Comparative analysis of histone H3K4me3 modifications between blastocysts and somatic tissues in cattle

Epigenetic changes induced in the early developmental stages by the surrounding environment can have not only short-term but also long-term consequences throughout life. This concept constitutes the “Developmental Origins of Health and Disease” (DOHaD) hypothesis and encompasses the possibility of controlling livestock health and diseases by epigenetic regulation during early development. As a preliminary step for examining changes of epigenetic modifications in early embryos and their long-lasting effects in fully differentiated somatic tissues, we aimed to obtain high-throughput genome-wide histone H3 lysine 4 trimethylation (H3K4me3) profiles of bovine blastocysts and to compare these data with those from adult somatic tissues in order to extract common and typical features between these tissues in terms of H3K4me3 modifications. Bovine blastocysts were produced in vitro and subjected to chromatin immunoprecipitation-sequencing analysis of H3K4me3. Comparative analysis of the blastocyst-derived H3K4me3 profile with publicly available data from adult liver and muscle tissues revealed that the blastocyst profile could be used as a “sieve” to extract somatic tissue-specific modifications in genes closely related to tissue-specific functions. Furthermore, principal component analysis of the level of common modifications between blastocysts and somatic tissues in meat production-related and imprinted genes well characterized inter- and intra-tissue differences. The results of this study produced a referential genome-wide H3K4me3 profile of bovine blastocysts within the limits of their in vitro source and revealed its common and typical features in relation to the profiles of adult tissues

H4K20 monomethylation inhibition causes loss of genomic integrity in mouse preimplantation embryos

Maintaining genomic integrity in mammalian early embryos, which are deficient in DNA damage repair, is critical for normal preimplantation and subsequent development. Abnormalities in DNA damage repair in preimplantation embryos can cause not only developmental arrest, but also diseases such as congenital disorders and cancers. Histone H4 lysine 20 monomethylation (H4K20me1) is involved in DNA damage repair and regulation of gene expression. However, little is known about the role of H4K20me1 during mouse preimplantation development. In this study, we revealed that H4K20me1 mediated by SETD8 is involved in maintaining genomic integrity. H4K20me1 was present throughout preimplantation development. In addition, reduction in the level of H4K20me1 by inhibition of SETD8 activity or a dominant-negative mutant of histone H4 resulted in developmental arrest at the S/G2 phase and excessive accumulation of DNA double-strand breaks. Together, our results suggest that H4K20me1, a type of epigenetic modification, is associated with the maintenance of genomic integrity and is essential for preimplantation development. A better understanding of the mechanisms involved in maintaining genome integrity during preimplantation development could contribute to advances in reproductive medicine and technology

Identification and characterization of an oocyte factor required for development of porcine nuclear transfer embryos.

Nuclear reprogramming of differentiated cells can be induced by oocyte factors. Despite numerous attempts, these factors and mechanisms responsible for successful reprogramming remain elusive. Here, we identify one such factor, necessary for the development of nuclear transfer embryos, using porcine oocyte extracts in which some reprogramming events are recapitulated. After incubating somatic nuclei in oocyte extracts from the metaphase II stage, the oocyte proteins that were specifically and abundantly incorporated into the nuclei were identified by mass spectrometry. Among 25 identified proteins, we especially focused on a multifunctional protein, DJ-1. DJ-1 is present at a high concentration in oocytes from the germinal vesicle stage until embryos at the four-cell stage. Inhibition of DJ-1 function compromises the development of nuclear transfer embryos but not that of fertilized embryos. Microarray analysis of nuclear transfer embryos in which DJ-1 function is inhibited shows perturbed expression of P53 pathway components. In addition, embryonic arrest of nuclear transfer embryos injected with anti-DJ-1 antibody is rescued by P53 inhibition. We conclude that DJ-1 is an oocyte factor that is required for development of nuclear transfer embryos. This study presents a means for identifying natural reprogramming factors in mammalian oocytes and a unique insight into the mechanisms underlying reprogramming by nuclear transfer

Altered hormonal milieu and dysregulated protein expression can cause spermatogenic arrest in ectopic xenografted immature rat testis

Testis tissue xenografting complemented with cryopreservation is a feasible technique for fertility preservation in children with malignancy receiving gonadotoxic therapy and for endangered species with high neonatal mortality rate. However, xenografted testis of human and most endangered species are known to undergo spermatogenic arrest. In this study, we xenografted immature rat testis onto immunodeficient male mice to investigate the plausible underlying causes of spermatogenic arrest. Histological analysis of xenografted testes collected 8-wk post-grafting showed incomplete spermatogenesis with pachytene-stage spermatocytes as the most advanced germ cells. Although the levels of serum luteinizing hormone and testosterone were normal in recipient mice, those of follicle stimulating hormone (FSH) were significantly high, and specific receptors of FSH were absent in the xenografts. The xenografts demonstrated dysregulated expression of Sertoli cell-transcriptional regulators (WT1 and SOX9) and secretory proteins (SCF and GDNF). In conclusion, results from our study suggested that an altered hormonal milieu in recipients and dysregulated protein expression in xenografts could be a potential cause of spermatogenic arrest in xenografted immature rat testis. Further stereological analysis of xenografts can demonstrate precise cellular composition of xenografts to decipher interactions between germ and somatic cells to better understand spermatogenic arrest in xenografted testis

Histone methyltransferase Smyd3 regulates early embryonic lineage commitment in mice.

SET and MYND domain-containing protein 3 (Smyd3) is a histone H3 lysine 4 (H3K4) di- and tri-methyltransferase that forms a transcriptional complex with RNA polymerase II and activates the transcription of oncogenes and cell cycle genes in human cancer cells. However, the study of Smyd3 in mammalian early embryonic development has not yet been addressed. In the present study, we investigated the expression pattern of Smyd3 in mouse preimplantation embryos and the effects of RNA interference (RNAi)-mediated Smyd3 repression on the development of mouse embryos. We showed that Smyd3 mRNA levels increased after the two-cell stage, peaked at the four-cell stage, and gradually decreased thereafter. Moreover, in two-cell to eight-cell embryos, SMYD3 staining was more intense in the nuclei than it was in the cytoplasm. In Smyd3-knockdown embryos, the percentage of inner cell mass (ICM)-derived colony formation and trophectoderm (TE)-derived cell attachment were significantly decreased, which resulted in a reduction in the number of viable offspring. Furthermore, the expression of Oct4 and Cdx2 during mid-preimplantation gene activation was significantly decreased in Smyd3-knockdown embryos. In addition, the transcription levels of ICM and epiblast markers, such as Oct4, Nanog, and Sox2, the transcription levels of primitive endoderm markers, such as Gata6, and the transcription levels of TE markers, such as Cdx2 and Eomes, were significantly decreased in Smyd3-knockdown blastocysts. These findings indicate that SMYD3 plays an important role in early embryonic lineage commitment and peri-implantation development through the activation of lineage-specific genes

The role of signaling pathways on proliferation and self-renewal of cultured bovine primitive germ cells

First online: 19 August 2014[Purpose]Gonocytes are primitive male germ cells residing in the neonatal testes and are unipotent in nature, but also have pluripotent stem cell ability in mice under appropriate culture conditions. This study was performed to elucidate the molecular mechanisms of self-renewal and survival of cultured bovine gonocytes. [Methods]Gonocytes were isolated from neonatal bull calves and were cultured in DMEM/F12 supplemented with 15 % knock-out serum replacement (KSR) and glial cell-derived neurotrophic factor (GDNF). Cells were analyzed six days after culturing for cell-signaling molecular markers. [Results]Colony formation was observed 3–4 days after being cultured. Addition of GDNF enhanced mitogen-activated protein kinase 1/2 (MAPK1/2) phosphorylation and activated the MAPK signaling pathway. Inhibition of MAPK signaling reduced cell proliferation and abolished colony formation. However, inhibition of phosphoinositide 3-kinase-AKT (PI3K-AKT) signaling, a dominant pathway for self-renewal of mouse germ cells, did not show any effects on cultured bovine gonocytes. Expression of cell cycle-related regulators cyclin D2 and cyclin-dependent kinase 2 (CDK2) was downregulated with inhibition of MAPK signaling. [Conclusions]These results indicate activation of MAPK plays a critical role in self-renewal and survival of bovine gonocytes via cyclin D1 and CDK2

ING3 Is Essential for Asymmetric Cell Division during Mouse Oocyte Maturation.

ING3 (inhibitor of growth family, member 3) is a subunit of the nucleosome acetyltransferase of histone 4 (NuA4) complex, which activates gene expression. ING3, which contains a plant homeodomain (PHD) motif that can bind to trimethylated lysine 4 on histone H3 (H3K4me3), is ubiquitously expressed in mammalian tissues and governs transcriptional regulation, cell cycle control, and apoptosis via p53-mediated transcription or the Fas/caspase-8 pathway. Thus, ING3 plays a number of important roles in various somatic cells. However, the role(s) of ING3 in germ cells remains unknown. Here, we show that loss of ING3 function led to the failure of asymmetric cell division and cortical reorganization in the mouse oocyte. Immunostaining showed that in fully grown germinal vesicle (GV) oocytes, ING3 localized predominantly in the GV. After germinal vesicle breakdown (GVBD), ING3 homogeneously localized in the cytoplasm. In oocytes where Ing3 was targeted by siRNA microinjection, we observed symmetric cell division during mouse oocyte maturation. In those oocytes, oocyte polarization was not established due to the failure to form an actin cap or a cortical granule-free domain (CGFD), the lack of which inhibited spindle migration. These features were among the main causes of abnormal symmetric cell division. Interestingly, an analysis of the mRNA expression levels of genes related to asymmetric cell division revealed that only mTOR was downregulated, and, furthermore, that genes downstream of mTOR (e.g., Cdc42, Rac1, and RhoA) were also downregulated in siIng3-injected oocytes. Therefore, ING3 may regulate asymmetric cell division through the mTOR pathway during mouse oocyte maturation

Effects of extracellular matrices and lectin Dolichos biflorus agglutinin on cell adhesion and self-renewal of bovine gonocytes cultured in vitro.

Surface molecules of primitive male germ cells, gonocytes, are essential components for regulating cell adhesion and maintaining self-renewal in mammalian species. In domestic animals, the stage-specific glycan epitope α-N-acetylgalactosamine (GalNAc) is recognised by the lectin Dolichos biflorus agglutinin (DBA) and is found on the surface of gonocytes and spermatogonia. Gonocytes from bovine testis formed mouse embryonic stem-like cell colonies on plates that had been coated with DBA or extracellular matrix (ECM) components, such as gelatin (GN), laminin (LN) and poly-L-lysine (PLL). The number of colonies on the DBA-coated plate was significantly higher than that on the GN-, LN- and PLL-coated plates. Pretreating gonocytes with DBA to neutralise the terminal GalNAc residues strongly suppressed colony formation. Furthermore, expression of a germ cell-specific gene and pluripotency-related transcription factors was increased considerably on the DBA-coated plates. These results suggest that the GalNAc residues on gonocytes can recognise precoated DBA on plates and the resulting GalNAc-DBA complexes support germ cell and stem cell potentials of gonocytes in vitro. These glycan complexes, through the GalNAc epitope, may provide a suitable microenvironment for the adhesion and cell proliferation of gonocytes in culture