Short-Term Storage and Transport at Cold Temperatures of 2-Cell Mouse Embryos Produced by Cryopreserved Sperm

Abstract

At refrigerated temperatures, mouse embryos can maintain developmental ability for short periods. Previously, we succeeded in transporting vitrified and warmed 2-cell mouse embryos while maintaining developmental ability at refrigerated temperatures for 50 h. Transport of nonfrozen embryos is an easier and more useful means of exchanging genetically engineered mice between laboratories than is transport of cryopreserved embryos. Here we examined the developmental ability of transported 2-cell embryos that were produced through in vitro fertilization using cryopreserved sperm. Results show that 2-cell embryos produced by cryopreserved sperm can develop into blastocysts after cold storage for 24, 48, and 72 h. Transported 2-cell embryos produced by cryopreserved sperm yielded a favorable number of pups in all of the receiving laboratories after transport lasting 48 to 52 h. In summary, cold storage and transport of 2-cell embryos derived from cryopreserved sperm at refrigerated temperatures provides a novel means of transporting genetically engineered mice as an alternative to the transport of cryopreserved embryos and sperm.

Recently, mouse resource banks have been established worldwide.² These banks play an important role in archiving and providing genetically engineered mice such as transgenic, knockout, and spontaneous mutant mice for the research community. The banks have efficiently generated numerous cryopreserved embryos and sperm from genetically engineered mice and have supplied these strains to researchers requesting them.⁵

For several reasons, transporting cryopreserved embryos and gametes is more efficient and advantageous than is transporting live animals.^8,10 Transport of live animals carries potential risks of death, escape, and viral or bacterial infection during transport, whereas cryopreserved embryos are not susceptible to these risks and can be easily transported in small containers at low cost. However, the transport of cryopreserved embryos has certain problems. Specifically, the receiving facility needs to have sufficiently mastered cryopreservation skills and must prepare special instruments for various freezing and thawing protocols.⁸ Therefore, not all institutes can receive and exchange cryopreserved embryos.

We previously developed a nonfreezing transport system using cold storage of vitrified and warmed 2-cell mouse embryos at refrigeration temperatures.¹³ In that study, we found that M2 medium excels at maintaining the development ability of 2-cell embryos under refrigerated temperatures compared with PBS or modified Whitten medium. In addition, we demonstrated that embryos transported from our laboratory by using the cold storage system developed well into live young at all receiving laboratories.¹³ The transport system that we developed has several advantages for the receiving laboratories compared with that used for cryopreserved embryos. First, it omits all thawing procedures, which means that facilities can easily receive and quickly use the embryos after arrival. Second, it allows the use of a simple container made of Styrofoam, rather than a special container for maintaining freezing temperatures during transport (a so-called ’dry-shipper) for embryo transport. Therefore, receiving laboratories need not return the container to the sender after receiving the embryos, thereby reducing shipping costs.

Recently, cryopreservation of sperm has been used to archive strains of genetically engineered mice based on several advantages.³ First, sperm cryopreservation can be performed via a simple freezing protocol. In addition, a large number of sperm can be retrieved from a single male animal, allowing researchers to efficiently preserve and maintain genetic resources compared with the cryopreservation of embryos. In our view, these considerations suggest that the transport of embryos produced by cryopreserved sperm will increase in the future.

In the present study, we performed in vitro fertilization by using cryopreserved mouse sperm and examined the developmental ability of the resultant 2-cell embryos after cold storage at refrigerated temperatures. Thereafter, the produced 2-cell embryos were transported from our laboratory to several research institutes in Japan, and their developmental ability after embryo transfer was examined.

Materials and Methods

Animals.

C57BL/6JJcl (C57BL/6) mice were purchased from CLEA Japan (Tokyo, Japan) and used as sperm (age, 12 to 15 wk) and oocyte (age, 8 to 10 wk) donors. Mice used as recipients for the transfer of 2-cell embryos were of the ICR strain (CLEA Japan, Tokyo, Japan) and were 8 to 16 wk old. All animals were kept under a 12:12-h dark:light cycle (lights on, 0700 to 1900) at a constant temperature of 22 ± 1 °C with free access to food and water. All animal experiments were carried out with the approval of The Animal Care and Use Committee of the Kumamoto University School of Medicine.

Media.

For sperm cryopreservation, a modified 18% raffinose pentahydrate and 3% skim milk (Difco, Becton Dickinson, Franklin Lakes, NJ) solution containing 100 mM L-glutamine (mR18S3 medium) was used and prepared according to the previously published method.^3,14

A modified Krebs–Ringer bicarbonate solution containing 0.75 mM methyl-β-cyclodextrin (THY medium with MBCD, product no. C4555, Sigma, St Louis, MO) and 1.0 mg/mL polyvinyl-alcohol (product no. P8136, Sigma) was used for sperm preincubation.^1,12 Human tubal fluid medium was used for in vitro fertilization.⁷ M2 medium (with HEPES but without penicillin and streptomycin; product no. M7167, Sigma) was used for cold storage of the 2-cell embryos.⁶ Modified Whitten medium was used for the culture of 2-cell embryos to the blastocyst stage.¹⁵ All media formulations are given in Table 1.

Table 1.

Composition of media for sperm preincubation, in vitro fertilization, cold storage, and embryo culture

Reagenta	TYH with MBCDb	Human tubal fluid medium	M2 mediumc	modified Whitten medium
NaCl	697.6	593.8	553.2	640.0
KCl	35.6	35.0	35.6	35.6
MgSO4	0	0	16.5	0
MgSO4•7 H2O	29.3	4.9	0	29.4
KH2PO4	16.2	5.4	16.2	16.2
NaHCO3	210.6	210.0	35.0	190.0
Sodium pyruvate	5.5	3.7	3.6	2.5
Glucose (D+)	100.0	50.0	100.0	100.0
CaCl2•2 H2O	25.1	0	25.1	0
CaCl2	0	57.0	0	0
HEPES	0	0	542.7	0
60% Sodium lactate (μL)	0	340	332.0	0
Calcium lactate •5 H2O	0	0	0	46.0
20 mM β-mercaptoethanol (μL)	0	0	0	10
Methyl-β-cyclodextrin	98.3	0	0	0
100 mM disodium EDTA (μL)	0	0	0	50
Penicillin G (potassium salt)	7.5	7.5	0	7.5
Streptomycin sulfate	5.0	5.0	0	5.0
Polyvinylalcohol	100.0	0	0	0
Bovine serum albumin	0	400.0	400.0	300.0
0.5% phenol red (μL)	0	40	0	200
Phenol red	0	0	1.1	0

^aIn mg/100 mL unless otherwise stated.

^bA modified Krebs–Ringer bicarbonate solution containing methyl-β-cyclodextrin.

^cPurchased from Sigma (product no. M7167).

Sperm freezing and thawing.

The cryopreservation of sperm was performed as described previously.^3,14 Aliquot of 60-μL of the mR18S3 was placed on a 35-mm culture dish and covered with paraffin oil. Thereafter, a 60-μL aliquot of the same solution was added to the drop (final volume: 120-μL) to make a tall, semispherical drop. Male mice were euthanized by cervical dislocation, and 2-tailed caudal epididymides removed from each male were transferred in the drop of mR18S3. The tissue was cut in the medium into 5 or 6 pieces by using microspring scissors. The dish was gently shaken every minute to disperse sperm from the tissue at room temperature. After 3 min, the sperm suspension was divided into 10 aliquots of 10-μL each on a culture dish. All 10 specimens from a single male mouse were put into a 0.25-mL plastic straw (IMV, Paris, France), and the straws were heat-sealed. The sealed straws were cooled by putting them into the neck (liquid nitrogen gas layer) of a container for 10 min and then plunged directly into liquid nitrogen for storage. After 5 d, the samples were removed from liquid nitrogen and thawed in a water bath at 37 °C for 10 min.

In vitro fertilization.

The procedures used for preincubation and in vitro fertilization using fresh or frozen/thawed sperm were essentially the same as those described previously.^12,14 Mature female mice were superovulated by using 7.5 IU equine chorionic gonadotropin (ASKA Pharmaceutical, Tokyo, Japan) IP followed by 7.5 IU human chorionic gonadotropin (ASKA Pharmaceutical) IP 48 h later. At 14 to 15 h after the injections, mice were euthanized by cervical dislocation, their oviducts removed, and 4 or 5 cumulus–oocyte complexes were obtained from the ampulla of the fallopian tube. These complexes were introduced into a 90-μL drop of human tubal fluid medium covered with paraffin oil.

A 10-μL aliquot of thawed sperm suspension was added to the center of a 90-μL drop of TYH medium with MBCD covered with paraffin oil. The thawed sperm were preincubated for 30 min at 37 °C with 5% CO₂ in air. After preincubation, a 10-μL aliquot of sperm suspension containing motile sperm was collected from the peripheral part of the drop by using a wedge-shaped pipette tip (0.5 to 10 μL, Quality Scientific Plastics, Petaluma, CA). The sperm suspension was transferred to the drop containing cumulus–oocyte complexes and incubated at 37 °C with 5% CO₂ in air (final motile sperm concentration, 200 to 400 sperm per microliter). After 5 to 6 h, the inseminated oocytes were washed 3 times in 100 μL human tubal fluid medium covered with paraffin oil and then cultured at 37 °C with 5% CO₂ in air. At 24 h after insemination, fertilization rates were calculated as the total number of 2-cell embryos divided by the total number of inseminated oocytes multiplied by 100%.

Cold storage of 2-cell embryos.

Cold storage of 2-cell embryos was performed as described previously.¹³ Briefly, after in vitro fertilization, 2-cell embryos were transferred, washed 3 times in a drop of 100 μL M2 medium, and then placed into 0.5-mL tubes (Fisherbrand Flip-Cap Microtubes 0.5 mL; Fisher Scientific, Pittsburgh, PA) containing 600 μL M2 medium. The tubes were placed in an embryo storage box and stored in a refrigerator at 4.0 ± 1.5 °C for 24, 48, or 72 h. After cold storage, the developmental ability of stored 2-cell embryos was examined through culture in modified Whitten medium.

Embryo culture.

After cold storage in M2 medium for 24, 48, or 72 h, the 2-cell embryos were retrieved and transferred in a 100 μL drop of modified Whitten medium as described earlier and incubated at 37 °C for 72 h with 5% CO₂ in air. The development rates were calculated by using the following equation:

Development rate = (no. of 4-cell embryos, morulae, or blastocysts) / (no. of morphologically normal 2-cell embryos) × 100%.

As a control, nonstored 2-cell embryos were cultured in the same manner.

Transport of 2-cell embryos.

The 2-cell embryos were transported as described.¹³ Briefly, embryo storage boxes containing 2-cell embryos were placed in embryo transport containers and transported from the Center for Animal Resources and Development, Kumamoto University, to several facilities in Japan (Asahikawa Medical College, RIKEN Research Center for Allergy and Immunology, International Medical Center of Japan, Experimental Research Center for Infectious Diseases, Kyoto University, Kinki University, Trans Genic, and Ehime University). The time required for transport did not exceed 52 h. During transport, the temperature in each embryo transport box was measured continuously by using a temperature data logger (Thermochron iButtons; Maxim Integrated Products, Sunnyvale, CA). During transport, the temperature gradually dropped and was maintained at 4.0 ± 1.5 °C.

Embryo transfer.

At the receiving institutions, 2-cell embryos were retrieved from M2 medium, moved into modified Whitten medium, and transferred into the oviducts of pseudopregnant female mice (19 to 21 embryos per female) on the day that a vaginal plug was found (day 1 of pseudopregnancy). After 19 d, we examined the incidence of embryos developing into live young (no. of live young/ no. transferred embryos × 100%). As a control, nontransported 2-cell embryos stored in M2 medium for 48 h were transferred to the oviducts of recipient females at the Center for Animal Resources and Development, Kumamoto University.

Statistical analysis.

Statistical analysis was performed by using Prism version 3.0 (GraphPad, San Diego, CA). All percentage data are given as mean ± 1 SD. Differences between the means for each treatment were compared by using ANOVA after arcsine transformation of the percentage data. Differences between the means were considered to be significant when a P value of less than 0.05 was achieved.

Results

Fertilization rate of fresh and frozen–thawed C57BL/6 mouse sperm.

Fertilization rates of frozen–thawed sperm (mean ± 1 SD, 64.7% ± 11.1%) were slightly lower than those for the fresh control group (79.9% ± 9.9%). However, frozen–thawed sperm showed stability and high rates of fertilization.

Comparison of the developmental abilities of 2-cell embryos derived from fresh and frozen–thawed sperm after cold storage.

After cold storage, 2-cell embryos derived from frozen–thawed sperm developed into blastocysts over 72 h (Table 2). The developmental rates of 2-cell embryos derived from frozen–thawed sperm initially were equivalent to that of fresh sperm but gradually decreased in a time-dependent manner.

Table 2.

Development of 2-cell embryos derived from fresh and frozen–thawed sperm to blastocysts in vitro

Sperm	Storage time (h)	No. of 2-cell embryosa	No. (%)b of 4-cell embryos at 24 h	No. (%)b of morulae at 48 h	No. (%)b of blastocysts at 72 h
Fresh	0	138	134 (97.1 ± 4.3)	130 (94.2 ± 4.5)	128 (92.8 ± 5.23)
	24	121	117 (96.7 ± 3.9)	114 (94.2 ± 1.6)	102 (84.3 ± 2.34)
	48	123	114 (92.7 ± 3.8)	109 (88.6 ± 3.8)	79 (64.2 ± 11.6)c
	72	128	121 (94.5 ± 6.8)	96 (75.0 ± 10.4)c	61 (47.7 ± 10.1)c
Frozen–thawed	0	117	115 (98.3 ± 1.5)	113 (96.6 ± 4.18)	104 (88.9 ± 7.2)
	24	127	124 (97.6 ± 2.1)	119 (93.7 ± 5.33)	107 (84.3 ± 4.8)
	48	126	119 (94.4 ± 4.5)	113 (89.7 ± 2.83)	82 (65.1 ± 16.5)c
	72	127	118 (92.9 ± 6.6)	89 (70.1 ± 5.69)c	56 (44.1 ± 7.7)c

Experiments were repeated 3 times for each group.

^aAll (100%) of embryos examined were viable.

^bPercentage data reflect mean ± 1 SD of 2-cell embryos that developed to the stage described.

^cValue is significantly different (P < 0.05) from that for fresh sperm at 0 h (control).

Developmental abilities of 2-cell embryos derived from frozen–thawed sperm after cold storage and transport to several laboratories.

We transported 2-cell embryos in M2 medium at cold temperatures to several laboratories within 52 h, where the embryos were transferred into pseudopregnant female mice. At all laboratories, live young were obtained from the embryos after transport (Table 3). The incidence of live young after transport of 2-cell embryos was equivalent to that of embryos stored for 48 h before transfer to recipients in our laboratory (control, 44% [106 of 241]; transport, 43% [274 of 557]).

Table 3.

Short-term storage and transport of 2-cell embryos produced by frozen–thawed sperm at cold temperature.

Institution	No. of 2-cell embryos examined	No. of 2-cell embryos viable after transport (%)a,b	No. of live young (%)b
Control	242	241 (99.6 ± 1.4)	106 (44.0 ± 18.8)
A	80	80 (100)	24 (30.0 ± 24.8)
B	80	80 (100)	51 (63.8 ± 21.0)
C	80	77 (96.3 ± 2.5)	41 (53.2 ± 10.0)
D	80	80 (100)	38 (47.5 ± 13.2)
E	80	80 (100)	30 (37.5 ± 22.2)
F	80	80 (100)	33 (41.3 ± 28.7)
G	80	80 (100)	21 (26.3 ± 18.0)
Total (A through G)	560	557 (99.5 ± 1.6)	238 (42.7 ± 22.0)

Duration of transport was 48 h in all cases.

Embryo transfer using the transported embryos was performed in the same manner at the receiving laboratories.

^aAll viable 2-cell embryos were transferred into pseudopregnant mice (n = 12 for control; n = 4 for all other institutions).

^bPercentage data reflect mean ± 1 SD of 2-cell embryos viable after transport and transferred.

Discussion

In the present study, frozen–thawed C57BL/6 mouse sperm show stability and high rates of fertilization and cold-stored 2-cell embryos derived from frozen–thawed sperm had high probability of developing into blastocysts over 72 h. The developmental ability of stored 2-cell embryos produced from fresh or frozen–thawed sperm is equivalent. In addition, the transported 2-cell embryos produced by frozen–thawed sperm using the cold storage and transport system maintain developmental ability and develop into live young at all receiving laboratories for up to 52 h.

In general, frozen–thawed C57BL/6 mouse sperm show extremely low fertility after in vitro fertilization.^4,9,11 Here we used a novel protocol involving methyl-β-cyclodextrin to yield stable and highly fertile (fertilization rate, 64.7%) frozen–thawed C57BL/6 mouse sperm. The rates are nearly equal to those achieved in our previous work (69.2%), thereby enabling us to verify that this protocol for in vitro fertilization using cryopreserved mouse sperm yields a high reproduction probability.¹⁴

Cold storage of 2-cell embryos produced from cryopreserved sperm retained their development potential (development rate, 44.1%) and developed into blastocysts after 72 h in the same manner as those derived from fresh sperm (47.7%). The developmental rate of vitrified–warmed 2-cell embryos into blastocysts after cold storage for 72 h (42.5%) in our previous study¹³ was the same as that in the present study. These figures suggest that 2-cell embryos that have been cryopreserved or derived from fresh or frozen–thawed sperm can be transported by means of our cold storage system at refrigeration temperatures.

In the current study, all receiving facilities succeeded in obtaining live young derived from the cold stored 2-cell embryos after transport. Transporting 2-cell embryos produced by cryopreserved sperm instead of cryopreserved sperm itself offers several advantages to researchers.⁸ Freezing and thawing procedures and their associated equipment are eliminated. Moreover, the cold transport system will provide an efficient means of transporting genetically engineered mice instead of the transport of cryopreserved embryos or gametes.

From a practical point of view, coordination between shipper and recipient is important to efficiently receive 2-cell embryos by using the cold transport system. In preparation for cold transport, the shipper should confirm a transit time of no more than 52 h to the receiving laboratory, with the embryos kept refrigerated. In addition, shippers should convey the arrival date to the recipient. After fixing the schedule, the recipient should prepare pseudopregnant mice in time for the arrival of the embryos. If embryo transfer cannot be performed on the arrival date, the received embryos can be stored in a refrigerator, as long as the total refrigeration time (including transport) does not exceed 52 h.

Many institutions and companies involved in regenerative medicine have begun providing a worldwide transport service for live cells and organs which can be maintained at refrigerated temperatures. This service enables the transport of these samples worldwide within 5 d. Accordingly, when it becomes possible to maintain the developmental ability of 2-cell embryos after cold storage for 5 d, it will be possible to transport the embryos through this network to wherever a researcher is located.

In conclusion, we have demonstrated that 2-cell embryos derived from cryopreserved mouse sperm and stored for 52 h at cold temperature have a high probability of developing into live young at recipient laboratories. This transport system meets a need for many users and promotes efficient distribution of genetically engineered mice to researchers.