Akira Iritani

Establishment of embryonic stem cell lines from cynomolgus monkey blastocysts produced by IVF or ICSI

Human embryonic stem (ES) cells are predicted to be a valuable source for producing ES‐derived therapeutic spare tissues to treat diseases by controlling their growth and differentiation. To understand the regulative mechanisms of their differentiation in vivo and in vitro, ES cells derived from nonhuman primates could be a powerful tool. We established four ES cell lines from cynomolgus monkey (Macaca fascicularis) blastocysts produced by in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI). The ES cells were characterized by the expression of specific markers such as alkaline phosphatase and stage‐specific embryonic antigen‐4. They were successfully maintained in an undifferentiated state and with a normal karyotype even after more than 6 months of culture. Pluripotential competence was confirmed by the formation of teratomas containing ectoderm‐, mesoderm‐, and endoderm‐ derivatives after subcutaneous injection into SCID mice. Differentiation to a variety of tissues was identified by immunohistochemical analyses using tissue‐specific antibodies. Therefore, we established pluripotent ES cell lines derived from monkeys that are widely used as experimental animals. These lines could be a useful resource for preclinical stem cell research, including allogenic transplantation into monkey models of disease.

Resurrection of a Bull by Cloning from Organs Frozen without Cryoprotectant in a −80°C Freezer for a Decade

Frozen animal tissues without cryoprotectant have been thought to be inappropriate for use as a nuclear donor for somatic cell nuclear transfer (SCNT). We report the cloning of a bull using cells retrieved from testicles that had been taken from a dead animal and frozen without cryoprotectant in a −80°C freezer for 10 years. We obtained live cells from defrosted pieces of the spermatic cords of frozen testicles. The cells proliferated actively in culture and were apparently normal. We transferred 16 SCNT embryos from these cells into 16 synchronized recipient animals. We obtained five pregnancies and four cloned calves developed to term. Our results indicate that complete genome sets are maintained in mammalian organs even after long-term frozen-storage without cryoprotectant, and that live clones can be produced from the recovered cells.

Expression and Functional Analyses of Circadian Genes in Mouse Oocytes and Preimplantation Embryos: Cry1 Is Involved in the Meiotic Process Independently of Circadian Clock Regulation

Abstract In mammals, circadian genes, Clock, Arntl (also known as Bmal1), Cry1, Cry2, Per1, Per2, and Per3, are rhythmically transcribed every 24 h in almost all organs and tissues to tick the circadian clock. However, their expression and function in oocytes and preimplantation embryos have not been investigated. In this study we found that the circadian clock may stop in mouse oocytes and preimplantation embryos. Real-time PCR analysis revealed the presence of transcripts of these genes in both oocytes and preimplantation embryos; however, their amounts did not oscillate every 24 h in one- to four-cell and blastocyst-stage embryos. Moreover, immunofluorescence analyses revealed that CLOCK, ARNTL, and CRY1 were localized similarly in the nuclei of germinal vesicle (GV) oocytes and one-cell- to four-cell-stage embryos. Because CRY1 is known to interact with the CLOCK-ARNTL complex to suppress transcription-promoting activity of the complex for genes such as Wee1, Cry2, Per1, Per2, and Per3 in cells having the ticking circadian clock, we hypothesized that if the circadian clock functions in GV oocytes and one-cell- to four-cell-stage embryos, CLOCK, ARNTL, and CRY1 might suppress the transcription of these genes in GV oocytes and one-cell- to 4-cell-stage embryos as well. As a result, knockdown of CRY1 in GV oocytes by RNA interference did not affect the transcription levels of Wee1, Cry2, Per1, Per2, and Per3, but it reduced maturation ability. Thus, it seems that circadian genes are not involved in circadian clock regulation in mouse oocytes and preimplantation embryos but are involved in physiologies, such as meiosis.

Early morphological events of in vitro fertilized bovine oocytes with frozen-thawed spermatozoa

Bovine follicular oocytes were matured and inseminated in vitro with spermatozoa capacitated in vitro. The first evidence of sperm penetration was observed at 3 h after insemination. The penetration rate increased until 5 h, and reached a maximum rate (92%) at 5 h. Decondensation of the sperm head and pronuclear formation were observed 4 h and 7 h after insemination, respectively. Female chromatins of all penetrated oocytes were activated at 3 h, and female pronuclei were formed at 7 h after insemination. Percentages of oocytes with male and female pronuclei at 9 h were 88 and 94%. Polyspermy (4, 7, 19 and 29% at 4, 5, 7 and 9 h after insemination, respectively) and abnormal development of male pronuclei (6 and 7% at 7 and 9 h after insemination, respectively) were also seen.

Meiotic Arresting Substance Separated from Porcine Ovarian Granulosa Cells and Hypothetical Arresting Mechanism of Meiosis

In mammals, oogonia enter prophase of the first meiotic division in prenatal life, and shortly after birth, all oocytes are arrested in a late prophase known as the dictyate stage, and when an oocyte in an ovary reaches the end of its growth period, it contains a single large nucleus called the germinal vesicle (Franchi et al., 1962). It is now generally accepted that the resumption of the arrested first meiotic division occurs as a result of the surge of luteinizing hormone (LH) at each ovarian cycle (Shuetz, 1969; Tsafriri, 1978a), although the ability to complete the first meiotic division is related as well to the stage of follicular development (Tsafriri and Channing, 1975a; Sorensen and Wassarman, 1976).

Effect of visual light on in vitro embryonic development in the hamster.

The effect of light exposure during collection and culture of hamster embryos on their subsequent development in vitro was examined. When embryos were collected under dark conditions (70 lux) within 10 minutes and then cultured in a HECM-1 medium in 5% CO2 in air, the developmental rates of 1-cell embryos to the 4- and 8-cell stages were 88.6% (93/105) and 66.7% (70/105), respectively. These rates were significantly higher than those under light conditions (1600 lux): 51.9% (56/108) and 34.3% (37/108). Light irradiation during the culture of 1-cell embryos suppressed subsequent development. The degree of suppression correlated inversely with duration of light irradiation, and light irradiation of 30 minutes or more completely blocked development to the 2-cell stage. When 1-cell embryos were irradiated through a yellow filter, cutting the light wavelengths to less than 500 nm, embryonic development was still suppressed. However, the degree of the suppression varied and 45.7% (53/116), 6.0% (7/116), and 0.9% (1/116) of the embryos developed to the 2-, 4-, and 8-cell stages, respectively, under 30 minute light irradiation. Inhibitory effects of light irradiation on the development of 2- and 8-cell embryos were also observed, showing an inverse correlation with duration; the developmental rates of 2-cell embryos to the 8-cell stage under 0, 10, and 30 minutes of irradiation were 85.6% (107/125), 1.6% (2/122), and 0% (0/129), respectively, and those of 8-cell embryos to the blastocyst stage were 79.8% (91/114), 74.8% (86/115), and 0% (0/110), respectively. These findings indicate that early-stage embryos are sensitive to light exposure; however, severe light exposure adversely affects the development of embryos at any stage. Thus, the protection of embryos from light exposure at all stages of embryo manipulation, from collection to culture, is essential.

Abnormal DNA methylation of the Oct-4 enhancer region in cloned mouse embryos

Oct‐4 is essential for normal embryonic development, and abnormal Oct‐4 expression in cloned embryos contributes to cloning inefficiency. However, the causes of abnormal Oct‐4 expression in cloned embryos are not well understood. As DNA methylation in regulatory regions is known to control transcriptional activity, we investigated the methylation status of three transcriptional regulatory regions of the Oct‐4 gene in cloned mouse embryos—the distal enhancer (DE), the proximal enhancer (PE), and the promoter regions. We also investigated the level of Oct‐4 gene expression in cloned embryos. Immunochemistry revealed that 85% of cloned blastocysts expressed Oct‐4 in both trophectoderm and inner cell mass cells. DNA methylation analysis revealed that the PE region methylation was greater in cloned morulae than in normal morulae. However, the same region was less methylated in cloned blastocysts than in normal blastocysts. We found abnormal expression of de novo methyltransferase 3b in cloned blastocysts. These results indicate that cloned embryos have aberrant DNA methylation in the CpG sites of the PE region of Oct‐4, and this may contribute directly to abnormal expression of this gene in cloned embryos. Mol. Reprod. Dev. 76: 342–350, 2009.

Cloning and sequencing of cDNA that encodes goat growth hormone

The cDNA that encodes goat growth hormone (gGH) was isolated from a goat pituitary cDNA library. The cDNA, about 880 base pairs long, had a coding sequence, 5′‐ and 3′‐untranslated regions and a poly(A) chain. The cDNA could encode a polypeptide of 217 amino acids. The amino acid sequence homology between gGH and the sequences of bovine GH, rat GH and human GH was 99, 83 and 66%, respectively. By Northern blot hybridization, we found that the possible gGH gene is transcribed in the goat pituitary.

Cis-acting elements (E-box and NBE) in the promoter region of three maternal genes (Histone H1oo, Nucleoplasmin 2, and Zygote Arrest 1) are required for oocyte-specific gene expression in the mouse.

We examined the promoter activities of three mouse maternal genes (H1oo, Npm2, and Zar1) in oocytes and pre‐implantation embryos, and examined the promoters for cis‐acting elements of 5′‐flanking region to obtain the best promoter for inducing oocyte‐specific gene expression. For the assay, we injected firefly luciferase gene constructs under the control of the promoters into the oocytes and embryos. Each promoter region showed transcriptional activity in oocytes, but not in fertilized embryos. Deletion analysis showed that a putative E‐box region at position −72 of the H1oo promoter and at the −180 of the Npm2 promoter were required for basal transcriptional activity in oocytes. Moreover, a putative NBE motif (NOBOX DNA binding elements) (−1796) was shown to enhance basal transcriptional activity of the Npm2 promoter. Thus, the E‐box and/or NBE may be key regulatory regions for the expression of the examined maternal genes (H1oo and Npm2) in growing mouse oocytes. Mol. Reprod. Dev. 75: 1104–1108, 2008.

Full-term development of bovine follicular oocytes matured in culture and fertilized in vitro

Follicular oocytes were cultured for 28h in vitro and 91% of the oocytes reached the the second metaphase in culture. The penetration rate after insemination in vitro using frozen-thawed spermatozoa was 81%. After cultivation for 48h in vitro, 18% of the in vitro fertilized oocytes developed to the three- to four-cell stages and 21% of these developed to the six- to eight-cell stages. Following in vivo culture in the rabbit oviduct, 18% of six- to eight-cell and 5% of three- to four-cell embryos developed to the blastocyst stage. To confirm the full developmental competence, 11 blastocysts were transferred to recipient cows, and six (55%) cows became pregnant or delivered calves.