Primordial germ cells (PGCs), origin of the germ cell lineage, arise from epiblast cells in response to BMP4 secreted by adjacent extraembryonic ectoderm. We recently reconstituted the PGC specification in vitro using mouse embryonic stem cells (mESCs) as well as induced pluripotent stem cells (iPSCs). In the culture system, mESCs/iPSCs first differentiated into epiblast-like cells (EpiLCs) and then induced PGC-like cells from the EpiLCs. This manner of differentiation from mESCs to PGCs reproduces the manner of PGC specification in vivo. PGCs produced from mESCs, termed PGC-like cells (PGCLCs), were fully potent, since they differentiated into spermatozoa and in turn the fertilized eggs with the spermatozoa gave rise to healthy individuals. Although many attempts have been made to produce fully potent PGCs, this study was the first study demonstrating the successful production of healthy individuals from PGCLCs. This achievement was made possible by knowledge accumulated on the manner of PGC specification in vivo, the nature of self-renewing pluripotent stem cells, and growth factors endowing EpiLC formation and PGCLC induction in vitro. This article reviews the research advance that made it possible to reconstitute PGC specification in vitro from mESCs.

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Despite recent astonishing advances in the treatment of infertility by assisted reproductive technology (ART), the fundamental approaches to solving complete infertility due to congenital absence of germ cells or impaired fertility arising from gonadotoxic therapies during prepubertal childhood still remain unclear. Current findings in stem cell biology have resulted in new possibilities for the treatment of reproductive diseases, i.e., germ cell formation may be inducible in vitro. The idea of generating artificial germ cells or gametes may lead to a solution for the treatment of infertility. In this article, we review the recent advances in research into formation of primordial germ cells (PGCs), which are the precursors of gametes, in vitro.

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Parthenogenesis is the process in which an oocyte develops into an embryo without fertilization. Parthenogenetic activation can be performed at various stages of meiosis, yielding embryos with distinct genetic patterns of homozygosity and heterozygosity. Parthenogenetic embryonic stem (pES) cells derived from such embryos have heterozygous patterns that can be identified using genome-wide single nucleotide polymorphism (SNP) analysis, to determine whether extrusion of the first or second polar body has been inhibited. Heterozygous pES cells carrying the full complement of major histocompatibility complex (MHC) antigens matched to the oocyte donor may provide a potential source of immune-matched cells and tissues for cell replacement therapy. In this review, we summarize the process of deriving heterozygous MHC-matched pES cells using mouse and human models.

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Embryonic stem (ES) cells have been known to differentiate into various progenitor cells. In this study, we investigated the differentiation capacity of mouse ES cells into pancreatic hormone-secreting cells, and insulin-secreting cells. ES cells were cultured in Dulbecco’s modified Eagle medium (DMEM) after removal of leukemia inhibitory factor (LIF) for 3 days and then transferred in DMEM supplemented with retinoic acid or activin A. When the culture of embryoid bodies (EBs) derived from ES cells in DMEM added with activin A (2 × 10^-9 M or 2 × 10^-10 M) for 5 days was performed, endoderm marker genes, GATA4, Sox17 and Foxa2 were expressed in the EBs. However, at 6 days of culture, expression of Sox17 was not observed. When EBs were cultured with activin A (2 × 10^-9 M) for 6 days, and followed by 6 days of culture with retinoic acid (10^-6 M), expression of the pancreatic cell marker genes, GATA4 and Fxa2, were continued, and pancreatic islet genes, insulins 1 and 2, glucagons and somatostatin, were expressed from 5 days of culture. Immunohistochemistrical analysis gave results similar to RT-PCR. We consider this differentiation method for definitive endoderm production and pancreatic hormone—secreting cells, by using activin A and, retinoic acid is considered to be effective.

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The decline of human fecundability stems from compromised quality and reduced quantity of gametes. The age-related female infertility and spermatogenic failure have been the major obstacle to overcome in ART with only limited success unless donated gametes are used. I review the different approaches to manufacturing and regenerating gametes. Attempts of nuclear transplantation for preventing oocyte aneuploidy thereby enhancing the developmental competence or for inducing haploidization of somatic cell nuclei aiming to generate oocytes and spermatozoa are explored by highlighting advantages and limitations of these strategies. In addition, approaches to differentiate precursor cells or pluripotent stem cells to their progenies or mature gametes are revisited. Finally, I also report preliminary data on enhancing the reproductive performance of a single spermatozoon by male genome cloning utilizing micromanipulation techniques.

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We evaluated the efficacy and safety of two chemical oocyte activators, strontium chloride (SrCl₂) and calcium ionophore A23187 (A23187), after ICSI for patients with low fertilization (PLF) (less than 30%). Eighty-five PLF were randomly divided two groups: 35 patients in the SrCl₂ group (SG) and 50 patients in the A23187 group (AG). The control group (CG) was 530 patients who had undergone ICSI without artificial oocyte activation (AOA). The fertilization rate after AOA significantly increased from 24.7% to 54.5% in SG and from 20.9% to 62.4% in AG. Without AOA, there were no clinical pregnancies, but with AOA, 6 of 22 in SG and 9 of 37 in AG achieved pregnancies. Twenty-two babies (twenty singletons and one pair of twins) born after AOA had no abnormalities at birth [CG: 3.0% (16/530), SG: 0% (0/12), AG: 0% (0/10)]. Also, none of the infants’ developmental characteristics showed any significant difference from the CG. Consequently, AOA using A23187 or SrCl₂ is beneficial for PLF following ICSI and does not adversely affect the growth or health of infants in their first 4 yr. Further studies of clinical tests for proper patient selection, efficacy and safety of AOA in larger samples are needed.

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Developmental competence of in vitro matured human oocytes is dependent on the morphology of the cumulus-oocyte complex (COC) just after collection from the follicle. We postulated that COCs categorized as having poor morphology (two or fewer less than two layers of cumulus cells) would not secrete a sufficient amount of matulation factors, resulting in low developmental competence of the matured oocytes. In the present study, the level of progesterone secreted from good morphology COCs with three or more layers of cumulus cells, 39.2 ± 12.8 ng/ml (n=31), was significantly higher than that secreted from poor morphology COCs (9.65 ± 1.34 ng/ml, n=22). The addition of 20 ng/ml progesterone to in vitro maturation culture of the poor morphology group significantly improved the fertilization ability of the oocytes. The rates of development to the morula and blastosyst stages were also increased by progesterone, however the differences were not significant. In conclusion, the secreted level of progesterone during in vitro maturation of human COCs was dependent on the number of cumulus cells attached to oocyte. When an oocyte is surrounded by two or fewer 2 layers of cumulus cells, the addition of progesterone to FSH- and hCG-containing medium appears to be a useful method for obtaining an oocyte with a high developmental competence.

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To facilitate pronuclear microinjection, we have been developing the Vibratory Microinjection Systems (VMSs) that provides a micropipette with longitudinal vibration. The current VMS utilizes any frequency up to 100 kHz. We compared 35-kHz vibratory microinjection (VM) with ordinary microinjection (OM). Fourteen micropipettes were used to inject 420 BDF1 zygotes. Each micropipette finished its injection quota of 30 eggs, which were manipulated one by one alternately using the two types of microinjections, even when it repeatedly pulled out nuclear components. All microinjections were conducted at a compensation pressure of 30 hPa and digitally recorded for subsequent image analysis. VM resulted in slightly better embryonic development in 4-day culture than OM, but significantly shortened the time spent on microinjection and the time spent swelling the pronucleus by 25% and 30%, respectively. These pronuclear swelling times, together with the almost identical degrees of pronuclear swelling in both groups, suggested that VM injected a GFP solution at a 42% higher speed. VM significantly reduced the incidence of pulling out nuclear components, suggesting VM’s capability of removing the nuclear components already adhering to the micropipettes. These results indicate VMS is a useful option which is capable of raising the efficiency of microinjection significantly by saving time, labor and cost of microinjection.

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The theoretical rate of Y chromosome-bearing sperm (Y-sperm) and X chromosome-bearing (X-sperm) in ejaculate is 50:50 in mammals, therefore the sex ratio of the embryos following fertilization is expected to be 50:50. The sex ratio of embryos produced in vitro, however, is skewed towards either males or females. The primary aim of the present study was to investigate the factors of in vitro fertilization (IVF) that affect hte attachment or binding rate of bovine Y chromosome-bearing sperm (Y sperm rate) to the zona pellucidae (ZPe). Oocytes collected from bovine ovaries were cultured for 21h in vitro under various conditions. ZPe were collected from the oocytes. After frozen-thawed bovine semen and ZPe were co-incubated, the ZPe were mounted on glass slides. Then, sperm attached or bound to the ZPe were subjected to in situ hybridization with a Y chromosome-specific probe to determine the Y-sperm rate. In a preliminary experiment, the Y-sperm rate of frozen-thawed sperm was 50.2% without deviation from the theoretical rate (50%), and the Y sperm rate in Y sperm sorted semen was 93.4% as expected. In Experiment 1, the effect of the sperm-ZP coincubation period on the Y-sperm rate was investigated. Short coincubation periods (5min and 8min) deviated the Y-sperm rate (55.1% and 54.9%) from the theoretical rate, but long coincubation periods (300min) did not affect the Y-sperm rate (49.2%). In Experiment 2, a combination of preincubation of sperm for 3h prior to sperm-ZP coincubation and extension of the maturation period to 36h did not change the skewed Y-sperm rate of coincubation of untreated sperm with ZPe derived from 21-h-matured COCs (53.0%, 53.9%, and 54.5%, respectively). In conclusion, the ability of frozen-thawed sperm to attach to the ZPe during the first 5min or 8min of coincubation was slightly higher in Y sperm than X sperm although neither sperm preincubation nor extension of the maturation period affected the Y-sperm rate.

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The objective of the present study was to evaluate the viability of vitrified biopsied bovine embryos. Bovine embryos ranging from the compacted morula to blastocyst stage on day 7 (day 0=day of estrus) were collected from donor females, biopsied for sexing, and vitrified with ethylene glycol (EG) 25% (v/v), dimethylsulfoxide (DMSO) 25% (v/v) and 0.3% (w/v) bovine serum albumin dissolved in PBS using 0.25-ml straws. We assessed the concentrations of cryoprotectants in the straw after in-straw dilution, and the percentages (v/v) were 6.9-7.7% for EG and 2.8-3.3% for DMSO. The pregnancy rate of vitrified-warmed embryos in the in-straw dilution group (ISD group) was 57.9% when embryos were expelled from the straws after diluting the cryoprotectant in the straws, and that the survival of vitrified-warmed embryos were observed before transfer. In contrast, the pregnancy rate in the directly transfer group (DIR group) was 62.5% when embryos were transferred directly to recipients without expelling embryos from the straws. There were no differences among the pregnancy rates of the ISD group, the DIR group and the non-vitrified biopsied embryos (NV group) (56.3%). These results suggest that it was possible to warm the vitrified-biopsied bovine embryos on farms and to transfer them immediately to recipients, yielding a practical pregnancy rate.

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Spermatozoa used for intracytoplasmic sperm injection (ICSI) are selected based on their motility and morphology. To explore other methods for selecting better spermatozoa, we developed a spermatozoa-sorting method using a physiologically natural selection system involving penetration into cervical mucus (CM). In addition, we analyzed the spermatozoa that penetrated the CM (CM-penetrating spermatozoa). The results were as follows. The CM-penetrating spermatozoa traveled a longer distance with better linear motility than spermatozoa in semen. Also, in comparison with spermatozoa obtained by density gradient centrifugation (DGC) and the swim-up method, the proportion of spermatozoa with normal morphology was higher, although, the proportion of spermatozoa with the head vacuole did not change. No DNA fragmentation was detected in the CM-penetrating spermatozoa. This method has several advantages. The technical procedure is simple and easy. Physical damage to spermatozoa is reduced because it does not require any centrifugation or washing procedure. A higher collection ratio of morphologically normal spermatozoon is achievable compared to the DGC and swim-up method, and it is a physiological selection method. We conclude that the CM penetration-based spermatozoa-sorting method is a promising new technique for ICSI because it is possible to collect physiologically better spermatozoa than those selected in the conventional selection method.

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As reported previously, berberine, the main component extracted from Coptis rhizome and Phellodendron, has potential as a contraceptive for animals since berberine has a strong inhibitory effect on the in vitro development of mouse zygotes and on fetal development in vivo. The present study was undertaken to examine the effect of berberine treatment on the reversibility of the development of zygotes and gametes, and on the fertilization and subsequent development in the mouse. The reversibility of the berberine-induced inhibition of the development of mouse zygotes was dependent on the concentration used and treatment period. Berberine treatment did not inhibit the fertilizing capacity of epididymal spermatozoa and the fertilizability of oocytes at the second metaphase stage. The present study demonstrated that in vitro development of mouse zygotes is about 100 times more sensitive to berberine than the in vitro growth of mouse fetal fibroblast cells. The high stability of berberine at low temperatures for at least for 12 months and the high sensitivity of preimplantation embryos to berberine is useful information when considering the administration of berberine to females as a contraceptive.

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Purpose: To investigate the impact of the volume of cytoplasm aspirated into the injection pipette on the fertilization, embryo development and implantation ability after intracytoplasmic sperm injection. Methods: This was a preliminary observational study conducted between October 2010 and December 2010. We divided oocytes into two groups based on the farthest point reached by the aspirated cytoplasm. The intersection of the ICSI pipette and the outer surface of the zona pellucida was used as a marker. When the farthest point reached by aspirated cytoplasm from the tip of the injection pipette was less than the marker, we classified the oocyte as group A, and when it was beyond the marker, we classified the oocyte as group B. Results: The oocyte degeneration rate of group A (7%) was higher than that of group B (3%), but the difference was not significant. The fertilization rate (91% versus 71%) was significantly higher in group A than in group B (P = 0.007). On the other hand, the rates of survival, cleavage, good quality day-3 embryo, good quality blastocyst and pregnancy following single day-3 embryo or day-5 embryo transfers were similar between the two groups. Conclusions: These results indicate that the volume of cytoplasm aspirated into the injection pipette affects the fertilization rate, but does not influence embryo development or implantation ability after intracytoplasmic sperm injection.

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