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P Collas

Publications and source records attributed to P Collas.

At least 55 records · Page 3Linked to original sources
PubMed1996

Targeting of membranes to sea urchin sperm chromatin is mediated by a lamin B receptor-like integral membrane protein.

We have identified an integral membrane protein of sea urchin gametes with an apparent molecular mass of 56 kD that cross-reacts with an antibody against the nucleoplasmic NH2-terminal domain of human lamin B receptor (LBR). In mature sperm, p56 is located at the tip and base of the nucleus from where it is removed by egg cytosol in vitro. In the egg, p56 is present in a subset of cytoplasmic membranes (MV2 beta) which contributes the bulk of the nuclear envelope during male pronuclear formation. p56-containing vesicles are required for nuclear envelope assembly and have a chromatin-binding capacity that is mediated by p56. Lamin B is not present in these vesicles and is imported into the nucleus from a soluble pool at a later stage of pronuclear formation. Lamin B incorporation and addition of new membranes are necessary for pronuclear swelling and nuclear envelope growth. We suggest that p56 is a sea urchin LBR homologue that targets membranes to chromatin and later anchors the membrane to the lamina.

Animals↗
PubMed1996

Distinct egg membrane vesicles differing in binding and fusion properties contribute to sea urchin male pronuclear envelopes formed in vitro.

We have identified three distinct membrane vesicle populations from sea urchin egg cytoplasm that cooperate in assembling the male pronuclear envelope in vitro. Membranes from sea urchin egg homogenates were separated by buoyant density into five vesicle fractions, three of which bind to demembranated sperm nuclei. Each requires a membranous element (lipophilic structure) derived from the sperm nuclear envelope at the tip and base (poles) of the nucleus in order to bind. Binding is differentially sensitive to protease, high salt and N-ethyl maleimide treatment of the membrane vesicles. MV1 binds at the poles and is required for fusion of the membrane vesicle fractions to each other and to the lipophilic structures. MV2 beta binds over the entire chromatin surface and is enriched in an endoplasmic reticulum marker enzyme. MV2 alpha binds at the nuclear poles, is enriched in a Golgi enzyme marker and is required for fusion of MV2 beta. All three fractions are required for nuclear envelope formation in vitro. The results suggest a multistep process for nuclear envelope formation involving contributions from both sperm and egg, roles for both endoplasmic reticulum and non-endoplasmic reticulum-derived vesicles, and the localization of a critical element of the fusion machinery in MV1.

Animals↗
PubMed1996

Conserved binding recognition elements of sperm chromatin, sperm lipophilic structures and nuclear envelope precursor vesicles.

Detergent-resistant, lipophilic structures (LSs) at the apex and base of the conical sea urchin sperm nucleus are targets for cytoplasmic membrane vesicle (MV) binding and fuse with these vesicles to form a nuclear envelope in vitro. We report similar LSs associated with trout, frog and mammalian (mouse and bovine) sperm nuclei. The LSs are located at the implantation fossae of all species examined, as well as in the ventral hook region of mouse sperm nuclei. LSs can be removed from, and reconstituted back to, their original sites on nuclei. LS removal prevents MV binding, and reconstitution restores MV binding and GTP-induced fusion activities. Binding of LSs to chromatin or to MVs is mediated, at least in part, by proteins on each structure. Inter-specific LS-chromatin reconstitutions using sea urchin, fish, frog, and mammalian LSs indicate that site-specific binding is not dependent on species. All LSs also bind to MVs of sea urchin or bovine origin, but sea urchin MVs will only fuse with sea urchin LSs, and mammalian MVs only with mammalian LSs. These results demonstrate the conservation between echinoderms, fish, amphibians, and mammals of recognition elements for LS-chromatin and LS-MV binding. The mechanism for fusion of LSs with cytoplasmic MVs, however, is apparently not conserved, and close apposition of MVs on the chromatin surface mediated by LSs is not sufficient to permit MV-MV fusion in the presence of GTP.

Animals↗
PubMed1995

Inactivation of histone H1 kinase by Ca2+ in rabbit oocytes.

The present study investigated the role of intracellular Ca2+ (Ca2+i) elevation on the inactivation of maturation promoting factor (MPF) in rabbit oocytes. The effects of the number of Ca2+ stimulations and of the amplitude of Ca2+i elevation on the profile of histone H1 kinase activity were determined. A Ca2+ stimulation consisted of transferring mature oocytes from culture medium to 0.3 M mannitol containing 0.1-1.0 mM CaCl2, and pulsing them at 1.25 kV/cm for 10 microseconds, or microinjecting 2-8 mM CaCl2 into the oocyte cytoplasm. The number of electrically-induced Ca2+ stimulations was varied, and amplitude of the Ca2+i rise was controlled by altering Ca2+ concentration in the pulsing medium or the injection pipette. Ca2+i concentration was determined with fura-2 dextran; oocytes were snap-frozen at indicated time points and assayed for H1 kinase activity. The activity was quantified by densitometry and expressed as a fraction of activity in nonstimulated oocytes. Electrically-mediated Ca2+i rises inactivated H1 kinase in a manner dependent on the number of Ca2+ stimulations. A single Ca2+ stimulation inactivated H1 kinase to 30-40% of its initial activity. However, H1 kinase inactivation was only transient, regardless of the amplitude of the electrically- or injection-mediated Ca2+i elevation. Increasing the number of Ca2+ stimulations helped to maintain H1 kinase activity at basal (pronuclear) levels. The results show the necessity of a threshold of Ca2+i concentration to trigger MPF inactivation, and suggest a role for the extended period of time over which Ca2+i oscillates at fertilization.

Animals↗
PubMed1995

Formation of the sea urchin male pronucleus in vitro: membrane-independent chromatin decondensation and nuclear envelope-dependent nuclear swelling.

We demonstrate that complete sea urchin male pronuclear development in vitro is a two-step process involving membrane-independent chromatin decondensation and nuclear envelope-dependent pronuclear swelling. In the absence of cytoplasmic membrane vesicles (MVs), permeabilized sperm chromatin decondenses into a spherical nucleus of approximately 4 microns in diameter. Pronuclear swelling to approximately 7 microns requires an intact nuclear envelope, and the degree of swelling is limited by the amount of MVs assembled on the chromatin. Furthermore, after a nuclear envelope is formed, swelling can occur in the absence of additional cytoplasmic MVs. Nuclear swelling also requires ATP hydrolysis, Ca2+ and cytosolic factors, some of which are sensitive to heat and to the sulfhydryl alkylating agent, N-ethylmaleimide. The requirement for a nuclear envelope and the rate of pronuclear swelling are consistent with previous in vivo observations.

Adenosine Triphosphate↗
PubMed1995

Lipophilic organizing structures of sperm nuclei target membrane vesicle binding and are incorporated into the nuclear envelope.

We report the existence of lipophilic structures (LS's) associated with demembranated sperm nuclei which together act as a pronuclear envelope organizing center. These structures can be visualized as objects which stain with lipophilic dyes but are resistant to solubilization in 0.1% of the non-ionic detergent Triton X (TX)-100. The structures, located at the acrosomal and centriolar poles of the sea urchin sperm nucleus, initiate ATP-dependent membrane vesicle binding in a fertilized egg extract. The lipophilic material in LS's is incorporated into the nuclear envelope during GTP-induced membrane fusion. Removal of the LS's from sperm nuclei with 1% TX-100 abolishes membrane vesicle binding to the nuclei. LS's recovered from supernatants of extracted nuclei can be reconstituted to their original locations on the stripped nuclei. Rebinding of isolated LS's occurs preferentially at the acrosomal pole. Such reconstituted nuclei direct membrane vesicle binding only to the acrosomal pole region and result in incomplete nuclear envelope assembly following membrane fusion. Binding of LS's and subsequently membrane vesicles to both nuclear poles allows complete nuclear envelope formation.

Acrosome↗
PubMed1995

Lamin dynamics during sea urchin male pronuclear formation in vitro.

The dynamics of lamin disassembly and reassembly during sea urchin male pronuclear development in vitro was investigated. Using five anti-lamin antibodies, we monitored by immunofluorescence and immunoblotting the changes in lamins during sperm chromatin decondensation, nuclear envelope (NE) formation, and male pronuclear swelling in fertilized sea urchin egg cytoplasmic extracts. We report the existence of five proteins in sperm nuclei and swollen male pronuclei (p49, p54, p65, p72, p84) which react with the antibodies. The sperm antigens resist membrane permeabilization with lysolecithin and 0.1% Triton X (TX)-100, but are removed from the lateral aspects of the nuclei by 1% TX-100. All five are completely removed from nuclei within 10 min of incubation in egg extracts. Initial chromatin decondensation and NE formation occur without reassembly of the putative lamins, but all lamins or lamin epitope-containing peptides assemble coordinately during pronuclear swelling promoted by adenosine 5'-triphosphate. Of the five pronuclear antigens, p49 and p54 appear to originate exclusively from the sperm. p65, p72, and p84 are also present in the egg cytoplasm and may be contributed to pronuclei by either source. Assembly of putative lamins and nuclear swelling, but not chromatin decondensation and nuclear envelope formation, are prevented in lamin-depleted cytoplasmic extracts. Our results indicate that p49 and p54 are not necessary for nuclear swelling, cytoplasmic p65 is required for swelling, and p72 and p84 are by themselves not sufficient but may be involved with p65 in nuclear swelling and full pronuclear development in vitro.

Animals↗
PubMed1995

Histone phosphorylation during sea urchin development.

Studies on histone phosphorylation during transitions in chromatin structure occurring in vivo during spermatogenesis and early embryogenesis in sea urchins are reviewed and evaluated in the light of recent studies on histone phosphorylation occurring during chromatin synthesis in frog egg extracts in vitro and evidence that protein kinases and phosphatases play direct roles in the regulation of cellular structure. Sperm-specific histone variants Sp H1 and Sp H2B are maintained as phosphorylated derivatives N and O/P throughout spermatogenesis and early embryogenesis and egg specific histone variants CS H1 and CS H2A are phosphorylated during early embryogenesis. These developmental correlations provide clues about the roles of histone phosphorylation in control of chromatin structure in vivo and provide a basis for the interpretation of data obtained from in-vitro sperm chromatin remodeling in egg extracts and from biochemical studies on the effects of histone phosphorylation on DNA binding. The potential consequences for chromatin structure of the various histone phosphorylation events observed in sea urchins and frog egg extracts are discussed.

Animals↗
PubMed1994

Nuclear transplantation by microinjection of inner cell mass and granulosa cell nuclei.

The developmental potential of bovine inner cell mass (ICM) and somatic differentiated (granulosa cell) nuclei was investigated using nuclear transplantation. ICM blastomeres were isolated after immunosurgery of day 7 in vitro produced blastocysts and cumulus granulosa cells recovered from in vitro matured oocytes. Nuclear transplantation was carried out by microinjection of the lysed donor cells into enucleated mature oocytes. Oocytes were activated by three 0.2 kVcm-1/20 microseconds pulses in mannitol containing 100 microM Ca2+, with each pulse 22 min apart. Embryos were cultured in vitro for 7 days and blastocysts were transferred into recipients. ICM and granulosa cell donor nuclei directed 7% (20/304) and 9% (19/213) development to blastocysts, respectively. Fifteen blastocysts from ICM donors resulted in four pregnancies (27%) and two births. No pregnancy was detected with granulosa cell donors. The results illustrate the totipotency of ICM nuclei and indicate that granulosa cell nuclei promote preimplantation development of nuclear transplant embryos.

Animals↗
PubMed1993

Chromatin and microtubule organization in the first cell cycle in rabbit parthenotes and nuclear transplant embryos.

Artificial activation and nuclear transfer in rabbit oocytes have been used in past years in an attempt to develop viable techniques for cloning in cattle. The procedures established in our laboratory, using the rabbit as a model, consistently lead to high rates of development to the blastocyst stage. However, the rate of embryos developing to term is considerably lower. In the present study, we undertook a detailed immunocytochemical study of the patterns of both microtubules and chromatin during the first cell cycle of electrical pulse-activated oocytes and of nuclear transfer embryos. Our goal was to investigate the responses of the cell to the different stimuli applied and to establish the sequence of events leading to first cleavage in the absence of normal fertilization. Our results show that, in both electrically activated oocytes and nuclear transfer embryos, although the initial development patterns are rather unusual, embryos become synchronized at the time of the formation of a pronuclear-like structure, and then organize metaphase spindles and cleave. These spindles consistently present small defects, suggesting that problems in the formation of the mitotic apparatus during the first cell cycle may have a long-term effect leading to embryo mortality.

Animals↗
PubMed1993

Electrically induced calcium elevation, activation, and parthenogenetic development of bovine oocytes.

The influence of electrical stimulation on the level of intracellular Ca2+ in bovine oocytes, as well as activation and extent of parthenogenetic development, was investigated. Mature oocytes were electrically stimulated at 29 hr of maturation, and intracellular Ca2+ concentration was determined with the Ca2+ indicator fura-2 dextran (fura-2 D). The Ca2+ response of oocytes to a given electrical pulse was variable. Oocytes responded with either no Ca2+ rise from baseline (approximately 12 nM), a short-duration Ca2+ rise (from 12 nM to 300 nM) that returned to baseline within 2 min of the pulse, or a long-duration Ca2+ rise (from 12 nM to 1,000-2,000 nM) that never returned to baseline during the 8 min period over which the oocytes were monitored. In these oocytes, Ca2+ level returned to baseline when oocytes were removed from 0.30 M mannitol and placed in an ionic medium. Increasing field strength or pulse duration tended to increase the proportion of oocytes displaying a Ca2+ rise, and at 1.0 kVcm-1 for 40 microseconds, all oocytes displayed a long-duration Ca2+ elevation. Direct transfer of oocytes from culture medium to mannitol also triggered a Ca2+ rise. Multiple stimulations, either electrical or by transferring to mannitol, produced multiple Ca2+ rises. This mannitol-induced Ca2+ rise could be inhibited by first washing the oocytes in medium containing equal parts of 0.30 M mannitol and phosphate buffered saline (PBS). The level of Ca2+ stimulation affected activation and development of oocytes. Insufficient, or, conversely, excessive Ca2+ stimulation impaired development. Optimum development was obtained with 1) three pulses of 0.2 kVcm-1 for 20 microseconds, each pulse 22 min apart, after direct transfer of oocytes from culture medium to mannitol (22% blastocysts) or 2) three pulses of 1.0 kVcm-1 for 20 microseconds after transfer of oocytes from culture medium to medium containing equal parts mannitol and PBS, then to mannitol (24% blastocysts). This procedure avoided induction of a Ca2+ rise prior to the pulse. The results indicate that the level of Ca2+ stimulation can be regulated by incubation conditions prior to the pulse and, to some extent, by field strength and pulse duration. The level of electrical stimulation influenced oocyte Ca2+ response, activation, and parthenogenetic development.

Animals↗
PubMed1993

Histone H1 kinase activity in bovine oocytes following calcium stimulation.

The influence of number of Ca2+ stimulations on the profile of histone H1 kinase activity in bovine oocytes was investigated. A CA2+ stimulation consisted of transferring oocytes directly from culture medium to mannitol containing 100 microM Ca2+ and pulsing oocytes with a 0.2 kVcm-1, 20 microseconds discharge. One, three, or six Ca2+ stimulations were given, each 22 min apart. Oocytes were frozen from 0 to 8 hr after the first stimulation at indicated time points and assayed for histone H1 kinase activity. H1 kinase activity was quantified using a densitometer and expressed as a percent of activity in nonpulsed metaphase II oocytes. Stimulating oocytes in the absence of Ca2+ in the pulsing medium did not inactivate H1 kinase. In the presence of Ca2+, however, H1 kinase was rapidly inactivated after stimulation. A single stimulation decreased H1 kinase activity to 44% +/- 11% of its initial level in 1 hr. However, H1 kinase was dramatically reactivated at 2 hr after the stimulation and reached 122% +/- 22% of the initial activity at 6 hr. With three stimulations, basal H1 kinase activity was 21% +/- 3% and was obtained in 30 min. H1 kinase reactivation started at 4 hr after the first stimulation and level of activity reached 38% +/- 15% at 8 hr. Six stimulations also led to rapid H1 kinase inactivation and to a basal activity of 14% +/- 0.4%. With six stimulations, however, basal H1 kinase activity was maintained over at least 8 hr, and no reactivation occurred during this period. Basal H1 kinase activity obtained after six stimulations was similar to that of fertilized oocytes.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1993

Influence of recipient oocyte cell cycle stage on DNA synthesis, nuclear envelope breakdown, chromosome constitution, and development in nuclear transplant bovine embryos.

Nuclear transplantations into metaphase II (MII) and S phase oocyte cytoplasm were performed to investigate the influence of recipient cell cycle stage on nuclear function and development of bovine nuclear transplant (NT) embryos. Rate of inactivation of histone H1 kinase and duration of DNA synthesis in activated oocytes were determined. The proportion of S phase blastomeres in in vivo produced day 5.5 bovine embryos was measured. DNA synthesis was also assessed in NT embryos after transfer into MII and S phase cytoplasm. MII NT embryos were produced by fusing a blastomere into a MII oocyte; the fusion pulse served to activate the oocyte. S NT embryos were produced by fusing a blastomere into an early S phase oocyte electrically activated 4 h prior to fusion. Nuclear envelope structure, chromosome constitution, and extent of development were examined in MII and S NT embryos. Histone H1 kinase activity dropped to baseline within 2 h of electrical activation. A second electrical pulse did not alter H1 kinase activity when delivered 4 h after the first pulse. The frequency of S phase blastomeres in day 5.5 bovine embryos ranged from 79% to 100%, depending on the duration of culture in 3H-thymidine. Nuclear transplantation into MII cytoplasm resulted in a transient drop in DNA synthesis over 3.5 h. DNA synthesis resumed at 4.5 h post activation (hpa), concomittantly with initiation of DNA replication in activated oocytes. In contrast, DNA synthesis was not interrupted after transfer into S phase cytoplasm. DNA synthesis persisted until 13.5 hpa, as in activated oocytes.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

Influence of cell cycle stage of the donor nucleus on development of nuclear transplant rabbit embryos.

We evaluated the influence of the stage of the cell cycle of the donor nucleus on development in vitro of nuclear transplant rabbit embryos. The developmental potential of nuclei in early, mid-, and late stages of the cell cycle was determined. Duration of the G1 phase in early embryos was determined, and a procedure for reversibly synchronizing donor embryos in the G1 phase was developed. In addition, the extent of development in vitro of nuclear transplant embryos with donor nuclei synchronized in the G1 phase was evaluated. Development to blastocysts was greatly affected by the stage of the cycle of the donor nucleus. Use of early-stage nuclei led to 59% nuclear transplant blastocysts, whereas 32% and 3% were obtained with mid- and late-stage nuclear donors, respectively (p less than 0.001). The short duration of the G1 phase in 16- and 32-cell-stage embryos (approximately 30 min) necessitated a procedure for synchronizing blastomeres in the G1 phase. This entailed, first, a 10-h incubation in 0.5 micrograms/ml colcemid to arrest embryos in metaphase. After release from colcemid, embryos were allowed to cleave in 0.1 microgram/ml of the DNA synthesis inhibitor, aphidicolin, and remained blocked at the G1/S transition. This treatment was reversible, as assessed by the resumption of DNA synthesis, cleavage rate, and development to blastocysts of treated embryos. The beneficial effect of using early-stage donor blastomeres was confirmed by the enhanced rate of development of manipulated embryos to blastocysts with donor nuclei in the G1 phase (71%), as opposed to the late S phase (15%, p less than 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

Effect of donor cell cycle stage on chromatin and spindle morphology in nuclear transplant rabbit embryos.

We investigated the influence of the cell cycle stage of the nuclear donor on prematurely condensed chromatin (PCC) and spindle morphology and on chromosome constitution in rabbit nuclear transplant embryos. The configuration of PCC following nuclear transplantation with G1, early S, and late S phase donor nuclei (G1, early S, and late S transplants, respectively) was characterized in whole mounts and chromosome spreads. In addition, the influence of the donor cell cycle stage on chromosome constitution in cleavage stage-manipulated embryos was determined. Within 2 h after fusion of the donor blastomere, the recipient oocyte cytoplasm was able to induce formation de novo of a metaphase plate associated with a spindle in G1, early S, and late S transplants. Metaphase chromosomes and spindle were intact in most cases of PCC in G1 transplants. However, these structures displayed minor abnormalities in early S transplants and gross abnormalities in late S transplants, such as incomplete or absent spindle formation and incomplete chromatin condensation. Normal chromosomes were present in G1 and early S transplants, whereas chromosome abnormalities were detected in late S transplants. The results indicate that morphology of prematurely condensed G1 and early S chromatin has a minor influence on chromosome constitution of manipulated embryos. That of late S chromatin, however, affects chromosome constitution in embryos and may account for reduced development of nuclear transplant embryos when late S phase donor nuclei are used.

Animals↗
PubMed1991

In vitro development of rabbit pronuclear embryos in rabbit peritoneal fluid.

The use of rabbit peritoneal fluid (PF) for the culture of rabbit embryos in vitro was evaluated. Development of zygotes cultured in PF and Earle's balanced salts solution (EBSS) + 10% fetal calf serum (EBSS/FCS) was compared. The effects of increasing the concentration of PF in EBSS and of culturing embryos in fractionated PF were also investigated. In addition, embryonic development in PF was compared to that in vivo. Development to hatching blastocysts was enhanced with PF (73%) compared to EBSS/FCS (3%, p less than 0.001). PF manifested greater mitogenic activity than EBSS/FCS, as indicated by higher cell number in embryos at 48, 72, and 96 h post-mating/hCG (p less than 0.001). PF also promoted blastocyst cell proliferation in a dose-dependent manner (r = 0.98, p less than 0.01); however, embryo growth remained slower than in vivo. Culture in the high (greater than 30,000 Da) molecular mass fraction of PF reduced incidence of hatching (56% vs. 92%, p less than 0.001) and mean cell number in Day 4 blastocysts (151 +/- 4 vs. 243 +/- 5, p less than 0.001). Rates of blastocyst hatching (10%) and cell number (110 +/- 3) were further reduced in the low (less than 30,000 Da) molecular mass fraction. When the high molecular mass fraction was dialyzed, embryos did not develop beyond the early morula stage. This suggests that the interaction or the synergy of high and low molecular mass components of PF is necessary for optimum development of rabbit embryos.

Animals↗
PubMed1991

Relationship between nuclear remodeling and development in nuclear transplant rabbit embryos.

The present study characterized the profile of nuclear remodeling in nuclear transplant rabbit embryos and investigated the relationship between chromatin behavior after transfer and embryo development. The developmental potential and pattern of remodeling of donor nuclei from cleavage-, morula-, and blastocyst- (inner cell mass ICM, and trophectoderm, TE) stage donors were evaluated. In addition, we determined whether a modification in the synchrony between blastomere fusion and oocyte activation altered the profile of nuclear remodeling and affected development of reconstituted embryos. Development to blastocysts was similar with 8- and 32-cell-stage donor nuclei (42% and 33%, respectively, p greater than 0.1). However, it was reduced with ICM transplants (17%, p less than 0.05), and development of TE transplants did not progress beyond the 8-cell stage. Upon blastomere fusion into nonactivated oocyte cytoplasm, nuclear remodeling was characterized by premature chromosome condensation (PCC), followed by pronuclear (PN) formation and swelling. PCC occurred synchronously within 1.2-1.5 h post-fusion with all stages of donor nuclei (p greater than 0.1). PN formation in 8- and 32-cell transplants occurred approximately 4 h after fusion, and was synchronous to that of female pronuclei in activated oocytes; however, it was delayed in ICM and TE transplants (p less than 0.01). With all stages of donor nuclei, final nuclear diameter was similar to, or larger than, that of female pronuclei. Fusion to activated oocyte cytoplasm, as opposed to nonactivated cytoplasm, prevented PCC and extensive nuclear swelling (16.0 +/- 0.7 vs. 30 +/- 0.7 microns, respectively, p less than 0.01). Nuclear diameter in early embryos was smaller (p less than 0.01), and development to blastocysts was reduced (p less than 0.05). The results indicate that remodeling of the donor nucleus is not essential for development to blastocysts; however, it is beneficial. Furthermore, complete reprogramming seems possible only after remodeling of the donor nucleus, i.e., PCC in nonactivated cytoplasm, followed by nuclear swelling upon activation of the oocyte.

Animals↗