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This study examined the role of cyclin-dependent kinase inhibitor 1a (CDK1A, p21) in response to exogenous stressors during mouse preimplantation embryo development. CDKN1A knockdown (KD) one-cell zygotes were exposed to 39 °C heat stress (HS) for 4 days or irradiated by 1 (1-Gy) or 3 (3-Gy) Gy X-rays, and their developmental competence and gene expression were compared with control embryos. CDKN1A KD and HS did not influence early cleavage or subsequent embryonic development; however, HS delayed cavitation and induced elevated Cdkn1a expression in control embryos. Exposure to 1- or 3-Gy had no effect on development to the morula stage; however, a significant number of morulae failed to develop to the blastocyst stage. Interestingly, under the 1-Gy condition, the blastocyst rate of CDKN1A KD embryos (77.7%) was significantly higher than that of the controls (44.4%). In summary, exposure to cellular stressors resulted in the upregulation of Cdkn1a in embryos exposed to HS or X-ray irradiation, particularly in response to heat stress or low-dose X-ray irradiation, and depleting Cdkn1a mRNA alleviated cell cycle arrest. These findings suggest that CDKN1A plays a vital role in cellular senescence during preimplantation embryo development.

The anti-Müllerian hormone (AMH) produced by the granulosa cells of growing follicles is critical for folliculogenesis and is clinically used as a diagnostic and prognostic marker of female fertility. Previous studies report that AMH-pretreatment in mice creates a pool of quiescent follicles that are released following superovulation, resulting in an increased number of ovulated oocytes. However, the quality and developmental competency of oocytes derived from AMH-induced accumulated follicles as well as the effect of AMH treatment on live birth are not known. This study reports that AMH priming positively affects oocyte maturation and early embryonic development culminating in higher number of live births. Our results show that AMH treatment results in good-quality oocytes with greater developmental competence that enhances embryonic development resulting in blastocysts with higher gene expression. The transcriptomic analysis of oocytes from AMH-primed mice compared with those of control mice reveal that AMH upregulates a large number of genes and pathways associated with oocyte quality and embryonic development. Mitochondrial function is the most affected pathway by AMH priming, which is supported by more abundant active mitochondria, mitochondrial DNA content and adenosine triphosphate levels in oocytes and embryos isolated from AMH-primed animals compared with control animals. These studies for the first time provide an insight into the overall impact of AMH on female fertility and highlight the critical knowledge necessary to develop AMH as a therapeutic option to improve female fertility.

N6-methyladenosine (m6A) catalyzed by METTL3 regulates the maternal-to-zygotic transition in zebrafish and mice. However, the role and mechanism of METTL3-mediated m6A methylation in blastocyst development remains unclear. Here, we show that METTL3-mediated m6A methylation sustains porcine blastocyst development via negatively modulating autophagy. We found that reduced m6A levels triggered by METTL3 knockdown caused embryonic arrest during morula-blastocyst transition and developmental defects in trophectoderm cells. Intriguingly, overexpression of METTL3 in early embryos resulted in increased m6A levels and these embryos phenocopied METTL3 knockdown embryos. Mechanistically, METTL3 knockdown or overexpression resulted in a significant increase or decrease in expression of ATG5 (a key regulator of autophagy) and LC3 (an autophagy marker) in blastocysts, respectively. m6A modification of ATG5 mRNA mainly occurs at 3’UTR, and METTL3 knockdown enhanced ATG5 mRNA stability, suggesting that METTL3 negatively regulated autophagy in an m6A dependent manner. Furthermore, single-cell qPCR revealed that METTL3 knockdown only increased expression of LC3 and ATG5 in trophectoderm cells, indicating preferential inhibitory effects of METTL3 on autophagy activity in the trophectoderm lineage. Importantly, autophagy restoration by 3MA (an autophagy inhibitor) treatment partially rescued developmental defects of METTL3 knockdown blastocysts. Taken together, these results demonstrate that METTL3-mediated m6A methylation negatively modulates autophagy to support blastocyst development.

CRISPR-mediated transcriptional activation, also known as CRISPR-on, has proven efficient for activation of individual or multiple endogenous gene expression in cultured cells from several species. However, the potential of CRISPR-on technology in preimplantation mammalian embryos remains to be explored. Here, we report for the first time the successful modulation of endogenous gene expression in bovine embryos by using the CRISPR-on system. As a proof of principle, we targeted the promoter region of either SMARCA4 or TFAP2C genes, transcription factors implicated in trophoblast lineage commitment during embryo development. We demonstrate that CRISPR-on provides temporal control of endogenous gene expression in bovine embryos, by simple cytoplasmic injection of CRISPR RNA components into one cell embryos. dCas9VP160 activator was efficiently delivered and accurately translated into protein, being detected in the nucleus of all microinjected blastomeres. Our approach resulted in the activation of SMARCA expression shortly after microinjection, with a consequent effect on downstream differentiation promoting factors, such as TFAP2C and CDX2. Although targeting of TFAP2C gene did not result in a significant increase in TFAP2C expression, there was a profound induction in CDX2 expression on day 2 of development. Finally, we demonstrate that CRISPR-on system is suitable for gene expression modulation during the preimplantation period, since no detrimental effect was observed on microinjected embryo development. This study constitutes a first step toward the application of the CRISPR-on system for the study of early embryo cell fate decisions in cattle and other mammalian embryos, as well as to design novel strategies that may lead to an improved trophectoderm development. Fil: Savy, Virginia. Universidad de Buenos Aires. Facultad de Agronomía. Pabellón de Zootecnica. Laboratorio de Biotecnología Animal; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Unidad Ejecutora de Investigaciones en Producción Animal. Universidad de Buenos Aires. Facultad de Ciencias Veterinarias. Unidad Ejecutora de Investigaciones en Producción Animal; Argentina Fil: Alberio, Virgilia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Unidad Ejecutora de Investigaciones en Producción Animal. Universidad de Buenos Aires. Facultad de Ciencias Veterinarias. Unidad Ejecutora de Investigaciones en Producción Animal; Argentina. Universidad de Buenos Aires. Facultad de Agronomía. Pabellón de Zootecnica. Laboratorio de Biotecnología Animal; Argentina Fil: Canel, Natalia Gabriela. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Unidad Ejecutora de Investigaciones en Producción Animal. Universidad de Buenos Aires. Facultad de Ciencias Veterinarias. Unidad Ejecutora de Investigaciones en Producción Animal; Argentina. Universidad de Buenos Aires. Facultad de Agronomía. Pabellón de Zootecnica. Laboratorio de Biotecnología Animal; Argentina Fil: Ratner, Laura Daniela. Universidad de Buenos Aires. Facultad de Agronomía. Pabellón de Zootecnica. Laboratorio de Biotecnología Animal; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Unidad Ejecutora de Investigaciones en Producción Animal. Universidad de Buenos Aires. Facultad de Ciencias Veterinarias. Unidad Ejecutora de Investigaciones en Producción Animal; Argentina Fil: Gismondi, Maria Ines. Instituto Nacional de Tecnologia Agropecuaria. Centro de Investigacion En Ciencias Veterinarias y Agronomicas. Instituto de Agrobiotecnologia y Biologia Molecular. - Consejo Nacional de Investigaciones Cientificas y Tecnicas. Oficina de Coordinacion Administrativa Pque. Centenario. Instituto de Agrobiotecnologia y Biologia Molecular; Argentina Fil: Ferraris, Sergio Raúl. Universidad Maimónides. Centro de Ciencias Veterinarias; Argentina Fil: Fernández y Martín, Rafael. Universidad de Buenos Aires. Facultad de Agronomía. Pabellón de Zootecnica. Laboratorio de Biotecnología Animal; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Unidad Ejecutora de Investigaciones en Producción Animal. Universidad de Buenos Aires. Facultad de Ciencias Veterinarias. Unidad Ejecutora de Investigaciones en Producción Animal; Argentina Fil: Knott, Jason G.. Department Of Animal Science; Estados Unidos Fil: Bevacqua, Romina Jimena. Universidad de Buenos Aires. Facultad de Agronomía. Pabellón de Zootecnica. Laboratorio de Biotecnología Animal; Argentina. University of Stanford; Estados Unidos Fil: Salamone, Daniel Felipe. Universidad de Buenos Aires. Facultad de Agronomía. Pabellón de Zootecnica. Laboratorio de Biotecnología Animal; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Unidad Ejecutora de Investigaciones en Producción Animal. Universidad de Buenos Aires. Facultad de Ciencias Veterinarias. Unidad Ejecutora de Investigaciones en Producción Animal; Argentina

Background: N6-methyladenosine (m6A) catalyzed by METTL3 regulates the maternal-to-zygotic transition in zebrafish and mice. However, the role and mechanism of METTL3-mediated m6A methylation in blastocyst development remains unclear. Results: We found that reduced m6A levels triggered by METTL3 knockdown caused embryonic arrest during morula-blastocyst transition and developmental defects in trophectoderm cells. Intriguingly, overexpression of METTL3 in early embryos resulted in increased m6A levels and these embryos phenocopied METTL3 knockdown embryos. Mechanistically, METTL3 knockdown or overexpression resulted in a significant increase or decrease in expression of ATG5 and LC3 (an autophagy marker) in blastocysts, respectively. m6A modification of ATG5 mRNA mainly occurs at 3’UTR, and METTL3 knockdown enhanced ATG5 mRNA stability, suggesting that METTL3 negatively regulated autophagy in an m6A dependent manner. Furthermore, single-cell analysis revealed that METTL3 knockdown only increased expression of LC3 and ATG5 in trophectoderm cells, indicating preferential inhibitory effects of METTL3 on autophagy activity in the trophectoderm lineage. Importantly, autophagy restoration by 3MA (an autophagy inhibitor) treatment partially rescued developmental defects of METTL3 knockdown blastocysts. Conclusions: Our results demonstrate that METTL3-mediated m6A methylation negatively modulates autophagy to support blastocyst development.

In mammals, the first cell-fate decision occurs during preimplantation embryo development when the inner cell mass (ICM) and trophectoderm (TE) lineages are established. The ICM develops into the embryo proper, while the TE lineage forms the placenta. The underlying molecular mechanisms that govern lineage formation involve cell-to-cell interactions, cell polarization, cell signaling and transcriptional regulation. In this review, we will discuss the current understanding regarding the cellular and molecular events that regulate lineage formation in mouse preimplantation embryos with an emphasis on cell polarity and the Hippo signaling pathway. Moreover, we will provide an overview on some of the molecular tools that are used to manipulate the Hippo pathway and study cell-fate decisions in early embryos. Lastly, we will provide exciting future perspectives on transcriptional regulatory mechanisms that modulate the activity of the Hippo pathway in preimplantation embryos to ensure robust lineage segregation.

Background: Long-term exposure to the heavy metals cadmium (Cd) and mercury (Hg) is known to increase the risk of chronic diseases. However, to our knowledge, exposure to Cd and Hg beginning at the periconception period has not been studied to date. Objective: We examined the effect of Cd and Hg that were co-administered during early development on indices of chronic diseases in adult male mice. Methods: Adult female CD1 mice were subcutaneously administered a combination of cadmium chloride (CdCl2) and methylmercury (II) chloride (CH3HgCl) (0, 0.125, 0.5, or 2.0 mg/kg body weight each) 4 days before and 4 days after conception (8 days total). Indices of anxiety-like behavior, glucose homeostasis, endocrine and molecular markers of insulin resistance, and organ weights were examined in adult male offspring. Results: Increased anxiety-like behavior, impaired glucose homeostasis, and higher body weight and abdominal adipose tissue weight were observed in male offspring of treated females compared with controls. Significantly increased serum leptin and insulin concentrations and impaired insulin tolerance in the male offspring of dams treated with 2.0 mg/kg body weight of Cd and Hg suggested insulin resistance. Altered mRNA abundance for genes associated with glucose and lipid homeostasis (GLUT4, IRS1, FASN, ACACA, FATP2, CD36, and G6PC) in liver and abdominal adipose tissues as well as increased IRS1 phosphorylation in liver (Ser 307) provided further evidence of insulin resistance. Conclusions: Results suggest that the co-administration of Cd and Hg to female mice during the early development of their offspring (the periconception period) was associated with anxiety-like behavior, altered glucose metabolism, and insulin resistance in male offspring at adulthood. Citation: Camsari C, Folger JK, McGee D, Bursian SJ, Wang H, Knott JG, Smith GW. 2017. Effects of periconception cadmium and mercury co-administration to mice on indices of chronic diseases in male offspring at maturity. Environ Health Perspect 125:643–650; http://dx.doi.org/10.1289/EHP481

Characterization of the molecular factors regulating early embryonic development and their functional mechanisms is critical for understanding the causes of early pregnancy loss in monotocous species (cattle, human). We previously characterized a stage specific functional role of follistatin, a TGF-beta superfamily binding protein, in promoting early embryonic development in cattle. The mechanism by which follistatin mediates this embryotropic effect is not precisely known as follistatin actions in cattle embryos are independent of its classically known activin inhibition activity. Apart from activin, follistatin is known to bind and modulate the activity of the bone morphogenetic proteins (BMPs), which signal through SMAD1/5 pathway and regulate several aspects of early embryogenesis in other mammalian species. Present study was designed to characterize the activity and functional requirement of BMP signaling during bovine early embryonic development and to investigate if follistatin involves BMP signaling for its stage specific embryotropic actions. Immunostaining and western blot analysis demonstrated that SMAD1/5 signaling is activated after embryonic genome activation in bovine embryos. However, days 1–3 follistatin treatment reduced the abundance of phosphorylated SMAD1/5 in cultured embryos. Inhibition of active SMAD1/5 signaling (8–16 cell to blastocyst) using pharmacological inhibitors and/or lentiviral-mediated inhibitory SMAD6 overexpression showed that SMAD1/5 signaling is required for blastocyst production, first cell lineage determination as well as mRNA and protein regulation of TE (CDX2) cell markers. SMAD1/5 signaling was also found to be essential for embryotropic actions of follistatin during days 4–7 but not days 1–3 of embryo development suggesting a role for follistatin in regulation of SMAD1/5 signaling in bovine embryos.

Problem Heightened maternal stress affects trophoblast function and increases risk for adverse pregnancy outcomes. Methods of Study Studies were performed using the first-trimester trophoblast cell line, Sw.71. Cytokines were quantified using qPCR and ELISA. Epigenetic regulation of cytokines was characterized by inhibiting histone deacetylation (1 μmol/L suberoylanilide hydroxamic acid [SAHA]) or methylation (5 μmol/L 5-azacytidine), or with chromatin immunoprecipitation (ChIP) with a pan-acetyl histone-3 antibody. Invasion assays used Matrigel chambers. Results Cortisol inhibited expression of CSF2 (GM-CSF) and CSF3 (G-CSF) in trophoblast cells. Cortisol-associated inhibition was dependent on DNA methylation and was not affected by acetylation. There was also a modest decrease in trophoblast invasion, not dependent on loss of CSFs. Conclusion In first-trimester trophoblast cells, the physiological glucocorticoid, cortisol, inhibited two cytokines with roles in placental development and decreased trophoblast invasion. Cortisol-associated changes in trophoblast function could increase the risk for immune-mediated abortion or other adverse pregnancy outcomes.

In mouse blastocysts, CDX2 plays a key role in silencing Oct4 and Nanog expression in the trophectoderm (TE) lineage. However, the underlying transcriptional and chromatin-based changes that are associated with CDX2-mediated repression are poorly understood. To address this, a Cdx2-inducible mouse embryonic stem (ES) cell line was utilized as a model system. Induction of Cdx2 expression resulted in a decrease in Oct4/Nanog expression, an increase in TE markers, and differentiation into trophoblast-like stem (TS-like) cells within 48 to 120 h. Consistent with the down-regulation of Oct4 and Nanog transcripts, a time-dependent increase in CDX2 binding and a decrease in RNA polymerase II (RNAPII) and OCT4 binding was observed within 48 h (P

The establishment of a functional trophectoderm (TE) epithelium is an essential prerequisite for blastocyst formation and placentation. Transcription coactivator yes-associated protein (YAP), a downstream effector of the hippo signaling pathway, is required for specification of both the TE and epiblast lineages in mice. However, the biological role of YAP in porcine blastocyst development is not known. Here, we report that maternally derived YAP protein is localized to both the cytoplasm and nuclei prior to the morula stage and is then predominantly localized to the TE nuclei in blastocysts. Functionally, maternal YAP knockdown severely impeded blastocyst formation and perturbed the allocation of the first two lineages. The treatment of embryos with verteporfin, a pharmacological inhibitor of YAP, faithfully recapitulated the phenotype observed in YAP deleted embryos. Mechanistically, we found that maternal YAP regulates multiple genes which are important for lineage commitment, tight junction assembly, and fluid accumulation. Consistent with the effects on tight junction gene expression, a permeability assay revealed that paracellular sealing was defective in the trophectoderm epithelium. Lastly, YAP knockdown in a single blastomere at the 2-cell stage revealed that the cellular progeny of the YAP+ blastomere were sufficient to sustain blastocyst formation via direct complementation of the defective trophectoderm epithelium. In summary, these findings demonstrate that maternal YAP facilitates porcine blastocyst development through transcriptional regulation of key genes that are essential for lineage commitment, tight junction assembly, and fluid accumulation.

The first cell-fate decision in mammalian preimplantation embryos is the segregation of the inner cell mass (ICM) and trophectoderm (TE) cell lineages. The ICM develops into the embryo proper, whereas the TE ensures embryo implantation and is the source of the extra-embryonic trophoblast cell lineages, which contribute to the functional components of the placenta. The development of a totipotent zygote into a multi-lineage blastocyst is associated with the generation of distinct transcriptional programs. Several key transcription factors participate in the ICM and TE-specific transcriptional networks, and recent studies indicate that post-translational histone modifications as well as ATP-dependent chromatin remodeling complexes converge with these transcriptional networks to regulate ICM and TE lineage specification. This review will discuss our current understanding and future perspectives related to transcriptional and epigenetic regulatory mechanisms that are implicated in the initial mammalian lineage commitment steps, with a focus on events in mice.

Background During implantation, the blastocyst trophectoderm attaches to the endometrial epithelium and continues to differentiate into all trophoblast subtypes, which are the major components of a placenta. Aberrant trophoblast proliferation and differentiation are associated with placental diseases. However, due to ethical and practical issues, there is almost no available cell or tissue source to study the molecular mechanism of human trophoblast differentiation, which further becomes a barrier to the study of the pathogenesis of trophoblast-associated diseases of pregnancy. In this study, our goal was to generate a proof-of-concept model for deriving trophoblast lineage cells from induced pluripotency stem (iPS) cells from human fibroblasts. In future studies the generation of trophoblast lineage cells from iPS cells established from patient’s placenta will be extremely useful for studying the pathogenesis of individual trophoblast-associated diseases and for drug testing. Methods and results Combining iPS cell technology with BMP4 induction, we derived trophoblast lineage cells from human iPS cells. The gene expression profile of these trophoblast lineage cells was distinct from fibroblasts and iPS cells. These cells expressed markers of human trophoblasts. Furthermore, when these cells were differentiated they exhibited invasive capacity and placental hormone secretive capacity, suggesting extravillous trophoblasts and syncytiotrophoblasts. Conclusion Trophoblast lineage cells can be successfully derived from human iPS cells, which provide a proof-of-concept tool to recapitulate pathogenesis of patient placental trophoblasts in vitro.

Our previous studies demonstrated that maternal (oocyte derived) follistatin (FST) expression is positively associated with bovine oocyte competence and exogenous follistatin treatment during the pre-compaction period of development (d 1-3 post insemination) is stimulatory to bovine early embryogenesis in vitro [blastocyst rates and cell numbers/allocation to trophectoderm (TE)]. In the present study, bovine embryos were treated with exogenous follistatin during d 1-3, d 4-7 and d 1-7 post insemination to test the hypothesis that embryotropic effects of exogenous follistatin are specific to the pre-compaction period (d 1-3) of early embryogenesis. Follistatin treatment during d 4-7 (peri-/post-compaction period) of embryo culture increased proportion of embryos reaching blastocyst and expanded blastocyst stage and total cell numbers compared to controls, but blastocyst rates and total cell numbers were lower than observed following d 1-3 (pre-compaction) follistatin treatment. Follistatin supplementation during d 1-7 of embryo culture increased development to blastocyst and expanded blastocyst stages and blastocyst total cell numbers compared to d 1-3 and d 4-7 follistatin treatment and untreated controls. A similar increase in blastocyst CDX2 mRNA and protein (TE cell marker) was observed in response to d 1-3, d 4-7 and d 1-7 follistatin treatment. However, an elevation in blastocyst BMP4 protein (TE cell regulator) was observed in response to d 1-3 and d 1-7, but not d 4-7 (peri-/post-compaction) follistatin treatment. In summary, our study revealed the potential utility of follistatin treatment for increasing the success rate of in vitro embryo production in cattle. Such results also expand our understanding of the embryotropic actions of follistatin and demonstrate that follistatin actions on blastocyst development and cell allocation to the TE layer are not specific to the pre-compaction period.

The CHD family of proteins is characterized by the presence of chromodomains and SNF2-related helicase/ATPase domains, which alter gene expression by modification of chromatin structure. Chd1-null embryos arrest at the peri-implantation stage in mice. However, the functional role of CHD1 during preimplantation development remains unclear, given maternal-derived CHD1 may mask the essential role of CHD1 during this stage in traditional knockout models. The objective of this study was to characterize CHD1 expression and elucidate its functional role in preimplantation development using the bovine model. CHD1 mRNA was elevated after meiotic maturation and remained increased through the 16-cell stage, followed by a sharp decrease at morula to blastocyst stage. Similarly, immunoblot analysis indicated CHD1 protein level is increased after maturation, maintained at high level after fertilization and declined sharply afterwards. CHD1 mRNA level was partially decreased in response to alpha-amanitin (RNA polymerase II inhibitor) treatment, suggesting that CHD1 mRNA in eight-cell embryos is of both maternal and zygotic origin. Results of siRNA-mediated silencing of CHD1 in bovine early embryos demonstrated that the percentages of embryos developing to the 8- to 16-cell and blastocyst stages were both significantly reduced. However, expression of NANOG (inner cell mass marker) and CDX2 (trophectoderm marker) were not affected in CHD1 knockdown blastocysts. In addition, we found that histone variant H3.3 immunostaining is altered in CHD1 knockdown embryos. Knockdown of H3.3 using siRNA resulted in a similar phenotype to CHD1-ablated embryos. Collectively, our results demonstrate that CHD1 is required for bovine early development, and suggest that CHD1 may regulate H3.3 deposition during this period.

Parthenogenetic embryonic stem cells (P-ESCs) offer an alternative source of pluripotent cells, which hold great promise for autologous transplantation and regenerative medicine. P-ESCs have been successfully derived from blastocysts of several mammalian species. However, compared with biparental embryonic stem cells (B-ESCs), P-ESCs are limited in their ability to fully differentiate into all 3 germ layers. For example, it has been observed that there is a differentiation bias toward ectoderm derivatives at the expense of endoderm and mesoderm derivatives-muscle in particular-in chimeric embryos, teratomas, and embryoid bodies. In the present study we found that H19 expression was highly upregulated in P-ESCs with more than 6-fold overexpression compared with B-ESCs. Thus, we hypothesized that manipulation of the H19 gene in P-ESCs would alleviate their limitations and allow them to function like B-ESCs. To test this hypothesis we employed a small hairpin RNA approach to reduce the amount of H19 transcripts in mouse P-ESCs. We found that downregulation of H19 led to an increase of mesoderm-derived muscle and endoderm in P-ESCs teratomas similar to that observed in B-ESCs teratomas. This phenomenon coincided with upregulation of mesoderm-specific genes such as Myf5, Myf6, and MyoD. Moreover, H19 downregulated P-ESCs differentiated into a higher percentage of beating cardiomyocytes compared with control P-ESCs. Collectively, these results suggest that P-ESCs are amenable to molecular modifications that bring them functionally closer to true ESCs.

For more than thirty years, the dog has been used as a model for human diseases. Despite efforts made to develop canine embryonic stem cells, success has been elusive. Here, we report the generation of canine induced pluripotent stem cells (ciPSCs) from canine adult fibroblasts, which we accomplished by introducing human OCT4, SOX2, c-MYC, and KLF4. The ciPSCs expressed critical pluripotency markers and showed evidence of silencing the viral vectors and normal karyotypes. Microsatellite analysis indicated that the ciPSCs showed the same profile as the donor fibroblasts but differed from cells taken from other dogs. Under culture conditions favoring differentiation, the ciPSCs could form cell derivatives from the ectoderm, mesoderm, and endoderm. Further, the ciPSCs required leukemia inhibitory factor and basic fibroblast growth factor to survive, proliferate, and maintain pluripotency. Our results demonstrate an efficient method for deriving canine pluripotent stem cells, providing a powerful platform for the development of new models for regenerative medicine, as well as for the study of the onset, progression, and treatment of human and canine genetic diseases.

Oct4 and Nanog are crucial for maintaining pluripotency in embryonic stem (ES) cells and early-stage embryos. In the present study, the status of DNA methylation and of histone modifications in the regulatory regions of Oct4 and Nanog in rhesus nuclear transfer-derived ES (ntES) cells was compared with in vitro fertilized embryo-derived ES (IVFES) cell counterparts. Dynamic changes in DNA methylation during differentiation into neural lineage were also monitored and correlated with mRNA abundance and protein levels of both genes. In ntES cells Oct4 exhibited mono-allelic methylation along with relatively lower mRNA levels, and its transcription was seen predominantly from the unmethylated allele. In contrast, in IVFES cells Oct4 was hypomethylated on both alleles and had relatively higher transcript levels, suggesting incomplete reprogramming of DNA methylation on the Oct4 gene following somatic cell nuclear transfer. During neuronal differentiation, Oct4 underwent biallelic methylation and reduced amounts of Oct4 mRNA were detected in both types of ES cells. Analysis of Nanog regulatory regions revealed that both alleles were hypomethylated and similar levels of Nanog transcripts were expressed in ntES cells and IVFES cells. During neuronal differentiation both alleles were methylated and reduced amounts of Nanog mRNA were detected. Other epigenetic modifications including histone 3 lysine 4, 9, and 27 trimethylation (H3K4me3, H3K9me3, and H3K27me3) showed similar patterns around the regulatory regions of Oct4 and Nanog in both kinds of ES cells. During neural differentiation, dramatic enrichment of H3K27me3 and H3K9me3 (repressive marks) was observed on Oct4 and Nanog regulatory regions. Differentiation of ntES and IVFES cells correlated with the silencing of Oct4 and Nanog, reactivation of the neural marker genes Pax6, N-Oct3, and Olig2, and dynamic changes in histone modifications in the upstream regions of Pax6 and N-Oct3. In short, although ES cells derived from somatic cell nuclear transfer showed a different epigenetic status in the Oct4 regulatory region than the IVF-derived counterparts, based on the parameters tested, the neural differentiation potential of ntES and IVFES cells is equivalent.

The SWI/SNF-Brg1 chromatin remodeling protein plays critical roles in cell-cycle control and differentiation through regulation of gene expression. Loss of Brg1 in mice results in early embryonic lethality, and recent studies have implicated a role for Brg1 in somatic stem cell self-renewal and differentiation. However, little is known about Brg1 function in preimplantation embryos and embryonic stem (ES) cells. Here we report that Brg1 is required for ES cell self-renewal and pluripotency. RNA interference-mediated knockdown of Brg1 in blastocysts caused aberrant expression of Oct4 and Nanog. In ES cells, knockdown of Brg1 resulted in phenotypic changes indicative of differentiation, downregulation of self-renewal and pluripotency genes (e.g., Oct4, Sox2, Sall4, Rest), and upregulation of differentiation genes. Using genome-wide promoter analysis (chromatin immunoprecipitation) we found that Brg1 occupied the promoters of key pluripotency-related genes, including Oct4, Sox2, Nanog, Sall4, Rest, and Polycomb group (PcG) proteins. Moreover, Brg1 co-occupied a subset of Oct4, Sox2, Nanog, and PcG protein target genes. These results demonstrate an important role for Brg1 in regulating self-renewal and pluripotency in ES cells.

Inositol 1,4,5-trisphosphate generated by the action of a phospholipase C (PLC) mediates release of intracellular Ca2+ that is essential for sperm-induced activation of mammalian eggs. Much attention currently focuses on the role of sperm-derived PLCzeta in generating changes in egg intracellular Ca2+ despite the fact that PLCzeta constitutes a very small fraction of the total amount of PLC in a fertilized egg. Eggs express several isoforms of PLC, but a role for an egg-derived PLC in sperm-induced Ca2+ oscillations has not been examined. Reducing egg PLCbeta1 by a transgenic RNAi approach resulted in a significant decrease in Ca2+ transient amplitude, but not duration or frequency, following insemination. Furthermore, overexpressing PLCbeta1 by microinjecting a Plcb1 cRNA significantly perturbed the duration and frequency of Ca2+ transients and disrupted the characteristic shape of the first transient. These results provide the first evidence for a role of an egg-derived PLC acting in conjunction with a sperm-derived PLCzeta in egg activation.

The TGF-beta-SMAD signaling pathway is involved in regulation of various aspects of female reproduction. However, the intrinsic functional role of SMADs in early embryogenesis remains poorly understood. Previously, we demonstrated that treatment with follistatin, an activin (TGF-beta superfamily ligand)-binding protein, is beneficial for bovine early embryogenesis and specific embryotropic actions of follistatin are dependent on SMAD4. Because SMAD4 is a common SMAD that can bind both SMAD2/3 and SMAD1/5, the objective of this study was to further determine the intrinsic role of SMAD2/3 in the control of early embryogenesis and delineate if embryotropic actions of follistatin in early embryos are SMAD2/3 dependent. By using a combination of pharmacological and small interfering RNA-mediated inhibition of SMAD2/3 signaling in the presence or absence of follistatin treatment, our results indicate that SMAD2 and SMAD3 are both required for bovine early embryonic development and stimulatory actions of follistatin on 8- to 16-cell and that blastocyst rates, but not early cleavage, are muted when SMAD2/3 signaling is inhibited. SMAD2 deficiency also results in reduced expression of the bovine trophectoderm cell-specific gene CTGF. In conclusion, the present work provides evidence supporting a functional role of SMAD2/3 in bovine early embryogenesis and that specific stimulatory actions of follistatin are not observed in the absence of SMAD2/3 signaling.

During mouse preimplantation development, the generation of the inner cell mass (ICM) and trophoblast lineages comprises upregulation of Nanog expression in the ICM and its silencing in the trophoblast. However, the underlying epigenetic mechanisms that differentially regulate Nanog in the first cell lineages are poorly understood. Here, we report that BRG1 (Brahma-related gene 1) cooperates with histone deacetylase 1 (HDAC1) to regulate Nanog expression. BRG1 depletion in preimplantation embryos and Cdx2-inducible embryonic stem cells (ESCs) revealed that BRG1 is necessary for Nanog silencing in the trophoblast lineage. Conversely, in undifferentiated ESCs, loss of BRG1 augmented Nanog expression. Analysis of histone H3 within the Nanog proximal enhancer revealed that H3 lysine 9/14 (H3K9/14) acetylation increased in BRG1-depleted embryos and ESCs. Biochemical studies demonstrated that HDAC1 was present in BRG1-BAF155 complexes and BRG1-HDAC1 interactions were enriched in the trophoblast lineage. HDAC1 inhibition triggered an increase in H3K9/14 acetylation and a corresponding rise in Nanog mRNA and protein, phenocopying BRG1 knockdown embryos and ESCs. Lastly, nucleosome-mapping experiments revealed that BRG1 is indispensable for nucleosome remodeling at the Nanog enhancer during trophoblast development. In summary, our data suggest that BRG1 governs Nanog expression via a dual mechanism involving histone deacetylation and nucleosome remodeling.

The retinoblastoma (RB) tumor suppressor and related family of proteins play critical roles in development through their regulation of genes involved in cell fate. Multiple regulatory pathways impact RB function, including the ubiquitin-proteasome system with deregulated RB destruction frequently associated with pathogenesis. With the current study we explored the mechanisms connecting proteasome-mediated turnover of the RB family to the regulation of repressor activity. We find that steady state levels of all RB family members, RB, p107, and p130, were diminished during embryonic stem cell differentiation concomitant with their target gene acquisition. Proteasome-dependent turnover of the RB family is mediated by distinct and autonomously acting instability elements (IE) located in their C-terminal regulatory domains in a process that is sensitive to cyclin-dependent kinase (CDK4) perturbation. The IE regions include motifs that contribute to E2F-DP transcription factor interaction, and consistently, p107 and p130 repressor potency was reduced by IE deletion. The juxtaposition of degron sequences and E2F interaction motifs appears to be a conserved feature across the RB family, suggesting the potential for repressor ubiquitination and specific target gene regulation. These findings establish a mechanistic link between regulation of RB family repressor potency and the ubiquitin-proteasome system. Background: RB family protein abundance is dynamic and sensitive to growth conditions. Results: RB family turnover is mediated by C-terminal degrons in a process that is phosphorylation-sensitive. Conclusion: The RB family degrons are important regulatory domains linking stability and transcriptional potency. Significance: Dual control of stability and potency by RB family degrons may contribute to embryonic development.

It is widely believed that stimulation of the phosphoinositide pathway and production of 1,4,5-inositol trisphosphate (IP3) underlies the oscillatory changes in the concentration of intracellular free calcium ions ([Ca 21 ]i) seen during mammalian fertilization. IP3 promotes Ca 21 release in eggs by binding to its receptor, the type-1 IP3 receptor (IP3R-1, also known as ITPR1), a ligand-gated Ca 21 channel located in the membrane of the endoplasmic reticulum, the main Ca 21 store of the cell. While IP3R1 has been shown to mediate all Ca 21 release during mouse fertilization, whether or not it plays such an essential role in fertilization-induced Ca 21 release in large domestic species such as bovine and porcine is presently not known. Accordingly, we have generated metaphase II bovine eggs with a ;70%‐80% reduction in the number of intact IP3R-1 by inducing receptor down-regulation during oocyte maturation. We did so by injecting the nonhydrolyzable IP3 analogue, adenophostin A. Functional Ca 21 release analysis revealed that IP3R-1 is the predom

A sperm-specific phospholipase (PL) C, termed PLCζ, is proposed to be the soluble sperm factor that induces Ca2+ oscillations in mammalian eggs and, thus, initiates egg activation in vivo. We report that sperm from transgenic mice expressing short hairpin RNAs targeting PLCζ mRNA have reduced amounts of PLCζ protein. Sperm derived from these transgenic mice trigger patterns of Ca2+ oscillations following fertilization in vitro that terminate prematurely. Consistent with the perturbation in patterns of Ca2+ oscillations is the finding that mating of transgenic founder males to females results in lower rates of egg activation and no transgenic offspring. These data strongly suggest that PLCζ is the physiological trigger of Ca2+ oscillations required for activation of development.

Cell fate decisions are fundamental to the development of multicellular organisms. In mammals the first cell fate decision involves segregation of the pluripotent inner cell mass and the trophectoderm, a process regulated by cell polarity proteins, HIPPO signaling and lineage-specific transcription factors such as CDX2. However, the regulatory mechanisms that operate upstream to specify the trophectoderm lineage have not been established. Here we report that transcription factor AP-2γ (TFAP2C) functions as a novel upstream regulator of Cdx2 expression and position-dependent HIPPO signaling in mice. Loss- and gain-of-function studies and promoter analysis revealed that TFAP2C binding to an intronic enhancer is required for activation of Cdx2 expression during early development. During the 8-cell to morula transition TFAP2C potentiates cell polarity to suppress HIPPO signaling in the outside blastomeres. TFAP2C depletion triggered downregulation of PARD6B, loss of apical cell polarity, disorganization of F-actin, and activation of HIPPO signaling in the outside blastomeres. Rescue experiments using Pard6b mRNA restored cell polarity but only partially corrected position-dependent HIPPO signaling, suggesting that TFAP2C negatively regulates HIPPO signaling via multiple pathways. Several genes involved in regulation of the actin cytoskeleton (including Rock1, Rock2) were downregulated in TFAP2C-depleted embryos. Inhibition of ROCK1 and ROCK2 activity during the 8-cell to morula transition phenocopied TFAP2C knockdown, triggering a loss of position-dependent HIPPO signaling and decrease in Cdx2 expression. Altogether, these results demonstrate that TFAP2C facilitates trophectoderm lineage specification by functioning as a key regulator of Cdx2 transcription, cell polarity and position-dependent HIPPO signaling.

Mammalian oocytes undergo significant growth during oogenesis and experience extensive cytoplasmic and nuclear modifications immediately before ovulation in a process commonly referred to as oocyte maturation. These changes are intended to maximize the developmental success after fertilization. Entry of a spermatozoon into the oocyte, which occurs a few hours after ovulation, initiates long-lasting oscillations in the free intracellular calcium ([Ca(2+)](i)) that are responsible for all events of oocyte activation and the initiation of the developmental programme that often culminates in the birth of young. Nevertheless, the cellular and molecular changes that occur during maturation to optimize development are transient, and exhibit rapid deterioration. Moreover, fertilization of oocytes after an extended residence in the oviduct (or in culture) initiates a different developmental programme, one that is characterized by fragmentation, programmed cell death, and abnormal development. Inasmuch as [Ca(2+)](i) oscillations can trigger both developmental programmes in mammalian oocytes, this review addresses one of the mechanism(s) possibly used by spermatozoa to initiate these persistent [Ca(2+)](i) responses, and the cellular and molecular changes that may underlie the postovulatory cellular fragmentation of ageing mammalian oocytes.

Human polyclonal antibodies (hPABs) are useful therapeutics, but because they are available only from human donors, their supply and application is limited. To address this need, we prepared a human artificial chromosome (HAC) vector containing the entire unrearranged sequences of the human immunoglobulin (hIg) heavy-chain (H) and lambda (lambda) light-chain loci. The HAC vector was introduced into bovine primary fetal fibroblasts using a microcell-mediated chromosome transfer (MMCT) approach. Primary selection was carried out, and the cells were used to produce cloned bovine fetuses. Secondary selection was done on the regenerated fetal cell lines, which were then used to produce four healthy transchromosomic (Tc) calves. The HAC was retained at a high rate (78-100% of cells) in calves and the hIg loci underwent rearrangement and expressed diversified transcripts. Human immunoglobulin proteins were detected in the blood of newborn calves. The production of Tc calves is an important step in the development of a system for producing therapeutic hPABs.

A study was undertaken to determine whether injection of porcine sperm factors (pSF), which trigger oscillations in intracellular calcium concentration ([Ca(2+)](i)) in mammalian oocytes, could be used to activate bovine oocytes during nuclear transfer. To date, only combined treatments that induce a monotonic rise in [Ca(2+)](i) and inhibit either phosphorylation or protein synthesis have been utilized in nuclear transfer. Several doses of pSF were tested. Injection of 5 mg/ml pSF triggered [Ca(2+)](i) oscillations that resembled those associated with fertilization with respect to amplitude and periodicity, and as a result, a high percentage of oocytes underwent activation. Furthermore, this concentration of pSF supported in vitro and in vivo development up to 60-90 days of gestation, comparable to development in control nuclear transfer embryos. Nevertheless, neither activation procedure supported development as well as did fertilization. The effectiveness of pSF as an activating agent in bovine oocytes may have been compromised because pSF was unable to support oscillations past 3-5 h postinjection and a second injection was necessary to extend the [Ca(2+)](i) oscillations. Likewise, a single injection of pSF failed to trigger downregulation of the inositol 1,4,5-trisphosphate receptor 1 subtype, whereas a second injection downregulated the receptor in a manner similar to that seen in fertilized oocytes. These results demonstrate that soluble factor(s) from porcine sperm can support early development in bovine nuclear transfer embryos; however, the efficacy may be limited because of the premature cessation of the induced oscillations.

Mammalian reproduction is critically dependent on the trophoblast cell lineage, which assures proper establishment of maternal–fetal interactions during pregnancy. Specification of trophoblast cell lineage begins with the development of the trophectoderm (TE) in preimplantation embryos. Subsequently, other trophoblast cell types arise with the progression of pregnancy. Studies with transgenic animal models as well as trophoblast stem/progenitor cells have implicated distinct transcriptional and epigenetic regulators in trophoblast lineage development. This review focuses on our current understanding of transcriptional and epigenetic mechanisms regulating specification, determination, maintenance and differentiation of trophoblast cells.

Transforming growth factor beta (TGFbeta) superfamily signaling controls various aspects of female fertility. However, the functional roles of the TGFbeta-superfamily cognate signal transduction pathway components (e.g., SMAD2/3, SMAD4, SMAD1/5/8) in early embryonic development are not completely understood. We have previously demonstrated pronounced embryotrophic actions of the TGFbeta superfamily member-binding protein, follistatin, on oocyte competence in cattle. Given that SMAD4 is a common SMAD required for both SMAD2/3- and SMAD1/5/8-signaling pathways, the objectives of the present studies were to determine the temporal expression and functional role of SMAD4 in bovine early embryogenesis and whether embryotrophic actions of follistatin are SMAD4 dependent. SMAD4 mRNA is increased in bovine oocytes during meiotic maturation, is maximal in 2-cell stage embryos, remains elevated through the 8-cell stage, and is decreased and remains low through the blastocyst stage. Ablation of SMAD4 via small interfering RNA microinjection of zygotes reduced proportions of embryos cleaving early and development to the 8- to 16-cell and blastocyst stages. Stimulatory effects of follistatin on early cleavage, but not on development to 8- to 16-cell and blastocyst stages, were observed in SMAD4-depleted embryos. Therefore, results suggest SMAD4 is obligatory for early embryonic development in cattle, and embryotrophic actions of follistatin on development to 8- to 16-cell and blastocyst stages are SMAD4 dependent.

Oocyte-expressed genes regulate key aspects of ovarian follicular development and early embryogenesis. We previously demonstrated a requirement of the oocyte-specific protein JY-1 for bovine early embryogenesis. Given that JY-1 is present in oocytes throughout folliculogenesis, and oocyte-derived JY-1 mRNA is temporally regulated postfertilization, we hypothesized that JY-1 levels in oocytes impact nuclear maturation and subsequent early embryogenesis. A novel model system, whereby JY-1 small interfering RNA was microinjected into cumulus-enclosed germinal vesicle-stage oocytes and meiotic arrest maintained for 48 h prior to in vitro maturation (IVM), was validated and used to determine the effect of reduced oocyte JY-1 expression on nuclear maturation, cumulus expansion, and embryonic development after in vitro fertilization. Depletion of JY-1 protein during IVM effectively reduced cumulus expansion, percentage of oocytes progressing to metaphase II, proportion of embryos that cleaved early, total cleavage rates and development to 8- to 16-cell stage, and totally blocked development to the blastocyst stage relative to controls. Supplementation with JY-1 protein during oocyte culture rescued effects of JY-1 depletion on meiotic maturation, cumulus expansion, and early cleavage, but did not rescue development to 8- to 16-cell and blastocyst stages. However, effects of JY-1 depletion postfertilization on development to 8- to 16-cell and blastocyst stages were rescued by JY-1 supplementation during embryo culture. In conclusion, these results support an important functional role for oocyte-derived JY-1 protein during meiotic maturation in promoting progression to metaphase II, cumulus expansion, and subsequent embryonic development.

Oocyte-expressed genes regulate key aspects of ovarian follicular development and early embryogenesis. We previously demonstrated a requirement of the oocyte-specific protein JY-1 for bovine early embryogenesis. Given that JY-1 is present in oocytes throughout folliculogenesis, and oocyte-derived JY-1 mRNA is temporally regulated postfertilization, we hypothesized that JY-1 levels in oocytes impact nuclear maturation and subsequent early embryogenesis. A novel model system, whereby JY-1 small interfering RNA was microinjected into cumulus-enclosed germinal vesicle-stage oocytes and meiotic arrest maintained for 48 h prior to in vitro maturation (IVM), was validated and used to determine the effect of reduced oocyte JY-1 expression on nuclear maturation, cumulus expansion, and embryonic development after in vitro fertilization. Depletion of JY-1 protein during IVM effectively reduced cumulus expansion, percentage of oocytes progressing to metaphase II, proportion of embryos that cleaved early, total cleavage rates and development to 8- to 16-cell stage, and totally blocked development to the blastocyst stage relative to controls. Supplementation with JY-1 protein during oocyte culture rescued effects of JY-1 depletion on meiotic maturation, cumulus expansion, and early cleavage, but did not rescue development to 8- to 16-cell and blastocyst stages. However, effects of JY-1 depletion postfertilization on development to 8- to 16-cell and blastocyst stages were rescued by JY-1 supplementation during embryo culture. In conclusion, these results support an important functional role for oocyte-derived JY-1 protein during meiotic maturation in promoting progression to metaphase II, cumulus expansion, and subsequent embryonic development.

The effects of cell cycle stage and the age of the cell donor animal on in vitro development of bovine nuclear transfer embryos were investigated. Cultures of primary bovine fibroblasts were established from animals of various ages, and the in vitro life span of these cell lines was analyzed. Fibroblasts from both fetuses and calves had similar in vitro life spans of approximately 30 population doublings (PDs) compared with 20 PDs in fibroblasts obtained from adult animals. When fibroblasts from both fetuses and adult animals were cultured as a population, the percentage of cells in G1 increased linearly with time, whereas the percentage of S-phase cells decreased proportionately. Furthermore, the percentage of cells in G1 at a given time was higher in adult fibroblasts than in fetal fibroblasts. To study the individual cells from a population, a shake-off method was developed to isolate cells in G1 stage of the cell cycle and evaluate the cell cycle characteristics of both fetal and adult fibroblasts from either 25% or 100% confluent cultures. Irrespective of the age, the mean cell cycle length in isolated cells was shorter (9.6-15.5 h) than that observed for cells cultured as a population. Likewise, the length of the G1 stage in these isolated cells, as indicated by 5-bromo-deoxyuridine labeling, lasted only about 2-3 h. There were no differences in either the number of cells in blastocysts or the percentage of blastocysts between the embryos reconstructed with G1 cells from 25% or 100% confluent cultures of fetal or adult cell lines. This study suggests that there are substantial differences in cell cycle characteristics in cells derived from animals of different ages or cultured at different levels of confluence. However, these factors had no effect on in vitro development of nuclear transfer embryos.

In mouse blastocysts segregation of the inner cell mass (ICM) and the trophectoderm (TE) is regulated by the mutually antagonistic effects of the transcription factors Oct4 and Cdx2 expressed in the ICM and TE, respectively. In contrast, in other species such as bovine and human, Oct4 is not restricted to the ICM and continues to be expressed in the Cdx2-positive TE. A recent comparative study of the bovine and mouse Oct4 promoters revealed that additional mechanisms might act in conjunction with Cdx2 to downregulate Oct4 expression in the mouse TE lineage. For instance, the mouse Oct4 distal enhancer contains an AP-2γ (Tcfap2c) binding motif that is absent in the bovine and human Oct4 distal enhancer. Nonetheless, the functional relevance of Tcfap2c in Oct4 repression during mouse preimplantation development was not tested. To elucidate the role of Tcfap2c in Oct4 expression an RNA interference approach was utilized. Depletion of Tcfap2c triggered a decrease in Oct4 expression at the 8-cell and morula stage. Remarkably, at the blastocyst stage depletion of Tcfap2c and/or its family member Tcfap2a had no effect on Oct4 repression. To test whether Tcfap2c interacts with Oct4 to positively regulate Oct4 expression, chromatin immunoprecipitation and in situ co-immunoprecipitation analyses were performed. These experiments revealed Tcfap2c and Oct4 binding were enriched at the Oct4 distal enhancer in embryonic stem (ES) cells, but were rapidly lost during differentiation into trophoblast-like cells when Oct4 became repressed. Moreover, Tcfap2c and Oct4 interactions were detected at the morula stage, but were lost during blastocyst formation. In summary, these data demonstrate that Tcfap2c is not required for Oct4 silencing in mouse blastocysts, but may be necessary for the maintenance of Oct4 expression during the 8 cell-to-morula transition. These findings support the notion Cdx2 is the predominant negative regulator of Oct4 expression during blastocyst formation in mice.

The trophectoderm epithelium is the first differentiated cell layer to arise during mammalian development. Blastocyst formation requires the proper expression and localization of tight junction, polarity, ion gradient and H2O channel proteins in the outer cell membranes. However, the underlying transcriptional mechanisms that control their expression are largely unknown. Here, we report that transcription factor AP-2γ (Tcfap2c) is a core regulator of blastocyst formation in mice. Bioinformatics, chromatin immunoprecipitation and transcriptional analysis revealed that Tcfap2c binds and regulates a diverse group of genes expressed during blastocyst formation. RNA interference experiments demonstrated that Tcfap2c regulates genes important for tight junctions, cell polarity and fluid accumulation. Functional and ultrastructural studies revealed that Tcfap2c is necessary for tight junction assembly and paracellular sealing in trophectoderm epithelium. Aggregation of control eight-cell embryos with Tcfap2c knockdown embryos rescued blastocyst formation via direct contribution to the trophectoderm epithelium. Finally, we found that Tcfap2c promotes cellular proliferation via direct repression of p21 transcription during the morula-to-blastocyst transition. We propose a model in which Tcfap2c acts in a hierarchy to facilitate blastocyst formation through transcriptional regulation of core genes involved in tight junction assembly, fluid accumulation and cellular proliferation.

During blastocyst formation the segregation of the inner cell mass (ICM) and trophectoderm is governed by the mutually antagonistic effects of the transcription factors Oct4 and Cdx2. Evidence indicates that suppression of Oct4 expression in the trophectoderm is mediated by Cdx2. Nonetheless, the underlying epigenetic modifiers required for Cdx2-dependent repression of Oct4 are largely unknown. Here we show that the chromatin remodeling protein Brg1 is required for Cdx2-mediated repression of Oct4 expression in mouse blastocysts. By employing a combination of RNA interference (RNAi) and gene expression analysis we found that both Brg1 Knockdown (KD) and Cdx2 KD blastocysts exhibit widespread expression of Oct4 in the trophectoderm. Interestingly, in Brg1 KD blastocysts and Cdx2 KD blastocysts, the expression of Cdx2 and Brg1 is unchanged, respectively. To address whether Brg1 cooperates with Cdx2 to repress Oct4 transcription in the developing trophectoderm, we utilized preimplantation embryos, trophoblast stem (TS) cells and Cdx2-inducible embryonic stem (ES) cells as model systems. We found that: (1) combined knockdown (KD) of Brg1 and Cdx2 levels in blastocysts resulted in increased levels of Oct4 transcripts compared to KD of Brg1 or Cdx2 alone, (2) endogenous Brg1 co-immunoprecipitated with Cdx2 in TS cell extracts, (3) in blastocysts Brg1 and Cdx2 co-localize in trophectoderm nuclei and (4) in Cdx2-induced ES cells Brg1 and Cdx2 are recruited to the Oct4 promoter. Lastly, to determine how Brg1 may induce epigenetic silencing of the Oct4 gene, we evaluated CpG methylation at the Oct4 promoter in the trophectoderm of Brg1 KD blastocysts. This analysis revealed that Brg1-dependent repression of Oct4 expression is independent of DNA methylation at the blastocyst stage. In toto, these results demonstrate that Brg1 cooperates with Cdx2 to repress Oct4 expression in the developing trophectoderm to ensure normal development.

It is widely believed that stimulation of the phosphoinositide pathway and production of 1,4,5-inositol trisphosphate (IP(3)) underlies the oscillatory changes in the concentration of intracellular free calcium ions ([Ca(2+)](i)) seen during mammalian fertilization. IP(3) promotes Ca(2+) release in eggs by binding to its receptor, the type-1 IP(3) receptor (IP(3)R-1, also known as ITPR1), a ligand-gated Ca(2+) channel located in the membrane of the endoplasmic reticulum, the main Ca(2+) store of the cell. While IP(3)R-1 has been shown to mediate all Ca(2+) release during mouse fertilization, whether or not it plays such an essential role in fertilization-induced Ca(2+) release in large domestic species such as bovine and porcine is presently not known. Accordingly, we have generated metaphase II bovine eggs with a approximately 70%-80% reduction in the number of intact IP(3)R-1 by inducing receptor down-regulation during oocyte maturation. We did so by injecting the nonhydrolyzable IP(3) analogue, adenophostin A. Functional Ca(2+) release analysis revealed that IP(3)R-1 is the predominant Ca(2+) release channel in bovine eggs, requiring as little as 20% of total intact receptor to mount persistent [Ca(2+)](i) oscillations in response to fertilization, expression of PLCzeta (also known as PLCZ1), and adenophostin A. However, lower concentrations of IP(3) and near-physiological concentrations of porcine sperm extract were unable to trigger [Ca(2+)](i) oscillations in this reduced IP(3)R-1 model. Furthermore, we present evidence that the sensitivity of bovine IP(3)R-1 is impaired at the first embryonic interphase. Together, these results demonstrate the essential role of IP(3)R-1-mediated Ca(2+) release during fertilization in bovine eggs, and identify cell cycle regulatory mechanisms of [Ca(2+)](i) oscillations at the level of IP(3)R-1.

Although the dynamics of oscillations of cytosolic Ca2+ concentration ([Ca2+]cyt) play important roles in early mammalian development, the impact of the duration when [Ca2+]cyt is elevated is not known. To determine the sensitivity of fertilization-associated responses [i.e., cortical granule exocytosis, resumption of the cell cycle, Ca2+/calmodulin-dependent protein kinase II (CaMKII) activity, recruitment of maternal mRNAs] and developmental competence of the parthenotes to the duration of a [Ca2+]cyt transient, unfertilized mouse eggs were subjected to a prolonged [Ca2+]cyt change for 15, 25, or 50 min by means of repetitive Ca2+ electropermeabilization at 2-min intervals. The initiation and completion of fertilization-associated responses are correlated with the duration of time in which the [Ca2+]cyt is elevated, with the exception that autonomous CaMKII activity is down-regulated with prolonged elevated [Ca2+]cyt. Activated eggs from 25- or 50-min treatments readily develop to the blastocyst stage with no sign of apoptosis or necrosis and some implant. Ca2+ influx into unfertilized eggs causes neither Ca2+ release from intracellular stores nor rapid removal of cytosolic Ca2+. Thus, the total Ca2+ signal input appears to be an important regulatory parameter that ensures completion of fertilization-associated events and oocytes have a surprising degree of tolerance for a prolonged change in [Ca2+]cyt.

A sperm-specific phospholipase (PL) C, termed PLCzeta, is proposed to be the soluble sperm factor that induces Ca(2+) oscillations in mammalian eggs and, thus, initiates egg activation in vivo. We report that sperm from transgenic mice expressing short hairpin RNAs targeting PLCzeta mRNA have reduced amounts of PLCzeta protein. Sperm derived from these transgenic mice trigger patterns of Ca(2+) oscillations following fertilization in vitro that terminate prematurely. Consistent with the perturbation in patterns of Ca(2+) oscillations is the finding that mating of transgenic founder males to females results in lower rates of egg activation and no transgenic offspring. These data strongly suggest that PLCzeta is the physiological trigger of Ca(2+) oscillations required for activation of development.

A cytosolic sperm protein(s), referred to as the sperm factor (SF), is thought to induce intracellular calcium ([Ca(2+)](i)) oscillations during fertilization in mammalian eggs. These oscillations, which are responsible for inducing complete egg activation, persist for several hours. Nevertheless, whether a protracted release of SF is responsible for the duration of the oscillations is unknown. Using a combination of intracytoplasmic sperm injection (ICSI), in vitro fertilization (IVF), sperm removal, reinjection of the withdrawn sperm, and [Ca(2+)](i) monitoring, we determined that 30 min was necessary for establishing oscillations. Importantly, a significant portion of the Ca(2+) activity became dissociated from the sperm within 15-60 min after entry, and by 120 min post-ICSI or IVF, sperm were unable to induce oscillations. The initiation of oscillations coincided with exposure and solubilization of the perinuclear theca (PT), as evidenced by transmission electron microscopy, although disassembly of the PT was not required for commencement of the [Ca(2+)](i) responses. Remarkably, despite its complete release into the ooplasm, SF associated with nuclear structures at the time of pronuclear formation. Lastly, release of SF was not affected by the cell cycle. We conclude that mouse sperm serves as a carrier for SF, which is rapidly and completely solubilized to establish [Ca(2+)](i) oscillations.

A study was undertaken to determine whether injection of porcine sperm factors (pSF), which trigger oscillations in intracellular calcium concentration ([Ca(2+)](i)) in mammalian oocytes, could be used to activate bovine oocytes during nuclear transfer. To date, only combined treatments that induce a monotonic rise in [Ca(2+)](i) and inhibit either phosphorylation or protein synthesis have been utilized in nuclear transfer. Several doses of pSF were tested. Injection of 5 mg/ml pSF triggered [Ca(2+)](i) oscillations that resembled those associated with fertilization with respect to amplitude and periodicity, and as a result, a high percentage of oocytes underwent activation. Furthermore, this concentration of pSF supported in vitro and in vivo development up to 60-90 days of gestation, comparable to development in control nuclear transfer embryos. Nevertheless, neither activation procedure supported development as well as did fertilization. The effectiveness of pSF as an activating agent in bovine oocytes may have been compromised because pSF was unable to support oscillations past 3-5 h postinjection and a second injection was necessary to extend the [Ca(2+)](i) oscillations. Likewise, a single injection of pSF failed to trigger downregulation of the inositol 1,4,5-trisphosphate receptor 1 subtype, whereas a second injection downregulated the receptor in a manner similar to that seen in fertilized oocytes. These results demonstrate that soluble factor(s) from porcine sperm can support early development in bovine nuclear transfer embryos; however, the efficacy may be limited because of the premature cessation of the induced oscillations.

Since the landmark study of Wilmut et al. describing the birth of a cloned lamb derived from a somatic cell nucleus, there has been debate about the donor nucleus cell cycle stage required for somatic cell nuclear transfer (NT). Wilmut et al. suggested that induction of quiescence by serum starvation was critical in allowing donor somatic cells to support development of cloned embryos. In a subsequent report, Cibelli et al. proposed that G0 was unnecessary and that calves could be produced from actively dividing fibroblasts. Neither study conclusively documented the importance of donor cell cycle stage for development to term. Other laboratories have had success with NT in several species, and most have used a serum starvation treatment. Here we evaluate methods for producing G0 and G1 cell populations and compare development following NT. High confluence was more effective than serum starvation for arresting cells in G0. Pure G1 cell populations could be obtained using a "shake-off" procedure. No differences in in vitro development were observed between cells derived from the high-confluence treatment and from the "shake-off" treatment. However, when embryos from each treatment were transferred to 50 recipients, five calves were obtained from embryos derived from "shake-off" cells, whereas no embryos from confluent cells survived beyond 180 days of gestation. These results indicate that donor cell cycle stage is important for NT, particularly during late fetal development, and that actively dividing G1 cells support higher development rates than cells in G0.