at risk of extinction in wild populations because of habitat destruction and hunting (Morrell and Hodges, 1998). In addition, many NHP in captive breeding programs have significant reproductive problems compared to NHP living in their natural habitats around the world (Gould and Johnson-Ward, 2000). The preservation of germplasm from these species could help captive breeding programs, which are aimed at conserving genetic diversity. Therefore, there is a need for the development of effective germplasm cryopreservation methods for many NHP species. Procedures for the retrieval of oocytes and embryos from mammalian species are relatively complicated, requiring exogenous gonadotropin stimulation, followed by invasive surgical collection procedure. Furthermore, oocyte cryopreservation has proven to be very challenging for many mammalian species, including NHP (Critser et al, 2002).
The ability to cryopreserve semen from NHP in combination with assisted reproductive technologies (ART) such as artificial insemination (AI) would provide flexibility in conservation efforts (Gould and Styperek, 1989; Gabriel Sanchez-Partida et al, 2000; Watson and Holt, 2001; VandeVoort, 2004). Although sperm cryopreservation technology in combination with more advanced ART such as in vitro fertilization and intracytoplasmic sperm injection are currently being developed for some NHP species (eg, rhesus macaques, cynomolgus) with poor motility (Wolf et al, 1989), the overall success of these ARTs would benefit from the development of improved sperm cryopreservation protocols.
Many studies have been conducted on human gamete cryobiology (Agca and Critser, 2002). Unfortunately, similar information for NHP, including the chimpanzee (Gopalakrishnan and Apkarian, 1990; Younis et al, 1998), vervet (Conradie et al, 1994), and rhesus monkey (Songsasen et al, 2002; Rutllant et al, 2003) is largely unknown. It is widely accepted that inappropriate semen handling before cooling is one of the prime reasons for low postthaw survival (Watson, 2000). Furthermore, factors such as individual differences, season, semen collection method (ie, penile electroejaculation, rectal probe), diluent components (eg, egg yolk, Tris buffer, sugars), and choice of permeating and nonpermeating cryoprotective agents (CPA) also have an impact on sperm survival (Holt, 2000).
During cryopreservation, cells experience osmotically driven volume changes as a result of CPA addition and removal before cooling, as well as during cooling and warming (Mazur, 1984). These transient volume excursions can be detrimental if CPA addition and removal is not performed in a manner that maintains the cell within the osmotic range that can be tolerated (Muldrew and McGann, 1994). Importantly, each CPA imposes different osmotic effects due to their membrane permeability coefficients (Gao et al, 1997; Gilmore et al, 1997). Therefore, using systematic quantitative approaches to cryopreservation that focus on the fundamental biophysical cell properties such as water and solute permeability coefficients and functional integrity is necessary to develop optimal cryopreservation protocols. In this study we performed a series of experiments that were aimed at providing a comprehensive understanding of the osmotic tolerance and permeability characteristics of chimpanzee spermatozoa to dimethyl sulfoxide (Me2SO), glycerol (Gly), propylene glycol (PG), and ethylene glycol (EG).
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Materials and Methods |
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Media
A NaCl-free TL-HEPES solution was prepared and both hyposmotic as well as
hyperosmotic TL-HEPES solutions were made by adding appropriate amounts of
NaCl to yield solutions of 80, 160, 220, 260, 290, 350, 400, 600, and 880,
mOsm. Phosphate-buffered saline (PBS) solutions were prepared by dilution of a
10x PBS stock to 160, 290, 600, and 860 mOsm. Solutions of
Me2SO, Gly, PG, and EG were prepared at a concentration of 1 M in
isosmotic (290 mOsm) TL-HEPES. All materials were purchased from Sigma
Chemical Co (St Louis, Mo) unless otherwise noted. Osmolalities were
determined using a vapor pressure osmometer (VAPRO 5520, Wescor, Logan,
Utah).
Experiment 1: Isosmotic (Viso) and Osmotically Inactive Cell Volume (Vb)
The goals of this experiment were: 1) to determine the isosmotic volume of
chimpanzee spermatozoa; 2) to analyze the osmotic behavior of chimpanzee
spermatozoa after exposure to anisosmotic solutions and to determine if the
cells behave as linear osmometers; and 3) to determine the osmotically
inactive volume of chimpanzee spermatozoa. Isosmotic cell volume was
determined by adding 100 µL of a sperm cell suspension to 15 mL of
isosmotic PBS (290 mOsm) and cell volumes were measured in triplicate at room
temperature. Osmotically driven cell volume responses and the osmotically
inactive cell volume (Vb) were determined by adding 100
µL of a cell suspension in isosmotic PBS to 15 mL of anisosmotic solutions
of PBS (160, 600, and 860 mOsm). A Coulter counter ZM model (Coulter
Electronics, Hialeah, Fla) with a 50-µm standard-resolution aperture tube
was used to determine cell volume as described previously
(Gilmore et al, 1995). The
cell volume changes were recorded kinetically during the shrink or swell
period and final cell volumes were determined by measuring the equilibrated
states. Sperm cell volumes were calibrated for each anisosmotic solution using
spherical styrene beads (Duke Scientific Corp, Palo Alto, Calif) with a
diameter of 3 µm (14.14 µm3). To record and analyze the data,
the Coulter counter was interfaced to a microcomputer using a CSA-1S interface
and the data were analyzed using "Cell Size Analyzer" software
(The Great Canadian Computer Company, Edmonton, Canada). Cell volume estimates
were based on the median value of the distribution of the osmotically active
population. Our hypothesis was that chimpanzee sperm behave as linear
osmometers. To test this hypothesis, equilibrium cell volumes were fitted to
the reciprocal of the extracellular osmolality of the solution, which is
described by the Boyle van't Hoff relationship:
 | (1) |
Experiment 2: Determination of Membrane Permeability Coefficients
A pair of coupled nonlinear differential equations that describe the cell
volume and amount of solute in the cell as functions of time were derived by
Kedem and Katchalsky (1958):
 | (2) |
 | (3) |
. An electronic particle counter (Coulter counter model ZM; Coulter Electronics) was used to measure the equilibrium volume of cells and changes in cell volume in response to anisosmotic conditions (McGann et al, 1982). In the kinetic experiments, a 100-µL aliquot of cells preloaded with 0.8 M CPA was abruptly diluted into 10 mL of an isotonic PBS solution and this extracellular concentration was assumed to be invariant with time. The electronic signals from cell debris and nondissociated cells (eg, cell doublets or triplets) accounted for less than 5% of total population and were separated from signals of single cells by using appropriate Coulter counter settings and by digital filtering. The data were averaged over 100-ms intervals to reduce the size of the data set of the experiments. A commercial software package, MLAB (Civilized Software, Bethesda, Md), was used to solve Equations 2 and 3 using the Gear method (Bunow and Knott, 1995). The Marquard-Levenberg curve-fitting method (Bunow and Knott, 1995), as implemented in MLAB, was used to fit the experimental data and determine the values of the hydraulic conductivity (Lp) and cryoprotectant permeability (PCPA, PMe2SO, PGly, PPG, and PEG) coefficients. A fixed value for Vb, determined independently from the Boyle van't Hoff plot, was used in the fitting calculation.
For this experiment we used fixed effects, randomized complete block
design, with the main effect of 4 levels of CPA. We tested the hypothesis that
the parameters of Lp, PCPA, and 
are dependent on
the type of CPA. An aliquot of an ejaculate from each of four animals was used
for all factor levels. These data were analyzed using the SAS system with the
-level chosen to be 0.05, and the Tukey's least significant difference
test was used to make pairwise comparisons
(Westfall et al, 1999).
Variance heterogeniety was not evident among the treatment levels.
Experiment 3: Effects of Anisosmotic Conditions on Chimpanzee Spermatozoa Motility
The goal of this experiment was to determine the effects of exposure to
anisosmotic solutions on chimpanzee spermatozoa motility. The osmotic
tolerance of chimpanzee sperm was determined using total motility as the
endpoint. Two treatments were used: 1) exposure of cells to a series of
anisosmotic solutions (80, 160, 220, 260, 350, 400, 600, and 880 mOsm); and 2)
a return of the cells to isosmotic conditions after anisosmotic exposure. To
determine the effects of exposure to anisosmotic solutions, a 10-µL aliquot
of each sperm suspension (n = 4 males) was transferred into a 1.5-mL Eppendorf
centrifuge tube containing 150 µL from one of the eight different osmotic
solutions, yielding a final concentration of approximately 10 x
106 spermatozoa/mL. A 5-µL aliquot from each treatment was
transferred to a preheated (37°C) microcell® (20 µL) stage and
allowed to equilibrate for 30 seconds. Motility was determined after 5 minutes
of incubation at room temperature using a computer-assisted semen analysis
system (Hamilton Thorne, model HT M2030; Beverley, Mass). A minimum of 200
cells was analyzed in six to nine fields and three replicates were analyzed
per male. Motility estimates were validated manually by the video playback
option of the instrument.
For the second treatment, spermatozoa were returned to near isosmolality
(approximately 290 mOsm) by transferring a calculated amount of an anisosmotic
solution into a 1.5-mL Eppendorf tube. Various amounts of stock solutions were
added to the tubes (see Table
2) to bring the cell suspensions back to isosmotic condition. For
example, for the Eppendorf tube that contained solution at 350 mOsm, 47 µL
of 80 mOsm stock solution was added to bring the cell suspension back to
isosmotic condition. Motility was determined after 5 minutes of incubation at
room temperature. For this experiment we used a two-way, mixed-effects model.
Osmotic treatment was a repeated-measures factor, having two levels, and
osmolality was a between-group factor having nine levels. These data were
analyzed using the SAS system with the -level chosen to be 0.05.
Comparisons of the anisosmotic treatments to the isosmotic control were
performed with the Dunnett's one-sided test
(Westfall et al, 1999).
Variance heterogeneity was not evident among the treatment levels.
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-level chosen to be .05.
Variance heterogeneity was not evident among the treatment levels. |
Results |
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Experiment 2
The changes in cell volume in the presence of Me2SO, Gly, PG,
and EG were measured over time, and these data were fitted to calculate
Lp and PCPA at 22°C. The values
for these coefficients are listed in Table
3. None of the values for Lp were
significantly different (P = .5855). However, the value of
PCPA for EG was significantly higher than that for
Me2SO (P = .0074).
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Experiment 3
Figure 2 shows the
percentage of motile spermatozoa, normalized to motility at 290 mOsm, as a
function of osmolality for the two osmotic stress treatments. There was a
significant interaction between the treatments (P = .0013). Loss of
sperm motility increased as the solution concentration diverged from
isosmotic. The osmolality levels of 80, 160, 600, and 880 caused a significant
reduction in motility of sperm compared to those only exposed to an isosmotic
solution. Few cells were motile after exposure to 80, 600, or 880 mOsm.
Although the levels of motility increased upon return to isosmotic conditions,
only the cells having been exposed to 160 mOsm regained a level of motility
comparable to the untreated sperm.
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Experiment 4
Figure 3 shows calculated
relative volume changes of chimpanzee spermatozoa during a one-step addition
and fivefold dilution of 1 M Me2SO, Gly, PG, and EG, on the basis
of the values obtained from Experiments 1 and 2.
Figure 4 shows chimpanzee sperm
motility (normalized to the isotonic treatment) during exposure to solutions
of 1 M Me2SO, Gly, PG, and EG, and after their dilution. Although
the average motility in each solution was lower than for the cells only
exposed to an isotonic treatment, this difference was not significant
(P > .11).
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Discussion |
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To develop successful cell cryopreservation methods, it is imperative to understand cryobiologically relevant cell characteristics. The cell surface-to-volume ratio and the osmotically inactive cell volume are important factors related to formation of lethal intracellular ice during freezing. The isosomotic cell volume determined for chimpanzee spermatozoa in this study (21.7 µm3) is smaller than that for human (28.2 µm3; Gilmore et al, 1995), boar (26.3 µm3; Gilmore et al, 1996), rhesus (36.8 µm3; Rutllant et al, 2003), and bull spermatozoa (23.5 µm3; Guthrie et al, 2002). The spermatozoa of most mammalian species studied to date behaved as "ideal osmometers," in that their cell volume changes are linearly related to the reciprocal of the extracellular nonpermeating solute osmolality. The present study has demonstrated that chimpanzee sperm are also ideal osmometers. The osmotically inactive cell volume for chimpanzee spermatozoa (69%) determined in this study was higher than human (50%; Gilmore et al, 1995), bull (61%; Guthrie et al, 2002), mouse (60.7%; Willoughby et al, 1996), and boar spermatozoa (67.4%; Gilmore et al, 1996). This information is important in enabling mathematical modeling of the chimpanzee spermatozoa cell volume changes during series of anisosmotic exposures inherent in cryopreservation.
When cells are exposed to CPA solutions, they undergo volume excursion, first shrinking in response to an extracellular hyperosmotic solution, then returning to slightly greater volume than their initial isosmotic volume as the CPA enters the cell and water follows to maintain its chemical potential. The extent of the initial shrinkage and the subsequent time course of the cell to return to near-isosmotic volume are directly related to the cell's Lp and PCPA coefficients. In general, higher Lp and PCPA lead to smaller initial shrinkage and shorter time course of the cell to return to near-isosmotic volume. Knowledge of these parameters provides the means to predict optimal CPA addition and removal procedures (Gao et al, 1995).
These values allow the calculation of the volume history of the cell after exposure to a given CPA during its addition and removal, as well as during cooling and warming if activation energies of Lp and PCPA are known (Mazur, 1970, 1984; Gao et al, 1995). This current finding is very consistent with human sperm permeability characteristics in the presence of the same CPAs, although the previously reported PEG value is 3.6 times higher for human sperm than chimpanzee sperm (Gilmore et al, 1995). Furthermore, the Lp values for chimpanzee sperm in the presence of these CPAs is much higher than human spermatozoa (0.84, 0.77, 1.23, and 0.74 µm/min per atmosphere, respectively). Previously determined boar spermatozoa Lp values in the presence of Me2SO, Gly, and EG (0.12, 0.13, and 0.20 µm/min per atmosphere, respectively) are also much lower than those for chimpanzee sperm, although PMe2SO and PEG values (0.93 x 10-3 cm/min and 2.0 x 10-3 cm/min) are comparable (Gilmore et al, 1998). Studies on sperm permeability characteristics of various mammalian species including mouse, boar, bull, and human show that spermatozoa from these species have different characteristics for different CPAs. However, it should be noted that one common characteristic of the sperm from all aforementioned species is the highest permeability to EG. During CPA dilution, cells may experience significant swelling due to rapid influx of water, which can be very damaging to sperm functional integrity. Rapid efflux of the CPA during its removal will reduce this swelling. The current results suggest that EG may be a superior CPA for cryopreserving chimpanzee sperm since the Lp and PCPA values are the largest among those tested.
In this study, we determined that chimpanzee sperm motility loss was moderate (20%–25%) after exposure to intermediate hyposmotic (160 mOsm) or hyperosmotic (400 mOsm) solutions. Additional 65% to 70% motility loss occurred when the hyposmotic (80 mOsm) and hyperosmotic (600 mOsm) range was broadened. These data suggest that chimpanzee sperm are less sensitive to hyposomotic exposure than human sperm (Gao et al, 1995). Although human sperm lose about 70% of their motility after exposure to 160 mOsm and return to isosmotic conditions, chimpanzee sperm experienced only 30% motility loss under the same osmotic conditions (Gao et al, 1995). In contrast, when human and chimpanzee sperm were exposed to a 600 mOsm solution and subsequently returned to an isosmotic condition, human sperm maintained about 90% motility, whereas chimpanzee sperm retained only about 35% of their motility. This suggests that human spermatozoa are more robust to hyperosmotic conditions than chimpanzee sperm. To date, among the species studied, boar spermatozoa demonstrated the highest sensitivity to deviations from an isosmotic condition compared to human, bull, and rhesus on the basis of motility assessment (Gao et al, 1995; Guthrie et al, 2002; Rutllant et al, 2003). According to Gilmore et al (1998), boar spermatozoa can only swell to within 1.02 times and shrink to within 0.97 times their isosmotic volume and maintain at least 70% motility. Another study conducted on mouse sperm showed that their motility is substantially more sensitive to osmotic stress than their mitochondrial or plasma membrane integrity and thus mouse sperm should be maintained within 76%–124% of their isosmotic volume to maintain about 80% of pretreatment motility (Willoughby et al, 1996).
Ozasa and Gould (1982) determined that harmful effects of osmotic stress on chimpanzee sperm could be reduced by supplementing the freezing media with as little as 2 mM taurine. Similar results were also reported for cynomolgus monkey sperm (Li et al, 2003). It was determined that the addition of 5 mM proline, 10 mM glutamine, and 10 mM glycine to Tes-Tris–egg yolk freezing medium significantly increased post-thaw survival of cynomolgus monkey sperm assessed by motility and acrosome integrity. Although the true nature of the protection provided by amino acids is not known, the authors attributed improved survival to a possible osmoregulatory role during cryopreservation. Others also suggested that the beneficial effects of amino acids may be due to reduction of the formation of reactive oxygen species during the cryopreservation procedure (Alvarez and Storey, 1983a,b). In the same context, a similar effect was also observed when mouse sperm was exposed to anisosmotic challenges in the presence of two commonly used semen extenders, skim milk and egg yolk (Agca et al, 2002). For sperm cryopreservation, a one-step addition of a CPA at a concentration of 0.5–1 M in combination with whole milk or egg yolk–Tris (10%–20%) semen extenders is commonly used for many species. On the basis of our general knowledge of fundamental cryobiology of spermatozoa, the optimal CPA will be the one that most rapidly permeates the cell, causing the least amount of volume excursion during its addition and removal. The current study has demonstrated that chimpanzee sperm are most permeable to EG and least permeable to Me2SO.
Using the permeability coefficients determined from the current study, theoretical volume excursions resulting from the addition and dilution of 1 M concentrations of Me2SO, Gly, PG, and EG were calculated (Figure 3). This estimation revealed that the predicted relative volume expansion was as high as 1.6-fold (Me2SO) and 1.4–1.5-fold for Gly, PG, and EG. It was previously shown that an abrupt addition of 1 M Gly caused less motility loss (20%) to human sperm compared to a one-step removal (60%) (Gao et al, 1995). Later studies conducted on other mammalian spermatozoa strongly supported this early observation, indicating that spermatozoa become dysfunctional when they experience high volume expansion. For example, it was shown for bull sperm that one-step addition and removal of 1 M concentration of Me2SO, Gly, and EG caused 90%, 31%, and 6% motility loss, respectively (Guthrie et al, 2002). The motility loss reported for stallion sperm was greater (55%, 92%, 51%, and 24%) for Me2SO, Gly, PG, and EG, respectively (Ball and Vo, 2001). It is notable that one of the common osmotic characteristics of the sperm from these species is their greater tolerance to one-step addition and removal of 1 M EG than one-step addition and removal of 1 M Gly, Me2SO, or PG. The current study has shown that chimpanzee sperm exposed to 160 mOsm NaCl have the ability to recover 70% of their pretreatment motility, which is much higher than human, boar, bull, and stallion sperm (37%, 10%, 25%, and 41%, respectively) (Gilmore et al, 1995, 1996; Guthrie et al, 2002; Ball and Vo, 2001).
On the basis of these observations, we hypothesized that chimpanzee sperm will tolerate a one-step addition and removal of 1 M Gly, Me2SO, PG, or EG without significant motility loss. The results demonstrated that this treatment resulted in a statistically insignificant reduction (approximately 20%) in motility compared to the control, supporting the hypothesis. In an early study by Sadleir (1966), chimpanzee sperm were frozen in either 7% Gly or Me2SO in extender containing 20% egg yolk and 1.5% fructose (approximately 1100–1150 mOsm). It was concluded that Gly was relatively better CPA than Me2SO for freezing chimpanzee sperm on the basis of post-thaw motility. Their observation is consistent with the current study in that the highest level of volume excursion is predicted for Me2SO. Gould and Styperek (1989) froze chimpanzee sperm in Ham F10 medium supplemented with 15% human cord serum and either 7.8% Me2SO or 7.8% Gly. They further demonstrated that despite the high post-thaw motility (70%), chimpanzee sperm had difficulty penetrating hamster oocytes under in vitro conditions (24.4%). After AI using frozen-thawed semen in their study, 2 of 11 (18%) recipient females conceived successfully. Younis et al (1998) investigated the effects of supplementing freezing media (7.8% Gly and test yolk buffer) with antifreeze peptide II (AFP) or insulin transferrin selenium (ITS) on chimpanzee sperm motility and acrosomal integrity. Although they found beneficial effects of supplementing freezing media with AFP and ITS on post-thaw sperm motility, acrosomal integrity was not protected at a similar level. Okada et al (2001) equilibrated cynomolgus monkey sperm in Tris buffer egg yolk containing 10% Gly (approximately 1470 mOsm). Post-thaw live fraction of the spermatozoa after Percoll separation had significantly higher acrosomal abnormalities (swollen, vesiculated, and ruptured) than control (79% vs 8.1%). The current finding is also in agreement with those previous studies in that high post-thaw motility recovery was achieved. Thus, the reduced ability to penetrate hamster oocytes of frozen-thawed chimpanzee sperm may be related to acrosomal integrity after freezing and thawing.
In conclusion, it appears from the present data that chimpanzee sperm have a high tolerance to anisosmotic conditions as assessed by maintenance of motility. Therefore, osmotic stress may not be the most likely reason for the poor fertility of frozen-thawed chimpanzee sperm observed in the previous studies. It may be that, although motility is protected to a high level, acrosomal integrity may be damaged by the same osmotic conditions. This raises an important aspect of chimpanzee sperm cryobiology that should be addressed in future studies.
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Acknowledgments |
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Footnotes |
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These authors contributed equally to this work 
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