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Reproductive Technology |
Center for Engineering in Medicine,3 Massachusetts General Hospital, Harvard Medical School, and Shriners Hospital for Children, Boston, Massachusetts 02115
Department of Cell Biology,4 Harvard Medical School, Boston, Massachusetts 02115
Beth Israel Deaconess Medical Center,5 Harvard Medical School, Boston, Massachusetts 02115
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSICSIRESULTSDISCUSSIONREFERENCES |
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assisted reproductive technology
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSICSIRESULTSDISCUSSIONREFERENCES |
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Cryopreservation is the state of the art for the preservation of mouse spermatozoa. The first report of successful cryopreservation of mouse sperm was published in 1990 by three independent groups of Japanese investigators [7–9]. The use of raffinose and glycerol appeared to be critical for success in these studies. Over the last 10 years, a number of investigators have reported successful cryopreservation of sperm from inbred [7, 10, 11], outbred [7,9], hybrid [8, 12–14], and transgenic [15] mice. The protocol described by Nakagata [16], with modifications using various combination of raffinose, glycerol, and skim milk as cryoprotectant, appears to produce the best results for mouse sperm cryopreservation. In addition, studies are underway for a better mechanistic understanding of the stresses that occur during cryopreservation of spermatozoa, including osmotic [17], thermal [12], mechanical, and oxidative stresses [14]. One drawback of cryopreservation is the requirement for low-temperature refrigerators for storage and transportation. Desiccation offers the possibility of the entire handling and subsequent storage and transportation of spermatozoa being done at ambient temperatures and therefore offers attractive logistical flexibility compared with cryopreservation.
Ever since the pioneering work of Polge et al. [18], where fowl sperm were successfully recovered after desiccation, results have been mixed and controversial, with only limited success in recovering motile sperm [19–22]. With recent advances in technology, microinjection of immotile sperm into eggs has been a strategy that has been shown to successfully produce offspring in animals like cattle [23]. This technique, also known as intracytoplasmic sperm injection (ICSI), has been extended to obtain live fetuses by injecting frozen [24] or freeze-dried [25] mouse sperm into oocytes. The spermatozoa have lost their motility and membrane integrity but presumably have genetic integrity. This has allowed the exploration of desiccation as a preservation protocol for mouse sperm storage. The benefits of desiccation include possible storage at room temperature, thereby significantly reducing the logistics associated with low-temperature preservation. In addition, desiccation significantly reduces the complications associated with removal of protectants. Recently, Kusakabe et al. [26] freeze dried various strains of mouse sperm in a tris-HCl buffer supplemented with EGTA at a high pH (8.2) and obtained live fetuses after injecting rehydrated sperm into oocytes. A simpler desiccation technique would be very useful in advancing desiccation as the preservation method of choice.
In the current study, we explored the feasibility of convective drying as a technique for desiccation. Convective drying using an inert gas offers a simpler and less expensive alternative to freeze drying without the requirement of any specialized equipment and the ability to perform the entire protocol at room temperature. For our study, we used the EGTA-supplemented Tris-HCl buffer [26] and isolated B6D2F1 sperm and dried them using different convective drying protocols and stored them overnight at 4°C prior to ICSI. We monitored the in vitro development of oocytes and used the protocol that gave the highest blastocyst formation rate to successfully obtain 15-day fetuses.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSICSIRESULTSDISCUSSIONREFERENCES |
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Nine- to 14-wk-old male B6D2F1 mice (Jackson Laboratories, Bar Harbor, ME) and 7–13-wk-old B6C3F1 female mice (Jackson Laboratories or Harlan Sprague Dawley, Indianapolis, IN) were used for the experiments. Animals were maintained in accordance with guidelines of the Committee on Care and Use of Laboratory Animal Resources, National Research Council.
Reagents and Media
All reagents were obtained from Sigma Chemicals (St. Louis, MO) unless otherwise stated. The media used for sperm isolation was prepared as outlined elsewhere [26]. Briefly, the solution consisted of 10 mmoles/L Tris-HCl buffer supplemented with 50 mmoles/L of each of NaCl and EGTA [ethylene glycol-bis (ß-aminoethyl ether)-N,N,N',N'-tetraacetic acid]. The final solution was adjusted to a pH of 8.2 and is referred to as EGTA solution in the rest of the paper.
Two versions of potassium simplex optimized medium (KSOM) [27] were used for oocyte isolation, injection, and eventual culture of embryos. Oocyte and embryo cultures were done using KSOMaag, which is medium KSOM supplemented with 0.5x modified eagle medium amino acids and 5.5 mmoles/L glucose. HEPES-buffered KSOM with 1 mg/ml polyvinyl alcohol in place of BSA (mFHM medium) [27] was used for oocyte collection and ICSI procedures.
Sperm Collection
Male mice were anesthetized with CO2 or halothane and then killed by cervical dislocation and used to obtain sperm. For each experiment, the caudal epididymides were excised from the male and collected in 0.5 ml EGTA solution. The epididymides were then punctured with a 22-gauge needle three to four times while squeezing with forceps in order to release the contents into the solution. The concentration of sperm for the experiments varied between 5 and 10 million/ml. The sperm suspension was incubated at 37°C for 10–20 min on a slide warmer prior to use either for the desiccation experiments or for injection as control (fresh) sperm. Spermatozoa used as fresh sperm were obtained from other mice using the same protocol. Motility was assessed in a hemocytometer and the membrane integrity was measured using propidium iodide and SYBR (Molecular Probes, Eugene, OR) under a microscope (Nikon Eclipse 800; Tokyo, Japan) by counting a total of 500 cells in at least 10 different representative fields of view.
Convective Drying
A convective drying system to achieve controlled drying of mouse sperm is shown schematically in Figure 1. Dry and highly purified nitrogen gas (grade 4.8) from a pressurized cylinder (BOC, Murray Hill, NJ) was blown through a chamber that was designed to hold a glass microslide with a 20-µl sperm droplet placed on it. Slides that were used for the current study had two 10-mm-diameter etched rings (Gold Seal, Portsmouth, NH), which allowed reproducible droplets to be created. Two different chambers were used for the study. The chamber used for forced convective drying consisted of a groove milled in a Plexiglass fixture to hold a standard glass microslide. The fixture was sealed by an identical (in dimensions) Plexiglass cover that had a transparent window as well as an inlet and outlet port for gas flow. A polydimethylsulfoxide gasket was placed between the chamber and its cover, and the system was held together by 10 screws. The chamber was 10 mm wide and 3 mm high. This enabled the accurate modeling of the velocity profile that helped ensure reproducible drying. Drying by natural convection was done in an airtight acrylic box (22 x 20 x 18 cm) (Sanplatec, Osaka, Japan), previously equilibrated with CaSO4/CoCl2 desiccant (W.A. Hammond Drierite, Xenia, OH). The nitrogen was only used to purge the system in this drying protocol. The flow of nitrogen was controlled by a flowmeter (Cole Parmer, Vernon Hills, IL) placed at the entrance of the flow chamber. The entire flow path and the flow chamber were tightly sealed to prevent leakage and to insure reproducible drying conditions. After completion of the drying protocols, silicone isolator spacers (Molecular Probes) were sealed on the microslide and the microslide sealed with a covering glass slide. Finally, the whole glass sandwich was sealed (Foodsaver, Compact II; Tilia Inc., San Francisco, CA) in a vacuum-sealed bag (Foodsaver VacLoc Roll; Tilia Inc.).
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Freeze Drying
The freeze-drying protocol was done to compare the results from the current study with those of Kusakabe et al. [26]. The experiment was done using a shelf-type freeze dryer (Genesis 12EL Virtis; Gardiner, NY). One hundred microliters of sperm suspension were loaded in an amber-colored borosilicate glass vial (Sun Brokers, Inc., Wilmington, NC) and plunged into liquid nitrogen for 30 sec and then placed on the shelf of the freeze dryer that had been precooled to -40°C. A small drop of mineral oil (E.R. Squibb and Sons, Inc., Princeton, NJ) was put between the shelf and the glass vial to ensure isothermal contact. Isothermal contact ensured that the bottom of the vial was at the same temperature as the shelf. Experimental readings using thermocouples have shown a maximum variation of 2.5°C in the frozen sample after it has been loaded on the tray. Immediately after the sample was loaded, the vacuum was set at 30 mtorr (30 mbar), with the shelf temperature held at -40°C. The first phase of drying (primary drying) was at 30 mtorr and -40°C for 4 h. This was followed by increasing the temperature to -20°C for 30 min and subsequently to 0°C, where the sample was held for 8 h. During the entire procedure, the vacuum setting remained unchanged at 30 mtorr. Following the procedure, the shelf was purged with gaseous nitrogen and the sample removed, capped, and stored in vacuum-packed bags at 4°C for 18–24 h prior to ICSI.
Rehydration
Sperm samples were rehydrated by restoring the starting sample volume (20 µl for convective drying and 100 µl for freeze drying) using deionized water at room temperature. The rehydration protocol was done in two steps, where half the total volume of water was added first and the rest added after 10 sec.
Moisture Measurement
Moisture measurements were made by gravimetric analysis using an analytical balance (AE 163; Mettler, Columbus, OH). The formula for percent moisture is
where the dry weight is the weight of the sample after the drying protocol has been completed, the wet weight is the starting weight of the sample, and the baked weight is the weight after an equivalent sample has been heated overnight at 90°C for 24 h in an oven (Precision, Winchester, VA). The baked weight is assumed to be entirely free of moisture.
Oocyte Collection
Female mice (B6C3F1 strain) were superovulated by intraperitoneal injections of 5 IU eCG (P.G. 600; Intervet Inc., Millsboro, DE) and 5 IU hCG 48 h later. Oocytes were collected from oviducts about 14 h after hCG injection and treated with 0.1% hyaluronidase (H-3506; Sigma) in mFHM medium to remove cumulus cells. The oocytes were incubated in KSOMaag at 37.5°C under 6% CO2 in air until micromanipulation.
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ICSI |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSICSIRESULTSDISCUSSIONREFERENCES |
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The procedure of micromanipulation was essentially similar to that described by Kimura and Yanagimachi [28] except that it was done at room temperature (20–24°C). The holding pipette was pulled from a glass capillary tube (Sutter Instrument, Novato, CA) using an automatic pipette puller (P-87; Sutter Instrument Co., Novato, CA) and cut and polished using a microforge (MT-1; TPI, St. Louis, MO). The diameter of the holding pipette was 80µm outside and 15µm inside. The injection pipette was pulled using the same glass capillary with a 6–8-µm inside diameter. The injection pipette was loaded with mercury and attached to a piezo-driving unit (Model PiezoDrill; Burleigh Instruments, Inc., Fishers, NY). After transfer of the eggs into the operating drop, about 2µl sperm suspension was thoroughly mixed with the PVP drop. Each sperm head was cut off using the injection pipette with piezo pulses. The heads were then washed in a second drop and injected into the oocytes after piezo drilling through the zona and oolema. For every protocol, 10–12 eggs were injected.
Culture of ICSI Oocytes and Embryos
After ICSI, the oocytes were cultured in KSOMaag medium at 37.5°C under 6% CO2 in air in a humidified incubator. After a 2-h culture, they were evaluated and the lysed oocytes were discarded. Intact, surviving zygotes were further cultured in KSOMaag medium for 1–4 days to evaluate preimplantation development or for embryo transfers to recipient females.
Embryo Evaluation
Embryos were observed and graded for stage of development every 24 h post-ICSI. The development stages were 2 cell, 3–4 cell, 5–8 cell, compacted morula, and blastocyst, respectively.
Embryo Transfer
Embryos were transferred either at 2-cell or 3–4-cell stages (24 or 48 h post-ICSI, respectively) into Day 0.5 pseudopregnant ICR females (Taconic Farms, Germantown, NY) that were mated in natural cycle with vasectomized males. For each experimental protocol, 4–6 embryos were transferred into each oviduct. Embryos that developed after injecting oocytes with freshly isolated sperm were transferred to one randomly chosen oviduct, while the embryos developed after injecting oocytes with desiccated sperm were transferred to the opposite oviduct of the same recipient female. Postimplantation development was assessed at Day 15 of pregnancy. The number of implantation sites was recorded and fetuses were collected for gross examination and wet weight measurements.
Statistics and Data Analysis
The exact Kruskal-Wallis significance test for singly ordered r x c tables was calculated using StatXact 5.0 [29]. Correlation coefficients between moisture content and blastocyst formation were determined using Microsoft Excel 2000 (Seattle, WA).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSICSIRESULTSDISCUSSIONREFERENCES |
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Isolation of sperm in nonphysiological EGTA solution (pH = 8.2) led to rapid loss in motility, resulting in less than 5% motility in 10 min. During this time, the mean membrane integrity was 70%. Over 1 h, almost all sperm became immotile (>99%) and the membrane integrity dropped to a mean value of 50%. Because all the experimental protocols were performed within 1 h, motility and membrane integrity were not observed beyond 1 h.
Convective Drying
The convective drying setup shown in Figure 1 was used to generate reproducible drying kinetics at different flow rates, as shown in Figure 2. Forced convective drying protocols were used at flow rates of 10 and 4 L/min and are labeled as rapid and moderate drying, respectively. Assuming no leakage, the free-stream velocities obtained in the chamber with a width of 25 mm and a height of 3 mm were 2.187 and 0.87 m/sec, respectively. The third protocol generated was the slow drying in a large acrylic chamber that generated natural convective drying conditions with negligible convective velocity. Figure 2 summarizes the weight fraction at different times of drying. All three drying rates showed a similar trend of convective drying. The initial part of each drying curve is approximately linear, demonstrating that the drying rate is approximately constant. The curve then falls off exponentially. The linear region was steepest for the rapid-drying protocol and the shallowest for the slow-drying protocol. An exponential fit was able to predict the mean weight percent for all the drying data within 7% accuracy. The estimated time constants (
) based on exponential regression analysis for the rapid, moderate, and slow drying were 4.6, 13.7, and 21.3 min, respectively. The data from rapid drying showed the largest variation in the data, as evidenced by the standard error bars; however, the variation in moisture content exceeded 10% for one specific data point only. For the moderate- and slow-drying protocols, the fluctuation in data point was relatively low at 
5%. Overall, the results demonstrate a quantitative and reproducible convective drying set-up that achieves controlled drying rates.
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Final Moisture Content
The goal was to obtain a final moisture content in the sperm of 5% for all three drying rates used in this study. Based on curves generated in Figure 2, the times required to generate weight fractions 0.05 were 10, 30, and 60 min for the rapid-, moderate-, and slow-drying protocols, respectively (shown by dotted line in Fig. 2). Also shown are the results from the freeze-dried experiments. The moisture fraction was calculated for each sample using Equation 1, and the results are summarized in Figure 3 using dot plots. There are nine sets of experiments for the rapid-drying protocol and six sets of experiments for the rest of the protocols. The sample spread is the highest for the rapid-dried samples and the least for the freeze-dried samples. There are three sets of experiments for the rapid-dried protocol for which the end moisture percent exceeded 5%. The resulting mean moisture percent for rapid-, moderate-, slow-, and freeze-drying protocols are 3.6% ± 1.1% (SEM), 1.5% ± 0.6%, 1.2% ± 0.3%, and 0.76% ± 0.1%, respectively.
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In Vitro Development of Embryos Post-ICSI
One spermatozoa that had been dried in EGTA solution, stored for 16–18 h at 4°C, and subsequently rehydrated were injected into each oocyte. The development of the injected ovum was followed for subsequent development over the next 96–120 h. The results are summarized in Table 1. Approximately 50% of the injected ova survived, while 
87% of the surviving oocytes cleaved in all experiments. The percentages of surviving ova and fertilized ova post-ICSI seemed to be independent of the drying protocol (or control). The percent of blastocysts that developed from ova injected with rapidly dried sperm was 63% and not significantly different from the control, which was 82% (P = 0.05). The percentage of blastocysts that developed using the moderate-dried and slow-dried protocols were 58% and 33%, respectively, and were significantly different from the control percentage (P < 0.05).
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The distributions of the final stages of development of the fertilized embryos are shown in Figure 4. The results were obtained by pooling together the data from all the experiments within a given protocol. The Kruskal-Wallis test for statistical homogeneity demonstrated that the data for the experiments for each protocol were homogeneous. The results indicate that a majority of the embryos progressed to blastocysts in the control, rapid-dried, moderate-dried, and freeze-dried groups, with the percentage being highest for the control group followed by the rapid-dried, moderate-dried, and freeze-dried groups. Less than 10% of the embryos stopped development at the 2-cell stage, while an increasing percentage was arrested at the 3–4-cell and morula stages. A different trend was observed using the slow-drying protocol. The tests resulted in the development of the lowest percentage of blastocysts compared with all other protocols, the majority of embryos becoming arrested at the 3–4-cell stage. Using the Kruskal-Wallis test for statistical analysis for significance of all the developmental stages, it was shown that the development of the embryos injected with sperm that have been rapid dried are not different from the control group (P = 0.06). The Kruskal-Wallis test also showed that embryos injected with moderate-dried and slow-dried sperm were significantly different from the control group (P < 0.05). Also, the degree of development results from the slow-dried samples was significantly different from both the rapid-dried and moderate-dried samples, whereas there was no significant difference between the rapid-dried and moderate-dried samples (P = 0.32).
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Effect of Drying Rate on Blastocyst Formation Rate
Figure 5 shows the mean and the standard error of the percent blastocysts that developed post-ICSI for different treatment protocols. Despite having lower mean values for the rapid-drying protocol (71.4%) compared with control (81.1%), the results are not statistically different from each other (P > 0.05). Again, the moderate-drying protocol has a mean (63.4%) that is significantly lower (P < 0.05) than the control but not significantly different from the rapid-drying protocol (P > 0.05). There is a statistically significant drop in the blastocyst formation rate for the slow-drying protocol (P < 0.05) compared with all other protocols, implying the importance of drying rate in the blastocyst formation rate. Results from the current study also indicate that the rapid- and moderate-drying protocols are comparable with the freeze-drying protocol, which has a slightly lower mean value (58%) of blastocyst formation rate but not significantly higher than the rapid- and moderate-drying protocols (P > 0.05).
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Effect of Final Moisture Content on Blastocyst Formation Rate
Figure 6 is a plot of the percent blastocyst growth vs. moisture content. The range of moisture content, as demonstrated in Figure 3, is between 0% and 8%, with all but three data points being above 5%. The correlation coefficient (r) values obtained for individual drying protocols are -0.41, -0.36, and 0.51 for rapid-, moderate-, and slow-drying protocols, respectively, none of which is significant (P > 0.05). Therefore, there is no significant correlation between the final moisture content (for a mean moisture <5%) and the rate of blastocyst formation.
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Development of Fetuses from Ova Fertilized with Rapidly Dried Sperm
The sperm samples were dried for 10 min using the rapid-drying protocol (Fig. 2) and stored overnight at 4°C. The average moisture content of the dried sperm was 
4.5% (data not shown). The results summarized in Table 2 show the number of implantations and number of fetuses observed on Day 15 of pregnancy after transfer of embryos 24 (two-cell) or 48 (four-cell) h after ICSI. The number of implantations is the sum of the number of resorbtion sites and the number of fetuses. Implantation rates resulted from transfer of embryos produced with control (70%) or dried sperm (53%). The percent of fetuses that developed were 37% and 11% for control and dried sperm when transferred at the two-cell stage. The percentage of fetuses obtained from dried sperm (11%) was significantly lower (P < 0.05) than the control (37%) for two-cell stage transfer. Similar results were obtained for the four-cell stage transfer. However, the apparent improvement using four-cell transfer compared with two-cell transfers is not statistically significant (P > 0.05). In addition, the final moisture content of the dried sperm sample from each experiment (range 1%–8%) was not significantly correlated with the percentage of implantations or the percentage of fetuses that developed (data not shown). Fetuses from both two- and four-cell embryos transferred into 0.5-day pseudopregnant recipients were at about the 14–14.5 developmental stage defined by Wahlsten and Wainwright [30] when collected on Day 15 of pregnancy.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSICSIRESULTSDISCUSSIONREFERENCES |
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The EGTA buffer used in the current study for convective drying was first used successfully to freeze dry mouse sperm by Kusakabe et al. [26], who obtained a 75% blastocyst formation rate. Similar freeze-drying studies performed by us resulted in a mean blastocyst formation rate of 56%. The primary drying protocol used by us was similar to that used by Kusakabe et al. [26], but in addition, our study had a secondary drying protocol in which the temperature was raised from -40°C to 0°C. The final moisture content was not reported by Kusakabe et al. [26], which made it difficult to compare the end moisture content with our current results. Although differences in the freeze dryer and the detailed protocol used in the two studies make comparisons difficult, our studies are in general agreement with Kusakabe et al. [26].
Despite being a nonphysiological solution for mouse sperm isolation, EGTA solution (pH = 8.2; 250 mOsm) offered protection to the sperm nucleus during the desiccation process. The results with the current EGTA solution were improved over convective and freeze-drying experiments performed in a supplemented physiological Dulbecco phosphate-buffered saline (DPBS) buffer (pH 7.2; 290 mOsm) that has been successfully used for freezing of mouse sperm [17]. The average motility and the membrane integrity after isolation in DPBS (until 60 min) were 66% and 80%, respectively, and therefore higher than the isolation in EGTA buffer. However, the average blastocyst formation rate using the rapid-drying and freeze-drying protocols were only 18% (data not shown) and therefore significantly lower than drying performed in EGTA buffer. Further comparisons of oocyte development reveal that while the division into two- and four-cell stages was comparable between oocytes injected with sperm dried in DPBS solution vs. EGTA solution, there was a drop off in morula formation rate and subsequent development into blastocysts. Thus, higher sperm motility and membrane integrity postisolation were not good indicators of eventual nuclear stability postdrying. The exact nature of the protective mechanism offered by the EGTA buffer is unknown. A possible explanation is that an EGTA solution with high pH may play an important role in repressing the activity of endogenous endonucleases that degrade DNA [26]. Further studies elucidating the mechanism of protection of mouse sperm DNA postdehydration are required.
The rate of drying may be a critical parameter in the optimization of a desiccation protocol for biological molecules as well as cells. Rapid convective drying resulted in a higher blastocyst formation rate compared with moderate- and slow-drying protocols, indicating that there may be a critical threshold of drying rate for preservation of mouse sperm nucleus. In the current study, where the isolation and subsequent desiccation were performed in a nonphysiological EGTA solution, the various stress vectors on the nuclear material are possibly minimized by rapid removal of moisture. Rapid drying has also been shown to be beneficial compared with slow drying for the preservation of proteins [31]. In contrast, preservation of the whole cell may be more complex. Prokaryotes [32] and plant seeds like maize [33] seem to retain their structure and thereby survive desiccation better by slow drying as opposed to rapid drying. It has been argued that, in nature, slow drying allows organisms to go through a series of complex molecular and physiological adaptations, including accumulation of sugars [34]. This situation may not be relevant in our work, where only the preservation of sperm nuclear material was the focus. Clearly, the membrane was damaged as shown by the dye-uptake studies; however, the function of the sperm nucleus was preserved. Optimal drying rate may therefore be influenced by a number of factors, including the particular biological structure(s)/molecule(s) being preserved, the cell type being desiccated, and the composition of the desiccation solution.
In most experiments involving desiccation of proteins, bacteria, or plants, the final moisture reported for stable storage has been 5%. In the present work, the final moisture content (0.5–7%) did not affect the rate of blastocyst formation as much as the rate of drying. A similar trend has been reported by others [35, 36]. Crowe et al. [35] showed that preservation of liposomes with sugar did not require any residual water because sugars alone provided adequate stability. Puhlev et al. [36] used Fourier transform infrared spectroscopy and reported negligible moisture content in human fibroblasts where the survival percent varied between 25% and 31%. In contrast, Chen et al. [37] showed that plasma membrane integrity of mouse fibroblasts dropped off rapidly for moisture content <15%, thereby compromising cellular survival. Gordon et al. [38] implied that moisture content may affect viability of human mesenchymal stem cells, although no correlation between viability and moisture content was demonstrated. Strong evidence that moisture content may have an effect on storage of desiccated cells came from the work of Buitink et al. [39]. Using electron paramagnetic resonance spectroscopy, they have shown that, in certain seeds and pollen, the molecular mobility decreases with lowering moisture content and then increases when the water content becomes very low. Because prevention of molecular mobility is critical for long-term storage, these results suggest that there may be a critical threshold of moisture content for storage. There appears to be varying thresholds of moisture content for the stable storage of different critical cellular components like the membrane, proteins, and DNA.
Studies indicate that an intact sperm nucleus may be a sufficient condition for successful embryonic development, even after motility and membrane integrity of the sperm have been compromised [24–26]. Thus, it becomes increasingly important to identify and subsequently optimize the critical components that are involved in the stable storage of the sperm nucleus. Ward et al. [40] showed that the stability of the sperm nuclear matrix may be critical in the development of oocytes into live offspring. The stability of the nuclear matrix seems to be influenced by the medium in which the sperm is dehydrated. Kusakabe et al. [26] demonstrated that more chromosomal aberrations were found in sperm that were freeze dried after isolation in CZB medium [41], which possibly reduced the rate of development of zygotes produced by ICSI. Their study optimized the concentration of EGTA solution that gave the least number of chromosomal aberrations per zygote, suggesting that calcium chelators like EGTA and a high pH may act synergistically to prevent chromosomal damage. Other studies have demonstrated that the activation of sperm nucleases caused sperm chromosomal DNA degradation [42] that may be partially prevented by nuclease inhibitor aurintricarboxylic acid [43], although these studies did not explore the effects on desiccation and subsequent long-term storage of sperm. Other mechanisms of possible DNA damage during drying include damage induced by reactive oxygen species or oxygen acting as a free-radical scavenger [32]. Because most of these damage mechanisms involve a series of biochemical pathways, a mechanism of prevention of these deleterious reactions may be essential for the long-term storage stability of DNA. This may be possible by using sugars as stabilizers that may bind to relevant molecules or form a glassy matrix, thereby preventing the damaging chemical reactions from progressing [33].
In summary, we used controlled convective drying protocols to desiccate B6D2F1 sperm that were stored overnight at 4°C. The results indicate that rapid drying gives the best in vitro development of ova postinjection of rehydrated sperm. We were able to obtain 15-day live fetuses, which demonstrates the feasibility of this simple technique for the desiccation of mouse sperm. Results with long-term storage stability are required in order to determine the true potential of convective drying as a desiccation technique for mouse sperm.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 The first two authors (S.B and L.Z.) contributed equally to this work
3 Correspondence: Mehmet Toner, Massachusetts General Hospital, SHC, HMS, 51 Blossom Street, Boston, MA 02114. FAX: 617 371 4950; mtoner{at}sbi.org
Received: 17 July 2002.
First decision: 20 August 2002.
Accepted: 3 December 2002.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSICSIRESULTSDISCUSSIONREFERENCES |
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M. A. Ward, T. Kaneko, H. Kusakabe, J. D. Biggers, D. G. Whittingham, and R. Yanagimachi Long-Term Preservation of Mouse Spermatozoa after Freeze-Drying and Freezing Without Cryoprotection Biol Reprod, December 1, 2003; 69(6): 2100 - 2108. [Abstract] [Full Text] [PDF]
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, rapid; 
, moderate; and 
, slow