MYC-MAX heterodimerization is essential for the induction of major zygotic genome activation and subsequent preimplantation development.

In mouse preimplantation development, zygotic genome activation (ZGA), which synthesizes new transcripts in the embryo, begins in the S phase at the one-cell stage, with major ZGA occurring especially at the late two-cell stage. Myc is a transcription factor expressed in parallel with ZGA, but its direct association with major ZGA has not been clarified. In this study, we found that developmental arrest occurs at the two-cell stage when mouse embryos were treated with antisense oligonucleotides targeting Myc or MYC-specific inhibitors from the one-cell stage. To identify when MYC inhibition affects development, we applied time-limited inhibitor treatment and found that inhibition of MYC at the one-cell, four-cell, and morula stages had no effect on preimplantation development, whereas inhibitor treatment at the two-cell stage arrested development at the two-cell stage. Furthermore, transcriptome analysis revealed that when MYC function was inhibited, genes expressed in the major ZGA phase were suppressed. These results suggest that MYC is essential for the induction of major ZGA and subsequent preimplantation development. Revealing the function of MYC in preimplantation development is expected to contribute to advances in assisted reproductive technology.

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.

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.

Oocyte-specific gene Oog1 suppresses the expression of spermatogenesis-specific genes in oocytes.

Oog1, an oocyte-specific gene that encodes a protein of 425 amino acids, is present in five copies on mouse chromosomes 4 and 12. In mouse oocytes, Oog1 mRNA expression begins at embryonic day 15.5 and almost disappears by the late two-cell stage. Meanwhile, OOG1 protein is detectable in oocytes in ovarian cysts and disappears by the four-cell stage; the protein is transported to the nucleus in late one-cell to early two-cell stage embryos. In this study, we examined the role of Oog1 during oogenesis in mice. Oog1 RNAi-transgenic mice were generated by expressing double-stranded hairpin Oog1 RNA, which is processed into siRNAs targeting Oog1 mRNA. Quantitative RT-PCR revealed that the amount of Oog1 mRNA was dramatically reduced in oocytes obtained from Oog1-knockdown mice, whereas the abundance of spermatogenesis-associated transcripts (Klhl10, Tekt2, Tdrd6, and Tnp2) was increased in Oog1 knockdown ovaries. Tdrd6 is involved in the formation of the chromatoid body, Tnp2 contributes to the formation of sperm heads, Tekt2 is required for the formation of ciliary and flagellar microtubules, and Klhl10 plays a key role in the elongated sperm differentiation. These results indicate that Oog1 down-regulates the expression of spermatogenesis-associated genes in female germ cells, allowing them to develop normally into oocytes.

Long-term culture of undifferentiated spermatogonia isolated from immature and adult bovine testes.

Undifferentiated spermatogonia eventually differentiate in the testis to produce haploid sperm. Within this cell population, there is a small number of spermatogonial stem cells (SSCs). SSCs are rare cells in the testis, and their cellular characteristics are poorly understood. Establishment of undifferentiated cell line would provide an indispensable tool for studying their biological nature and spermiogenesis/spermatogenesis in vitro. However, there have been few reports on the long-term culture of undifferentiated spermatogonia in species other than rodents. Here, we report the derivation and long-term in vitro culture of undifferentiated spermatogonia cell lines from immature and adult bovine testes. Cell lines from immature testes were maintained in serum-free culture conditions in the presence of glial-cell-line-derived neurotropic factor (GDNF) and bovine leukemia inhibitory factor (bLIF). These cell lines have embryonic stem (ES)-like cell morphology, express pluripotent-stem-cell-specific and germ-cell-specific markers at the protein and mRNA levels, and contributed to the inner cell mass (ICM) of embryos in the blastocyst stage. Meanwhile, cell lines established from adult testes were maintained in low-serum media in the presence of 6-bromoindirubin-3'-oxime (BIO). These cell lines have characteristics resembling those of previously reported male mouse germ cell lines as confirmed by their botryoidally aggregated morphology, as well as the expression of germ-cell-specific markers and pluripotent stem cell markers. These findings could be useful for the development of long-term culture of undifferentiated spermatogonia, which could aid in conservation of species and improvement of livestock production through genome editing technology.

Derivation of Induced Trophoblast Cell Lines in Cattle by Doxycycline-Inducible piggyBac Vectors.

Trophectoderm lineage specification is one of the earliest differentiation events in mammalian development. The trophoblast lineage, which is derived from the trophectoderm, mediates implantation and placental formation. However, the processes involved in trophoblastic differentiation and placental formation in cattle remain unclear due to interspecies differences when compared with other model systems and the small repertoire of available trophoblast cell lines. Here, we describe the generation of trophoblast cell lines (biTBCs) from bovine amnion-derived cells (bADCs) using an induced pluripotent stem cell technique. bADCs were introduced with piggyBac vectors containing doxycycline (Dox)-inducible transcription factors (Oct3/4(POU5F1), Sox2, Klf4, and c-Myc). Colonies that appeared showed a flattened epithelial-like morphology similar to cobblestones, had a more definite cell boundary between cells, and frequently formed balloon-like spheroids similar to trophoblastic vesicles (TVs). biTBCs were propagated for over 60 passages and expressed trophoblast-related (CDX2, ELF5, ERRß, and IFN-τ) and pluripotency-related genes (endogenous OCT3/4, SOX2, KLF4, and c-MYC). Furthermore, when biTBCs were induced to differentiate by removing Dox from culture, they formed binucleate cells and began to express pregnancy-related genes (PL, PRP1, and PAG1). This is the first report demonstrating that the induction of pluripotency in bovine amniotic cells allows the generation of trophoblastic cell lines that possess trophoblast stem cell-like characteristics and have the potential to differentiate into the extra-embryonic cell lineage. These cell lines can be a new cell source as a model for studying trophoblast cell lineages and implantation processes in cattle.

Generation of Naïve Bovine Induced Pluripotent Stem Cells Using PiggyBac Transposition of Doxycycline-Inducible Transcription Factors.

Generation of pluripotent stem cells (PSCs) in large domestic animals has achieved only limited success; most of the PSCs obtained to date have been classified as primed PSCs, which possess very little capacity to produce chimeric offspring. By contrast, mouse PSCs have been classified as naïve PSCs that can contribute to most of the tissues of chimeras, including germ cells. Here, we describe the generation of two different types of bovine induced pluripotent stem cells (biPSCs) from amnion cells, achieved through introduction of piggyBac vectors containing doxycycline-inducible transcription factors (Oct3/4, Sox2, Klf4, and c-Myc). One type of biPSCs, cultured in medium supplemented with knockout serum replacement (KSR), FGF2, and bovine leukemia inhibitory factor (bLIF), had a flattened morphology like human PSCs; these were classified as primed-type. The other type biPSCs, cultured in KSR, bLIF, Mek/Erk inhibitor, GSK3 inhibitor and forskolin, had a compact morphology like mouse PSCs; these were classified as naïve-type. Cells could easily be switched between these two types of biPSCs by changing the culture conditions. Both types of biPSCs had strong alkaline phosphatase activity, expressed pluripotent markers (OCT3/4, NANOG, REX1, ESRRß, STELLA, and SOCS3), and formed embryoid bodies that gave rise to differentiated cells from all three embryonic germ layers. However, only naïve-type biPSCs showed the hallmarks of naïve mouse PSCs, such as LIF-dependent proliferation, lack of FGF5 expression, and active XIST expression with two active X chromosomes. Furthermore, naïve-type biPSCs could contribute to the inner cell mass (ICM) of host blastocysts and most tissues within chimeric embryos. This is the first report of generation of biPSCs with several characteristics similar to those of naïve mouse PSCs and a demonstrated potential to contribute to chimeras.

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.

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.

Characterization and in vitro culture of male germ cells from developing bovine testis.

The transition from male primitive germ cells (gonocytes) to type A spermatogonia in the neonatal testis is the initial process and a crucial process in spermatogenesis. However, in large domestic animals, the physiological and biochemical characteristics of germ cells during the developmental processes remain largely unknown. In this study, we characterized bovine germ cells in the developing testis from the neonatal stage to the adult stage. The binding of the lectin Dolichos biflorus agglutinin (DBA) and the expression of ubiquitin carboxyl-terminal hydrolase 1 (UCHL1) were restricted to gonocytes in the neonatal testis and spermatogonia in the adult testis. Gonocytes also expressed a germ cell marker (VASA) and stem cell markers (NANOG and OCT3/4), while the expressions of these markers in the adult testis were restricted to differentiated spermatic cells and were rarely expressed in spermatogonia. We subsequently utilized these markers to characterize gonocytes and spermatogonia after culture in vitro. Spermatogonia that were collected from the adult testis formed colonies in vitro only for one week. On the other hand, gonocytes from the neonatal testis could proliferate and form colonies after every passage for 1.5 months in culture. These colonies retained undifferentiated states of gonocytes as confirmed by the expression of both germ cell and stem cell markers. Moreover, a transplantation assay using immunodeficient mice testes showed that long-term cultured cells derived from gonocytes were able to colonize in the recipient testis. These results indicated that bovine gonocytes could maintain germ cell and stem cell potential in vitro.

Multipotential ability of primitive germ cells from neonatal pig testis cultured in vitro.

Gonocytes are progenitor-type germ cells that arise from primordial germ cells and differentiate further into spermatogonia, thereby initiating spermatogenesis. In the present study, freshly isolated gonocytes were found to have either weak or no expression of pluripotency determining transcription factors, such as POU5F1, SOX2 and C-MYC. Interestingly, the expression of these transcription factors, as well as other vital transcription factors, such as NANOG, KLF4 and DAZL, were markedly upregulated in cultured cells. Cells in primary cultures expressed specific germ cell and pluripotency markers, such as lectin Dolichos biflorus agglutinin (DBA), KIT, ZBTB16, stage-specific embryonic antigen (SSEA-1), NANOG and POU5F1. Using a monoclonal antibody to specifically identify porcine germ cells, the stem cell potential of fresh and cultured cells was determined with a testis xenotransplantation assay. Colonised porcine germ cells were detected only in mouse testes that were either transplanted with fresh testicular cells or with cells from primary cultures. Interestingly, testes transplanted with cells from primary cultures showed colonisation of germ cells in the interstitial space, reflecting their tumourigenic nature. The formation of teratomas with tissues originating from the three germinal layers following the subcutaneous injection of cells into nude mice from primary cultures confirmed their multipotency. The results of the present study may provide useful information for the establishment of multipotent germ stem cell lines from neonatal pig testis.

Expression of NANOG, but not POU5F1, points to the stem cell potential of primitive germ cells in neonatal pig testis.

Gonocytes are primitive germ cells that are present in the neonatal testis and are committed to male germline development. Gonocytes differentiate to spermatogonia, which establish and maintain spermatogenesis in the postnatal testis. However, it is unknown whether large animal species have pluripotency-specific proteins in the testis. Nanog and Pou5f1 (Oct3/4) have been identified as transcription factors essential for maintaining pluripotency of embryonic stem cells in mice. Here, we show that NANOG protein was expressed in the germ cells of neonatal pig testes, but was progressively lost with age. NANOG was expressed in most of the lectin Dolichos biflorus agglutinin- and ZBTB16-positive gonocytes, which are known gonocyte-specific markers in pigs. NANOG was also expressed in Sertoli and interstitial cells of neonatal testes. Interestingly, POU5F1 expression was not detected at either the transcript or the protein level in neonatal pig testis. In the prepubertal testis, NANOG and POU5F1 proteins were primarily detected in differentiated germ cells, such as spermatocytes and spermatids, and rarely in undifferentiated spermatogonia. By using a testis transplantation assay, we found that germ cells from 2- to 4-day-old pigs could colonize and proliferate in the testes of the recipient mice, suggesting that primitive germ cells from neonatal pig testes have stem cell potential.

Cryopreservation in liquid nitrogen of gonocytes from neonatal porcine testes stored at 4°C.

Aim: Gonocytes are primitive germ cells in neonatal male testes. Germ cells from the neonatal testes of mice have a self-renewal activity and have pluripotential characteristics in established stem-cell lines. Therefore, these germ cells are reliable sources for the preservation of genetic resources of domestic animals and endangered species. The aim of the present study was to examine the cryopreservation of porcine gonocytes in liquid nitrogen (LN2) from neonatal testes that were freshly collected or stored at 4°C for 24 h. Methods: Gonocytes were isolated as lectin Dolichos biflorus agglutinin (DBA) positive cells from porcine testes 2-5 days after birth. The effects of the cryoprotectants used in the cryopreservation of the gonocytes, which were isolated from testes stored at 4°C in various solutions for 24 h, were examined on the results of cell viability after cryopreservation and cell proliferation in culture. Testis tissues from stored testes were transplanted into immunodeficient mice to evaluate the ability of the gonocytes to differentiate 5 weeks after transplantation. Results: The portion of the gonocytes that was isolated from stored testes at 4°C was approximately 70%. The viability of the gonocytes from stored testes was significantly higher in HEPES-supplemented Dulbecco's Modified Eagle's Medium/F12 (DMEM/F12) and HEPES-supplemented phosphate-buffered saline than from stock solutions without HEPES. The addition of 10% dimethylsulfoxide (DMSO) and 0.07 mmol/L sucrose to cryopreservation solutions supported high viability of gonocytes after freezing and thawing. The cryopreserved gonocytes formed colonies with DBA activity in DMEM/F12 supplemented with 10% fetal bovine serum 3 days after culture and continued to proliferate for at least 12 days in culture. The germ cells in the testis tissues that were xenografted into immunodefficient mice differentiated into primitive spermatogonia. Conclusion: Gonocytes in the testis stored at 4°C for at least 24 h, isolated and cryopreserved can survive. The cryopreserved gonocytes differentiated in immunodefficient mice and proliferated along with the formation of colonies in vitro. (Reprod Med Biol 2008; 7: 153-160).

Zygotically activated genes are suppressed in mouse nuclear transferred embryos.

Mammalian oocytes have the ability to confer totipotency to terminally differentiated somatic cell nuclei. Viable cloned animals have been produced by somatic cell nuclear transfer (NT) into oocytes in many mammalian species including mouse. However, the success rates of the production were quite low in all species. Many studies have measured differences in gene expression between NT and fertilized embryos in relatively advanced stages of development such as pre- and post-natal stages or the blastocyst stage. In the present study, we compared gene expression patterns using differential display RT-PCR (DDRT-PCR) between the NT and IVF embryos at the 2-cell stage to detect some abnormalities affecting later development of NT embryos. Aberrant gene expression was detected in NT embryos compared with IVF embryos, and MuERV-L and Dnaja2 genes were down-regulated and Inpp5b and Chst12 genes were up-regulated in the NT embryos. Further analysis showed that the expression of zygotically activated genes such as Interferon-gamma, Dub-1, Spz1, DD2106 (unknown gene), and DD2111 (unknown gene) were suppressed in NT embryos, suggesting that the cellular process involved in the nuclear reprogramming of somatic nucleus is not appropriately regulated.

Developmental competence of somatic cell nuclear transfer embryos reconstructed from oocytes matured in vitro with follicle shells in miniature pig.

The developmental competence of domestic pig oocytes that were transferred to somatic cell nuclei of miniature pig was examined. A co-culture system of oocytes with follicle shells was used for the maturation of domestic pig oocytes in vitro. Co-cultured oocytes progressed to the metaphase II stage of meiosis more quickly and more synchronously than non co-cultured oocytes. Oocytes were enucleated and fused with fibroblast cells of Potbelly miniature pig at 48 h of maturation. The blastocyst formation rate of nuclear transfer (NT) embryos using cocultured oocytes (24%) was significantly higher (p < 0.05) than that of non-co-cultured oocytes (13%). Cleaved embryos at 48 h after nuclear transfer using co-cultured oocytes were transferred to the oviducts of 14 Göttingen miniature pigs and four Meishan pigs. Estrus of all Göttingens returned at around 20-31 days of pregnancy. Two of the four Meishans became pregnant. Three and two cloned piglets were born after modest number of embryo transfer (15 and 29 embryos transferred), respectively. These results indicated that oocytes co-cultured with follicle shells have a high developmental competence after nuclear transfer and result in full-term development after embryo transfer.

Cyclin A2 is phosphorylated during the G2/M transition in mouse two-cell embryos.

In the present study, we investigated the expression of cyclin A2 in mouse two-cell embryos to elucidate the role of cyclin A2 at the G2/M transition. Two forms of cyclin A2 on SDS-PAGE (an upper and a lower band) were detected in two-cell embryos synchronized at the M phase by nocodazole. To investigate the nature of this shift, embryos synchronized at the M phase were treated with alkaline phosphatase (AP). The upper band of cyclin A2 was fainter in AP-treated embryos than in nontreated embryos. This result indicates that cyclin A2 in mouse two-cell embryos is phosphorylated and the band on SDS-PAGE shifts up during the G2/M transition. In addition, we examined the sequential expression of cyclin A2 in two-cell blocked embryos after OA treatment. The upper band of cyclin A2 was first detected at 2 hr after the treatment, corresponding to the timing of Cdc2 kinase activation. In two-cell embryos after removal from nocodazole treatment, the phosphorylated form of cyclin A2 protein decreased abruptly just before cytokinesis. These results suggest that the mechanism of cyclin A2 degradation in mouse two-cell embryos may be different from that in somatic cells.

MuERV-L is one of the earliest transcribed genes in mouse one-cell embryos.

The expression pattern and function of the murine endogenous retrovirus-like (MuERV-L) gene in mouse preimplantation embryos was investigated. MuERV-L was rapidly transcribed from the beginning of S phase (8 h after fertilization) in the first cell cycle. MuERV-L expression was completely repressed when transcription from the zygotic genome was inhibited by alpha-amanitin. These results reveal that MuERV-L is transcribed from the zygotic genome and that it is expressed earlier than any other genes previously reported. In addition, MuERV-L was expressed even when the first round of DNA synthesis was inhibited by aphidicolin, suggesting that its expression is controlled by the zygotic clock. The function of MuERV-L in the development of mouse embryos was also examined using antisense oligonucleotides. The developmental competence of embryos was markedly suppressed after the 4-cell stage when they were treated with antisense oligonucleotides. This result suggests that MuERV-L plays an important role in the development of mouse embryos at the early preimplantation stage.