Introduction
Assisted reproductive technologies (ARTs) such as in vitro fertilization (IVF) and cryopreservation have become important techniques for the efficient maintenance of animals and production of novel genetically modified animals (Thornton et al., Reference Thornton, Brown and Glenister1999). According to the 3R Principles of animal welfare, successful IVF conditions resulting in normal fertilization and subsequent development to term lead to reduction of animal usage in animal-based investigations. However, IVF conditions in previous studies were not necessarily satisfactory for all inbred mice, including the 129, BALB/c and C3H strains (Byers et al., Reference Byers, Payson and Taft2006; Vasudevan and Sztein, Reference Vasudevan and Sztein2012). We previously showed that using medium with a high calcium concentration (5.13 mM) during sperm:ova co-incubation resulted in successful fertilization in BALB/cA mice (Kito and Ohta, Reference Kito and Ohta2008) but not in C3H/He mice (more specifically C3H/HeNrs) in which such medium induced disturbed PBII extrusion in 30–50% of ova (Ohta et al., Reference Ohta, Nagao, Minami, Tsukamoto and Kito2016). Although digynic triploid embryos developed to blastocysts that were indistinguishable from normal diploid embryos, they never developed into newborns. Therefore, conditions leading to abnormal fertilization should be avoided for efficient use of animals and gametes.
C3H/He inbred mice are used as a general-purpose strain in several biomedical research areas. In addition, C3H/He mice have various substrains (Whitmore and Whitmore, Reference Whitmore and Whitmore1985). Therefore, it is important to check the phenotype of C3H/He substrains in ART such as IVF. To examine the substrain specificity of disturbed PBII extrusion induced by high calcium concentrations, we compared the disturbance frequencies under 1.71–6.84 mM calcium concentrations among six C3H/He mouse substrains commercially available in Japan.
Materials and Methods
The six substrains examined were C3H/HeJJcl, C3H/HeNJcl (CLEA Japan, Tokyo, Japan), C3H/HeSlc, C3H/HeJYokSlc, C3H/HeNSlc (Japan SLC, Shizuoka, Japan) and C3H/HeNCrlCrlj (Oriental Yeast, Tokyo, Japan). Mice were maintained in a conventional facility at 23 ± 2°C and 50 ± 10% humidity under a light regimen of 12L:12D conditions (lights on at 07:00 am). All animals were housed and maintained as previously described (Ohta et al., Reference Ohta, Nagao, Minami, Tsukamoto and Kito2016).
Thirty 10–12-week-old females and four to six 12–16-week-old males with >60% sperm motility were used for each substrain. Detailed procedures of IVF using cumulus-free ova were the same as those described previously (Kito and Ohta, Reference Kito and Ohta2008; Ohta et al., Reference Ohta, Nagao, Minami, Tsukamoto and Kito2016). To control variability among females, equal numbers of ova (10–20 ova per drop) from individual females were distributed in human tubal fluid with 1.71, 2.57, 3.42, 5.14 or 6.84 mM calcium. At 5 h post insemination, fixed ova were examined for PBII extrusion and formation of male and female pronuclei (PNs). Fertilized ova were those that had incorporated sperm nuclei or male PN and ova with disturbed PBII were those with two female pronuclei instead of extruded PBII (Ohta et al., Reference Ohta, Nagao, Minami, Tsukamoto and Kito2016).
For each substrain, percentage data from four to six replicates, in which one male was used in each replicate, were transformed using arcsin transformation to analyse variance with a random block design, with each male assigned as a block (Ohta et al., Reference Ohta, Nagao, Minami, Tsukamoto and Kito2016; Kito and Ohta, Reference Kito and Ohta2008). Multiple comparisons within each substrain were made using Tukey’s test at a probability of P < 0.05.
Results and Discussion
Fertilization was not affected by calcium concentration because more than 80% of the ova from all six substrains were fertilized at the various calcium concentrations. As shown in Fig. 1, only the two J substrains showed no significant increase in disturbed PBII extrusion at the various calcium concentrations (<10% of fertilized ova, P > 0.05). Ova of C3H/HeNJcl and C3H/HeSlc were extremely sensitive because more than 40% of fertilized ova were abnormal at calcium concentrations of 5.13 and 6.84 mM. We concluded that this phenomenon is specific for all the C3H/He substrains except for the J substrains (Fig. 1).
Percentages of ova with disturbed PBII extrusion out of fertilized ova in six C3H/He substrains. Data for C3H/HeNrs were extracted from table 1 in the publication by Ohta et al. (Reference Ohta, Nagao, Minami, Tsukamoto and Kito2016) with permission. n: Total number of fertilized ova examined. a–cPercentages with different superscripts within each substrain are significantly different (P < 0.05).
C3H/HeJ substrains are known to have a point mutation in Toll-like receptor 4 (Tlr4) alleles that causes cells with Tlr4 to become unresponsive to endotoxic lipopolysaccharide (Festing and Blackmore, Reference Festing and Blackmore1971; Poltorak et al., Reference Poltorak, He, Smirnova, Liu, Van Huffel, Du, Birdwell, Alejos, Silva, Galanos, Freudenberg, Ricciardi-Castagnoli, Layton and Beutler1998). However, there is no report of detailed genetic analysis for genes other than Tlr4 in the C3H/He substrains. Genetic differences have been investigated in detail among the C57BL/6 substrains, including C57BL/6J and C57BL/6N (Simon et al., Reference Simon, Greenaway, White, Fuchs, Gailus-Durner, Wells, Sorg, Wong, Bedu and Cartwright2013). In addition, phenotypic differences have been reported among the C57BL/6 substrains, especially in sperm freezing and in vitro fertilization (Liu et al., Reference Liu, Nutter, Law and McKerlie2009). Therefore, this study was performed to evaluate other phenotypic differences between C3H/He substrains.
In mouse ARTs, optimization of IVF conditions to produce normally fertilized ova with high consistency and efficiency allows for estimation of the number of animals required and the concomitant reduction in the number of animals used for zygote or embryo manipulation. Our study indicated that conditions such as high calcium levels during sperm:ova co-incubation are not recommended for IVF in C3H/He mice except for the J substrains. In the future, detailed genetic analysis and genotyping of single nucleotide polymorphisms (SNPs) to demonstrate genetic differences in C3H/He substrains need to be performed.
Acknowledgements
We thank Y. Kaneko, H Yano and M. Hayashi for taking care of animals. We are also grateful to C. Steger and D. Cookfair for critically reviewing this manuscript.
Financial support
This research received no specific grant from any funding agency, commercial or not-for-profit sectors.
Conflicts of interest
None.
Ethical standards
All animals were handled and treated according to the Recommendations for Handling of Laboratory Animals for Biomedical Research, compiled by the Institutional Animal Care and Use Committee for Laboratory Animal Experiments of the National Institute of Radiological Sciences.
