Robert M. Moor

Mammalian leukocytes contain all the genetic information necessary for the development of a new individual.

We have used leukocytes and oocytes from commercially slaughtered animals to clone a progeny tested Brown Swiss bull. Mononuclear cells were separated from the heparinized blood of the donor male on a Histopaque gradient and cryopreserved. The nuclei of thawed leukocytes were directly microinjected into enucleated Holstein Friesian oocytes that were subsequently activated. Development to morula was 23% and to blastocysts was 17%. Some of the cloned compacting morulae were subjected to a second round of nucleus transfer by fusion of individual blastomeres to enucleated oocytes. Development of these second generation embryos to the blastocyst stage was 19%. Following embryo transfer of 50 blastocysts to 50 recipient heifers (31 from first generation and 19 from second generation), 28 pregnancies were established as evidenced by fetal heartbeat at 35 days. A high proportion of the pregnancies established were lost by day 45. One fetus from a second generation embryo developed to term. The phenotype (Brown Swiss) and DNA analysis (11 microsatellites on 11 different chromosomes) of the resultant normal healthy calf confirmed its identity to the donor sire. The ability to clone animals from hematopoietic cells that can be easily collected and cryopreserved from any donor irrespective of species, age, or sex has important implications for the preservation of genetic resources from a wide variety of animals in the animal breeding and artificial insemination industries and for human medicine.

Spindle Formation and Dynamics of γ-Tubulin and Nuclear Mitotic Apparatus Protein Distribution During Meiosis in Pig and Mouse Oocytes

Abstract This work focuses on the assembly and transformation of the spindle during the progression through the meiotic cell cycle. For this purpose, immunofluorescent confocal microscopy was used in comparative studies to determine the spatial distribution of α- and γ-tubulin and nuclear mitotic apparatus protein (NuMA) from late G2 to the end of M phase in both meiosis and mitosis. In pig endothelial cells, consistent with previous reports, γ-tubulin was localized at the centrosomes in both interphase and M phase, and NuMA was localized in the interphase nucleus and at mitotic spindle poles. During meiotic progression in pig oocytes, γ-tubulin and NuMA were initially detected in a uniform distribution across the nucleus. In early diakinesis and just before germinal vesicle breakdown, microtubules were first detected around the periphery of the germinal vesicle and cell cortex. At late diakinesis, a mass of multi-arrayed microtubules was formed around chromosomes. In parallel, NuMA localization changed from an amorphous to a highly aggregated form in the vicinity of the chromosomes, but γ-tubulin localization remained in an amorphous form surrounding the chromosomes. Then the NuMA foci moved away from the condensed chromosomes and aligned at both poles of a barrel-shaped metaphase I spindle while γ-tubulin was localized along the spindle microtubules, suggesting that pig meiotic spindle poles are formed by the bundling of microtubules at the minus ends by NuMA. Interestingly, in mouse oocytes, the meiotic spindle pole was composed of several γ-tubulin foci rather than NuMA. Further, nocodazole, an inhibitor of microtubule polymerization, induced disappearance of the pole staining of NuMA in pig metaphase II oocytes, whereas the mouse meiotic spindle pole has been reported to be resistant to the treatment. These results suggest that the nature of the meiotic spindle differs between species. The axis of the pig meiotic spindle rotated from a perpendicular to a parallel position relative to the cell surface during telophase I. Further, in contrast to the stable localization of NuMA and γ-tubulin at the spindle poles in mitosis, NuMA and γ-tubulin became relocalized to the spindle midzone during anaphase I and telophase I in pig oocytes. We postulate that in the centrosome-free meiotic spindle, NuMA aggregates the spindle microtubules at the midzone during anaphase and telophase and that the polarity of meiotic spindle microtubules might become inverted during spindle elongation.

Association between p34cdc2 levels and meiotic arrest in pig oocytes during early growth

The molecules involved in determining meiotic competence were determined in porcine oocytes isolated from preantral and antral follicles of different sizes. Oocytes isolated from preantral follicles had a mean diameter of 78 microns, contained diffuse filamentous chromatin in the germinal vesicle and were incapable of progressing from the G2 to the M phase of the cycle even after 72 h in culture. Oocytes from early antral follicles had a mean diameter of 105 microns, showed a filamentous chromatin configuration and about half resumed meiosis but arrested at metaphase I (MI) when cultured. Oocytes from mid-antral (3-4 mm) and large antral follicles (5-6 mm) had mean oocyte diameters of 115 and 119 microns respectively, contained condensed chromatin around the nucleolus and progressed to metaphase II (MII) in 48% and 93% of instances respectively. Analysis of p34cdc2, the catalytic subunit of maturation promoting factor (MPF), by immunoblotting indicates that the inability of small (78 microns) oocytes to resume meiosis is due, at least in part, to inadequate levels of the catalytic subunit of MPF. On the other hand, the inability of intermediate-sized (105 microns) oocytes from antral follicles to complete the first meiotic division by progressing beyond MI appears not to be limited by levels of p34cdc2, which are maximal by this stage. We postulate that an inadequacy of molecules other than p34cdc2 limits progression of MI to MII; the acquisition of these molecules during the final stages of growth may be correlated with the formation of the perinucleolar chromatin rim in the germinal vesicle.

Localisation of phosphorylated MAP kinase during the transition from meiosis I to meiosis II in pig oocytes.

Mitogen-activated protein kinase (MAPK) has been reported to be involved in oocyte maturation in all animals so far examined. In the present study we investigate the expression and localisation of active phosphorylated MAPKs (p44ERK1/p42ERK2) during maturation of pig oocytes. In immunoblot analysis using anti-p44ERK1 antibody which recognised both active and inactive forms of p44ERK1 and p42ERK2, we confirmed that MAPKs were phosphorylated around the time of germinal vesicle breakdown (GVBD) and the active phosphorylated MAPKs (pMAKs) were maintained until metaphase II, as has been reported. On immunofluorescent confocal microscopy using anti-pMAPK antibody which recognised only phosphorylated forms of MAPKs, pMAPK was localised at the spindle poles in pig mitotic cells. On the other hand, in pig oocytes, no signal was detected during GV stage. After GVBD, the area around condensed chromosomes was preferentially stained at metaphase I although whole cytoplasm was faintly stained. At early anaphase I, the polar regions of the meiotic spindle were prominently stained. However, during the progression of anaphase I and telophase I pMAPK was detected at the mid-zone of the elongated spindle, gradually becoming concentrated at the centre. Finally, at the time of emission of the first polar body, pMAPK was detected as a ring-like structure between the condensed chromosomes and the first polar body, and the staining was maintained even after the metaphase II spindle was formed. The inhibition of MAPK activity with the MAPK kinase inhibitor U0126 during the meiosis I/meiosis II transition suppressed chromosome separation, first polar body emission and formation of the metaphase II spindle. From these results, we propose that the spindle-associated pMAPKs play an important role in the events occurring during the meiosis I/meiosis II transition, such as chromosome separation, spindle elongation and cleavage furrow formation in pig oocytes.

Nuclei of Nonviable Ovine Somatic Cells Develop into Lambs after Nuclear Transplantation

Abstract Here we report on the successful reprogramming of nuclei from somatic cells rendered nonviable by heat treatment. Granulosa cells from adult sheep were heated to nonphysiological temperatures (55°C or 75°C) before their nuclei were injected into enucleated metaphase II oocytes. Reprogramming was demonstrated by the capacity of the reconstructed embryos to develop to the blastocyst stage in vitro and into fetuses and viable offspring in suitable foster mothers. To our knowledge, this is the first report of cloned mammalian offspring originating from nonviable cells. In addition, our experiments show that heat-treating donor nuclei destabilizes higher-order features of chromatin (but leaves intact its nucleosomal organization) and results in a high proportion of reconstructed embryos developing to the blastocyst stage and beyond.

Cloning by somatic cell nuclear transfer

The birth of the first cloned mammals, produced by the introduction of somatic cell nuclei into enucleated oocytes, was an impressive and surprising development.(1) Although the ethical debate has been intense, the important scientific questions raised by this work have been inadequately discussed and are still unresolved. In this essay we address three questions about nuclear transplantation in the eggs of mice and domestic animals. First, why were the recent experiments on somatic cell cloning successful, when so many others have failed? Second, were these exceptional cases, or is somatic cloning now open to all? Third, what are the future possibilities for increasing the efficiency and wider applicability of the cloning process? BioEssays 20:847–851, 1998.

Nuclear transfer in sheep embryos: The effect of cell-cycle coordination between nucleus and cytoplasm and the use of in vitro matured oocytes

The developmental ability of nuclear transplant sheep embryos derived from in vitro matured oocytes was studied by controlling cell‐cycle coordination of donor embryonic nuclei and recipient cytoplasts. Oocytes were recovered from nonatretic antral follicles of adult sheep ovaries and cocultured with follicle shells in M199‐based medium supplemented with gonadotrophins in a nonstatic system. Effective activation of IVM oocytes was obtained by applying two pulses of 1.0 kv/cm 22 min apart in inositol‐based electroporation medium to oocytes matured in vitro for 27 hr. Synthesis of DNA (S‐phase) was assessed by BrdU incorporation and was found to initiate around 5 hpa (hours postactivation) and to persist until 18 hpa. Mitotic blastomeres were induced by treating embryos with 6.6 μM nocodazole for 14–17 hr. Three types of transfers were compared directly: “S → S,” early embryonic nuclei (mostly in S‐phase) were transferred to presumptive S‐phase cytoplasts; “M → MII,” nocodazole‐treated embryonic nuclei (most in M‐phase) were transferred to MII‐phase cytoplasts; and control (S → MII), conventional nuclear transfer of fusion and activation simultaneously. The results showed that fusion and recovery rates did not differ among the three groups. However, after 6 days of in vivo culture, the morula and blastocyst formation rate was significantly higher for the M → MII combination than for the control (28.3% vs. 8.1%, P < 0.05), while no significant differences in developmental rate were observed between S → S and M → MII, and between S → S and control, though developmental rate was also increased for S → S compared to control (20.9% vs. 8.1%, P > 0.05). Transfer of blastocysts derived from M → MII or S → S nuclear cytoplasmic reconstitution to synchronized recipient ewes resulted in the birth of lambs. These data suggest that in vitro matured oocytes can support full‐term development of nuclear transplant sheep embryos when the cell cycle of nucleus and cytoplasm is coordinated, and that M → MII nuclear transfer might be an efficient and simple way to improve the developmental competence of the reconstituted embryos. Mol. Reprod. Dev. 47:255–264, 1997.

Specific regulation of CENP-E and kinetochores during meiosis I/meiosis II transition in pig oocytes

To understand the mechanisms which regulate meiosis‐specific cell cycle and chromosome distribution in mammalian oocytes, the level and the localization of CENP‐E and the kinetochore number and direction on a half bivalent were examined during pig oocyte maturation. CENP‐E is a kinetochore motor protein whose intracellular level and localization are strictly regulated in the somatic cell cycle. The localizations of CENP‐E on meiotic chromosomes from diakinesis stage to anaphase I and at the spindle midzone at telophase I were shown by immunofluorescent confocal microscopy to be similar to those in somatic cells of pig and other species. Further, ultrastructural analysis revealed the presence of CENP‐E on fibrous corona and outer plate of kinetochores of the meiotic chromosomes. However, unlike mitosis, CENP‐E staining was continuously detected either at the spindle midzone or on the kinetochores of segregated chromosomes during the first polar body emission. Consistent with this, immunoblot analysis revealed that CENP‐E level remained high during meiosis I/meiosis II (MI/MII) transition and that some of CENP‐E survived through the transition even in cycloheximide‐treated oocytes in which cyclin B1 was completely degraded. Furthermore, examinations of CENP‐E signals in confocal microscopy and kinetochores in electron microscopy in MI and MII oocytes provide the cytological evidence in mammalian oocytes which suggests that each sister chromatid in a pair has its own kinetochore which localizes side‐by‐side so that two sister chromatids on a half bivalent are oriented toward and connected to the same pole in MI. Mol. Reprod. Dev. 56:51–62, 2000.

Selective Requirement for Cdc25C Protein Synthesis During Meiotic Progression in Porcine Oocytes

Abstract Fundamental differences between meiosis and mitosis suggest that the shared central cell cycle machinery may be regulated differently during the two division cycles. This paper focuses on unique features of Cdc25C protein function during meiotic progression. We report on the existence of oocyte-specific CDC25C transcripts that differ from their somatic counterparts in the 3′ untranslated region. While CDC25C mRNA levels remain constant in fully-grown oocytes, corresponding protein levels increase progressively during maturation to a maximum at metaphase II. Elevation of Cdc25C protein levels in G2-oocytes by mRNA injection failed to increase MPF-kinase levels or to induce premature entry into M-phase. Likewise, antisense-induced arrest of translation (translational arrest) had no effect on chromosome condensation, nucleolar disassembly, or nuclear membrane contraction. By contrast, translational arrest inhibited subsequent events including membrane disassembly and spindle formation. Neither up- nor down-regulation of Cdc25C synthesis after metaphase I plate formation influenced progression to metaphase II. However, translational arrest during metaphase resulted in incomplete chromosome decondensation and abnormal pronuclear membrane assembly after activation. We conclude that Cdc25 protein, translated from unique transcripts, is preferentially located in the oocyte nucleus and is essential for progress through late diakinesis. Subsequently, new synthesis of Cdc25C protein is required for the orderly transition from meiotic to mitotic cell division.

Embryonic stem cells in farm animals

Embryonic stem cell technology is now well established in the mouse (reviewed by Robertson, 1987). This technology implies the isolation from the preimplantation embrao of a cell line (ES) that is cultured in vitro in an undifferentiated state. Embryonal carcinoma cells (EC) lines obtained from malignant tumours (Martin, 1975), together with all the information available on their culture requirements (reviewed by Heath, 1987), represented a very important starting point for the establishment of ES cells (Martin, 1981). ES cells share many characteristics with EC cells such as the ability to contribute to somatic tissues of animals obtained following injection of cells into a host blastocyst, to differentiate in vitro under appropriate stimuli (Rudnicki & McBurney, 1987) and to form retransplantable tumours. ES cells, however, have substantial advantages over EC cells in that they can be derived directly from a normal embryo, they maintain a normal karyotype and when reintroduced into a host blastocyst they can colonise the germ line (Bradley, 1987). ES cells are maintained in an undifferentiated state by the presence of feeder layers producing various factor(s) that prevent to the cells from differentiating. It has been shown that glycoproteins are responsible for this effect and these have been named according to their different activities: DIA, differentiation inhibitory activity (Smith & Hooper, 1987); LIF, leukaemia inhibiting factor (Smith et al, 1988; Williams et al, 1988); HILDA, human interleukin for DA cells (Moreau et al., 1988). It is now possible to establish and maintain ES cells in culture in the absence of feeders cells but in the presence of such factors (Nichols et al., 1990).