Cytoplasm Of Early Embryos Research Articles

Polo-like kinase 4 regulates spindle and actin assembly in meiosis and influence of early embryonic development in bovine oocytes

PLK4, a polo-like kinase (PLK) family member that accumulates in the cytoplasm, has been identified as a crucial regulator of centriole formation. PLK4 also controls several essential cellular functions, including cytokinesis and gene expression. In this study, we investigated the expression and function of PLK4 during bovine oocyte meiotic maturation and subsequent embryo development. The PLK4 mRNA was detected in bovine oocytes at all developmental stages during meiotic maturation. Immunofluorescence staining showed that PLK4 protein exhibited a dynamic localization pattern in the oocyte cytoplasm during meiotic maturation, and fluorescence immunostaining markedly increased in metaphase II. When an interfering double-stranded RNA targeting PLK4 was injected into germinal vesicle–stage oocytes, PLK4 transcript levels decreased significantly in metaphase II oocytes (P < 0.05). The PLK4 knockdown caused spindle defects and chromosome misalignment and considerably reduced the amount of cortical and cytoplasmic actin. PLK4 was localized in the cytoplasm of early embryos, and PLK4 knockdown in germinal vesicle–stage oocytes led to failure in the early development of in vitro fertilized embryos (P < 0.05). Taken together, these results indicated that PLK4 plays crucial roles in bovine oocyte meiotic maturation and subsequent early embryo development.

Gonadogenesis and expression pattern of the vasa gene in the sea cucumber Apostichopus japonicus during early development

Vasa has been extensively used as a germ-line marker to trace the origin and migration pathway of primordial germ cells (PGCs) in many organisms, but little work has been reported on vasa genes and the origin of PGCs in holothurians. Using in situ hybridization and immunohistochemistry, vasa mRNA and protein of the sea cucumber Apostichopus japonicus (Aj-vasa) was detected in the cytoplasm of the unfertilized egg and was equally distributed in the cytoplasm of early embryos, from the two-cell embryo to the blastula, indicating that Aj-vasa mRNA is maternally supplied. Later, expression of both Aj-vasa mRNA and protein centralizes gradually in newly organized structures from blastula to five-tentacle larva, and then is restricted to PGC-like cells of the original gonad in juveniles with 0.1-cm body length. The structure of the gonad develops further from a simple tubular gonad in 0.5-cm-length juveniles to a branched gonad in 3-cm-length juveniles. Our findings showed that the maternal supply of the vasa gene products in A. japonicus is different from that in sea urchin Strongylocentrotus purpuratus, of echinoderm, and suggested that the specialization of PGCs is an epigenesis mechanism in A. japonicus.

Developmental expression pattern of the Fc-vasa-like gene, gonadogenesis and development of germ cell in Chinese shrimp, Fenneropenaeus chinensis

The vasa gene, a member of the DEAD-box gene family, has been reported to be restricted exclusively to the germ cell lineage in most animals, and thus is the most reliable molecular marker for studies on germ cell development. In this study, the developmental expression pattern of the vasa-like gene in Fenneropenaeus chinensis ( Fc-vasa-like) is presented. The origin and migration of the germ cell are revealed by whole mount in situ hybridization (ISH). Furthermore, gonadogenesis is observed by histological techniques and ISH. Fc-vasa-like mRNA is located in the cytoplasm of oocytes before and after spawning, and equally distributed in the cytoplasm of early embryos, from the 2-cell embryo to the blastula, indicating that Fc-vasa-like mRNA is maternally supplied. Later on, the expression of the Fc- vasa-like gene centralizes gradually, and is restricted to five cell clusters as precursor cells of primordial germ cells (PGCs) at the limb bud stage. The five cell clusters are located at the base of the antennules and antenna, as well as the cephalic lobe, implying an epigenetic mechanism of germ cell specification in this species. These cells migrate to a dorsolateral position in naupliar and zoeal stages, and gradually enter the genital ridge at the mysis 1 stage. The gonad is first detected histologically at the post-larval 1 stage (PL 1, 1st day postlarva), and germ stem cells (GSCs) are distinguished in cluster in juveniles with the body length of 30 mm. Our findings show that the developmental expression pattern of Fc-vasa-like is different from that of other Crustaceans, and suggest an epigenetic mechanism of germ cell development in Chinese shrimp.

Gene Expression of Dnmt1 Isoforms in Porcine Oocytes, Embryos, and Somatic Cells

In the mouse, the dynamics of genomic methylation and the initial events of gametic imprinting are controlled by the activity of an oocyte isoform of the DNA methyltransferase-1 (Dnmt1o) enzyme. The objectives of this study were to identify the alternative splicing variants of Dnmt1 in porcine oocytes and determine the gene expression pattern of the different Dnmt1 isoforms during embryo development. A rapid amplification of cDNA ends (RACE ) system was used to amplify the 5' cDNA end of Dnmt1 isoforms in porcine oocytes. RNA levels of the Dnmt1 isoforms were analyzed in porcine oocytes and embryos. DNMT1 protein expression of oocytes and somatic cells were analyzed by western blot and immunostaining. Two new Dnmt1o RNA isoforms were identified--Dnmt1o1 and Dnmt1o2. The previously reported somatic Dnmt1 isoform (Dnmt1s) was expressed at low but constant levels in oocytes and embryos from the two-cell to the blastocyst stage. Abundant RNA levels of Dnmt1o1 and Dnmt1o2 were detected in oocytes and embryos from the two- to the eight- to 16-cell stage. Levels of these Dnmt1o transcripts were low at the morula and blastocyst stages. Although Dnmt1s was present in all the somatic cell types analyzed, Dnmt1o1 and Dnmt1o2 were not detected in any somatic tissues. As predicted by the RNA sequence and verified by western blot analysis, Dnmt1o1 and Dnmt1o2 RNAs translate one DNMT1o enzyme. Western blot analysis confirmed that both the oocyte and the somatic forms of DNMT1 protein are present in porcine oocytes and early embryos, whereas somatic cells produce only DNMT1s protein. DNMT1o is localized mainly in the nuclei of oocytes and early embryos, whereas DNMT1s is expressed in the ooplasm cortex of oocytes and cytoplasm of early embryos.

Follow-up of Exogenous DNA by Sperm-mediated Gene Transfer via Liposome

To examine the feasibility of using a sperm vector system for gene transfer, we have investigated the binding and the uptaking of foreign DNA into the sperm nucleus by PCR, in situ hybridization and LSC. We have also examined the transportation of exogenous DNA into oocytes by immunofluorescene via PCR. Sperm cells were incubated with DNA/liposome complexes (1:4 ratio) in fertilization medium with BSA or without BSA. In situ hybridization demonstrated that the transfection rate of sperm cells with and without BSA was 41 and 68% respectively, when the cells were treated with liposome/DNA complexes and 13% for DNA alone. LSC analysis showed that the binding of exogenous DNA was greatly reduced by DNase I treatment which digests DNA bound onto spermatozoa, suggesting that some of the DNA was internalized into the sperm membrane. To find out whether transfected DNA was internalized into sperm intracytomembrane, sperm DNA was amplified by inverse PCR. No PCR products were detected from sperm cells, indicating that the foreign DNA was simply bound onto the sperm membrane. To investigate transfer rates of exogenous DNA into oocytes via sperm cells, we used immunofluorescene method to follow the distribution of foreign DNA via spermatozoa: a few exogenous DNA was located in the cytoplasm of early embryos (13/60, 21.7% for DNA+/liposome+/BSA) and was not located in the pronucleus and/or nucleus. These results suggest that most of the transfected sperm cells could carry the foreign DNA into the egg by in vitro fertilization, but that the transferred DNA is degraded in the developing embryos without stable integration into the zygote genome. Therefore, we have directly injected with transfected sperm cell into oocyte cytoplasm and observed that some of the exogenous DNA was detected in preimplantation embryonic cytoplasm and expressed at preimplantation stages, suggesting that exogenous DNA in early zygote has their integrity. In this study, we have not identified a noble mechanism that interfering transportation of foreign DNA into zygote genome via spermatozoa. Our data, however, demonstrated that inverse PCR and immunofluorescene methods would be used as a new tool for follow-up of gene distribution in oocyte via sperm cells.

Regulation of SR protein localization during development.

Ser-Arg-rich (SR) proteins play numerous roles in spliceosome assembly and the regulation of splice-site selection. Whereas considerable attention has focused on the mechanistic details of SR protein activities, little is known concerning how these splicing regulators are controlled by the cell. Here we examined the subcellular localization of precursor mRNA splicing factors during early development of the nematode Ascaris lumbricoides. In the early embryo, before major zygotic gene activation, most SR proteins, along with RNA polymerase II, are localized in the cytoplasm. As development proceeds, we observe a significant decrease in the cytoplasmic levels of these factors and a concomitant increase in nuclear localization. In contrast, trimethylguanosine-capped small nuclear ribonucleoproteins are predominantly localized in the nucleus throughout this period. We previously showed that the phosphorylation state and activity of SR proteins are regulated during A. lumbricoides embryogenesis. These changes correlate with the onset of precursor mRNA splicing and zygotic transcription. Thus, a coordinate change in the subcellular localization of SR proteins and RNA polymerase II occurs at the transition from reliance on maternally deposited factors to embryonic expression. We propose that before zygotic gene activation, SR proteins and RNA polymerase II are stockpiled in the cytoplasm of early embryos, awaiting signals that lead to their activation.

DNA methyltransferase is actively retained in the cytoplasm during early development.

The overall DNA methylation level sharply decreases from the zygote to the blastocyst stage despite the presence of high levels of DNA methyltransferase (Dnmt1). Surprisingly, the enzyme is localized in the cytoplasm of early embryos despite the presence of several functional nuclear localization signals. We mapped a region in the NH(2)-terminal, regulatory domain of Dnmt1 that is necessary and sufficient for cytoplasmic retention during early development. Altogether, our results suggest that Dnmt1 is actively retained in the cytoplasm, which prevents binding to its DNA substrate in the nucleus and thereby contributes to the erasure of gamete-specific epigenetic information during early mammalian development.

Transgenesis in fish

Gene transfer into fish embryo is being performed in several species (trout, salmon, carps, tilapia, medaka, goldfish, zebrafish, loach, catfish, etc.). In most cases, pronuclei are not visible and microinjection must be done into the cytoplasm of early embryos. Several million copies of the gene are generally injected. In medaka, transgenesis was attempted by injection of the foreign gene into the nucleus of oocyte. Several reports indicate that the injected DNA was rapidly replicated in the early phase of embryo development, regardless of the origin and the sequence of the foreign DNA. The survival of the injected embryos was reasonably good and a large number reached maturity. The proportion of transgenic animals ranged from 1 to 50% or more, according to species and to experimentators. The reasons for this discrepancy have not been elucidated. In all species, the transgenic animals were mosaic. The copy number of the foreign DNA was different in the various tissues of an animal and a proportion lower than 50% of F1 offsprings received the gene from their parents. This suggests that the foreign DNA was integrated into the fish genome at the two cells stage or later. An examination of the integrated DNA in different cell types of an animal revealed that integration occurred mainly during early development. The transgene was found essentially unrearranged in the fish genome of the founders and offsprings. The transgenes were therefore stably transmitted to progeny in a Mendelian fashion. Southern blot analysis revealed the presence of possible junction fragments and also of minor bands which may result from a rearrangement of the injected DNA. In all species, the integrated DNA appeared mainly as random end-to-end concatemers. In adult trout blood cells, a small proportion of the foreign DNA was maintained in the form of non-integrated concatemers, as judged by the existence of end fragments. The transgenes were generally only poorly expressed. The majority of the injected gene constructs contained essentially mammalian or higher vertebrates sequences. The comparison of the expression efficiency of these constructs in transfected fish and mammalian cells indicates that some of the mammalian DNA sequences are most efficiently understood by the fish cell machinery. Chloramphenicol acetyl transferase gene under the control of promoters from Rous sarcoma virus, and human cytomegalovirus, was expressed in several tissues of transgenic fish. Chicken delta-crystallin gene was expressed in several tissues of transgenic fish.(ABSTRACT TRUNCATED AT 400 WORDS)

The graded distribution of the dorsal morphogen is initiated by selective nuclear transport in Drosophila

The maternal morphogen dorsal ( dl) plays a key role in the establishment of dorsal-ventral polarity in Drosophila. We present evidence that the graded distribution of dl protein is initiated by selective nuclear transport. The dl protein is uniformly distributed throughout the cytoplasm of early embryos, but approximately 90 min after fertilization, dl protein present in ventral but not dorsal regions is selectively transported to the nucleus. Mutations in maternally active genes that regulate dl disrupt this transport process, resulting in an inactive, cytoplasmically localized form of the dl protein. Selective nuclear transport of dl protein was reproduced in tissue culture cells. The wild-type dl protein is largely restricted to the cytoplasm, while truncated proteins are predominantly localized within the nucleus. Transient cotransfection assays suggest that dl activates expression from several promoters in an apparently sequence-independent manner. We discuss the role of nuclear transport as a regulated process in gene expression and development.