Superovulation is a method used to reduce the number of mice used per experiment by increasing the egg number. Conventionally, superovulation for obtaining mouse eggs involves the use of equine chorionic gonadotropin (eCG) for stimulation and human CG for induction. Female mice of the C57BL/6 inbred strain spontaneously ovulate approximately 10 eggs. The average number of eggs ovulated using the conventional superovulation method is approximately twice as high as that obtained by spontaneous ovulation. Here, we describe the conventional and non-conventional methods of intraperitoneal injection of superovulation reagents in mice and subsequent egg collection. The non-conventional superovulation method combining anti-inhibin serum (AIS) plus eCG for stimulation is more efficient than conventional superovulation. Appropriate intervals from each injection to sampling induce large numbers of high-quality eggs. Immediately after ovulation, eggs are surrounded by cumulus cells, forming an egg-cumulus complex. These cumulus cells are then removed from the egg-cumulus complex by treatment with hyaluronidase to obtain the exact number of eggs. This protocol is suitable for further manipulations such as intracytoplasmic sperm injection and cryopreservation of eggs, as well as for the analyses of responsivity to superovulation reagents in genetically modified mice obtained by genome editing.

Multiple genes, whose functions or expression are overlapping, compensate for the loss of one gene. A gene cluster in the mouse genome encodes five seminal vesicle proteins (SVS2, SVS3, SVS4, SVS5, and SVS6). These proteins are produced by male rodents and function in formation of the copulatory plug following mating. SVS2 plays an essential role in the successful internal fertilization by protecting the sperm membrane against a uterine immune attack. We hypothesized that the four remaining seminal vesicle proteins (SVPs) of this gene cluster may partially/completely compensate for the deficiency of SVS2. For confirming our hypothesis, we generated mice lacking the entire SVP-encoding gene cluster and compared their fecundity with Svs2-deficient (Svs2^−/−) mice; that is, mice deficient in Svs2 alone. A single loxP site remained after the deletion of the Svs2 gene. Therefore, we inserted another loxP site by combining the CRISPR/Cas9 system with single-stranded oligodeoxynucleotides (ssODN). Male mice lacking the entire SVP-encoding gene cluster (Svs2–6^−/− mice) and thereby all five SVP proteins, generated by the deletion of 100kbp genomic DNA, showed low fecundity. However, the fecundity level was comparable with that from Svs2^−/− male mice. Our results demonstrate that SVS3, SVS4, SVS5, and SVS6 do not function in the protection of sperm against a uterine immune attack in the absence of SVS2. Thus, Svs2 is the critical gene in the SVP gene cluster.

RUNX2 is a transcription factor crucial for bone formation. Mutant mice with varying levels of Runx2 expression display dosage-dependent skeletal abnormalities, underscoring the importance of Runx2 dosage control in skeletal formation. RUNX2 activity is regulated by several molecular mechanisms, including epigenetic modification such as DNA methylation. In this study, we investigated whether targeted repressive epigenome editing including hypermethylation to the Runx2-DMR/CpG island shore could influence Runx2 expression using Cas9based epigenome-editing tools. Through the transient introduction of CRISPRoff-v2.1 and gRNAs targeting Runx2-DMR into MC3T3-E1 cells, we successfully induced hypermethylation of the region and concurrently reduced Runx2 expression during osteoblast differentiation. Although the epigenome editing of Runx2-DMR did not impact the expression of RUNX2 downstream target genes, these results indicate a causal relationship between the epigenetic status of the Runx2-DMR and Runx2 transcription. Additionally, we observed that hypermethylation of the Runx2-DMR persisted for at least 24 days under growth conditions but decreased during osteogenic differentiation, highlighting an endogenous DNA demethylation activity targeting the Runx2-DMR during the differentiation process. In summary, our study underscore the usefulness of the epigenome editing technology to evaluate the function of endogenous genetic elements and revealed that the Runx2-DMR methylation is actively regulated during osteoblast differentiation, subsequently could influence Runx2 expression.

Purpose: The synchronization of the estrous cycle is crucial for optimizing reproductive procedures and improving experimental consistency in female mice. Traditional progesterone (P4) administration methods, such as oral or subcutaneous routes, have limitations including invasiveness and variable efficacy. We aimed to develop intravaginal P4 application as a new method for synchronizing the mouse estrous cycle. Methods: A c ustom‐prepared P4 cream was applied to the vagina and washed out the following day. A vaginal smear test was conducted for 4–5 consecutive days. A combination of an anti‐inhibin antibody (AIMA) and P4 was used to improve reproductive performance. The P4‐treated, synchronized female mice were tested to determine whether they could serve as pseudopregnant recipients for embryo transfer (ET). Results: Intravaginal P4 application effectively synchronized the estrous cycle and did not adversely affect pregnancy rate or litter size. The combined use of AIMA and P4 significantly increased the number of offspring compared to natural mating ( p = 0.006). Successful offspring production was achieved with improved procedural consistency when this protocol was used to produce pseudopregnant females for ET. Conclusions: Our method reduces animal discomfort and the need for vasectomized males, offering a practical approach for enhancing reproductive efficiency and animal welfare in research settings.