Spectrin and calmodulin in spreading mouse blastomeres

The role of spectrin and its association with calmodulin in spreading mouse blastomeres was investigated. Embryonic spectrin binds 125I-calmodulin in a calcium-dependent fashion in the blot overlay technique. Double-labeling experiments show coordinate redistribution of spectrin and calmodulin in blastomeres preparing to undergo active spreading movement. At this stage cortical spectrin staining is lost from the region of cell-substrate contact and spectrin and calmodulin become concentrated in two structures closely associated with the contacted region: a group of spherical bodies located on the cytoplasmic side of the cortical layer and a subcortical ring that marks the perimeter of the contacted region. The localization pattern of spectrin and calmodulin is also coordinated with that of actin and myosin. The results suggest that spectrin plays a role in the spreading of blastomeres and that this function may involve linkage of spectrin, calmodulin, and the cortical contractile apparatus.     

Expression of Xenopus Daz-like protein during gametogenesis and embryogenesis

Using a monoclonal antibody raised against Xenopus Daz-like protein (Xdazl), we showed that Xdazl is present in all stages of male and female germ cells except mature spermatozoa. Xdazl is not localized to any specific regions in early-stage embryos, in contrast to the strict localization of its mRNA in the germ plasm. Xdazl disappears after gastrulation but reappears in the primordial germ cells situated at the genital ridge. This is the first detailed report on the protein expression of a Daz-like gene during gametogenesis and embryogenesis in Xenopus, showing the difference in expression patterns of its mRNA and protein.     

On the gametogenesis and early embryogenesis in some olive tree cultivars

A comparative embryological study on two Spanish cultivars and one growing in Bulgaria of Olea europaea was carried out. The embryo sac develops according to Allium (bisporic)-type. The critical phases during the development of male and female gametophyte that lead to sterility and abortiveness of the embryo sacs and ovules are reported. The embryogenesis follows the Asterad-type. The endosperm passes a free nuclear stage and later on becomes cellular.     

Spectrin synthesis in the preimplantation mouse embryo

The preimplantation mouse embryo expresses two polypeptides, Mr 240,000 and Mr 235,000, that are immunologically cross-reactive with antibody to the alpha and beta subunits of mouse brain spectrin. We investigated the synthesis of the spectrin subunits in the Triton-soluble and Triton-insoluble fractions of fertilized eggs, two-cell embryos, compacted morulae, and blastocysts labeled with L-[35S]methionine. Synthesis of embryonic spectrin began in the Triton-soluble fraction with significant levels of alpha-spectrin synthesis first detected in the morula stage and significant levels of beta-spectrin synthesis detected in the blastocyst stage. Incorporation of newly synthesized alpha- and beta-spectrin into the cytoskeletal fraction took place in the blastocyst when equal amounts of both subunits were assembled. Previous studies have shown Triton-insoluble spectrin to be concentrated in regions of cell-cell contact in the embryo (J. S. Sobel and M. A. Alliegro, 1985, J. Cell Biol. 100, 333-336). The temporal and spatial correlation between the assembly of newly synthesized spectrin and its concentration in regions of cell apposition is consistent with the hypothesis that cell contact may influence the assembly of embryonic spectrin.     

Histone synthesis in the cells of proliferating and nonproliferating tissues during gametogenesis and early embryogenesis

A review of available data on the replication-dependent and replication-independent histone synthesis in the proliferating and nonproliferating (quiescent) cells during gametogenesis and early embryogenesis. In each of the considered models the replication-dependent and replication-independent histone synthesis play different roles in the chromatin organization and metabolism. The transition from replication-dependent to replication-independent histone synthesis during gametogenesis is a regular process directed to the formation of a highly compacted metabolically inert chromatin (males) and to the formation of histone protein pool in order to provide the chromatin nucleosome structure in the sperm nucleus during fertilization, as well as the nuclear chromatin in zygotes and blastomeres (females). A suggestion is put forward that the coupling of histone and DNA syntheses should arise not simultaneously in all cells of the embryo but have a regional pattern, due, possibly, to the asynchrony of cell cycle in the early embryos.     

YAO is a nucleolar WD40-repeat protein critical for embryogenesis and gametogenesis in Arabidopsis

Background  

In flowering plants, gametogenesis generates multicellular male and female gametophytes. In the model system Arabidopsis, the male gametophyte or pollen grain contains two sperm cells and a vegetative cell. The female gametophyte or embryo sac contains seven cells, namely one egg, two synergids, one central cell and three antipodal cells. Double fertilization of the central cell and egg produces respectively a triploid endosperm and a diploid zygote that develops further into an embryo. The genetic control of the early embryo patterning, especially the initiation of the first zygotic division and the positioning of the cell plate, is largely unknown.     

Disruption of the Arabidopsis SMC4 gene, AtCAP-C, compromises gametogenesis and embryogenesis

In higher eukaryotes, the condensin complex is a multisubunit apparatus that plays a pivotal role in the coordinated condensation of chromatin during mitosis. The catalytic subunits, CAP-E and CAP-C, members of the SMC family of ATPases, form a heterodimer, the activity of which is controlled by the non-SMC subunits CAP-D2, CAP-G and CAP-H. Here, we report the characterization of a T-DNA insertion mutant of the Arabidopsis CAP-C gene. Analysis of the progeny of selfed heterozygotes revealed that the homozygous null genotype is embryo lethal, with arrest occurring at or before the globular stage of development. Patterning defects associated with altered planes of cytokinesis were found in both the embryo and the suspensor. Crosses of heterozygotes with wild type plants revealed both male and female gametophytic defects. Stretched chromatin was observed between segregating mitotic chromosomes in pollen produced by selfed heterozygotes. Additionally, some plants heterozygous for the T-DNA insertion exhibited loss of apical dominance and mild fasciation, indicating a semi-dominant effect of the mutation. These results reveal a critical role for AtCAP-C during cell division and, unlike our previous studies on the AtCAP-E genes, suggest that no redundant factors for AtCAP-C exist in the Arabidopsis genome.     

Parental-specific methylation of an imprinted transgene is established during gametogenesis and progressively changes during embryogenesis.

Genomic imprinting is a regulatory process that requires a cell to recognize the parental origin of alleles. To understand how these alleles are distinguished, we have assessed changes in the DNA methylation of an imprinted transgene as it switches from one inheritance pattern to another while moving through gametogenesis and embryogenesis. We find that both maternally and paternally inherited methylation patterns are erased in primordial germ cells and that distinctive patterns emerge during germ cell maturation. In the case of the maternal allele, the methylation pattern is fully acquired during oogenesis. In the case of the paternal allele, the methylation pattern found in sperm undergoes further modification during embryogenesis. Thus, the distinction between "erased" maternal and paternal alleles is first established during their residence in different germ cells and then may be maintained by the recognition of the distinctive patterns that each allele displays in the zygote.     

Modification of in vitro mouse embryogenesis by X-rays and fluorochromes

In view of planned studies using single particle irradiation at the Institute for Medical Radiobiology (IMR), confocal microscopy will become an important tool to visualise subtle changes in embryo morphology. Therefore, the influence of X-rays and that of three different fluorochromes on the in vitro development of murine 2-cell stage embryos was investigated. Embryos of B₆C₃F₁ mice were cultured at 32 h post-conception (pc) and treated with X-rays, acridine orange (AO), ethidium bromide (EB) or propidium iodide (PI), respectively, in various doses (0.0–3.0 Gy) or concentrations (AO: 0.05–2.00 µg/ml; EB and PI: 0.50–10.00 µg/ml). Additional experiments using combinations of AO and irradiation were performed. The embryos were cultivated for a total of 7 days and checked for their vital status by morphological endpoints such as the percentage of embryos that reached the blastocyst stage and the hatching rate. After treatment of the 2-cell embryos with AO, EB and PI, the dose-effect curves with the endpoint ‘hatching rate’ showed a 23-fold reduction of the ED₅₀ for AO (0.23 µg/ ml) compared with EB (5.30 µg/ml). Despite the higher toxicity, AO had much better staining qualities in subtoxic concentrations than EB. PI showed no toxicity in these experiments and was used as an inverse control for embryo vitality. No synergistic modification of the radiogenic effects could be seen in combination experiments (AO+X-rays).     

Expression of inhibin α-subunit gene during mouse gametogenesis

Mammalian gametogenesis is regulated through complex interactions between germ and somatic cells. To investigate the mechanism underlying the differentiation of functional gametes, some genes specifically expressed during gametogenesis have been isolated and characterized. In a search for further examples of such genes, we have isolated from a newborn mouse testis cDNA library, a clone corresponding to mouse inhibin alpha-subunit. Although it is known that the inhibin alpha-subunit molecule is abundantly produced in ovarian follicle and in testicular Sertoli cells, the spatial and temporal patterns of expression of this gene remain to be elucidated. In this study, the patterns of expression of inhibin alpha-subunit mRNA during mouse gametogenesis were examined by RNA blot, cytoplasmic dot and in situ hybridization techniques. In the testis, the concentration of inhibin alpha-subunit mRNA increased from about 16 dpc (days post coitum), peaked at birth and then gradually decreased, paralleling testicular development. Inhibin alpha-subunit mRNA was localized in Sertoli cells of wild type as well as W/Wv testes. In adult testis, mRNA was restricted to the perinuclear cytoplasm of Sertoli cells. Inhibin alpha-subunit mRNA was expressed in follicle cells of adult ovary more abundantly than in adult testis. Analysis of expression during folliculogenesis showed that the accumulation of this mRNA began in preantrum follicles and the level of expression reached a maximum in Graafian follicles.     

Spectrin subtypes in mammalian brain

Mammalian neural cells contain at least two forms of brain spectrin: brain spectrin (240/235) which is located primarily in the axons and presynaptic terminals of neurons, and brain spectrin (240/235E) which is found in the cell bodies, dendrites and postsynaptic terminals of neurones. Brain spectrin (240/235E) is also found in certain glial cell types. Antibodies against red blood cell spectrin detect only brain spectrin (240/235E), while antibodies against brain spectrin isolated from axonal and synaptic membranes detect brain spectrin (240/235). Previous apparent discrepancies in the literature concerning brain spectrin localization at the light microscope level were undoubtedly due to different laboratories detecting distinct brain spectrin subtypes, based on the particular antibody being utilized for immunohistochemistry. In this review we (1) discuss the data supporting the presence of at least two distinct subtypes of mammalian brain spectrin, (2) explain how these results reconcile previous discrepancies concerning the localization of spectrin within neural cells, and (3) suggest the future implications of these findings.     

Growth factors and proto-oncogenes in early mouse embryogenesis and tumorigenesis.

Growth factors and proto-oncogenes play an important role in the regulation of embryonic growth and differentiation as well as in tumorigenesis. Insulin and insulin-like growth factor I (IGF I) are secreted by embryonic tissues during the prepancreatic stage of mouse development. Measureable amounts of these factors were found in 8- to 12-day-old embryos. Embryonic cells derived from 8- to 10-day-old embryos secrete insulin and IGF I in serum-free medium. Relatively high levels of c-myc, c-fos and c-H-ras oncoproteins were also detected in 8- to 12-day-old embryos. Insulin and IGF I, when added to the culture of embryonic cells, stimulate their proliferation. Similar results were obtained in some animal or human tumors. Murine myeloid leukemias and melanoma B 16 secrete a substance immunologically cross reactive with insulin (SICRI) both in vivo and in serum-free media. In culture, the DNA synthesis rate per leukemic or melanoma cell is proportional to cell density and is reduced by antiinsulin serum in case of leukemic cells. Human hemangiosarcoma secrete IGF I, which also plays a role as an autocrine factor. Purified IGF I efficiently induce c-myc and c-fos mRNA, which is among the earliest events following growth factor stimulation, leading to mitosis. These results lead us to the conclusion that IGF I and insulin together with oncoproteins stimulate the growth of embryonic and tumor cells, which is indirect evidence for a paracrine (or autocrine) type of action.