Journal of Andrology, Vol. 22, No. 2, March/April 2001 Copyright 䉷 American Society of Andrology
Dialysis Addition of Trehalose/Glycerol Cryoprotectant Allows Recovery of Cryopreserved Mouse Spermatozoa With Satisfactory Fertilizing Ability as Assessed by Yield of Live Young KATHLEEN A. THOMPSON, JEAN RICHA, STEPHEN A. LIEBHABER, AND BAYARD T. STOREY From the Center for Research on Reproduction and Women’s Health, Transgenic Mouse Facility, and Department of Genetics, University of Pennsylvania Medical Center, Philadelphia, Pennsylvania.
ABSTRACT: Mouse sperm cryopreservation provides a means for storing the genetic information in genetically modified mice (mutants, transgenics, and ‘‘knockouts’’) in a cost- and space-effective manner. Sperm from this species are highly sensitive to cryodamage, which has impeded their cryopreservation in the past. The cryoprotectant used in this study was 6% glycerol (0.65 M) plus 7.5% trehalose (0.22 M), which was added to a concentrated suspension of sperm from B6SJLF1/J mice in bicarbonate-free buffer by dialysis to minimize osmotic stress on the cells. Sperm suspensions were frozen in 0.25 mL straws and stored in liquid N2. Eggs were obtained from B6SJLF1/J superovulated females. For in vitro fertilization (IVF), 15–25 L of sperm suspension post-thaw from one straw was added directly to each of three 1.5 mL drops of fertilization medium containing 30 eggs each, for 3 replicates per experiment. The fertilized eggs were scored for blastocyst forma-
tion, after which 12 blastocysts from each drop were implanted into pseudopregnant CD-1 females. The number of live pups were then scored at birth. Ten experiments yielded 21.7 ⫾ 1.4 (SD) blastocysts per 30 eggs inseminated (72%) and 7.3 ⫾ 0.4 (SD) live pups per 12 blastocysts implanted (61%). The overall yield of live pups was 44 per 100 eggs inseminated (44%). This yield should be satisfactory for maintaining a mouse strain through sperm cryostorage, with restart of the strain through IVF and embryo transfer. The method should also provide improvement in human sperm cryopreservation, as human sperm are less sensitive to cryodamage than are mouse sperm. Key words: Sperm recovery post-thaw, direct post-thaw in vitro insemination, zygote to blastocyst yield, blastocyst to pup yield, B6SJLF1/J male mice, CD-1 female mice. J Androl 2001;22:339–344
s the mouse continues to prove itself to be the mammalian model of choice for studying the physiological effects of modified gene expression, accomplished by discovery of new mutants, insertion of transgenes, and ‘‘knockout’’ of intrinsic genes, the motivation to preserve mouse lines with modified genes continues to increase. One particularly effective means to this end is cryopreservation of sperm from the males of the gene-modified strain (Thornton et al, 1999). The obstacle to achieving this means is the great sensitivity to cryodamage exhibited by mouse sperm (Mazur et al, 2000). Despite this sensitivity, cryopreserved mouse sperm have been shown to have the ability to fertilize eggs and
to produce embryos that develop to live pups. In a recent short review, Nakagata (2000) listed 56 reports of successful fertilization and 32 reports of the birth of live young from cryopreserved mouse sperm. A wide range of cryoprotectants have been examined. The first successes were achieved by Okuyama et al (1990), Tada et al (1990), Yokoyama et al (1990), and Takashima et al (1991) with a cryoprotectant of raffinose plus one other cryoprotectant, dimethyl sulfoxide (DMSO), glycerol, or skim milk. The cryoprotectant mix of 18% raffinose plus 3% skim milk is the one still utilized by Nakagata (2000). Difficulties in obtaining sperm survival with these systems led Penfold and Moore (1993) to formulate an improved cryoprotectant medium of Tes/Tris-buffered 25% egg yolk plus 1.25% glycerol. Songsasen et al (1997) reported the birth of live young using a cryprotectant solution with a composition of 0.45 M raffinose plus 0.3 M glycerol in Dulbeccos phosphate-buffered saline (DPBS) supplemented with 25% egg yolk. These reports show that mouse sperm can retain fertilizing ability after cryopreservation; this survival, however, is strain-dependent (Songsasen and Leibo, 1997). The survival rate in all cases leaves much to be desired. These freezing media con-
A
Supported by grant HD-31757 from the National Institute of Child Health and Human Development, National Institutes of Health. Correspondence to: Bayard T. Storey, PhD, CRRWH, University of Pennsylvania Medical Center, 1314 BRB II/III, 421 Curie Blvd, Philadelphia, PA 19104–6142 (e-mail:
[email protected]). A preliminary report of this study was presented at the 24th annual meeting of the American Society of Andrology, Louisville, Kentucky, April 10–13, 1999. Received for publication August 29, 2000; accepted for publication October 30, 2000.
339
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340 tain either milk or egg derivatives that may differ in different locations, thus impeding reproducibility; and they contain particulates resulting in turbid media, from which the sperm must be removed by centrifugation. An et al (2000) omitted these derivatives and showed that a medium of 16.6% raffinose plus 10% metrizamide resulted in poor survival rate of the sperm post-thaw as assessed by motility of ⬍10%; those sperm that did survive could fertilize eggs to yield live young. None of the studies cited above addressed the possible factors underlying the sensitivity of mouse sperm to cryopreservation. Mazur et al (2000) have done just this in an extensive study to dissect out the different factors contributing to this sensitivity. The factors are include low tolerance to volume changes induced by osmotic imbalance; fragility when subjected to mechanical stress induced by stirring, pipetting, and centrifugation; the need for glycerol as a permeant cryoprotectant but eventual sensitivity to glycerol toxicity at higher concentrations; and exacerbation of cryodamage by O2-derived free radicals. Damage from mechanical stress can be ameliorated by careful handling of the sperm suspensions, and free radical damage can be reduced by incorporating an O2consuming system, a bacterial membrane preparation designated Oxyrase (Mazur et al, 2000). Determination of the optimal concentration of glycerol for cryoprotection is linked to the cell damaging factor most difficult to deal with, that of volume changes induced by osmotic imbalance. Mouse sperm suspended in an aqueous medium containing impermeant solutes such as buffer salts are in osmotic equilibrium with the medium due to high permeability of the plasma membrane to water (Noiles et al, 1997). Addition of glycerol, to which the membrane is less permeable, produces a sudden osmotic imbalance, which then declines with time. Mouse sperm can tolerate volume changes due to this imbalance only to the extent of ⫾24% of their volume in isosmotic medium (Willoughby et al, 1996). Osmotic imbalance can be greatly lessened if the given amount of glycerol is added in multiple increments (Gao et al, 1995; Gilmore et al, 1997). Calculation of optimal freezing rates also encounters the problem of osmotic imbalance as the permeability coefficients of water and glycerol are each affected by the other and by temperature, as shown in elegant studies with sperm from mouse (Devireddy et al, 1999; Phelps et al, 1999) and human (Gilmore et al, 1995, 1997, 2000; Devireddy et al, 2000). The freezing protocol of Tao et al (1995), based on the earlier of these studies, has proven particularly efficacious in promoting post-thaw survival of mouse sperm. A recent study in this laboratory (Storey et al, 1998) examined the effect of glycerol and other polyols in the presence of raffinose and trehalose as a cryoprotectant on the survival and fertilizing ability of mouse sperm. The
Journal of Andrology · March/April 2001 optimum cryoprotectant medium was found to be a bicarbonate-free buffer based on piperazine-N,N⬘(bis)ethanesulfonic acid (PIPES) containing 6% (w/v) glycerol (0.65 M) and 7.5% (w/v) trehalose (0.22 M) with regard to recovery of sperm motility post-thaw. The cryoprotectant was added by dialysis to a concentrated suspension of sperm, and the suspension was frozen using the program described by Tao et al (1995). The suspension was added post-thaw in a small volume to eggs in the appropriate insemination medium without prior removal of cryoprotectant. Percentage of eggs fertilized, as assessed at the 8-cell stage, was 62% normalized to controls with fresh unfrozen sperm. In this paper, we report the use of this procedure, with modifications, to produce live young in 44% yield from superovulated eggs by in vitro fertilization (IVF), development to blastocyst stage, and implantation of blastocysts in surrogate mothers.
Materials and Methods Reagents Cell culture grade water was obtained from Gibco Life Technologies (Gaithersburg, Md). HEPES and PIPES were from Research Organics (Cleveland, Ohio). Diethylenetriaminepentaacetic acid (DTPA) was from Lancaster Synthesis (Windham, NH). Bovine serum albumin (BSA; fraction V; Pentex 81-068-3, very low endotoxin grade) was from Miles Laboratories (Kankakee, Ill). Glycerol (cell culture grade, G5150), trehalose dihydrate (cell culture grade, T0167), tris(hydroxymethyl)aminomethane (Tris; SigmaUltra, T6791), D-(⫹)-glucose (G6152), sodium pyruvate (P4562), sodium L-lactate (L7028), gentamycin (10 mg/mL, (G1272), hyaluronidase (H3506), human chorionic gonadotropin (hCG, CG10), and polyvinylpyrrolidone (PVP, P2307) were purchased from Sigma Chemical Company (St Louis, Mo). Pregnant mare serum gonadotropin (PMSG, 367222) was obtained from Calbiochem (San Diego, Calif). Mineral oil (O121), and reagent grade inorganic chemicals were from Fisher Scientific (Pittsburgh, Pa).
Media The medium used to isolate mouse cauda epididymal sperm was the bicarbonate-free medium NTP (Storey et al, 1998) with the following composition: 113 mM NaCl, 10 mM Tris, 15 mM PIPES, 5 mM MgCl2, 2 mM CaCl2, 1.5 mM glucose, 0.4 mM DTPA containing 5 mg/mL BSA and adjusted to pH 7.4 with 1 N NaOH; just prior to use, the medium was sterilized by passage through a 0.22 m filter. The medium for collection of eggs (Moore et al, 1993) was minimum essential medium (MEM) with Earles salts supplemented with 25 mM HEPES, 1 mM sodium pyruvate, 10 g/mL gentamycin, and 3 mg/mL PVP pH 7.3. Whitten medium (Whitten, 1971) containing 15 mg/mL BSA was used as the insemination medium for sperm and eggs. The culture of fertilized eggs for embryo development utilized the medium described by Chatot et al (1989), designated CZB medium.
Thompson et al · Mouse Sperm Cryopreservation
Preparation of Sperm Suspensions Suspensions of mouse sperm from the caudae epididymides were obtained by modification of the previously described procedure (Storey et al, 1998). The 2 caudae epididymides from 1 retired breeder B6SJLF1/J (Jackson Laboratory, Bar Harbor, Me) were carefully incised at 3 separate points so as to avoid blood contamination and were placed near one wall of a Petri dish containing 0.5 mL NTP medium. The dish was placed in an incubator at 37⬚C under an atmosphere of air at 100% humidity, and the sperm were allowed to swim out for 20 minutes across the dish. From the side of the dish opposite the epididymal tissue, 0.4 mL of the concentrated sperm suspension, containing ⬃4 ⫻ 107 cells/mL, free of tissue contamination, was taken up slowly into a 1 mL syringe with an 18-gauge needle (Becton Dickinson, Franklin Lakes, NJ) and discharged slowly into a 0.5 mL SlideA-Lyzer dialysis cassette (Pierce, Rockford, Ill) through the cassette inlet designed for a 18-gauge needle. The rate of uptake and discharge through the 18-gauge needle were tested in preliminary experiments to ensure that shear forces on the sperm during the process did not harm the cells. The dialysis cassette consists of 2 dialysis membranes with a molecular weight cutoff of 10 kilodaltons held together by a frame to form a chamber with a maximum volume of 0.5 mL.
Addition of Cryoprotectant by Dialysis The cassette containing the sperm suspension was placed in the slotted foam rubber holder, supplied with the cassette, that acts as flotation device to hold the cassette inverted in the cryoprotectant solution. For each cassette, 200 mL of cryoprotectant solution made up of NTP medium containing 6% (w/v) glycerol (0.65 M) and 7.5% (w/v) trehalose calculated as anhydrous trehalose (0.22 M) was used; the cryoprotectant solution containing the inverted dialysis cassette dipping into the solution was gently stirred for 120 minutes at room temperature (22⬚C). The reverse process, trehalose efflux from the cassette, was previously shown (Storey et al, 1998) to reach equilibrium in 100 minutes under these conditions. This method of cryoprotectant addition minimizes the osmotic stress initially imposed on the cells due to osmotic imbalance between cryoprotectant solution and intracellular cytosol originally in osmotic equilibrium with suspending medium (Gao et al, 1995; Gilmore et al, 1997).
Freeze-Thaw Protocol As the trehalose/glycerol cryoprotectant solution equilibrates with the sperm suspension in the cassette, the volume decreases from 0.4 mL to ⬃0.35 mL. From this volume, 0.25 mL containing ⬃3 ⫻ 107 cells was withdrawn, using the 1 mL syringe with 18-gauge needle, and placed in a straw designed for this volume (Cassou type; IMV Corp, Minneapolis, Minn); the ends of the straw were then heat-sealed. The straw was cooled to ⫺80⬚C in a programmable Planer Cell Freezer (Model R204; TS Scientific, Perkasie, Pa) using the program described by Tao et al (1995). The program is as follows: cool from 22⬚C to 4⬚C at 3⬚C per minute, hold at 4⬚C for 2 minutes, cool to ⫺5⬚C at 3⬚C per minute then to ⫺30⬚C at 1⬚C per minute, then to ⫺80⬚C at 3⬚C per minute. The straw was then placed directly in liquid N2 for storage. Just prior to use in the IVF procedure, the sample was thawed by removing the straw from liquid N2; a transit time of
341 15 seconds at room temperature (22⬚C) was taken to place the straw in an incubator at 37⬚C, where it was held for 5 minutes. The straw was then allowed to cool to 22⬚C prior to use in IVF.
In Vitro Fertilization Procedure Fertilization of mouse eggs with cryopreserved mouse sperm in vitro followed the protocol, with modifications, described by Moore et al (1993). For each experiment, 5 female B6SJLF1/J mice, 6 weeks old, were used as egg donors after superovulation with 10 units PMSG followed 48 hours later by 10 units of hCG. Eggs enclosed in cumulus cells were obtained by efflux from excised oviducts 12 hours after hCG injection; the eggs from the 2 oviducts of each mouse were placed in a 0.1-mL drop of MEM containing 0.05% hyaluronidase under oil at 37⬚C. Removal of the cumulus cells by hyaluronidase treatment was accomplished by incubation for 5 minutes with gentle pipetting to loosen the cumulus. The eggs were washed by passage through six 0.1-mL drops of MEM to remove residual cumulus cells and hyaluronidase. The eggs were then transferred to the Whitten medium for insemination in three 1.5-mL drops containing 30 eggs each, under oil, so that each IVF experiment could be scored in triplicate. This drop volume provided sufficient dilution of MEM and also provided for high dilution of the sperm suspension medium during insemination. The sperm suspension was used directly without removal of cryoprotectant, as this was shown to give superior results compared with cryoprotectant removal by serial dilution (Storey et al, 1998). The sperm concentration for insemination was adjusted to be 5 ⫻ 105 cells/mL, requiring 15– 20 L of the thawed sperm suspension per 1.5 mL drop. Sperm and eggs were incubated for 3 hours at 37⬚C in an atmosphere of 5% O2, 5% CO2, and 90% N2. The 30 putative 1-cell embryos were then removed from the drop, washed 3 times in the CZB medium, and transferred to CZB in 1.5 mL drops under oil for embryo culture for 5 days in the same atmosphere used for sperm/egg incubation. Only those embryos that had attained the blastocyst stage, as assessed by morphology, at this time point were scored.
Embryo Transfer Recipients of the cultured blastocysts were 6-week-old CD-1 females (Charles River, Wilmington, Mass). Three females were placed with one 8-week-old vasectomized CD-1 male (Charles River) overnight; the females were examined for vaginal plugs the next day to assess pseudopregnancy. Three days after establishment of vaginal plugs, the females were anesthetized with an intraperitoneal Avertin injection (0.5 g/kg), a 1 cm2 area of their backs was shaved and swabbed with 2% Chlorhexidine (DVM Pharmaceuticals, Miami, Fla) solution, and a ⬍1 cm paracostal incision was made near the dorsal midline at the level of the last rib. The uterus, oviduct, ovary, and fat pad were exposed through the incision, and 12 blastocysts obtained from the culture of 30 fertilized eggs were transferred by pipette into 1 uterine horn of the pseudopregnant female through a hole made by a 26-gauge needle 3–5 mm down from the uterotubal junction. The organs were returned to their position in the body cavity and the incision closed by suturing the muscle layer and skin. The females were then ear-tagged and placed 3 to a cage until day 18 of gestation, at which time they were placed in individual
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Journal of Andrology · March/April 2001
Table 1. Yield of blastocysts from mouse eggs inseminated in vitro with cryopreserved mouse sperm and yield of pups from implantation of blastocysts from the same egg sample. For each numbered experiment, eggs from 5 superovulated mice were pooled and subdivided into three 1.5 mL volumes containing 30 eggs in each volume; the sperm for each experiment was from one 0.25 mL straw representing 1 male Yield* Egg Sample
Experiment No. 1 2 3 4 5 6 7 8 9 10
Stage
1
2
3
Mean†
Blastocysts Pups Blastocysts Pups Blastocysts Pups Blastocysts Pups Blastocysts Pups Blastocysts Pups Blastocysts Pups Blastocysts Pups Blastocysts Pups Blastocysts Pups
20 7 15 5 20 8 22 8 19 8 20 9 22 8 24 7 22 7 20 7
22 9 20 8 25 5 18 5 22 7 22 7 24 7 22 6 19 9 25 5
24 7 22 8 22 9 25 9 26 9 20 5 26 9 21 8 20 6 22 8
22.0 7.7 19.0 7.0 22.3 7.3 21.7 7.3 22.3 8.0 20.7 7.0 24.0 8.0 22.3 7.0 20.3 7.3 22.3 6.7
* Blastocysts indicates the yield of blastocysts per 30 eggs inseminated; Pups indicates the yield of live pups per 12 blastocysts implanted into surrogate mothers. In each row, the blastocysts implanted were obtained from the inseminated egg sample immediately above. † Mean of 3 replicates in each row.
cages to await the birth of pups. For each experiment, 3 drops each containing 30 eggs were used for IVF; embryo transfer of 12 blastocysts from each drop was to 1 pseudopregnant female, giving 3 surrogate mothers for a given experiment.
Statistics The single factor analysis of variance (ANOVA) was performed with the Statistics software program (Macintosh), obtained from Blackwell Scientific Publications Ltd (Oxford, United Kingdom). Significance was taken as P ⬍ .05.
Results Ten IVF experiments, with eggs pooled from 5 superovulated females and sperm from 1 male frozen in one 0.25 mL straw in each experiment, were undertaken to assess the fertilizing ability of sperm cryopreserved using the dialysis protocol for cryoprotectant addition. The mouse strain used was B6SJLF/J, as this is the strain used for gene modification studies in the Transgenic Mouse Facility of the University of Pennsylvania School of Medicine.
Table 2. Summary of yield of blastocysts and live pups from mouse eggs inseminated in vitro with cryopreserved mouse sperm Yield
Blastocyst
Live Pups
Mean of means ⫾ SD (%)* Overall† (%) Per 100 eggs‡
21.7 ⫾ 1.4 (72) 651:900 (72) 72
7.3 ⫾ 0.4 (61) 220:360 (61) 44
* Mean of the 10 means from the experiments in Table 1. † Ratio of the total yield to total entries of experiments in Table 1. ‡ Yield per 100 eggs inseminated.
Enough eggs were recovered that 3 replicates utilizing 30 eggs apiece could be realized for each experiment. More than enough of the 30 eggs fertilized with the cryopreserved sperm in each replicate developed to the blastocyst stage that 12 blastocysts were available for implantation from that replicate, this number being the practical limit for implantation. The results from the individual experiments are tabulated in Table 1. A summary of the overall results is given in Table 2. The yield of live pups per 100 eggs inseminated was 44, which should be satisfactory for restarting a gene-modified strain, particularly if the entire 0.25 mL of sperm suspension in the freezing straw is used. Because 20 L of sperm suspension was adequate for IVF with 30 eggs, 1 straw could, conservatively, suffice for IVF of 300 eggs, yielding 132 live pups. Because all experimental manipulations were the same in all 10 experiments, the variables between experiments are the pooled eggs and the post-thaw sperm. Single-factor ANOVA showed no difference between experiments for the yield of blastocysts (f ⫽ 0.89, P ⫽ .48) and for the yield of live pups (f ⫽ 0.61, P ⫽ .78). This outcome indicates that the protocol utilizing cryopreserved mouse sperm, from this mouse strain at least, gives consistent results.
Discussion Efficacy of Dialysis/Direct Insemination for Cryoprotectant Addition The use of dialysis to add cryoprotectant was prompted by the demonstration that multiple additions of cryoprotectant to reach a desired level was beneficial to sperm survival because of reduced volume change due to osmotic imbalance at each addition (Gao et al, 1995; Gilmore et al, 1997). Dialysis, in effect, provides addition in an infinite number of steps and so should reduce the volume excursion to a minimum. Centrifugation to concentrate the suspension is avoided, thus eliminating a major source of mechanical stress. The only source of mechanical stress in the procedure is adding and removing the sperm suspension to and from the dialysis cassette through an 18-gauge needle. This stress is under the control of the operator. The rate of flow in and out of the
Thompson et al · Mouse Sperm Cryopreservation cassette through the needle that does not damage the cells is slow, but can still be completed in 2–3 minutes. Once the cryoprotectant has been loaded into the mouse sperm, one can undertake the freeze/thaw procedures and use the sperm directly for insemination in IVF without subsequent removal of cryoprotectant. In our previous study (Storey et al, 1998), it was found that cryoprotectant addition by dialysis prefreeze followed by cryoprotectant removal by dialysis post-thaw gave a threefold lower fertilization percentage in IVF than did cryoprotectant addition by dialysis followed by direct insemination postthaw. The use of serial dilution to add and remove cryoprotectant gave a twofold lower fertilization percentage. That study (Storey et al, 1998) thus established the greater effectiveness of the present protocol. Direct insemination requires that the sperm suspension be sufficiently concentrated that dilution of sperm suspension in the insemination drop be ⬃1:100, which could be deleterious due to excess volume excursion. Glycerol as a permeant cryoprotectant has been shown to lower the water permeability, Lp, of the sperm plasma membrane (Phelps et al, 1999). We propose that this lowering of Lp allows the rate of glycerol efflux to remain sufficiently balanced to the rate of water influx that cell volume excursion remains within the ⫾24% limit (Willoughby et al, 1996). We had proposed earlier (Storey et al, 1998) that trehalose adsorbed to the plasma membrane could provide additional stabilization against cell volume excursion. A systematic investigation of these membrane effects was deemed beyond the scope of this study. It suffices to point out that the dialysis procedure described in this report (hereinafter referred to as the ‘‘dialysis procedure’’) apparently goes far enough in addressing the factors leading to the sensitivity of mouse sperm toward cryodamage (Mazur et al, 2000) that a satisfactory yield of live pups can be obtained through IVF with cryopreserved sperm.
Problems and Possible Improvements These experiments were carried out with the B6SJLF1/J strain of mice because these have proved most useful for the Transgenic Mouse Facility. It has been shown that sperm obtained from different strains of mice have different sensitivities to cryodamage (Songsasen and Leibo, 1997; Phelps et al, 1999). It is therefore premature to claim that the dialysis procedure utilizing the glycerol/ trehalose cryoprotectant used in this study will be effective with other strains; this must be tested for each strain. Related to the mouse strain question is that of transgenic mice. As long as the transgenic males from the B6SJLF/ J strain are fertile, it is a reasonable presumption that their sperm will be successfully cryopreserved by the dialysis procedure. If transgenic and knockout mice are marginally fertile, then the procedure may have to be fine-tuned on a case-by-case basis. In cases of marginal fertility or
343 infertility in which testicular sperm are still obtainable, resort may be had to intracytoplasmic sperm injection (ICSI) to fertilize the eggs. It has been shown that survival after cryopreservation is not necessary in the case of mouse sperm to be used for ICSI, as injection of the sperm heads suffices to induce fertilization (Kuretake et al, 1996; Wakayama et al, 1998). The IVF and embryo culture media used in this study were developed more than a decade ago. An enriched medium, suitable both for mouse IVF and embryo culture was recently described by Summers et al (2000), after the completion of this study. The medium is the simplex optimized medium of Lawitts and Biggers (1993) supplemented with glucose, BSA, and 19 of the 20 naturally occurring amino acids, cystine providing the cysteine. Use of this improved medium should enhance the yield of live pups from cryopreserved mouse sperm. The 2-step protocol used in this study, in which eggs fertilized by IVF were cultured to the blastocyst stage prior to implantation in surrogate mothers, was used to provide a control checkpoint during development of the embryo for the quality of the cryopreserved sperm. It may prove more efficacious to transfer embryos at the 2- or 4-cell stage. This remains to be tested on a case-by-case basis.
Relevance to Human Sperm Cryopreservation Human sperm are less sensitive to cryodamage than are mouse sperm, but still show sublethal damage both to freeze/thaw and mechanical stress from centrifugation (Alvarez et al, 1993; Alvarez and Storey, 1993; Lasso et al, 1994). The dialysis procedure uses only glycerol and trehalose as cryoprotectant; the use of egg yolk is eliminated and with it the need for additional centrifugation. Given the satisfactory success achieved with cryopreserved mouse sperm using this procedure, it is to be expected that its use with human sperm could improve survival and fertilizing ability of these sperm after cryopreservation.
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