Inositol trisphosphate-induced calcium release in the generation of calcium oscillations in bovine eggs.

Bovine eggs exhibit repetitive rises in intracellular calcium concentration ([Ca2+]i) in response to fertilization. The signaling pathways and Ca2 release mechanisms involved in their generation are not well characterized. This study examined the presence of a GTP-binding protein (G-protein) signaling pathway as well as the role of inositol 1,4,5-trisphosphate (InsP3) receptor (InsP3R)-mediated Ca2+ release and ryanodine receptor (RyR)-mediated Ca2+ release, the two Ca2+ receptors/channels most often thought to participate in the generation of [Ca2+]i oscillations, by injecting appropriate agonists and antagonists and monitoring their effects on Ca2+ release and pronucleus formation, injection of guanosine 5'-0-(3-thiotriphosphate) (GTP gamma [S]), which promotes G-protein-mediated phosphoinositide turnover, induced, at high concentrations, repetitive [Ca2+]i rises. Low concentrations of GTP gamma [S] were ineffective. Injection of inositol trisphosphorothioate (InsP3S3), a nonmetabolizable analogue of InsP3, evoked an immediate Ca2+ release followed by [Ca2R]i oscillations. The GTP gamma [S]- and InsP3S3-induced oscillations showed a rapid attenuation in amplitude and were terminated in about 30-60 min. Thimerosal, a thiol oxidizing agent, caused repetitive rises in [Ca2+]i by sensitizing Ca2+ injection-induced Ca2+ release. Injection of ryanodine, which stimulates Ca(2+)-induced Ca2+ release via the RyR, did not induce [Ca2+]i oscillations; and eggs into which it was preinjected exhibited normal [Ca2+]i oscillations in response to thimerosal. Preinjection of heparin, a competitive InsP3R antagonist, blocked in a dose-dependent manner the Ca2+ response to InsP3 and thimerosal, and its injection into fertilized oscillating eggs inhibited [Ca2+]i oscillations in all eggs.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

Identification of an M(r) 60,000 polypeptide unique to the meiotic spindle of the mouse oocyte.

The mouse oocyte expresses an M(r) 60,000 (p60) polypeptide that is associated with the first and second meiotic spindles. Immunoreactive p60 was not detectable in the meiotic spindles of male germ cells or in mitotic spindles. P60 was identified with a polyclonal antibody whose predominant activity is directed against ankyrin. However, immunoadsorption experiments demonstrated that p60 is not an ankyrin isoform and represents a secondary activity of the polyclonal antibody. Circumstantial evidence suggest that p60 may be a microtubule-associated protein. Since the most obvious difference between the female meiotic spindle and other spindles is the long half-life of the former, we hypothesize that p60 may function in the maintenance of the long-lived female meiotic apparatus.

Factors involved in nuclear reprogramming during early development in the rabbit.

Mechanisms of nuclear reprogramming and assessment of potential malfunctions that could be deleterious for development were evaluated in rabbit zygotes, parthenotes, and nuclear transfer embryos by analysis of DNA replication, nucleolar fibrillarin label, and localization of nuclear material reactive to the MPM-2 antibody. Nuclear transfer embryos were derived from G1/early S-phase donor nuclei and MII oocytes. In nuclear transfer embryos, DNA rereplication was likely to have occurred because label was incorporated, possibly in the centromeric regions of the chromosomes, prior to premature chromosome condensation and again following pronuclear formation. In parthenotes, DNA replication began very late in the cell cycle, which may be due to deficiencies in the artificial activation stimulus. The presence of fibrillarin label in the nucleolus was used as an indication of nucleolar transcriptional activity. Fibrillarin label was absent in embryos of all types up to the 16-32-cell stage. Although fibrillarin reappeared in nuclear transfer and parthenote embryos at the appropriate stage, not all blastomeres showed label indicating impaired development in these embryos. Labelling of phosphorylated epitopes by MPM-2 antibody showed a change in pattern of labelling during early development. Early cleavage stage embryos did not exhibit labelling over the spindle poles as did blastomeres from 32-cell embryos and tissue culture cells. All cell types exhibited labelling during interphase as dots located primarily over the nucleus in blastomeres from 32-cell embryos and in tissue culture cells, together with cytoplasmic label in embryos at early cleavage stages. Nuclear transplant embryos had a normal pattern of MPM-2 label. In contrast, the appearance of MPM-2 label in parthenotes depended on the type of calcium stimulation. These results demonstrate defects in DNA synthesis, nucleolar activity, and specific phosphorylation events, likely resulting from an improper activation stimulus and chromosome condensation in the transplanted nucleus.

Morphology and subsequent development in culture of bovine oocytes matured in vitro under various conditions of fertilization.

The objective of these experiments was to evaluate factors affecting in vitro fertilization of bovine oocytes matured in vitro, and their subsequent development to blastocysts. In Expts 1 and 2, sperm concentration, spermatozoa and oocyte incubation time, motility enhancers and semen source were manipulated. Fluorescent microscopy of microtubules and chromatin was used to observe sperm penetration rate, sperm aster formation and chromatin decondensation. Oocyte penetration rates were affected by sperm concentration but not by spermatozoa and oocyte incubation time. The effect of sperm concentration was due primarily to changes in polyspermy and not monospermy. Motility enhancers had no effect on any parameter measured. In Expt 3, oocytes were matured for 17, 22, 28 and 34 h before fertilization and evaluated for fertilization rates, morphology of cortical granules and exocytosis and blastocyst development. A domain free of cortical granules that was associated with the metaphase chromatin was not observed in mature bovine oocytes. As oocytes matured from 17 to 34 h, the distribution of cortical granules progressed from clustered to diffuse. Although monospermic fertilization rates were similar and cortical granule exocytosis occurred in all groups, polyspermy increased with maturation time. Development to blastocysts increased from 17 to 22 h of maturation but decreased thereafter with increasing maturation time. These results suggest that polyspermy can be reduced by adjusting sperm concentration and spermatozoa and oocyte incubation time with little effect on monospermic fertilization. Increased polyspermy with increased maturation time was not due to a lack of cortical granule exocytosis.

Dephosphorylation of sperm midpiece antigens initiates aster formation in rabbit oocytes.

During fertilization in most mammals, the penetrating sperm organizes an aster of microtubules. We have investigated the mechanisms underlying this function of the sperm by a series of experiments based on microinjection of isolated sperm midpieces into unfertilized oocytes. These midpieces contain antigens recognized by the MPM-2 antibody. These antigens, which are absent from the rest of the tail fraction, correspond to three phosphorylated polypeptides of 77, 81, and 85 kDa. Dephosphorylation with alkaline phosphatase abolishes antigenicity on blots and in whole sperm. Reactivity to the antibody disappears between 1 and 3 hr after calcium stimulation of oocytes, following the decline in H1 kinase activity and coincident with aster formation. In unactivated oocytes, no aster forms and the antigen remains unchanged. MPM-2 treatment of midpieces prior to injection blocks their ability to form asters in oocytes activated by calcium stimulation. The epitope also disappears in 6-methyl-aminopurine-treated oocytes, implying that maintenance of the phosphorylated state requires kinase activity. A result that confirms this view is that sperm midpieces dephosphorylated by alkaline phosphatase can be rephosphorylated after injection into oocytes or by exposure in vitro to a Xenopus oocyte cytoplasmic fraction high in H1 kinase activity. We suggest that the microtubule nucleation activity of sperm midpieces after fertilization is triggered by the calcium-induced decrease in maturation promoting factor, which results in dephosphorylation of specific sperm centrosomal proteins.

Chromatin and microtubule morphology during the first cell cycle in bovine zygotes.

Chromatin and microtubule configurations during the first cell cycle of bovine zygotes were analyzed by DNA staining and microtubule immunolocalization using an IVM/IVF system and oocytes matured and fertilized in vivo, in order to investigate the origin of the active centrosome and to characterize the nuclear and the cytoplasmic changes following bovine fertilization. Our results suggest that the paternal centrosome is active during early zygotic development, forming a conspicuous sperm aster soon after fertilization. We also report that polyspermy in bovine eggs, leads to the formation of numerous sperm asters with different degrees of association with the chromatin. The maternal structures in both monospermic and polyspermic zygotes can be lost or degenerate. Consequently, these cells may resume the first cell cycle as androgenotes, very often with several types of mitotic activity taking place in different regions of the cell cytoplasm at the same time. As indicated by a comparison of monospermic and polyspermic fertilization rates to rates of development, it is possible that some androgenetic embryos cleave and develop to the blastocyst stage.

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.

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.

Immunofluorescence localization of an adducin-like protein in the chromosomes of mouse oocytes.

The mouse oocyte expresses a polypeptide of Mr 120,000 that cross-reacts with an antibody to the brain membrane skeletal protein adducin. Immunofluorescence localization showed a bright chromosomal staining reaction in metaphase I and metaphase II oocytes. Following in vitro fertilization the maternal chromosomes lost their immunoreactivity during pronuclear development. The fertilizing sperm chromatin and male pronucleus did not show any detectable staining reaction. Bright chromosomal fluorescence was again observed in the first mitotic metaphase when both maternal and paternal chromosomes gave a positive staining reaction. In contrast to the immunoreactivity of the maternal meiotic chromosomes, the meiotic chromosomes of male germ line cells failed to exhibit any detectable staining reaction and this difference was confirmed by immunolabeling of oocyte and spermatocyte karyotypes. Mitotic chromosomes in preimplantation embryos, fetal liver, adult intestinal epithelium, and MDCK cells also failed to show any detectable labeling reaction. The results suggest that expression of the immunoreactive chromosomal adducin may be a unique feature of oogenesis.