Journal of Reproduction and Development, Vol. 68, No 4, 2022
Original Article
Production of offspring from vacuum-dried mouse spermatozoa and assessing the effect of drying conditions on sperm DNA and embryo development
Natsuki USHIGOME1', Sayaka WAKAYAMA2', Kango YAMAJI1', Daiyu ITO1', Masatoshi OOGA1) and Teruhiko WAKAYAMA1'2)
1>Faculty of Life and Environmental Science, University of Yamanashi, Kofu 400-8510, japan ^Advanced Biotechnology Center, University of Yamanashi, Kofu 400-8510, japan
Abstract. Freeze-dried sperm (FD sperm) are of great value because they can be stored at room temperature for long periods of time, However, the birth rate of offspring derived from FD sperm is low and the step in the freeze-drying process particularly responsible for low offspring production remains unknown. In this study, we determined whether the drying process was responsible for the low success rate of offspring by producing vacuum-dried sperm (VD sperm), using mouse spermatozoa dried in a vacuum without being frozen. Transfer of embryos fertilized with VD sperm to recipients resulted in the production of several successful offspring. However, the success rate was slightly lower than that of FD sperm. The volume, temperature, and viscosity of the medium were optimized to improve the birth rate. The results obtained from a comet assay indicated that decreasing the drying rate reduced the extent of DNA damage in VD sperm. Furthermore, even though the rate of blastocyst formation increased upon fertilization with VD sperm, full-term development was not improved. Analysis of chromosomal damage at the two-cell stage through an abnormal chromosome segregation (ACS) assay revealed that reduction in the drying rate failed to prevent chromosomal damage. These results indicate that the lower birth rate of offspring from FD sperm may result from the drying process rather than the freezing process.
Keywords: Abnormal chromosomal segregation (ACS), Freeze-dry, Intracytoplasmic sperm injection (ICSI), Spermatozoa, Vacuum-dry
(J. Reprod. Dev. 68: 262-270, 2022)
Long-term spermatozoa preservation plays a significant role in assisted reproductive technology, maintenance of genetically modified animals, and preservation of genetic resources for rare animal species. In general, liquid nitrogen (LN2) is used to permanently preserve spermatozoa, as they regain motility after thawing; however, the use of LN2 has several limitations. Most importantly, the maintenance costs are high because of the need for a constant supply, making it difficult for some countries to maintain their specimens. Furthermore, transporting a small number of spermatozoa samples to another laboratory requires expensive dry shippers or a large amount of dry ice, which results in high costs.
The use of freeze-dried spermatozoa (FD sperm) was developed as a preservation method to solve the problems associated with LN2-based preservation. Although mouse spermatozoa are unable to survive after freeze-drying, their DNA is still viable, and normal offspring may be obtained when the head of the FD sperm is injected into oocytes [1]. Subsequently, this technique has been successfully employed to obtain offspring from the FD sperm of rats [2], hamsters [3], rabbits [4], and horses [5] and has been applied to domestic animals, such as sheep [6] and cattle [7, 8]. In addition, several studies have aimed at long-term FD sperm preservation [9-13]. In our previous study, we succeeded in producing healthy offspring from
Received: April 8, 2022 Accepted: April 28, 2022 AdvancedEpub: June 7, 2022
©2022 by the Society for Reproduction and Development Correspondence: T Wakayama (e-mail: twakayama@yamanashi.ac.jp) This is an open-access article distributed under the terms of the Creative Commons Attribution Non-Commercial No Derivatives (by-nc-nd) License. (CC-BY-NC-ND 4.0: https://creativecommons.Org/licenses/by-nc-nd/4.0/)
FD mouse sperm stored for more than one year at room temperature (RT) by improving the vacuum conditions of the ampoules [12]. Under refrigerated conditions, FD rat sperm may be stored for five years [14]. Interestingly, offspring have been obtained from FD mouse sperm exposed to high or low temperatures [15], high radiation [16] or even those stored for six years on the International Space Station [17, 18]. In addition, FD sperm can be stored in thin plastic sheets, allowing the sperm to be mailed on postcards [19]. These studies suggest that FD sperm are highly tolerant to extreme environments and enable long-term preservation of sperm DNA. However, more than 20 years after the first success with FD mouse sperm, the birth rate remains low at approximately one-third of that of fresh spermatozoa [12,20, 21]. Although improving the quality of FD sperm and the success rate of offspring production to facilitate stable storage and reliable transport is crucial, the underlying cause of the low birth rate remains unclear [22].
The procedure for FD sperm production comprises five steps: preculture, freezing, drying, preservation, and rehydration. However, the step that causes the highest damage to spermatozoa has not yet been examined in detail. Previous studies have demonstrated that freezing spermatozoa without a cryoprotectant kills all spermatozoa; however, when the sperm head is injected into oocytes after thawing, healthy offspring may be obtained with only a slight decrease in birth rate [21, 23, 24]. Therefore, the freezing process of FD sperm is possibly not the main reason for the low birth rate of fertilized embryos.
To obtain clear results regarding the effects of the drying process on spermatozoa using FD sperm is challenging. To date, evaporative (or convective) drying of mouse sperm and heat-drying of rat sperm have been employed as methods of drying spermatozoa without
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freezing [25-27, 28]. Each method has been successfully used in offspring production, and microwave drying of feline spermatozoa also produces blastocysts [29]. However, it is possible that evaporation, heating, and microwave treatment used in these studies damaged sperm DNA; therefore, the results cannot be directly compared with those of FD sperm. To elucidate whether DNA damage occurs in FD sperm during the drying process, it is important to examine vacuum-dried (VD sperm) sperm, which are dried under conditions similar to those of FD sperm, without freezing them. To date, VD sperm has only been produced from rhesus monkey sperm, and it has been confirmed that monkey embryos fertilized with VD sperm can develop into blastocysts [30, 31].
In this study, we investigated whether offspring can be produced from embryos fertilized with mouse VD sperms. We optimized the drying conditions, including the volume of the medium, drying temperature, and viscosity of the medium to determine how the drying process affects the integrity of sperm DNA and embryonic development.
Materials and Methods
Animals
Female and male ICR mice (8-10 weeks old) were obtained from SLC, Inc. (Hamamatsu, Japan). Surrogate pseudo-pregnant ICR females were prepared for use as recipients of the embryos by mating with vasectomized ICR males whose sterility had been previously demonstrated. On the day of the experiment or after completion of all experiments, the mice were euthanized via C02 inhalation or cervical dislocation and used in the experiments described further. All animal-based experiments were conducted in accordance with the Guide for the Care and Use of Laboratory Animals and approved by the Institutional Committee of Laboratory Animal Experimentation of the University of Yamanashi (reference number: A29-24), which followed the ARRIVE guidelines.
Media
HTF medium was used for capacitation, freeze-thawing, freeze-drying, and vacuum-drying of spermatozoa [32]. HEPES-CZB [33] and CZB [34] media were used for oocyte/embryo manipulation and were incubated in 5% C02 at 37°C.
Preparation ofVD spermatozoa
Both epididymides were collected from male ICR mice and the ducts were severed using sharp scissors. A few drops of dense spermatozoa mass were then placed into a centrifuge tube containing 1-ml HTF medium, which was then incubated for 30 min at 37°C and 5% C02. The concentration and motility of spermatozoa were determined using a microscope, and 800 ul of the supernatant was collected. Aliquots (10-200 ul) of the spermatozoa suspension were dispensed into glass ampoules. The ampoules were connected to an FDU-2200 freeze dryer (EYELA, Tokyo, Japan) for vacuum-drying. The cork of the freeze-dryer was opened for at least 6 h until all samples were completely dry. Subsequently, the ampoules were sealed by melting the ampoule necks using a gas burner under vacuum, as described previously [35] (Figs. 2A-C). All ampoules were stored in a freezer at -30°C until further use to avoid the effects of damage suffered during storage and preserved for 3 days to 2 weeks before use [15]. No additional protective measures were taken to preserve spermatozoa.
Experimental design of vacuum-drying
The experimental procedure for vacuum-drying of spermatozoa has been described in Fig. 1. The effects of the following methods were analyzed.
ExperimentA. Effect of freezing, freeze-drying, or vacuum drying on the spermatozoa used for fertilization and development after Intracytoplasmic sperm injection (ICSI). After dispensing 50 ul of sperm suspension into ampoules, they were treated as follows: no treatment (fresh sperm), freeze-thawed (FT sperm), freeze-dried (FD sperm), and vacuum-dried (VD sperm) (Fig. 1A).
Experiment B. Effect of the volume of the drying medium on VD sperm: 10, 25, 50, 100, or 200 ul of the sperm suspension was placed in glass ampoules and then vacuum-dried at RT (Fig. IB).
Experiment C. Effect of temperature during vacuum-drying on VD sperm: 50 ul sperm suspension was transferred to glass ampoules, placed in a water bath at 37°C, in iced water (0.5°C), or at RT, and then vacuum-dried (Fig. 1C).
Experiment D. Effect of viscosity of the drying medium on VD sperm: The sperm suspension was centrifuged at 2300 x g for 10 min. Different quantities of the supernatant (most, 1/2, 3/4, or 1/8) were removed and replaced with the same amount of 12% polyvinylpyrrolidone (PVP; 360 kDa) and mixed by pipetting. The concentration of spermatozoa remained constant, whereas the concentrations of PVP were 12%, 6%, 3%, and 1.5%. Then, 50 ul of sperm suspension with PVP was placed in glass ampoules and vacuum-dried at RT (Fig. ID).
E. Effect of combined method: Methods of Experiment B and C above were combined by transferring 50 ul sperm suspension with or without PVP into glass ampoules and vacuum-drying at 37°C, RT, or0.5°C (Fig. IE).
Measurement of drying speed
The drying speed of each sample mixture was determined and compared, using the following procedure. First, empty 1.5 mlPCR tubes or 1 ml glass ampoules were weighed and the instrument set to zero. Next, the medium or medium with PVP was added to the tubes or glass ampoules and then vacuum-dried using the procedure described above. The weight was measured every 2 min for the first 10 min of drying and every 5 min thereafter for up to 30 min after turning off the vacuum drier and removing the tubes or ampoules from the machine.
Detection of trapped air in ampoules using a Tesla coil leak detector
Ampoules containing air were identified using a Tesla coil leak detector (Sanko Electronic Laboratory, Kanagawa, Japan), according to the manufacturer's protocols. Ideally, when the tip of the Tesla coil is brought near the ampoule, sparks are observed around the glass. If a substantial amount of air is trapped inside the ampoule, it cannot be ionized. However, if the ampoule contains only a small amount of residual air, ionization generates a spark inside the ampoule. Only Tesla-positive ampoules, considered highly vacuumed ampoules, were used in the experiments [12].
Measurement of sperm heads severance rate
After rehydration, 5-10 ul of sperm suspension was placed into a drop of PVP medium. About 15-20 photographs were taken for each drop, and 150-300 sperm were observed using microscope (Olympus EX-73, Tokyo, Japan). The severance rate was measured by dividing the number of sperm without heads by the total number of sperm.
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A. Effect of freezing and drying Fresh sperm
ICSI      Embryo transfer
•5-
Freeze-Thawed ~ttt (FT) sperm LN'
Freeze-Dried Sperm     "^(FD) sperm suspension
ICSI      Embryo transfer
LN,
		
Vacuum dryer		
ICSI      Embryo transfer
Without freezing
Vacuum-Dried (VD) Sperm
ICSI      Embryo transfer
B. Effect of volume of drying medium
.1
Sperm suspension (10uL-200uL)
Without freezing
Room Temp.
C. Effect of ambient temperature during drying
50 uL of
sperm
suspension
U
Without freezing
m
m
Drying with iced or hot water
D. Effect of PVP in drying medium
50 uL of
sperm
suspension
U
Without freezing
E. Mixed effect
50 uL of
sperm
suspension
Without freezing
Adding PVP
Room Temp.
Adding PVP
Drying with iced or hot water
Fig. 1. Schematic diagram of the preparation of vacuum-dried (VD) sperm and the type of experiments conducted.
Analysis and scoring of comet slides
Spermatozoa DNA damage, potentially caused by single- and double-strand breaks [36], was measured using the CometAssay™Kit (Trevigen, Gaithersburg, MD, USA) according to the manufacturer's protocol. Briefly, spermatozoa specimens were collected from the ampoules immediately after opening and rehydrated in water. The specimen was mounted on the slide, and 100-300 spermatozoa heads on each slide were analyzed via electrophoresis. To standardize the
results obtained from different conditions under which the spermatozoa were produced, the length of each DNA comet tail was divided by the mean length of the one-sided results for each experiment. Fresh and FT spermatozoa could not be used as controls, since the requirement of a different preparation technique would prevent adequate comparison between specimens on the same slide.
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Oocyte preparation
Female mice were superovulated via inj ection with 5 IU of equine chorionic gonadotropin, followed by 5 IU of human chorionic gonadotropin injected after 48 h. After 14-16 h, cumulus-oocyte complexes (COCs) were collected from the oviducts of female mice and transferred to a Falcon dish containing HEPES-CZB medium. To disperse the cumulus, COCs were transferred to a 50 ul droplet of HEPES-CZB medium containing 0.1% bovine testicular hyaluronidase for 3 min. Cumulus-free oocytes were washed twice and added to a 20 ul droplet of CZB for culture.
ICSI and embryo transfer
ICSI was performed as described previously [33]. Immediately prior to ICSI, the neck of the ampoule was broken off, and 50 jxl of sterile distilled water was immediately added and mixed using a pipette. Then, 1-2 jxl of the sperm suspension was transferred directly to the injection chamber. The sperm suspension was replaced every 30 min during ICSI. Several piezo pulses were applied to separate the sperm head from the tail and the head was then injected into the oocyte. The oocytes that survived ICSI were incubated in CZB medium at 37°C in 5% C02. Pronuclear formation was verified 6 h after ICSI. The embryos at the two-cell stage were transferred to a pseudo-pregnant mouse at day 0.5 that had been mated with a vasectomized male the night prior. Five to 12 embryos were transferred into each oviduct. On day 18.5, the offspring were delivered via cesarean section and allowed to mature. The remaining unused embryos were cultured for up to four days to evaluate their potential to develop into blastocysts.
Detection of abnormal chromosome segregation (ACS)
The day after ICSI, embryos in the two-cell stage were fixed and permeabilized with 4% PFA and 0.5% Triton X-100 for 15 min. The embryos were observed via fluorescence microscopy (Olympus IX-73, Tokyo, Japan) in PBS containing DAPI and 1% BSA. ACS was categorized into four groups: light, moderate, heavy, and lethal, as previously described [18]. Light ACS was considered when only one micronucleus was detected. Moderate ACS was noted when two small, one to two medium micronuclei, or a single large micronucleus were detected. Heavy ACS was designated when three small or medium or two or three large micronuclei were detected. Lethal ACS was considered when the embryos contained multiple micronuclei. In several instances, two conditions occurred simultaneously, making the more severe evaluation. For instance, if one medium and two small micronuclei were observed in an embryo, it was considered as "heavy".
Statistical analysis
The results of the comet assay were analyzed using the Wilcoxon-Mann-Whitney nonparametric test. Blastocyst and birth rates were evaluated using the chi-square test or Tukey's WSD test. The statistical significance of the differences between variables was set at P< 0.05.
Results
DNA damage ofVD sperm and its developmental potential after fertilization
To determine the quality of VD sperm, we compared the extent of DNA damage, fertilization rate, in vitro development to the blastocyst stage, and full-term development between fresh, FT, FD, and VD sperm (Fig. 1A). The morphology of VD sperm after rehydration did not differ from that of FD sperm (Figs. 2 D-F); however, more spermatozoa displayed separated heads and tails
than fresh spermatozoa. The extent of DNA damage between FD and VD sperms was similar, as determined by the comet assay (Figs. 2G-I). When spermatozoa were injected into oocytes (Fig. 2J), most of the oocytes were fertilized normally (Table 1). The rate of blastocyst generation did not differ between FD (39%) and VD (32%) sperms; however, both were significantly lower than that of the fresh (58%) and FT (63%) sperms (Table 1, Fig. 2K, L, P < 0.05). After transferring the embryos at two-cell stage to the recipient females, healthy offspring were obtained from embryos derived from VD sperm (Fig. 2M); however, the success rate was lower than that of FD sperm (11% vs. 23%, P < 0.05).
Relationship between drying rate and DNA damage to VD sperm
The factors that regulate the drying rate during vacuum drying include the amount of medium, drying temperature, and viscosity of the medium. We investigated the drying rate and degree of DNA damage in VD sperm obtained using each method. When VD sperm was prepared with different volumes of the medium, varying from 10 to 200 jxl (Fig. 2N), the drying rate decreased with increasing volume of the medium. In contrast, DNA damage tended to decrease with increasing volume of the medium, and the lowest DNA damage was observed when 200 jxl of medium was added (Fig. 20).
Next, we examined the drying rate of the medium under different temperatures, varying from 37°C to 0.5°C. The drying rate decreased concomitantly with temperature, with the lowest rate observed at 0.5°C (Fig. 2P). DNA damage evaluated using the comet assay showed the same pattern. As the medium temperature decreased, DNA damage also decreased, and the lowest DNA damage was observed when spermatozoa were vacuum-dried at 0.5°C (Fig. 2Q).
When PVP was added to the medium, the viscosity increased and the drying rate decreased with increasing PVP concentration (Fig. 2R). At 6% PVP or higher, complete drying of the medium was not achieved even after 30 min of vacuum drying. The DNA damage of VD sperm also exhibited a similar pattern; with increased concentrations of PVP or viscosity, DNA damage decreased, and the lowest DNA damage was observed when VD sperm was generated in 6% PVP (Fig. 2S).
Finally, we combined the aforementioned optimized parameters to produce VD sperm, in which 50 (xL of medium with 6% PVP was vacuum-dried at 37°C to 0.5°C. The drying rate and DNA damage decreased with decreasing temperature (Figs. 2T, U).
Relationship between drying rate and in vitro developmental potential of embryos derived from VD sperm
Next, we investigated the in vitro development of embryos obtained after fertilization with VD sperms prepared using these methods. When the VD sperm produced in different volumes of medium were used, the blastocyst formation rate increased with an increase in the volume of the medium and reached a maximum at 100 jxl (30%) (Table 2). However, since further samples could not be generated with such high volume of sperm suspension within a single ampoule, and considering that comet assay revealed similar extents of DNA damage with 100 and 50 jxl, we selected 50 jxl of sperm suspension for subsequent experiments.
VD sperm produced at different temperatures did not display any significant difference; however, the rate of blastocyst formation increased as temperature decreased (37°C: 21%, RT: 29%, and 0.5°C: 29%). Furthermore, when 6% PVP was added to the medium and the temperature was varied as above, the highest rate of blastocyst formation (44%) was obtained from the embryos derived from the
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Fig. 2. Examination of the sperm and DNA damage after vacuum-drying. (A) Ampoule carrying vacuum-dried (VD) sperm. (B, C) Freeze-dried (FD) and VD sperm inside the ampoule. (D, E) Fresh and VD sperm after rehydration. (F) Higher magnification of the image presented in E. (G, H) Comet DNA assays of FD and VD sperm. (I) Comparison of comet tail lengths of FD and VD sperm. The lengths of the comet tails were standardized against the average lengths of the FD sperm. Each plot shows the relative value of each sperm, and the bars indicate the average of the relative values. Error bar indicates standard deviation. (J) Image representing VD sperm injected into fresh ICR oocytes. (K, L) Blastocysts derived from embryos fertilized with fresh and VD sperm. (M) Offspring obtained from VD sperm. (N, P, R, T) Measurement of the sample drying rate. Horizontal axis indicates drying time and vertical axis shows weight ratio compared to before drying. (O, Q, S, U) Comet DNA assays of VD sperm with different amounts of medium, temperatures, and viscosities, and with a combined method. (V) The rate of sperm head severance from the tail. (W) Embryo at the two-cell stage derived from FD sperm showing normal chromosomal segregation (NCS) detected by DAPI staining. (X) Embryo at the two-cell stage derived from VD sperm showing abnormal chromosome segregation (ACS). (Y) The ratio of NCS and ACS in embryos derived from different types of sperm. Embryos showing light ACS were included in NCS. Different characters show significant difference (P < 0.05).
Table 1. Rate of blastocyst formation and full-term development of embryos after fertilization with fresh, freeze-thawed (FT), freeze-dried (FD), and vacuum-dried (VD) spermatozoa
		No. of oocytes	No. (%) of	No. (%) of embryos developed to				No. of transferred	No. (%)
Treatment	Observation	surviving after ICSI	fertilized Zygotes	2 cell	4-8 cell	Morula	Blastocyst	embryos [no. of recipients]	[min-max] of offspring
Fresh	In vitro culture	116	111(96)3	103 (93)	84 (76)	66 (59)	64 (58)3	—	—
	Full-term	142	130 (92)	117 (90)	—	—	—	82 [8]	39 (48)3 [32-67]
Freeze Thaw	In vitro culture	118	112 (95)3	108 (96)	95 (85)	86 (77)	70 (63)3	—	—
	Full-term	168	149 (89)	125(84)	—	—	—	82 [8]	38 (46)3 [14-77]
Freeze Dry	In vitro culture	160	135 (84)b	98 (73)	89(66)	81 (60)	52 (39)b	—	—
	Full-term	187	171 (91)	132(77)	—	—	—	125 [10]	29 (23)b [0-43]
Vacuum Dry	In vitro culture	156	130 (83)b	90 (69)	77 (59)	66 (51)	41 (32)b	—	—
	Full-term	252	224 (89)	87 (39)	—	—	—	84 [8]	9 (11)c [0-20]
a vs. b, c; b vs. c: P < 0.05.
Table 2. Effect of different drying rates on fertilization rate and the in vitro development of vacuum-dried (VD) spermatozoa
Volume	Temperature	PVP (6%)	No. of oocytes	No. (%) of	No. (%) of embryos developed to			
			surviving after ICSI	fertilized zygotes	2 cell	4-8 cell	Morula	Blastocyst
10	RT	No	65	61 (94)	28 (46)	15(25)	7(11)	4 (7)3
25			79	76 (96)	42 (55)	25 (33)	11 (14)	10 (13)
50			136	133 (98)	99 (74)	62 (47)	48 (36)	24 (18)
100			83	79(95)	68 (86)	51(65)	40 (51)	24 (30) b
200			84	84 (100)	68 (81)	58 (69)	38 (45)	24 (29)
50	37°C	No	114	103 (90)	50 (49)	28 (27)	24 (23)	22 (21)
	RT		246	207 (84)	135 (65)	109 (53)	82 (40)	59 (29)
	0.5°C		271	227 (84)	141 (62)	114 (50)	92 (41)	65 (29)
50	37°C	Yes	65	62 (95)3	49 (79)	34 (55)	24 (39)	17 (27)
	RT		209	169 (81) b	129 (76)	110 (65)	100 (59)	56 (33)
	0.5°C		143	121 (85) b	85 (70)	76 (63)	74 (61)	53 (44)
a vs. b: P < 0.05. RT, Room temperature.
VD sperm produced with PVP at 0.5°C.
Relationship between drying rate and full-term development of embryos derived from VD sperm
To determine whether the increase in the rate of development of blastocysts was associated with an enhanced rate of offspring production, embryos derived from VD sperm produced using various methods were transferred to recipient females at the two-cell stage. As presented in Table 3, offspring were obtained in all experimental groups. Although the rate of offspring production was significantly higher at 0.5°C than at RT (20% vs. 7%, P < 0.05), no obvious difference was noted between the offspring production rates of embryos derived from VD sperm obtained at different temperatures
with PVP in the medium.
Rate of sperm head severance from the tail
To determine the reason for the decrease in DNA damage without a reduction in the offspring production rate, we measured other types of sperm damage, including the rate of sperm head severance from the tail after rehydration (Fig. 2V). The results indicated that when VD sperm were prepared by adding PVP to the medium, the percentage of severed sperm heads was significantly reduced compared with VD sperm obtained without PVP. This clearly suggests that PVP in the medium can prevent physical damage to the spermatozoa during the drying process.
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Table 3. Effect of ambient temperature and addition of 6% PVP during the production of vacuum-dried (VD) sperm onfull-term development
Temperature	PVP (6%)	No. of oocytes surviving after ICSI	No. (%) of fertilized zygotes	2 cell (%)	No. of transferred embryos [no. of recipients]	No. (%) [min-max] of offspring
37°C	No	134	130 (97)	104 (80)	89 [5]	11 (12) [0-20]
RT		281	257 (91)	169 (66)	161 [15]	12 (7)3 [0-50]
0.5°C		182	170(93)	108(64)	92 [6]	18 (20)b [5-44]
37°C RT 0.5°C	Yes	275 161 140	249 (91)b 155 (96)3 119 (85)b	88 (35) 115(74) 83 (70)	66 [4] 95 [7] 72 [6]	4 (6) [0-38] 7 (7) [0-15] 7 (10) [0-25]
a vs. b: P < 0.05. RT, Room temperature.
Adverse effects of vacuum-drying treatment on chromosome segregation
To identify the cause of failure in offspring formation and to improve the offspring production rate, which were persistent issues even when the blastocyst formation rate was improved and physical damage was reduced; we examined the incidence of abnormal chromosome segregation (ACS) at the two-cell stage. The ACS incidence was considered to be a more profound DNA damage that could not be detected via the comet assay. In this study, we focused on moderate level of ACS, because embryos with a low level of ACS still have the potential to reach full-term development [37]. As presented in Figs. 2W and 2Y, when FD sperm were used, 71% of the embryos exhibited normal chromosome segregation (NCS); however, the rate was reduced to 47% when VD sperm were used (Figs. 2X, Y). Furthermore, the NCS rate was similar when the VD sperm was prepared by adding PVP to the medium and drying was performed at 0.5°C. This suggests that vacuum-drying treatment causes more severe DNA damage to the sperm nuclei compared with that of FD sperm even when slow drying is performed.
Discussion
Until now, whether pre-incubation, freezing, drying, preservation, or rehydration is the specific step responsible for the decline in sperm quality after freeze-drying has not been determined. In this study, we succeeded in producing offspring from mouse VD sperm. However, the success rate was lower than that for FD sperm. Indicating that the freezing process may produce dried sperm with less damage.
When oocytes and embryos are freeze-thawed without cryoprotec-tants, they do not survive due to ice crystal formation in the cytoplasm, in addition to severe osmotic stress [38]. We hypothesized that if the freezing process is omitted during the production of dry sperm, the offspring rate would increase. Contrary to our prediction, however, a lower birth rate resulting from VD sperm compared with FD sperm was noted, which was probably caused by the drying process rather than the freezing process. Damage to spermatozoa, such as the immobilization of spermatozoa and separation between the head and tail, was observed when the sperm was dried with or without the freezing process. However, it has been reported that ice crystals were not observed in horse spermatozoa frozen at various freezing rates [39]. Although we did not determine whether ice crystal formation occurred in mouse sperm after freezing, we suspect that because the DNA of spermatozoa was tightly packaged with protamine [40], the freezing process may not have damaged sperm DNA.
In the case of vacuum-drying, boiling occurs at RT because the vacuum conditions reduce the boiling point to approximately -30°C [41]. This may also cause significant physical damage to the intracellular organelles and DNA in spermatozoa. In contrast to freeze-drying,
in vacuum-drying, if the drying rate of the medium under vacuum is high, evaporation occurs rapidly and causes a swift change in the osmotic pressure around the spermatozoa. A radical change in osmotic pressure can disrupt the lipid membrane structure and membrane proteins of spermatozoa, thereby causing leakage of membrane ions, which leads to morphological abnormalities [42, 43]. We previously demonstrated that when epididymides were placed directly into salt powder and rapidly dehydrated, spermatozoa could be stored for only 1 day [44], whereas mouse spermatozoa could be stored for 2 months when slowly dehydrated in a hyperosmotic solution [45]. These results indicate that when spermatozoa are rapidly vacuum-dried, the effect of boiling on intercellular organelles and rapid osmotic pressure changes may severely damage the sperm DNA, resulting in a lower success rate of offspring formation after fertilization. Therefore, in the present study, when VD sperm were produced via a slower drying process, DNA damage in the spermatozoa was reduced (Figs. 20, Q, S, U). Interestingly, in the case of spermatozoa produced via evaporative drying, rapid drying resulted in a higher blastocyst formation rate than slow drying [46]. Although the sperm quality is clearly affected by the drying speed, it may be optimized for each method.
In the present study, we succeeded in significantly reducing DNA damage in VD sperm and improving the in vitro formation of blastocysts after fertilization by adjusting the drying speed. However, we were unable to improve the success rate of offspring production. Fertilized embryos derived from FD sperm with chromosomal abnormalities can develop into blastocysts relatively efficiently [20, 21, 47], and embryos fertilized with VD monkey sperm can develop into blastocysts with the same results as those using fresh sperm [30]. This indicates that the degree of DNA damage in VD sperm is not associated with the rate of development of embryos into blastocysts. We employed a comet assay for a comprehensive analysis of sperm DNA integrity as an indicator of global DNA damage, although it cannot measure damage such as chromosome breaks. Therefore, we also conducted an ACS assay that detects severe DNA damage in spermatozoa, which has been correlated with the offspring production rate [48]. In our study, ACS was more prominently observed in embryos with VD sperm than in those with FD sperm. Thus, even though reducing the drying rate decreased the overall DNA damage in VD sperm, no reduction was observed in severe DNA damage. Thus, to improve the success rate of offspring derived from VD or FD sperm, identification of the factor causing the severe DNA damage that occurs during the drying process is necessary.
In conclusion, we demonstrated for the first time that mouse embryos fertilized with VD sperm developed into offspring, although the success rate was inferior to that of FD sperm. We suspect that even though the freezing process is not essential for dry preservation of sperm, and can cause damage to the sperm, also reduce the damage
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caused due to drying. Reducing the severe DNA damage caused by the drying process is crucial for improving the success rate of the offspring produced from FD spermatozoa.
Conflict of interests: The authors declare no conflicts of interest.
Acknowledgments
We thank Dr. S. Kishigami, Dr. Y. Fujimoto, Ms. C. Yamagu-chi, Ms. Y. Kanda, Ms. Li Yang, and Ms. Rina Emura for their assistance in preparing this manuscript. This work was partially funded by the Japan Society for the Promotion of Science to D. I. (JP20J23364) andM.O. (17K08134); Naito Foundation and Takahashi-Sangyo Foundation (189) to S.W.; and Asada Science Foundation, Watanabe Foundation, and the Canon Foundation (M20-0008) to T.W.
References
1. Wakayama T, Yanagimachi R. Development of normal mice from oocytes injected with freeze-dried spermatozoa. NatBiotechnol 1998; 16; 639-641. [Medline] [CrossRef]
2. Hirabayashi M, Kato M, Ito J, Hochi S. Viable rat offspring derived from oocytes intracytoplasmically injected with freeze-dried sperm heads. Zygote 2005; 13; 79-85. [Medline] [CrossRef]
3. Muneto T, Horiuchi T. Full-term development of hamster embryos produced by inj ecting freeze-dried spermatozoa into oocytes. JMamm Ova Res 2011; 28; 32-39. [CrossRef]
4. Liu J-L, Kusakabe H, Chang C-C, Suzuki H, Schmidt DW, Julian M, Pfeffer R, Bormann CL, Tian XC, Yanagimachi R, Yang X. Freeze-dried sperm fertilization leads to full-term development in rabbits. Biol Reprod 2004; 70: 1776-1781. [Medline] [CrossRef]
5. Choi YH, Varner DD, Love CC, Hartman DL, Hinrichs K. Production of live foals via intracytoplasmic injection of lyophilized sperm and sperm extract in the horse. Reproduction 2011; 142: 529-538. [Medline] [CrossRef]
6. Palazzese L, Anzalone DA, Turri F, Faieta M, Donnadio A, Pizzi F, Pittia P, Mat-sukawa K, Loi P. Whole genome integrity and enhanced developmental potential in ram freeze-dried spermatozoa at mild sub-zero temperature. Sci Rep 2020; 10; 18873. Medline] [CrossRef]
7. Hara H, Tagiri M, Hwang IS, Takahashi M, Hirabayashi M, Hochi S. Adverse effect of cake collapse on the functional integrity of freeze-dried bull spermatozoa. Cryobiology 2014; 68: 354-360. [Medline] [CrossRef]
8. Keskintepe L, Pacholczyk G, Machnicka A, Norris K, Curuk MA, Khan I, Brackett BG. Bovine blastocyst development from oocytes injected with freeze-dried spermatozoa. Biol Reprod 2002; 67: 409-415. [Medline] [CrossRef]
9. Kawase Y, Suzuki H. A study on freeze-drying as a method of preserving mouse sperm. J Reprod Dev 2011; 57: 176-182. [Medline] [CrossRef]
10. Kaneko T, Serikawa T. Long-term preservation of freeze-dried mouse spermatozoa. Cryobiology 2012; 64: 211-214. [Medline] [CrossRef]
11. Kaneko T, Nakagata N. Relation between storage temperature and fertilizing ability of freeze-dried mouse spermatozoa. CompMed 2005; 55; 140-144. [Medline]
12. Kamada Y, Wakayama S, Shibasaki I, Ito D, Kamimura S, Ooga M, Wakayama T. Assessing the tolerance to room temperature and viability of freeze-dried mice spermatozoa over long-term storage at room temperature under vacuum. Sci Rep 2018; 8; 10602. [Medline] [CrossRef]
13. Kaneko T, Nakagata N. Improvement in the long-term stability of freeze-dried mouse spermatozoa by adding of a chelating agent. Cryobiology 2006; 53; 279-282. [Medline] [CrossRef]
14. Kaneko T, Serikawa T. Successful long-term preservation of rat sperm by freeze-drying. PLoSOne 2012; 7: e35043. [Medline] [CrossRef]
15. Wakayama S, Ito D, Kamada Y, Yonemura S, Ooga M, Kishigami S, Wakayama T. Tolerance of the freeze-dried mouse sperm nucleus to temperatures ranging from-196 CC to 150 °C. Sci Rep 2019; 9: 5719. [Medline] [CrossRef]
16. Kusakabe H, Kamiguchi Y. Chromosomal integrity of freeze-dried mouse spermatozoa after 137Cs gamma-ray irradiation. MutatRes 2004; 556: 163-168. [Medline] [CrossRef]
17. Wakayama S, Kamada Y, Yamanaka K, Kohda T, Suzuki H, Shimazu T, Tada MN, Osada I, Nagamatsu A, Kamimura S, Nagatomo H, Mizutani E, Ishino F, Yano S, Wakayama T. Healthy offspring from freeze-dried mouse spermatozoa held on the International Space Stationfor 9 months. Proc Natl Acad Sci USA 2017; 114: 5988-5993. Medline] [CrossRef]
18. Wakayama S, Ito D, Kamada Y, Shimazu T, Suzuki T, Nagamatsu A, Araki R, Ishikawa T, Kamimura S, Hirose N, Kazama K, Yang L, Inoue R, Kikuchi Y, Hayashi E, Emura R, Watanabe R, Nagatomo H, Suzuki H, Yamamori T, Tada MN, Osada I, Umehara M, Sano H, Kasahara H, Higashibata A, Yano S, Abe M, Kishigami S,
Kohda T, Ooga M, Wakayama T. Evaluating the long-term effect of space radiation on the reproductive normality of mammalian sperm preserved on the International Space Station. SciAdv 2021; 7: 5554-5565. [Medline] [CrossRef]
19. Ito D, Wakayama S, Emura R, Ooga M, Wakayama T. Mailing viable mouse freeze-dried spermatozoa on postcards. iScience 2021; 24; 102815. [Medline] [CrossRef]
20. Kusakabe H, Szczygiel MA, Whittingham DG, Yanagimachi R. Maintenance of genetic integrity in frozen and freeze-dried mouse spermatozoa. Proc Natl Acad Sci USA 2001; 98: 13501-13506. [Medline] [CrossRef]
21. Ward MA, Kaneko T, Kusakabe H, Biggers JD, Whittingham DG, Yanagimachi R. Long-term preservation of mouse spermatozoa after freeze-drying and freezing without cryoymscxion. Biol Reprod 2003; 69: 2100-2108. [Medline] [CrossRef]
22. Gil L, Olaciregui M, Lufio V, Malo C, Gonzalez N, Martinez F. Current status of freeze-drying technology to preserve domestic animals sperm. Reprod DomestAnim 2014; 49(Suppl4): 72-81. [Medline] [CrossRef]
23. Kuretake S, Kimura Y, Hoshi K, Yanagimachi R. Fertilization and development of mouse oocytes injected with isolated sperm heads. Biol Reprod 1996; 55; 789-795. [Medline] [CrossRef]
24. Wakayama T, Whittingham DG, Yanagimachi R. Production of normal offspring from mouse oocytes injected with spermatozoa cryopreserved with or without cryoprotection. J ReprodFertil 1998; 112: 11-17. [Medline] [CrossRef]
25. Liu J, Lee GY, Lawitts JA, Toner M, Biggers JD. Live pups from evaporatively dried mouse sperm stored at ambient temperature for up to 2 years. PLoS One 2014; 9; e99809. [Medline] [CrossRef]
26. Li MW, Biggers JD, Elmoazzen HY, Toner M, McGinnis L, Lloyd KCK. Long-term storage of mouse spermatozoa after evaporative drying. Reproduction 2007; 133; 919-929. [Medline] [CrossRef]
27. McGinnis LK, Zhu L, Lawitts JA, Bhowmick S, Toner M, Biggers JD. Mouse sperm desiccated and stored in trehalose medium without freezing. Biol Reprod 2005; 73; 627-633. [Medline] [CrossRef]
28. Lee KB, Park K-E, Kwon IK, Tripurani SK, Kim KJ, Lee JH, Niwa K, Kim MK. Develop to term rat oocytes injected with heat-dried sperm heads. PLoS One 2013; 8; e78260. [Medline] [CrossRef]
29. Patrick JL, Elliott GD, Comizzoli P. Structural integrity and developmental potential of spermatozoa following microwave-assisted drying in the domestic cat model. Therio-genology20\l\ 103: 36-43. [Medline] [CrossRef]
30. Meyers SA, Li M-W, Enders AC, Overstreet JW. Rhesus macaque blastocysts resulting from intracytoplasmic sperm injection of vacuum-dried spermatozoa. J Med Primatol 2009; 38: 310-317. [Medline] [CrossRef]
31. Klooster KL, Burruel VR, Meyers SA. Loss of fertilization potential of desiccated rhesus macaque spermatozoa following prolonged storage. Cryobiology 2011; 62; 161-166. [Medline] [CrossRef]
32. Quinn P, Moinipanah R, Steinberg JM, Weathersbee PS. Successful human in vitro fertilization using a modified human tubal fluid medium lacking glucose and phosphate ions. FertilSteril 1995; 63: 922-924. [Medline] [CrossRef]
33. Kimura Y, Yanagimachi R. Intracytoplasmic sperm injection in the mouse. Biol Reprod 1995; 52: 709-720. [Medline] [CrossRef]
34. Chatot CL, Lewis JL, Torres I, Ziomek CA. Development of 1-cell embryos from different strains of mice in CZB medium. Biol Reprod 1990; 42: 432-440. [Medline] [CrossRef]
35. Ito D, Wakayama S, Kamada Y, Shibasaki I, Kamimura S, Ooga M, Wakayama T.
Effect of trehalose on the preservation of freeze-dried mice spermatozoa at room temperature. J Reprod Dev 2019; 65: 353-359. [Medline] [CrossRef]
36. Haines G, Marples B, Daniel P, Morris I. DNA damage in human and mouse spermatozoa after in vitro-irradiation assessed by the comet assay. Adv Exp Med Biol 1998; 444; 79-91, discussion :92-93. [Medline] [CrossRef]
37. Mashiko D, Ikeda Z, Yao T, Tokoro M, Fukunaga N, Asada Y, Yamagata K. Chromosome segregation error during early cleavage in mouse pre-implantation embryo does not necessarily cause developmental failure after blastocyst stage. Sci Rep 2020; 10; 854. [Medline] [CrossRef]
38. Kasai M. Advances in the cryopreservation of mammalian oocytes and embryos: Development of ultrarapid vitrification. Reprod Med Biol 2002; 1; 1-9. [Medline] [CrossRef]
39. Morris GJ, Faszer K, Green JE, Draper D, Grout BWW, Fonseca F. Rapidly cooled horse spermatozoa: loss of viability is due to osmotic imbalance during thawing, not intracellular ice formation. Theriogenology 2007; 68; 804-812. [Medline] [CrossRef]
40. Bao J, Bedford MT. Epigenetic regulation of the histone-to-protamine transition during spermiogenesis. Reproduction 2016; 151; R55-R70. [Medline] [CrossRef]
41. Davies JF, Miles REH, Haddrell AE, Reid JP. Temperature dependence of the vapor pressure and evaporation coefficient of supercooled water. J Geophys Res D Atmospheres 2014;119:10931-10940. [CrossRef]
42. Holt WV, North RD. Thermotropic phase transitions in the plasma membrane of ram spermatozoa. J Reprod Fertil 1986; 78: 447-457. [Medline] [CrossRef]
43. Hezavehei M, Sharafi M, Kouchesfahani HM, Henkel R, Agarwal A, Esmaeili V, Shahverdi A. Sperm cryopreservation; A review on current molecular cryobiology and advanced approaches. ReprodBiomed Online 2018; 37; 327-339. [Medline] [CrossRef]
44. Ono T, Mizutani E, Li C, Wakayama T. Preservation of sperm within the mouse cauda epididymidis in salt or sugars at room temperature. Zygote 2010; 18; 245-256. [Medline] [CrossRef]
270
USHIGOME et al.
45. Van Thuan N, Wakayama S, Kishigami S, Wakayama T. New preservation method for mouse spermatozoa without freezing. Biol Reprod 2005; 72; 444-450. [Medline] [CrossRef]
46. Bhowmick S, Zhu L, McGinnis L, Lawitts J, Nath BD, Toner M, Diggers J. Desicca-tiontolerance of spermatozoa dried at ambient temperature: production of fetal mice. Biol Reprod 2003; 68: 1779-1786. [Medline] [CrossRef]
47. Kusakabe H, Yanagimachi R, Kamiguchi Y. Mouse and human spermatozoa can be freeze-dried without damaging their chromosomes. Hum Reprod 2008; 23: 233-239. [Medline] [CrossRef]
48. Yamagata K, Suetsugu R, Wakayama T. Assessment of chromosomal integrity using a novel live-cell imaging technique in mouse embryos produced by intracytoplasmic sperm injection. Hum Reprod 2009; 24: 2490-2499. [Medline] [CrossRef]