The localization of LAP2 beta during pronuclear formation in bovine oocytes after fertilization or activation

We have shown that the assembly of lamin-associated polypeptide (LAP) 2beta was detected surrounding the chromatin mass around the time of extrusion of the second polar body (PB) in some fertilized oocytes, but not in most activated oocytes, by using A23187 and cycloheximide (CaA + CH). Here, we immunohistologically analysed the correlation between LAP2beta assembly and chromatin condensation in fertilized and activated oocytes during the second meiosis. In bovine cumulus cells, the onset of LAP2beta assembly was observed around anaphase chromosomes with strongly phosphorylated histone H3. No LAP2beta assembled around the chromosomes in the first and second polar bodies and the alternative oocyte chromatin (oCh) if histone H3 was phosphorylated. Only histone H3 of oCh was completely dephosphorylated during the telophase II/G1 transition (Tel II/G1), and then LAP2beta assembled around only the oCh without phosphorylated histone H3. In the oocytes activated by CaA + CH, LAP2beta did not assemble around the condensed oCh during the Tel II/G1 transition, although their histone H3 dephosphorylation occurred rather rapidly compared with that of the fertilized oocytes. The patterns of histone H3 dephosphorylation and LAP2beta assembly in oocytes activated by CaA alone showed greater similarity to those in fertilized oocytes than to those in oocytes activated by CaA + CH. These results show that LAP2beta assembles around only oCh after complete dephosphorylation of histone H3 after fertilization and activation using CaA alone, and that the timing of histone H3 dephosphorylation and LAP2beta assembly in these oocytes is different from that of somatic cells. The results also indicate that CH treatment inhibits LAP2beta assembly around oCh but not histone H3 dephosphorylation.     

Dynamics of lamin A/C in porcine embryos produced by nuclear transfer

This study was conducted to investigate the presence of lamin A/C in porcine nuclear transfer embryos and to determine whether lamin A/C can serve as a potential marker for nuclear reprogramming. First, lamin A/C was studied in oocytes and embryos produced by fertilization or parthenogenetic oocyte activation. We found that lamin A/C was present in the nuclear lamina of oocytes at the germinal vesicle stage while it was absent in mature oocytes. Lamin A/C was detected throughout preimplantation development in both in vivo-derived and parthenogenetic embryos. Incubation of the activated oocytes in the presence of alpha-amanitin (an inhibitor of RNA polymerase II), or cycloheximide (a protein synthesis inhibitor) did not perturb lamin A/C assembly, indicating that the assembly resulted from solubilized lamins dispersed in the cytoplasm. In nuclear transfer embryos, the lamin A/C signal that had previously been identified in fibroblast nuclei disappeared soon after fusion. It became detectable again after the formation of the pronucleus-like structure, and all nuclear transfer embryos displayed lamin A/C staining during early development. Olfactory bulb progenitor cells lacked lamin A/C; however, when such cells were fused with enucleated oocytes, the newly formed nuclear envelopes stained positive for lamin A/C. These findings suggest that recipient oocytes remodel the donor nuclei using type A lamins dispersed in the ooplasm. The results also indicate that lamin A/C is present in the nuclear envelope of pig oocytes and early embryos and unlike in some other species, its presence after nuclear transfer is not an indicator of erroneous reprogramming.     

体外受精和孤雌活化过程中小鼠胚胎细胞骨架的动态变化

为研究微管在体外受精与孤雌活化过程中的动态变化,本实验比较了体外受精胚胎、SrCl2激活的孤雌胚胎和体内受精的原核期胚胎在体外发育的情况,采用免疫荧光化学与激光共聚焦显微术检测卵母细胞孤雌活化过程中及体外受精后微管及核的动态变化,以分析微管在减数分裂过程中的作用及其对早期发育的影响.结果显示,体内受精胚胎的发育率显著高于体外受精和孤雌激活胚胎体外发育率(P<0.05),而体外受精与孤雌激活胚胎在各阶段发育率差异均不显著.在体外受精中,精子入卵,激活卵母细胞,减数分裂恢复,纺锤丝牵拉赤道板卜致密排列的母源染色体向纺锤体两侧迁移;后期将染色体拉向两极;末期时,微管分布于两组已去凝集的母源染色体之间,卵母细胞排出第二极体(the second polarbody,Pb2),解聚的母源染色体形成雌原核.同时,在受精后5～8 h精子染色质发生去浓缩与再浓缩,形成雄原核.在原核形成的同时,胞质星体在雌、雄原核的周围重组形成长的微管,负责雌、雄原核的迁移靠近.孤雌活化过程中,卵母细胞恢复减数分裂,姐妹染色单体分离,被拉向两极,经细胞松弛素B处理后,活化4～6 h,卵周隙中未见Pb2,而在胞质中出现两个混合的单倍体原核,之间由微管相连接,负责两个单倍体原核的迁移靠近.与体外受精相比较,孤雌活化时卵母细胞更容易被激活,减数分裂期间微管的发育早且更完善.     

Nuclear lamin antigen and messenger RNA expression in bovine in vitro produced and nuclear transfer embryos

The nuclear lamina is a complex meshwork of nuclear lamin filaments that lies on the interface of the nuclear envelope and chromatin and is important for cell maintenance, nucleoskeleton support, chromatin remodeling, and protein recruitment to the inner nucleolus. Protein and mRNA patterns for the major nuclear lamins were investigated in bovine in vitro fertilized (IVF) and nuclear transfer embryos. Expression of lamins A/C and B were examined in IVF bovine germinal vesicle (GV) oocytes, metaphase II oocytes, zygotes, 2-cell, 8-cell, 16-32-cell embryos, morulae, and blastocysts (n = 10). Lamin A/C was detected in 9/10 immature oocytes, 10/10 zygotes, 8/10 2-cell embryos, 4/10 morulae, 10/10 blastocysts but absent during the maternal embryonic transition. Lamin B was ubiquitously expressed during IVF preimplantation development but was only detected in 4/10 GV oocytes. Messenger RNA expression confirms that the major lamins, A/C and B1 are expressed throughout preimplantation development and transcribed by the embryo proper. Lamin A/C and B expression were observed (15 min, 30 min, 60 min, 120 min) following somatic cell nuclear transfer using adult fibroblasts and at the 2-cell, 8-cell, 16-32-cell, morula and blastocyst stage (n = 5). Altered expression levels and localization of nuclear lamins A/C and B was determined in nuclear transfer embryos during the first 2 hr post fusion, coincidental with only partial nuclear envelope breakdown as well as during the initial cleavage divisions, but was restored by the morula stage. This mechanical and molecular disruption of the nuclear lamina provides key evidence for incomplete nuclear remodeling and reprogramming following somatic cell nuclear transfer.     

Detergent-salt resistance of LAP2alpha in interphase nuclei and phosphorylation-dependent association with chromosomes early in nuclear assembly implies functions in nuclear structure dynamics.

Lamina-associated polypeptide (LAP) 2 of the inner nuclear membrane (now LAP2beta) and LAP2alpha are related proteins produced by alternative splicing, and contain a common 187 amino acid N-terminal domain. We show here that, unlike LAP2beta, LAP2alpha behaved like a nuclear non-membrane protein in subcellular fractionation studies and was localized throughout the nuclear interior in interphase cells. It co-fractionated with LAP2beta in nuclear lamina/matrix-enriched fractions upon extraction of nuclei with detergent, salt and nucleases. During metaphase LAP2alpha dissociated from chromosomes and became concentrated around the spindle poles. Furthermore, LAP2alpha was mitotically phosphorylated, and phosphorylation correlated with increased LAP2alpha solubility upon extraction of cells in physiological buffers. LAP2alpha relocated to distinct sites around chromosomes at early stages of nuclear reassembly and intermediarily co-localized with peripheral lamin B and intranuclear lamin A structures at telophase. During in vitro nuclear assembly LAP2alpha was dephosphorylated and assembled into insoluble chromatin-associated structures, and recombinant LAP2alpha was found to interact with chromosomes in vitro. Some LAP2alpha may also associate with membranes prior to chromatin attachment. Altogether the data suggest a role of LAP2alpha in post-mitotic nuclear assembly and in the dynamic structural organization of the nucleus.     

Cytoplasmic modification of the nuclear lamina during pronuclear-like transformation of mouse blastomere nuclei.

During the successive interphases of cleaving mouse embryos the nuclear periphery diminishes its reactivity to anti-lamin A and C antibodies. This developmentally regulated characteristic can be modified by exposure of the blastomere nuclei to metaphase II (M II) oocyte cytoplasm followed by activation. In the current study we define the cytoplasmic conditions necessary for this modification of 8-cell and 16-cell stage nuclei in hybrids obtained by fusion with metaphase II arrested oocytes, oocytes at various time points after parthenogenetic activation, naturally fertilized eggs (zygotes) and interphase 2-cell embryo blastomeres. The intensity of fluorescence obtained with anti-lamins A/C in the blastomere nuclei increases as a result of fusion with freshly activated oocytes or early zygotes (first 3.0-5.5 h in the case of parthenogenetic activation), and not when eggs or 2-cell blastomeres advanced in interphase are used as partners for fusion. This transformation of the A/C lamin pattern is correlated with the ability to promote pronucleus-like growth of blastomere nuclei in hybrids. Blastomere nuclei introduced into M II-arrested oocytes undergo premature chromatin condensation and dissolution of the nuclear lamina. The results are discussed with regard to certain particularities of the first embryonic interphase of the mouse and the potential involvement of nuclear lamins in pronuclear growth.     

Nuclear lamin antigens are developmentally regulated during porcine and bovine embryogenesis

The nuclear lamins, proteins that reside on the inner face of the nuclear envelope, are thought to provide attachment sites for anchoring the chromatin to the nuclear envelope, thus facilitating the overall organization of the nucleus. The composition of the nuclear lamin proteins changes during differentiation and development in a variety of mammalian and nonmammalian tissues. Bovine and porcine oocytes and early embryos were prepared for immunocytochemical detection of nuclear lamins using three different antibodies (recognizing lamin B, lamins A/B/C, or lamins A/C). In both species, germinal vesicle nuclei and early cleavage stage nuclei react positively with the antibodies. However, on nuclei of bovine embryos, the A/C epitope was not detectable at the 16-cell stage, compact morula, spherical blastocyst, or the chorionic cell nuclei of a Day 35 conceptus, but was detectable on both amniotic and embryonic ectodermal cell nuclei of a Day 35 conceptus. All three antibodies reacted with nuclei from two bovine tissue culture cell lines (bovine embryonic cells and Madin-Darby bovine kidney cells) and one porcine kidney cell line. Nuclei in porcine embryos followed a similar pattern, except the loss of the A/C epitope occurred at the 8-cell stage and the epitope was absent from compact morula and spherical blastocyst stage nuclei. All interphase nuclei in both species reacted with both anti-lamin A/B/C and anti-lamin B antibodies, whereas metaphase chromosomes did not react with any of the lamin antibodies tested. The change in recognizing the lamin epitope occurred one cell cycle after the expected transition from maternal control to zygotic control of development. Nuclear transplantation showed that 16-cell stage porcine nuclei, which are lamin A/C negative, acquired the A/C epitope after transfer to an enucleated metaphase II oocyte. These results suggest that the A/C epitope is developmentally regulated.     

The kinetics of oocyte activation and dynamics of polar body formation in Calomys musculinus and Calomys laucha (Rodentia-Sigmodontinae)

The objective of this study was to determine whether Calomys laucha and Calomys musculinus superovulated oocytes undergo parthenogenetic activation following activation stimuli. Cumulus-intact or denuded oocytes were treated with medium containing ethanol (7%), medium containing strontium chloride, or medium alone. They were then incubated for 6-8 h to allow for activation. A group of oocytes was fixed immediately after maturation to serve as a control. The nuclear status of the oocytes was examined after staining with Hoechst 33342, to determine the timing of pronuclear progression from metaphase II to anaphase II or telophase II or to the pronuclear stage. The proportion of oocytes that underwent activation was higher for oocytes treated with ethanol or strontium chloride than in those incubated in medium alone, for the two species studied (p < 0.001). There was little evidence of spontaneous activation occurring in oocytes during the treatments. Most of the activated oocytes contained a single haploid pronucleus, but it was possible to find immediate cleavage and two pronuclei. The different classes of activated oocytes were cultured for 5 days. The type of activating treatment had a marked effect on the ability of the resulting C. musculinus and C. laucha parthenogenetic embryos to develop to the preimplantation stages. Incubation with ethanol produced only 8-cell embryos while the embryos induced with strontium chloride reached the blastocyst stage. This is the first report of parthenogenesis in C. musculinus and C. laucha. The ability of strontium ions to induce matured secondary oocytes to initiate parthenogenesis and obtain further development of Calomys provides opportunities to use Calomys oocytes in vitro and, therefore, to study the genetics, cell biology and virology of development.     

Review: lamina-associated polypeptide 2 isoforms and related proteins in cell cycle-dependent nuclear structure dynamics

The lamina-associated polypeptide (LAP) 2 family comprises up to six alternatively spliced proteins in mammalian cells and three isoforms in Xenopus. LAP2beta is a type II integral protein of the inner nuclear membrane, which binds to lamin B and the chromosomal protein BAF, and may link the nuclear membrane to the underlying lamina and provide docking sites for chromatin. LAP2alpha shares only the N-terminus with the other isoforms and contains a unique C-terminus. It is a nonmembrane protein associated with the nucleoskeleton and may help to organize higher order chromatin structure by interacting with A-lamins and chromosomes. Recent studies using mutant proteins have just begun to unravel functions of LAP2 isoforms during postmitotic nuclear reassembly. LAP2alpha associates with chromosomes via an alpha-specific domain at early stages of assembly, possibly providing a structural framework for chromosome reorganization. The subsequent interaction of both LAP2alpha and LAP2beta with the chromosomal BAF may stabilize chromatin structure and target membranes to the chromosomes. At later stages LAP2 may regulate the assembly of lamins. LAP2 isoforms have been found to share a homologous approximately 40 amino acid long region, the LEM domain, with nuclear membrane proteins MAN1 and emerin, which has been implicated in Emery-Dreifuss muscular dystrophy.     

Detection of nuclear lamin B epitopes in oocyte nuclei from mice, sea urchins, and clams using a human autoimmune serum

Somatic nuclei typically contain two or three major proteins, the lamins A, B, and C or their antigenically related equivalents, interspersed between the chromatin and its attachment site, the inner nuclear membrane. The late oocyte nuclear envelopes of the previously investigated Xenopus and Spisula germinal vesicles, however, have no chromatin attached and only one lamin-like protein. Since mouse and sea urchin germinal vesicles have chromatin attached, we tested them for the possible presence of more than one lamin. In both species we found two different lamins incorporated in their nuclear envelope structure. One lamin is recognized by anti-lamin B and the other by anti-lamin AC antibodies. Spisula germinal vesicles were found to contain not only the nuclear envelope-bound lamin (clamin), but also a 65-kDa protein cross-reactive with anti-lamin B antibodies. This protein is present unattached to any structure and is apparently soluble. Our findings provide a possible explanation of the early presence of lamin B in pronuclei of mouse and sea urchin contrary to the late appearance of a lamin B equivalent in amphibian embryos. In Spisula, as in Xenopus, the presence of a lamin B equivalent could not be documented in the nuclear envelopes of early embryos, indicating that a separate lamin B equivalent is not essential for chromatin binding to the envelope in these species during early embryogenesis. The results also indicate that the nuclear complement of structural proteins might vary substantially in the same cell type of different species.     

Nuclear envelope and chromatin compositional differences comparing undifferentiated and retinoic acid- and phorbol ester-treated HL-60 cells

The human leukemic cell line (HL-60) can be induced to differentiate in vitro to granulocytic form with retinoic acid (RA), or to monocytic/macrophage form with phorbol ester (TPA). The granulocytic form acquires nuclear lobulation, nuclear envelope-limited chromatin sheets (ELCS), and cytoskeletal polarization, none of which are acquired following treatment with TPA. Immunoblotting analyses and capillary zone electrophoresis demonstrated that following RA treatment: lamins A/C and B1, and vimentin decreased to negligible amounts; LAP2 beta, lamin B2 and emerin remained essentially unchanged; lamin B receptor (LBR) increased markedly; histone subtypes H1.4 and 1.5 exhibited dephosphorylation. Following TPA treatment: lamins A/C and B1, B2 and vimentin increased in amount; LAP2 beta and emerin remained essentially unchanged; LBR increased markedly; histone subtypes H1.4 and 1.5 exhibited dephosphorylation. Emerin, which was cytoplasmic in undifferentiated or granulocytic cells, localized into the nuclear envelope following TPA. Normal human granulocytes revealed compositional differences compared to granulocytic forms of HL-60, namely increased vimentin and appearance of histone subtype H1.3. A working hypothesis for nuclear lobulation postulates a combination of: increased nuclear envelope deformability due to lamins A/C and B1 deficiency; an increase in nuclear surface area/volume; an increase in chromatin-nuclear envelope interactions.     

Membrane-associated lamins in Xenopus egg extracts: identification of two vesicle populations

Nuclear lamin isoforms of vertebrates can be divided into two major classes. The B-type lamins are membrane associated throughout the cell cycle, whereas A-type lamins are recovered from mitotic cell homogenates in membrane-free fractions. A feature of oogenesis in birds and mammals is the nearly exclusive presence of B-type lamins in oocyte nuclear envelopes. In contrast, oocytes and early cleavage embryos of the amphibian Xenopus laevis are believed to contain a single lamin isoform, lamin LIII, which after nuclear envelope breakdown during meiotic maturation is reported to be completely soluble. Consequently, we have reexamined the lamin complement of Xenopus oocyte nuclear envelopes, egg extracts, and early embryos. An mAb (X223) specific for the homologous B-type lamins B2 of mouse and LII of Xenopus somatic cells (Hoger, T., K. Zatloukal, I. Waizenegger, and G. Krohne. 1990. Chromosoma. 99:379-390) recognized a Xenopus oocyte nuclear envelope protein biochemically distinct from lamin LIII and very similar or identical to somatic cell lamin LII. Oocyte lamin LII was detectable in nuclear envelopes of early cleavage embryos. Immunoblotting of fractionated egg extracts revealed that approximately 20-23% of lamin LII and 5-7% of lamin LIII were membrane associated. EM immunolocalization demonstrated that membrane-bound lamins LII and LIII are associated with separate vesicle populations. These findings are relevant to the interpretation of nuclear reconstitution experiments using Xenopus egg extracts.     

Somatic cell nuclear transfer in the pig: control of pronuclear formation and integration with improved methods for activation and maintenance of pregnancy

To clone a pig from somatic cells, we first validated an electrical activation method for use on ovulated oocytes. We then evaluated delayed versus simultaneous activation (DA vs. SA) strategies, the use of 2 nuclear donor cells, and the use of cytoskeletal inhibitors during nuclear transfer. Using enucleated ovulated oocytes as cytoplasts for fetal fibroblast nuclei and transferring cloned embryos into a recipient within 2 h of activation, a 2-h delay between electrical fusion and activation yielded blastocysts more reliably and with a higher nuclear count than did SA. Comparable rates of development using DA were obtained following culture of embryos cloned from ovulated or in vitro-matured cytoplasts and fibroblast or cumulus nuclei. Treatment of cloned embryos with cytochalasin B (CB) postfusion and for 6 h after DA had no impact on blastocyst development as compared with CB treatment postfusion only. Inclusion of a microtubule inhibitor such as nocodozole with CB before and after DA improved nuclear retention and favored the formation of single pronuclei in experiments using a membrane dye to reliably monitor fusion. However, no improvement in blastocyst development was observed. Using fetal fibroblasts as nuclear donor cells, a live cloned piglet was produced in a pregnancy that was maintained by cotransfer of parthenogenetic embryos.     

Ultraviolet-irradiated spermatozoa activate oocytes but arrest preimplantation development after fertilization and nuclear transplantation in cattle

Artificial means of parthenogenetically activating mammalian oocytes are believed to lack an essential sperm epigenetic component required for normal development. The main goal of this study was to examine the potential of ultraviolet (UV)-irradiated sperm as a means of functionally eliminating the chromatin component of spermatozoa without affecting the ability to induce activation and support parthenogenetic development in cattle. Spermatozoa were stained with a DNA dye, exposed to various UV irradiation doses, and used to fertilize secondary oocytes. Although the percentage of pronuclei at 18 h postinsemination was similar using treated and control sperm, most oocytes fertilized by UV-irradiated sperm failed to develop beyond the 2-cell stage, suggesting that UV irradiation can functionally destroy the genomic component of spermatozoa with limited effects on the ability to induce oocyte activation. However, when oocytes activated with UV-irradiated sperm were used as hosts for nuclear transfer, developmental rates to cleavage and to blastocyst improved only marginally and remained lower than in the controls, indicating that UV-treated spermatozoa blocked development even in the presence of a diploid donor nucleus. Although DNA replication was not inhibited by UV irradiation treatment, abnormal chromatin morphology after cleavage suggests improper segregation of chromatin to daughter blastomeres during the first mitotic division. Together, these results indicate that although sperm exposed to UV can activate oocytes, a developmental block occurs at or soon after the first mitosis in parthenotes and oocytes reconstructed by nuclear transfer.     

Roles of LAP2 proteins in nuclear assembly and DNA replication: truncated LAP2beta proteins alter lamina assembly, envelope formation, nuclear size, and DNA replication efficiency in Xenopus laevis extracts

Humans express three major splicing isoforms of LAP2, a lamin- and chromatin-binding nuclear protein. LAP2beta and gamma are integral membrane proteins, whereas alpha is intranuclear. When truncated recombinant human LAP2beta proteins were added to cell-free Xenopus laevis nuclear assembly reactions at high concentrations, a domain common to all LAP2 isoforms (residues 1-187) inhibited membrane binding to chromatin, whereas the chromatin- and lamin-binding region (residues 1-408) inhibited chromatin expansion. At lower concentrations of the common domain, membranes attached to chromatin with a unique scalloped morphology, but these nuclei neither accumulated lamins nor replicated. At lower concentrations of the chromatin- and lamin-binding region, nuclear envelopes and lamins assembled, but nuclei failed to enlarge and replicated on average 2. 5-fold better than controls. This enhancement was not due to rereplication, as shown by density substitution experiments, suggesting the hypothesis that LAP2beta is a downstream effector of lamina assembly in promoting replication competence. Overall, our findings suggest that LAP2 proteins mediate membrane-chromatin attachment and lamina assembly, and may promote replication by influencing chromatin structure.     

A phosphorylation cluster in the chromatin-binding region regulates chromosome association of LAP2alpha

LAP2alpha is a LEM family protein associated with nucleoplasmic A-type lamins and chromatin in interphase. Like lamins and other lamina proteins LAP2alpha is cytoplasmic in metaphase, but it associates with chromosomes prior to nuclear envelope formation in late anaphase to telophase. In vitro phosphorylation analysis and mass spectrometry identified a cluster of at least three mitotic cyclin-dependent kinase 1 phosphorylation sites in the C-terminal chromatin-binding region of LAP2alpha as well as four additional potential sites in the cluster, some of which were targeted alternatively in LAP2alpha mutated at the major sites. LAP2alpha mutants containing serine --> alanine mutations at all seven sites revealed a clear phenotype. Mutated LAP2alpha remained associated with chromosomes throughout mitosis, but the dissociation of lamins into the cytoplasm and nuclear envelope disassembly were not affected. These data demonstrate the in vivo significance of mitotic phosphorylation for the dynamic behavior of LAP2alpha in the cell cycle and show that, unlike the interaction with lamins, the chromatin association of LAP2alpha is regulated by multiple mitosis-specific phosphorylation at sites clustered within a defined region in the C terminus of the protein.     

Expression of nuclear lamins during mouse preimplantation development

The expression of nuclear lamins during mouse preimplantation development was studied by immunofluorescence, immunoblotting and immunoprecipitation. Two sera were used, specific either for lamin B or lamins A and C. Both sera gave a positive staining of the nuclear periphery throughout preimplantation development (fertilized eggs to late blastocysts). Immunoblots revealed that the three lamins were present in eggs and blastocysts. However, lamin A from eggs was found to have a higher apparent Mr than lamin A from blastocysts and other mouse cells. Using immunoprecipitation, synthesis of lamin A was detected in eggs while synthesis of lamin B was detected in 8-cell embryos and blastocysts, indicating that at least some of the lamins used during early development do not come from a store in the egg. These results are discussed in relation to the possible role of lamins during cell differentiation.     

Structural basis for dimerization of LAP2alpha, a component of the nuclear lamina

Lamina-associated polypeptides (LAPs) are important components of the nuclear lamina, the dense network of filaments that supports the nuclear envelope and also extends into the nucleoplasm. The main protein constituents of the nuclear lamina are the constitutively expressed B-type lamins and the developmentally regulated A- and C-type lamins. LAP2alpha is the only non-membrane-associated member of the LAP family. It preferentially binds lamin A/C, has been implicated in cell-cycle regulation and chromatin organization, and has also been found to be a component of retroviral preintegration complexes. As an approach to understanding the role of LAP2alpha in cellular pathways, we have determined the crystal structure of the C-terminal domain of LAP2alpha, residues 459-693. The C-terminal domain is dimeric and possesses an extensive four-stranded, antiparallel coiled coil. The surface involved in binding lamin A/C is proposed based on results from alanine-scanning mutagenesis and a solid-phase overlay binding assay.     

Development of sheep androgenetic embryos is boosted following transfer of male pronuclei into androgenetic hemizygotes

Androgenetic embryos are useful model for investigating the contribution of the paternal genome to embryonic development. Little work has been done with androgenetic embryo production in domestic animals. The aim of this study was the production of diploid androgenetic sheep embryos. In vitro matured sheep oocytes were enucleated and fertilized in vitro; parthenogenetic and normally fertilized embryos were also produced as a control. Fifteen hours after in vitro fertilization (IVF), presumptive zygotes were centrifuged and scored for the number of pronucleus. IVF, parthenogenetic, and androgenetic embryos (haploid, diploid, and triploid) were cultured in SOFaa medium with bovine serum albumin (BSA). The proportion of oocytes with polyspermic fertilization increased linearly with increasing sperm concentration. After IVF, there was no significant difference in early cleavage and morula formation rates between the groups, while there was a significant difference on blastocyst development between IVF, parthenogenetic, and androgenetic embryos, the last ones displaying poor developmental potential (IVF, parthenogenetic, and haploid, diploid, and triploid androgenetic embryos: 43%, 38%, 0%, 2%, and 2%, respectively). In order to boost androgenetic embryonic development, we produced diploid androgenetic embryos through pronuclear transfer. Single pronuclei were aspirated with a bevelled pipette from haploid or diploid embryos and transferred into the perivitelline space of other haploid embryos, and the zygotes were reconstructed by electrofusion. Fusion rates approached 100%. Pronuclear transfer significantly increased blastocyst development (IVF, parthenogenetic, androgenetic: Diploid into Haploid, and Haploid into Haploid: 42%, 42%, 19%, and 3%, respectively); intriguingly, the Haploid + Diploid group showed the highest development to blastocyst stage. The main findings of our study are: (1) sheep androgenetic embryos display poor developmental ability compared with IVF and parthenogenetic embryos; (2) diploid androgenetic embryos produced by pronuclear exchange developed in higher proportion to blastocyst stage, particularly in the Diploid-Haploid group. In conclusion, pronuclear transfer is an effective method to produce sheep androgenetic blastocysts.     

Bovine parthenogenetic blastocysts following in vitro maturation and oocyte activation with ethanol

The appropriate in vitro bovine oocyte maturation and ethanol activation conditions for preimplantation bovine embryo parthenogenetic development to the blastocyst stage were investigated. A 7% ethanol concentration significantly enhanced (P<0.05) the proportion of activated, in vitro-matured bovine oocytes (7% ethanol, 83.4 +/- 3.2% versus 0% ethanol, 63.9 +/- 2.0%). The proportion of activated oocytes was significantly higher (P<0.05) by treatment with 7% ethanol for a minimum of 2 minutes (2 minutes, 89.8 +/- 4.0% versus 0.5 minutes 63.4 +/- 4.9%). Oocyte maturation for periods ranging from 30, 34, 38 and 44 hours resulted in a significant increase (P<0.05) in the proportion of activated oocytes, and in oocytes displaying 2 or 3 pronuclei versus oocytes matured for 26 hours. The proportion of cleaved, activated oocytes (2-cell stage), 4 -cell stage and parthenogenetic morula/blastocysts was significantly higher (P<0.05) within the 34-hour oocyte maturation treatment group. Although the 44-hour oocyte maturation treatment group displayed the highest proportion of activated oocytes with 2 pronuclei, it did not display the highest cleavage frequency, possibly due to the effects of postovulatory aging. Several morphologically normal parthenogenetic bovine blastocysts developed from oocytes that were in vitro matured for 34 hours. The ability to produce such parthenogenetic embryos will eventually facilitate investigation into the role(s) of the maternal and paternal genomes during bovine early development.