Direct evidence that KNDy neurons maintain gonadotropin pulses and folliculogenesis as the GnRH pulse generator.

The gonadotropin-releasing hormone (GnRH) pulse is fundamental for mammalian reproduction: GnRH pulse regimens are needed as therapies for infertile women as continuous GnRH treatment paradoxically inhibits gonadotropin release. Circumstantial evidence suggests that the hypothalamic arcuate KNDy neurons expressing kisspeptin (encoded by Kiss1), neurokinin B (encoded by Tac3), and dynorphin A serve as a GnRH pulse generator; however, no direct evidence is currently available. Here, we show that rescuing >20% KNDy neurons by transfecting Kiss1 inside arcuate Tac3 neurons, but not outside of these neurons, recovered folliculogenesis and luteinizing hormone (LH) pulses, an indicator of GnRH pulses, in female global Kiss1 knockout (KO) rats and that >90% conditional arcuate Kiss1 KO in newly generated Kiss1-floxed rats completely suppressed LH pulses. These results first provide direct evidence that KNDy neurons are the GnRH pulse generator, and at least 20% of KNDy neurons are sufficient to maintain folliculogenesis via generating GnRH/gonadotropin pulses.

LGI1-ADAM22-MAGUK configures transsynaptic nanoalignment for synaptic transmission and epilepsy prevention.

Physiological functioning and homeostasis of the brain rely on finely tuned synaptic transmission, which involves nanoscale alignment between presynaptic neurotransmitter-release machinery and postsynaptic receptors. However, the molecular identity and physiological significance of transsynaptic nanoalignment remain incompletely understood. Here, we report that epilepsy gene products, a secreted protein LGI1 and its receptor ADAM22, govern transsynaptic nanoalignment to prevent epilepsy. We found that LGI1-ADAM22 instructs PSD-95 family membrane-associated guanylate kinases (MAGUKs) to organize transsynaptic protein networks, including NMDA/AMPA receptors, Kv1 channels, and LRRTM4-Neurexin adhesion molecules. Adam22ΔC5/ΔC5 knock-in mice devoid of the ADAM22-MAGUK interaction display lethal epilepsy of hippocampal origin, representing the mouse model for ADAM22-related epileptic encephalopathy. This model shows less-condensed PSD-95 nanodomains, disordered transsynaptic nanoalignment, and decreased excitatory synaptic transmission in the hippocampus. Strikingly, without ADAM22 binding, PSD-95 cannot potentiate AMPA receptor-mediated synaptic transmission. Furthermore, forced coexpression of ADAM22 and PSD-95 reconstitutes nano-condensates in nonneuronal cells. Collectively, this study reveals LGI1-ADAM22-MAGUK as an essential component of transsynaptic nanoarchitecture for precise synaptic transmission and epilepsy prevention.

Kisspeptin neurons as a key player bridging the endocrine system and sexual behavior in mammals.

Reproductive behaviors are sexually differentiated: for example, male rodents show mounting behavior, while females in estrus show lordosis behavior as sex-specific sexual behaviors. Kisspeptin neurons govern reproductive function via direct stimulation of gonadotropin-releasing hormone (GnRH) and subsequent gonadotropin release for gonadal steroidogenesis in mammals. First, we discuss the role of hypothalamic kisspeptin neurons as an indispensable regulator of sexual behavior by stimulating the synthesis of gonadal steroids, which exert "activational effects" on the behavior in adulthood. Second, we discuss the central role of kisspeptin neurons that are directly involved in neural circuits controlling sexual behavior in adulthood. We then focused on the role of perinatal hypothalamic kisspeptin neurons in the induction of perinatal testosterone secretion for its "organizational effects" on masculinization/defeminization of the male brain in rodents during a critical period. We subsequently concluded that kisspeptin neurons are key players in bridging the endocrine system and sexual behavior in mammals.

Cellular and molecular mechanisms regulating the KNDy neuronal activities to generate and modulate GnRH pulse in mammals.

Accumulating findings during the past decades have demonstrated that the hypothalamic arcuate kisspeptin neurons are supposed to be responsible for pulsatile release of gonadotropin-releasing hormone (GnRH) to regulate gametogenesis and steroidogenesis in mammals. The arcuate kisspeptin neurons express neurokinin B (NKB) and dynorphin A (Dyn), thus, the neurons are also referred to as KNDy neurons. In the present article, we mainly focus on the cellular and molecular mechanisms underlying GnRH pulse generation, that is focused on the action of NKB and Dyn and an interaction between KNDy neurons and astrocytes to control GnRH pulse generation. Then, we also discuss the factors that modulate the activity of KNDy neurons and consequent pulsatile GnRH/LH release in mammals.

Germline development in rat revealed by visualization and deletion of Prdm14.

Primordial germ cells (PGCs), the founder cells of the germline, are specified in pre-gastrulating embryos in mammals, and subsequently migrate towards gonads to mature into functional gametes. Here, we investigated PGC development in rats, by genetically modifying Prdm14, a unique marker and an essential PGC transcriptional regulator. We trace PGC development in rats, for the first time, from specification until the sex determination stage in fetal gonads using Prdm14 H2BVenus knock-in rats. We uncover that the crucial role of Prdm14 in PGC specification is conserved between rat and mice, by analyzing Prdm14-deficient rat embryos. Notably, loss of Prdm14 completely abrogates the PGC program, as demonstrated by failure of the maintenance and/or activation of germ cell markers and pluripotency genes. Finally, we profile the transcriptome of the post-implantation epiblast and all PGC stages in rat to reveal enrichment of distinct gene sets at each transition point, thereby providing an accurate transcriptional timeline for rat PGC development. Thus, the novel genetically modified rats and data sets obtained in this study will advance our knowledge on conserved versus species-specific features for germline development in mammals.

Characterization of homozygous Foxn1 mutations induced in rat embryos by different delivery forms of Cas9 nuclease.

BACKGROUND: The Cas9 nuclease is delivered in the form of either Cas9 protein or mRNA along with CRISPR guide RNA (gRNA: dual-crRNA:tracrRNA or chimeric single-guide RNA) or in a plasmid package encoding both Cas9 and the CRISPR gRNA. METHODS AND RESULTS: We directly compared the efficiency of producing rat blastocysts with homozygous mutations of the Foxn1 locus by pronuclear injection of Cas9 in the form of protein, mRNA, or plasmid DNA. For highly efficient production of rat blastocysts with homozygous Foxn1 mutations, pronuclear injection of Cas9 protein at 60 ng/µl was likely optimal. While blastocyst harvest in the mRNA groups was higher than those in the protein and plasmid DNA groups, genotype analysis showed that 63.6%, 8.7-20.0%, and 25.0% of the analyzed blastocysts were homozygous mutants in the protein, mRNA, and plasmid DNA groups, respectively. The high efficiency of producing homozygous mutant blastocysts in the 60 ng/µl protein group may be associated with primary genome editing being initiated before the first cleavage. In most cases, homozygous mutations at the target Foxn1 locus are triggered by deletion and repair via nonhomologous end joining or microhomology-mediated end joining. Deletion downstream of the Cas9 break site was more likely than deletion in the upstream direction. CONCLUSIONS: The Cas9 nuclease in protein form, when coinjected with the CRISPR gRNA (ribonucleoprotein) into a rat zygote pronucleus, can access the target genome site and induce double-strand breaks promptly, resulting in the efficient production of homozygous mutants.

Interspecies organogenesis generates autologous functional islets.

Islet transplantation is an established therapy for diabetes. We have previously shown that rat pancreata can be created from rat pluripotent stem cells (PSCs) in mice through interspecies blastocyst complementation. Although they were functional and composed of rat-derived cells, the resulting pancreata were of mouse size, rendering them insufficient for isolating the numbers of islets required to treat diabetes in a rat model. Here, by performing the reverse experiment, injecting mouse PSCs into Pdx-1-deficient rat blastocysts, we generated rat-sized pancreata composed of mouse-PSC-derived cells. Islets subsequently prepared from these mouse-rat chimaeric pancreata were transplanted into mice with streptozotocin-induced diabetes. The transplanted islets successfully normalized and maintained host blood glucose levels for over 370 days in the absence of immunosuppression (excluding the first 5 days after transplant). These data provide proof-of-principle evidence for the therapeutic potential of PSC-derived islets generated by blastocyst complementation in a xenogeneic host.

Sex difference in developmental changes in visualized Kiss1 neurons in newly generated Kiss1-Cre rats.

Hypothalamic kisspeptin neurons are master regulators of mammalian reproduction via direct stimulation of gonadotropin-releasing hormone and consequent gonadotropin release. Here, we generated novel Kiss1 (kisspeptin gene)-Cre rats and investigated the developmental changes and sex differences in visualized Kiss1 neurons of Kiss1-Cre-activated tdTomato reporter rats. First, we validated Kiss1-Cre rats by generating Kiss1-expressing cell-specific Kiss1 knockout (Kiss1-KpKO) rats, which were obtained by crossing the current Kiss1-Cre rats with Kiss1-floxed rats. The resulting male Kiss1-KpKO rats lacked Kiss1 expression in the brain and exhibited hypogonadotropic hypogonadism, similar to the hypogonadal phenotype of global Kiss1 KO rats. Histological analysis of Kiss1 neurons in Kiss1-Cre-activated tdTomato reporter rats revealed that tdTomato signals in the anteroventral periventricular nucleus (AVPV) and arcuate nucleus (ARC) were not affected by estrogen, and that tdTomato signals in the ARC, AVPV, and medial amygdala (MeA) were sexually dimorphic. Notably, neonatal AVPV tdTomato signals were detected only in males, but a larger number of tdTomato-expressing cells were detected in the AVPV and ARC, and a smaller number of cells in the MeA was detected in females than in males at postpuberty. These findings suggest that Kiss1-visualized rats can be used to examine the effect of estrogen feedback mechanisms on Kiss1 expression in the AVPV and ARC. Moreover, the Kiss1-Cre and Kiss1-visualized rats could be valuable tools for further detailed analyses of sexual differentiation in the brain and the physiological role of kisspeptin neurons across the brain in rats.

Establishment of embryo transfer in the musk shrew (Suncus murinus).

The present study established techniques to induce pseudopregnancy, in vitro oocyte cultures from pronuclear to 2- to 4-cell stages, and embryo transfer in musk shrews, a reflex ovulator. Offspring were subsequently obtained by transferring in vivo-developed or in vitro-cultured embryos. Female musk shrews received human chronic gonadotropin (hCG), with or without mating stimuli, from vasectomized males to produce pseudopregnant recipients. Embryos at the 2- to 4-cell stage were collected 44-48 h after mating. Another set of embryos was collected 26-27 h after mating and then cultured for 20 h from the pronuclear to 2- to 4-cell stages. Subsequently, embryos were transferred into the oviducts of pseudopregnant recipients 24 or 48 h after the induction of pseudopregnancy. Offsprings were successfully obtained from recipients that received hCG 24 h before embryo transfer, regardless of mating stimuli. These techniques may be valuable for producing transgenic musk shrews.

Inducible Kiss1 knockdown in the hypothalamic arcuate nucleus suppressed pulsatile secretion of luteinizing hormone in male mice.

Accumulating evidence suggests that kisspeptin-GPR54 signaling is indispensable for gonadotropin-releasing hormone (GnRH)/gonadotropin secretion and consequent reproductive functions in mammals. Conventional Kiss1 knockout (KO) mice and rats are reported to be infertile. To date, however, no study has investigated the effect of inducible central Kiss1 KO/knockdown on pulsatile gonadotropin release in male mammals. Here we report an in vivo analysis of inducible conditional Kiss1 knockdown male mice. The mice were generated by a bilateral injections of either adeno-associated virus (AAV) vectors driving Cre recombinase (AAV-Cre) or AAV vectors driving GFP (AAV-GFP, control) into the hypothalamic arcuate nucleus (ARC) of Kiss1-floxed male mice, in which exon 3 of the Kiss1 gene were floxed with loxP sites. Four weeks after the AAV-Cre injection, the mice showed a profound decrease in the both number of ARC Kiss1-expressing cells and the luteinizing hormone (LH) pulse frequency. Interestingly, pulsatile LH secretion was apparent 8 weeks after the AAV-Cre injection despite the suppression of ARC Kiss1 expression. The control Kiss1-floxed mice infected with AAV-GFP showed apparent LH pulses and Kiss1 expression in the ARC at both 4 and 8 weeks after the AAV-GFP injection. These results with an inducible conditional Kiss1 knockdown in the ARC of male mice suggest that ARC kisspeptin neurons are responsible for pulsatile LH secretion in male mice, and indicate the possibility of a compensatory mechanism that restores GnRH/LH pulse generation.

Conditional kisspeptin neuron-specific Kiss1 knockout with newly generated Kiss1-floxed and Kiss1-Cre mice replicates a hypogonadal phenotype of global Kiss1 knockout mice.

The present study aimed to evaluate whether novel conditional kisspeptin neuron-specific Kiss1 knockout (KO) mice utilizing the Cre-loxP system could recapitulate the infertility of global Kiss1 KO models, thereby providing further evidence for the fundamental role of hypothalamic kisspeptin neurons in regulating mammalian reproduction. We generated Kiss1-floxed mice and hypothalamic kisspeptin neuron-specific Cre-expressing transgenic mice and then crossed these two lines. The conditional Kiss1 KO mice showed pubertal failure along with a suppression of gonadotropin secretion and ovarian atrophy. These results indicate that newly-created hypothalamic Kiss1 KO mice obtained by the Cre-loxP system recapitulated the infertility of global Kiss1 KO models, suggesting that hypothalamic kisspeptin, but not peripheral kisspeptin, is critical for reproduction. Importantly, these Kiss1-floxed mice are now available and will be a valuable tool for detailed analyses of roles of each population of kisspeptin neurons in the brain and peripheral kisspeptin-producing cells by the spatiotemporal-specific manipulation of Cre expression.

Pluripotent stem cell-derived organogenesis in the rat model system.

Rats make an excellent model system for studying xenotransplantation since, like mice pluripotent stem cell lines, such as embryonic stem cells and induced pluripotent stem cells as well as gene knock-outs are also available for rats, besides rats have larger organs. The emergence of new genome-editing tools combined with stem cell technology, has revolutionized biomedical research including the field of regenerative medicine. The aim of this manuscript is to provide an overview of the recent progresses in stem cell-derived organ regeneration involving "gene knock-out" and "blastocyst complementation" in the rat model system. Knocking-out Pdx1, Foxn1, and Sall1 genes have successfully generated rat models lacking the pancreas, thymus, and kidney, respectively. When allogeneic (rat) or xenogeneic (mouse) pluripotent stem cells were microinjected into blastocyst-stage rat embryos that had been designed to carry a suitable organogenetic niche, devoid of specific organs, the complemented blastocysts were able to develop to full-term chimeric rat offspring containing stem cell-derived functional organs in their respective niches. Thus, organs with a tridimensional structure can be generated with pluripotent stem cells in vivo, accelerating regenerative medical research, which is crucial for organ-based transplantation therapies. However, to address ethical concerns, public consent after informed discussions is essential before production of human organs within domestic animals.

Haploid embryonic stem cell lines derived from androgenetic and parthenogenetic rat blastocysts.

The present study was conducted to establish haploid embryonic stem (ES) cell lines using fluorescent marker-carrying rats. In the first series, 7 ES cell lines were established from 26 androgenetic haploid blastocysts. However, only 1 ES cell line (ahES-2) was found to contain haploid cells (1n = 20 + X) by fluorescence-activated cell sorting (FACS) and karyotypic analyses. No chimeras were detected among the 10 fetuses and 41 offspring derived from blastocyst injection with the FACS-purified haploid cells. In the second series, 2 ES cell lines containing haploid cells (13% in phES-1 and 1% in phES-2) were established from 2 parthenogenetic haploid blastocysts. Only the phES-2 cell population was purified by repeated FACS to obtain 33% haploid cells. Following blastocyst injection with the FACS-purified haploid cells, no chimera was observed among the 11 fetuses; however, 1 chimeric male was found among the 47 offspring. Although haploid rat ES cell lines can be established from both blastocyst sources, FACS purification may be necessary for maintenance and chimera production.

Hypomorphic phenotype of Foxn1 gene-modified rats by CRISPR/Cas9 system.

The Foxn1 gene is known as a critical factor for the differentiation of thymic and skin epithelial cells. This study was designed to examine the phenotype of Foxn1-modified rats generated by the CRISPR/Cas9 system. Guide-RNA designed for first exon of the Foxn1 and mRNA of Cas9 were co-injected into the pronucleus of Crlj:WI zygotes. Transfer of 158 injected zygotes resulted in the birth of 50 offspring (32 %), and PCR identified five (10 %) as Foxn1-edited. Genomic sequencing revealed the deletion of 44 or 60 bp from and/or insertion of 4 bp into the Foxn1 gene in a single allele. The number of T-cells in the peripheral blood lymphocytes of mutant rats decreased markedly. While homozygous deleted mutant rats had no thymus, the mutant rats were not completely hairless and showed normal performance in delivery and nursing. Splicing variants of the indel-mutation in the Foxn1 gene may cause hypomorphic allele, resulting in the phenotype of thymus deficiency and incomplete hairless. In conclusion, the mutant rats in Foxn1 gene edited by the CRISPR/Cas9 system showed the phenotype of thymus deficiency and incomplete hairless which was characterized by splicing variants.

Molecular and Epigenetic Mechanism Regulating Hypothalamic Kiss1 Gene Expression in Mammals.

After the discovery of hypothalamic kisspeptin encoded by the Kiss1 gene, the central mechanism regulating gonadotropin-releasing hormone (GnRH) secretion, and hence gonadotropin secretion, is gradually being unraveled. This has increased our understanding of the central mechanism regulating puberty and subsequent reproductive performance in mammals. Recently, emerging evidence has indicated the molecular and epigenetic mechanism regulating hypothalamic Kiss1 gene expression. Here we compile data regarding DNA and histone modifications in the Kiss1 promoter region and provide a hypothetic scheme of the molecular and epigenetic mechanism regulating Kiss1 gene expression in two populations of hypothalamic kisspeptin neurons, which govern puberty and subsequent reproductive performance via GnRH/gonadotropin secretion.

Direct comparison of Cryotop® vitrification and Bicell® freezing on recovery of functional rat pancreatic islets.

Two protocols, Bicell® freeze-thawing and Cryotop® vitrification-warming, were compared for suitability in cryopreserving rat pancreatic islets (101-150 µm in mean diameter). Immediate survival rates of post-thaw and post-warm islets (50 and 57%, respectively), assessed by FDA/PI double staining, were lower than that of fresh control islets (90%). Most of the PI-positive dead cells were detected in peripheral area of post-warm islets, and were removed after subsequent 24 h culture (survival rate; 85% vs 59% in post-thaw islets). Quantitative PCR analysis showed that Bicell® freeze-thawing compromised expression of genes relating to ß-cell function (Pdx1 and Glut2), but not to one of apoptotic pathways (Bax/Bcl2 ratio). Expression of these genes was maintained in islets before and after the Cryotop® vitrification-warming. Values of stimulus index (SI) for 20 mM/3 mM glucose-stimulated insulin secretion were 6.7, 1.9 and 3.9 in fresh control, post-thaw and post-warm islets, respectively. The SI values after 24 h culture were 4.1, 1.9 and 3.1, respectively. Larger islets (>150 µm in diameter) had comparable survival rates, but lower SI values after Cryotop® vitrification-warming when compared to smaller counterparts. These results suggest that rat pancreatic islets can be cryopreserved by Cryotop® vitrification-warming rather than Bicell® freeze-thawing, without considerable loss of in vitro ß-cell function.

Epigenetic regulation of Kiss1 gene expression mediating estrogen-positive feedback action in the mouse brain.

This study aims to determine the epigenetic mechanism regulating Kiss1 gene expression in the anteroventral periventricular nucleus (AVPV) to understand the mechanism underlying estrogen-positive feedback action on gonadotropin-releasing hormone/gonadotropin surge. We investigated estrogen regulation of the epigenetic status of the mouse AVPV Kiss1 gene locus in comparison with the arcuate nucleus (ARC), in which Kiss1 expression is down-regulated by estrogen. Histone of AVPV Kiss1 promoter region was highly acetylated, and estrogen receptor α was highly recruited at the region by estrogen. In contrast, the histone of ARC Kiss1 promoter region was deacetylated by estrogen. Inhibition of histone deacetylation up-regulated in vitro Kiss1 expression in a hypothalamic non-Kiss1-expressing cell line. Gene conformation analysis indicated that estrogen induced formation of a chromatin loop between Kiss1 promoter and the 3' intergenic region, suggesting that the intergenic region serves to enhance estrogen-dependent Kiss1 expression in the AVPV. This notion was proved, because transgenic reporter mice with a complete Kiss1 locus sequence showed kisspeptin neuron-specific GFP expression in both the AVPV and ARC, but the deletion of the 3' region resulted in greatly reduced GFP expression only in the AVPV. Taken together, these results demonstrate that estrogen induces recruitment of estrogen receptor α and histone acetylation in the Kiss1 promoter region of the AVPV and consequently enhances chromatin loop formation of Kiss1 promoter and Kiss1 gene enhancer, resulting in an increase in AVPV-specific Kiss1 gene expression. These results indicate that epigenetic regulation of the Kiss1 gene is involved in estrogen-positive feedback to generate the gonadotropin-releasing hormone/gonadotropin surge.

Effect of leukemia inhibitory factor and forskolin on establishment of rat embryonic stem cell lines.

This study was designed to investigate whether supplementation of 2i medium with leukemia inhibitory factor (LIF) and/or forskolin would support establishment of germline-competent rat embryonic stem (ES) cell lines. Due to the higher likelihood of outgrowth rates, supplementation of forskolin with or without LIF contributed to the higher establishment efficiency of ES cell lines in the WDB strain. Germline transmission competency of the chimeric rats was not influenced by the profile of ES cell lines until their establishment. When the LIF/forskolin-supplemented 2i medium was used, the rat strain used as the blastocyst donor, such as the WI strain, was a possible factor negatively influencing the establishment efficiency of ES cell lines. Once ES cell lines were established, all lines were found to be germline-competent by a progeny test in chimeric rats. In conclusion, both LIF and forskolin are not essential but can play a beneficial role in the establishment of "genuine" rat ES cell lines.

Dynamics of pulsatile activities of arcuate kisspeptin neurons in aging female mice.

Reproductive senescence is broadly observed across mammalian females, including humans, eventually leading to a loss of fertility. The pulsatile secretion of gonadotropin-releasing hormone (GnRH), which is essential for gonad function, is primarily controlled by kisspeptin neurons in the hypothalamic arcuate nucleus (ARCkiss), the pulse generator of GnRH. The pulsatility of GnRH release, as assessed by the amount of circulating gonadotropin, is markedly reduced in aged animals, suggesting that the malfunctions of ARCkiss may be responsible for reproductive aging and menopause-related disorders. However, the activity dynamics of ARCkiss during the natural transition to reproductive senescence remain unclear. Herein, we introduce chronic in vivo Ca2+ imaging of ARCkiss in female mice by fiber photometry to monitor the synchronous episodes of ARCkiss (SEskiss), a known hallmark of GnRH pulse generator activity, from the fully reproductive to acyclic phase over 1 year. During the reproductive phase, we find that not only the frequency, but also the intensities and waveforms of individual SEskiss, vary depending on the stage of the estrus cycle. During the transition to reproductive senescence, the integrity of SEskiss patterns, including the frequency and waveforms, remains mostly unchanged, whereas the intensities tend to decline. These data illuminate the temporal dynamics of ARCkiss activities in aging female mice. More generally, our findings demonstrate the utility of fiber-photometry-based chronic imaging of neuroendocrine regulators in the brain to characterize aging-associated malfunction.

Ability of tetraploid rat blastocysts to support fetal development after complementation with embryonic stem cells.

This study was undertaken to generate rat offspring via tetraploid blastocyst complementation with embryonic stem (ES) cells. Tetraploid blastocysts were prepared by electrofusion of blastomeres from two-cell stage embryos, and subsequent in vivo culture for 4 days. Microinjection into the tetraploid blastocoel of an inner cell mass isolated by immunosurgery resulted in the generation of rat offspring, suggesting the successful contribution of tetraploid blastocysts to their placenta. Tetraploid blastocyst complementation was attempted with a total of 4 ES cell lines (2 lines of female karyotype and 2 lines of male karyotype). In the rESWIv-3i-5 (XX) cell line, normal-sized fetuses with heartbeats were harvested on E11.5 (12.1%), E12.5 (9.5%), and E13.5 (9.1%), but no viable fetuses were detected on E14.5. Similarly, use of the rESWIv-3i-1 (XX) cell line resulted in no viable fetus production on E14.5. Using the rESBLK2i-1 (XY) cell line, viable fetuses were harvested not only on E11.5-E13.5 (2.6-5.5%), but also on E14.5 (3.0%). The transfer of a total of 487 tetraploid blastocysts complemented with rESBLK2i-1 cells resulted in 256 implantation sites (52.6%) on E21.5, but no viable offspring was detected. Use of the rESBLK2i-1/huKO (XY) cell line also resulted in no viable offspring production on E21.5. Analyses of the methylation pattern in differentially methylated regions and transcript level of genes that are imprinted in mice (H19, Meg3, Igf2r, Peg5, and Peg10) in the E14.5 conceptuses indicated a marked difference between the ES cell-derived and control normal fetuses, but not between the tetraploid and control diploid placenta.