The TYH medium was first reported as a medium for in vitro fertilization (IVF) of mouse eggs with epididymal spermatozoa, by Toyoda, Yokoyama and Hosi, in 1971. It was a modified Krebs-Ringer bicarbonate solution, supplemented with glucose, Na-pyruvate, antibiotics and bovine serum albumin. In TYH medium, almost all eggs are fertilized within 1 h, if the spermatozoa are pre-incubated for 2 h before insemination, while the sperm penetration is delayed for approximately 1 h when fresh epididymal spermatozoa are used. These findings showed that sperm capacitation can be induced in a chemically defined medium without requiring the female reproductive tract. Although the medium was not specifically named in the original paper, it was later called “TYH” after the initials of the three authors of the original paper. The IVF method using TYH medium is widely used due to its high reproducibility. In this mini-review, we describe the early efforts to develop the TYH medium and briefly discuss the related areas.

Rat 1-cell embryos fertilized in vivo or in vitro show a complete developmental block at the 2- to 4-cell stage in conventional culture media. To develop a culture medium supporting early embryonic development in rats, we examined the effects of some chemical and physical factors on the development of rat 1-cell embryos. The results indicated that (1) the developmental block is caused by phosphate, (2) glucose and amino acids are necessary, (3) the optimal osmolarity is 244–246 mOsm, and (4) the presence of bovine serum albumin and relatively high (110.0–130.0 mM) NaCl concentrations in the early stages preceding pronucleus formation is necessary. On the basis of these results, novel culture media designated as modified rat 1-cell embryo culture medium (mR1ECM) and mR1ECM for in vitro fertilization (mR1ECM-IVF) were developed. In cultures using these media, 90% or more of rat 1-cell embryos fertilized in vivo or in vitro successfully developed to the blastocyst stage in vitro.

Systematic studies of mouse embryo culture beginning in 1949 led to an understanding of essential medium components for early mammalian embryos, and embryo culture from the zygote to the blastocyst stage was achieved in 1968. Since then, medium components that are either beneficial or detrimental for embryo culture have been identified. A variety of culture media that mimic the female reproductive tract, such as human tubal fluid medium and sequential media, were developed from the 1970s to the 1990s, and a single medium in which the concentrations of components were determined by a simplex optimization method was introduced for clinical use in 2002. While either sequential media or a single medium is currently used in most cases, no conclusion has yet been reached as to which of the two approaches is the best. That we are now easily able to culture embryos is the result of the work of pioneers. This review presents a chronological overview of media development from initial attempts at mouse embryo culture using synthetic media to the human embryo culture media used today. It also presents the characteristics of sequential media and a single medium. Finally, problems observed with current embryo culture media are discussed, along with future development in this area.

Assisted reproductive technology (ART) has greatly benefited numerous infertile couples who would never have had their babies without this technique. However, in vitro culture is reported to cause epigenetic and transcriptomic changes on preimplantation embryos, leading to adverse effect on development, and little is known about the molecular mechanisms underlying these changes. Here, we first introduce key studies that designate the effectiveness of an omic strategy to explore the molecular mechanisms governing preimplantation development of in vitro-cultured embryos. Furthermore, we review how in vitro culture components facilitate genomic reprogramming and zygotic genome activation (ZGA) contributing to preimplantation development after somatic cell nuclear transfer (SCNT). From these different perspectives, we would search for a breakthrough to further optimize preimplantation embryo culture conditions and improve ART.

Mouse parthenogenetic (PG) embryos do not survive beyond day 9.5 of pregnancy. In this study, to understand the molecular mechanisms underlying the failure of parthenogenesis at the early developmental stage, we performed global gene expression profiling of the PG inner cell mass (ICM) and trophectoderm (TE) using microarray analysis, compared the results with those from in vitro-fertilized embryos, and identified genes whose expression levels showed more than a 4-fold change as a cutoff. Eighty probe sets were up-regulated and 59 probe sets down-regulated in the PG ICM, while 169 and 43 probe sets were respectively up-regulated and downregulated in PG TE. We selected two genes (Sfmbt2 and Gab1) that were down-regulated in both the PG ICM and TE, one gene (Nat1) that was down-regulated in the PG ICM, and one gene (Lysmd2) that was up-regulated in the PG TE, and analyzed the gene expression levels using real-time PCR. The quantitative expression levels of these four genes were confirmed by real-time PCR. In the present study, we identified differentially expressed genes in PG embryos and also identified those that were ICM- or TE-specific in PG embryos.

The survival probability of mouse blastocysts developed from nuclear transfer (NT) was estimated prior to embryo transfer using a novel biopsy method. NT embryos were separated into two at the 2-cell stage to produce monozygotic twin blastocysts from a single NT oocyte. We first examined efficient culture methods to produce monozygotic twin blastocysts. Then one of the two blastocysts was used for gene expression analysis, and the other was transferred to a recipient mouse. Co-culture of monozygotic twin blastomeres with fertilized embryos or culture in a small well in vitro improved the developmental potential to the blastocyst stage and increased the blastocyst cell number. Although 21.9% of twin somatic cell NT embryos had the same expression levels of Oct4 and Sox2 genes as fertilized embryos, no fetuses were obtained after transfer to recipients.

Recently, the quality of in vitro matured (IVM) oocytes has been improved by numerous studies using animal models. However, the implantation rates of IVM oocytes remain lower than those of in vitro fertilization (IVF). Chromosome abnormalities, which mainly occur during the first meiotic division, are often observed in IVM oocytes. To clarify how the properties of the cumulus cell layers relate to chromosome abnormalities of oocytes during IVM, we investigated the occurrence of aneuploidy and developmental competence using two different media in a mouse model. Maturation rates were significantly lower in denuded oocytes at oocyte collection in both types of maturation media as compared with those of oocytes mechanically denuded just after collection of cumulus-oocyte complex. Fertilization rates and blastocyst rates were lower in Waymouth's MB752/1 medium than in HTF medium. The occurrence of premature chromatid separation (PCS) remarkably increased in Waymouth's MB752/1 medium compared to HTF medium. This demonstrates that denuded oocytes at oocyte collection have low ability to progress to nuclear maturation under IVM conditions. However, the fertilization ability and developmental competence to the blastocyst stage of denuded oocytes were fulfilled under a suitable culture condition. We suggest that oocytes that are already denuded, before or during oocyte collection, have the potential to be matured in vitro if the denuded oocytes are cultured in a suitable culture media.

Although vitrification and follicular survival subsequent to xenotransplantation of canine ovarian tissues have been reported, the degree of cryoinjury after the vitrification has not yet been fully examined. Since cryoinjury after vitrification of ovaries has been reported in bovine and humans, this study evaluated canine ovarian tissues after vitrification by immunohistochemistry and TUNEL assay. Ovaries were collected from immature bitches. The ovarian tissue cubes were pretreated with 1 M DMSO. Subsequently, DAP 213 solution (2 M DMSO, 1 M acetamide, 3 M propylene glycol) was added. The cryotubes containing the ovarian tissues were placed on ice for 5 min, then plunged directly into liquid nitrogen. After warming with 0.25 M sucrose, cryopreserved ovarian tissues were fixed and subsequently stained with hematoxylin-eosin, proliferation cell nuclear antigen (PCNA) antibody, or active caspase-3 (AC-3) antibody, and evaluated by the TUNEL assay. There were no differences in the numbers of primordial, primary, secondary and antral follicles/mm² between the fresh and vitrified-warmed ovarian tissues. Percentages of PCNA and AC-3 antibody positive cells were similar regardless of the developmental stage of the follicles and experimental group. A few TUNEL positive cells were detected in both experimental groups. These results suggest that vitrification with DAP213 does not induce cryoinjury in ovarian tissues from immature bitches.