Changes in levels of polymeric tubulin associated with activation and dorsoventral polarization of the frog egg

The level of polymeric tubulin was measured during the first cell cycle of the electrically activated and the fertilized egg of Xenopus laevis. Eggs were homogenized in a microtubule-stabilizing medium, and the amount of tubulin pelleted by centrifugation was determined by quantitative Western blots. The pelleted tubulin (polymer) was in the form of microtubules based on the presence of microtubules in the pellet and on the effects of cold, nocodazole, and D2O. Unactivated eggs had a high level of polymer (greater than 0.1 microgram/egg) which disappeared within minutes of activation. The level of polymer stayed low (less than 0.02 microgram/egg) until halfway through the cell cycle (0.5 on a normalized time scale) when the level rose to the preactivation value. There was a decrease associated with metaphase (0.85 normalized time) and a return to a high level at first cleavage (1.0 normalized time). Fertilized eggs showed a similar pattern although the amount of polymer increased earlier (0.3-0.5 normalized time), presumably due to the spermaster. The depolymerization of microtubules at activation indicates that there is a dramatic change of the cytoskeleton at this time. The polymerization at 0.5 normalized time coincides with the start of the cytoplasmic shift leading to dorsoventral polarity. This result, together with previous inhibitor studies, shows that microtubules are involved in dorsoventral polarization of the embryo.     

Dissociation of intracellular lysosomal rupture from the cell death caused by silica

The relationship between intracellular lysosomal rupture and cell death caused by silica was studied in P388d(1) macrophages. After 3 h of exposure to 150 μg silica in medium containing 1.8 mM Ca(2+), 60 percent of the cells were unable to exclude trypan blue. In the absence of extracellular Ca(2+), however, all of the cells remained viable. Phagocytosis of silica particles occurred to the same extent in the presence or absence of Ca(2+). The percentage of P388D(1) cells killed by silica depended on the dose and the concentration of Ca(2+) in the medium. Intracellular lyosomal rupture after exposure to silica was measured by acridine orange fluorescence or histochemical assay of horseradish peroxidase. With either assay, 60 percent of the cells exposed to 150 μg silica for 3 h in the presence of Ca(2+) showed intracellular lysosomal rupture, was not associated with measureable degradation of total DNA, RNA, protein, or phospholipids or accelerated turnover of exogenous horseradish peroxidase. Pretreatment with promethazine (20 μg/ml) protected 80 percent of P388D(1) macrophages against silica toxicity although lysosomal rupture occurred in 60-70 percent of the cells. Intracellular lysosomal rupture was prevented in 80 percent of the cells by pretreatment with indomethacin (5 x 10(-5)M), yet 40-50 percent of the cells died after 3 h of exposure to 150 μg silica in 1.8 mM extracellular Ca(2+). The calcium ionophore A23187 also caused intracellular lysosomal rupture in 90-98 percent of the cells treated for 1 h in either the presence or absence of extracellular Ca(2+). With the addition of 1.8 mM Ca(2+), 80 percent of the cells was killed after 3 h, whereas all of the cells remained viable in the absence of Ca(2+). These experiments suggest that intracellular lysosomal rupture is not causally related to the cell death cause by silica or {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187. Cell death is dependent on extracellular Ca(2+) and may be mediated by an influx of these ions across the plasma membrane permeability barrier damaged directly by exposure to these toxins.     

Site of sperm entry and a cortical contraction associated with egg activation in the frog Rana pipens

The region of the frog egg that is receptive to fertilization was determined. As an approximation to the site of sperm entry, the start of the male pronuclear penetration path within the egg was made visible externally by bleaching fixed eggs. A bleached egg had a pigment accumulation on its surface corresponding to the start of the penetration path. The accumulation characteristically changed shape with cortical movements prior to first cleavage, and most accumulations (path starts) were within 60° of the animal pole.Localized inseminations and an analysis of the distribution of failures of fertilization at the egg plasma membrane demonstrated that few if any sperm entered the vegetal region of the egg. Localized inseminations, however, demonstrated that sperm entered between 60° from the animal pole and the animal-vegetal margin.Although sperm entry occurred throughout the animal region, most penetration paths started within 60° of the animal pole. To account for this, the sperm nucleus must move towards the animal pole prior to starting the penetration path. This movement appeared to be due to a contraction of the cortex towards the animal pole that occurred 3–4 min after activation of the egg.     

Paracrine effects of oocyte secreted factors and stem cell factor on porcine granulosa and theca cells <Emphasis Type="Italic">in vitro</Emphasis>

Oocyte control of granulosa and theca cell function may be mediated by several growth factors via a local feedback loop(s) between these cell types. This study examined both the role of oocyte-secreted factors on granulosa and thecal cells, cultured independently and in co-culture, and the effect of stem cell factor (SCF); a granulosa cell derived peptide that appears to have multiple roles in follicle development. Granulosa and theca cells were isolated from 2–6 mm healthy follicles of mature porcine ovaries and cultured under serum-free conditions, supplemented with: 100 ng/ml LR3 IGF-1, 10 ng/ml insulin, 100 ng/ml testosterone, 0–10 ng/ml SCF, 1 ng/ml FSH (granulosa), 0.01 ng/ml LH (theca) or 1 ng/ml FSH and 0.01 ng/ml LH (co-culture) and with/without oocyte conditioned medium (OCM) or 5 oocytes. Cells were cultured in 96 well plates for 144 h, after which viable cell numbers were determined. Medium was replaced every 48 h and spent medium analysed for steroids.     

Frogs without polliwogs: evolution of anuran direct development

Direct development is the assumption of the adult morphology without progression through an intervening, morphologically distinct, free-living larval phase. We discuss the ecological factors contributing to the evolution of this derived life-history strategy in frogs, and the developmental modifications that facilitate such an unusual mode of embryogenesis. Studies on the Puerto Rican tree frog, Eleutherodactylus coqui, have identified several such modifications, including developmental adaptations for dealing with increased egg size, and loss of tadpole structures. Surprisingly, this direct developer still undergoes a thyroid hormone-dependent metamorphosis, which occurs before hatching. We suggest how the ancestral biphasic developmental pattern may have been rearranged during the evolution of direct development.     

Phylogenetic analysis of the tenascin gene family: evidence of origin early in the chordate lineage

Background  

Tenascins are a family of glycoproteins found primarily in the extracellular matrix of embryos where they help to regulate cell proliferation, adhesion and migration. In order to learn more about their origins and relationships to each other, as well as to clarify the nomenclature used to describe them, the tenascin genes of the urochordate Ciona intestinalis, the pufferfish Tetraodon nigroviridis and Takifugu rubripes and the frog Xenopus tropicalis were identified and their gene organization and predicted protein products compared with the previously characterized tenascins of amniotes.     

Hormone replacement therapy: a survey of Ontario physicians' prescribing practices

BACKGROUND: Although much has been written about hormone replacement therapy (HRT), there are few clearcut recommendations on its use. The purpose of this study was to determine Ontario physicians'' patterns of and reasons for prescribing HRT, their use of pretreatment investigations and their surveillance of HRT users, and to determine whether physicians'' reported practice is consistent with existing recommendations. METHODS: A self-administered questionnaire was mailed to a nonproportional stratified sample of 327 Ontario physicians (23.9% gynecologists, 76.1% general practitioners/family physicians [GP/FPs]). Outcome measures were ranking of reasons for prescribing HRT, nature of preliminary testing, regimens prescribed, duration of HRT and frequency of follow-up. RESULTS: The response rate was 60.9% overall (70.9% of the gynecologists, 58.3% of the GP/FPs). Prevention of osteoporosis was reported by 97.4% as an important or very important reason for prescribing HRT; prevention of coronary artery disease was important or very important for 89.3%. When considering whether or not to prescribe HRT, 97.3% stated that breast cancer was an important or very important factor. When presented with hypothetical cases, 97.0% stated that they would prescribe combined estrogen-progestin for a symptomatic woman with an intact uterus; 13.6% stated that they would do so for a woman with no uterus. Most reported that they would prescribe HRT for 12 or more years (73.3%) and would follow up patients every 1 to 2 years (70.6%). INTERPRETATION: Despite controversy about HRT in the published literature, the Ontario physicians surveyed reported similar reasons and patterns of prescribing, pretreatment investigations, and surveillance of postmenopausal women using HRT. These results suggest that Ontario physicians'' knowledge about HRT is consistent with recommendations in the published literature.     

Cytoplasmic phases in the first cell cycle of the activated frog egg

The first cell cycle of the activated frog egg is longer than subsequent cycles and several developmentally important events such as the determination of bilateral symmetry occur at this time. When eggs of Rana pipiens or Xenopus laevis are dissected at times after activation, differences in the consistency of the animal half cytoplasm can be detected visually, and the first cell cycle has been divided into four cytoplasmic phases on this basis. Phase 1 includes the events of activation and lasts about one-third of the first cycle. In phase 2, the cytoplasm becomes fluid except for the rigid, growing sperm aster, and most of the migration of the pronuclei occurs in phase 2. In phase 3, the cytoplasm becomes firm whether or not a sperm aster had been present, and the grey crescent forms, indicating the plane of bilateral symmetry. The firmness of the cytoplasm is colchicine sensitive but cytochalasin B insensitive as is grey crescent formation. In phase 4, the cortex detaches from the firm cytoplasm, and the firmness is now cytochalasin B sensitive and colchicine insensitive. The changes in cytoplasmic consistency during the first cell cycle probably reflect changes in the cytoskeleton, and the cytoplasmic consistency is functionally correlated with developmental events in the first cell cycle.