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The success of somatic nuclear transfer depends upon the cell cycle stage of the donor nucleus and the recipient cytoplast. Recently, we established efficient cell cycle synchronization protocols for porcine fetal fibroblasts and found that serum withdrawal leads to cell death. Here, we examined whether the specific cell death induced by serum deprivation follows the conventional apoptotic pathway in porcine fibroblasts. Terminal deoxynucleotidyl transferase mediated dUTP nick end-labeling analysis revealed that serum deprivation induced DNA fragmentation in a concentration and time dependent manner. Semiquantitative RT-PCR and Western blotting revealed activation of cell death-related genes Bak and Bax of the Bcl-2 family. However, electrophoretic analysis of genomic DNA from serum deprived cells did not provide evidence for the internucleosomal DNA cleavage which is characteristic of conventional apoptosis. Thus, serum deprivation triggers initial steps in the apoptotic pathway, but does not lead to the typical oligonucleosome-sized DNA ladder. These findings contribute to a better understanding of apoptotic pathways and aid to define essential parameters of the donor nucleus for successful somatic cloning.

Effective activation of a recipient oocyte and its compatibility with the nuclear donor are critical to the successful nuclear reprogramming during nuclear transfer. We designed a series of experiments using various activation methods to determine the optimum activation efficiency of bovine oocytes. We then performed nuclear transfer (NT) of embryonic and somatic cells into cytoplasts presumably at G1/S phase (with prior activation) or at metaphase II (MII, without prior activation). Oocytes at 24 hr of maturation in vitro were activated with various combinations of calcium ionophore A23187 (A187) (5 μM, 5 min), electric pulse (EP), ethanol (7%, 7 min), cycloheximide (CHX) (10 μg/ml, 6 hr), and then cultured in cytochalasin D (CD) for a total of 18 hr. Through a series of experiments (Exp. 1–4), an improved activation protocol (A187/EP/CHX/CD) was identified and used for comparison of NT efficiency of embryonic versus somatic donor cells (Exp. 5). When embryonic cells from morula and blastocysts (BL) were used as nuclear donors, a significantly higher rate of blastocyst development from cloned embryos was obtained with G1/S phase cytoplasts than with MII-phase cytoplasts (36 vs. 11%, P

Background The aim of our study was to evaluate the possibility of embryonic or somatic cell haploidization after fusion with intact or enucleated immature oocytes which were subsequently cultured in vitro. Embryonic or somatic cell nuclei do not undergo premature chromosome condensation when fused to intact or enucleated immature oocytes whose maturation is prevented by dibutyryl cyclic AMP (dbcAMP). The presence of dbcAMP permits, however, the completion of DNA replication in somatic cell nuclei. Methods and results The chromosomes condensed when the reconstructed cells were released from the dbcAMP block. When somatic or embryonic nuclei were introduced into intact immature meiotically competent oocytes and subsequently cultured their chromosomes assembled on a common spindle with meiotic chromosomes and proceeded through the meiotic-like division, judged according to the presence of the first polar body extruded. When embryonic cell nuclei were introduced into cytoplasts obtained from immature meiotically competent oocytes, polar bodies were extruded in about 75% of reconstructed cells but the metaphase plates were abnormal in almost all cases. When somatic cell nuclei were inserted into the above cytoplasts, polar bodies were extruded only very exceptionally and in these cells chromosomes were arranged in abortive metaphase plates. Conclusions Our results suggest that somatic cell nuclei are unable to proceed through the reduction division (haploidization) when introduced into an immature oocyte meiotic cytoplasm.

The effect of the stage of the cell cycle of donor cells and recipient cytoplasts on the timing of DNA replication and the developmental ability in vitro of bovine nuclear transfer embryos was examined. Embryos were reconstructed by fusing somatic cells with unactivated recipient cytoplasts or with recipient cytoplasts that were activated 2 h before fusion. Regardless of whether recipient cytoplasts were unactivated or activated, the embryos that were reconstructed from donor cells at the G0 phase initiated DNA synthesis at 6-9 h postfusion (hpf). The timing of DNA synthesis was similar to that of parthenogenetic embryos, and was earlier than that of the G0 cells in cell culture condition. Most embryos that were reconstructed from donor cells at the G1/S phase initiated DNA synthesis within 6 hpf. The developmental rate of embryos reconstructed by a combination of G1/S cells and activated cytoplasts was higher than the rates of embryos in the other combination of donor cells and recipient cytoplasts. The results suggest that the initial DNA synthesis of nuclear transfer embryos is affected by the state of the recipient oocytes, and that the timing of initiation of the DNA synthesis depends on the donor cell cycle. Our results also suggest that the cell cycles of somatic cells synchronized in the G1/S phase and activated cytoplasts of recipient oocytes are well coordinated after nuclear transfer, resulting in high developmental rates of nuclear transfer embryos to the blastocyst stage in vitro.

Foraminifera are usually between 0.1 and 1 mm in size, thus falling within the range of the largest eukaryotic cells. However, some fossil and extant foraminiferal species reach diameters of more than 100 mm. One hypothesis of how these gigantic sizes could have been attained by these unicellular organisms is the temporary compartmentalization of cytoplasm into smaller volumes of effective metabolism, as reported for several recent species. Evidence of this phenomenon is shown in fossil genera of larger foraminifera belonging to five families of Cretaceous to Oligocene age. Alternative interpretations are discussed.

Somatic cell nuclear transfer was used to produce live piglets from cultured fetal fibroblast cells. This was achieved by exposing donor cell nuclei to oocyte cytoplasm for approximately 3 h before activation by chemical means. Initially, an experiment was performed to optimize a cell fusion system that prevented concurrent activation in the majority of recipient cytoplasts. Cultured fibroblast cells were fused in medium with or without calcium into enucleated oocytes flushed from superovulated gilts. Cybrids fused in the presence of calcium cleaved at a significantly (P < 0.05) greater rate (69%, 37 out of 54) after 2 days of culture compared with those fused without calcium (10%, 7 out of 73), suggesting that calcium-free conditions are needed to avoid activation in the majority of recipient cytoplasts during fusion. In the second experiment, cybrids fused in calcium-free medium were activated approximately 3 h later with ionomycin, followed by incubation in 6-dimethylaminopurine to determine development in vitro. Following 2 days of culture, cleavage rates of chemically activated and unactivated cybrids (fusion without activation control) were 93% (100 out of 108) and 7% (2 out of 27), respectively. After an additional 5 days of culture, activated cloned embryos formed blastocysts at a rate of 23% (25 out of 108) with an average inner cell mass and trophectoderm cell number of 10 (range, 3 to 38) and 31 (range, 16 to 58), respectively. In the third experiment, activated nuclear transfer embryos were transferred to the uteri of synchronized recipients after 3 days of culture to assess their development in vivo. Of 10 recipients receiving an average of 80 cleaved embryos (range, 40 to 107), 5 became pregnant (50%) as determined by ultrasound between Day 25 and Day 35 of gestation. Of the five pregnant recipients, two subsequently farrowed one piglet per litter originating from two different cell culture lines. In this study, efficient reprogramming of porcine donor nuclei by fusing cells in the absence of calcium followed by chemical activation of recipient cytoplasts was reflected in high rates of development to blastocyst and pregnancy initiation leading to full term development.

Oocytes enucleated at the second metaphase stage (MII) are often used as recipient cytoplasts for nuclear transfer. The oocyte's nuclear material has been traditionally removed blindly by aspirating the first polar body (Pb1) along with a portion of the cytoplasm. However, the Pb1-guided enucleation method is unreliable because the position of the Pb1 is variable. A previous study showed that pretreatment of mouse oocytes with 3% (0.09 M) sucrose allowed visualization of the metaphase spindle and chromosomes under standard light microscopy and led to a 100% enucleation rate. The same sucrose treatment, however, did not produce the same effect in bovine oocytes. In this study, we increased the concentration of sucrose to 0.3-0.9 M in PBS containing 20% fetal bovine serum (SPF) and found that the majority of the treated bovine oocytes (75%-86%) formed a small transparent bud into the perivitelline space, as compared with the 0.1 M sucrose (6%) or the no sucrose (0%) control groups. Staining of DNA with Hoechst 33342 revealed that these projections coincided with the position of the metaphase chromosomes in 100% of sucrose-treated oocytes, whereas only 31% of oocytes showed alignment of the position of Pb1 with their nuclear materials. Furthermore, 95% of oocytes treated in 0.3 M SPF were successfully enucleated by removing a small amount of cytoplasm adjacent to the projection. This is a significantly higher enucleation rate than that obtained by conventional Pb1-guided enucleation, even when a larger amount of cytoplasm was removed. For nuclear transfer, the enucleated oocytes treated with sucrose did not differ from the control oocytes in rates of fusion, cleavage, or development to blastocysts, or in the average cell numbers in blastocysts. This study demonstrated that 0.3 M sucrose treatment of bovine oocytes facilitates the localization of metaphase chromosomes under normal light microscopy and hence increases enucleation efficiency without compromising the in vitro development potential of cloned embryos by nuclear transfer.

This study investigates the effects of spermatogenic germ cells on inhibin alpha-subunit and beta B-subunit expression, and inhibin alpha-subunit and inhibin B production by rat Sertoli cells in vitro. Sertoli cells isolated from 19-day-old rats were cultured for 48 h at 32 degrees C, in the presence or absence of FSH (2.3-2350 mIU/ml), and in the presence of pachytene spermatocytes, round spermatids or cytoplasts of elongated spermatids purified from adult rat testis by elutriation and density gradient separation. Sertoli cell secretion of inhibin alpha-subunit and inhibin B, as measured by immunoassay, was dose-dependently stimulated by FSH (maximal stimulation 13- and 2-fold, respectively). Round spermatids or cytoplasts co-cultured with Sertoli cells had no effect on basal or FSH-induced secretion of inhibin alpha-subunit or inhibin B. When Sertoli cells were co-cultured with pachytene spermatocytes, inhibin alpha-subunit secretion was unaltered, while inhibin B secretion was suppressed in a cell concentration-dependent manner to reach a maximal suppression of 45% compared with Sertoli cells alone (P

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.

We examined the effects of butyrolactone I (BL-I), a specific cdc2 kinase inhibitor, on the inhibition of meiotic resumption of porcine oocytes and subsequent developmental competence after somatic cell nuclear transfer (NT). Porcine follicular oocytes were cultured in a medium containing BL-I during maturation culture. BL-I suppressed germinal vesicle (GV) breakdown in a dose-dependent manner and most (98.3%) oocytes were arrested at the GV stage when they were cultured for 28 h with 100 μM BL-I, but after 48 h of culture with 100 μM BL-I half of the oocytes underwent GV breakdown. Twenty hours after release from the BL-I treatment (28-h culture), most (95.9%) oocytes reached the metaphase II stage, whereas most non-treated oocytes reached the metaphase II stage after 40 h of maturation culture. There were no differences between BL-I-treated and non-treated oocytes in meiotic progression. When oocytes were enucleated and fused with serum-starved cumulus cells, the development of NT embryos to the blastocyst stage with the BL-I-treated oocytes (12.3%) was similar to that achieved with non-treated oocytes (12.5%). The results demonstrate that BL-I could reversibly inhibit meiotic resumption of porcine oocytes, and that BL-I-treated porcine oocytes can be used as recipient cytoplasts for NT without compromising developmental competence.

Ooplasmic transplantation is based on the premise that ooplasmic components are compromised in some individuals. In theory, the transfer of small amounts of healthy ooplasm can correct such deficits, allowing for improved development and implantation. The technique is based on a well-established background of experimental embryology demonstrating that cytoplasmic manipulation in oocytes and early embryos can be entirely compatible with normal development. Cytoplasm has been manipulated via karyoplast and cytoplast transfer and by cytoplasmic injection. Term development has been obtained following such manipulations in a variety of mammalian species. While some manipulative scenarios have exhibited compromised development, others have exhibited improved development. Developmental problems involving specific epigenetic and mitochondrial incompatibilities have been observed in a very limited subset of animal studies. These studies are based on genetic and physical models that have little relation to the actual substance of ooplasmic transplantation in the human. In fact, the majority of animal studies suggest that ooplasmic transplantation is well-founded and unlikely to result in negative developmental consequences. Furthermore, there are considerable physical, physiological and developmental differences between human and rodent eggs and embryos. These differences suggest that potentially negative issues raised by rodent results may not be relevant in the human.

The type of donor cell most suitable for producing cloned animals is one of the topics under debate in the field of nuclear transfer. To provide useful information to answer this question, G2/M- and G0/G1-stage fetal fibroblasts were used as donor cells for nuclear transfer. In vitro-matured oocytes derived from abattoir ovaries were used as recipient cytoplasts. In both groups, nuclear envelope breakdown and premature chromosome condensation were completed within 1-2 h after donor cells were injected into the cytoplasm of oocytes. Microtubules were organized around condensed chromosomes and formed a spindle within 1-1.5 h after activation. Decondensation of chromosomes could be seen within 2-4 h after activation. Reformation of the new nuclear envelope occurred 4-6 h after activation and was followed by nuclear swelling and formation of a pronucleus-like structure (PN) 8-12 h after activation. Most (80.6%) of the reconstructed oocytes derived from G2/M cells extruded polar body-like structures (PB). However, a much lower frequency of PB (21.7%) was observed in the reconstructed oocytes derived from G0/G1 donors. A variety of PN and PB combinations were observed in reconstructed oocytes derived from G2/M-stage donors, including 1PN+0PB, 1PN+1PB, 1PN+2PB, 2PN+0PB, 2PN+1PB, 2PN+2PB, and 3PN+1PB. Chromosomes of most embryos (10/13) derived from G2/M stage were diploid. The percentage of cleavage and blastocysts and the average nuclear number of blastocysts in the G2/M and G0/G1 groups were not different. These results demonstrate that the G2/M stage can be morphologically remodeled by cytoplasm of MII oocytes in pigs. To maintain normal ploidy, the extra chromosomes derived from G2/M-stage cells could be expelled by oocytes as a second polar body. G2/M-stage fibroblast nuclei could direct reconstructed embryos to develop to the blastocyst stage.

The use of nuclear transfer (NT) techniques to create transgenic offspring capable of producing valuable proteins may have a major impact on the pharmaceutical market. Our objective was to compare the in vivo developmental potential of NT embryos produced from the fusion of transgenic donor cells with cytoplasts prepared from either FSH-stimulated ovaries or nonstimulated abattoir-derived ovaries. Donor cells were prepared from a transgenic fetus carrying the gene for human antithrombin III as a marker and used within four to eight subpassages. Cells were serum deprived for 4 days prior to cytoplast transfer. Oocytes were enucleated by removing the metaphase plate using a DNA stain and epifluorescent illumination. Donor cells were fused to enucleated oocytes by electric pulse and then chemically activated. There was no difference in the number of transferable embryos produced from cytoplasts of FSH-stimulated ovaries or from the fusion of cytoplasts from abattoir ovaries, nor was there a difference in the number of pregnancies established per recipient with either treatment. All pregnancies from both groups culminated in the births of healthy female kids (five total). To our knowledge, this is the first report of cloned goats produced from NT using cytoplasts derived from abattoir ovaries.

A set of exercises aimed at the discovery of basic structures and functions of living cells are described. The exercises are intended for university students and can be carried out in one intensive day. The students will learn that: the cell wall has a specific architecture of cellulose — the fibrillar component of the cell wall which is anisotropic in its structural organisation; Microtubules form distinct parallel arrays in the vicinity of the plasma membrane and this orientation is reflected in the architecture of the cell wall; The plasma membrane is intimately linked to the cell wall at hundreds of spots per cell; The endoplasmic reticulum forms a network close to the cell membrane. This becomes very clear at cell membrane/cell wall linkage sites during plasmolysis. Mitochondria move very actively in the cell, as do other small organelles, plastids, and Golgi bodies. This movement is based on the presence of an elaborate actin filament network, and involves the activity of myosin-type molecular motor...

Successful nuclear transfer (NT) of somatic cell nuclei from various mammalian species to enucleated bovine oocytes provides a universal cytoplast for NT in endangered or extinct species. Buffalo fetal fibroblasts were isolated from a day 40 fetus and were synchronized in presumptive G(0) by serum deprivation. Buffalo and bovine oocytes from abattoir ovaries were matured in vitro and enucleated at 22 h. In the first experiment, we compared the ability of buffalo and bovine oocyte cytoplasm to support in vitro development of NT embryos produced by buffalo fetal fibroblasts as donor nuclei. There were no significant differences (p0.05) between the NT embryos derived from buffalo and bovine oocytes, in fusion (74% versus 71%) and cleavage (77% versus 75%) rates, respectively. No significant differences were also observed in blastocyst development (39% versus 33%) and the mean cell numbers of day 7 cloned blastocysts (88.5 +/- 25.7 versus 51.7 +/- 5.4). In the second experiment, we evaluated the effects of activation with calcium ionophore A23187 on development of NT embryos after electrical fusion. A significantly higher (p0.05) percentage of blastocyst development was observed in the NT embryos activated by calcium ionophore and 6-DMAP when compared with 6-DMAP alone (33% versus 17%). The results indicate that the somatic nuclei from buffalo can be reprogrammed after transfer to enucleated bovine oocytes, resulting in the production of cloned buffalo blastocysts similar to those transferred into buffalo oocytes. Calcium ionophore used in conjunction with 6-DMAP effectively induces NT embryo development.

Different factors are believed to influence the outcome of nuclear transfer (NT) experiments. Besides the cell cycle stage of both recipient cytoplast and donor karyoplast, the origin of the donor cells (embryonic, fetal, and adult) is of interest. We compared in vitro development of NT embryos derived from small serum-starved (G0) or small cycling (G1) porcine fetal fibroblast cells. Serum starvation did not have a positive effect on cleavage rate or the percentage of embryos that developed to the morula and blastocyst stages. Next, we investigated the development of porcine NT embryos derived from different transgenic clonal cell lines that had originated from the same fetus. When different clonal lines of fetal fibroblasts were fused to enucleated metaphase II oocytes, differences in fusion rates as well as in development to the morula and blastocyst stages were observed (P < 0.05). When oocytes derived from sow ovaries were used as recipient cytoplasts, significantly better cleavage (P = 0.03) and blastocyst formation (P < 0.014) was obtained when compared with oocytes derived from gilts. Our data indicate that not only different cell lines, but also different clones derived from one primary cell line, result in different development when used for NT. In addition, the use of sow oocytes as a cytoplast source also improves the efficiency of NT experiments.

The subcellular origin of ceramide signaling in ionizing radiation-triggered apoptosis was investigated using two previously described subclones of the autonomous erythro-myeloblastic cell line TF-1, radio-resistant and -sensitive TF-1-34 and TF-1-33, respectively. We show in nuclei-free lysates and cytoplasts that both cell lines failed to generate ceramide in response to ionizing radiation. Moreover, whereas cytoplasts did respond to anti-Fas stimulation through phosphatidylserine externalization, no effect was observed with ionizing radiation. Only in highly purified nuclei preparations did we observe ceramide generation, neutral sphingomyelinase activation, and apoptotic features (PARP cleavage, nuclear fragmentation, DNA laddering) in TF-1-33, but not in TF-1-34 cells. These observations suggest that nuclear sphingomyelinase and ceramide formation may contribute to ionizing radiation-triggered apoptosis.

This research was to study the in vitro and in vivo development of cloned embryos derived from adult rabbit fibroblasts following various activation protocols. Effects of serum starvation and passage number of donor cells on the efficiency of cloning were also examined. In experiment I, oocytes were activated either by electric pulses or by electric pulses followed by culture with 6-dimethylaminopurin (DMAP). For experiment II, the best activation protocol from experiment I was employed for cloning using adult rabbit fibroblasts that were cultured for 0-15 passages. In experiment III, the effect of serum starvation of the donor cells on cloning was examined. Finally, in experiment IV, embryo transfers were conducted. These experiments showed that combined electrical pulse and DMAP treatment resulted in superior parthenogenetic blastocyst development (up to 29%), and that activation of the cytoplast before versus after fusion was not different in supporting the in vitro development of nuclear transferred embryos (16%-18% blastocysts). Adult fibroblasts derived from nonpassaged cells were less capable of developing into blastocysts than passaged cells (6% vs. 17%). Serum starvation of donor cells improved cleavage (up to 71%) but did not improve blastocyst development (13%), and no progeny was obtained, irrespective of the treatment. Cell-cycle analysis of adult rabbit fibroblast cells showed that passage 6 and 12 cells were more likely to be in G(0)/G(1) than passage 0 cells, which agrees with the improved embryo development in the passaged-cell groups.

To determine the effect of maturational age of porcine oocytes on the induction of activation and development following nuclear transfer, we investigated maturation rate and efficacy of oocyte activation treatment with an electrical stimulus (ES) and cycloheximide (CHX) at various timing of maturation culture. Most oocytes developed to the metaphase II (MII) stage after 32 hr of maturation culture. Both in newly matured (32 and 36 hr of age) and MII-arrested (42 and 48 hr of age) oocytes, ES followed by exposure to CHX for 6 hr caused higher rates of pronuclear formation than ES alone. Effect of maturational age of oocytes on the development of parthenotes activated with ES and CHX was then examined. The highest percentage of parthenotes developed to blastocysts was obtained when ES was added at 42 hr of culture. Finally, newly matured (33 hr of age) and MII-arrested oocytes (44 hr of age) were enucleated, fused with serum-starved pig fetal fibroblasts and activated with ES and CHX around suitable timing (39 to 40 hr of age) or later (50 to 51 hr of age), respectively. The frequencies of cleavage (58.8%) and blastocyst formation (13.6%) of newly matured oocytes were significantly (P

A series of experiments were designed to evaluate the meiotic competence of mouse oocyte germinal vesicle (GV) in rabbit ooplasm. In experiment 1, an isolated mouse GV was transferred into rabbit GV-stage cytoplast by electrofusion. It was shown that 71.8% and 63.3% of the reconstructed oocytes completed the first meiosis as indicated by the first polar body (PB1) emission when cultured in M199 and M199 + PMSG, respectively. Chromosomal analysis showed that 75% of matured oocytes contained the normal 20 mouse chromosomes. When mouse spermatozoa were microinjected into the cytoplasm of oocytes matured in M199 + PMSG and M199, as many as 59.4% and 48% finished the second meiosis as revealed by the second polar body (PB2) emission and a few fertilized eggs developed to the eight-cell stage. In experiment 2, a mouse GV was transferred into rabbit MII-stage cytoplast. Only 13.0–14.3% of the reconstructed oocytes underwent germinal vesicle breakdown (GVBD) and none proceeded past the MI stage. When two mouse GVs were transferred into an enucleated rabbit oocyte, only 8.7% went through GVBD. In experiment 3, a whole zona-free mouse GV oocyte was fused with a rabbit MII cytoplast. The GVBD rates were increased to 51.2% and 49.4% when cultured in M199 + PMSG and M199, respectively, but none reached the MII stage. In experiment 4, a mouse GV was transferred into a partial cytoplasm-removed rabbit MII oocyte in which the second meiotic apparatus was still present. GVBD occurred in nearly all the reconstructed oocytes when one or two GVs were transferred and two or three metaphase plates were observed in ooplasm after culturing in M199 + PMSG for 8 hr. These data suggest that cytoplasmic factors regulating the progression of the first and the second meioses are not species-specific in mammalian oocytes and that these factors are located in the meiotic apparatus and/or its surrounding cytoplasm at MII stage. J. Exp. Zool. 289:322–329, 2001. © 2001 Wiley-Liss, Inc.

To enable both the multiplication of elite livestock and the engineering of transgenic animals for various agricultural and biochemical purposes, scientists around the world are intensively studying efficient ways of improving developmental competency of bovine embryos reconstructed by somatic cell nuclear transfer. Because it is widely accepted that culture conditions along with many other factors contribute to the developmental competency of reconstructed embryos, this preliminary study was designed to test whether or not bovine reconstructed embryos could develop in vitro using a simple portable CO(2) incubator. CO(2) and O(2) gas tensions and air pressure can be varied using this system. The parameters used in the five conducted trials were low CO(2) (2-5%) and O(2) (8-10%) gas tensions, and negative air pressure (of 300 mm Hg). Chamber temperature was maintained at 38.5 degrees C. Bovine fetal fibroblasts were used as donor karyoplasts and were fused into microsurgically enucleated M II oocytes followed by activation and culture. From the 250 enucleated oocytes, 217 (86.8%) fused, 183 (73.2%) cleaved, and 43 (17.2%) developed to the blastocyst stage. While relatively low developmental rates were achieved, technical proficiency may have been a contributing factor. Further studies using this system are needed to determine optimal levels of O(2), CO(2), and air pressure.

We report a method for introducing mtDNA mutations into the mouse female germ line by means of embryonic stem (ES) cell cybrids. Mitochondria were recovered from the brain of a NZB mouse by fusion of synaptosomes to a mtDNA-deficient (ρ°) cell line. These cybrids were enucleated and the cytoplasts were electrofused to rhodamine-6G (R-6G)-treated female ES cells. The resulting ES cell cybrids permitted transmission of the NZB mtDNAs through the mouse maternal lineage for three generations. Similarly, mtDNAs from a partially respiratory-deficient chloramphenicol-resistant (CAP R ) cell line also were introduced into female chimeric mice and were transmitted to the progeny. CAP R chimeric mice developed a variety of ocular abnormalities, including congenital cataracts, decreased retinal function, and hamaratomas of the optic nerve. The germ-line transmission of the CAP R mutation resulted in animals with growth retardation, myopathy, dilated cardiomyopathy, and perinatal or in utero lethality. Skeletal and heart muscle mitochondria of the CAP R mice were enlarged and atypical with inclusions. This mouse ES cell-cybrid approach now provides the means to generate a wide variety of mouse models of mitochondrial disease.

This study was designed to examine the developmental ability of porcine embryos after somatic cell nuclear transfer. Porcine fibroblasts were isolated from fetuses at Day 40 of gestation. In vitro-matured porcine oocytes were enucleated and electrically fused with somatic cells. The reconstructed eggs were activated using electrical stimulus and cultured in vitro for 6 days. Nuclear-transferred (NT) embryos activated at a field strength of 120 V/mm (11.6 ± 1.6%) showed a higher developmental rate as compared to the 150-V/mm group (6.5 ± 2.3%) (P 24.6 ± 10.1 > 16.5 ± 7.4 per blastocyst) as exposure time (2, 4, and 6 h) of nuclei to oocyte cytoplast before activation was prolonged. There was a significant difference in the cell number between the 2- and 6-h groups (P < 0.05). Nuclear-transferred embryos (9.4 ± 0.9%) had a lower developmental rate than in vitro fertilization (IVF)-derived (21.4 ± 1.9%) or parthenogenetic embryos (22.4 ± 7.2%) (P < 0.01). The mean cell number (28.9 ± 11.4) of NT-derived blastocysts was smaller than that (38.6 ± 10.4) of IVF-derived blastocysts (P < 0.05) and was similar to that (29.9 ± 12.1) of parthenogenetic embryos. Our results suggest that porcine NT eggs using somatic cells after electrical activation have developmental potential to the blastocyst stage, although with smaller cell numbers compared to IVF embryos.

Since the first report of live mammals produced by nuclear transfer from a cultured differentiated cell population in 1995 (ref. 1), successful development has been obtained in sheep, cattle, mice and goats using a variety of somatic cell types as nuclear donors. The methodology used for embryo reconstruction in each of these species is essentially similar: diploid donor nuclei have been transplanted into enucleated MII oocytes that are activated on, or after transfer. In sheep and goat pre-activated oocytes have also proved successful as cytoplast recipients. The reconstructed embryos are then cultured and selected embryos transferred to surrogate recipients for development to term. In pigs, nuclear transfer has been significantly less successful; a single piglet was reported after transfer of a blastomere nucleus from a four-cell embryo to an enucleated oocyte; however, no live offspring were obtained in studies using somatic cells such as diploid or mitotic fetal fibroblasts as nuclear donors. The development of embryos reconstructed by nuclear transfer is dependent upon a range of factors. Here we investigate some of these factors and report the successful production of cloned piglets from a cultured adult somatic cell population using a new nuclear transfer procedure.

Apoptosis manifests in two major execution programs downstream of the death signal: the caspase pathway and organelle dysfunction. An important antiapoptosis factor, Bcl-2 protein, contributes in caspase pathway of apoptosis. Calcium, an important intracellular signal element in cells, is also observed to have changes during apoptosis, which maybe affected by Bcl-2 protein. We have previously reported that in Harringtonine (HT) induced apoptosis of HL-60 cells, there's a change of intracellular calcium distribution, moving from cytoplast especially Golgi's apparatus to nucleus and accumulating there with the highest concentration. We report here that caspase-3 becomes activated in HT-induced apoptosis of HL-60 cells, which can be inhibited by overexpression of Bcl-2 protein. No sign of apoptosis or intracellular calcium movement from Golgi's apparatus to nucleus in HL-60 cells overexpressing Bcl-2 or treated with Ac-DEVD-CHO, a specific inhibitor of caspase-3. The results indicate that activated caspase-3 can promote the movement of intracellular calcium from Golgi's apparatus to nucleus, and the process is inhibited by Ac-DEVD-CHO (inhibitor of caspase-3), and that Bcl-2 can inhibit the movement and accumulation of intracellular calcium in nucleus through its inhibition on caspase-3. Calcium relocalization in apoptosis seems to be irreversible, which is different from the intracellular calcium changes caused by growth factor.

Eggs must be the major locus of reproductive aging in women, because donation of eggs from younger to middle-aged women abrogates the effects of age on fertility. Oxidative stress, mitochondrial dysfunction, and apoptosis are associated with senescence. To develop an animal model of egg senescence, we treated mouse zygotes with 175 microM H(2)O(2) that induced mitochondrial dysfunction and developmental arrest, followed by delayed cell death, consistent with apoptosis. We reconstructed zygotes with nuclei and cytoplasm from treated or untreated zygotes, then followed development and apoptotic cell death in the reconstituted embryos. Pronuclear exchange between untreated, normal zygotes served as nuclear transfer controls. Rates of cleavage and development to morula and blastocysts were significantly lower (P

Cytoplasts from cotton ( Gossypium hirsutum L.) fiber cells retain microtubule and microfilament cytoskeletons through extraction with non-ionic detergent and ethylene glycol bis-(β-aminoethyl ether) N,N,N',N'-tetraacetic acid. Tubulin and actin are the most abundant proteins in extracted cytoplasts; however, many other less abundant proteins are also present. To determine if minor proteins were associated with the cytoskeleton, microtubules and microfilaments were selectively removed from extracted cytoplasts by detergent extraction in an alkaline Ca 2+ solution. Under these extraction conditions, microtubules and microfilaments were fragmented and depolymerized unless previously stabilized by taxol and phalloidin. Associated proteins were identified by their loss in conjunction with either microtubules or microfilaments. As judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, one protein, of roughly 115 kDa, appeared to be associated with microfilaments since it was present in Ca 2+ -extracted preparations only when microfilaments were stabilized with phalloidin. The failure of most minor proteins to associate with microtubules and microfilaments suggests that caution must be used when interpreting co-isolation as evidence for an association of low abundance proteins with cytoskeletons.

The success of somatic nuclear transfer critically depends on the cell cycle stage of the donor nucleus and the recipient cytoplast. In this study we tested serum deprivation as well as two reversible cell cycle inhibitors, aphidicolin and butyrolactone I, for their ability to synchronize porcine fetal fibroblasts at either G0 stage or G1/S or G2/M transition. The synchronization efficiency of the various protocols was determined by fluorescence-activated cell sorting (FACS), cell proliferation assays, and semiquantitative multiplex reverse transcription-polymerase chain reaction detection of the cell cycle-regulated porcine Polo-like kinase mRNA (Plk-p). FACS measurements revealed that 66.6-73.3% of the porcine fetal fibroblasts were in G0/G1 stage (2C DNA content) in serum-supplemented medium. Short periods of 24-72 h of serum deprivation significantly increased the proportion of cells at G0/G1 phase to 77.9-80.2%, and mitotic activity had already terminated after 48 h. Prolonged culture in serum-deprived medium induced massive DNA fragmentation. Aphidicolin treatment led to an accumulation of 81.9 +/- 4.9% of cells at the G1/S transition. Butyrolactone I arrested 81.0 +/- 5.8% of the cells at the end of G1 stage and 37.0 +/- 6.8% at the G2/M transition. The effects of both chemical inhibitors were fully reversible, and their removal led to a rapid progression in the cell cycle. The measurement of Plk-p expression allowed discrimination between the presumptive G0 phase induced by serum deprivation and the G1/S transition arrest achieved by chemical inhibitors. These data indicate that porcine fetal fibroblasts can be effectively synchronized at various cell cycle stages without compromising their proliferation capacity.

Abnormal oocyte spindle due to the improper function of ooplasm is associated with female infertility of advanced maternal age. A possible way to overcome this problem is to transfer an oocyte germinal vesicle (GV) which contains genetic materials of a patient with a history of poor embryo development to the cytoplast from a donor oocyte. Here we demonstrate that GV transfer is feasible using a rabbit model. When the GVs were transferred to auto- or hetero-cytoplasts of GV stage oocytes, around 80% of the reconstructed oocytes could mature in vitro and 7.1-9.4% of the oocytes developed to blastocyst stage after intracytoplasmic sperm injection (ICSI). Transfer of 93 fertilized eggs reconstructed via GV transfer into six recipients resulted in two live offspring. Results of this experiment indicate that GV transfer can potentially become a new approach in treatment of infertility because of advanced maternal age.

The cloning of mammals originated with the production of limited numbers of genetically identical offspring by blastomere separation or embryo splitting. In the past few years, remarkable progress has been reported in cloning by nuclear transfer (NT) with donor nuclei recovered from embryonic, fetal or adult cells. Factors that contribute to the successful reprogramming of the transferred nucleus and the normal term development of the newly reconstructed embryo include the cell cycle stage of both the donor nucleus and recipient cytoplast, the timing of fusion and cytoplast activation, and the source of donor nuclei. The possibility of producing live offspring by somatic cell NT carries potential applications in animal husbandry, biotechnology, transgenic and pharmaceutical production, biomedical research, and the preservation of endangered species. However, the low efficiencies of cloning by NT coupled with high embryonic, fetal and neonatal losses may restrict immediate commercial applications in agriculture. These limitations notwithstanding, the greatest benefits and practical implications of this new technology could be in transplantation medicine and therapeutic cloning.

Mitochondria are endosymbiotic organelles responsible for energy production in practically every eukaryotic cell. Their uniparental fashion of inheritance, maternally inherited in mammals, and the homogeneity of mitochondrial DNA (mtDNA) within individuals and matrilineages, are biological phenomena that remain unexplained. This paper reviews some of the recent findings on mitochondrial influences on the manner in which embryos develop and how their genotypes are inherited in mammals, with particular emphasis on the genetic “bottleneck” effect. Animal models carrying a mix of mtDNAs (heteroplasmic) have been produced by karyoplast and cytoplast transplantation to analyze the segregation patterns at different stages during embryogenesis, in fetuses and offspring. Comparisons performed between murine and bovine reveal interesting changes in segregation and replication of transplanted mtDNAs. We have recently obtained Bos indicus and Bos taurus fetuses and calves from embryos reconstructed using enucleated polymorphic oocytes of Bos taurus origin. These and other findings on mitochondrial biology will have important implications in determining the cytoplasmic genotype of clones and in the preservation of endangered breeds and species.

The utility of cryopreserved bovine oocytes as recipient cytoplasts for nuclear transfer (NT) was examined. In vitro-matured (IVM), metaphase-II oocytes were enucleated by mechanical suction and activated parthenogenetically. The cytoplasts were fused with blastomeres of in vitro-produced day-5 morulae by a DC electropulse, and then cultured up to 8 days (non-frozen controls; group I). Oocytes were frozen-thawed in 1.5-M ethylene glycol and 0.1-M sucrose before enucleation (group II), after enucleation (group III), after enucleation and aging culture (group IV), or after activation (group V). In group I, 91% of IVM oocytes could be used for NT and 89% of them fused successfully. Finally, 36% of the fused zygotes developed into blastocysts. The proportions of morphologically normal oocytes after thawing in groups IV and V (70 and 69%, respectively) were higher than in group III (56%), and the proportion of IVM oocytes used for NT in group IV (56%) was higher than those in groups II, III, and V (33%, 35%, and 38%, respectively). Fusion rates of the NT zygotes in groups III, IV, and V (90%, 88%, and 88%, respectively) were higher than the rate in group II (75%). Rates of development into blastocysts of the fused zygotes in groups II, III, IV, and V were 0%, 3%, 2%, and 6%, respectively (P < 0.05, group II vs. groups III, IV, and V). Developmental kinetics and cell numbers of the blastocysts were similar among the groups. It was suggested that timing of oocyte cryopreservation is among the factors influencing efficiency of production of cloned embryos in cattle.

We evaluated the maturational competence of mouse oocytes reconstructed by the transfer and electrofusion of germinal vesicles (GV) into anuclear cytoplasts of GV stage oocytes (both auto- and hetero-transfers), metaphase II stage oocytes or zygotes. Following in-vitro culture, the maturation rates of the reconstructed oocytes to metaphase II did not significantly differ between auto- and hetero-transfers (40/70 versus 95/144 respectively); these rates also did not differ from those of control oocytes (57/97) which were matured in vitro without micromanipulation and electrofusion. In contrast, when a GV was transferred into an enucleated metaphase II oocyte or a zygote, only a few reconstructed oocytes underwent germinal vesicle breakdown (5/30 and 2/21 respectively); moreover, none reached metaphase II stage. Cytogenetic and immunofluorescence analyses were conducted on hetero-GV oocytes that extruded a first polar body. Each oocyte showed two groups of chromosomes, one in the cytoplast and one in the polar body, as well as a bipolar spindle with twenty univalent chromosomes. Our findings suggest that oocytes reconstructed by GV transfer into a cytoplast of the same developmental stage mature normally in vitro through metaphase II. Such oocytes may be a useful research model to elucidate the cytoplasmic and nuclear mechanisms regulating meiosis and the relationships between meiotic errors and age-related changes in the oocyte.

Although technically established in mammalian species more than 15 year ago, the efficiency of embryo cloning by nuclear transfer in terms of offspring production has been invariably low. This article reviews a series of interrelated experiments carried out from 1992 to 1997 regarding the co-ordination of nuclear and cytoplasmic events after embryo reconstruction. The experiments were undertaken at the Istituto Zootecnico e Caseario per la Sardegna (IZCS) and the sheep used belonged to the Sardinian dairy breed. The use of enucleated, pre-activated recipient cytoplasts resulted in an increased frequency of development to blastocyst of embryos reconstructed with unsynchronized blastomere nuclei. This increase in development was due to the absence of chromatin damage and unbalanced ploidy in nuclei transferred after the decay of MPF (Maturation Promoting Factor). The combination of synchronous S-phase cytoplast-karyoplast nuclear transfer together with the reduction of embryo losses from the oviduct of temporary recipients allowed for the first time the production of large numbers of clones of genetically identical lambs, thus bringing nuclear transfer closer to practical application. Finally, the last series of experiments described deals with the establishment of an alternative protocol for embryonic nuclear transfer where the combined use of a chemical activating agent, ionomycin, with a protein kinase inhibitor, 6-Dimethylaminopurine, resulted in the highest development rates to blastocyst stage of metaphase II enucleated oocytes reconstructed with embryonic nuclei described so far.

The enucleation of recipient oocytes in nuclear transfer experiments is generally carried out by aspirating one third of the ooplasm adjacent to the first polar body. It was supposed that this enucleation step affects the ultrastructure of the remaining cytoplast, resulting in a decline or destruction of its cellular compartments. Even if the transferred nucleus had the potential to support the development of a single-cell nucleus transfer embryo to the blastocyst stage, meiotic division could be stopped at any stage if the destruction of the ultrastructure of host cytoplasm resulted in a limited metabolism. The present study was conducted to investigate the influence of the enucleation procedure on the ultrastructure of the remaining ooplast. In vitro matured oocytes; in vitro matured and enucleated oocytes; and in vitro matured and enucleated oocytes that were subsequently cultivated in vitro for additional 4 h were prepared for transmission electron microscopy (TEM). An examination of ultra-thin sections showed that the arrangement of organelles in all matured oocytes was in accordance with that already described for normal oocyte development. Immediately after enucleation no major differences in the arrangement of cortical granules, mitochondria, smooth endoplasmic reticulum (SER), lipid droplets and vacuoles were found compared with nonmanipulated oocytes. After enucleation and 4 h of culture, 24- and 36-h matured oocytes differed from each other in the arrangement of large aggregates of SER surrounded by a wall of mitochondria and lipid droplets. These complexes were still found in the 24-h but not in 36-h matured, enucleated and cultivated oocytes. Clusters of SER, mitochondria and lipid droplets were described by different authors as having metabolic activity. The results of this study in connection with results from nuclear transfer experiments suggest that these aggregates and their metabolic activity can be transferred with cytoplasm from 24- but not 36-h matured oocytes. Only cytoplasm from the 24-h matured oocytes showed a development-supporting effect when fused to enucleated recipient cells before nuclear transfer.

Cell-cycle phase of the donor and recipient cells at the moment of nuclear transfer influences subsequent development of the reconstituted embryo. In order to study this effect, the precise cell-cycle phase of the recipient oocyte at the time of fusion must be known and this depends on reliable activation of oocytes in a protocol that has a low incidence of spontaneous activation. Mouse oocytes recovered before (8-10 hours post-human chorionic gonadotropin [hCG]) and after ovulation (14 and 18 hours post-hCG) were exposed to strontium ions in calcium magnesium-free M16 culture medium. The effect on development of haploid parthenotes of post-hCG age of the oocyte, the duration of exposure, and strontium concentration in the medium was determined. These experiments established a reliable method of parthogenetic activation of recently ovulated mouse oocytes, involving the culture of oocytes for 60 minutes in 25 mM strontium in a calcium magnesium-free M16 medium. This method of activation was also able to induce activation of preovulatory oocytes after a preincubation period in vitro. Only a low incidence of spontaneous activation was observed if oocytes were recovered before or immediately after ovulation (14 hours after hCG).

The technology of cloning involves transplanting a diploid nucleus into a mature oocyte cytoplast. The cytoplast is then activated to initiate the first cell cycle of development as a nuclear transplant embryo. Initiation and regulation of events during the first cell cycle are, therefore, critical for proper reprogramming of the donor nucleus and development as a cloned embryo. Activation is normally induced by the sperm and is mediated by a series of intracellular free calcium ([Ca(2+)](i)) oscillations that last for several hours. Although it is not known precisely how the sperm induces activation, current evidence favors the delivery, by the sperm, of a soluble protein factor that causes the production of IP3. IP3 acts to open a Ca(2+) channel in the endoplasmic reticulum and release Ca(2+) into the cytosol. A variety of methods have been used to duplicate or replace the sperm-induced [Ca(2+)](i) increase to cause activation in nuclear transplant embryos. It has been found that treatments that cause a single transient [Ca(2+)](i) activate some oocytes with the level of activation increasing as the oocyte ages. Attempts have been made to extend the period of time over which [Ca(2+)](i) oscillations occur. This has been successful in increasing activation rates of less mature oocytes but the techniques are still cumbersome. An alternative method, that has been very successful, is the combination of a treatment that elevates [Ca(2+)](i) and a treatment that maintains low levels of maturation promoting factor for several hours after the initial [Ca(2+)](i) elevation. The sperm also contributes the centrosome that organizes microtubules during the first cell cycle. One current hypothesis for regulation of sperm centrosomal activity consists of a dephosphorylation of sperm connecting piece proteins following sperm entry into the oocyte and activation of the oocyte. Dephosphorylation of these proteins results in the disassembly of the connecting piece and assembly of a functional centrosome. In nuclear transfer, centrosomal components are contributed by the donor cell. If the cell is fused to the cytoplast before centriole replication then a single aster forms. If the cell is fused after centriole replication then two asters form. In either case and even in parthenogenetic oocytes, which do not have centrioles, the first cell cycle progresses to metaphase. However, progress is slow and some defects are observed in the assembly of chromosomes into a metaphase plate.

Cloning mammalian species from cell lines of adult animals has been demonstrated. Aside from its importance for cloning multiple copies of genetically valuable livestock, cloning now has the potential to salvage endangered or even extinct species. The aim of this study was to investigate the effect of the bovine and domestic (Ovis aries) ovine oocyte cytoplasm on the nucleus of an established cell line from an endangered argali wild sheep (Ovis ammon) after nuclear transplantation. A fibroblast cell line was established from skin biopsies from an adult argali ram from the People's Republic of China. Early karyotype analysis of cells between 3-6 passages revealed a normal diploid chromosome number of 56. The argali karyotype consisted of 2 pairs of biarmed and 25 pairs of acrocentric autosomes, a large acrocentric and minute biarmed Y. Bovine ovaries were collected from a local abattoir, oocytes aspirated, and immediately placed in maturation medium consisting of M-199 containing 10% fetal bovine serum, 100 IU/mL penicillin, 100 microg/mL streptomycin, 0.5 microg/mL follicle-stimulating hormone (FSH), 5.0 microg/mL luetinizing hormone (LH) and 1.0 microg/mL estradiol. Ovine (O. aries) oocytes were collected at surgery 25 hours postonset of estrus from the oviducts of superovulated donor animals. All cultures were carried out at 39 degrees C in a humidified atmosphere of 5% CO2 and air. In vitro matured MII bovine oocytes were enucleated 16-20 hours after onset of maturation and ovine oocytes within 2-3 hours after collection. Enucleation was confirmed using Hoechst 33342 and UV light. The donor argali cells were synchronized in G0-G1 phase by culturing in Dulbecco's modified Eagle's medium (DMEM) plus 0.5% fetal bovine serum for 5-10 days. Fusion of nuclear donor cell to an enucleated oocyte (cytoplast) to produce nuclear transfer (NT) embryos was induced by 2 electric pulses of 1.4 kV/cm for 30 microsc. Fused NT embryos were activated after 24 hours of maturation by exposure to ionomycin (5 microM, 4 minutes) followed by incubation in 6-dimethylaminopurine (0.2 mM, 4 hours) and cultured in microdrops of CR1aa medium. From a total of 166 constructed nuclear donor cell-bovine cytoplasm NT couples, 128 (77%) successfully fused, 100 (78%) developed to 8-16 cell stage, and 2 (1.56%) developed to the blastocyst stage. The presence of argali nuclei in 8-16 cell stage embryo clones was confirmed after observation of Hoechst 33342 stained embryos under UV light and chromosome analysis of metaphase spreads from blastomeres. A total of 127 constructed nuclear donor cell-ovine cytoplasm NT couples were produced, 101 (80%) successfully fused, 81 (80% of fused) developed to the 16- to 32-cell stage. A total of 28 hybrid (argali-sheep) and 21 sheep-sheep NT embryos were transferred into 6 recipients and 4 recipients, respectively. Two of these recipients, 1 carrying argali-sheep and 1 sheep-sheep, were confirmed pregnant at 49 days by ultrasound, but both pregnancies terminated by 59 days. The results of this study demonstrate the possibility of using xenogenic oocytes to produce early-stage embryos and pregnancies from an established fibroblast cell line of an endangered species.

In early 1997, the birth of a lamb after transfer of the nucleus from an adult mammary gland cell into an enucleated oocyte, along with the production of rhesus monkeys by nuclear transfer of embryonic cells, marked a reemergence of the field of mammalian cloning. Clonally derived rhesus monkeys would be invaluable in biomedical research, and the commercial interests in transgenic sheep and cattle propagated by cloning are substantial. Nuclear transfer technology is under consideration in human in vitro fertilization clinics to overcome infertility secondary to advanced maternal age or mitochondrial-based genetic disease. Nuclear transfer involves preparing a cytoplast as a recipient cell, in most cases a mature metaphase II oocyte from which the chromosomes have been removed. A donor nucleus cell is then placed between the zona and the cytoplast, and fusion, as well as cytoplast activation, is initiated by electrical stimulation. Successful reprogramming of the donor cell nucleus by the cytoplast is critical--a step that may be influenced by cell cycle stage. Embryos produced by nuclear transfer are cultured in vitro for several cell divisions before cryopreservation or transfer to the oviduct or uterus of a host mother. The efficiency of producing live young by nuclear transfer in domestic species is low, with a high frequency of developmental abnormalities in both preterm and term animals. However, a number of pregnancies have now been established using fetal cells as the source of donor nuclei. The use of cell lines not only allows large clone sizes but also supports the ability to genetically manipulate cells in vitro before nuclear transfer. Ongoing research focused on the production of clonally derived rhesus monkeys using fetal fibroblasts and embryonic stem cells as the source of donor nuclei will be reviewed.

In many animal cells, minus ends of microtubules (MTs) are thought to be capped by the centrosome whereas plus ends are free and display dynamic instability. We tested the role of the centrosome by examining MT behavior in cytoplasts from which the centrosome was removed. Cells were injected with Cy3–tubulin to fluorescently label MTs and were enucleated by using a centrifugation procedure. Enucleation resulted in a mixture of cytoplasts containing or lacking the centrosome. Fibroblast (CHO-K1) and epithelial (BSC-1) cells were investigated. In fibroblast cytoplasts containing the centrosome, MTs showed dynamic instability indistinguishable from that in intact cells. In contrast, in cytoplasts lacking the centrosome, MTs treadmilled—shortened at the minus end at about 12 μm/min while growing at the plus end at the same rate. The change in behavior of the plus end from dynamic instability to persistent growth correlated with an elevated level of free tubulin subunits (78% in centrosome-free cytoplasts vs. 44% in intact cells) generated by minus-end depolymerization. In contrast to fibroblast cells, in centrosome-free cytoplasts prepared from epithelial cells, MTs displayed dynamic instability at plus ends and relative stability at minus ends presumably because of specific minus-end stability factors distributed throughout the cytoplasm. We suggest that, in fibroblast cells, a minus-end depolymerization mechanism functions to eliminate errors in MT organization and that dynamic instability of MT plus ends is a result of capping of minus ends by the centrosome.

The activation of M-phase promoting factor (MPF) in one-cell mouse embryo is independent from the nucleus. Other autonomous phenomena include the cortical activity observed at the end of the first cell cycle and the reorganization of the microtubule network. Here, we observed that the autonomous control of MPF activation is present also in two-cell mouse embryos (H1 kinase activity being higher in the first than in the second cell cycle). Moreover, the disappearance of the cortical activity in anucleated halves is observed when MPF activation takes place. The rounding up of the cytoplast and the mitotic reorganization of the microtubule network correlates with the maximum activity of H1 kinase in anucleated halves from one-cell embryos. In anucleated halves of two-cell stage blastomeres neither the cortical activity nor the microtubule reorganization were observed. The degree of activation of histone H1 kinase, and, as a consequence, the cortical activity and the microtubule reorganization, does not depend on the distribution of cyclin B. Finally, the level of cyclin B synthesis is similar in anucleated and nucleated halves derived from both one-and two-cell embryos.

Cryopreservation of bovine oocytes would be beneficial both for nuclear transfer and for preservation efforts. The overall objective of this study was to evaluate the viability as well as the cryodamage to the nucleus vs. cytoplasm of bovine oocytes following freezing-thawing of oocytes at immature (GV) and matured (MII) stages using in vitro fertilization (IVF), parthenogenetic activation, or nuclear transfer assays. Oocytes were collected from slaughterhouse ovaries. Oocytes at the GV, MII, or MII but enucleated (MIIe) stages were cryopreserved in 5% (v/v) ethylene glycol; 6% (v/v) 1,2-propanediol; and 0.1-M sucrose in PBS supplemented with 20% (v/v) fetal bovine serum. Frozen-thawed oocytes were subjected to IVF, parthenogenetic activation, or nuclear transfer assays. Significantly fewer GV oocytes survived (i.e., remained morphologically intact during freezing-thawing) than did MII oocytes (47% vs. 84%). Subsequent development of the surviving frozen-thawed GV and MII oocytes was not different (58% and 60% cleavage development; 7% and 12% blastocyst development at Day 9, respectively, P > 0.05). Parthenogenetic activation of frozen-thawed oocytes resulted in significantly lower rates of blastocyst development for the GV than the MII oocyte groups (1% vs. 14%). Nuclear transfer with cytoplasts derived from frozen-thawed GV, MII, MIIe, and fresh-MII control oocytes resulted in 5%, 16%, 14%, and 17% blastocyst development, respectively. However, results of preliminary embryo transfer trials showed that fewer pregnancies were produced from cloned embryos derived from frozen oocytes or cytoplasts (9%, n = 11 embryos) than from fresh ones (19%, n = 21 embryos). Transfer of embryos derived by IVF from cryopreserved GV and MII oocytes also resulted in term development of calves. Our results showed that both GV and MII oocytes could survive freezing and were capable of developing into offspring following IVF or nuclear transfer. However, blastocyst development of frozen-thawed oocytes remains poorer than that of fresh oocytes, and our nuclear transfer assay suggests that this poorer development was likely caused by cryodamage to the oocyte cytoplasm as well as to the nucleus.

A clone can be defined as a set of genetically identical animals. Small clones of two or occasionally up to four identical animals can be obtained by embryo splitting or blastomere separation. Embryo cloning by nuclear transfer involves the transfer of genetic material from a donor cell (karyoplast) to the cytoplasm of an oocyte or zygote from which the genetic material has been removed (cytoplast). In farm animals, metaphase II oocytes are most widely used as cytoplasts. There are now many factors known to influence the efficiency of embryo cloning by nuclear transfer. These include stage of development and cell cycle of donor cells, the choice of the recipient cell, the methods for activation of oocytes, the cell cycle coordination between donor cell and recipient cytoplast, and the method for fusion between nuclear donor and recipient cytoplast. Recent progress in cloning embryos and animals from cultured cells of embryonic, fetal, or adult origin offers a wide spectrum of potential applications of nuclear transfer, such as the unlimited multiplication of elite embryos or animals from selected matings and the potential for precise genetic modification of farm animals for gene farming or xenotransplantation.

Over the last 25 yr, success in characterizing the individual protein components of animal cytoskeletons was possible, in part, due to technical advances in the isolation and purification of anucleate cytoskeletons from animal cells. As a step towards characterizing protein components of the plant cytoskeleton, we have isolated cytoskeletons from cytoplasts (anucleate protoplasts) prepared from cotton fiber cells grown in ovule culture. Cytoplasts isolated into a hypertonic, Ca2+-free medium at pH 6.8 retained internal structures after extraction with the detergent, Triton X-100. These structures were shown to include microtubule and microfilament arrays by immunofluorescence and electron microscopy. Actin and tubulin were the only abundant proteins in these preparations, suggesting that microfilaments and microtubules were the major cytoskeleta elements in the isolated cytoskeletons. The absence of additional, relatively abundant proteins suggests that (a) other cytoskeletal arrays potentially present in fiber cells (e.g., intermediate filaments) were either lost during detergent extraction or were minor components of the fiber cell cytoskeleton; and (b) high ratios of individual cytoskeletal-associated proteins relative to actin and tubulin were not required to maintain microtubules and microfilaments in organized structures.

It has been demonstrated previously that removal of acellular debris from the preimplantation mouse embryo is beneficial for subsequent development to the hatched blastocyst stage. We have studied the impact of cellular fragmentation induced in the mouse embryo during the late pronuclei and 8-cell stages on the hatching frequency and total cell number at the blastocyst stage. At the late pronuclei stage about one-quarter of the cytoplasm was removed from embryos in the experimental group, in four to six steps, thus creating four to six cytoplasts that were subsequently returned as anucleated fragments under the zona pellucida. Embryos with one-quarter of the cytoplasm removed and with intact cytoplasm after partial zona dissection (PZD) served as controls. At the 8-cell stage, embryos with their nucleoplast removed from two blastomeres served as an experimental group. Groups of embryos with part of the cytoplast removed from two blastomeres (nucleated fragments), embryos with two blastomeres removed and embryos after PZD alone served as controls. After manipulation all embryos were left in culture and analysed at about 100 h after human chorionic gonadotrophin administration. Fragments induced at the late pronuclei stage did not participate in compaction and were often spontaneously expelled from the embryo during hatching. Neither embryo hatching rate nor total cell number was affected when compared with zygotes with reduced cytoplasm. Although both nucleated and anucleated fragments induced at the 8-cell stage participated in recompaction, hatching was not compromised and there was no interference in further development as assessed by the cell number or hatching rate at the blastocyst stage, as compared with embryos with blastomeres removed. We conclude that anucleated cellular fragments formed in an otherwise healthy embryo, both before and after acquisition of the ability for compaction, are benign and that their removal provides no benefit for embryo development, at least to the hatched blastocyst stage.

The enucleation of oocytes to be used as host cytoplasts for embryo reconstruction by nuclear transfer is an important limiting step when cloning mammals. We propose an enucleation technique based on the removal of chromatin after oocyte activation, at the telophase stage, by aspirating the second polar body and surrounding cytoplasm. In a preliminary experiment to determine an optimal activation protocol, oocytes were matured for 26 and 30 hr and exposed for 5 min to 7% ethanol and/or for 3 hr at either 25 or 4 degrees C. Relative to most activation treatments tested, oocytes matured for 30 hr and exposed to ethanol alone showed highest activation rates, as determined by low levels of H1 kinase activity within 90 min from exposure and high pronuclear formation (82%) after 12 hr of culture. No synergistic effect on activation rates was observed when oocytes also were exposed to reduced temperature after ethanol treatment. Microsurgical removal of the telophase-stage chromatin in a small volume of cytoplasm adjacent to the second polar body was significantly more effective in enucleating than aspiration of a larger cytoplasm volume surrounding the first polar body of metaphase-arrested oocytes (98% versus 59%; P < 0.01). Moreover, compared with a nuclear transfer protocol based on enucleation of metaphase-arrested oocytes followed by aging and cooling, more (38% versus 16%; P < 0.001) and better-quality blastocytes (126 versus 84 nuclei per blastocyst; P < 0.02) were obtained from embryos reconstructed using the telophase procedure. Higher development potential of embryos reconstructed by the telophase procedure may be attributed to (1) the selection of oocytes that activate and respond by extruding the second polar body, (2) avoiding the use of DNA dyes and ultraviolet irradiation, and (3) the limited removal of cytoplasm during enucleation. The ease with which telophase enucleation can be performed is likely to render this technique widely useful for research and practice on mammalian cloning.

On immunoblots of human neutrophil cytoplasts (U-CYT), a previously undescribed 97kDa protein was revealed by intense and selective reaction with an antibody that was initially raised to recognize PKC-γ. Denoted “γ-rp” for gamma-related protein, this acidic cytosolic protein somewhat resembled the classic forms of PKC in several biochemical respects. Appearing as a doublet on low-percentage SDS-PAGE gels, both its mobility and staining pattern were rapidly altered by treatment of U-CYT with either phorbol ester or chemotactic peptide. Whole neutrophil γ-rp was detectable only after TCA precipitation of intact cells. It was also detectable in human platelets, lymphocytes, and neutrophil-like differentiated HL60 cells, but not in fibroblasts, erythrocytes, monocytes, or monocyte-like differentiated HL60 cells. Our data suggest that γ-rp merits further study as a potential participant in cellular activation, and as a possible structural or functional relative of PKC.

Neutrophils utilize Fcγ Receptors (FcγR) to bind and internalize antibody coated cells or immune complexes. We have compared ultrastructurally FcγR-mediated phagocytosis by neutrophil cytoplasts (enucleated and granule-free neutrophils) prepared either by treatment with cytochalasin B (CB-cytoplasts) followed by ultracentrifugation or by brief heating at 45°C in suspension followed by ultracentrifugation. The phagocytosis of antibody-coated erythrocytes (EIgG) or the internalization of crosslinked IgG complexes by cytoplasts prepared by brief heating was comparable to that of untreated neutrophils. In contrast, CB-cytoplasts bound but failed to internalize EIgG or crosslinked IgG complexes. Comparison of these two methods for the preparation of neutrophil cytoplasts may assist in clarifying the signal transduction requirements involved following ligand binding to FcγR which initiate phagocytosis.

The objective of the study was to characterize the ultrastructure changes and biochemical mechanisms underlying the expulsion of the entire chromosome complement in chemically enucleated mouse oocytes. The ultrastructural studies demonstrated that the morphology of cytoplasts produced by etoposide-cycloheximide treatment were indistinguishable from intact metaphase I and II oocytes. Moreover, polar bodies formed by chemical enucleation were in almost all cases completely separated from the parent cytoplast and differed from normal polar bodies only in their chromatin content morphology and because they contained a slightly higher number of cytoplasmic organelles. The mode of polar body formation, however, in normal and chemically enucleated oocytes differs substantially: spindle involvement is important for normal polar body extrusion but plays no part in the protracted expulsion of chromosomes during chemical enucleation. After etoposide-cycloheximide treatment, histone H1 kinase activity remains high for the ensuing 6-8 h before declining gradually to basal levels 14 h after treatment. The expulsion of the polar body occurred only after the slowly declining H1 kinase activity reached basal levels. The activity of this kinase rose sharply to reach maximal levels within 4 h when the enucleated oocytes were removed from the inhibitor-supplemented medium and placed in normal medium. The findings in this paper indicate that cytoplasts produced by chemical enucleation are morphologically normal, thus suggesting that these enucleated cells are suitable for cloning studies. Although effective in mouse oocytes, we postulate that certain modifications to the enucleation technology are necessary before a reliable non-invasive protocol for ungulate oocytes will be available.

Self-amputation of body appendages, such as severing of the tail by lizards or a leg by long-legged harvestmen (Phalangida), is widespread in nature. This phenomenon is observed in hydroid polyps, actinia, nemertines, annelids, starfishes, mollusks, and crustaceans and is regarded as a physiological protective mechanism [1]. However, the first data on the amputation of neuronal processes [2] were debated, although natural separation of fragments from the nucleus-containing soma and vice versa was described in other cells. The cell fragments separated from megakaryocytes were referred to as platelets [3]. Mammalian erythrocytes separate the nucleus-containing cell fragment and continue to live and function for a long time. Viable cytosomes (cytoplasts) were separated in experiments with the use of cytochalasin in all cells studied [4]. Although self-amputation as a physiological process has not been studied in neurons, we have no doubts that it can be observed in these cells. In this work we studied the autotomy and self-amputation of neurites under normal conditions in cell culture and in vivo. We discovered and analyzed the autotomy of neuronal processes and self-amputation of terminals of nonsynaptic dendrites in the tissue culture and in vivo. It was found that development of both processes is determined by the contractile activity of the processes in combination with adhesion of the terminals to a substrate or to other neurons. We showed that these processes take place continuously during activity of neurons and alternate with protruding growth processes. Self-amputation proceeds by the apocrine mechanism. The data obtained suggest that the dendrites that undergo self-amputation of cytoplasts filled with activated lysosomes have a certain trophic function. The study was performed with the tissue culture of isolated neurons of the pond snail ( Lymnaea stagnalis ). Single neurons were isolated via enzymatic and mechanical processing of the peripharyngeal nerve ring of mature pond snails (for details, see [5]). The cells were cultured for 5‐6 days in a serum-free medium (RPMI-1640 or Eagle’s MEM). The dynamics of regenerating neurons was recorded with an automated microvideoinstallation using time-lapse photography (shooting speed, 1 frame per 10 min) under an inverted phase-contrast microscope (objectives, 20 × , 40 × ; eyepiece, 10 × ). The illustrations are presented as a series of frames from the films. A comparison of the results of in vitro and in vivo studies was performed with the use of Dogel’s type II cells of the enteric plexus of healthy mature cats. The neurons were impregnated according to Bil’shovskii and Gros. The sensor terminals of nonsynaptic dendrites were studied with the use of the conventional electron microscopy.