Mouse sperm cryopreservation has greatly developed and spread throughout the world since it was the first successfully performed in 1990. The technology of sperm cryopreservation is widely used for storage of genetically engineered mice produced by global large-scale knockout mice or mouse mutagenesis projects in mouse banks. There are several benefits to using sperm cryopreservation. These include the ability to archive a great number of genetically engineered mice at a low cost in a small space; the ease with which freezing and thawing procedures can be carried out; and the convenience of performing mouse reproduction via in vitro fertilization. Sperm cryopreservation provides the research community with permanent access to genetically engineered mice and as such is an essential part of research activities. Many researchers have actively been investigating the techniques surrounding mouse sperm cryopreservation and embryo production using cryopreserved sperm. In particular, the topic of improving the low fertility rate of cryopreserved sperm in the C57BL/6 strain of mouse, the most common inbred strain used as a base for genetically engineered mice, has been garnering much attention. This review focuses on recent work regarding various attractive technologies in the fields of sperm cryopreservation and efficient embryo production using cryopreserved C57BL/6 mouse sperm.

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A procedure to cryopreserve mammalian embryos resulting in the birth of offspring was first described almost forty years ago. This procedure resulting in the birth of a child was reported 26 years ago. Since then, the preservation of human oocytes and embryos by cooling them to low subzero temperatures has become an integral part of Assisted Reproductive Technologies (ART). Hundreds of thousands of children have now been born after having been cryopreserved as oocytes or embryos. These results owe as much to the fundamental understanding of cryobiology as to the application of reproductive medicine. This brief review summarizes the history of embryo cryobiology, and presents a synopsis of basic cryobiology as it applies to present methods to improve the cryopreservation of human oocytes.

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Cryopreservation of embryos is an important strategy for the conservation of species and valuable strains of laboratory species. Historically, in the mouse, the technical development of cryopreservation started with slow freezing methods in the 1970’s, which was then followed by vitrification method developed in the 1980’s. Vitrification is advantageous in its quickness and simplicity, because it does not need programmable controlled-rate freezing machines. Furthermore, the survivability of embryos recovered after vitrification is significantly improved by avoiding chilling injury and intra- and extra-cellular ice formation. Recently, a series of new vitrification methods using a minimal volume of cryopreservation agent (CPA) and extremely high osmolality CPAs have been developed. For laboratory rats and mice, there is a long history of embryo cryopreservation and huge numbers of embryos have been kept frozen in cryobanks. In contrast, the techniques have only recently been optimized for other laboratory animals, e.g., rabbits, Syrian hamsters, Mongolian gerbils and mastomys (African rodent). Besides safety in cryopreservation, simple transportation of vitrified embryos on dry ice has been a challenging issue. By using the extremely high osmolality CPAs, we are now examining the feasibility and effectiveness of a simple method for the transportation of vitrified embryos. Development of new cryopreservation methods for embryos of laboratory species should be an integral part of the technological logistics supporting the development of biomedical sciences.

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Cloning by somatic cell nuclear transfer (SCNT) can be used for the conservation of endangered and extinct animals and elite livestock, and their somatic cells have often been cryopreserved as genetic resources. However, viable cells are not always available when individuals are already dead or when their species have extinct. If the intact genome could be retrieved from such animals, cloned animals could be produced by SCNT technology. Recently, we have demonstrated that viable somatic cells can be obtained from certain bovine organs frozen without cryoprotectant for a decade and that viable cloned animals can be produced from the retrieved cells by SCNT technology. In this mini-review, we discuss recent attempts to rescue animal genetic resources from tissues, organs or bodies frozen without cryoprotectant for a long time.

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The removal, cryopreservation, and subsequent reimplantation of ovaries would make it possible to treat a young cancer patient and improve her quality of life by preserving her fertility. The current technology requires cutting the ovary into pieces before freezing and does not support preservation of the whole ovary. The ovary has a complex endocrinologic function. It is composed of cells of different form and character and contains oocytes at various stages of development. Successful cryopreservation, transplantation, and functional rehabilitation of the whole ovary would have broad significance, not only for ovaries but also for other organs such as the liver, kidney, and heart. Ovarian cryopreservation technology would lead the way to the establishment of a biological bank for frozen internal organs.

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The fine structure of human oogonia in foetal ovaries and the stages of folliculogenesis in fertile ovaries are documented with digital images. The human oogonia show progressive changes in fine structure during folliculogenesis and oogenesis until they are ovulated and ready for fertilization. Parallel changes also occur in their relationship with the surrounding follicle cells, which support and nurse the growing and maturing oocytes untill ovulation. The inactivation of the maternal centrosome is the most significant event during human oogenesis

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To demonstrate chromosomal injuries associated with in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI), sister chromatid exchange (SCE), an indicator of DNA damage, was investigated and the cytogenetic influence of embryonic manipulation on chromosomes of early embryos and offspring was investigated. SCE analysis was performed in early embryos and offspring obtained by IVF, ICSI, or in vivo fertilization (control). To label chromosomes of early embryos, the embryos were cultured in BrdU-supplemented medium for 2 cell cycles. For offspring, BrdU solution was repeatedly injected intraperitoneally. Chromosome samples prepared from the embryos and the bone marrow cells of offspring were stained by the Fluorescence plus Giemsa (FPG) method. In the IVF and ICSI groups, the rates of early development, implantation and offspring were lower than those in the control group. The SCE frequency of early embryos was significantly higher in the IVF and ICSI groups than in the control group (P < 0.05). In contrast, the SCE frequency of offspring in the IVF and ICSI groups was not significantly different from that in the control group. These findings suggest that embryos having serious DNA damage due to embryonic manipulation may be eliminated in a relatively early developmental step and may not reach term.

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Mongolian gerbil 1-cell embryos can develop into blastocysts in vitro in a co-culture with oviductal cells in mTCM199 medium. When 1-cell embryos are cultured in a chemically defined medium, they do not develop into blastocysts. Thus, the objective of this study was to examine the effects of amino acids on the in vitro development of Mongolian gerbil embryos. Amino acid depletion/accumulation by the Mongolian gerbil preimplantation embryo was analyzed with an amino acid analyzer. The amino acids of threonine, valine, isoleucine, leucine, lysine, serine, glutamine, glycine, and alanine were depleted in the medium at all the developmental stages. Two-cell stage embryos were cultured in mM16 medium supplemented with each amino acid. We found that 37.1% and 11.4% of 2-cell embryos reached the morula and blastocyst stages, respectively, after supplementation of the medium with valine. Three amino acids (threonine, aspartic acid, and glutamine) promoted development to the 4-cell stage (P < 0.05). Although the combination of valine and threonine, aspartic acid, or glutamine significantly increased development to the 4-cell and 8-cell stages, no improvement of the blastocyst rates was observed. This study has demonstrated that amino acids can support preimplantation gerbil embryo development in vitro.

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Sperm-zona pellucida (ZP) binding is the initial step in fertilization. In this study, to examine sperm-ZP binding properties, we collected ZPs from oocytes obtained from gilt ovaries after 0, 20, 44, and 68 h of maturation culture, and counted the number of sperm bound to the ZP (NSBZ) after 2 min of sperm-ZP co-incubation. Culture of cumulus oocyte complexes (COCs) for 44 h produced significantly higher NSBZ than the other culture durations. Culture of denuded oocytes (DO) produced the same result. To examine the effect of cumulus cells (CCs) and the maturity of oocytes on the NSBZ after maturation culture for 44 h, three oocyte culture conditions (COCs, DO, and combination of DO and CCs) were established, and oocytes were categorized according to the presence of a polar body (PB). The NSBZ did not differ among the culture conditions. NSBZ of oocytes with a PB was greater than that of oocytes without a PB. The addition of tunicamycin, a potent inhibitor of N-linked glycosylation, to the COC maturation medium significantly decreased the NSBZ, although the maturation rate was not affected. In conclusion, NSBZ increases during oocyte maturation and oocytes play a key role in increasing the NSBZ via modification of N-linked glycosylation.

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Sperm express the Toll-like receptor (TLR) family and have an innate immune function defending against bacterial infection in semen. It has been known that virus infection is also observed in semen with low motility of sperm, suggesting that sperm are also able to respond to virus infection. However, there is little information about the expression and roles of TLRs that recognize virus-released pathogens in semen. The present study clearly showed that sperm express functional TLR9 that recognizes the unmethylated CpG DNA sequence. Using a specific antibody, we detected the expression of TLR9 in sperm. However, the positive band was disappeared by progesterone treatment that induced the acrosome reaction. When sperm were cultured with the TLR9 ligand, sperm motility significantly decreased in a time-dependent manner. The treatment of sperm with the TLR9 ligand affected the acrosomal status and suppressed the BSA-induced capacitation of sperm. Additionally, sperm were used for in vitro fertilization and injection into the uterus of super-ovulated female mice following pre-culture with the TLR9 ligand. The fertilization rates were significantly suppressed both in vitro and in vivo. From these results, we conclude that sperm express and have functional TLR9 that potentially recognizes virus infection in semen.

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Mongolian gerbil 1-cell embryos do not develop into blastocysts in vitro because of 8-cell block. Recently, we reported that 2-cell embryos of Mongolian gerbils developed into blastocysts in vitro in mM16 supplemented with valine. However, the role of valine in the in vitro development of Mongolian gerbil embryos is unclear. Thus, the objective of this study was to investigate of the role of valine in the in vitro development of Mongolian gerbil embryos. Mongolian gerbil embryos at the 1-cell, 2-cell, 8-cell, morula, and blastocyst stages were cultured in mM16 medium containing ¹⁴C-valine or ¹⁴C-glucose. A 2.5 mM NaOH solution was used as a trap for the evolved ¹⁴CO₂. After incubation, all samples were analyzed using a liquid scintillation counter. The incorporation of ¹⁴C-valine significantly increased at the 8-cell stage. The oxidation of ¹⁴C-valine significantly increased at the 8-cell and blastocyst stages. The incorporation of ¹⁴C-glucose in mM16+valine was significantly higher than that in mM16 at the blastocyst stage. The oxidation of ¹⁴C-glucose in mM16 was significantly higher than that in mM16+valine at the 8-cell stage, although that in mM16+valine was significantly higher than that in mM16 at the blastocyst stage. The carbon skeleton of ¹⁴C-valine was metabolized into lipid. Our results suggest that valine was used and metabolized as an energy source in preimplantation Mongolian gerbil embryos.

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The fate of human oocytes with impaired stretchability of the oolemma during piezo-ICSI was investigated in vitro (development into blastocysts in 5-day culture) and in vivo (implantation 4 weeks after embryo transfer). Oolemma of metaphase-II human oocytes was penetrated either before application of a piezo-pulse at stretching of less than 75% of the diameter (category-Low), or by a piezo-pulse when the oolemma was stretched over 75% of the diameter (category-High). Following in vitro culture, oocyte survival and developmental capacity to the blastocyst stage were compared between the two categories of oolemma stretchability. Moreover, we investigated whether oolemma stretchability was independently related as a risk factor of implantation using multivariate logistic regression. The post-injection survival rate of category-Low oocytes (73.6%) was significantly (P < 0.01) lower than that of category-High oocytes (99.4%). The blastocyst yield of category-Low oocytes (36.7%; calculated from surviving oocytes) was similar to that of category-High oocytes (44.5%). The low oolemma stretchability was found to be an independent risk factor of implantation (odds ratio 4.18, 95% confidence interval 1.04–16.74, P = 0.043). In conclusion, stretchability of human oolemma during piezo-ICSI affects post-injection survival, but not the developmental potential to blastocysts. Since oolemma stretchability also affects the implantation, we propose this parameter as a criterion for embryo selection in single embryo transfer.

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The present study focused on determining the incidence of chromosomal abnormalities in river buffalo embryos produced in vitro to reveal the causes of the low conception rate of transferred embryos. River buffalo oocytes were collected from 2- to 8-mm ovarian follicles of slaughtered river buffaloes in the Philippines and India, matured in vitro, and inseminated with frozen-thawed river buffalo spermatozoa at a final concentration of 1 × 10⁶ sperm/ml. At 36–40 h of in vitro culture, 2- to 8-cell stage embryos were treated with vinblastine sulphate for 6 to 10 h, and prepared as chromosomal samples. The rate of chromosomally normal embryos (diploidy 2n = 50) was 52.2% (48/92), while the incidence of chromosomal abnormalities reached 47.8% (44/92) in the analyzed embryos. Polyploidy was the most frequent abnormality (22 embryos; 23.9%), while the incidences of mixoploid (11 embryos, 12%) and haploid embryos (11 embryos, 12%) were the same. The polyploidy and mixoploidy observed in the present study may be the result of polyspermy. The early-stage river buffalo embryos derived from in vitro production displayed a high incidence of chromosomal abnormalities that might affect their subsequent development.

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