Monosodium glutamate enhances freeze-dried mouse sperm quality during room-temperature storage

Abstract

Although freeze-dried (FD) sperm can be stored at room temperature, making it cheaper and safer to store and transport, freeze-drying can damage the sperm’s DNA, which can decrease the birth rate compared to frozen sperm. In this study, we examined whether adding monosodium glutamate (MSG), a compound used to dry and preserve yeast, to the drying medium could improve the quality of mouse FD sperm. FD sperm stored using MSG showed lower morphological and DNA damage, and the birth rate of embryos fertilized using those sperm improved to a similar level (50%) to that obtained with frozen-thawed sperm. The offspring showed normal growth and reproductive performance. Similar results were obtained for embryos derived from other mouse strains, even using FD sperm stored for 3 months at room temperature. These results suggest that MSG can protect not only microorganisms but also the DNA of mammalian FD sperm and improve the birth rate to a practical level. This method is not only inexpensive and safer than cryopreservation using liquid nitrogen but has expansion potential to maintain a large number of genetically modified mouse strains.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-025-16635-9.

Introduction

Sperm preservation plays an important role in the lineage maintenance of genetically modified mice, conservation of wildlife or rare animal species, and assisted reproductive technology. Typically, long-term sperm preservation is achieved using liquid nitrogen^1,2. Sperm preserved by this method can be stored semi-permanently, and since they maintain motility after thawing, they can fertilize and develop normal embryos after in vitro fertilization. However, regular replenishment of liquid nitrogen is necessary, and if not properly controlled, there is a risk of frostbite and asphyxiation. Furthermore, if the liquid nitrogen cannot be replenished after natural disasters, valuable samples can get lost³. Further, it is a costly preservation method because it requires special containers such as dry sippers and large amounts of dry ice to transport small amounts of samples⁴.

Freeze-dried sperm (FD sperm) is a room-temperature preservation method that addresses the issues of cryopreservation. Mouse FD sperm lose their motility after rehydration, but can produce normal offspring by intracytoplasmic sperm injection (ICSI), if the nuclei are undamaged^5,6. This technique has been successfully used to produce offspring from the FD sperm of rabbits⁷rats⁸horses⁹and hamsters¹⁰and it has been used in domestic animals such as cattle¹¹pigs¹²sheep¹³dogs¹⁴and cats¹⁵ as well. Interestingly, the chromosomal integrity of mouse FD sperm has been demonstrated after storage at high temperatures^16,17 and exposure to radiation^18,19. In fact, offspring can be produced even from mouse FD sperm exposed to space radiation for extended periods at the International Space Station^19,20. Recently we have successfully produced offspring from mouse FD sperm stored at room temperature for > 6 years without decreased birth rate²¹ which suggests the feasibility of longer storage at room temperature. Further, simplified preservation methods are being investigated; now, it is possible to obtain normal offspring from mouse FD sperm stored simply in plastic microtubes²² or thin plastic film, which allows mailing sperm attached to a postcard⁴. Similarly, sheep FD sperm can be preserved in stainless steel mini capsules²³. Although ICSI is necessary to produce offspring because FD sperm do not move after rehydration, this technique is used worldwide, including for humans^24,25. However, the birth rate obtained with FD sperm remains low compared with that of fresh sperm^3,5,21,26,27. Given the common occurrence of DNA damage in FD sperm²⁸for this technology to become practical, more effective desiccant protectants are needed to reduce DNA damage in FD sperm and increase birth rates.

There are four steps involved in the preparation and use of FD sperm: freezing, drying, storage at room temperature, and rehydration. The damage caused during room-temperature storage and rehydration can be improved by adding trehalose²⁹ or rehydration with high-temperature water (submitted). However, the major cause of the decreased birth rate of embryos derived from FD sperm is drying rather than freezing³⁰. Thus, to prevent the DNA damage caused by FD, sperm can be treated with protective agents such as EDTA, a chelating agent with a retention effect on endonuclease activity during storage³¹; silica gel as a dehydrating agent³²; and rosmarinic acid as an antioxidant³³. However, these agents only achieved minor improvements and did not substantially increase birth rates. Therefore, we focused on freeze-drying and vacuum-drying methods for microorganism preservation^34,35. Numerous drying media have been optimized for different microbial species, typically incorporating monosodium glutamate (MSG), an amino acid. MSG not only acts as a cryoprotectant^36–38 but also protects cell membranes and acts as an antioxidant³⁹. Moreover, it stabilizes the protein structure through reaction of amino groups with the carboxyl groups of proteins in microorganisms and can retain a large amount of residual moisture⁴⁰making it useful for drying and storage of microorganisms. Since microorganisms such as dried yeast are viable after dry storage, MSG may be an effective desiccation protectant for mammalian FD sperm.

In mammals, it has been reported that sperm produced from rats that consumed large amounts of MSG exhibited functional disorders⁴¹. However, to the best of our knowledge, there are no examples of MSG being added to sperm culture medium or cryoprotectant agents. Thus, this study aimed to determine whether MSG addition acts as a protective agent in mouse FD sperm. Accordingly, 1–10% (w/v) MSG was added to the drying medium during FD sperm production to determine its protective effect on sperm and its influence on embryonic development after ICSI. Additionally, the effects of MSG on several mouse strains, including C57BL/6 N (B6N), were examined (Fig. 1).

Fig. 1.

Schematic representation of the preparation of freeze-dried spermatozoa and other experiments.

Results

Effect of MSG on the separation rate of sperm heads and Tails

The ampoules of FD sperm preserved in the desk drawer (Fig. 2a) were tested with a Tesla coil leak detector, and only those ampoules that showed a positive reaction were used (Fig. 2b).To determine the protective effect of MSG on FD sperm, we first evaluated the physical damage caused by FD by quantifying the sperm head and tail separation rate. The percentage of tailless sperm in FD sperm was 23%, much higher than the fresh control (3%). Conversely, MSG addition significantly decreased the percentage of tailless sperm for FD sperm in a concentration-dependent manner, up to 6% (Fig. 2c). Similar results were obtained for freeze-thaw sperm (Table S1).

Fig. 2.

Analysis of FD sperm produced with MSG. (a) A glass ampoule containing FD sperm (left) stored in a desk blower (right). (b) Vacuum level determination using a Tesla coil leak detector. A glowing ampoule (right ampoule) with high vacuum was used. (c) Ratio of tailless FD sperm produced by different MSG concentrations. Error bars indicate SE. (d) Detection of acrosomes in FD sperm produced with 5% MSG. Acrosomes were stained with lectin PNA. Upper left; DAPI. Upper right; lectin PNA. Bottom left; merge. Bottom right; merge + bright field (BF). Arrows indicate PNA negatives. Arrowheads indicate PNA positives. (e) Ratio of PNA-positive sperm with and without Triton X-100 (Tx) treatment. Yellow; with Tx. Red; without Tx. Error bars indicate SE. (f–h) Comet assay with 0%, 3%, and 10% MSG-added FD sperm, respectively. (i) Relative values of the comet tail of FD spermatozoa at different MSG concentrations. Different characters indicate significant differences (P < 0.05).

Effect of MSG on the sperm membrane and sperm DNA

To observe the acrosome, fresh and FD sperm treated with 0–10% MSG-HTF were examined for fluorescence using lectin PNA labeled with a fluorescent dye that targets the acrosome matrix. In this study, Triton X-100 (Tx) treatment was performed to slightly permeabilize the cell membranes of some spermatozoa. To confirm that the acrosome had not been lost by this permeabilization treatment, Tx treatment and PNA staining were performed using Acro-green fluorescent protein (GFP) Tg mice that express GFP in the acrosome. Subsequently, it was confirmed that sperm acrosomes did not disappear even after Tx treatment using this method, and sperm acrosomes could be observed by PNA staining (Fig. S1a; Table S2). The PNA-positive sperm rate of intact sperm for fresh sperm was 17%, but permeabilization treatment by Tx treatment increased the PNA-positive sperm rate to 92%. Similarly, intact FD sperm had a low PNA-positive sperm rate (31%), but Tx treatment increased the PNA-positive sperm rate to 97%. In contrast, increasing MSG concentrations increased the PNA-positive sperm rate even without Tx treatment, and the rate became as high as that with Tx treatment at MSG concentrations ≥ 5% (Fig. 2d,e, and S1b; Table S3).

Next, the comet assay was used to measure DNA damage in MSG-treated FD sperm at different MSG concentrations by comparing their comet tail lengths. This analysis evaluates overall DNA damage in sperm, including minor DNA damage. All four individuals showed a concentration-dependent reduction in comet tail length, and correspondingly, less DNA damage (Fig. 2f–i; Table S4).

MSG toxicity for embryo development

To confirm that MSG does not adversely affect embryo development, mature oocytes were injected with a small amount of MSG-HTF medium and activated artificially for parthenogenesis. Our results show that 3% and 10% MSG-HTF-injected oocytes could develop blastocysts at similar development rates as control HTF medium injected oocytes and intact fresh oocytes (Table 1). In addition, ICSI was performed using fresh sperm with 3% or 10% MSG-HTF medium; in both cases, normal fertilization and blastocyst rates were similar to those of 0% and fresh sperm (Table 1). These results indicate that MSG is not toxic to embryo development at concentrations up to 10%. However, sperm motility decreased in a concentration-dependent manner with MSG addition and completely lost motility with 10% of added MSG (Table S5). After three washes and returning them to normal HTF medium, up to 3% of MSG-treated sperm regained a motility similar to that of fresh sperm, while 10% of MSG-treated sperm maintained their lost motility.

Table 1.

Development rate of parthenogenesis performed by injecting MSG-HTF into unfertilized oocytes and ICSI performed with sperm replaced by MSG-HTF.

Type of embryo	Concentration of MSG	No. of oocytes surviving after activation/ICSI	No. (%) of 2PN formed embryos	2-cell (%)	4-8cell (%)	morula (%)	Blastocyst (%)
Parthenogenesis	Fresh*1	70	69 (99)	69 (100)	66 (96)	60 (87)	56 (81)
	0%	64	61 (95)	60 (98)	57 (93)	57 (93)	50 (82)
	3%	71	71 (100)	69 (97)	65 (92)	59 (83)	57 (80)
	10%	75	75 (100)	73 (97)	71 (95)	65 (87)	55 (73)
ICSI	Fresh*2	58	50 (86)	48 (96)	36 (72)	31 (62)	29 (58)
	0%	50	41 (82)	39 (95)	35 (85)	33 (80)	30 (73)
	3%	71	60 (85)	56 (93)	50 (83)	45 (75)	43 (72)
	10%	73	66 (90)	59 (89)	52 (79)	44 (67)	39 (59)

PN: pronucleus.

All data within each column are not statistically different (P < 0.05).

*1: no injection.

*2: no centrifugation and no replacement.

Effect of MSG on chromosomal segregation abnormalities at the 2-cell stage

Clustered and heavier DNA damage cannot be detected by the comet assay but could be detected at the time of cell division as abnormal chromosomal segregation (ACS)¹⁹. Therefore, to evaluate heavier DNA damage at the chromosome level, we examined the incidence of ACS at the two-cell stage via DNA staining (Fig. 3a,b). ACS rates were not significantly different among groups; however, they were lower in the 1% and 3% groups than in untreated groups. Conversely, at 5% and 10% MSG, ACS rates tended to be higher than in the untreated group and increase the percentage of “lethal” (Fig. 3c; Table S6).

Fig. 3.

Analysis of embryos fertilized with MSG-treated FD sperm. (a,b) Chromosomal segregation error detected by DAPI staining of two-cell stage embryos derived from FD sperm. A single large nucleus in each blastomere suggests normal chromosomal segregation (NCS) (a) while a large nucleus with several micronuclei in blastomeres indicate abnormal chromosomal segregation (ACS) (b). Arrows indicate the micronucleus. (c) NCS to ACS ratio in embryos derived from FD sperm at different MSG concentrations. Embryos showing light ACS in NCS. N; NSC, A; ACS. (d) Blastocysts derived from embryos fertilized with 5% MSG-treated FD sperm. (e) Developmental rate of embryos derived from FD sperm with different MSG concentrations up to the blastocyst stage. 2 C; two-cell stage, 4–8 C; four-eight-cell stage, Mor; morula stage, Bla; blastocyst stage. (f) Immunostaining images of blastocysts derived from MSG-treated FD sperm. Embryo nuclei detected by nuclear staining with DAPI (left, blue); Nanog-positive cells (inner cell mass [ICM]; second row from left, green), CDX2-positive cells (trophectoderm [TE]; second row from right, red) and merged images (right). (g) Cell number of blastocysts at different MSG concentrations in immunostaining images. Blue-only fluorescence was considered to be unknown, while both green and red fluorescence was considered coexpression. Error bars indicate the SD of the total cell number.

In vitro developmental ability of embryos derived from FD sperm treated with MSG and stored at room temperature

To verify the protective effect of MGS on FD sperm, embryos derived from FD sperm were cultured to blastocysts after ICSI. Most oocytes injected with FD sperm were normally fertilized (Table 2). Conversely, the percentage of blastocysts increased in a concentration-dependent manner, with a maximum of 68% of the embryos developed when 5% MSG was added, which was about 2 times higher than that of the control untreated embryos (Fig. 3d,e). These blastocysts were used for immunostaining, and embryo quality was evaluated based on the inner cell mass (ICM) and trophectoderm (TE) cell numbers. Although the number of TE cells tended to decrease with MSG treatment compared with untreated group, there was no significant difference in ICM and TE cell counts among groups (Fig. 3f,g; Table S7).

Table 2.

The rate of blastocyst development and full-term development of embryos injected with MSG-treated FD sperm from ICR male mice stored at room temperature into ICR oocytes.

Concentration	Observation	No. of oocytes surviving after ICSI	No. (%) of fertilized zygotes	No. (%) of embryos developed to				No. of transferred embryos [no. of recipients]	No. (%) [min-max] of offspring
				2 cell	4–8 cell	Morula	Blastocyst
0%	In vitro culture	90	80 (89)ab	69 (86)	59 (74)	56 (70)	30 (38)a	–	–
	Full-term	206	193 (94)	178 (92)	–	–	–	171 [11]	44 (26)a [5–69]
1%	In vitro culture	83	77 (93)ab	67 (87)	61 (79)	59 (77)	41 (53)ab	–	–
	Full-term	173	154 (89)	147 (95)	–	–	–	147 [9]	37 (25)a [18–63]
3%	In vitro culture	78	77 (99)a	73 (95)	69 (90)	65 (84)	51 (66)b	–	–
	Full-term	222	211 (95)	186 (88)	–	–	–	159 [11]	75 (47)b [29–65]
5%	In vitro culture	79	68 (86)b	60 (88)	58 (85)	56 (82)	46 (68)b	–	–
	Full-term	180	161 (89)	139 (86)	–	–	–	127 [9]	44 (35)ab [15–67]
10%	In vitro culture	88	78 (89)ab	70 (90)	65 (83)	57 (73)	50 (64)b	–	–
	Full-term	153	139 (91)	110 (79)	–	–	–	110 [8]	43 (39) ab [20–64]
Fresh*	In vitro culture	116	111 (96)	103 (93)	84 (76)	66 (59)	64 (58)	–	–
	Full-term	142	130 (92)	117 (90)	–	–	–	82 [8]	39 (48) [32–67]
Freeze thaw*	In vitro culture	118	112 (95)	108 (96)	95 (85)	86 (77)	70 (63)	–	–
	Full-term	168	149 (89)	125 (84)	–	–	–	82 [8]	38 (46) [14–77]

Different superscript letters within a column indicate significantly different values (P < 0.05).

FD sperm was stored at room temperature for up to 3 months.

*These data have been previously reported (Ushigome et al., J Reprod Dev, 2022).

Offspring production from embryos fertilized with MSG-treated ICR mouse FD sperm stored at room temperature

Offspring production is the most reliable way to confirm the protective effect of MSG on FD sperm. Therefore, 2-cell embryos were obtained after ICSI with ICR mouse strain FD sperm and birth rates compared between control (without MSG) FD sperm and 1–10% MSG-added FD sperm preserved at room temperature for up to 3 months. The birth rate of the control was 26%, but 3% MSG addition produced the highest birth rate (47%, Table 2). Based on sperm DNA damage, MSG toxicity, and embryo development rate, 3% MSG was chosen as the optimal concentration for future experiments.

Effects of MSG treatment on FD sperm storage time in different mouse strains

To study the effect of MSG on FD sperm from different mouse strains during extended room-temperature preservation, FD sperm with or without 3% MSG were produced from B6D2F1 (BDF1) and C57BL/6 N (B6N) mice and stored for up to 3 months. As shown in Fig. 4a, b, and Table 3, MSG addition significantly increased the birth rates compared with samples stored without MSG (BDF1 strain: 37–50% vs. 19–29%; B6N strain: 49% vs. 32%, respectively). Further, the sex ratio of the offspring obtained in these experiments was within normal ranges (Table S8). After the offspring grew to full term, they were randomly selected and mated with each other. All these couples produced offspring, showed normal reproductive performance, and grew normally up to the 3rd generation (Fig. S2, Table S9).

Fig. 4.

Birth rate of embryos fertilized with FD sperm from various mouse strains treated with MSG. (a) Healthy offspring derived from FD sperm preserved at room temperature for 3 months with 3% MSG. (b) Birth rates of embryos derived from BDF1 and B6N FD sperm stored with 3% MSG or without MSG for up to 3 months at room temperature. Error bars indicate SE. Asterisks indicate significant differences between MSG-treated and untreated samples in each period (P < 0.05).

Table 3.

Full-term development of embryos fertilized with MSG-treated FD sperm from BDF1 or B6N mice stored at room temperature for up to 3 months.

Storage period	Sperm strain	MSG treatment	No. of oocytes surviving after ICSI	No. (%) of fertilized Zygotes	No. (%) of 2-cell	No. of transferred embryos [no. of recipients]	No. (%) of implantation	No. (%) [min-max] of offspring
~ 2 weeks	BDF1	−	170	160 (94)	134 (84)a	123 [8]	64 (52)a	23 (19)a [0–41]
		+	152	136 (89)	127 (93)b	92 [7]	65 (71)b	41 (45)b [38–63]
1 month	BDF1	−	144	141 (98)a	126 (89)	119 [7]	67 (56)a	34 (29)a [9–43]
		+	175	163 (93)b	146 (90)	146 [11]	115 (79)b	73 (50)b [31–77]
3 months	BDF1	−	222	208 (94)	159 (76)	157 [12]	86 (55)a	33 (21)a [4–32]
		+	250	238 (95)	193 (81)	140 [13]	94 (67)b	52 (37)b [18–54]
1 month	B6N	−	157	137 (87)a	133 (97)	116 [8]	67 (58)a	37 (32)a [18–59]
		+	171	163 (95)b	158 (97)	131 [10]	101 (77)b	64 (49)b [20–57]

Different superscript between MSG and non-MSG groups in each storage periods indicate significantly different values (P < 0.05).

Discussion

In this study, we significantly improved the birth rate from embryos fertilized with FD sperm preserved at room temperature by adding MSG to the medium. This method obtained reproducible results in various mouse strains and can serve to maintain a vast number of strains of genetically modified mice and become an alternative to the conventional method using liquid nitrogen.

Sperm heads and tails are connected by a strong sperm head-tail coupling apparatus (HTCA) composed of multiple proteins⁴². MSG treatment significantly reduced the tailless rate of FD sperm, probably because the HTCA was protected by the stabilizing effect of MSG on the protein structure. However, it is well known that oxidative stress is correlated with DNA damage⁴³. MSG is involved in antioxidant glutathione production⁴⁴; therefore, it cannot be denied that it may have functioned as an indirect antioxidant, resulting in a concentration-dependent decrease in DNA damage (Fig. 2). Furthermore, although the free water in the sperm is completely removed during drying, the water-holding capacity of MSG⁴⁰ may allowed to maintain some bound water on the sperm, protecting its DNA structure. In fact, the comet and ACS assays showed decreased DNA damage with MSG treatment.

However, the damage to the cell membrane of FD sperm observed here contradicts previous experiments. In a previous study, electron microscopic observations showed strong damage to the surface of FD sperm⁵. In this study, however; over half of both FD sperm and fresh sperm showed PNA-negative (Fig. 2e, S1b), which suggest that the cell membrane of FD sperm does not appear to get damaged by FD, or if so, the damage is not so bad as to expose the inside of the sperm. In contrast, adding MSG during FD treatment increased the percentage of PNA-positive sperm in a concentration-dependent manner. At 10% MSG, the acrosome detected FD sperm was more than in fresh sperm whose cell membrane was permeabilized artificially by the Tx treatment.

Nevertheless, when those sperm were injected into oocytes, most oocytes were activated, indicating that the oocyte activation factors in the sperm head are not damaged by MSG. These results suggest that adding MSG to the medium protects the sperm inner structure, including the DNA or the acrosome, but damages the sperm cell membrane.

However, DNA damage decreased and birth rates improved with MSG concentrations up to 3% but worsened at concentrations > 5%. The sperm membrane also showed a concentration-dependent damage increase from MSG, suggesting that MSG may worsen FD sperm quality. In fact, > 5% MSG applied to fresh sperm made them lose their motility, this motility was not fully recovered even after washing. As high concentrations of MSG may substantially increase the osmolarity, it cannot be ruled out that high osmotic pressure had a negative effect rather than MSG itself. The addition of 3% MSG to the drying medium may have yielded the highest birth rate because it balanced the reduction of morphological and minor DNA damage caused by MSG and the increase in the cell membrane and severe DNA damage caused by high concentrations of MSG.

Herein, we demonstrated that adding MSG to the drying medium successfully improved the birth rate from FD sperm, to levels comparable to fresh sperm (46–60%) or freeze-thaw sperm (23–48%) in previous studies^30,45,46. However, in this study, the birth rate from FD sperm stored for 3 months was lower than that stored for 1 month. If the addition of MSG caused the birth rate to decrease, this method would not be suitable for long-term storage at RT. However, as we already reported that storing FD sperm in a desk drawer at room temperature for up to 6 years does not decrease the birth rate²¹. Furthermore, it has been found that even when using sperm from the same male to prepare FD sperm ampoules simultaneously, there are significant differences in the birth rate between ampoules³². Thus, further detailed verification of the protective effect of MSG is necessary, but it is likely that the decrease in birth rate is due to differences between ampoules rather than the storage period. Whether permanent preservation is possible remains unclear, but it appears possible to reliably preserve FD sperm with a high birth rate for a sufficient time. Although FD of oocytes is not yet possible, healthy offspring have been produced from embryos fertilized with FD round spermatid⁴⁷ or cloned embryos with FD somatic cells⁴⁸indicating that FD can be applied not only to sperm but also to other cells. If MSG had a protective effect on oocytes as well, it may allow producing offspring from FD oocytes.

Currently, researchers are paying high costs to maintain a growing number of genetically modified mice strains being produced around the world. However, using this method not only eliminates the cost of maintaining mouse strains and makes it easy to store them anywhere, it also eliminates the fear of losing all strains due to the inability to replenish liquid nitrogen in the event of a disaster²⁷. As FD sperm is feasible in other species²⁶it could be used to safely and inexpensively preserve the genetic resources of endangered species and original livestock species in developing countries³. When humans expand into space in the future, they will take their livestock and pets with them to other planets. FD sperm can be transported in large numbers in a spacecraft, thus avoiding the inbreeding of livestock and pets at the destination²⁰. In addition, if the Earth’s genetic resources were stored under the moon in FD, it would be possible to revive all species in the event of any major disaster on Earth⁴⁹. The preservation of genetic resources by FD technology could contribute greatly to the future of humankind.

Materials and methods

Animals

ICR and B6D2F1 (BDF1) female and male mice and C57BL/6 N (B6N) male mice (8–10 weeks of age) were obtained from SLC Inc (Hamamatsu, Japan). Surrogate pseudopregnant ICR females, used as embryo recipients, were mated with vasectomized ICR males with proven sterility. The GFP transgenic mice carrying the acrosin/eGFP (Acr3-EGFP) transgenes⁵⁰ was kindly provided by Dr. M. Okabe. On the day of the experiment or after all experiments were completed, the mice were euthanized by CO₂ inhalation or cervical dislocation and used for further experiments. All animal experiments were conducted according to 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: A4-10) following ARRIVE guidelines. All mice were maintained under SPF conditions, with controlled temperature (25 °C), relative humidity (50%), and photoperiod (14 L-10D). They were fed a commercial diet and provided distilled water ad libitum.

Media

HTF medium⁵¹ was used for capacitation and FD of spermatozoa. HEPES-CZB medium⁵² and CZB medium⁵³ were used for oocyte/embryo manipulation and for incubation in 5% CO₂ at 37 °C, respectively.

Preparation of FD spermatozoa

Both epididymides were collected from male mice (ICR, BDF1, and B6N) and the ducts severed with sharp scissors. A few drops of dense spermatozoa mass were then placed into a centrifuge tube containing 1 mL HTF medium and incubated for 60 min at 37 °C in 5% CO₂. Spermatozoa concentration and motility were determined, and 800 µL supernatant was collected. Then, the sperm suspension was centrifuged at 2300 g for 10 min before removing the supernatant and replacing it with HTF medium containing 1% (w/v), 3% (w/v), 5% (w/v), and 10% (w/v) MSG. The 50 µL aliquots of the spermatozoa suspension were then dispensed into glass ampoules and connected to an FDU-2200 freeze dryer (EYELA, Tokyo, Japan) for vacuum drying. After freezing in liquid nitrogen for 1 min, the cork of the freeze dryer was open for ≥ 6 h until all samples were completely dry. After drying, the ampoules were sealed by melting the ampoule necks using a gas burner under vacuum, as previously described²⁹. All ampoules were then stored at room temperature until further use (Fig. 2a).

Detection of trapped air in ampoules using the Tesla coil leak detector

Ampoules containing air were identified using a Tesla coil leak detector (Sanko Electronic Laboratory, Kanagawa, Japan) according to manufacturer’s instructions. When the tip of the Tesla coil comes near the ampoule, the tip will spark around the glass. If a lot of air is trapped inside the ampoule, the air cannot be ionized. However, if the ampoule contains only a small amount of residual air, its ionization produces a spark inside the ampoule (Fig. 2b). Only Tesla-positive ampoules indicating highly vacuumed ampoules were used for all experiments³².

Measurement of sperm head and tail separation rate

The sperm suspension was replaced with MSG-HTF as for the FD sperm preparation described above, and 50 µL was dispensed into plastic tubes. The plastic tubes were then frozen in liquid nitrogen for 1 min and incubated at room temperature until completely thawed. The freeze-thawed sperm suspension was diluted 3–5 times with distilled water, and 150–300 sperm were counted in the frame using a blood cell calculator. The sperm head-tail separation rate corresponded to the amount of sperm heads divided by the total sperm count. For FD sperm, water was added within 3 days after FD to avoid the effects of the storage period and measured as described above.

Observation of the acrosome

We optimized the methods based on previous study⁵⁴. After water addition, ICR FD sperm were centrifuged at 800 g for 5 min. For Tx (+) treatment, the sperm supernatant was replaced by 0.1% Triton X-100 and incubated for 15 min at room temperature. For Tx (−) treatment, this treatment was skipped. After centrifugation, they were then replaced with lectin PNA and Alexa Flur 568 conjugate (1:500, 2 µg/mL, Invitrogen, MA, USA) and incubated for 30 min at room temperature. After centrifugation, the fluid was replaced by a 4′,6-diamidino-2-phenylindole (DAPI) solution (1:1000), the samples were mounted on a slide and observed under a confocal microscope (FV1200, Olympus, Tokyo, Japan). When fresh sperm were used, they were fixed in paraformaldehyde (PFA) for 15 min on ice and washed with chilled PBS. They were then treated in the same way as FD sperm.

Analysis and scoring of comet slides

Spermatozoa DNA damage, potentially caused by single- and double-stranded breaks⁵⁵was measured using the CometAssay^® Kit (Trevigen, MD, USA), according to manufacturer’s instructions. In brief, spermatozoa specimens were collected from ampoules immediately after opening and rehydrated in water. Both the specimen and its counterpart were mounted on the same slide, and 100–300 spermatozoa heads on each slide were analyzed by electrophoresis. To standardize the results across conditions under, the length of each DNA comet tail was divided by the mean length of the one-side results in each experiment. In this comet assay, fresh and freeze-thaw spermatozoa were unsuitable as controls because their preparation techniques differ. This discrepancy would have hindered an accurate comparison between specimens on the same slide.

Oocyte preparation

Female ICR or BDF1 mice were superovulated by injection of 7.5 IU equine chorionic gonadotropin, followed by7.5 IU human chorionic gonadotropin after 48 h. Cumulus-oocyte complexes (COCs) were collected from the oviducts of females after 14–16 h and moved to a Falcon dish containing HEPES-CZB media. To disperse the cumulus, COCs were transferred into a 100 µL droplet of HEPES-CZB medium containing 0.1% bovine testicular hyaluronidase for 3 min. Cumulus-free oocytes were then washed twice and transferred to a 20 µL droplet of CZB for culture.

ICSI and embryo transfer

ICSI was performed as previously described⁵². Just before starting the ICSI, the ampoule neck was broken and 50 µL of sterile distilled water was added and mixed with a pipette. For ICSI, 1–2 µL of the sperm suspension was transferred directly to the injection chamber. The sperm suspension was replaced every 30 min during ICSI. The application of several piezo pulses separated the sperm head from the tail, and the head was then injected into ICR or BDF1 oocytes. The oocytes that survived ICSI were incubated in CZB medium at 37 °C with 5% CO₂. Pronuclear formation was verified 6 h after ICSI. Next, embryos at the 2-cell stage were transferred into day 0.5 pseudopregnant mice mated with a vasectomized male the night before transfer. At that point, 5–12 embryos were transferred into each oviduct. On day 18.5 of gestation, the offspring were delivered by cesarean section and allowed to mature. The remaining unused embryos were cultured for up to 4 days to evaluate their potential for developing into blastocysts.

Toxicity assessment of MSG

The effect of MSG on oocytes was observed by its injection into oocytes before activation for parthenogenesis. As previously reported, 3–5 pL of 0%, 3%, and 10% MSG-HTF were injected into fresh oocytes using piezo-driven micropipettes (Prime Tech, Ibaraki, Japan)⁵⁶. Briefly, microinjection was performed in HEPES-buffered CZB on an inverted microscope (Olympus) with a micromanipulator (Narishige, Tokyo, Japan). The zona pellucida and cell membrane were penetrated with a piezo drive. After MSG-HTF was injected into the oocytes, they were diploidized by incubation for 5–6.5 h in activated medium with 5 µg/mL cytochalasin B⁵⁷. After washing three times in CZB medium, embryos were incubated in CZB medium for 4 days to evaluate the developmental potential of the blastocyst.

The impact of MSG on sperm was assessed by examining motility and embryo development following ICSI. Sperm was precultured for 1 h in HTF medium before replacing it with 0%, 3%, or 10% MSG-HTF incubated for 1 h. For the sperm motility assay, sperm were washed three times in HTF medium and motility visually evaluated under an inverted microscope. For the developmental potential assay, ICSI and embryo culture were performed using the method described above.

ACS detection

The day after ICSI, two-cell stage embryos were fixed and permeabilized with 4% PFA and 0.5% Tx for 15 min. These embryos were observed using fluorescence microscopy (BZ-X710, KEYENCE, Osaka, Japan) in DAPI and 1% BSA containing PBS. ACS was categorized into four groups: “light,” “moderate,” “heavy,” and “lethal,” as described previously¹⁹. Light ACS was considered when a single micronucleus was detected. Moderate ACS was considered when two small, 1–2 medium, or one large micronucleus was detected. Heavy ACS was considered when three small, medium, or 2–3 large micronuclei were detected. Lethal ACS was considered when embryos had multiple micronuclei. Importantly, when both conditions co-occurred, the evaluation was more severe. For example, a moderate micronucleus and two small micronuclei were detected in the embryo and classified as “heavy.”

Immunostaining

We modified previous methods⁵⁸and performed immunofluorescence staining on blastocysts. After imaging with BZ-X710 (KEYENCE, Osaka, Japan), we counted the total number of nuclei, as well as the number of TE cells and ICM blastomeres. At 96 h after ICSI, blastocysts were fixed in 4% PFA with 0.2% Tx for 15 min at room temperature. Fixed embryos were washed three times in PBS containing 1% (w/v) BSA (BSA-PBS) for 15 min. The primary antibodies used were an anti-CDX2 mouse monoclonal antibody (1:500, BioGenex, CA, USA) to detect TE cells and an anti-Nanog rabbit polyclonal antibody (1:500, abcam, Cambridge, UK) to detect ICM cells. The secondary antibodies used were an Alexa Fluor^® 488-labeled goat anti-mouse IgG (1:500, invitrogen, MA, USA) and an Alexa Fluor^® 568-labeled goat anti-rabbit IgG (1:500 invitrogen, MA, USA). DNA was stained with DAPI. The total cell number was counted as DAPI-positive cells.

Statistical analysis and reproducibility

All experiments were repeated at least thrice, and similar results were obtained irrespective of the experimentalists. Results of the comet assay were analyzed using the Bonferroni-Dunn test. Blastocyst cell counts were analyzed using the Tukey-Kramer’s HSD test. Sperm head-tail separation rate, lectin-positive rate, blastocyst rate, and birth rates were evaluated using the Tukey’s WSD test or chi-square test. Statistical significance of differences between variables was determined at P of < 0.05.

Supplementary Information

Below is the link to the electronic supplementary material.

Author contributions

N.U., and T.W. conceived and designed the study. N.U., M.M., D.I., S.W., and T.W. performed experiments, analyzed the data, and interpreted the results. N.U. and T.W. wrote the manuscript. All authors read and edited the manuscript.

Data availability

All data generated or analyzed during this study are included in this published article.

Declarations

Competing interests

The authors declare no competing interests.