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D1Pas1 is a mouse autosomal DEAD-box RNA helicase expressed predominantly in the testis. To assess its possible function, we generated D1Pas1-deficient mice using embryonic stem cells with a targeted D1Pas1 allele. Deletion of D1Pas1 did not cause noticeable embryonic defects or death, indicating that D1Pas1 is not essential for embryogenesis. Whereas homozygous knockout female mice showed normal reproductive performance, homozygous knockout male mice were completely sterile. The seminiferous epithelium of D1Pas1-deficient males contained no spermatids or spermatozoa because of spermatogenic arrest at the late pachytene stage. Upregulation of retrotransposons such as LINE-1 was not found in D1Pas1-deficient males, unlike males lacking Mvh, another testicular DEAD-box RNA helicase. Meiotic chromosome behavior in developing spermatocytes of D1Pas1-deficient males was indistinguishable from that in wild-type males, at least until synaptonemal complex formation. Thus, mouse D1Pas1 is the first-identified DEAD-box RNA helicase that plays critical roles in the final step of the first meiotic prophase in male germ cells., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Faithful transcriptome regulation is important in development and also crucial for applications in reproductive and regenerative medicine. Intracytoplasmic sperm injection (ICSI), one of the human assisted reproductive technologies (ART), has long raised concerns about its influence on development. No clear consensus has been reached, however, in spite of many cohort studies carried out in the last two decades on the children conceived by ICSI and/or in vitro fertilization (IVF). In this study, the pre- and postnatal effects of ICSI were assessed using comprehensive transcriptome and phenotypic analyses in mice under strict conditions. Here we demonstrate that, in contrast to IVF, ICSI induces distinct long-lasting transcriptome change that remains at the neonatal stage. Importantly, no remarkable differences were observed in the ICSI adults in either the gene expression or phenotypic profiles, and there was no indication of transmission to the next generation via natural mating. Our results suggest there are no lifelong or transgenerational effects of ICSI, but the ICSI effects during neonatal period remain to be evaluated., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Somatic cell cloning by nuclear transfer (NT) in mice is associated with hyperplastic placentas at term. To dissect the effects of embryonic and extraembryonic tissues on this clone-associated phenotype, we constructed diploid (2n) fused with (<-->) tetraploid (4n) chimeras from NT- and fertilization-derived (FD) embryos. Generally, the 4n cells contributed efficiently to all the extraembryonic tissues but not to the embryo itself. Embryos constructed by 2n NT<-->4n FD aggregation developed hyperplastic placentas (0.33+/-0.22 g) with a predominant contribution by NT-derived cells. Even when the population of FD-derived cells in placentas was increased using multiple FD embryos (up to four) for aggregation, most placentas remained hyperplastic (0.36+/-0.13 g). By contrast, placentas of the reciprocal combination, 2n FD<-->4n NT, were less hyperplastic (0.15+/-0.02 g). These nearly normal-looking placentas had a large proportion of NT-derived cells. Thus, embryonic rather than extraembryonic tissues had more impact on the onset of placental hyperplasia, and that the abnormal placentation in clones occurs in a noncell-autonomous manner. These findings suggest that for improvement of cloning efficiency we should understand the mechanisms regulating placentation, especially those of embryonic origin that might control the proliferation of trophoblastic lineage cells.

Although embryonic stem (ES) cell lines derived from mice and primates are used extensively, the development of such lines from other mammals is extremely difficult because of their rapid decline in proliferation potential and pluripotency after several passages. This study describes the establishment of rabbit ES cell lines with indefinite proliferation potential. It was found that the feeder cell density determines the fate of rabbit ES cells, and that maximum proliferation potential was obtained when they were cultured on a feeder cell density of one-sixth of the density at confluency. Higher and lower densities of feeder cells induced ES cell differentiation or division arrest. Under optimized conditions, rabbit ES cells were passaged 50 times, after which they still possessed high telomerase activity. This culture system enabled efficient gene transduction and clonal expansion from single cells. During culture, rabbit ES cells exhibited flattened monolayer cell colonies, as reported for monkey and human ES cells, and expressed pluripotency markers. Embryoid bodies and teratomas formed readily in vitro and in vivo respectively. These ES cell lines can be safely cryopreserved for later use. Thus, rabbit ES cells can be added to the list of stable mammalian ES cells, enabling the rabbit to be used as a small animal model for the study of human cell transplantation therapy.

Hyperplastic placentas have been reported in several experimental mouse models, including animals produced by somatic cell nuclear transfer, by inter(sub)species hybridization, and by somatic cytoplasm introduction to oocytes followed by intracytoplasmic sperm injection. Of great interest are the gross and histological features common to these placental phenotypes--despite their quite different etiologies--such as the enlargement of the spongiotrophoblast layers. To find morphological clues to the pathways leading to these similar placental phenotypes, we analyzed the ultrastructure of the three different types of hyperplastic placenta. Most cells affected were of trophoblast origin and their subcellular ultrastructural lesions were common to the three groups, e.g., a heavy accumulation of cytoplasmic vacuoles in the trophoblastic cells composing the labyrinthine wall and an increased volume of spongiotrophoblastic cells with extraordinarily dilatated rough endoplasmic reticulum. Although the numbers of trophoblastic glycogen cells were greatly increased, they maintained their normal ultrastructural morphology, including a heavy glycogen deposition throughout the cytoplasm. The fetal endothelium and small vessels were nearly intact. Our ultrastructural study suggests that these three types of placental hyperplasias, with different etiologies, may have common pathological pathways, which probably exclusively affect the development of certain cell types of the trophoblastic lineage during mouse placentation.

Germ cells have unique features strikingly distinguishable from somatic cells. The functional divergence between these two cell lineages has been postulated to result from epigenetic mechanisms. Here we show that the chromosomal centric and pericentric (C/P) regions in male and female germline cells are specifically DNA-hypomethylated, despite the hypermethylation status in somatic cells. In multipotent germline stem cells, the C/P region was initially hypomethylated and then shifted to the hypermethylation status during differentiation into somatic lineage in vitro. Moreover, the somatic-type hypermethylation pattern was maintained in the somatic cell-derived nuclear transfer embryos throughout preimplantation development. These results imply that the identity of germ cell lineage may be warranted by the hypomethylation status of the C/P region as an epigenetic signature.

Spermatogonial stem cells can convert into embryonic stem (ES) cell-like multipotent germline stem (mGS) cells in vitro and produce germline chimeras by blastocyst microinjection. Although homologous recombination was previously demonstrated in mGS cells, spermatogenesis was not found in chimeras, suggesting that they are not competent for germline modification. Here we conducted detailed analysis of chimeric animals to determine whether mGS cells retain germline potential after genetic manipulation. Spermatozoa that were deficient in the occludin gene could be recovered from animals that were chimeric with mGS cells that underwent homologous recombination. The phenotypes of the occludin knockout (KO) mice were similar to those reported for KO mice produced using ES cells, and the animals showed growth retardation, gastritis and male infertility. Furthermore, we found that heterozygous mGS cells acquire two copies of the G418-resistant genes and become homozygous for the targeted allele by culturing at high concentrations of G418. Cytogenetic analysis showed that the aneuploid mGS cells observed during genetic manipulation were trisomic for chromosome 8 or 11, which is a common chromosomal abnormality in ES cells. Thus, mGS cells can be used to produce KO animals, and this novel method of germline manipulation may prove useful in diverse mammalian species.

Somatic cell nuclear transfer technology has been applied to produce live clones successfully in several mammalian species, but the success rates are very low. In mice, about half of the nuclear transfer embryos undergo implantation, but very few survive to term. We undertook detailed histological analyses of placentas from cloned mouse embryos generated from cumulus cells at 10.5 dpc of pregnancy, by which stage most clones have terminated their development. At 10.5 dpc, the extraembryonic tissues displayed several defined histological patterns, each reflecting their stage of developmental arrest. The most notable abnormality was the poor development of the spongiotrophoblast layer of diploid cells. This is in contrast to the placental hyperplasia frequently observed in somatic clones at 12.5 dpc or later stages. A variety of structural abnormalities were also observed in the embryos. Both placental and embryonic defects likely contribute to the low success rate of the mouse clones.

Since the development of a reliable mouse intracytoplasmic sperm injection (ICSI) technique in 1995, microinsemination techniques have been widely applied in several laboratory species. As gametes and embryos have specific biological and biochemical features according to the species, technical improvements are necessary for successful microinsemination that subsequently leads to normal fetal development in several species. Recent advanced reproductive research involving genetic engineering often depends on microinsemination techniques that require a high degree of skill, and new human assisted reproductive technology (ART) requires experimental models using laboratory animals. The accumulation of technical improvements in these fields should accelerate the development of microinsemination techniques in mammals, including humans., (Copyright 2002 Elsevier Science Inc.)

Six simple methods for short-term (up to 8 d), nonfrozen (5 to 20 degrees C) storage of mouse epididymides were compared with respect to the motility and fertility of spermatozoa. A high percentage of progressively motile spermatozoa was obtained from epididymis stored for 8 d at 5 degrees C in mineral oil (78.3%), covered with body fat (80.0%), or stored in the intact body of the euthanized donor animal (77.5%). Fertilized eggs (6.4% fertilization rate) were obtained by IVF using spermatozoa that had been stored in mineral oil at 5 degrees C for at least 8 d, and offspring were obtained from 77.5% of transferred eggs that were fertilized by spermatozoa stored for 2 d. These methods inhibited moisture loss from the preserved epididymal spermatozoa, thereby allowing spermatozoa to be stored for a few days without loss of either motility or fertility. These methods make possible such wide-ranging applications as the long-distance transport of epididymis spermatozoa. While in storage at 5 degrees C, the tail of each recovered spermatozoon was bent midway along the tail, possibly owing to damage to the plasma membranes and due to the spermatozoa's hardening in the phospholipid by exposure to the low temperature.