A simplified protocol for vitrification of hamster embryos

Abstract

Golden hamsters (Mesocricetus auratus) have been extensively used in biomedical research. With the advent of genome-editing technology, it is now possible to generate gene-knockout hamsters, providing unique research models that cannot be achieved with mice or rats. Therefore, the development of cryopreservation techniques for hamster embryos is in high demand. In this study, we present a simplified vitrification protocol for hamster embryo preservation. In vivo-derived 8-cell or morula embryos (Day 3) were vitrified using Cryotop in modified HECM-3 medium containing ethylene glycol, DMSO, and sucrose. After warming, the embryos were transferred into the uteri of Day 3-pregnant females with a different coat color. The results showed that 21–26% of the transferred embryos developed to the term. The experiments were conducted in a conventional laboratory setting, avoiding direct light exposure. Given the reproducibility of our vitrification protocol, it has broad applicability in laboratories that use hamsters.

The golden or Syrian hamster (Mesocricetus auratus) is one of the most widely used rodent species that provides experimental models for oncology, immunology, physiology, microbiology, and reproductive biology [1]. It belongs to the Cricetidae family of rodents, which are taxonomically distinct from laboratory Muridae rodents such as mice and rats. Hamsters possess several advantages in reproductive physiology, including a consistent 4-day estrous cycle, high responsiveness to conventional superovulation regimens with eCG and hCG, the shortest gestation period (16 days) among eutherian mammals, and a large litter size of approximately 10 pups per litter [2]. These animals can be maintained in conventional animal rooms as easily as mice and rats and exhibit high reproductive performance under long-light photoperiods [2]. Owing to these advantages, hamsters have significantly contributed to studies on developmental and reproductive biology, particularly in elucidating the mechanisms underlying fertilization. Notably, successful in vitro fertilization (IVF) using epididymal spermatozoa and intracytoplasmic sperm injection (ICSI) was first established in hamsters [3, 4]. However, the strong developmental block in vitro of hamster embryos impedes the production of offspring from in vitro-manipulated embryos. Hamster embryos are highly sensitive to various environmental factors, including visible light, temperature fluctuations, pH changes, CO₂ concentration variations, and glucose and phosphate levels in the culture media [5,6,7]. These technical challenges in manipulating hamster embryos in vitro have hindered the generation of genetically modified hamsters.

In 2014, researchers demonstrated the feasibility of generating gene knockout (KO) hamsters using genome editing. However, the technique remained impractical due to the requirement for highly skilled embryo handling and a dark room to prevent developmental blocks in hamster embryos [8, 9]. To address the challenges associated with hamster embryo culture, we recently implemented an intraoviductal transfection technique [10]. The protocol, named i-GONAD, involves injecting guide RNA (gRNA) and Cas9 protein into zygote-bearing oviducts and applying electric pulses to them [11]. This approach eliminates the need for in vitro embryo handling procedures, including collection, injection, culture, and transfer. Using the hamster i-GONAD method, we successfully produced KO hamster strains deficient in acrosin [12], Mov10l1 [13], Ovgp1 [14], Irs2 [15], and AANOT [16]. These KO mutant hamsters exhibited gene functions that could not be achieved in mice or provided disease models that differed from their mouse counterparts. The number of KO hamster strains has rapidly increased. Consequently, to conserve space, reduce costs, and minimize the effort required to maintain these valuable hamster strains, it is imperative to cryopreserve embryos safely and efficiently.

According to a previous report, the conventional slow-freezing method is not suitable for cryopreserving hamster embryos because of their low viability after freezing and thawing [17]. To explore the possibility of cryopreserving hamster embryos through vitrification, we investigated their sensitivities to different cryoprotectants. Despite optimizing the vitrification procedure, obtaining hamster pups after vitrification remained challenging [18]. Lane et al. addressed the low survival rate of hamster embryos after freeze–thaw cycles by using cryoloops instead of conventional plastic straws, successfully developing a protocol for hamster embryo vitrification [19]. However, this method has not gained widespread adoption because it is specifically optimized for vitrifying 1-cell or 2-cell hamster embryos, which are the most sensitive to in vitro handling. Fan et al. examined different vitrification conditions, considering factors such as embryonic stage, cryoprotectant concentration, and treatment duration for both vitrification and warming [20]. They achieved a 29% birth rate of healthy pups from vitrified 2-cell embryos following transfer into pregnant females with different coat colors.

Therefore, we examined whether the method described by Fan et al. could be applied to the vitrification of 8-cell hamster embryos, which are less sensitive to in vitro handling than 2-cell embryos. Initially, when we performed 8-cell embryo vitrification/warming and transferred the embryos to recipient females in our conventional embryo manipulation room, no pups were obtained (0/92 embryos transferred). However, when we shielded the embryo manipulation bench from direct ceiling light (Fig. 1A), we achieved vitrification-derived litter (13% [4/30] embryos transferred). This indicates that simple shading of light is sufficient, and a specialized darkroom is not necessary for at least 8-cell embryo vitrification. We placed red or orange filter glass in the optical path of the stereomicroscope, following a previous hamster ICSI study [21] (Fig. 1B). Although the method proposed by Fan et al. could be applied to 8-cell hamster embryos, we observed that a proportion of embryos (33%, 10/30) were damaged after vitrification/warming, exhibiting blastomere lysis or zona pellucida removal. This damage may be due to insufficient equilibration with the highly viscous vitrification solution containing a high concentration of Ficoll (300 mg/ml), compared to the 10 mg/ml concentration used by Lane et al. [19]. This high Ficoll concentration presents challenges in handling embryos during the vitrification and warming processes.

Fig. 1.

Simplified vitrification protocol for hamster embryos. (A) Direct light from the ceiling was prevented by a black plastic sheet, which enabled us to handle hamster embryos in a conventional experimental room. (B) A red or orange filter glass was placed in the optical path of the stereomicroscope to minimize damage to the hamster embryos. (C) Procedures for vitrification and warming of hamster Day-3 embryos (8-cells). Cryotop was used as a container for the vitrified embryos.

To simplify the vitrification medium for a more practical and easier experimental setting, we omitted Ficoll from the vitrification solution. This modification aimed to reduce embryo damage by increasing the penetration of the vitrification solution and facilitating embryo manipulation during the vitrification/warming procedures. We used a simple, modified HECM-3 as the base medium instead of HECM-9 (containing 11 amino acids) (Formula A in Table 1), based on our previous results using HECM-3 for hamster embryo transfer experiments [22]. The entire vitrification and warming procedure is illustrated in Fig. 1C. As expected, the rate of damaged embryos after warming decreased significantly to 1.5% (2/134), likely due to the removal of Ficoll. Following the transfer of embryos into the uteri of recipient females, 21% (12/56) reached term (Table 2, Supplementary Fig. 1A). Additionally, we observed that another modified HECM-3 (Formula B), containing pantothenate (which enhances in vitro development of hamster embryos into blastocysts [23]) and non-essential amino acids (100×), resulted in a comparable birth rate of 26% (20/78) (Tables 1 and 2, Supplementary Fig. 1B).

Table 1. Modified HECM-3 used in this study.

	Formula A (mM)	Formula B (mM)	(HECM-9 **)
Polyvinyl alcohol	0.1 mg/ml	0.1 mg/ml	0.1 mg/ml
NaCl	110.6	113.8	113.6
KCl	3.2	3.0	3.0
MgCl2,6H2O	0.5	0.5	0.5
NaHCO3	15	25	25
CaCl2,2H2O *	2	2	1.9
Hepes	10	10
Glycine-HCl	2.2
1M HCl		4.5 µl/ml	1.4 µl/ml
Hypotaurine	1
Taurine		5	0.5
Glutamine *	0.2	0.2	0.2
Ca Pantothenate		0.003	0.003
Na lactate (60% syrup)	64 µl/ml	64 µl/ml	4.5
MEM non-essential amino acid		×100
Asparagine			0.01
Cysteine			0.01
Histidine			0.01
Lysine			0.01
Proline			0.01
Serine			0.01
Aspartic acid			0.01
Glycine			0.01
Glutamic acid			0.01
Bovine serum albumin	2 mg/ml	2 mg/ml

* CaCl₂,2H₂O and glutamine were added on the day of experiment. ** McKiernan and Bavister [23].

Table 2. Results of transfer of vitrified/warmed hamster embryos into pregnant recipients.

Protocol	No. of embryos transferred per recipient	No. (%) of embryos that developed to term	No. of pups/embryos from recipient mother
Formula A	9	1 (11)	7
	10	1 (10)	1
	9	0 (0)	7
	9	6 (67)	9
	10	1 (10)	10
	9	3 (33)	13
Total	56	12 (21)	47
Formula B	14	5 (36) *	NR
	5	2 (40)	4
	9	2 (22)	2
	18	3 (17)	2
	16	4 (25)	5
	16	4 (25)	5
Total	78	20 (26)

Live pups were retrieved by caesarian section except for one case (* natural delivery).

In this study, we simplified the vitrification protocol for hamster embryos to enable the safe cryopreservation of hamster strains. The vitrification solution consists of simple HECM-3 as the base medium and does not contain Ficoll, making it easy to prepare and manage. Furthermore, vitrification/warming procedures can be performed in a conventional laboratory room by simply avoiding direct light, as 8-cell embryos are less sensitive to visible light than early-stage embryos. Our vitrification protocol can be widely used in laboratories working with genetically modified hamsters for embryo preservation.

Materials and Methods

Animals

Wild-type golden hamsters (Syrian) were purchased from Japan SLC, Inc. (Hamamatsu, Japan). For embryo transfer experiments, we used pregnant females as recipients and prepared hamsters of different colors to identify pups derived from the transferred embryos. We generated white (albino) and black hamsters by deleting the tyrosinase (Tyr) and agouti protein (Asip) genes, respectively (Supplementary Fig. 2). Albino hamsters demonstrated easier handling and higher reproductive performance than black hamsters; therefore, they were used in most experiments. In some experiments, we used a natural mutant strain with a white coat and black ear tips (Supplementary Fig. 1B). All hamsters were housed under controlled lighting conditions (daily light period, 0500–1900) and provided water and food ad libitum. The cells were maintained under specific pathogen-free conditions. All animal experiments were approved by the Animal Experimentation Committee of the RIKEN Tsukuba Institute and were performed in accordance with the committee’s guiding principles (T2024-EP004). A total of 45 female hamsters were included in this study. Each experimental group (corresponding to one of the three vitrification protocols) comprised 12–20 animals. The number of embryos obtained from each female was highly variable, ranging from 5–40. Additionally, 20 male hamsters were mated with females. The sample size was determined based on a previous study [20] to minimize the number of animals used while maintaining statistical significance.

Embryo collection

Female hamsters were mated with males on the evening of Day 4 (Day 1 was the day of estrus vaginal discharge) and euthanized on the afternoon of Day 3. Eight-cell-stage embryos were collected by flushing the oviducts and uterus with modified HECM-3. The embryos were then cultured in modified HECM-3 at 37.5°C under 6% CO₂ in air until vitrification (less than 15 min). All embryo handling procedures were performed using a black plastic sheet to prevent exposure of the hamster embryos to direct ceiling light (Fig. 1A). The embryos were observed under a stereomicroscope equipped with an orange glass filter (Fig. 1B).

Vitrification of embryos

As a preliminary experiment, we vitrified hamster embryos following the method developed by Fan et al. [20], with the exception of using 8-cell embryos instead of 2-cell embryos in the embryo transfer experiments (Fig. 1C). Vitrification experiments were performed according to our original protocol (Fig. 1C). Approximately 10 embryos were transferred to 50 μl of equilibrium solution (Solution I) containing 7.5% ethylene glycol (EG) and 7.5% dimethyl sulfoxide (DMSO) in modified HECM-3 at room temperature. After 2 min, the embryos were transferred to 100 μl of vitrification solution at room temperature (Solution II, comprising 15% EG, 15% DMSO, and 0.5 M sucrose in modified HECM-3). After 15–30 sec, the embryos were placed on a Cryotop (Kitazato Co., Fuji, Japan) and immersed directly in liquid nitrogen (LN₂). On the day of embryo transfer, the Cryotop was removed from LN₂ and immediately immersed in 0.8 M sucrose-modified HECM-3 at 37.5°C. After < 1 min, the embryos were transferred to 0.5 M sucrose-modified HECM-3 at room temperature. Following a 3-min incubation, the embryos were transferred to 0.25 M sucrose-modified HECM-3 and kept for 5 min at room temperature. Subsequently, embryos were placed in modified HECM for 5 min and incubated at 37.5°C under 6% CO₂ in air until embryo transfer (less than 10 min). In some experiments, modified HECM-3 containing pantothenate (3 μM) and MEM non-essential amino acid solution (100×; Thermo Fisher Scientific, Waltham, MA, USA) was used as the base medium [24]. Both modified HECMs had a pH of 7.2–7.3 inside the CO₂ incubator.

Embryo transfer

Vitrified-warmed embryos were transferred to the uteri of Day 3 pregnant females under xylazine and ketamine anesthesia. Recipient females that became pregnant were either euthanized on Day 15 (one day before delivery) or allowed to deliver pups. The fetuses/pups derived from donor embryos were distinguished by their coat color, which differed from that of the recipient females.

Conflict of interests

The authors declare no conflict of interest.