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D F Albertini

Publications and source records attributed to D F Albertini.

At least 37 records · Page 2Linked to original sources
PubMed1994

Patterns of intercellular connectivity in the mammalian cumulus-oocyte complex.

Electron and fluorescence microscopic techniques have been used in a complementary fashion to study the patterns of follicle cell-oocyte interactions within cumulus-oocyte-complexes of various mammals. The principal findings are: (1) two distinct types of transzonal processes exist that are distinguishable on the basis of cytoskeletal composition; (2) in some of the species examined (pig, goat, primate), corkscrew-shaped processes rich in tubulin, traverse the zona pellucida and are invaginated into the oocyte cortex; (3) actin-rich processes either ramify as a network at the outer surface of the zona pellucida or penetrate the zona and make contact with the oolemma in a species specific manner. These results are discussed with respect both to the need to employ complementary optical methods in assessing connectivity patterns within COC and to the possible role that extracellular matrix-cell interactions play in the homeostatic control of oocyte growth and maturation.

Animals↗
PubMed1994

Hormonal regulation of meiotic maturation in the hamster oocyte involves a cytoskeleton-mediated process.

Maturation of the mammalian oocyte involves hormone-induced meiotic cell cycle progression from prophase I to metaphase II and extrusion of the first polar body (PB). In this study, the effects of gonadotrophins on meiotic cell cycle progression in cultured hamster oocytes were analyzed with respect to changes in cumulus cell-oocyte interactions and the oocyte cytoskeleton. Cumulus-oocyte complexes were obtained from large antral follicles (> or = 700 microns in diameter) of eCG-primed animals and were cultured in the presence or absence of gonadotropins alone (FSH, hCG, LH) or in combination (FSH + hCG or FSH + LH). Oocytes were analyzed using conventional, digital, and confocal fluorescence microscopy to monitor chromatin, actin, and tubulin organization under different culture conditions. Most oocytes (83%) cultured with FSH alone progressed to and arrested at metaphase II and extruded the first PB; in contrast, meiotic progression and PB extrusion were impaired (45-85%) in all other groups. The presence of cumulus cells associated with the oocyte was found necessary for progression to metaphase I and for first PB emission. Completion of meiotic maturation in the presence of FSH alone was correlated with enhanced cortical actin polymerization in the oocyte and the retraction of actin-containing transzonal cumulus processes. The results demonstrate that gonadotropins exert specific effects on meiotic progression, PB emission, cumulus-oocyte interactions, and oocyte cytoskeletal organization during in vitro maturation of hamster oocytes, indicating that the hormonal control of meiosis involves cytoskeletal changes in both the somatic and germ cells.

Actins↗
PubMed1993

Meiotic maturation in cultured bovine oocytes is accompanied by remodeling of the cumulus cell cytoskeleton.

Mammalian oocyte maturation and cumulus expansion are hormone-induced contemporaneous processes that involve changes in cumulus cell shape and in the interactions between cumulus cells and the oocyte. Laser scanning confocal and conventional fluorescence microscopy have been used to study the relationship between reorganization of the cumulus cell cytoskeleton and meiotic progression in bovine cumulus oocyte complexes (COCs) matured in vitro. Inclusion of hormone supplements (FSH, LH, E2) in the maturation media was required for meiotic progression to metaphase 2 and maximal cumulus expansion. Under these conditions, stage-specific changes in the cytoskeletal content and organization of cumulus cell transzonal processes was observed. Specifically, abundant, actin-filled transzonal processes were observed in GV stage COCs. In stages intermediate in the maturation process (10 hr culture), fewer actin-filled transzonal processes were seen and microtubule-filled transzonal processes became apparent. At the end of maturation (24 hr culture), numerous, elongate microtubule rich transzonal processes were evident and actin-filled processes were rarely observed. Additionally, a spatial relationship between microtubule rich transzonal processes and oocyte chromatin was consistently observed in all stages of oocytes examined. These results indicate that bovine oocyte maturation in culture involves modifications of the cumulus cell cytoskeleton that may regulate the onset, progression, and completion of oocyte maturation.

Actins↗
PubMed1992

Regulation of meiotic maturation in the mammalian oocyte: interplay between exogenous cues and the microtubule cytoskeleton.

Mammalian oocytes exhibit a series of cell cycle transitions that coordinate the penultimate events of meiosis with the onset of embryogenesis at fertilization. The execution of these cell cycle transitions, at G2/M of meiosis-I and metaphase/anaphase of meiosis I and II, involve both biosynthetic and post-translational modifications that directly modulate centrosome and microtubule behavior. Specifically, somatic cells alter the signal transduction pathways in the oocyte and influence the expression of maturation promoting factor (MPF) and cytostatic factor (CSF) activity through a microtubule-dependent mechanism. The regulation of the oocytes' cell cycle machinery by hormone-mediated somatic cell signals, involving both positive and negative stimuli, ensures that meiotic cell cycle progression is synchronized with the earliest pivotal events of mammalian reproduction.

Animals↗
PubMed1992

Protein synthesis requirements during resumption of meiosis in the hamster oocyte: early nuclear and microtubule configurations.

The organization of chromatin and cytoplasmic microtubules changes abruptly at M-phase entry in both mitotic and meiotic cell cycles. To determine whether the early nuclear and cytoplasmic events associated with meiotic resumption are dependent on protein synthesis, cumulus-enclosed hamster oocytes were cultured in the presence of 100 micrograms/ml puromycin or cycloheximide for 5 hr. Both control (untreated) and treated oocytes were analyzed by fluorescence microscopy after staining with Hoechst 33258 and tubulin antibodies. Freshly isolated oocytes exhibit prominent nucleoli and diffuse chromatin within the germinal vesicle as well as an interphase network of cytoplasmic microtubules. After 4-4.5 hr in culture, most oocytes were in prometaphase I of meiosis as characterized by a prominent spindle with fully condensed chromosomes and numerous cytoplasmic asters. After 5-5.5 hr in culture, microtubule asters are no longer detected in most cells, and the spindle is the only tubulin-positive structure. Incubation for 5 hr in the presence of inhibitors does not impair germinal vesicle breakdown, chromatin condensation, kinetochore microtubule assembly, or cytoplasmic aster formation in the majority of oocytes examined; however, under these conditions, a population of oocytes retains a germinal vesicle, exhibiting variable degrees of chromatin condensation and cytoplasmic aster formation. Meiotic spindle formation is inhibited in all oocytes. These effects are fully reversible upon culture of treated oocytes in drug-free medium for 5 hr. The data indicate that meiotic spindle assembly is dependent on ongoing protein synthesis in the cumulus-enclosed hamster oocyte; in contrast, chromatin condensation and aster formation are not as sensitive to protein synthesis inhibitors during meiotic resumption.

Animals↗
PubMed1992

Centrosome phosphorylation and the developmental expression of meiotic competence in mouse oocytes.

Previous studies suggested that the transition from an incompetent to a competent meiotic state during the course of oogenesis in the mouse involved a G2/M-like cell cycle transition (Wickramasinghe et al, 1991. Dev. Biol. 143, 162). The present studies tested the hypothesis that centrosome phosphorylation, an event normally induced by MPF, is required for this developmental transition and the expression of meiotic competence in cultured growing mouse oocytes. Multiple fluorescence labeling techniques were used to evaluate centrosome number, phosphorylation status, and microtubule nucleating capacity in competent and incompetent oocytes. Experimental conditions were established for reversibly altering the phosphorylation status of the centrosomes and the effects of these treatments on meiotic resumption were examined. Phosphorylated centrosomes nucleating short microtubules were observed in competent oocytes, whereas nonphosphorylated centrosomes and interphase microtubule arrays were found in incompetent oocytes. Upon recovery from nocodazole-induced microtubule depolymerization, short microtubules formed from centrosomes in competent oocytes, whereas long microtubules reappear in the cytoplasm of incompetent oocytes. Perturbation of the phosphorylation state of oocytes with activators of protein kinase A or protein kinase C resulted in the formation of long interphase microtubules in competent oocytes while centrosome phosphorylation was maintained. Treatment of competent oocytes with the phosphorylation inhibitor 6-dimethylaminopurine also led to formation of long microtubules, although under these conditions centrosomes were dephosphorylated. When competent oocytes were treated simultaneously with puromycin and the phosphodiesterase inhibitor isobutyl methylxanthine (IBMX) for 6 hr, centrosomes became dephosphorylated; centrosomes were rephosphorylated when competent oocytes were further cultured in IBMX without puromycin. Conditions that induced centrosome dephosphorylation in competent oocytes resulted in the loss of the ability to express meiotic competence in culture, whereas maintenance of centrosome phosphorylation in these oocytes was correlated with the ability to resume meiosis. These results suggest that the G2/M transition that occurs when mouse oocytes progress from an incompetent to a competent state in vivo involves the phosphorylation of centrosomes and that the maintenance of centrosome phosphorylation is required for the in vitro expression of meiotic competence.

1-Methyl-3-isobutylxanthine↗
PubMed1992

Cytoplasmic microtubular dynamics and chromatin organization during mammalian oogenesis and oocyte maturation.

A chronological series of coordinated alterations in oocyte chromosome and microtubule disposition occur during oogenesis and oocyte maturation in the mammal. Timely transitions in meiotic spindle and cytoplasmic microtubules, due to modifications in both the assembly competence of the tubulin pool and nucleation capacity of centrosomes, underscore key nuclear events during the progressive stages of meiosis I and II. The regulation of these transitional states during meiosis is discussed with respect to hormonal influences imparted to the oocyte within the follicular microenvironment, and the possible ways in which environmental perturbations may result in defective chromosomal partitioning during meiosis.

Animals↗
PubMed1991

Meiotic competence acquisition is associated with the appearance of M-phase characteristics in growing mouse oocytes.

To determine whether the acquisition of meiotic competence during the growth phase of oogenesis is associated with the appearance of M-phase characteristics, oocytes obtained from 13- to 30-day-old mice were evaluated by fluorescence microscopy with respect to chromatin and microtubule organization , in vitro maturation ability, and the distribution of M-phase phosphoproteins. Meiotically incompetent oocytes were distinguished from their competent counterparts in displaying elaborate interphase-like arrays of cytoplasmic microtubules and dispersed germinal vesicle chromatin. Meiotically competent oocytes were larger in size, exhibited condensation of chromatin around the nucleolus, and displayed a progressive diminution of cytoplasmic microtubules in conjunction with the appearance of multiple microtubule organizing centers. After 24 hr in culture, medium- to large-sized oocytes exhibiting perinucleolar chromatin condensation resume meiosis whereas smaller meiotically incompetent oocytes retain GVs with diffuse chromatin. Moreover, indirect immunofluorescence studies using the M-phase phosphoprotein specific monoclonal antibody MPM-2 indicate that the appearance of reactive cytoplasmic foci is directly correlated with nuclear changes characteristic of meiotically competent oocytes. Thus, the earliest transition to a meiotically competent state during oocyte growth in the immature mouse ovary is characterized by stage-specific and coordinated modifications of nuclear and cytoplasmic components.

Animals↗
PubMed1991

Quality of oocytes from superovulated rhesus monkeys.

The parameters, serum oestradiol (E2) response, follicle size and cumulus morphology, which are commonly used to determine in-vivo oocyte maturation in human in-vitro fertilization and embryo transfer (IVF-ET) programmes, were shown to be unreliable predictors of maturation of rhesus oocytes. In two groups of rhesus, one stimulated with pregnant mares' serum gonadotrophin (PMSG) and human chorionic gonadotrophin (HCG) and the other with human menopausal gonadotrophin (HMG) and HCG, the three parameters varied widely within and between protocols. Triple fluorochrome staining (TFS) for chromatin, microtubules and filamentous actin (f-actin) in oocytes at the time of collection and following 24 h in culture showed major differences in their maturation both in vivo and in vitro following priming with PMSG and HMG. In evaluating IVF protocols, TFS provides a valuable assay for the meiotic status of fixed oocytes of non-human primates.

Animals↗
PubMed1991

Centrosome and microtubule dynamics during meiotic progression in the mouse oocyte.

The disposition, function and fate of centrosomes were analysed in mouse oocytes undergoing in vitro meiotic maturation, using multiple-label fluorescence microscopy. Oocytes fixed at various points during meiotic progression were double labeled with either human centrosome-specific antibody, 5051, and anti-tubulin antibodies or 5051 and MPM-2 antibodies in order to evaluate the microtubule nucleation capacity and phosphorylation status of centrosomes during this process. Double labeling with anti-tubulin antibodies revealed two populations of centrosomes that undergo stage-specific changes in number, location and microtubule nucleation capacity in relation to spindle assembly and cytoplasmic events. Specifically, one population was consistently associated with chromatin throughout meiotic maturation whereas a second population of cytoplasmic centrosomes exhibited maximal numbers and nucleation capacity at prometaphase and anaphase of meiosis-I. Quantitative evaluation of cytoplasmic centrosomes indicated increased numbers during the transition from diakinesis to prometaphase and metaphase to anaphase and total disappearance during telophase. Colocalization studies with MPM-2 revealed that centrosomes were always phosphorylated. However, at metaphase of meiosis I and II the microtubule nucleation capacity of centrosomes was diminished. These results suggest the existence of two discrete populations of centrosomes in the mouse oocyte that are coordinately regulated to subserve aspects of microtubule organization relative to both nuclear and cytoplasmic events.

Anaphase↗
PubMed1990

Oogenesis: chromatin and microtubule dynamics during meiotic prophase.

Changes in the organization of germinal vesicle chromatin in mouse oocytes have been analyzed by fluorescence microscopy with respect to progressive stages of follicular development and the disposition of oocyte cytoplasmic microtubules. Four discrete patterns of chromatin organization exist in germinal vesicle (GV)-stage oocytes isolated from the ovaries of 21-25-day-old gonadotropin-primed mice. Analysis of ovarian cryosections stained with the DNA-binding fluorochrome Hoechst 33258 indicates that sequential changes in GV chromatin occur during folliculogenesis that result in the formation of a continuous perinucleolar chromatin sheath at the time of antrum formation. Specific alterations in the cytoplasmic microtubule complex of GV-stage oocytes were observed that correlate with chromatin patterns. The extensive cytoplasmic microtubule complex seen in oocytes of preantral follicles initially localizes to perinuclear areas of the ooplasm. This is followed by a progressive reduction in cytoplasmic microtubules and the appearance of prominent microtubule-organizing centers at the nuclear periphery. Coordinated nuclear and microtubular alterations also occur under in vitro conditions prior to progression of meiosis to prometaphase-1. The results are discussed with respect to the ongoing differentiation of the oocyte nucleus and the microtubule cytoskeleton during folliculogenesis in preparation for the resumption of meiosis.

Animals↗
PubMed1990

Transitions in trophectoderm cellular shape and cytoskeletal organization in the elongating pig blastocyst.

Changes in cellular shape and filamentous actin (f-actin) organization within the trophectoderm of pig embryos have been studied by fluorescence microscopy during the transitions from spherical to filamentous blastocysts. Cells comprising the trophectoderm of spherical, ovoid, tubular, and filamentous blastocysts are distinctive in their shape, size, and organization of membrane-associated f-actin. Trophectodermal cells of spherical and ovoid embryos are both generally circular in shape. However, as the spherical embryo acquires an ovoid shape, uniformally distributed apical cell surface microvilli relocate to the apical intercellular margins of adjoining trophectodermal cells. Transitional modifications in cellular shape and f-actin organization are observed in tubular blastocysts when apical cell surface microvilli reappear. In elongating filamentous blastocysts, trophectodermal cells assume a spindle-shaped morphology. The f-actin associated with the apical surface is diminished whereas the associated with the basolateral membrane predominates, especially in constricted regions of the blastocyst. These observations, in conjunction with morphometric parameters of trophectodermal cells and whole blastocysts, are discussed in relation to the role of the actin cytoskeleton in processes that modify trophectodermal cell shape and function in the elongating pig blastocyst.

Actins↗
PubMed1988

Quantitative studies of changes in cortical granule number and distribution in the mouse oocyte during meiotic maturation.

Cortical granules (CGs) undergo a substantial change in distribution in the mouse oocyte cortex during meiotic maturation. In order to determine the mechanism of their change in distribution near the time of ovulation, CG density, total number per oocyte, and domain areas were quantitated. CGs were visualized microscopically by Lens culinaris agglutinin-biotin and Texas red-strepavidin fluorescence as well as by electron microscopy. Immature germinal vesicle stage (GV) oocytes from adult mice had a continuous cortical localization with some interior granules. Mature oocytes had an asymmetric cortical distribution with a CG-free domain, overlying the meiosis II metaphase spindle, occupying 40% of the cortex. The mean CG densities of the granule-occupied cortex of mature oocytes and the entire cortex of GV oocytes were 43 and 34 CGs/100 micron 2, respectively. The mean total numbers of CGs/oocyte were 4127 (mature) and 7440 (GV), and staining was absent in fertilized oocytes with two pronuclei. Calcium ionophore (A23187)-activated mature oocytes had a mean total number of 1235 CGs, some of which may have been in the process of exocytosis. The first polar body had few CGs, and thus was unlikely to account for the difference in CG number between GV and mature oocytes. The smaller total number and higher density of CGs in mature mouse oocytes suggests that both exocytosis and redistribution are plausible mechanisms for the development of the CG-free domain. Prefertilization exocytosis could account for the locus of sperm penetration which others have reported to occur in the hemisphere opposite the meiotic spindle in the mouse.

Animals↗
PubMed1987

Patterns of organelle distribution in mouse embryos during preimplantation development.

We have evaluated the distribution of mitochondria and acidic organelles using, respectively, the specific vital fluorescent dyes rhodamine 123 and acridine orange during preimplantation embryonic development in the mouse. Under conditions used to visualize organelles in living embryos, staining with either dye was found to have no effect on either the rate or extent of in vitro development of five- to eight-cell embryos up to the blastocyst stage. Mitochondria were randomly distributed throughout the cytoplasm and located around nuclei in blastomeres of uncompacted embryos. During compaction, mitochondria initially reorganized to the blastomere cortex; however, these organelles were later confined to the perinuclear region in the trophectoderm (TE) of expanded blastocysts. Acidic organelles were randomly distributed in the cytoplasm of uncompacted embryos, but following compaction, they were concentrated in cortical and perinuclear locations. Moreover, in TE cells of expanded blastocysts, acidic organelles were found exclusively in a tight perinuclear pattern. Microtubules and microfilaments in TE cells were localized in fixed embryos stained with antitubulin antibodies and rhodamine phalloidin, respectively; these structures were found primarily in the cortical cytoplasm at areas of cell-cell contact and secondarily in a perinuclear location. Thus mitochondria and acidic organelles undergo stage-specific redistributions from a diffuse or cortical pattern at the eight-cell stage to a tight perinuclear localization in the TE. We conclude that the polarized distributions of some organelles and cytoskeletal proteins during compaction may not be reliable permanent markers of the mature TE.

Acridine Orange↗
PubMed1987

Cytoplasmic reorganization during the resumption of meiosis in cultured preovulatory rat oocytes.

Changes in organelle topography and microtubule configuration have been studied during the resumption and progression of meiosis in cultured preovulatory rat oocytes. Germinal vesicle breakdown (GVBD) was reversibly inhibited by dibutyryl cAMP (DcAMP) or nocodazole, a microtubule-disrupting agent. The microtubule stabilizing agent taxol did not inhibit GVBD, but did impair further maturation. The migration of acidic organelles and chromatin in living oocytes was analyzed using the vital stains acridine orange and Hoechst 33258, respectively. Germinal vesicle stage oocytes undergo perinuclear aggregation of acidic organelles during GVBD and these organelles subsequently disperse into the cell cortex as the first meiotic spindle migrates to the oocyte periphery. DcAMP and nocodazole block the perinuclear aggregation of acidic organelles, whereas, in taxol-treated oocytes, organelle aggregation and GVBD occur but the dispersion of acidic organelles was arrested. Dose-response studies on the effects of nocodazole showed that GVBD was generally retarded and that a 50% inhibition of GVBD was achieved at concentrations in excess of 1.0 microM. Concentrations of taxol at 10 microM or above effectively inhibited both chromatin condensation and meiotic spindle formation. Indirect immunofluorescence microscopy with anti-tubulin antibodies revealed dissolution of microtubules with 1.0 microM nocodazole. Taxol had little effect on microtubule organization in germinal vesicle or chromatin condensation stage oocytes; however, when oocytes that had formed first meiotic spindles were treated with taxol, numerous microtubule asters appeared which were preferentially associated with the oocyte cortex. The changes in organelle topography, microtubule configuration, and drug sensitivity are discussed with respect to the regulation of cytoplasmic reorganization during the meiotic maturation of rat preovulatory oocytes.

Alkaloids↗
PubMed1987

Changes in the organization of the actin cytoskeleton during preimplantation development of the pig embryo.

The organization of the actin cytoskeleton was studied in unfertilized porcine oocytes and preimplantation stage embryos from Day 1 through Day 8 of development. Fixed and detergent-extracted oocytes and embryos were analyzed by fluorescence microscopy after staining with either rhodamine-phalloidin to localize filamentous actin or with affinity-purified anti-actin antibodies to localize the total immunodetectable actin. Whereas unfertilized oocytes contain immunoreactive cytoplasmic actin, rhodamine-phalloidin binding is not detected until fertilization when a prominent cortical staining pattern becomes apparent. In early cleavage stage embryos, filamentous actin is concentrated in the cell cortex of blastomeres especially at sites of cell-cell contact. Compacting morulae exhibit a marked accumulation of actin at the margins of blastomeres where numerous interdigitating cell processes are located. The predominantly pericellular distribution of actin becomes a distinguishing feature of trophectodermal cells in the expanding blastocyst at Day 6 of development; these cells form a prominent actin-limited zone circumscribing the inner cell mass. In Day 8 blastocysts, three cell types are present that are readily distinguishable based upon their actin displays among other cytological features. Trophectodermal cells exhibit continuous actin-rich lateral borders and stress fibers along their basal surface. Inner cell mass cells contain a discontinuous actin boundary and prominent foci of actin along their blastocoelic surface. Lining the blastocoel are patches of endodermal cells in which the actin is exclusively cortical. The data are discussed with respect to differences between species and the chronology of actin rearrangements during preimplantation development of the porcine embryo.

Actins↗
PubMed1985

Binding and internalization of heparin by vascular smooth muscle cells.

Previous work from our laboratory has demonstrated that heparin specifically inhibits the proliferation of vascular smooth muscle cells in vivo and in vitro. In this paper, we examine the binding and mode of internalization of heparin by smooth muscle cells. For these studies, radiolabeled and fluoresceinated (FITC) heparin probes were synthesized that retained their antiproliferative capacity. Binding of 3H-heparin to these cells occurs via specific, high-affinity binding sites (Kd = 10(-9) M, 100,000 binding sites per cell). Approximately 80% of the heparin bound to the cell surface was shed into the culture medium within 2 hr. The heparin that was left on the cell surface was internalized with biphasic kinetics. Approximately 50% of the bound material was internalized within 2 hr. After this initial rapid uptake, the rate slowed substantially, with the remaining heparin requiring 1-2 days to be internalized. Binding and uptake of FITC heparin was monitored using video image intensification fluorescence microscopy. When smooth muscle cells were exposed to FITC heparin at 4 degrees C, a diffuse surface staining pattern was observed. After warming the cells to 37 degrees C, intensely fluorescent vesicles were seen superimposed over the diffuse surface staining within 2 min. After 15 min at 37 degrees C, numerous large punctate vesicles were seen inside the cell. After 2 hr these vesicles had concentrated in the perinuclear region. This pattern of uptake, when considered along with the presence of specific, high-affinity binding sites and the initial rapid uptake of 3H-heparin, suggests that heparin enters smooth muscle cells by both receptor-mediated and other endocytic pathways.

Animals↗