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BOR - Papers in Press, published online ahead of print June 1, 2005.
Biol Reprod 2005, 10.1095/biolreprod.105.042291
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BIOLOGY OF REPRODUCTION 73, 627–633 (2005)
DOI: 10.1095/biolreprod.105.042291
© 2005 by the Society for the Study of Reproduction, Inc.

Mouse Sperm Desiccated and Stored in Trehalose Medium Without Freezing1

Lynda K. McGinnis 3,4 , Liben Zhu 3,4 , Joel A. Lawitts 5, Sankha Bhowmick 4,6 , Mehmet Toner 4, and John D. Biggers 2, 3,4 

Department of Cell Biology,3 Harvard Medical School, Boston, Massachusetts 02115 Center for Engineering in Medicine and Surgical Services,4 Massachusetts General Hospital, Harvard Medical School and Shriners Hospital for Children, Boston, Massachusetts 02114 Transgenic Mouse Facility,5 Beth Israel Deaconess Medical Center, Boston, Massachusetts 02115 Department of Mechanical Engineering,6 University of Massachusetts, Dartmouth, Massachusetts 02747


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Mouse sperm with and without trehalose were desiccated under nitrogen gas and stored at 4°C and 22°C. After rehydration, sperm were injected into oocytes using intracytoplasmic sperm injection and embryonic development was followed. Sperm were dried for 5.0, 6.25, or 7.5 min, stored at 22°C for 1 wk with and without trehalose. The percentages of blastocysts that developed from sperm with trehalose were 51%, 31%, and 20%, respectively, which was significantly higher than sperm without trehalose (10%, 3%, and 5%, respectively). Desiccation and storage in medium with trehalose significantly increased sperm developmental potential compared to medium without trehalose. Sperm dried for 5 min produced more blastocysts than sperm dried for 6.25 or 7.5 min. When sperm were dried in trehalose for 5 min and stored for 1 wk, 2 wk, 1 mo, or 3 mo at 4°C, the percentages of blastocysts were 73%, 84%, 63%, and 39%; whereas those stored at 22°C for 1 wk, 2 wk, or 1 mo were significantly lower (53%, 17%, and 6%, respectively). Embryos from sperm partially desiccated in trehalose for 5 min and stored at 4°C for 1 or 3 mo were transferred to 10 pseudopregnant recipients. Implantation rates were 81% and 48%; live fetuses were 26% and 5%, respectively. One of the recipients delivered three live fetuses. The results show that trehalose has a significant beneficial effect in preserving the developmental potential of mouse sperm following partial desiccation and storage at temperatures above freezing.

embryo, fertilization, gamete biology, ICSI, sperm, sperm desiccation, trehalose


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Since the early days of research on the preservation of mammalian sperm, there has been an interest in storing sperm in a dry state. Sperm from cattle, humans, hamsters [1–5], mice [6], rabbits [7], and swine [8] have all been preserved by freeze-drying with varying levels of success. The methods used in freeze-drying have varied. In general, these techniques require elaborate protocols of freezing and vacuum-drying or purchase of expensive freeze-drying equipment [9]. In 2003, we reported a simplified method for convectively drying mouse sperm at ambient temperature [10]. Sperm were desiccated under nitrogen gas and stored at 4°C. Live fetuses were produced, proving the feasibility of the convective drying procedure.

Anhydrobiotic organisms survive extreme loss of cellular water and desiccation. Recognition of anhydrobiotic life dates back to 1702, when Anthony van Leeuwenhoek described dry animalcules, which could be resurrected by adding water to dry dust from a gutter [11]. These organisms survive the potentially damaging effects of desiccation by intracellular synthesis of protective agents such as proteins and sugars [12–14]. One commonly synthesized intracellular protectant is trehalose [13, 15, 16], a nonreducing disaccharide formed by the linkage of two glucose molecules. Although mammals appear to lack the enzymatic pathways required to produce trehalose, many species of animals, insects, fungi, bacteria, and yeast generate trehalose both as an energy metabolite and as a protective agent against desiccation, heat, cold, hypoxia, and general cellular stress [15, 17]. Trehalose has been used with some success for the desiccation of mammalian cells [18–26]. In the current paper, we report the use of trehalose in a controlled, convective drying technique for the desiccation preservation of mouse sperm.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Donors

Seven- to 13-wk-old B6C3F1 female mice and 2- to 11-mo-old B6D2F1 male mice (Jackson Laboratories, Bar Harbor, ME; or Harlan Sprague-Dawley, Indianapolis, IN) were used as oocyte and sperm donors. The males were selected from our breeding colony, having been mated at least 1 wk before use for sperm collection. Mice of the ICR strain (Taconic Farms, Germantown, NY) were used to produce vasectomized males and pseudopregnant females for embryo transfer. 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 Chemical Co. (St. Louis, MO) unless otherwise stated. The media used for sperm isolation were prepared as outlined elsewhere [10]. Briefly, the solution contained 10 mmol/L Tris-HCl buffer supplemented with 50 mmol/L each of NaCl and EGTA. The final solution was adjusted to a pH of 8.2 and is referred to as EGTA medium [27]. A second drying medium was also formulated: EGTA medium supplemented with 0.5 M trehalose (trehalose-EGTA medium). Two versions of potassium simplex optimized medium (KSOM) were used for oocyte isolation, injection, and eventual culture of embryos. Oocytes and embryos were cultured in KSOMgAA, which is KSOM supplemented with half-strength, modified Eagle medium amino acids (AAs) and 5.5 mmol/ L glucose [28]. Hepes-buffered KSOM with 1 mg/ml polyvinyl alcohol in place of BSA (H-KSOMAA medium) was used for oocyte collection and intracytoplasmic sperm injection (ICSI) procedures. Both KSOMgAA and H-KSOMAA media contained 1 mmol/L glycyl-glutamine in place of standard glutamine [29]. {alpha}-Hemolysin was used to porate sperm in order to load trehalose, and was prepared as a 2x stock solution (25 µl ml–1; Sigma H9395) in HBSI medium (10 mM NaCl, 120 mM KCl, 5 mM glucose, and 20 mM Hepes; pH to 7.4) and stored at –20°C.

Sperm Treatment

Mice were anesthetized with CO2 or halothane and then killed by cervical dislocation. For each experiment, the caudal epididymides were excised from the male, collected in 0.5 ml EGTA solution, and punctured with a sterile needle to release the sperm into the solution. The sperm suspension was incubated at 37°C for 10 min to allow for sperm dispersion. Sperm were permeabilized for 15 min at room temperature by adding an equal volume of {alpha}-hemolysin stock solution. After permeabilization, 100 µl of the sperm sample was removed and mixed with an equal volume of 1.0 M trehalose in EGTA medium. The final concentration of trehalose was 0.5 M. Another sample of sperm was diluted with EGTA solution to produce a second sperm sample of approximately the same concentration without trehalose. The final sperm concentration ranged from 5 to 10 million/ml depending on the male. All sperm were then held at room temperature and desiccated within 2 h of the time of collection.

Convective Drying

The Plexiglass drying chamber and procedures were the same as those described previously [10]. Briefly, sperm were prepared in EGTA or trehalose-EGTA medium, and 20-µl samples were placed onto a glass slide of known weight, and weighed again to determine the original sample weight (W), using an analytical balance (M5; Mettler, Columbus, OH). The slide was then placed into the drying chamber and convectively dried by blowing dry nitrogen gas through the chamber. The length of drying time was varied (4.4, 5.0, 6.25, 7.5, 10.0, and 12.5 min). After drying, each slide was weighed again to determine the dried weight (D). A silicone isolator (Press-to-seal without adhesive; Grace Bio-Labs, Bend, OR) was placed around the dried sperm and covered with a glass coverslip. This sandwich was vacuum-sealed (Gamatech, Montebellow, CA) in a plastic bag (Food Saver Vac-Loc, Tilia International, Inc., San Francisco, CA). This package was then vacuum-packed into a mylar foil bag (Impak, Los Angeles, CA) and stored either at ambient temperature (~22°C) or in a refrigerator at 4°C. Aliquots of each sperm sample were weighed on the same day as stored sperm and were used to determine the anhydrous weight (baked weight) (DW) by drying in an oven (for further details see [10]). The water content of the samples was expressed in two ways as follows:

Sperm samples used for ICSI were reweighed on the day of ICSI to de termine moisture content after storage. The dry sperm packages were cut open, the cover glass and gaskets were removed, and the glass slide with dried sperm was reweighed before rehydration.

Oocyte Collection

Female mice (B6C3F1 strain) were superovulated with 5 IU eCG (P.G. 600; Intervet Inc., Millsboro, DE) and with 5 IU hCG 48 h later. Oocytes were collected from oviducts about 14 h after hCG injection and treated with 0.1% hyaluronidase in H-KSOMAA medium to remove cumulus cells. The oocytes were incubated in KSOMgAA at 37.5°C under 6% CO2 in air until micromanipulation.

Intracytoplasmic Sperm Injection

ICSI procedures have been previously reported [10, 30, 31]. Briefly, dried sperm were rehydrated by addition of sterile water at ambient tem perature. Oocytes were placed into H-KSOMAA for oocyte injection, and rehydrated sperm were mixed 1:1 with 12% (w/v) polyvinyl pyrrolidone in Dulbecco PBS [10]. Sperm heads were cut off by applying piezo pulses through the injection pipette. The sperm heads were then injected into the oocytes. Sperm exposed to three different treatments were injected on each day of ICSI. These treatments were randomized across days of ICSI so as to limit injections to a window of 14–16 h post-hCG. Groups of 15–25 oocytes were injected for each treatment on each day of ICSI. Oocytes were evaluated 2–4 h after ICSI, and broken oocytes were discarded. Overall ICSI survival was 50%.

Embryo Culture

After ICSI, the zygotes were cultured in 50-µl drops of KSOMgAA medium overlayered with mineral oil 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 2–4 days to evaluate preimplantation development or for embryo transfers to recipient females. Embryos were graded for stage of development every 24 h post-ICSI. The developmental stages were classified as 2-cell (fertilized), 3- to 4-cell, 5- to 8-cell, compacted, and blastocyst, respectively.

Embryo Transfer

Embryos were transferred at the 3- to 4-cell stage (48 h post-ICSI) into Day 0.5 pseudopregnant ICR females, which had been produced by natural mating with vasectomized males [10]. For each experimental protocol, 6–15 embryos were transferred into each of eight recipients. The number of embryos transferred depended on the number of 3- to 4-cell embryos and the number of recipients available on a given day. 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 [29]. In a separate experiment, two recipients received 19 and 21 embryos, respectively, to determine whether live pups could be born and develop to adulthood. Pups were allowed to mature to 2 mo of age. Photos of fetuses and adult mice were taken with an Olympus C-3040ZOOM digital camera using automatic settings. Images were adjusted for light and contrast using Photoshop 6.0 software (Adobe Software, San Jose, CA).

Statistics and Data Analysis

Singly ordered contingency tables were analyzed using the exact Kruskal-Wallis test of significance (StatXact 6; Cytel Statistical Software, Cambridge, MA). Logistic regression was applied using the exact computations available in LogXact 6 (Cytel). TableCurve 5.01 (Systat Software Co., Point Richmond, CA) was used to fit the curves to the data in Figure 1, a and b. They were selected arbitrarily and are not based on any physical theory.



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  		FIG. 1. Plots of water content after varying periods of drying of mouse sperm in a suspension of EGTA medium with and without 0.5 M trehalose. a) Residual water (%) versus drying time (min); (b) moisture (g water/ g dry weight) versus drying time (min)


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Drying Kinetics of EGTA Medium with and Without Trehalose

Sperm samples were prepared in EGTA medium ± 0.5 M trehalose and dried for varying times: 4.5, 5.0, 6.25, 7.5, 10.0, and 12.5 min. A minimum of five samples were dried for each treatment. The residual water in the media used for suspending the sperm without and with trehalose declined exponentially during drying and at similar rates (Fig. 1a). Sperm containing trehalose reached a steady-state residual water of 3%–4% higher than the sample with no trehalose, showing the ability of trehalose to hold onto water. The water content after drying relative to the total dry weight of the other components in the sample (cells, proteins, biomolecules, etc.) is expressed as grams of H2O/g dry weight (moisture; Fig. 1b). The trehalose-EGTA samples retained measurable water after drying for up to 12.5 min, whereas the medium without trehalose dried to moisture content below detectable levels by 10.0 min of drying time (Fig. 1b).

Effect of Trehalose on Blastocyst Development from Sperm Dried for 5.0, 6.25, and 7.5 Min

A 2 x 3 factorial experimental design was used. The effect of three drying times (5.0, 6.25, and 7.5 min) on sperm suspended in EGTA and trehalose-EGTA media were compared. The selected drying times were expected to produce final residual water contents in the range of 0%– 10% water. The vacuum-packaged sperm were stored at ambient room temperature (approximately 22°C) for 1 wk (±2 days). The results are summarized in Table 1.


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  		TABLE 1. Percentage of 2-cell embryos and blastocysts from sperm dried at 5.00, 6.25, and 7.50 min and stored for 1 wk at room temperature

There was no significant effect of drying time on the percentages of zygotes cleaving to the 2-cell stage in the absence of trehalose (Kruskal-Wallis test, P = 0.100), giving a pooled average of 96%. There was no effect of drying time on the development of these 2-cell embryos to blastocysts (Kruskal-Wallis test, P = 0.418), but the overall average development was only 7%. There was a significant effect of drying time on the percentages of zygotes cleaving to the 2-cell stage (P = 0.001), apparently due to fewer zygotes developing in the group that was dried for 7.5 min in trehalose-EGTA medium. The low percentage of development in this group was due to a single day of ICSI, which yielded poor cleavage to the 2-cell stage. The percentages of 2-cell embryos that developed into blastocysts were considerably greater when trehalose was in the drying medium. Increasing the length of drying time, however, significantly reduced the percentage of 2-cell embryos that developed into blastocysts from 51% with 5 min of drying to 20% with 7.5 min of drying, as shown by the significant negative regression (b) of the logit (proportion of blastocysts) on drying time (b = –0.60; 95% confidence limits –1.05 to –0.19; P = 0.003).

Blastocyst from Sperm Partially Desiccated and Stored in Trehalose at 4°C or 22°C

Based on the results from the previous experiment, sperm were partially dried for 5 min in medium with trehalose and stored either at 4°C or 22°C for 1 wk to 3 mo. The percentage of zygotes that cleaved to the 2-cell stage was very high overall, except for the storage for 1 mo in both storage temperatures (Table 2). The ~20% drop in average development that occurred after 1 mo of storage at both storage temperatures is statistically significant (4°C: Kruskal-Wallis test, P = 0.0002; 22°C: P = 0.023). However, this decline is likely due to the partial failure of ICSI on a single day, which similarly affected the responses using sperm stored at both temperatures.


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  		TABLE 2. Effect of storage temperature and storage time on the devel opmental rate of ova injected with sperm dried for five min in trehalose solution

The percentage of blastocysts that developed from 2-cell embryos was higher when sperm were stored for 1 wk, 2 wk, and 1 mo at 4°C than for the same storage times at 22°C (Table 2). Because of the low percentage of blastocyst development from sperm stored at 22°C for 1 mo, we did not store these sperm for 3 mo. The percentage of blastocysts produced from sperm stored at 4°C from 1 wk to 3 mo declined significantly over time from 74% after 1 wk of storage to 39% after 3 mo of storage. This is shown by the negative regression of logit (proportion of blastocysts) on storage time (b = –0.16; 95% confidence limits –0.23 to –0.09; P < 10–5). The percentage of blastocysts produced from sperm stored at 22°C was much lower and declined from 53% to 6%. This decline was also significant (b = –1.05; 95% confidence limits –1.61 to –0.58; P < 10–6).

Pregnancy and Live-Born Pups from Partially Dried Sperm Stored in Medium with Trehalose

Embryos (3–4 cells) produced from sperm stored at 4°C for 1 mo or 3 mo in trehalose-EGTA medium were transferred to pseudopregnant recipient female mice. Females were killed on Day 15 of pregnancy, the numbers of implantations and fetuses were counted, and the fetuses were weighed. Sperm stored for 1 mo produced 81% (34/42) implantation with 26% (11/42) live fetuses (Fig. 2, upper panel). Fetal weight averaged 0.138 g (range 0.099–0.175 g). Forty-eight percent (10/21) implantations were obtained from sperm stored for 3 mo with one live fetus on Day 15 (0.173 g).



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  		FIG. 2. Mouse fetus on the 15th day of pregnancy (upper panel) and a live-born mouse 1 mo old (lower panel) produced from sperm convectively dried for 5 min in EGTA medium with 0.5 M trehalose and stored for 1 mo at 4°C

In a separate experiment, 40 embryos (3–4 cells) from sperm stored for 1 mo at 4°C in trehalose-EGTA medium were transferred into two pseudopregnant recipient females and allowed to develop to term. Three live pups (3/19, 16%) were born from one female. One pup died within the first week after birth of unknown causes. The remaining two pups, both males, developed to adulthood and appeared normal (Fig. 2, lower panel). The second female gave birth to no pups (0/21). This recipient was killed 2 days after a litter was expected to examine her uterus for implantations. No implantations were found.

Water Content of Sperm in Trehalose-EGTA Medium During Storage

Before rehydration, sperm samples were reweighed to determine the moisture content after storage (Table 3). Only samples in which the coverglass could be removed without touching the sperm sample could be used for reweighing. A plot of the moisture at the time the sperm sample was stored versus the moisture after storage (at the time of ICSI) is shown in Figure 3a. All sperm samples lost moisture during storage. Sperm samples stored at 22°C lost more water than sperm stored at 4°C, especially when the samples were stored for more than 2 wk. The percentage of blastocysts that developed from each sample of sperm was plotted against the moisture content in each sample on the day of ICSI (Fig. 3b). The set of all data points in the graph can be partitioned into four subsets consisting of two pairs of nonoverlapping subsets. One pair relates to the sperm stored at 4°C (solid circles, solid lines). This pair consists of subset I (Fig. 3b), which had moisture contents ≥0.3 g H2O/g dry weight, and resulted in high yields of blastocysts (40%–100% blastocysts; average =68%); and subset II, which had moisture contents ≤0.2 g H2O/g dry weight, and resulted in lower yields of blastocysts (20%–42% blastocysts; average =29%). The other pair relates to the sperm stored at 22°C (open circles, dotted lines). This pair consists of subset III, which had moisture contents ≤0.1 g H2O/g dry weight, and resulted in very low blastocysts development (0%–29% blastocysts; average =10%); and subset IV, which had moisture contents ≥0.2 g H2O/g dry weight, and resulted in higher yields of blastocysts (0%–88% blastocysts; average =44%). The low responses in subset II are associated with storage for 1 and 3 mo with decreased moisture, whereas the responses in subset I are associated primarily with the shorter storage times and higher moisture. The high responses in subset IV were all associated with storage for only 1 wk with higher moisture levels.


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  		TABLE 3. The average moisture (g water/g dry weight) lost from sperm suspended in trehalose medium, dried for 5 min, vacuum packaged, and stored at either 4°C or 22°C for 1 wk to 3 mo



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  		FIG. 3. Effect of time storage on the moisture content of dried sperm. a) A plot of the moisture content of dried sperm at the time stored, against the moisture content at the time of ICSI (the diagonal line is the expected locus of points if no loss of water occurred). b) A plot of the percentage of development of ova fertilized with dried sperm against the moisture content at the time of ICSI. The open circles indicate sperm stored at 22°C; the solid circles indicate sperm stored at 4°C. The increasing size of the circles indicate the four times of storage (1 wk, 2 wk, 1 mo, and 3 mo, respectively). Four subsets of the data are enclosed by boxes—see text)


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The objective of most cell desiccation research is to recover an intact viable cell after rehydration. Viability is usually assessed by live/dead staining techniques that measure cell membrane integrity, cell replication, or both. By most measures of sperm viability, the desiccated sperm are dead. We have porated the cell membrane using {alpha}-hemolysin to allow trehalose to enter the cell. Thus, the desiccated sperm are neither motile nor do they have an intact membrane. Nevertheless, the sperm heads, when rehydrated and subsequently injected into ova, can activate and fertilize the oocyte, initiating the production of grossly normal fetuses and newborns. The addition of trehalose to this system provided a protective effect on the genetic material and the proteins required for oocyte activation and embryonic development.

Our previous research [10] showed that mouse sperm could survive desiccation by rapid convective drying to anhydrobiotic levels (≤0.1 g H2O/g dry weight, ~4.5% RW) with storage overnight at 4°C. These convectively dried sperm, stored for a brief period, produced blastocysts (71%) at rates similar to those of fresh sperm (81%), although development to live fetuses was reduced for desiccated sperm compared to fresh (11% and 17% versus 37% and 48% for 2-cell-stage and 4-cell-stage embryo transfers, respectively). In the present studies, when moisture content remained above ~0.2 g H2O/g dry weight, sperm dried and stored at 4°C in 0.5 M trehalose retained a high level of developmental potential to blastocysts even when stored for 3 mo (Table 1 and Fig. 3b). On the other hand, sperm had significantly lower developmental potential when dried and stored without trehalose (Table 1). The protective effect that we have observed of trehalose is significantly diminished when moisture content declines to an anhydrobiotic level (≤0.1–0.3 g H2O/g dry weight).

The beneficial effects of trehalose to preserve partially desiccated sperm (i.e., ≥0.2 g H2O/g dry weight) is probably related to its activity as an osmolyte [32–34]. As desiccation proceeds and water content declines, osmolarity of the sperm-drying solution will increase sharply. The EGTA medium alone will increase from 270 mOsm in the original solution to 900 mOsm at 30% residual water. In hyperosmotic conditions, trehalose is believed to protect biomolecules through a mechanism known as preferential exclusion [35, 36] (for a review of preferential exclusion see [37]). Biomolecules such as proteins incorporate many molecules of critically bound water. Trehalose is mostly excluded from direct contact with the protein, but as the free water in the outer solution decreases, and the relative concentration of solutes and trehalose increase, the trehalose is believed to form a protective milieu immediately surrounding the biomolecules and to keep the biomolecules in their native state.

The decreased ability of trehalose to afford protection in sperm samples dried below 0.2 g H2O/g dry weight may be multifactorial. In fact, reports of trehalose used for the in vitro desiccation of cells and biomolecules have been contradictory. Trehalose has been shown to protect isolated proteins [38], chromatin [34], and in vitro cell lines [20– 23, 25, 39]. However, trehalose could not prevent degradation of lyophilized lipid/DNA complexes during long-term storage [40]. Other researchers have found trehalose alone unable to preserve desiccated cells [32].

The material being dried consists of two parts, the sperm and the suspending fluid, which have very different initial properties with respect to their water content. The suspending fluid is dominated by the large concentration of trehalose. Ignoring the other constituents, the moisture content of this phase is about 5.29 g water/g dry weight. The sperm head is a unique cellular structure that consists almost entirely of a nucleus with very little surrounding cytoplasm. The nucleus is made up of two main components, the chromatin and the nuclear matrix. The chromatin consists of DNA with protamines wrapped around the major groove, which is packed in dense toroidal structures in an almost crystalline state (reviews: [41–43]). Individual chromosomes have been located within the nucleus with their centromeres centrally placed and the telomeric ends attached to the nuclear membrane. The nuclear matrix contains a variety of proteins, some of which are organized in fibrillar structures. Because the cell membrane pores created by {alpha}-hemolysin are too small for the monomers of {alpha}-hemolysin to go through, it is unlikely that {alpha}-hemolysin would porate nuclear membrane. It is not known at present whether trehalose penetrated into the sperm nucleus through the nuclear pores. Thus, it is possible that there maybe nonuniform distribution of trehalose in the injected sperm heads.

The volume of the mouse sperm head has been measured using atomic force microscopy [44]. The mean volume of nondried mouse sperm head was found to be about 10.48 µm3 [44]. The mean volume of mouse sperm heads after air-drying overnight at ambient temperature was found to be 6.28 µm3. The buoyant density of fresh mouse sperm was estimated to be 1.43 g/cm3 [45]. Using these estimates the moisture content of a fully hydrated mouse sperm head is calculated to be about 0.67 g water/g dry weight. The sperm chromatin is thus in a much drier state than the suspending fluid. In fact, it forms a coherent structure because it does not unravel after the nuclear membrane is removed [44]. Evidence suggests that preservation of the intact nuclear matrix and sperm chromatin structure are required for fertilization and full term development [46, 47]. Presumably, during the drying process, the water will be first removed from the suspending fluid before being withdrawn from the sperm and particularly the dense sperm nucleus. Removal of water without proper penetration of trehalose into the nuclear matrix and sperm chromatin may lead to damage to sperm as observed in this study for sperm dried below 0.2 g H2O/g dry weight.

Van Thuan et al. [48] reported recently that mouse sperm could be preserved for 2 mo in a high-salt solution. Increasing the concentration of NaCl in KSOMAA medium to produce a solution with osmolarity of 800 mOsm–1 protected sperm developmental ability during storage at 4°C for 1 mo. The range of osmolarity, which afforded protection, was narrow (about 800–1000 mOsm–l), and developmental potential declined rapidly after 1 mo of storage. Because addition of high NaCl is simply another method to induce cellular dehydration, it is interesting to compare the kinetics of this system versus desiccation. When intact sperm are desiccated by blowing nitrogen gas over the sample (convective drying) [10], water in the suspending fluid is removed and causes an increase in the concentration of solute in the surrounding fluid. Water is then drawn from inside of the cell, leading to dehydration. Similarly, when sperm are placed in a high concentration of NaCl [48], water is drawn out of the cell until equilibrium is reached. In both conditions, the intracellular biomolecules maybe partially protected by the increasing concentration of naturally occurring cytosolic osmolytes such as glutamine and glycine. Eventually, the cytosol will reach equilibrium with the surrounding environment. On the other hand, when the permeabilized sperm is placed in a suspending fluid containing a high concentration of trehalose then convectively dried, equilibrium between the inside and outside of the cell will occur almost immediately. As discussed previously, the presence of trehalose inside of the cell should afford protection to the internal biomolecules such as proteins, organelles, and chromatin. In our study, the developmental ability of sperm stored for 3 mo at 4°C was significantly reduced from storage for only 1 mo. However, this decrease is directly correlated with moisture loss. It is assumed that the sperm stored in high NaCl remained at a constant level of hydration, but still lost developmental ability after 2 mo. Further studies will be needed to understand the mechanisms underlying these differences.

Sperm cells preserved by freeze-drying in similar EGTA medium as in our current system but without trehalose have also been stored for extended periods of time at 4°C [27, 43] and produced embryonic development comparable to our partially desiccated sperm stored at 4°C in trehalose. However, the actual moisture content of the freeze-dried sperm before and after storage was not reported. Therefore, the results of the freeze-dried sperm cannot be directly compared against the current study of convective drying with regards to moisture content.

The original design of our packaging and storage system was intended to protect the cells during long-term storage. However, it is now obvious that the system needs to be improved. The intent was that vacuum packaging in both plastic and foil would remove air from the package and prevent the passage of moisture, air, and light into or out of the sperm samples. As we have observed retrospectively, the moisture level was not preserved, suggesting that the double packaging did not prevent movement of moisture out of the package, and thus also may not have inhibited transfer of oxygen into the sperm sample. This forces the consideration that mechanisms other than moisture loss may have contributed to degradation of the sperm viability. In a study of the lyophilization and long-term (2 yr) storage of lipid/DNA complexes, Molina et al. [40] found a high level of reactive oxygen species built up within the lyophilized cake even in the presence of trehalose. If this same build-up of reactive oxygen species occurs in sperm during storage, this may have added to the loss of sperm viability over time. Equally plausible is that the change in moisture level in our vacuum packaging may not be optimal for recovery of intact sperm head. A faster drying rate may be necessary for trehalose to afford protection in the dried state [10].

In the current research, we have produced live-born pups from mouse sperm partially desiccated at ambient temperature in a 0.5 M trehalose solution and stored for 1 mo in a refrigerator. These results suggest that an effective method can be developed for the simple convective drying of mouse sperm with long-term storage at temperatures above freezing.


   ACKNOWLEDGMENTS
 
We thank Dr. Betsey Williams for helpful criticism of the manuscript.


   FOOTNOTES
 
1 Supported by grant 1R24RR018934 from the National Center for Research Resources (NCRR). The work was initially conducted as part of the National Cooperative Program on Mouse Sperm Cryopreservation, which is funded by the National Institute of Child Health and Human Development and NCRR. A preliminary report of this work was presented at the 37th annual meeting of the Society for the Study of Reproduction, Vancouver, BC, Canada, August 1–4, 2004. Back

2 Correspondence: John D. Biggers, Department of Cell Biology, Harvard Medical School, 240 Longwood Avenue, Boston, MA 02115. FAX: 781 274 9988; john_biggers{at}hms.harvard.edu Back

Received: 23 March 2005.

First decision: 26 April 2005.

Accepted: 23 May 2005.


   REFERENCES
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 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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