Improvement of survivability and developmental ability in vitrified rat oocytes.

Oocyte cryopreservation is useful for human fertility treatment and strain preservation in both experimental and domestic animals. However, the embryonic development of vitrified rat oocytes was lower than that of vitrified embryos. To increase the viability of vitrified oocytes, intracellular ice formation during cooling and warming must be prevented. Rapid warming is important to prevent ice formation. Furthermore, suppressing the spontaneous activation of oocytes is also important because vitrification promotes the spontaneous activation of rat oocytes, and thus compromise developmental competence of the gametes. MG132, a proteasome inhibitor, suppresses the spontaneous activation of rat oocytes. Here, we examined the effects of rapid warming and MG132 treatment on the survival and embryonic development of vitrified rat oocytes. The warming rate was adjusted by changing the vitrification solution volume and warming solution temperature. The survival rate of oocytes vitrified in 10 µL solution and warmed at 50 °C (94%) was significantly higher than that of oocytes vitrified in 100 µL and 10 µL solution and warmed at 37 °C (49% and 81%, respectively). Furthermore, the rate of embryonic development of vitrified oocytes treated with MG132 during vitrification, warming, and intracytoplasmic sperm injection (ICSI) (44%) was significantly higher than that of untreated gametes (10%). Offspring were obtained after transferring embryos derived from MG132-treated vitrified oocytes (14%). Altogether, the survivability of vitrified rat oocytes increased by rapid warming, and MG132 improved embryonic development after ICSI.

Importance of nuclear localization signal-fused Cas9 in the production of genome-edited mice via embryo electroporation.

Previously, to generate genome-edited animals by introducing CRISPR-associated protein 9 (Cas9) into embryos, we developed the Technique for Animal Knockout system by Electroporation (TAKE). Additionally, by fluorescently labeling Cas9, we successfully visualized the Cas9 introduced into the pronuclei of embryos; however, whether Cas9 was introduced directly into the pronuclei by electric pulse or transferred from the cytoplasm by nuclear localization signal (NLS) remained unknown. Herein, we evaluated the localization of Cas9 with (Cas9-NLS) or without NLS (Cas9-noNLS) in mice embryos following electroporation by fusing them with GFP. Furthermore, we visually studied their effects on genome-editing rates in offspring by targeting tyrosinase gene. Fluorescence intensity in pronuclei of Cas9-NLS-electroporated embryos and genome-editing rates of offspring were significantly higher than those of Cas9-noNLS-electroporated embryos. Furthermore, fluorescence in Cas9-NLS-electroporated embryos in which pronuclei had not yet appeared 2.5 h after insemination was observed in the pronuclei of embryos appearing 3.5 h after electroporation. We demonstrated the effective transportation of Cas9 from the cytoplasm to pronuclei by the NLS following TAKE, which resulted in increased genome-editing rates in offspring. The TAKE along with fluorescently labeled nucleases can be used to verify nuclease delivery into individual embryos prior to embryo transfer for efficiently producing genome-edited animals.

Potential function of Scx+/Sox9+ cells as progenitor cells in rotator cuff tear repair in rats.

Tendons and their attachment sites to bone, fibrocartilaginous tissues, have poor self-repair capacity when they rupture, and have risks of retear even after surgical repair. Thus, defining mechanisms underlying their repair is required in order to stimulate tendon repairing capacity. Here we used a rat surgical rotator cuff tear repair model and identified cells expressing the transcription factors Scleraxis (Scx) and SRY-box 9 (Sox9) as playing a crucial role in rotator cuff tendon-to-bone repair. Given the challenges of establishing stably reproducible models of surgical rotator cuff tear repair in mice, we newly established Scx-GFP transgenic rats in which Scx expression can be monitored by GFP. We observed tissue-specific GFP expression along tendons in developing ScxGFP transgenic rats and were able to successfully monitor tissue-specific Scx expression based on GFP signals. Among 3-, 6-, and 12-week-old ScxGFP rats, Scx+/Sox9+ cells were most abundant in 3-week-old rats near the site of humerus bone attachment to the rotator cuff tendon, while we observed significantly fewer cells in the same area in 6- or 12-week-old rats. We then applied a rotator cuff repair model using ScxGFP rats and observed the largest number of Scx+/Sox9+ cells at postoperative repair sites of 3-week-old relative to 6- or 12-week-old rats. Tendons attach to bone via fibrocartilaginous tissue, and cartilage-like tissue was seen at repair sites of 3-week-old but not 6- or 12-week-old rats during postoperative evaluation. Our findings suggest that Scx+/Sox9+ cells may function in rotator cuff repair, and that ScxGFP rats could serve as useful tools to develop therapies to promote rotator cuff repair by enabling analysis of these activities.

Brca1L63X /+ rat is a novel model of human BRCA1 deficiency displaying susceptibility to radiation-induced mammary cancer.

Women who are heterozygous for deleterious BRCA1 germline mutations harbor a high risk of hereditary breast cancer. Previous Brca1-heterozygous animal models do not recapitulate the breast cancer phenotype, and thus all currently used knockout models adopt conditional, mammary-specific homozygous Brca1 loss or addition of Trp53 deficiency. Herein, we report the creation and characterization of a novel Brca1 mutant rat model harboring the germline L63X mutation, which mimics a founder mutation in Japan, through CRISPR-Cas9-based genome editing. Homozygotes (Brca1L63X/L63X ) were embryonic lethal, whereas heterozygotes (Brca1L63X/+ ) showed apparently normal development. Without carcinogen exposure, heterozygotes developed mammary carcinoma at a comparable incidence rate with their wild-type (WT) littermates during their lifetime. Intraperitoneal injection of 1-methyl-1-nitrosourea (25 or 50 mg/kg) at 7 weeks of age induced mammary carcinogenesis at comparable levels among the heterozygotes and their littermates. After exposure to ionizing radiation (0.1-2 Gy) at 7 weeks of age, the heterozygotes, but not WT littermates, displayed dose-dependent mammary carcinogenesis with 0.8 Gy-1 excess in hazard ratio during their middle age; the relative susceptibility of the heterozygotes was more prominent when rats were irradiated at 3 weeks of age. The heterozygotes had tumors with a lower estrogen receptor α immunopositivity and no evidence of somatic mutations of the WT allele. The Brca1L63X/+ rats thus offer the first single-mutation, heterozygous model of BRCA1-associated breast cancer, especially with exposure to a DNA break-inducing carcinogen. This implies that such carcinogens are causative and a key to breast cancer prevention in individuals who carry high-risk BRCA1 mutations.

Survivability and subsequent development of vitrified early-stage mouse embryos after warming at different temperatures.

Cryopreservation of embryos is a useful method for stably preserving various strains for a long time, and the cryopreserved embryos can be used at any time by simple warming. However, the viability of cryopreserved embryos, particularly vitrification at an early stage, is low compared to that of fresh embryos. As the warming process during vitrification is known to affect the survivability and subsequent development of embryos, the present study aimed to examine the viability and subsequent development of vitrified early-stage mouse embryos after warming at different temperatures. The survival rate of pronuclear and 2-cell stage embryos warmed at 60 °C (97% and 88%, respectively) was significantly higher than that of the embryos warmed at 37 °C (46% and 48%, respectively). The pronuclear and 2-cell stage embryos warmed at 60 °C (86% and 100%) showed better development to the blastocyst stage than the embryos warmed at 37 °C (72% and 84%, respectively). The development of offspring of the surviving embryos was similar at both the warming temperatures. These results showed that the survivability and subsequent development of vitrified early-stage mouse embryos were obviously increased upon rapid warming. This improved warming process could be helpful for the maintenance and reproduction of genetic resources.

Amount of Cas9 protein introduced into mouse embryos via electroporation affects the genome-editing rate.

Genetically engineered animals can be produced quickly using genome editing technology. A new electroporation technique, technique for animal knockout system by electroporation (TAKE), aids in the production of genome-edited animals by introducing nucleases into intact embryos using electroporation instead of microinjection. It is difficult to confirm nuclease delivery into embryos after electroporation using the conventional TAKE method. We previously reported the successful visualization of fluorescently-labeled tracrRNA in embryos after electroporation Cas9 paired with the crRNA:tracrRNA-ATTO550 duplex. However, the amount of fluorescence signal from labeled tracrRNA in embryos did not correlate with the genome editing rate of the offspring. This study examined the visualization of Cas9 protein in embryos after electroporation and its correlation with the genome editing rate of the offspring using a fluorescent Cas9 fusion protein. The fluorescent Cas9 protein was observed in all embryos that survived following electroporation. We found that the efficiency of Cas9 protein delivery into embryos via electroporation depended on the pulse length. Furthermore, we demonstrated that the amount of fluorescent Cas9 protein detected in the embryos correlated with the genome editing efficiency of the embryos. These data indicate that the TAKE method using fluorescently-labeled nucleases can be used to optimize the delivery conditions and verify nuclease delivery into individual embryos prior to embryo transfer for the efficient production of genome-edited animals.

Humanized UGT2 and CYP3A transchromosomic rats for improved prediction of human drug metabolism.

Although "genomically" humanized animals are invaluable tools for generating human disease models as well as for biomedical research, their development has been mainly restricted to mice via established transgenic-based and embryonic stem cell-based technologies. Since rats are widely used for studying human disease and for drug efficacy and toxicity testing, humanized rat models would be preferred over mice for several applications. However, the development of sophisticated humanized rat models has been hampered by the difficulty of complex genetic manipulations in rats. Additionally, several genes and gene clusters, which are megabase range in size, were difficult to introduce into rats with conventional technologies. As a proof of concept, we herein report the generation of genomically humanized rats expressing key human drug-metabolizing enzymes in the absence of their orthologous rat counterparts via the combination of chromosome transfer using mouse artificial chromosome (MAC) and genome editing technologies. About 1.5 Mb and 700 kb of the entire UDP glucuronosyltransferase family 2 and cytochrome P450 family 3 subfamily A genomic regions, respectively, were successfully introduced via the MACs into rats. The transchromosomic rats were combined with rats carrying deletions of the endogenous orthologous genes, achieved by genome editing. In the "transchromosomic humanized" rat strains, the gene expression, pharmacokinetics, and metabolism observed in humans were well reproduced. Thus, the combination of chromosome transfer and genome editing technologies can be used to generate fully humanized rats for improved prediction of the pharmacokinetics and drug-drug interactions in humans, and for basic research, drug discovery, and development.

Pathophysiological significance of Stim1 mutation in sympathetic response to stress and cardiovascular phenotypes in SHRSP/Izm: In vivo evaluation by creation of a novel gene knock-in rat using CRISPR/Cas9.

Genetic approach using rat congenic lines between SHRSP/Izm and WKY/Izm identified stromal interaction molecule 1 (Stim1), an essential component of store-operated Ca2+ entry (SOCE), as a promising candidate gene responsible for the exaggerated sympathetic response to stress in SHRSP. Since SHRSP has a nonsense mutation in Stim1 resulting in the expression of a truncated form of STIM1 that caused reduction of SOCE activity in primary cultured cerebral astrocytes, we created SHRSP/Izm knocked-in with the wild-type Stim1 (KI SHRSP) by the CRISPR/Cas9 method to investigate whether the functional recovery of STIM1 would mitigate sympatho-excitation to stress in vivo in SHRSP. No potential off-target nucleotide substitutions/deletions/insertions were found in KI SHRSP. Western blotting and fluorescent Ca2+ imaging of astrocytes confirmed wild-type STIM1 expression and restored SOCE activity in astrocytes from KI SHRSP, respectively. Blood pressure (BP) measured by the tail-cuff method at 12, 16, and 20 weeks of age did not significantly differ between SHRSP and KI SHRSP, while the heart rate of KI SHRSP at 16 and 20 weeks of age was significantly lower than that of age-matched SHRSP. Unexpectedly, the sympathetic response to stress (evaluated with urinary excretion of norepinephrine under cold stress and BP elevation under cold/restraint stress) did not significantly differ between SHRSP and KI SHRSP. The present results indicated that the functional deficit of STIM1 was not a genetic determinant of the exaggerated sympathetic response to stress in SHRSP and that it would be necessary to explore other candidates within the congenic fragment on chromosome 1.

LOX-1 (Lectin-Like Oxidized Low-Density Lipoprotein Receptor-1) Deletion Has Protective Effects on Stroke in the Genetic Background of Stroke-Prone Spontaneously Hypertensive Rat.

Background and Purpose- oxLDL (oxidized low-density lipoprotein) has been known for its potential to induce endothelial dysfunction and used as a major serological marker of oxidative stress. Recently, LOX-1 (lectin-like oxidized low-density lipoprotein receptor-1), a lectin-like receptor for oxLDL, has attracted attention in studies of neuronal apoptosis and stroke. We aim to investigate the impact of LOX-1-deficiency on spontaneous hypertension-related brain damage in the present study. Methods- We generated a LOX-1 deficient strain on the genetic background of stroke-prone spontaneously hypertensive rat (SHRSP), an animal model of severe hypertension and spontaneous stroke. In this new disease model with stroke-proneness, we monitored the occurrence of brain abnormalities with and without salt loading by multiple procedures including T2 weighted magnetic resonance imaging and also explored circulatory miRNAs as diagnostic biomarkers for cerebral ischemic injury by microarray analysis. Results- Both T2 weighted magnetic resonance imaging abnormalities and physiological parameter changes could be detected at significantly delayed timing in LOX-1 knockout rats compared with wild-type SHRSP, in either case of normal rat chow and salt loading (P<0.005 in all instances; n=11-20 for SHRSP and n=13-23 for LOX-1 knockout rats). There were no significant differences in the form of magnetic resonance imaging findings between the strains. A number of miRNAs expressed in the normal rat plasma, including rno-miR-150-5p and rno-miR-320-3p, showed significant changes after spontaneous brain damage in SHRSP, whereas the corresponding changes were modest or almost unnoticeable in LOX-1 knockout rats. There appeared to be the lessening of correlation of postischemic miRNA alterations between the injured brain tissue and plasma in LOX-1 knockout rats. Conclusions- Our data show that deficiency of LOX-1 has a protective effect on spontaneous brain damage in a newly generated LOX-1-deficient strain of SHRSP. Further, our analysis of miRNAs as biomarkers for ischemic brain damage supports a potential involvement of LOX-1 in blood brain barrier disruption after cerebral ischemia. Visual Overview- An online visual overview is available for this article.

Improvement of genome editing by electroporation using embryos artificially removed cumulus cells in the oviducts.

Many genome-edited animals have been produced using the CRISPR/Cas system. Genome-edited strains were produced by introducing nucleases into pronuclear stage embryos. Recently, a new electroporation technique (TAKE: Technique for Animal Knockout system by Electroporation) was developed for the production of genome-edited animals by introducing nucleases into intact embryos using electroporation instead of the microinjection method. Furthermore, this method, which can introduce nucleases into intact embryos, enables genome editing of mouse embryos in the oviducts. However, the present protocol required improvements for low litter size and restriction of operation time. In this study, the influence on the development and genome editing of mouse embryos in the oviducts by electroporation and operation time was examined. The genome-editing rate was higher in the embryos electroporated at 16:00-17:00 (PM) (54%) on the following day of natural mating compared to that of embryos at 10:00-11:00 (AM) (27%). The embryos at AM formed a complex with cumulus cells, and cumulus cells were freed from embryos by treatment with hyalronidase before electroporation. The results showed that the genome-editing rate was significantly increased in the embryos treated with hyalronidase at AM, because the cumulus cells surrounding the embryos interfered with the introduction of nucleases into embryos. This study demonstrated that it was possible to adjust the operation time for the introduction of nucleases into embryos in the oviducts by treatment with hyalronidase before electroporation. However, litter size and development of embryos after electroporation was quite low in all experiments (5-7) compared with the control without operation (11).

Genome editing of rodents by electroporation of CRISPR/Cas9 into frozen-warmed pronuclear-stage embryos.

Genome edited animals can now be easily produced using the clustered regularly interspaced short palindromic repeat (CRISPR) and CRISPR-associated protein 9 (Cas9) system. Traditionally, these animals have been produced by the introduction of endonucleases into pronuclear-stage embryos. Recently, a novel electroporation method, the "Technique for Animal Knockout system by Electroporation (TAKE)," has been established as a simple and highly efficient tool to introduce endonucleases into embryos instead of methods such as microinjection. Use of frozen-warmed pronuclear-stage embryos in this method has further contributed to efficient production of genome edited animals. However, early developmental stage embryos, including pronuclear-stage embryos, especially those of rats, sometimes show low resistance to physical damage by vitrification and introduction of endonucleases during microinjection. In this study, we propose an ethanol-free, slow-freezing method to reduce physical damage to pronuclear-stage embryos followed by the TAKE method. All mouse and rat frozen embryos were survived after electroporation, and 18% and 100% of offspring were edited target gene, respectively. The resulting protocol is an efficient method for producing genome edited animals.

Production of genome-edited mice by visualization of nucleases introduced into the embryos using electroporation.

Genome editing technology contributes to the quick and highly efficient production of genetically engineered animals. These animals are helpful in clarifying the mechanism of human disease. Recently, a new electroporation technique (TAKE: Technique for animal knockout system by electroporation) was developed to produce genome-edited animals by introducing nucleases into intact embryos using electroporation instead of the microinjection method. The aim of this study was to increase the efficiency of production of genome-edited animals using the TAKE method. In the conventional protocol, it was difficult to confirm the introduction of nucleases into embryos and energization during operation. Using only embryos that introduced nucleases for embryo transfer, it will lead to increased efficiency in the production of genome-edited animals. This study examined the visualization in the introduction of nucleases into the embryos by using nucleases fluorescent labeled with ATTO-550. The embryos were transfected with Cas9 protein and fluorescent labeled dual guide RNA (mixture with crRNA and tracrRNA with ATTO-550) targeted tyrosinase gene by the TAKE method. All embryos that survived after electroporation showed fluorescence. Of these embryos with fluorescence, 43.7% developed to morphologically normal offspring. In addition, 91.7% of offspring were edited by the tyrosinase gene. This study is the first to demonstrate that the introduction of nucleases into embryos by the TAKE method could be visualized using fluorescent-labeled nucleases. This improved TAKE method can be used to produce genome-edited animals and confirm energization during operation.

Rapid and efficient production of genome-edited animals by electroporation into oocytes injected with frozen or freeze-dried sperm.

Sperm preservation is a useful technique for maintaining valuable animal strains. Rat sperm could be frozen or freeze-dried in a simple Tris-EDTA solution (TE buffer), and oocytes that were fertilized with these sperm by intracytoplasmic sperm injection (ICSI) developed into offspring. Genome editing with the clustered regularly interspaced short palindromic repeat (CRISPR) and CRISPR-associated protein 9 (Cas9) system enables the rapid production of genetically modified rats. The recent innovative method, named the TAKE method, could easily produce genome edited rats by electroporation of endonucleases into embryos. Although various rat strains have been applied for genome editing, genome editing using strains that were preserved as sperm took longer because it required collecting embryos after maturation of animals regenerated from sperm. To reduce the production period, we directly electroporated Cas9 protein and gRNA into oocytes that were injected with frozen or freeze-dried sperm in TE buffer. No effect of electroporation until 30 V to ICSI-embryos derived from frozen or freeze-dried sperm were shown in the development of offspring. Furthermore, the rate of genome editing in offspring was high (56% for frozen and 50% for freeze-dried sperm). These results concluded that the combination of ICSI and the TAKE method was useful for the rapid production of genome-edited animals from sperm that have been preserved as genetic resources.

CHD1 acts via the Hmgpi pathway to regulate mouse early embryogenesis.

The protein CHD1 is a member of the family of ATPase-dependent chromatin remodeling factors. CHD1, which recognizes trimethylated histone H3 lysine 4, has been implicated in transcriptional activation in organisms ranging from yeast to humans. It is required for pre-mRNA maturation, maintenance of mouse embryonic stem cell pluripotency and rapid growth of the mouse epiblast. However, the function(s) of CHD1 in mouse preimplantation embryos has not yet been examined. Here, we show that loss of CHD1 function led to embryonic lethality after implantation. In mouse embryos in which Chd1 was targeted by siRNA microinjection, the expression of the key regulators of cell fate specification Pou5f1 (also known as Oct4), Nanog and Cdx2 was dramatically decreased, starting at mid-preimplantation gene activation (MGA). Moreover, expression of Hmgpi and Klf5, which regulate Pou5f1, Nanog and Cdx2, was also significantly suppressed at zygotic gene activation (ZGA). Suppression of Hmgpi expression in Chd1-knockdown embryos continued until the blastocyst stage, whereas suppression of Klf5 expression was relieved by the morula stage. Next, we rescued HMGPI expression via Hmgpi mRNA microinjection in Chd1-knockdown embryos. Consequently, Pou5f1, Nanog and Cdx2 expression was restored at MGA and live offspring were recovered. These findings indicate that CHD1 plays important roles in mouse early embryogenesis via activation of Hmgpi at ZGA.

Tolerance to vitrification of rat embryos at various developmental stages.

Numerous genetically engineered rat strains have been produced via genome editing. Although freezing of embryos is helpful for the production and storage of these valuable strains, the tolerance to freezing of embryos varies at each developmental stage of the embryo. This study examined the tolerance to freezing of rat embryos at various developmental stages, particularly at the pronuclear stage. Embryos that had developed to the pronuclear, 2-cell, and morula stages were frozen via vitrification using ethylene glycol- and propylene glycol-based solutions. More than 90% of the embryos at all developmental stages survived after warming. The developmental rates to offspring of thawed embryos at the pronuclear, 2-cell, and morula stages were 19%, 41%, and 52%, respectively. Pronuclear stage embryos between the early and late developmental stages were then vitrified. The developmental rates to offspring of the thawed pronuclear stage embryos collected at 24, 28, and 31 h after the induction of ovulation were 17%, 21%, and 23%, respectively. These results indicated that the tolerance to vitrification of rat embryos increased with the development of embryos. The establishment of vitrification method of rat embryos at various developmental stages is helpful for improving the production and storage of valuable rat strains used for biomedical science.

Reproductive technologies for the generation and maintenance of valuable animal strains.

Many types of mutant and genetically engineered strains have been produced in various animal species. Their numbers have dramatically increased in recent years, with new strains being rapidly produced using genome editing techniques. In the rat, it has been difficult to produce knockout and knock-in strains because the establishment of stem cells has been insufficient. However, a large number of knockout and knock-in strains can currently be produced using genome editing techniques, including zinc-finger nuclease (ZFN), transcription activator-like effector nuclease (TALEN), and the clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein 9 (Cas9) system. Microinjection technique has also contributed widely to the production of various kinds of genome edited animal strains. A novel electroporation method, the "Technique for Animal Knockout system by Electroporation (TAKE)" method, is a simple and highly efficient tool that has accelerated the production of new strains. Gamete preservation is extremely useful for maintaining large numbers of these valuable strains as genetic resources in the long term. These reproductive technologies, including microinjection, TAKE method, and gamete preservation, strongly support biomedical research and the bio-resource banking of animal models. In this review, we introduce the latest reproductive technologies used for the production of genetically engineered animals, especially rats, using genome editing techniques and the efficient maintenance of valuable strains as genetic resources. These technologies can also be applied to other laboratory animals, including mice, and domestic and wild animal species.

Depdc5 knockout rat: A novel model of mTORopathy.

DEP-domain containing 5 (DEPDC5), encoding a repressor of the mechanistic target of rapamycin complex 1 (mTORC1) signaling pathway, has recently emerged as a major gene mutated in familial focal epilepsies and focal cortical dysplasia. Here we established a global knockout rat using TALEN technology to investigate in vivo the impact of Depdc5-deficiency. Homozygous Depdc5(-/-) embryos died from embryonic day 14.5 due to a global growth delay. Constitutive mTORC1 hyperactivation was evidenced in the brains and in cultured fibroblasts of Depdc5(-/-) embryos, as reflected by enhanced phosphorylation of its downstream effectors S6K1 and rpS6. Consistently, prenatal treatment with mTORC1 inhibitor rapamycin rescued the phenotype of Depdc5(-/-) embryos. Heterozygous Depdc5(+/-) rats developed normally and exhibited no spontaneous electroclinical seizures, but had altered cortical neuron excitability and firing patterns. Depdc5(+/-) rats displayed cortical cytomegalic dysmorphic neurons and balloon-like cells strongly expressing phosphorylated rpS6, indicative of mTORC1 upregulation, and not observed after prenatal rapamycin treatment. These neuropathological abnormalities are reminiscent of the hallmark brain pathology of human focal cortical dysplasia. Altogether, Depdc5 knockout rats exhibit multiple features of rodent models of mTORopathies, and thus, stand as a relevant model to study their underlying pathogenic mechanisms.

Sperm freeze-drying and micro-insemination for biobanking and maintenance of genetic diversity in mammals.

Breeding by natural mating is ideal for maintaining animal populations. However, the lack of breeding space resulting from an increased number of strains and the decline in fertility caused by inbreeding inhibits the reproduction of subsequent generations. Reproductive technologies, such as gamete preservation and artificial fertilisation, have been developed to overcome these problems. These approaches efficiently produce offspring of laboratory, domestic and wild animals, and can also be used to treat human infertility. Gamete preservation using sperm contributes to improvements in reproductive systems and enables the use of smaller breeding spaces. Although cryopreservation with liquid nitrogen has been used to preserve spermatozoa, freeze-drying without liquid nitrogen, a novel method, facilitates long-term storage of spermatozoa. This method has recently been applied to maintain animal strains. Micro-insemination techniques, such as intracytoplasmic sperm injection (ICSI), are exceptional for improving assisted reproduction. ICSI can be used to fertilise oocytes, even with immotile and immature spermatozoa that are unsuitable for AI and IVF. Reproductive technologies provide a substantial advantage for biobanking and maintaining the genetic diversity of laboratory, domestic and wild animals. This review covers the latest method of sperm freeze-drying and micro-insemination, and future possibilities for maintaining animal strains and populations.

In vitro maturation of immature rat oocytes under simple culture conditions and subsequent developmental ability.

Rat oocytes can be produced artificially by superovulation. Because some strains show low sensitivity to superovulation treatment, in vitro maturation is an alternative method to produce numerous matured oocytes. Furthermore, establishment of an in vitro maturation system with simple culture conditions is cost effective and leads to easy handling of oocytes. This study examined developmental ability of rat germinal vesicle (GV) oocytes maturing in vitro under simple culture conditions. Significantly different numbers of ovulated oocytes reached the second metaphase of meiosis (MII) among Jcl:Wistar (17.0), F344/Stm (31.0), and BN/SsNSlc (2.2) rats in whom superovulation was induced by pregnant mare serum gonadotropin (PMSG) and human chorionic gonadotropin. However, similar numbers of GV oocytes were obtained from ovaries of PMSG-injected Wistar (27.7), F344 (34.7), and BN (24.7) rats. These GV oocytes were cultured in vitro in HTF, αMEM, and a 1:1 HTF + αMEM or TYH + αMEM mixture. High proportions of Wistar and F344 oocytes that matured to MII in αMEM were parthenogenetically activated by strontium chloride treatment (78% and 74%, respectively). Additionally, 10% of matured oocytes of both strains developed into offspring after intracytoplasmic sperm injection and embryo transfer to foster mothers. Although BN oocytes cultured in αMEM could be parthenogenetically activated and developed into offspring, the success rate was lower than that for Wistar and F344 oocytes. This study demonstrated that numerous GV oocytes were produced in rat ovaries by PMSG injection. This simple in vitro maturation system of immature oocytes could be further developed to maintain valuable rat strains experiencing reproductive difficulties.

Histone methyltransferase Smyd3 regulates early embryonic lineage commitment in mice.

SET and MYND domain-containing protein 3 (Smyd3) is a histone H3 lysine 4 (H3K4) di- and tri-methyltransferase that forms a transcriptional complex with RNA polymerase II and activates the transcription of oncogenes and cell cycle genes in human cancer cells. However, the study of Smyd3 in mammalian early embryonic development has not yet been addressed. In the present study, we investigated the expression pattern of Smyd3 in mouse preimplantation embryos and the effects of RNA interference (RNAi)-mediated Smyd3 repression on the development of mouse embryos. We showed that Smyd3 mRNA levels increased after the two-cell stage, peaked at the four-cell stage, and gradually decreased thereafter. Moreover, in two-cell to eight-cell embryos, SMYD3 staining was more intense in the nuclei than it was in the cytoplasm. In Smyd3-knockdown embryos, the percentage of inner cell mass (ICM)-derived colony formation and trophectoderm (TE)-derived cell attachment were significantly decreased, which resulted in a reduction in the number of viable offspring. Furthermore, the expression of Oct4 and Cdx2 during mid-preimplantation gene activation was significantly decreased in Smyd3-knockdown embryos. In addition, the transcription levels of ICM and epiblast markers, such as Oct4, Nanog, and Sox2, the transcription levels of primitive endoderm markers, such as Gata6, and the transcription levels of TE markers, such as Cdx2 and Eomes, were significantly decreased in Smyd3-knockdown blastocysts. These findings indicate that SMYD3 plays an important role in early embryonic lineage commitment and peri-implantation development through the activation of lineage-specific genes.