THERE IS a renewed or, at least, an accelerated interest in the clinical applica

Transcription

1 Improved Methods of Preservation of Human Spermatozoa by Freezing and Freeze-Drying J. K. SHERMAN, Ph.D. THERE IS a renewed or, at least, an accelerated interest in the clinical applica. tions of frozen human semen. This has been stimulated in part by awareness of the advantages offered by therapeutic insemination of stored semen from husband or donor. Ejaculates from oligospermic husbands can be collected and stored over several months, thawed, pooled, concentrated by centrifugation, and then inseminated at concentrations many times greater than the original. Stored semen of husband or donor can be used in four or five consecutive daily attempts to deliver large numbers of motile spermatozoa at times most likely to coincide with ovulation. Donor semen banks storing sperm cells bearing desired genetic characteristics for unusual blood types and the like facilitate availability for specific needs. By storing his ejaculates, a man can induce conception in the absence of testes, in old age, and after death. Also~ the promise of future fertility potential may encourage voluntary submission to vasectomy in proposed attempts at population control. Banks of frozen human germ cells protected from radiation may minimize or obviate the potential genetic dangers of man's exposure to radiation on earth and in space. Recently, frozen semen banks have been proposed as part of a program for genetic selection in controlled reproduction for the human race. 9 Carrying this further but not advocating it, the author believes that it is theoretically possible to eliminate the male and female in reproduction by preservation of spermatogonia and oogonia by freezing or freeze-drying, cultivation of these gonia on demand, controlled selection of sex and other genetic characteristics, and finally in-vitro fertilization and development. From the Department of Anatomy, School of Medicine, University of Arkansas Medical Center, Little Rock, Ark. The investigation reported here was supported by Research Grant RG-6418 from the U. S. Public Health Service. The author gratefully acknowledges the superior technical assistance of Mrs. James Mulkey, especially in the storage experiments, and the help of Mr. Robin Jones in collecting glycerolation data. The improved method of preservation by freezing in liquid nitrogen vapor was presented at the 46th Annual Meeting of the Federation of American Societies for Experimental Biology, Atlantic City, Apr. 14, 1962."' 49

2 50 SHERMAN FERTILITY & STERILITY Discussion of these applications, in practical or theoretical terms, is beyond the scope of this report. However, appreciation of their usage, either actual or proposed, should stimulate efforts toward making existing methods for banking frozen semen more practical and efficient. It is in this regard that the author was encouraged to present this report. The purpose of this communication is to summarize briefly the author's salient experimental findings on certain aspects of freezing and freeze-drying of human spermatozoa since his initial reports Improved methods of preservation of human spermatozoa by freezing will be presented. Attempts at preservation of a particular cell type should involve experimental evaluation of certain factors associated with life and death at low temperatures. These factors include rate of cooling above freezing, rate of cooling below freezing, final temperature reached, length of time stored at final temperature, rate of rewarming, glycerol pretreatment and site of action, and site and appearance of ice crystallization. GLYCEROL PRETREATMENT The poly hydric alcohol glycerol affords many different cells various degrees of protection from the adverse effects of freezing and thawing. Opinions differ as to the length of time during which glycerol must be in contact with bull spermatozoa prior to low-temperature exposure for maximal freeze-thaw survival. 14 It has been established that a pretreatment exposure of as little as 25 sec. at room temperature is sufficient for glycerol to protect human spermatozoa against the adverse effects of freezing and thawing.u Although the intimate direct relationship between temperature and cell permeability to glycerol is well known, the effect of glycerolation temperature on freeze-thaw survival of human spermatozoa has not been reported. Red blood cells 7 and skin 21 have been shown to require full permeation by glycerol for maximal survival on freeze-thawing, and such penetration has become an assumed but untested requisite for all cells. However, an inverse relationship between glycerol penetration and post-thaw survival of unfertilized mouse eggs and bull spermatozoa has been demonstrated. 20 This suggests that intracellular glycerol may not be essential and possibly is detrimental to maximal survival of certain cells during freezing and thawing. We have found that unlike red blood cells, on the one hand, or bull spermatozoa, on the other, human spermatozoa appear independent of the effect.on pe1meability of pre-freeze glycerolation temperature with respect to freeze-thaw survival. Thus far, human spermatozoa are interestingly unque in this regard. (".

3 VoL. 14, No. 1, 1963 SPERM FREEZE-DRYING 51 Method As in our experiments with bull spermatozoa, glycerolation temperature was the only variable. Split samples of human semen were exposed to the same temperatures for the same length of time, and treatment was varied only with respect to the temperature at which glycerol was added. The major portion of each of 24 ejaculates from 12 healthy subjects was placed in four or six 10 X 7S-mm. Kahn test tubes, 9 drops to each tube. Half the tubes (A) were placed in a S C. water bath, and the others (B) in a 37 C. bath. After 10 min., the A tubes were transferred to a 37 C. bath, and the B tubes to a soc. bath. for a 10-min. stay. The semen was then glycerolated by adding 1 drop of glycerol at the corresponding temperature to each tube and gently mixing. Following a 10-min. wait, all tubes were brought to soc. and then cooled to -7S o C. according t() the two-step method of Sherman.18 Semen was thawed in a room-temperature bath, and triplicate readings on motility were made at 37 C. Results No difference in post-thaw motility was observed between "penetrationfacilitating"-370c. and "penetration-inhibiting''-soc. prefreeze glycerolation (Table 1). RATES OF COOLING ABOVE FREEZING Temperature shock is that low-temperature phenomenon which results in injury to living cells during rapid cooling to temperatures at or near 0 C. Irreversible loss in motility is its most obvious manifestation in spermatozoa of the bull, ram, mouse, rat, hamster, guinea pig, and dog. In 19SS we reported that human spermatozoa from the S subjects tested were not susceptible to temperature shock. 12 Since then we tested over 200 samples from 101 individ- TABLE 1. Effect of Glycerolation Temperature on Survival of Human Spermatozoa during Freeze-Thawing Glyc. temp. ( c.) %motility Pre freeze Post-thaw o/o survivalt *Based on studies of 24 samples from 12 donors; samples were frozen to -75"C. and thawed to22 post-thaw o/o motility X 100. tcaleulated by means of the relation -=------:------'--.-,~ pre-freeze o/o motility

4 52 SHERMAN FERTILITY & STERILITY uals and found that semen from some do show as much as a 15 per cent drop in the number of motile cells during rapid cooling to 0 C. However, in terms of the number of samples showing no loss and the over-all average, human spermatozoa generally exhibit little or no susceptibility to temperature shock. Method One milliliter of semen from each ejaculate was placed in a 10 X 75-mm. Kahn test tube or in a 12 X 35-mm. shell vial. The tube or vial container was corked and placed in a 0 C. ice bath for 10 min. This standard test of temperature shock for spermatozoa results in an average cooling rate of 0.09 per second to ooc. The semen was quickly rewarmed by transfering the container to a room-temperature bath. Triplicate motility readings were made at 37 C. before cooling and after rewarming. Results Of the 101 semen specimens, 52 showed no motility loss, 30, a 5 per cent loss, 13, a 10 per cent loss, and 6, a 15 per cent loss. The average loss was 3.7 percent. The author has examined hundreds of samples of dairy bull spermatozoa with respect to their susceptibility to temperature shock. There were differences as to extent, but without exception, a striking, sometimes near complete, loss of motility was observed. This is in sharp contrast to the individual variability in the exhibition of temperature shock by human ejaculates as reflected by absence of loss in 52 per cent of the cases. We found that rabbit spermatozoa also do not show temperature shock In addition to cellular differences between species and individuals, variation extends to levels of the reproductive tract in the same animal: Sensitivity to rapid cooling to 0 C. increases from epididymal to vas to ejaculated spermatozoa Our results in nine experimental runs with 20% egg yolk (in 0.9% saline) indicated that this protective substance did not prevent temperature shock in susceptible human samples. There was a suggestion that it may reduce motility loss slightly, but the average difference between samples with and without egg yolk was only 2 per cent. Earlier, we found that while egg yolk is a protective substance for bull spermatozoa, it was ineffective during the rapid cooling of temperature-shock-sensitive mouse, rat, hamster, guinea pig, and dog spermatozoa It is quite evident, therefore, that certain phenomena exhibited during the cooling may have different effects on mammalian spermatozoa obtained from different sources. Indeed, within the same population, heterogeneous response to rapid cooling is evident, perhaps reflecting protoplasmic differences due to age, development, and so forth.

5 VoL. 14, No. 1, 1963 SPERM FREEZE-DRYING 53 RATES OF COOLING BELOW FREEZING Since 193()28 emphasis has been placed upon the need, in preserving life by freezing, for cooling rapidly enough either to vitrify the water present or to permit the formation of as small ice crystals as possible. Slower speeds favor production of larger ice crystals, which are assumed to be more apt to injure protoplasm and thus decrease chances for survival. However, with other factors constant, survival of human spermatozoa during freezing and thawing varies with the rate of cooling to subfreezing temperatures but does not follow predictions of survival based upon vitrification procedures in rapid cooling.u The slowest of the rates compared proved the least injurious and was incorporated into our initial methods for preservation of human semen. During the past several years, other rates of freezing have periodically been evaluated in this laboratory. The three screened as the most favorable for survival, in terms of post-thaw motility, were then compared. The methods and results are summarized below. Method A total of 30 ejaculates, two from each of 15 medical students, was used. Each ejaculate was glycerolated at room temperature by stepwise addition of glycerol to a final concentration of 10% by volume. One-milliliter quantities were placed in each of three H~-ml. glass ampules which were tip-sealed by flame. Within 15 min. ampules clipped to separate metal canes were exposed to the selected freezing conditions. Semen from every ejaculate was frozen by each of the three methods described below, one ampule per method. Three ejaculates usually were frozen at the same time. Thawing was accomplished by dropping ampules into a room-temperature water bath. Temperature changes during cooling and rewarming were recorded by means of an electronic strip recorder (thermograph). The thermograph's attached thermocouple lead was located in the center of the glycerolated 1-ml. semen mass in a 1~-ml. glass ampule on the cane. In each method ampules were placed in the thaw bath when the temperature reached -75 C. Motility observations were made at 37 C. in triplicate, before freezing and after thawing. Freezing Method 1. This method is essentially the same as that first proposed in Canes with ampules of semen were placed in the center of a hollowed-out cylindric area, 3 in. in diameter and 12 in. deep, in slabs of solid carbon dioxide (dry ice). The canes were kept in a vertical position free from direct contact with dry ice by placing their bottom ends into a 1-in.-thick styrofoam base. The average rate of cooling to -75 C. was 3.2 C. per minute. Freezing Method 2. Ampules on canes were immersed in a -42 C. isopro-

6 54 SHERMAN FERTILITY & STERILITY TABLE 2. Effect of Freezing Method on Survival of Human Spermatozoa 0 Method Cooling rate to -75 C. ( C./min.) Pre freeze o/o Motility Post-thaw o/o survival *Based upon average of motility data, read in triplicate, for 30 ejaculates from 15 individuals. panol bath until the core temperature of the semen reached -30 C. Canes were then quickly plunged into a bath of isopropanol at -78 C. This is the two-step method used by the author since 1955 and used successfully with bull spermatozoa. 10 The average rate of cooling to -75 C. was 24 C. per minute: Freezing Method 3. Canes with ampules were suspended by means of a copper wire from a styrofoam cover, over the surface of liquid nitrogen in a LNR-25B Linde refrigerator container. 0 The temperature range from the bottom to the top of the cane varied from -194 to -172 o C. The average rate of cooling to -75 C. was 25 C. per minute, and 16 C. per minute to final temperature. Results Table 2 summarizes motility data relative to survival of spermatozoa subjected to each of the three freezing methods. All three methods involved relatively slow cooling rates. Method 1 was shown to be inferior to Methods 2 and 3, which produced essentially identical survival. Cooling curves were made to compare the methods with respect to rates of temperature change between any desired intervals. Results with Method 3 were the same when cooling was allowed to proceed to final temperature ( -172 to -194 o C.) before thawing. EFFECT OF STORAGE TEMPERATURE ON SURVIVAL OF HUMAN SPERMATOZOA IN THE FROZEN STATE Experience with many types of cells has taught us that lower temperatures usually minimize viability loss with time during frozen storage. In England bull spermatozoa were shown to be capable of fertilizing and inducing the development of normal calves following storage at -75 C. for periods exceeding 7 years. However, we and others found that the number of bull spermatozoa which during frozen storage lost their ability to resume motility on *Linde Company, Division of Union Carbide Corporation, New York, N.Y.

7 VoL. 14, No. 1, 1963 SPERM FREEZE-DRYING 55 thawing was much greater at -75 than -196 C. The motility loss of 10 per cent evident after 3 days of storage increases to 26 per cent during a 12-month period at -75 C., as compared to an over-all loss of 4 per cent at -196 C. 27 Human semen was stored at -75 and -196 C. in our storage facilities in a split-sample comparison study involving ejaculates from 17 donors. Semen was glycerolated, placed in ampules, and frozen by Method 3 described above. Observations for 1 year are summarized in Table 3. Results were obtained from the average of triplicate readings on each of the samples. TABLE 3. Post-Thaw Motility of Human Spermatozoa During 12 Months' Storage at -75 and -196 C. Storage temp. (oo.) 0 hr. 3days 3wk. 3mo. 6mo. 12mo We found that, as in the instance of bull spermatozoa, -196 C. was far superior to -75 C. as a storage temperature. Specifically, when compared to 0-hr. freeze-thaw motility: ( 1) There was no apparent loss in motility of human spermatozoa stored in liquid nitrogen for periods up to 1 year; ( 2) a loss of 10 per cent was exhibited by split samples of semen stored for periods up to 1 year at -75 C.; ( 3) most ( 8 per cent) of the motility loss at -75 C. occurred during the first 3 days of storage. FINAL TEMPERATURE We established that human spermatozoa resemble bull spermatozoa in that neither are affected by lowering the final temperature reached after cooling to -75 C. Cooling from -75 to -196 C. and rewarming from -196 to -75 C. has no effect on post-thaw motilities of frozen samples. Of course, as shown above, exposure time at final temperature is definitely a factor, with loss on storage being more pronounced at -75 C. than at lower temperature. RATE OF THAWING The rate of rewarming from subfreezing temperatures is another factor which should be evaluated in attempts at preservation by freezing. On the basis of minimal hemolysis, the superiority of rapid rewarming over slow rewarming is recognized in establishing frozen-blood banks. 5 Our experiments during the past 3 years revealed that rapid rates of rewarming are superior to slow rates in freeze-thawing of: ( 1) ascites tumor cells, whh increase in total

8 56 SHERMAN FERTILITY & STERILITY cell population and live-dead staining following homotransplantation as criteria; ( 2) mouse skin, with permanent takes after autotransplantation as a criterion; 21 and ( 3) mouse kidney and intestine, with nuclear and cytoplasmic structure as the criterion. However, the rate of rewarming does not appear to be a factor in the post-thawmotility of bull spermatozoa. 27 The effects of six different rates of rewarming on post-thaw motility were evaluated in 31 ejaculates from 14 donors. The average rates compared, from -77 to 0 C., were 1.1, 4.3, 3.4, 7.4, 35, and 59 C. per minute, produced by using air at 5 and 22 C., and water baths at 0, 5, 22, and 37 C., respectively. Glycerolated semen samples, pooled and suitably split into groups for comparison, were frozen to -77 C. in ampules by the two-step method described above and thawed from this temperature within an hour. Rewarming curves were traced with a recording potentiometer (thermograph). No differences in percentage and type of post-thaw motility at room temperature and at 37 C. were found among the rates compared, so that we can consider that, as in the instance of bull spermatozoa, the rate of rewarming is not a vital factor in the freezing and thawing of human spermatozoa. This was found to be true also in thawing from -196 C. before and after storage. IMPROVED METHOD FOR PRESERVING HUMAN SPERMATOZOA BY FREEZING Investigations of certain factors associated with freeze-thaw survival of human spermatozoa, summarized above, have resulted in the formulation of a simple, practical, and efficient method of freezing and storing these.cells at subfreezing temperatures which we have used for over 3 years. We have found the essentials of freezing, storage, and thawing in this method applicable also to the preservation of bull spermatozoa, as well as cells such as those from Ehrlich ascites tumors, Sarcoma 37, and parakeet tumors (MCSA, No. 440, and No. 73). Its basic steps should prove applicable to many other cells and tissues, with suitable modifications in glycerol content, glycerolation temperature, medium, and the like. Comparison by the author of this nitrogenvapor-freezing method with others with the use of semen from dairy bulls in Wisconsin and Santa Gertrudis beef bulls in Arkansas has demonstrated its efficiency in terms of survival and simplicity. At the author's suggestion, such experiments were repeated and his results recently confirmed. 4 An outline of the steps in this method for human spermatozoa follows. 1. Semen is collected by masturbation into sterile 2-oz. ointment jars. 2. Following liquefaction ( min.), the volume of semen is measured by drawing it up into a 5-ml hypodermic syringe. At this point, when prescribed, a 0.5-ml. portion is taken for complete semen analysis.

9 VoL. 14, No. 1, 1963 SPERM FREEZE-DRYING Enough sterile glycerol is added to undiluted semen in usual stepwise mixing routine to make a final concentration of 10% by volume in a 10-ml. Erlenmeyer flask. 4. One-milliliter volumes of glycerolated semen are placed into 1~-ml. suitably labeled glass ampules (Kimball Glass S37 or equivalent) by means of a 5-ml. hypodermic syringe with an 18-gage needle. 5. Ampules are tip-sealed with flame by means of an ampule sealer Sealed ampules are placed on suitably labeled metal or fiberglass clipon canes which are suspended from a styrofoam cover over the surface of liquid nitrogen in a half-filled (or less) LNR-25B Linde refrigerator container (Fig. 1). Cooling temperature is between -194 and -172 o C., with an over-all rate of cooling of 25 C. per minute to -75 C. and of 16 C. per minute to final temperature. The cooling rate will be slower, of course, if ampules on a closely packed group of canes are to be cooled in this way. 7. After as short a time as 5-10 min., canes can be quickly transferred to a suitably labeled storage basket which hangs partially immersed in the liquid of the same LNR-25B refrigerator container. If liquid nitrogen is not available, the following workable freezing and storage method should be used. The method of freezing is as good, but storage is less efficient at -77 C. than at -196 C. Immerse the cane with ampules into a -42 C.-isopropanol-dry ice bath in a vacuum bottle or special styrofoam container. After 5 min., remove cane and plunge into -77 C.-isopropanol-dry ice slush storage container (vacuum or styrofoam) Thaw semen by dropping ampules into a room-temperature water bath. 9. Dry the ampules with a clean cloth, open with a diamond-point pencil or small triangular file, and pour the semen into a suitable sterile test tube for future processing for insemination, and the like. 10. If prescribed, centrifuge thawed semen for 5 min. at 3000 rpm for concentration of pooled samples. Essentially, the only significant expense is the initial cost of a LNR-25B refrigerator container (about $450). The cost of about 1 month's supply of liquid nitrogen, 25 L. in the container, will vary with locale and amount purchased, with a range of $0.20-$1.00 per liter. The author can suggest a practical banking arrangement on a small scale using styrofoam containers and dry ice in isopropanol (the second method described above) which would cost less than $100 initially and about $8 a month for dry ice. Although the advantages of liquid nitrogen in the combined freezing and storage operation with minimal storage loss are evident, they may not be essential to a functional Popper and Sons, Inc., New York, N.Y.

10 58 SHERMAN FERTILITY & STERILITY Fig. 1. Liquid nitrogen refrigerator container (Linde LNR-25B) as a combined freezing and storage unit. A. (left) Schematic drawing showing internal arrangement for seating 6 storage canisters in liquid nitrogen, providing uncrowded storage space for about 850 ampules with human semen or other cells. B. (right) Simple technic of lowering one or more metal canes with ampules in nitrogen vapor above surface of liquid nitrogen for favorable freezing procedure. After freezing, cane is placed in one of canisters in same refrigerator container, for storage. semen bank if alternate methods are properly considered in planning the storage facility. FREEZE-DRYING The theoretical predictions of preservation by freeze-drying of human spermatozoa have yet to be realized. Under conditions of incomplete dehydration in the presence of glycerop andjor evaporative freezing in the absence of glycerol, 8 13 motility of fowl, 10 bull, and human 13 spermatozoa has been restored upon rehydration after having been "dried" (dehydrated to various extents) by freeze-drying. One way to accomplish this is to freeze-dry a relatively thick smear of semen on a glass slide or in a beaker for short periods. During drying by sublimation, cells farthest from the glass surface will

11 VoL. 14, No. 1, 1963 SPERM FREEZE-DRYING 59 be dried first, and drying will progress toward the slide. In this situation it is true that the water content of the smear may become low. However, cells restored after freeze-drying will be those with appreciable water content nearest the glass while those in layers above will have perished due to extensive drying. Nevertheless, resistance of cells to the process of freeze-drying is quite different from their preservation by this process. The criterion for preservation of life by freeze-drying should be revival of freeze-dried cells upon rehydration, subsequent to storage for appreciable periods in a vacuum sealed container or in a desiccator, at temperatures above freezing. Time in storage must exceed that possible with fresh or untreated cells at the same temperature. Failure to meet this criterion makes all claims of such preservation invalid as they represent merely resistance to a certain degree of dehydration by freeze-drying.h However, an article in 1957 should have created widespread acclaim as a significant accomplishment in this regard. Yushtchenko reported that freeze-dried rabbit spermatozoa stored in sealed ampules for days at C. induced the development of normal progeny. Although this work has not been confinned by repetition because of incomplete details in methods, it remains the only reported example of true preservation of mammalian spermatozoa by freeze-drying. Human spermatozoa were used in what appears to be the first application of freeze-drying in evaluating the relationship between ice formation and functional cellular survival.u Survival was found to vary with rate of freezing and presence or absence of glycerol. Although these factors altered the size, number, and shape of ice formations, there was no evidence of physical injury of spermatozoa by ice. In this case, freeze-drying does not preserve viability of cells but rather their structure in the frozen state relative to the site and character of artifacts of extracellular and intracellular ice crystallization induced at various rates of cooling 17 (Fig. 2). Although similar in principle, the method has been improved so that not only a suspension of cells, but also an organ or its parts can be studied. It has research application, for example, in the evaluation of factors associated with survival of testicular cells during freezing and thawing. The testis of the mouse, with other tissues, is being studied in our laboratory by means of the modified method which follows. 1. Testis is quickly removed from animal and placed in suitable physiologic medium, if prescribed. 2. All or part of testis is transferred to a precooled freezing bath or chest, either directly or in a container, depending upon cooling rate desired. 3. Frozen tissues are quickly placed into a pedorated aluminum basket covered by cold isopentane ( -77 C.) in an ointment jar surrounded by dry ice. 4. The jar is carried to freeze-drying apparatus (Fig. 3), where basket is

12 60 FERTILITY & STERILITY SHERMAN I0 C./SEC. 40 C./SEC. I00 C./SEC. Fig. 2. Use of freeze-drying in preserving site and character of ice formations in smear of suspended cells (top row, without glycerol; bottom row, with glycerol). In human semen size of extracellular ice formations is seen to vary with cooling rate; the faster the rate, the smaller the ice formation. Also, glycerol modifies shape of ice formations, rounding off sharp comers, and the like. Survival of human spermatozoa is greatest during slowest rate compared, in presence of glycerol. There is no evidence on thawing, however, of physical injury to spermatozoa in any of situations tested.ll (X 1000) removed, drained on dry ice, and quickly lowered into the cooled ( -45 C.) drying chamber. Freeze-drying is then initiated and continued for 1-4 days, depending upon tissue type and size. 5. After drying period, diffusion pump is disconnected and cooling flask is removed from around the drying chamber, which is. allowed to warm to room temperature. When diffusion pump cools, the vacuum is broken. 6. The basket is removed and quickly immersed in melted paraffin (60 C.), from which tissue is gently transferred to the infiltration unit with a long wide-mouthed pipet; forceps are not used, to avoid undue contact pressure. 7. Paraffin infiltration under vacuum of forepump takes about 10 min., after which tissues are placed in paraffin blocks. 8. Three- to six-section units from 6-p. ribbon are then spread with fine brushes on the surface of a warm ( 47 C.) filtered mercury bath to prevent contact with water. 9. A slightly albuminized 3X1-in. glass microscope slide is placed over spread sections and depressed slightly in mercury to favor adhesion as slide is removed. The usual procedure is to place the ribbon with freeze-dried sections on a slide with ribbons of sections from Bouin's fixed untreated (control)

13 VoL. 14, No. 1, 1963 SPERM FREEZE-DRYING 61 Fig. 3. Freeze-drying apparatus. A, Refrigerator drying chamber for frozen tissues; B, cooling trap ( -77 C.) for water from tissues and oil vapors from pump; C, Dewar cooling flasks; D, combined mechanical (fore) and condensation (diffusion) vacuum pump; E, paraffin infiltration unit; and F, chemical desiccator, which dries incoming air when vacuum is broken. and frozen-thawed tissues. This permits ready comparison of microscopic structure before freezing, while frozen, and after rewarming.. It also insures that tissues compared are treated identically during subsequent steps related to staining and mounting. 10. Slides with sections are placed in two changes of xylol for paraffin removal, in absolute alcohol for xylol removal, and then into fresh absolute alcohol for 15- to 30-min. fixation. 11. Routine dehydration to distilled water follows. Sections are then stained for 3-5 min. in Delafield's hematoxylin, dehydrated ( counterstained if desired), cleared, and mounted in balsam. This technic, coupled with auto- and homotransplantation, has provided the first convincing experimental evidence for the compatibility of formation and dissolution of intracellular ice with functional survival of cells of higher animals Contrary to the popular assumption that cellular damage increases as the size of ice formed increases, functional survival of mammalian skin has been shown to vary directly with the size of intracellular ice forma-

14 62 SHERMAN FERTILITY & STERILITY. tions preserved by this method. 21 Also, it has made possible our observation that unlike other mammalian cells evaluated thus far, testicular cells appear to be exceptional as to the relationship between size of intracellular ice formations and structural alterations in freeze-thawing. Quite unexpectedly, slower rates of freezing, with the coincident, larger, more distorting ice formations in the frozen state, result in less physical damage to testicular cells than faster rates with smaller ice formations 23 (Fig. 4). SUMMARY Various factors considered vital to the preservation of human spermatozoa by freezing were evaluated. These were glycerol pretreatment, rates of cool- SLOW RAPID Fig. 4. In mouse testis, contrary to popular assumption, least post-thaw structural damage is observed to accompany slow rate ( l C, per minute) of freezing, which produces larger intracellular ice formations. Top row shows frozen specimens; bottom row shows thawed specimens. Pycnosis and nuclear crenation, chromosomal clumping, and cytoplasmic granulation and vacuolation are more pronounced on thawing in rapidly frozen (50 C. per second) testicular tissue with small ice formations. 23 ( X 500)

15 VoL. 14, No. 1, 1963 SPERM FREEZE-DRYING 63 ing above freezing, rates of cooling below freezing, storage time and temperature, and final temperature. Insensitivity of human spermatozoa to temperature shock was confirmed. Human spermatozoa glycerolated at either 5 or 37 C. survived best during freezing atrates of C. per minute, thawing at either slow or rapid rates between 1 and 59 C. per minute, and during storage at -196 C. Improved methods of preservation, based upon these findings, are presented. While the freeze-drying technic does not preserve human spermatozoa, it does have important applications as a tool in studying ice formation relative to survival of cells in suspensions or in tissues. University af Arkansas Medical Center Little Rock, Ark. REFERENCES 1. ALBRIGHT, J. L., ERB, R. E., and EHLERS, M. H. Freeze-drying bovine spermatozoa. /. Dairy Sc. 41:206, BUNGE, R. G., KEETTEL, W. C., and SHERMAN, J. K. Clinical use of frozen semen. Fertil. & Steril. 5:520, BUNGE, R. G., and SHERMAN, J. K. Fertilizing capacity of frozen human spermatozoa. Nature 172:161, FoRGASON, J. L., BERRY, W. T., and GooDWIN, D. E. Freezing bull semen in nitrogen vapor without instrumentation. ]. Animal Sc. 20:910, HAYNES, L. L., TURVILLE, W. C., SPROUL, M. T., and ZEMP, J. W. Long term blood preservation-a reality. /. Trauma 2:3, LEIDL, W. Experiments in freeze-drying of bull spermatozoa. Proc.111rd Intern. Congr. on Animal Reproduction. Cambridge, LovELOCK, J. E. The mechanism of the protective action of glycerol against haemolysis by freezing and thawing. Biochim. et Biophys. Acta 11:28, MERYMAN, H. Survival of spermatozoa following drying. Nature 184:410, MULLER, H. J. The guidance of human evolution. Perspect. Biol. & Med. 3: I, PoLGE, C., SMITH, A. U., and PARKES, A. S. Revival of spermatozoa after vitrification and dehydration at low temperatures. Nature 164:666, SHERMAN, J. K. Freezing and freeze-drying of human spermatozoa. Fertil. & Steril. 5:351, SHERMAN, J. K. Temperature shock in human spermatozoa. Proc. Soc. Exper. Biol. & Med. 88:6, SHERMAN, J. K. Freeze-drying of bull spermatozoct. A.F.B.R. Quart. Rep. No. 9, No. 10, SHERMAN, J. K. Freezing and freeze-drying of bull spermatozoa. Am.]. Physiol. 190: 281, SHERMAN, J. K. Temperature shock in mouse spermatozoa. A.F.B.R. Quart. Rep. No. 11, SHERMAN, J. K. Temperature shock ir. spermatozoa from different animals. A.F.B.R. Quart. Rep. No. 13, SHERMAN, J. K. Freeze-drying in the study of ice formation. Fed. Proc. 17:148, SHERMAN, J. K. Effect of repeated freeze-thaw cycles on survival of bull spermatozoa. ]. Dairy Sc. 42:94, SHERMAN, J. K. Temperature shock in mammalian spermatozoa. Fed. Proc. 19:178, SHERMAN, J. K. Protection of structure and function by intracellular glycerol during freeze-thawing. Anat. Rec. 136:216, 1960.

16 64 SHERMAN FERTILITY & STERILITY SHERMAN, J. K. Factors in survival of mouse skin during freezing and thawing. Fed. Proc. 20:134, SHERMAN, J. K. Functional survival of cells during the formation and dissolution of intracellular ice. Am. Zool. 1:388, SHERMAN, J. K. Effects of intracellular ice on structural damage to testicular cells. Anat. Rec. 142:219, SHERMAN, J. K. Improved method for frozen-storage of human spermatozoa. Fed. Proc. 21:436, SHERMAN, J. K., and BuNGE, R. G. Observations on preservation of human spermatozoa at low temperatures. Proc. Soc. Exper. Biol. & Med. 82:686, SHERMAN, J. K., and BUNGE, R. G. Effect of glycerol and freezing on some staining reactions of human spermatozoa. Proc. Soc. Exper. Biol. & Med. 84:179, SHERMAN, J. K., and ELLIOTT, F. I. Unpublished observations, STILES, W. On the cause of cold death in plants. Protoplasma 9:459, YUSHTCHENKO, N. P. Proof of the possibility of preserving spermatozoa of animals in dry state. Dokl. Akad. Seljak. Nauk. 6:31, The American Society for the Study of Sterility-1963 Awards The following three awards will be presented at the annual meeting of the American Society for the Study of Sterility in New York on Apr. 19, 20, and 21, The Ortho Medal The Ortho Medal and the sum of $1,000 will be awarded to a recipient selected by the Awards Committee of the Society on the basis of his outstanding contributions in fertility and sterility or related subjects during the three years 1960, 1961, and The money is to be used by the recipient for expenses in visiting medical and research centers of his choice. The Rubin Award The Rubin Award, consisting of a certificate of merit and the sum of $250, will be awarded to the author( s) whose paper is deemed by the Awards Committee to be the most significant contribution among those appearing in the society's journal, FERTILITY AND STERILITY, during the year The Carl G. Hartman Grant-in-Aid The Carl G. Hartman Grant-In-Aid in the amount of $500 will be awarded to the most meritorious research project in fertility and sterility or related subjects, as chosen by the Awards Committee. Applications for this grant-inaid should be sent to MICHAEL NEWTON, M.D., Chairman, Awards Committee, American Society for the Study of Sterility, 2500 North State Street, Jackson 6, Miss., before Mar.1, The application should be accompanied by an original and one carbon copy of an outline of the proposed research. This should be brief but sufficiently detailed for the committee to evaluate.