The roles of parathyroid hormone-like hormone during mouse preimplantation embryonic development

Parathyroid hormone-like hormone (PTHLH) was first identified as a parathyroid hormone (PTH)-like factor responsible for humoral hypercalcemia in malignancies in the 1980s. Previous studies demonstrated that PTHLH is expressed in multiple tissues and is an important regulator of cellular and organ growth, development, migration, differentiation, and survival. However, there is a lack of data on the expression and function of PTHLH during preimplantation embryonic development. In this study, we investigated the expression characteristics and functions of PTHLH during mouse preimplantation embryonic development. The results show that Pthlh is expressed in mouse oocytes and preimplantation embryos at all developmental stages, with the highest expression at the MII stage of the oocytes and the lowest expression at the blastocyst stage of the preimplantation embryos. The siRNA-mediated depletion of Pthlh at the MII stage oocytes or the 1-cell stage embryos significantly decreased the blastocyst formation rate, while this effect could be corrected by culturing the Pthlh depleted embryos in the medium containing PTHLH protein. Moreover, expression of the pluripotency-related genes Nanog and Pou5f1 was significantly reduced in Pthlh-depleted embryos at the morula stage. Additionally, histone acetylation patterns were altered by Pthlh depletion. These results suggest that PTHLH plays important roles during mouse preimplantation embryonic development.     

The effect of oxygen on the development of preimplantation mouse embryos in vitro

The optimal oxygen tension for development of preimplantation mouse embryos to the blastocyst stage in vitro was found to be between 2.5% and 5%. One- and two-cell embryos had a more sharply defined range of oxygen tension capable of supporting development than 8-cell and morula stages. At all stages of development, more embryos developed to the blastocyst stage under 5% O2 compared to the numbers of developing under higher oxygen tensions (20% and 40% O2). The blastocysts developing under 20% O2 had fewer blastomeres than those which developed under 5% O2. As the time required for development to the blastocyst stage in vitro increased, there were fewer blastomeres present at the blastocyst stage. These results indicate that the cleaving mouse embryo has an optimal oxygen requirement in vitro of about 5%. At higher oxygen tensions, fewer embryos develop to the blastocyst stage and in those which do develop, there are fewer cell divisions. If a gradient of oxygen tension exists across the blastomeres from the outside of the embryo to its centre, the blastomeres might be using this gradient to obtain imformation about their location within the embryo and respond accordingly. Thus blastomeres on the outside at a higher oxygen tension would divide at a slower rate and form trophectoderm whereas those on the inside at a lower oxygen tension would divide more rapidly and contribute to the inner cell mass.     

Disruption of the cytokeratin filament network in the preimplantation mouse embryo

The distribution of the cytokeratin network in the intact preimplantation mouse embryo and the role of cytokeratin filaments in trophectoderm differentiation were investigated by means of whole-mount indirect immunofluorescence microscopy and microinjection of anti-cytokeratin antibody. Assembled cytokeratin filaments were detected in some blastomeres as early as the compacted 8-cell stage. The incidence and organization of cytokeratin filaments increased during the morula stage, although individual blastomeres varied in their content of assembled filaments. At the blastocyst stage, each trophectoderm cell contained an intricate network of cytokeratin filaments, and examination of sectioned blastocysts confirmed that extensive arrays of cytokeratin filaments were restricted to cells of the trophectoderm. Microinjection of anticytokeratin antibody into individual mural trophectoderm cells of expanded blastocysts resulted in a dramatic rearrangement of the cytokeratin network in these cells. Moreover, antibody injection into 2-cell embryos inhibited assembly of the cytokeratin network during the next two days of development. Despite this disruption of cytokeratin assembly, the injected embryos compacted and developed into blastocysts with normal morphology and nuclear numbers. These results suggest that formation of an elaborate cytokeratin network in preimplantation mouse embryos is unnecessary for the initial stages of trophectoderm differentiation resulting in blastocyst formation.     

Laser Fusion of Mouse Embryonic Cells and Intra-Embryonic Fusion of Blastomeres without Affecting the Embryo Integrity

Manipulation with early mammalian embryos is the one of the most important approach to study preimplantation development. Artificial cell fusion is a research tool for various biotechnological experiments. However, the existing methods have various disadvantages, first of them impossibility to fuse selected cells within multicellular structures like mammalian preimplantation embryos. In our experiments we have successfully used high repetition rate picosecond near infrared laser beam for fusion of pairs of oocytes and oocytes with blastomeres. Fused cells looked morphologically normal and keep their ability for further divisions in vitro. We also fused two or three blastomeres inside four-cell mouse embryos. The presence of one, two or three nuclei in different blastomeres of the same early preimplantation mouse embryo was confirmed under UV-light after staining of DNA with the vital dye Hoechst-33342. The most of established embryos demonstrated high viability and developed in vitro to the blastocyst stage. We demonstrated for the first time the use of laser beam for the fusion of various embryonic cells of different size and of two or three blastomeres inside of four-cell mouse embryos without affecting the embryo’s integrity and viability. These embryos with blastomeres of various ploidy maybe unique model for numerous purposes. Thus, we propose laser optical manipulation as a new tool for investigation of fundamental mechanisms of mammalian development.     

Allocation of cells to inner cell mass and trophectoderm lineages in preimplantation mouse embryos

Inner cell mass (ICM) and trophectoderm cell lineages in preimplantation mouse embryos were studied by means of iontophoretic injection of horseradish peroxidase (HRP) as a marker. HRP was injected into single blastomeres at the 2- and 8-cell stages and into single outer blastomeres at the 16-cell and late morula (about 22- to 32-cell) stages. After injection, embryos were either examined immediately for localization of HRP (controls) or they were allowed to develop until the blastocyst stage (1 to 3.5 days of culture) and examined for the distribution of labeled cells. In control embryos, HRP was confined to one or two outer blastomeres. In embryos allowed to develop into blastocysts, HRP-labeled progeny were distributed into patches of cells, showing that there is limited intermingling of cells during preimplantation development. A substantial fraction of injected blastomeres contributed descendants to both ICM and trophectoderm (95, 58, 44, and 35% for injected 2-cell, 8-cell, 16-cell, and late morula stages, respectively). Although more than half of the outer cells injected at 16-cell and late morula stages contributed descendants only to trophectoderm (53 and 63%, respectively), some outer cells contributed also to the ICM lineage even at the late morula stage. Although the mechanism for allocation of outer cells to the inner cell lineage is unknown, our observation of adjacent labeled mural trophectoderm and presumptive endoderm cells implicated polarized cell division. This observation also suggests that mural trophectoderm and presumptive endoderm are derived from common immediate progenitors. These cells appear to separate into inner and outer layers during the fifth cleavage division. Our results demonstrate the usefulness of HRP as a cell lineage marker in mouse embryos and show that the allocation of cells to ICM or trophectoderm begins after the 2-cell stage and continues into late cleavage.     

Effect of Homologous Peptides of Differentiation Factors HLDF and PEDF on Preimplantation Development of Mice in vitro

We studied the effect of synthetic peptides PEDF-6 and HLDF-6 on preimplantation development of mouse embryos in vitro. PEDF-6 peptide corresponds to fragment 351–356 and of pigment epithelium-derived differentiation factor (PEDF), while HLDF-6 peptide corresponds to fragment 84–89 of differentiation factor HLDF isolated from HL-60 cell line. Despite high homology, these peptides had different effects on the early development. PEDF-6 had no effect on the cleavage of 2–4-cell embryos but decelerated blastocyst formation from such embryos and disturbed their structure. In the presence of HLDF-6 the blastomeres divided more actively as compared to the control and a higher number of embryos developed to the blastocyst stage. The effects of both peptides were stage-specific: the affect the embryos at early cleavage stages and, apparently, determine their further development at that moment although do not directly affect formation of the blastocysts.     

Development of bovine-ovine interspecies cloned embryos and mitochondria segregation in blastomeres during preimplantation

The objective of the study was to investigate interspecies somatic cell nuclear transfer (iSCNT) embryonic potential and mitochondrial DNA (mtDNA) segregation during preimplantation development. We generated bovine-ovine reconstructed embryos via iSCNT using bovine oocytes as recipient cytoplasm and ovine fetal fibroblast as donor cells. Chromosome composition, the total cell number of blastocyst and embryonic morphology were analyzed. In addition, mtDNA copy numbers both from donor cell and recipient cytoplasm were assessed by real-time PCR in individual blastocysts and blastomeres from 1- to 16-cell stage embryos. The results indicated the following: (1) cell nuclei of ovine fetal fibroblasts can dedifferentiate in enucleated bovine ooplasm, and the reconstructed embryos can develop to blastocysts. (2) 66% of iSCNT embryos had the same number of chromosome as that of donor cell, and the total cell number of iSCNT blastocysts was comparable to that of sheep parthenogenetic blastocysts. (3) RT-PCR analysis in individual blastomeres revealed that the ratio of donor cell mtDNA: recipient cytoplasm mtDNA remained constant (1%) from the one- to eight-cell stage. However, the ratio decreased from 0.6% at the 16-cell stage to 0.1% at the blastocyst stage. (4) Both donor cell- and recipient cytoplasm-derived mitochondria distributed unequally in blastomeres with progression of cell mitotic division. Considerable unequal mitochondrial segregation occurred between blastomeres from the same iSCNT embryos.     

Characterization of intercellular junctions in the preimplantation mouse embryo by freeze-fracture and thin-section electron microscopy

Intercellular junction formation in preimplantation mouse embryos was investigated with thin-section and freeze-fracture electron microscopy. At the four-cell stage, regions of close membrane apposition with focal points of membrane contact and occasional underlying cytoplasmic densities were observed between blastomeres of thin-sectioned embryos. Corresponding intramembrane specializations were not, however, observed in freeze-fractured embryos. At the 8- to 16-cell stage, small gap and macula occludens junctions and complexes of these junctions were observed at all levels between blastomeres of freeze-fractured embryos. As development progressed from the early to mid 8- to 16-cell stage, the size of the occludens/gap junction complexes increased, forming fascia occludens/gap junction complexes. At the morula stage, gap junctions and occludens/gap junction complexes were observed on both presumptive trophoblast and inner cell-mass cells. Zonula occludens junctions were first observed at the morula stage on presumptive trophoblast cells of freeze-fractured embryos. The number of embryos possessing zonula occludens junctions increased at the mid compared to the early morula stage. At the blastocyst stage, junctional complexes consisting of zonula occludens, macula adherens, and gap junctions were observed between trophoblast cells of freeze-fractured and thin-sectioned embryos. Isolated gap and occludens junctions, adherens junctions, and occludens/gap junction complexes were observed on trophoblast and inner cell-mass cells.     

Effect of homologous peptides of differentiation factors HLDF and PEDF on preimplantation development of mice in vitro

We studied the effect of synthetic peptides PEDF-6 and HLDF-6 on preimplantation development of mouse embryos in vitro. PEDF-6 peptide corresponds to fragment 351-356 and of pigment epithelium-derived differentiation factor (PEDF), while HLDF-6 peptide corresponds to fragment 84-89 of differentiation factor HLDF isolated from HL-60 cell line. Despite high homology, these peptides had different effects on the early development. PEDF-6 had no effect on the cleavage of 2-4-cell embryos but decelerated blastocyst formation from such embryos and disturbed their structure. In the presence of HLDF-6 the blastomeres divided more actively as compared to the control and a higher number of embryos developed to the blastocyst stage. The effects of both peptides were stage-specific: the affect the embryos at early cleavage stages and, apparently, determine their further development at that moment although do not directly affect formation of the blastocysts.     

Inhibition of implantation in vitro of frozen-thawed mouse embryos by the signal molecule diadenosine tetraphosphate

The dinucleotide polyphosphate, diadenosine 5', 5'-P(1), P(4)-tetraphosphate (Ap4A), has been identified in mammalian and non-mammalian cells as a signal molecule that initiates the process of DNA replication and cell division. The objective of this study was to determine the function of this messenger molecule in preimplantation mouse embryonic cells. Frozenthawed two-cell mouse embryos were incubated in the presence of 0, 0.1 and 1.0 mM Ap4A at 37 degrees C in moist 5% CO(2) in air mixture for 5 d. The developmental stages of the embryos in terms of hatching and implantation were evaluated. The data showed dose-dependent inhibition of blastocyst implantation; however, there were no differences observed in the number of embryos developing to the blastocyst stage. The results suggest that Ap4A neither promotes nor inhibits the development of early stage embryos except at the implantation stage, where it exerts inhibitory control.     

Evaluation of cyclooxygenase 2 derived endogenous prostacyclin in mouse preimplantation embryo development in vitro

Cyclooxygenase (COX) plays an important role in prostaglandin (PG) synthesis and has two isoforms, COX1 and COX2. PGI synthase (PGIS) catalyzes the isomeization of PGH(2) to prostacyclin (PGI(2)). It is reported that COX2 derived PGI2(2) plays a critical role in blastocyst implantation and decidualization and PGI2 mediates its function via PPARdelta receptor. It is also known that cyclooxygenase derived prostaglandins play an important role in mouse blastocyst hatching in vitro. In this study we hypothesized that COX2 derived PGI2 plays an important role in preimplantation embryonic development by increasing the cell number. To examine this hypothesis, 8-cell stage mouse embryos were cultured in the presence of selective inhibitors of COX1 (SC560), COX2 (NS398) and PGIS (U51605) respectively. COX2 and PGIS inhibitor significantly reduced the blastocyst development and presence of PGI2 analogue along with these inhibitors restored the blastocyst development by increasing the total number of embryonic cells. Our immunohistochemical analysis showed that COX1 is expressed at 2-cell, 8-cell, compaction and blastocyst stage whereas COX2 expression starts from eight cell stage embryos. PGIS and PPARdelta expression starts at 2-cell stage of development. Our results suggest that PGI(2) may affect blastomeres number via the so called hypothesis of PPARdelta nuclear receptor in autocrine manner.     

Zonula occludens-1 (ZO-1) is involved in morula to blastocyst transformation in the mouse

It is unknown whether or not tight junction formation plays any role in morula to blastocyst transformation that is associated with development of polarized trophoblast cells and fluid accumulation. Tight junctions are a hallmark of polarized epithelial cells and zonula occludens-1 (ZO-1) is a known key regulator of tight junction formation. Here we show that ZO-1 protein is first expressed during compaction of 8-cell embryos. This stage-specific appearance of ZO-1 suggests its participation in morula to blastocyst transition. Consistent with this idea, we demonstrate that ZO-1 siRNA delivery inside the blastomeres of zona-weakened embryos using electroporation not only knocks down ZO-1 gene and protein expressions, but also inhibits morula to blastocyst transformation in a concentration-dependent manner. In addition, ZO-1 inactivation reduced the expression of Cdx2 and Oct-4, but not ZO-2 and F-actin. These results provide the first evidence that ZO-1 is involved in blastocyst formation from the morula by regulating accumulation of fluid and differentiation of nonpolar blastomeres to polar trophoblast cells.     

Influence of the preimplantation-embryo-development (ped) gene on mouse blastocyst differentiation

Mouse preimplantation embryonic cleavage rate is dependent upon the presence or absence of the Preimplantation-embryo-development (Ped) gene; which is linked to the Qa-2 subregion of the H-2 complex. Expression of Qa-2 antigens by fast developing mouse embryos correlates with Ped gene pheno-type: Qa-2(a). It is not known if the Ped gene (Qa-2(a)) participates in cell differentiation in the preimplantation mouse blastocyst. Therefore, the study objective was to determine the differentiation of cells to the inner cell mass (ICM) and trophectoderm (TE) in Qa-2(a) positive (Ped +) and Qa-2(a) negative (Ped -) mouse blastocysts. One-cell stage embryos were recovered from the excised oviducts of PMSG (5 IU) and hCG (5 IU) primed virgin female (3-4 weeks) BALB/cByJ (Qa-2(a): Ped -) and BALB/cJ (Qa-2(a): Ped +) mice mated to fertile males (12+ weeks). Embryos were collected, 14 hr after hCG, and cultured in modified alpha-MEM, to the hatched blastocyst stage in an atmosphere of 5% CO2 in air, 95% relative humidity at 37 degrees C. Cell differentiation was determined by differential staining (bis-benzimide and propidium iodide) and fluorescence microscopy. Data were analyzed by Students t-test. There was no significant difference in total cell number between BALB/cJ (mean 139) and BALB/cByJ (mean 143) embryos. A significant difference (p < 0.001) was found in the number of cells differentiating to the ICM between BALB/cJ (mean 59.0) and BALB/cByJ (mean 29.0) mouse embryos. The number of cells differentiating to the TE, between BALB/cJ (mean 80.0) and BALB/cByJ (mean 114) embryos, approached significance (p = 0.062). The results suggest that the Ped gene (Qa-2(a)) may have an influential role in preimplantation blastocyst cell differentiation. Additional studies are warranted to further elucidate the role of the Ped gene in preimplantation embryo development and blastocyst formation.     

Chemical enhancement in embryo development and stem cell derivation from single blastomeres

Several chemicals targeting the mitogen-activated protein (MAP) kinase signaling pathway, which play an important role in regulating cell growth and differentiation, have shown enhancing effects on the development of the inner cell mass (ICM) and the derivation of ES cells. However, investigation of such chemicals on early embryonic development and the establishment of ES cell lines has not been elucidated. This study was aimed to determine if ACTH, MAP2K1 inhibitor [MAP2K1 (I)], and MAPK14 inhibitor [MAPK14 (I)] could enhance the development of the ICM in preimplantation mouse embryos and blastocyst outgrowths, and the establishment of ES cell lines from blastomeres of early embryos. We have demonstrated that both MAP2K1 (I) and MAPK14 (I) delay early embryo development and inhibit the development of embryos from early blastomeres. On the other hand, ACTH had a positive effect on embryos derived from early blastomeres. As a result, 17 ES cell lines were established. Among these ES cell lines, nine and five ES cell lines were established from single blastomeres of two-cell embryos with and without the supplement of ACTH, respectively. In addition to two-cell isolated blastomeres, three ES cell lines were established from blastomeres of four-cell embryos only with the supplement of ACTH. Our results suggest that ACTH can enhance the derivation of ES cells from single blastomere-derived embryos.     

Studies on the susceptibility of the mouse preimplantation embryo to infection with cytomegalovirus.

Intact and zona-free mouse preimplantation embryos were exposed to murine cytomegalovirus in vitro at various stages of development. The embryos developed normally to the blastocyst stage, and there was no evidence of embryonic infection. Intraperitoneal inoculation of female mice with this virus produced an acute generalized infection, and embryonic development was retarded in vivo. The embryos themselves were not productively infected, and they developed into apparently normal fetuses when transferred to uninfected mice.