1 Asymptote Ltd, St John's Innovation Centre, Cowley Road, Cambridge CB4 4WS, UK, and 2 Bourn Hall Clinic, Bourn, Cambridge CB3 7TR, UK
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: cryopreservation/electron microscopy/freeze fracture/freeze substitution/spermatozoa
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Improvements in cryopreservation of human spermatozoa have been attempted in the past by the use of different cryoprotectants and extenders, and in particular, by altering the cooling rate, usually a linear reduction in temperature with time (Serafini and Marrs, 1986; Ragni et al., 1990
; Henry et al., 1993
; Gilmore et al., 1997
). With many cell types, and mammalian embryos provide a well documented example, a well defined `optimum' rate of cooling exists, with survival decreasing at both faster and slower rates. Intriguingly, similar studies demonstrate that spermatozoa are relatively insensitive to the magnitude of the linear rate of cooling during freezing. With human spermatozoa, a very broad response curve exists with little difference in survival observed following cooling at 1°C/min up to 100°C/min (Henry et al., 1993
). This response is unusual for mammalian cell types, but has received surprisingly little comment. Furthermore, the recovery of viability is comparatively low, with typically less than 60% of cells retaining motility on thawing, which given that this is achieved with such a wide range of linear cooling rates would suggest that the linearity of temperature reduction may not be appropriate.
The cooling rate dependency of cell recovery of many cell types may be predicted from computer models of their osmotic behaviour during freezing. However the predicted results with spermatozoa have not been in agreement with experimental observations (Noiles et al., 1993; Curry et al., 1995
). For example, although conventional models have suggested that human sperm cells should survive cooling rates up to 10 000°C/min (Noiles et al., 1993
), experimentally the survival rate begins to decline beyond 100°C/min (Henry et al., 1993
). It is clear that human spermatozoa have unusual cryobiological behaviour and improvements in their survival have not been amenable to conventional approaches of cryobiology.
Many of the changes in physical properties which occur in an aqueous cryoprotectant following ice nucleation are not linear with temperature. Parameters such as the ice fraction, concentration of ionic species, osmolality, pH, viscosity and gas solubility, all vary in a non-linear manner with temperature (e.g. Franks, 1985). In addition, the biophysical characteristics of cells which determine the response to freezing, for example the cellular permeability to water, also change in a non-linear manner with temperature. Conventional approaches to cryopreservation thus impose a linear change of temperature with time whilst the stresses that cells are encountering are all non-linear with time. It is therefore appropriate to examine whether improved methods of cryopreservation may be developed by specifically manipulating the manner in which cells experience physical changes rather than imposing a linear temperature reduction. In order to implement the required control of external conditions a new cell freezer has been specifically developed to achieve the desired protocols.
In this investigation, human spermatozoa suspended in a standard cryoprotectant were frozen using various protocols that manipulated different aspects of the physical conditions and the effects on post-thaw survival and function were assessed in comparison with conventional techniques. In order to increase our understanding of the physical behaviour of both the spermatozoa and the cryoprotectant during freezing, fractured straws were examined using the cryostage of a scanning electron microscope and freeze substitution. In addition, some simple computational modelling of the osmotic behaviour of the spermatozoa during freezing was carried out.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Cryoprotectant
Cryoprotectant medium was prepared according to Richardson (1976). A primary buffer was prepared by adding 3 volumes of 0.1 M sodium citrate to 1 volume 0.33 M fructose and 1 volume of 0.33 M glucose. Four millilitres of fresh egg yolk was added to 3 ml glycerol, and 13 ml of primary buffer. The solution was heat inactivated at 56°C for 30 min and allowed to cool. Finally, 200 mg glycine was added, and the pH adjusted to between 7.2 and 7.3.
Freezing
Semen samples were diluted with equal volumes of cryoprotectant. The osmolality of the diluted semen was measured by freezing point osmometry to be 1430 mOsm/kg water. Samples were frozen in 0.25 ml straws, sealed with polyvinylalcohol powder. Ten straws were frozen using each experimental protocol. Freezing was carried out using a variety of methods:
Method 1. Suspension in nitrogen vapour, 2025 cm above the surface of liquid nitrogen in an open Dewar, no manual nucleation of ice.
Method 2. Within a vapour phase controlled rate freezer (Planer cryo10/16; Planer Products, Sunbury-on-Thames, UK). Straws were held vertically and cooled from 20°C to 5°C at a rate of 2°C/min. Straws were maintained at 5°C for 10 min , during which time they were nucleated manually by touching the wall of each straw with forceps previously cooled in liquid nitrogen. Straws were then cooled at a programmed linear rate of cooling of 10°C/min to 100°C and then transferred to liquid nitrogen for storage.
Method 3. Using the Planer controlled rate freezer programmed as in Method 2, but with no manual nucleation of ice.
Method 4. In the Asymptote SF100 freezer (Cook IVF, Letchworth, Herts, UK), in which straws are held horizontally, programmed as in Method 2, with manual nucleation.
Method 5. In the Asymptote SF100 freezer as Method 4, but without manual ice nucleation.
Method 6. In the Asymptote SF100 freezer to control various extracellular physical conditions following nucleation of ice at 5°C.
To simplify these preliminary studies it was assumed that the extracellular solution was entirely glycerol, and from the measured osmolality the concentration was equivalent to a 13.5% solution. From the phase diagram of glycerol (Lane, 1925) it was then possible to calculate the required temperature change in the sample during cooling in order that the cells were exposed to defined external physical changes. The temperature profiles were programmed into an Asymptote SF100 freezer to control the temperature of the aluminium sample plate during cooling. The close contact of the straws with the sample ensured good heat transfer, and the temperature within samples closely followed the programmed temperature. The following three protocols were compared. (i) Controlled rate of change in extracellular concentrationreferred to in the text as `controlled concentration'. This was chosen such that the rate of change of solute concentration in the liquid phase decreases for more than 90% of the time taken to lower the temperature from 5°C to 45°C. (ii) Linear change in ice fraction with time. (iii) Constant heat extraction with time.
The required temperature changes to achieve these extracellular conditions were controlled in these experiments from the nucleation temperature (5°C) to 45°C, (close to the eutectic temperature for an aqueous solution of glycerol). In all tests the elapsed time taken to cool from 5°C to 45°C was 4 min (i.e. an average cooling rate of 10°C/min, consistent with the linear cooling rate, Method 2 above). All samples were then cooled from 45°C to 100°C at a linear rate of cooling of 10°C/min and then transferred to liquid nitrogen for storage.
Thawing and post-thaw semen assessment
Straws were thawed at room temperature after 72 h storage in liquid nitrogen. Straws from each experimental treatment were pooled for estimation of sperm concentration, motility, viability staining, hypo-osmotic swelling (HOS) testing, and the acrosome reaction ionophore challenge (ARIC) test.
Semen analysis and sperm function testing
Sperm concentration and motility were assessed according to the methods described by the WHO, (1992). Viability was assessed by staining with 5% eosin (WHO, 1992).
Membrane function was assessed using the HOS test (Jeyendran et al., 1984). Briefly, an aliquot of spermatozoa was diluted 10:1 with hypo-osmotic medium (7.35g sodium citrate and 13.51g fructose in 1000 ml, 150 mOsm/kg), and incubated at 37°C for 30 min. Spermatozoa were scored for the presence or absence of swelling in the tail region.
Acrosome function was assessed using a modified ARIC test (Cummins et al., 1991). Briefly, washed sperm were incubated in 10 µM calcium ionophore, A23187. The presence or absence of the acrosome was identified by staining with FITC linked to Pisum sativum lectin, and viable spermatozoa identified using bis-benzimide (Hoechst H33258). A score was derived by subtracting the number of viable, acrosome-reacted spermatozoa in the control sample from the percentage of viable, reacted spermatozoa in the ionophore-treated sample.
Computational modelling
Estimates of the local undercooling were made by simple computation of the mass transport across the cell membrane. The membrane permeability was assumed to be temperature dependent, and the mass transport was assumed to be driven by the difference between the intracellular and extracellullar concentrations of solute. The intracellular solute concentration changed only as a result of mass transport.
Freeze fracture electron microscopy
Straws were prepared as described above and following the various methods of solidification were cross fractured under liquid nitrogen and then loaded onto the cryostage of a scanning electron microscope (cryoSEM; Oxford Instruments XL30-FEG; Oxford Instruments Ltd, Abingdon, Oxon, UK). The stage was warmed from 145°C to 90°C over 6 min and the sample etched at 90°C for 6 min before cooling to 145°C. The sample was then transferred to a preparation stage and coated with 1015 nm gold and then loaded back onto the cryoSEM stage for image recording.
Freeze substitution
Straws were refractured into 1 mm thick segments and transferred under liquid nitrogen to substitution chambers in a Reichert automated freeze substitution device. The substitution medium contained 2% osmium tetroxide and 1% uranyl acetate in methyl alcohol. Samples were maintained at 90°C for 24 h, warmed to 70°C at 3°C/h and then maintained at 70°C for 24 h. Samples were warmed to room temperature at 3°C/h, rinsed in methyl alcohol and embedded in Spurr's epoxy resin. Sections 0.5 µm in thickness were prepared with a Reichert Ultracut S microtome and stained with methylene blue. Photomicrographs were taken using a Zeiss Axiophot microscope.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
Control of extracellular freezing (Methods 6.i, 6.ii, 6.iii)
The highest recovery of grade A motility was seen in all three experiments when cooled using the `controlled concentration' method described above (method 6.i). A modification of this protocol, giving an even greater initial rise in solute concentration, was attempted in experiment #3, but gave rise to slightly lower recovery rates.
Those samples cooled such that the ice fraction increased at a linear rate (method 6.ii) had the poorest recovery in terms of motility. In addition, this protocol appeared to have a significant effect on membrane function with hypo-osmotic swelling apparent in all samples immediately on thawing (100% in experiment #1), and a significant decrease in the ARIC score in all three experiments.
Linear heat extraction (method 6.iii), which was carried out only in experiment #2, gave rise to a significantly lower recovery rate in terms of motility, viability and function, compared with passive cooling, and `controlled concentration'. It should also be noted that the sample used in experiment #2 appeared to be more susceptible to cryodamage than the samples used in experiments #1 and #3.
Experimental series #4non-pooled sperm
Spermatozoa from three patients were frozen by both the controlled concentration (method 6.1) and conventional linear cooling (method 4) and the results are presented in Table II. These three samples varied in their sensitivity to freezing injury when frozen by conventional linear cooling. When frozen by the controlled concentration method these samples retained their relative ranking, however motility compared with linear cooling was increased by a factor of at least 50%.
|
|
|
|
Light microscopy of freeze-substituted sections showed that spermatozoa were entrapped within the freeze-concentrated material (Figure 4a, b). Occasional spermatozoa were observed to bridge across two freeze-concentrated zones. At this magnification the freeze-concentrated matrix was observed to be relatively homogeneous in appearance following both linear cooling and linear ice fraction solidification (methods 4 and 6.ii). However following `controlled concentration' (method 6.i) areas of granularity were evident (Figure 4b
), which may be interpreted as substituted ice crystals within the freeze-concentrated matrix.
|
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
When linear cooling was applied the recovery was significantly less than that achieved with `controlled concentration' and was similar to that reported in other studies (Serafini and Marrs 1986; Ragni et al., 1990
; Henry et al., 1993
). It is significant to note that using the vapour phase cooling apparatus samples which were not manually nucleated had significantly lower motility than nucleated samples, which is consistent with the report by Crister et al (1987). It has been suggested that different sub-populations of spermatozoa may differ in their freezing sensitivity (Gao et al., 1993
; Curry and Watson, 1994
) and it appears that the `controlled concentration' protocol may provide successful cryopreservation of sensitive sub-populations.
This investigation provides significant insights into the response to freezing and thawing of spermatozoa. Freeze fracture electron microscopy and freeze substitution extends previous observations made by light cryomicroscopy (Korber et al., 1984; Holt et al., 1992
) that spermatozoa and solutes migrate either entirely into the freeze-concentrated matrix or are entrapped near to the interface of the ice and the freeze-concentrated material. In some cases sperm tails may be associated with ice crystals whilst the sperm heads are within the freeze-concentrated matrix. In extreme cases the head of the sperm was situated in one region of the freeze-concentrated matrix with the end of the sperm tail in another zone with the tail bridging through an ice crystal. This may occur because the dimensions of the freeze-concentrated matrix make it difficult to accommodate the sperm head and tail except when lying in the plane of the matrix, or because the tail section of the spermatozoon has surface properties which made it less likely to be excluded from the ice crystal matrix. It is important to note that the relative sperm cell recovery on thawing is not correlated with the structure of the ice crystal network because this structure is essentially fixed by the temperature of ice nucleation in the undercooled straws. All samples nucleated at the same temperature (5°C) formed a similar initial ice structure upon which all additional ice was subsequently deposited.
Major differences in the eutectic structure between different treatments were made apparent by freeze substitution. Following `controlled concentration' freezing the freeze-concentrated matrix contained large ice crystals, which were absent from the matrix of the other less successful freezing treatments. Further studies are required to determine at what temperature these ice crystals form within the freeze-concentrated matrix, but it is apparent that sperm cells within this matrix are in close association with ice crystals (Figure 4b). It is of interest that gross ice formation within the eutectic is observed in the sample with the highest recovery on thawing. The establishment of spatial gradients within freeze-concentrated materials has been clearly demonstrated by light cryomicroscopy (Korber et al., 1984
) and is well documented in metals (e.g. Davies, 1973).
Studies of freeze-substituted lymphocytes (Farrant et al., 1977) have demonstrated that intracellular ice may be visualized and correlates with loss of viability on thawing. Freeze-substitution electron microscopy of frozen sperm has been restricted to an analysis of mouse spermatozoa frozen in the tail of the epididymis (Sherman and Liu, 1982
). In this study, intracellular ice crystals were apparent and cellular dehydration was evident, particularly as voids between the acrosome and nuclear membrane and in the midpiece and tail. However as there were no motile cells in any sample following thawing it was not possible to correlate injury with cellular structure. In the current study freezing appeared to have little effect on the cell morphology as revealed by freeze fracture and freeze substitution. No osmotic shrinkage was evident nor was intracellular ice apparent. The possibility exists that cells contain micro-crystalline intracellular ice, beyond the limits of resolution of the ultrastructural techniques employed. Further studies are being undertaken to thermally cycle the samples, which would be expected to increase the dimensions of any intracellular ice present. The only significant structure observed in these studies was the ring of material surrounding sectioned spermatozoa, apparent by electron microscopy. The nature of this material and its formation require further investigation.
It has been demonstrated (Du et al., 1993) that human spermatozoa behave as ideal osmometers, within the range 250 to 1500 mOsm of sodium chloride and that 13% of the isotonic water is osmotically inactive. Using models of the osmotic behaviour of spermatozoa during freezing it has been suggested (Curry et al., 1995
) that following a linear cooling at 10°C/min, less than 10% of the cellular water would remain in the cell at 10°C. However, this major loss is not observed here: spermatozoa in Figures 3 and 5
exhibit no osmotic shrinkage. Although the low water content of sperm cells, combined with their flat, non-spherical shape, could allow large changes in cellular water content to cause little modification in the surface area, there is no evidence of any membrane alterations consistent with osmotic dehydration in any of the micrographs examined. However, in this study spermatozoa were frozen in the presence of glycerol, which has a high permeability to human spermatozoa (Gao et al., 1992
), and has also been demonstrated to reduce the water permeability of human spermatozoa (Noiles et al., 1992
). It is also of potential significance that an aquaporin (AQP7) which mediates water permeability in spermatozoa is also involved in glycerol transport (Ishibashi et al., 1997
). The lack of cellular shrinkage would be consistent with the cells effectively being in equilibrium with glycerol at all temperatures during freezing. Similarly the apparent absence of intracellular ice (Figure 5
) could also be associated with a high intracellular glycerol concentration.
Whilst experimental treatments give significantly different levels of viability any correlation with cell morphology in the frozen state is lacking, and it is the controlled parameter, namely the rate of change in solute concentration, which is the major factor affecting sperm recovery. It is of interest to speculate precisely why this is the case, and Figure 6a and b shows the estimated form of the local undercooling of the cells during the freezing process for the three distinct cases `controlled concentration' (method 6.i), linear ice fraction (method 6.ii) and standard linear cooling (method 4), whose concentration histories are shown in Figure 1
. These estimates of local undercooling have been made by a simple computation of the mass transport across the cell membrane. Figure 6a
shows the form of the undercooling when the mass transfer is dominated by water transport, and Figure 6b
shows the effect when the glycerol transport dominates.
|
However, it must be noted that intracellular ice is not apparent and it is therefore likely that other physical events determine viability during freezing and thawing. The corresponding estimated changes in mass transport are shown in Figure 7a and b for water movement and glycerol movement respectively. These values are again comparable for similar membrane permeabilities, but would scale relatively for different relative permeabilities. These estimates show the relative form of mass transport where the transport is dominated by either the water or glycerol transfer, and it would be expected that the cells may experience a combination of the two effects over the course of the freezing. Cell viability may be determined by a combination of potential cytotoxic events at high sub-zero temperatures together with restrictions on transport at low temperatures due to low permeability, high viscosity etc. The `controlled concentration' treatment would minimize the time of exposure at high sub-zero temperatures and allow extended periods at lower temperatures to compensate for reduction in transport processes.
|
![]() |
Acknowledgments |
---|
![]() |
Notes |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Cummins, J.M., Pember, S.M., Jequier, A.M. et al. (1991) A test of the human sperm acrosome reaction, following ionophore challenge: Relationship to fertility and other seminal parameters. J. Androl., 12, 98103.
Curry, M.R. and Watson, P.F. (1994) Osmotic effects on ram and human sperm membranes in relation to thawing injury. Cryobiology, 31, 3946[ISI][Medline]
Curry, M.R., Redding, B.J. and Watson, P.F. (1995) Determination of water permeability coefficient and its activation energy for rabbit spermatozoa. Cryobiology, 32, 175181.[ISI][Medline]
Crister, J.K., Huse-Benda, A.R., Aaker, D.V. (1987) Cryopreservation of human spermatozoa. I. Effect of holding procedure and seeding on motility, fertilizability and acrosome reaction. Fertil. Steril., 47, 656663.[ISI][Medline]
Davies, G.J. (1973) Solidification and Casting. Applied Science Publishers, London, pp. 7094.
Du, J., Kleinhans, F.W., Mazur, P. et al. (1993) Osmotic behaviour of human spermatozoa studied by EPR. Cryo-Letters, 14, 285294.[ISI]
Farrant, J., Lee, H. and Walter, C.A. (1977) Effects of interactions between cooling and rewarming conditions on survival of cells. In The Freezing of Mammalian Embryos. Ciba Foundation Symposium, 52, Elsevier, London, pp. 4962.
Franks, F. (1985) Biophysics and Biochemistry at Low Temperatures. Cambridge University Press, Cambridge, pp. 3761.
Gao, D.Y., Mazur, P., Kleinhans, F.W. et al. (1992) Glycerol permeability of human spermatozoa and its activation energy. Cryobiology, 29, 657667.[ISI][Medline]
Gao, D.Y., Ashworth, E., Watson, P.F. et al. (1993) Hyperosmotic tolerance of human spermatozoa: effects of glycerol, sodium chloride and sucrose on spermolysis. Biol. Reprod., 49, 112123.[Abstract]
Gilmore, J.A., Liu, L., Gao, D.Y. et al. (1997) Determination of optimal cryoprotectants and procedures for their addition and removal from human spermatozoa. Hum. Reprod., 12, 112118.[ISI][Medline]
Henry, M.A., Noiles, E.E., Gao, D. et al. (1993) Cryopreservation of human spermatozoa. IV The effects of cooling rate and warming rate on the maintenance of motility, plasma membrane integrity and mitochondrial function. Fertil. Steril., 60, 911918.[ISI][Medline]
Holt, W.V., Head, M.F. and North, R.D. (1992) Freeze-induced membrane damage in ram spermatozoa is manifested after thawing: Observations with experimental cryomicroscopy. Biol. Reprod., 46, 10861094.[Abstract]
Ishibashi, K., Kuwahara, M., Gu, Y. et al. (1997). Cloning and functional expression of a new water channel abundantly expressed in the testis permeable to water, glycerol and urea. J. Biol. Chem., 272, 2078220788.
Jeyendran, R.S, Van der Ven, H.H., Perez-Pelaez, M. et al. (1984). Development of an assay to assess functional integrity of the human sperm membrane and its relationship to other sperm characteristics. J. Reprod. Fertil., 70, 219228.[Abstract]
Korber, K.R., Scheiwe, M.W. and Wollhover, K. (1984) A cryomicroscope for the analysis of solute polarization during freezing. Cryobiology, 21, 6880.[ISI]
Lane, L.B. (1925) Freezing points of glycerol and its aqueous solutions. Ind. Eng. Chem., 17, 924.
Noiles, E.E., Mazur, P., Kleinhans, F.W. et al. (1992) Water permeability of human sperm in the presence of intracellular glycerol. Cryobiology, 29, 736.
Noiles, E.E., Mazur, P., Watson, P.F. et al. (1993) Determination of water permeability coefficient for human spermatozoa and its activation energy. Biol. Reprod., 48, 99109.[Abstract]
Ragni, G., Caccamo, A.M., Della Serra, A. and Guercilena, S. (1990) Computerized slow-staged freezing of semen from men with testicular tumors or Hodgkins disease preserves sperm better than standard vapour freezing. Fertil. Steril., 53, 10721075.[ISI][Medline]
Richardson, D.W. (1976) Techniques in sperm storage. In Richardson, D.W., Joyce, D. and Symonds, E.M. (eds) Artificial Insemination, Royal College of Obstetricians and Gynaecologists, London pp. 97125.
Royere, D., Barthelemy, C., Hamamah, S. and Lansac, J. (1996) Cryopreservation of spermatozoa: a 1996 review. Hum. Reprod. Update, 2, 553559.
Serafini, P. and Marrs, R.P. (1986) Computerized staged freezing technique improves sperm survival and preserves penetration of zona-free hamster ova. Fertil. Steril., 45, 854858.[ISI][Medline]
Sherman, J.K. and Liu, K.C. (1982) Ultrastructure before freezing, while frozen, and after thawing in assessing cryoinjury of mouse epididymal spermatozoa. Cryobiology, 19, 503510.[ISI]
World Health Organization (1992) Laboratory Manual for the Examination of Human Semen and Semen Cervical Mucus Interaction, 3rd edn. Cambridge University Press, Cambridge.
Submitted on June 12, 1998; accepted on December 17, 1998.