Overexpression of Let-7a mitigates diploidization in mouse androgenetic haploid embryonic stem cells.

Mouse androgenetic haploid embryonic stem cells (mAG-haESCs) can be utilized to uncover gene functions, especially those of genes with recessive effects, and to produce semicloned mice when injected into mature oocytes. However, mouse haploid cells undergo rapid diploidization during long-term culture in vitro and subsequently lose the advantages of haploidy, and the factors that drive diploidization are poorly understood. In this study, we compared the small RNAs (sRNAs) of mAG-haESCs, normal embryonic stem cells (ESCs), and mouse round spermatids by high-throughput sequencing and identified distinct sRNA profiles. Several let-7 family members and miR-290-295 cluster microRNAs (miRNAs) were found significantly differentially transcribed. Knockdown and overexpression experiments showed that let-7a and let-7g suppress diploidization while miR-290a facilitates diploidization. Our study revealed the unique sRNA profile of mAG-haESCs and demonstrated that let-7a overexpression can mitigate diploidization in mAG-haESCs. These findings will help us to better understand mAG-haESCs and utilize them as tools in the future.

Appropriate glycemic management protects the germline but not the uterine environment in hyperglycemia.

Emerging evidence indicates that parental diseases can impact the health of subsequent generations through epigenetic inheritance. Recently, it was shown that maternal diabetes alters the metaphase II oocyte transcriptome, causing metabolic dysfunction in offspring. However, type 1 diabetes (T1D) mouse models frequently utilized in previous studies may be subject to several confounding factors due to severe hyperglycemia. This limits clinical translatability given improvements in glycemic control for T1D subjects. Here, we optimize a T1D mouse model to investigate the effects of appropriately managed maternal glycemic levels on oocytes and intrauterine development. We show that diabetic mice with appropriate glycemic control exhibit better long-term health, including maintenance of the oocyte transcriptome and chromatin accessibility. We further show that human oocytes undergoing in vitro maturation challenged with mildly increased levels of glucose, reflecting appropriate glycemic management, also retain their transcriptome. However, fetal growth and placental function are affected in mice despite appropriate glycemic control, suggesting the uterine environment rather than the germline as a pathological factor in developmental programming in appropriately managed diabetes.

Transcriptome profiles and chromatin states in mouse androgenetic haploid embryonic stem cells.

Haploid embryonic stem cells (haESCs) are derived from the inner cell mass of the haploid blastocyst, containing only one set of chromosomes. Extensive and accurate chromatin remodelling occurs during haESC derivation, but the intrinsic transcriptome profiles and chromatin structure of haESCs have not been fully explored. We profiled the transcriptomes, nucleosome positioning, and key histone modifications of four mouse haESC lines, and compared these profiles with those of other closely-related stem cell lines, MII oocytes, round spermatids, sperm, and mouse embryonic fibroblasts. haESCs had transcriptome profiles closer to those of naïve pluripotent stem cells. Consistent with the one X chromosome in haESCs, Xist was repressed, indicating no X chromosome inactivation. haESCs and ESCs shared a similar global chromatin structure. However, a nucleosome depletion region was identified in 2056 promoters in ESCs, which was absent in haESCs. Furthermore, three characteristic spatial relationships were formed between transcription factor motifs and nucleosomes in both haESCs and ESCs, specifically in the linker region, on the nucleosome central surface, and nucleosome borders. Furthermore, the chromatin state of 4259 enhancers was off in haESCs but active in ESCs. Functional annotation of these enhancers revealed enrichment in regulation of the cell cycle, a predominantly reported mechanism of haESC self-diploidization. Notably, the transcriptome profiles and chromatin structure of haESCs were highly preserved during passaging but different from those of differentiated cell types.

Distinct H3K9me3 and DNA methylation modifications during mouse spermatogenesis.

DNA methylation and histone modifications critically regulate the expression of many genes and repeat regions during spermatogenesis. However, the molecular details of these processes in male germ cells remain to be addressed. Here, using isolated murine sperm cells, ultra-low-input native ChIP-Seq (ULI-NChIP-Seq), and whole genome bisulfite sequencing (WGBS), we investigated genome-wide DNA methylation patterns and histone 3 Lys-9 trimethylation (H3K9me3) modifications during mouse spermatogenesis. We found that DNA methylation and H3K9me3 have distinct sequence preferences and dynamics in promoters and repeat elements during spermatogenesis. H3K9me3 modifications in histones at gene promoters were highly enriched in round spermatids. H3K9me3 modification on long terminal repeats (LTRs) and long interspersed nuclear elements (LINEs) was involved in silencing active transcription from these regions in conjunction with reestablishment of DNA methylation. Furthermore, H3K9me3 remodeling on the X chromosome was involved in meiotic sex chromosome inactivation and in partial transcriptional reactivation of sex chromosomes in spermatids. Our findings also revealed the DNA methylation patterns and H3K9me3 modification profiles of paternal and maternal germline imprinting control regions (gICRs) during spermatogenesis. Taken together, our results provide a genome-wide map of H3K9me3 modifications during mouse spermatogenesis that may be helpful for understanding male reproductive disorders.

Mammalian haploid stem cells: establishment, engineering and applications.

Haploid embryonic stem cells (haESCs) contain only one set of genomes inherited from the sperm or egg and are termed AG- or PG-haESCs, respectively. Mammalian haESCs show genome-wide hypomethylation and dysregulated imprinting, whereas they can sustain genome integrity during derivation and long-term propagation. In addition, haESCs exhibit similar pluripotency to traditional diploid ESCs but are unique because they function as gametes and have been used to produce semi-cloned animals. More strikingly, unisexual reproduction has been achieved in mice by using haESCs. In combination with a gene editing or screening system, haESCs represent a powerful tool for studies of underlying gene functions and explorations of mechanisms of genetic and epigenetic regulation not only at the cellular level in vitro but also at the animal level in vivo. More importantly, genetically edited AG-haESC lines may further serve as an ideal candidate for the establishment of a sperm bank, which is a highly cost-effective approach, and a wide range of engineered semi-cloned mice have been produced. Here, we review the historical development, characteristics, advantages and disadvantages of haESCs. Additionally, we present an in-depth discussion of the recent advances in haESCs and their potential applications.

Reduced Self-Diploidization and Improved Survival of Semi-cloned Mice Produced from Androgenetic Haploid Embryonic Stem Cells through Overexpression of Dnmt3b.

Androgenetic haploid embryonic stem cells (AG-haESCs) hold great promise for exploring gene functions and generating gene-edited semi-cloned (SC) mice. However, the high incidence of self-diploidization and low efficiency of SC mouse production are major obstacles preventing widespread use of these cells. Moreover, although SC mice generation could be greatly improved by knocking out the differentially methylated regions of two imprinted genes, 50% of the SC mice did not survive into adulthood. Here, we found that the genome-wide DNA methylation level in AG-haESCs is extremely low. Subsequently, downregulation of both de novo methyltransferase Dnmt3b and other methylation-related genes was determined to be responsible for DNA hypomethylation. We further demonstrated that ectopic expression of Dnmt3b in AG-haESCs could effectively improve DNA methylation level, and the high incidence of self-diploidization could be markedly rescued. More importantly, the developmental potential of SC embryos was improved, and most SC mice could survive into adulthood.

Allelic reprogramming of the histone modification H3K4me3 in early mammalian development.

Histone modifications are fundamental epigenetic regulators that control many crucial cellular processes. However, whether these marks can be passed on from mammalian gametes to the next generation is a long-standing question that remains unanswered. Here, by developing a highly sensitive approach, STAR ChIP-seq, we provide a panoramic view of the landscape of H3K4me3, a histone hallmark for transcription initiation, from developing gametes to post-implantation embryos. We find that upon fertilization, extensive reprogramming occurs on the paternal genome, as H3K4me3 peaks are depleted in zygotes but are readily observed after major zygotic genome activation at the late two-cell stage. On the maternal genome, we unexpectedly find a non-canonical form of H3K4me3 (ncH3K4me3) in full-grown and mature oocytes, which exists as broad peaks at promoters and a large number of distal loci. Such broad H3K4me3 peaks are in contrast to the typical sharp H3K4me3 peaks restricted to CpG-rich regions of promoters. Notably, ncH3K4me3 in oocytes overlaps almost exclusively with partially methylated DNA domains. It is then inherited in pre-implantation embryos, before being erased in the late two-cell embryos, when canonical H3K4me3 starts to be established. The removal of ncH3K4me3 requires zygotic transcription but is independent of DNA replication-mediated passive dilution. Finally, downregulation of H3K4me3 in full-grown oocytes by overexpression of the H3K4me3 demethylase KDM5B is associated with defects in genome silencing. Taken together, these data unveil inheritance and highly dynamic reprogramming of the epigenome in early mammalian development.

Xist repression shows time-dependent effects on the reprogramming of female somatic cells to induced pluripotent stem cells.

Although the reactivation of silenced X chromosomes has been observed as part of the process of reprogramming female somatic cells into induced pluripotent stem cells (iPSCs), it remains unknown whether repression of the X-inactive specific transcript (Xist) can greatly enhance female iPSC induction similar to that observed in somatic cell nuclear transfer studies. In this study, we discovered that the repression of Xist plays opposite roles in the early and late phases of female iPSCs induction. Our results demonstrate that the downregulation of Xist by an isopropyl ß-d-1-thiogalactopyranoside (IPTG)-inducible short hairpin RNA (shRNA) system can greatly impair the mesenchymal-to-epithelial transition (MET) in the early phase of iPSC induction but can significantly promote the transition of pre-iPSCs to iPSCs in the late phase. Furthermore, we demonstrate that although the knockdown of Xist did not affect the H3K27me3 modification on the X chromosome, macroH2A was released from the inactivated X chromosome (Xi). This enables the X chromosome silencing to be a reversible event. Moreover, we demonstrate that the supplementation of vitamin C (Vc) can augment and stabilize the reversible X chromosome by preventing the relocalization of macroH2A to the Xi. Therefore, our study reveals an opposite role of Xist repression in the early and late stages of reprogramming female somatic cells to pluripotency and demonstrates that the release of macroH2A by Xist repression enables the transition from pre-iPSCs to iPSCs.

A pre-breeding screening program for transgenic boars based on fluorescence in situ hybridization assay.

For efficient transgenic herd expansion, only the transgenic animals that possess the ability to transmit transgene into next generation are considered for breeding. However, for transgenic pig, practically lacking a pre-breeding screening program, time, labor and money is always wasted to maintain non-transgenic pigs, low or null transgenic transmission pigs and the related fruitless gestations. Developing a pre-breeding screening program would make the transgenic herd expansion more economical and efficient. In this technical report, we proposed a three-step pre-breeding screening program for transgenic boars simply through combining the fluorescence in situ hybridization (FISH) assay with the common pre-breeding screening workflow. In the first step of screening, combined with general transgenic phenotype analysis, FISH is used to identify transgenic boars. In the second step of screening, combined with conventional semen test, FISH is used to detect transgenic sperm, thus to identify the individuals producing high quality semen and transgenic sperm. In the third step of screening, FISH is used to assess the in vitro fertilization embryos, thus finally to identify the individuals with the ability to produce transgenic embryos. By this three-step screening, the non-transgenic boars and boars with no ability to produce transgenic sperm or transgenic embryos would be eliminated; therefore only those boars could produce transgenic offspring are maintained and used for breeding and herd expansion. It is the first time a systematic pre-breeding screening program is proposed for transgenic pigs. This program might also be applied in other transgenic large animals, and provide an economical and efficient strategy for herd expansion.

Asymmetric reprogramming capacity of parental pronuclei in mouse zygotes.

It has been demonstrated that reprogramming factors are sequestered in the pronuclei of zygotes after fertilization, because zygotes enucleated at the M phase instead of interphase of the first mitosis can support the development of cloned embryos. However, the contribution of the parental pronucleus derived from either the sperm or the oocyte in reprogramming remains elusive. Here, we demonstrate that the parental pronuclei have asymmetric reprogramming capacities and that the reprogramming factors reside predominantly in the male pronucleus. As a result, only female pronucleus-depleted (FPD) mouse zygotes can reprogram somatic cells to a pluripotent state and support the full-term development of cloned embryos; male pronucleus-depleted (MPD) zygotes fail to support somatic cell reprogramming. We further demonstrate that fusion of an additional male pronucleus into a zygote greatly enhances reprogramming efficiency. Our data provide a clue to further identify critical reprogramming factors in the male pronucleus.

Generation and developmental characteristics of porcine tetraploid embryos and tetraploid/diploid chimeric embryos.

The aim of this study was to optimize electrofusion conditions for generating porcine tetraploid (4n) embryos and produce tetraploid/diploid (4n/2n) chimeric embryos. Different electric field intensities were tested and 2 direct current (DC) pulses of 0.9 kV/cm for 30 µs was selected as the optimum condition for electrofusion of 2-cell embryos to produce 4n embryos. The fusion rate of 2-cell embryos and the development rate to blastocyst of presumably 4n embryos, reached 85.4% and 28.5%, respectively. 68.18% of the fused embryos were found to be 4n as demonstrated by fluorescent in situ hybridization (FISH). Although the number of blastomeres in 4n blastocysts was significantly lower than in 2n blastocysts (P<0.05), there was no significant difference in developmental rates of blastocysts between 2n and 4n embryos (P>0.05), suggesting that the blastocyst forming capacity in 4n embryos is similar to those in 2n embryos. Moreover, 4n/2n chimeric embryos were obtained by aggregation of 4n and 2n embryos. We found that the developmental rate and cell number of blastocysts of 4-cell (4n)/4-cell (2n) chimeric embryos were significantly higher than those of 2-cell (4n)/4-cell (2n), 4-cell (4n)/8-cell (2n), 4-cell (4n)/2-cell (2n) chimeric embryos (P<0.05). Consistent with mouse chimeras, the majority of 4n cells contribute to the trophectoderm (TE), while the 2n cells are mainly present in the inner cell mass (ICM) of porcine 4n/2n chimeric embryos. Our study established a feasible and efficient approach to produce porcine 4n embryos and 4n/2n chimeric embryos.

[Cloning and gene expression of sall4b gene in pig].

Sall4, a member of sall4 gene family, plays important roles in embryo development; organogenesis as well as pluripotency maintenance and re-establishment. There are two isoforms of Sall4, Sall4A and Sall4B. The sequence of porcine sall4 gene is still not reported. Because of its distinct role in maintaining the pluripotent state of stem cells, we cloned and sequenced porcine sall4 gene and assessed its expression in pig tissues and embryos. One 2 372 bp nucleotide sequence representing the full-length cDNA of pig sall4 was obtained by 5'and 3'RACE. Analyses of putative protein sequence showed a 70% to 80% identity with isoform Sall4B of human and mouse. Comparing with Sall4A, the identity reduced to 30% to 55% because of the loss of a zinc-finger domain-rich fragment. Assessment of sall4b expression in porcine tissues by Real-time PCR showed that it expressed most strongly in ovary and stronger in spleen, lung, heart and testis. For preimplantation embryos, the expression level was lower in 4-cell embryos compared with other stages. Immuno-fluorescence analysis of Sall4 on porcine preimplantation embryos indicated that it expressed in all the preimplantation embryos and located in nucleus, in blastocyst it preferentially limited in ICM cells. Expression pattern in early embryos suggest that pig sall4b is associated with pluripotency and might be a new and useful reprogramming factor for establishing pig induced pluripotent stem cell lines.