Toshitaka Horiuchi

Long-Term Culture of Male Germline Stem Cells From Hamster Testes

Abstract Spermatogonial stem cells provide the foundation for spermatogenesis in male animals. We recently succeeded in culturing and genetically engineering mouse spermatogonial stem cells, but little is known regarding the culture and growth requirements of spermatogonial stem cells in other animal species. In this study, we report the successful long-term culture of spermatogonial stem cells from hamster testes. Spermatogonial stem cells were purified using an anti-ITGA6 antibody and cultured in the presence of glial cell line-derived neurotrophic factor. The cells continued to proliferate for at least 1 year. During this period, they were genetically modified using a lentivirus and underwent spermatogenesis after transplantation into the testes of immunodeficient nude mice. However, germ cells generated in the surrogate xenogeneic recipients did not differentiate beyond the spermatid stage, and these round spermatids could not produce offspring through in vitro microinsemination. These results suggest that the germ cells may not have acquired characteristics necessary for fertility in the xenogeneic microenvironment. Nevertheless, the successful establishment of culture conditions conducive for hamster spermatogonial stem cell growth and maintenance indicates that this technique can be extended to other animal species in which current genetic modification techniques are impossible or inefficient.

Effects of light on development of mammalian zygotes.

It is generally assumed that light has no effect on the physiology of oocytes, zygotes, or early embryos. Therefore, little or no attention has been paid to lighting conditions during the handling of these cells in vitro. Here we show that cool white fluorescent light, rich in short-wavelength visible light and commonly used in research and clinical laboratories, produces more reactive oxygen species in mouse and hamster zygotes than does warm white fluorescent light. Mouse blastocysts that developed from zygotes shielded from light best developed to term fetuses followed by those exposed to warm white fluorescent light and then by those exposed to cool white fluorescent light. We hypothesized that light is one of the physical factors affecting embryonic environment and that its effects on cultured mammalian zygotes and embryos should not be overlooked.

Full-Term Development of Golden Hamster Oocytes Following Intracytoplasmic Sperm Head Injection

Abstract The golden hamster is the mammalian species in which intracytoplasmic sperm injection (ICSI) was first tried to produce fertilized oocytes. Thus far, however, there are no reports of full-term development of hamster oocytes fertilized by ICSI. Here we report the birth of hamster offspring following ICSI. Keys to success were 1) performing ICSI in a dark room with a small incandescent lamp and manipulating both oocytes and fertilized eggs under a microscope with a red light source and 2) injecting sperm heads without acrosomes. All oocytes injected with acrosome-intact sperm heads died within 3 h after injection, while those oocytes injected with acrosomeless sperm heads survived injection. Under illumination with red light in a dark room, the majority of the oocytes injected with acrosomeless sperm heads were fertilized normally (77%), cleaved (91%), and developed into morulae (49%). Of the 47 morulae transferred to five recipient females, nine (19%) developed to live offspring.

Visualization of cell cycle in mouse embryos with Fucci2 reporter directed by Rosa26 promoter

Fucci technology makes possible the distinction between live cells in the G1 and S/G2/M phases by dual-color imaging. This technology relies upon ubiquitylation-mediated proteolysis, and transgenic mice expressing Fucci provide a powerful model system with which to study the coordination of the cell cycle and development. The mice were initially generated using the CAG promoter; lines expressing the G1 and S/G2/M phase probes that emitted orange (mKO2) and green (mAG) fluorescence, respectively, were separately constructed. Owing to cell type-biased strength of the CAG promoter as well as the positional effects of random transgenesis, however, we noticed some variability in Fucci expression levels. To control more reliably the expression of cell cycle probes, we used different genetic approaches to create two types of reporter mouse lines with Fucci2 and Rosa26 transcriptional machinery. Fucci2 is a recently developed Fucci derivative, which emits red (mCherry) and green (mVenus) fluorescence and provides better color contrast than Fucci. A new transgenic line, R26p-Fucci2, utilizes the Rosa26 promoter and harbors the G1 and S/G2/M phase probes in a single transgene to preserve their co-inheritance. In the other R26R-Fucci2 approach, the two probes are incorporated into Rosa26 locus conditionally. The Cre-mediated loxP recombination technique thus allows researchers to design cell-type-specific Fucci2 expression. By performing time-lapse imaging experiments using R26p-Fucci2 and R26-Fucci2 in which R26R-Fucci2 had undergone germline loxP recombination, we demonstrated the great promise of these mouse reporters for studying cell cycle behavior in vivo.

Human Sperm Aster Formation and Pronuclear Decondensation in Bovine Eggs Following Intracytoplasmic Sperm Injection Using a Piezo-Driven Pipette: A Novel Assay for Human Sperm Centrosomal Function

Abstract In human fertilization, the sperm introduces the centrosome; the microtubule-organizing center and microtubules are organized within the inseminated egg from the sperm centrosome. These microtubules form a radial array, called the sperm aster, the functioning of which is essential to pronuclear movement for union of male and female genome. The sperm centrosomal function is considered to be necessary for the normal human fertilization process. Therefore, the dysfunction of sperm centrosome is a possible cause of human fertilization failure. However, little information is available regarding human sperm centrosomal function during fertilization in clinically assisted reproductive technology. To assess the human sperm centrosomal function, we examined sperm aster formation and pronuclear decondensation following intracytoplasmic sperm injection (ICSI) with human sperm into the bovine egg using a Piezo-driven pipette and ethanol activation of eggs. After human sperm incorporation into bovine egg, we observed that the sperm aster was organized from sperm centrosome, and that the sperm aster was enlarged as the sperm nuclei underwent pronuclear formation. The sperm aster formation rate at 6 h post-ICSI and the male pronuclear formation rate at 8–12 h post-ICSI were 60.0% and 83.3%, respectively. No difference of the sperm aster formation rate and the male pronuclear formation rate was observed between eggs activated with ethanol and eggs without artificial activation. We concluded that this heterologous Piezo-ICSI system into bovine egg can be a novel assay for human sperm centrosomal function, and it is possible to explicate a course of fertilization failure that was unknown until now.

A successful pregnancy and live birth after intracytoplasmic sperm injection with globozoospermic sperm and electrical oocyte activation

OBJECTIVE To present the effectiveness of intracytoplasmic sperm injection (ICSI) using globozoospermic sperm and assisted oocyte activation by electrical stimulation. DESIGN A case report. SETTING A private IVF center in Japan. PATIENT(S) A man with globozoospermia. INTERVENTION(S) Acridine orange (AO) test, mouse oocyte activation test, and ICSI with electrical oocyte activation. MAIN OUTCOME MEASURE(S) Fertilization, pregnancy, and live birth. RESULT(S) In the first ICSI attempt, neither of the two injected oocytes fertilized. Staining of the patients sperm with AO showed that only 2.9% of the sperm emitted a green fluorescence at the characteristic round head (sperm with native DNA content). The mouse oocyte activation test using the roundheaded sperm showed that the normal fertilization rate was 78.9% when SrCl(2) was used for assisted oocyte activation; however, it was 6.0% without assisted oocyte activation. We confirmed that the sperm had defective ability to activate oocytes. In the second ICSI attempt, human oocytes were activated electrically with use of a single square direct current pulse after microinjection. All the seven injected oocytes fertilized normally, and two eight-cell embryos were transferred on day 3. Clinical pregnancy was confirmed, and a healthy girl weighing 2362 g was delivered at 37 weeks of gestation by cesarean section. CONCLUSION(S) This is the first successful outcome of ICSI using globozoospermic sperm and electrical oocyte activation. The electroactivation obviates the need for the use of potentially harmful drugs for activation.

Impact of the size of zona pellucida thinning area on vitrified-warmed cleavage-stage embryo transfers: a prospective, randomized study

PurposeThe aim of this study was to determine if the size of zona pellucida thinning area by laser assisted hatching could affect the rates of pregnancy and implantation for vitrified-warmed embryo transfers at the cleavage-stage.MethodsA total of 120 vitrified-warmed cleavage-stage embryo transfers were randomly assigned to either quarter or half of zona pellucida thinning group.ResultsThe rates of clinical pregnancy (46.7 versus 25.0%) and implantation (32.0 versus 16.2%) were significantly greater in the half thinning group than in the quarter thinning group (P = 0.0218 and P = 0.0090, respectively).ConclusionsThe results of this investigation show that, in vitrified-warmed embryo transfers at the cleavage-stage, the size of zona pellucida thinning area by laser assisted hatching impacts the rate of clinical pregnancy and implantation and that half of zona pellucida thinning significantly increases both of these results compared with quarter of zona pellucida thinning.

Full-Term Development of Hamster Embryos Produced by Injection of Round Spermatids into Oocytes

Abstract The golden hamster is a mammal in which microinjection of round spermatids into oocytes (ROSI) was first attempted. However, no live ROSI offspring have ever been obtained in this species. This is the first report of live hamster offspring obtained by round spermatid injection. Over 90% of oocytes, injected with round spermatids, were activated without any additional stimulation. The proportion of the oocytes that were fertilized normally and that developed to morulae and blastocysts was higher when the plasma membranes of the spermatids were broken before injection, as compared with when the membranes were left intact. Five percent of 57 ROSI morulae/blastocysts developed into live offspring after transfer to foster mothers.

Production efficiency of Japanese black calves by transfer of bovine embryos produced in vitro

The objective of this study was to examine the production efficiency of Japanese Black beef calves after transfer of bovine embryos derived from an in vitro procedure. In vitro-produced (IVP) embryos were obtained from in vitro maturation and fertilization and in vitro development by co-culture with cumulus cells until 7 or 8 days after insemination. In vivo-developed (IVD) embryos from superovulated Japanese Black heifers and cows 7 days after artificial insemination were used as a control group. Bovine embryos were transferred nonsurgically to recipient cows on Day 7 +/- 1 of the estrous cycle. Pregnancy was diagnosed by palpation per rectum at Day 60 to 70 after estrus. Pregnancy, abortion, perinatal accident and birth rates were examined according to the origin of embryos (IVP or IVD), the number of transferred embryos (single or twin) and the storage status (fresh or frozen-thawed). In Experiment 1, production efficiency by twin transfer of fresh IVP embryos was examined. Higher pregnancy rates (52 1% vs 42 9%, P < 0.05) and birth rates (47.0% vs. 33.0%, P < 0.05) were obtained by twin transfer than by single transfer of fresh IVP embryos. Thus, the twin transfer of fresh IVP embryos was effective for production of calves, although the birth rates for single and twin transfers of fresh IVD embryos were still higher (55.5% and 76.1%, P < 0.05). But the abortion and perinatal accident rates for twin transfer of fresh IVP embryos were also significantly greater than those for single and twin transfer of fresh IVD embryos (P < 0.05). In Experiment 2, production efficiency by twin transfer of frozen-thawed IVP embryos was examined. Either single or twin transfer of frozen-thawed IVP embryos resulted in a similar pregnancy rate (41.3% vs. 46.7%, P > 0.05) and birth rate (34.1% vs. 41.1%, P>0.05). Thus, in combination with frozen-thawed IVP embryos, the twin transfer did not enhance production efficiency. In conclusion, Japanese Black beef calves could effectively produce calves by twin transfer to Holstein recipients when using fresh IVP embryos, and by single transfer when using frozen-thawed IVP embryos.

Zona pellucida removal and vitrified blastocyst transfer outcome: a preliminary study

The objective of this study was to investigate whether a change in assisted hatching technique from partial opening to total removal of the zona pellucida improved the outcome of vitrified blastocyst transfer. This was a preliminary observational study conducted from November 2003 to April 2006. Partial opening using acid Tyrodes solution was performed in 45 cycles, while total removal using a laser and mechanical pipetting was performed in 57 cycles. The clinical pregnancy, implantation, and delivery rates were higher in the total removal group than in the partial opening group (67% versus 42%, P < 0.02; 55% versus 30%, P < 0.01; 56% versus 36%, P < 0.04, respectively). These results suggest that total removal of the zona pellucida is associated with higher pregnancy, implantation and delivery rates compared with partial opening for vitrified blastocyst transfer.