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C M Luetjens

Publications and source records attributed to C M Luetjens.

At least 19 recordsLinked to original sources
PubMed2006

Association of three isoforms of the meiotic BOULE gene with spermatogenic failure in infertile men.

The complex process of spermatogenesis requires the expression and precise coordination of a multitude of genes. Abnormal function of such genes is frequently associated with male infertility. Among these candidates is the human BOULE gene that is a possible fundamental mediator of meiotic transition. In this study, we describe for the first time the existence of three BOULE transcript variants (B1, B2 and B3). We investigated their tissue specificity and mRNA transcript levels in 23 testis biopsies from infertile men. B1, B2 and B3 differed solely in their N-terminal sequences, which are encoded by three alternatively spliced exons 1. In humans, all three isoforms are exclusively expressed in the testes in a relative proportion of 80:220:1 for B1, B2 and B3, respectively. RT-PCR quantification revealed significantly reduced mRNA expression of all three variants in testicular biopsies with meiotic arrest (MA) compared with those with qualitatively complete spermatogenesis. Alteration of the B1/B2 and B1/B3 transcript ratios was correlated with reduced meiotic capacity of spermatocytes to produce round spermatids as assessed by flow cytometry. Furthermore, BOULE mRNA reduction in biopsies with MA paralleled the absence of BOULE protein as analysed by immunohistochemistry. In conclusion, the relative proportions of B1, B2 and B3 may serve as predictive markers for meiotic efficiency and thus the probability of finding haploid cells in the human testis. Among the three isoforms, B2 might have the major role for meiotic completion.

Amino Acid Sequence↗
PubMed2005

Mosaic status in lymphocytes of infertile men with or without Klinefelter syndrome.

BACKGROUND: Gonosomal aneuploidies such as Klinefelter syndrome (47,XXY) are the most frequent chromosomal aberration in infertile men. Normally the chromosomal status of patients is detected by karyotyping of up to 20 metaphase spreads of lymphocyte nuclei, whereby low grade mosaicism may be overlooked. To test whether Klinefelter patients with 47,XXY karyotype or infertile men with 46,XY karyotype represent gonosomal mosaicisms, we performed meta- and interphase fluorescence in situ hybridization (FISH) on 45 men. METHODS AND RESULTS: A total of 400 interphase and 40 metaphase lymphocyte nuclei per patient were scored after hybridization with DNA probes specific for chromosomes X and Y, and chromosome 9 as a control. On the basis of conventional karyotype, hormone levels and clinical appearance, patients were subdivided into 18 Klinefelter syndrome patients with 47,XXY (group I), 11 Klinefelter syndrome-like patients with normal karyotype, 46,XY (group II) and six non-Klinefelter-like infertile patients with normal 46,XY karyotype (group III). Ten normal men (group IV) served as controls. Testicular volume in the Klinefelter group I was smaller compared with group II (P = 0.016), group III (P < 0.001) and group IV (P < 0.001). In addition, testicular volumes in group II were lower compared with group III and group IV (P < 0.004). No significant differences between the aneuploidy rate analysed by FISH in interphase nuclei and metaphases were found in either single patients or groups. Patients with Klinefelter syndrome, 47,XXY (group I) or with symptoms similar to those in Klinefelter patients 46,XY (group II) showed a similar aneuploidy rate (group I 7.1 +/- 4.0% and group II 4.6 +/- 3.4%) and two 47,XXY patients with a high prevalence for normal 46,XY lymphocytes had sperm in their ejaculate. However, in general, no correlations between FISH mosaic status and serum hormone parameters, nor with ejaculate parameters were found. CONCLUSIONS: The results suggest that 47,XXY patients with an increased incidence of XY cells (average of 4.2 +/- 2.3) may have a higher probability of germ cells as we found sperm only in the ejaculate of Klinefelter syndrome patients with mosaic 46,XY cells (6.0 and 7.0%). On the other hand, 46,XY patients with mosaic sex chromosome aneuploidies detected by FISH analysis more often show symptoms of hypogonadism phenotypically resembling Klinefelter syndrome.

Adult↗
PubMed2004

Case report: natural transmission of an AZFc Y-chromosomal microdeletion from father to his sons.

Y-chromosomal microdeletions, associated with oligozoospermia or azoospermia, are usually de novo deletions in the affected patients. We report here the rare case of an affected father who transmitted a Y-chromosomal microdeletion to at least two of his three sons naturally and who also fathered a daughter. The extent of the deletion, which was determined with new STS-primers and covers 3.5 Mb, was identical in the father and his azoospermic sons. To determine any possibly modifying influence of other genes involved in spermatogenesis, we analysed two polymorphisms of the DAZL gene, the autosomal homologue of the deleted DAZ gene. DAZL and DAZ might be functionally related to each other. However, we found identical polymorphisms in exon 2 and 3 of the DAZL gene, in both father and his sons, corresponding to the most prevalent genotype in fertile men. Thus, other genes or environmental factors must modify spermatogenesis in men with identical Y-chromosomal microdeletions.

Adult↗
PubMed2004

Chorionic gonadotrophin beta subunit mRNA but not luteinising hormone beta subunit mRNA is expressed in the pituitary of the common marmoset (Callithrix jacchus).

The pituitary gonadotrophins LH and FSH are responsible for regulation of gametogenesis in the testis and ovary. Chorionic gonadotrophin (CG), a third closely related glycoprotein hormone derived by gene duplication of the LHbeta gene and secreted by the placenta in primates, is essential for the rescue of the corpus luteum and maintenance of pregnancy. We have recently shown that marmoset (m) CGbeta mRNA is highly expressed in the pituitary of the common marmoset (Callithrix jacchus) and that LH is less active than human CG in activating the human LH receptor lacking exon 10. To investigate further which gonadotrophin is the actual ligand of the LH receptor (LHR) of the marmoset monkey that naturally lacks exon 10, we identified and characterised the genomic organisation of the mLHbeta gene and its expression. Intergenic PCR amplification of the region encompassing the mLHbeta and the mCGbeta genes revealed that, surprisingly, mCGbeta is located 20 kbp upstream of the LHbeta gene, whereas in other species the intergenic distance is approximately 2-3 kbp. Sequence analysis of the mLHbeta coding region showed 70% identity to mCGbeta and 90% identity to human LHbeta at the amino acid level. Both gonadotrophin beta subunits are present at the genomic level, but RT-PCR of pituitary and placental total RNA using specific oligonucleotides for mCGbeta and mLHbeta showed high expression of mCGbeta mRNA in both tissues, whereas LHbeta was expressed neither in the pituitary nor in the placenta. Thus mLHbeta mRNA is lacking in the marmoset pituitary. Immunohistochemistry of marmoset pituitaries showed that mCG was confined to the gonadotrophes, and partly co-localised in cells stained positively for FSH. Western blot analysis confirmed the presence of mCG in the pituitary. Northern blot analysis using mCGbeta as a probe displayed one transcript of 0.7 kb in the pituitary and detected two transcripts of 1.1 kb and 2 kb in the marmoset placenta. Our results suggest that, in the common marmoset, CG is the only gonadotrophin with luteinising function that is present in the pituitary. We postulate that, owing to an unknown mutational event in evolution, expression of mLH was completely abolished, and CG - which, unlike LH, acts normally even when exon 10 is missing from the LHR - took over its function.

Amino Acid Sequence↗
PubMed2002

Analysis of DNA fragmentation of in vitro cultured bovine blastocysts using TUNEL.

Programmed cell death (apoptosis) characteristically affects the single cells of blastocysts whereas necrosis affects cluster of cells in both the inner cell mass (ICM) and the trophectoderm (TE). This study uses the trophectodermrminal deoxynucleotidyl transferase (TdT) mediated dUTP nick-end labeling (TUNEL) assay as a way of evaluating the proportion of apoptotic cells and, thus, bovine blastocyst quality during in vitro culture at Days 6,7, and 8. Furthermore, parthenogenetic blastocysts were compared to in vitro fertilized blastocysts at Day 7. Confocal microscopy was used to generate three-dimensional reconstructions of the blastocysts. Apoptosis was observed in both early (Day 6) and late (Day 8) developing blastocysts. The dead cell index (DCI, total number of apoptotic nuclei/total number of nuclei) tend to increase as the in vitro culture time increases, and apoptosis is proportionately higher in the ICM than in the TE. The ratio of ICM to TE cells remains relatively constant even as the blastocysts cell number increases (Day 6 = 11.9 +/- 2.2, Day 7 = 11.2 +/- 0.5, Day 8 = 11.7 +/- 0.4). The overall cell number is significantly reduced in parthenogenetic blastocysts compared to Day 7 in vitro produced blastocysts (P = 0.037). The parthenogenetic blastocysts also show an increase of apoptosis over Day 7 controls. The decrease in cell number in the parthenogenetic blastocysts may be due to the increase of apoptotic nuclei observed. Based on these results we found the TUNEL assay to be a useful method for evaluating in vitro culture conditions of pre-implantation bovine embryos.

Animals↗
PubMed2002

Sperm aneuploidy rates in younger and older men.

BACKGROUND: In order to assess the possible risk of chromosomal abnormalities in offspring from older fathers, we investigated the effects of age on the frequency of chromosomal aneuploidy rates of human sperm. METHODS AND RESULTS: Semen samples were collected from 15 men aged <30 years (24.8 +/- 2.4 years) and from eight men aged >60 years (65.3 +/- 3.9 years) from the general population. No significant differences in ejaculate volume, sperm concentration and sperm morphology were found, whereas sperm motility was significantly lower in older men (P = 0.002). For the hormone values, only FSH was significantly elevated in the older men (P = 0.004). Multicolour fluorescence in-situ hybridization was used to determine the aneuploidy frequencies of two autosomes (9 and 18); and of both sex chromosomes using directly labelled satellite DNA probes on decondensed sperm nuclei. A minimum of 8000 sperm per donor and >330 000 sperm in total were evaluated. The disomy rates per analysed chromosomes were 0.1-2.3% in younger men and 0.1-1.8% in older men. The aneuploidy rate determined for both sex chromosomes and for the autosomes 9 and 18 were not significantly different between the age groups. CONCLUSIONS: The results suggest that men of advanced age still wanting to become fathers do not have a significantly higher risk of procreating offspring with chromosomal abnormalities compared with younger men.

Adult↗
PubMed2002

Manifestation of Y-chromosomal deletions in the human testis: a morphometrical and immunohistochemical evaluation.

BACKGROUND: Deletions of the AZF (azoospermia factor) subregions on the Y chromosome are accompanied by a diverse spectrum of spermatogenic disturbances ranging from hypospermatogenesis to total depletion of germ cells causing infertility. The AZF region encodes gene products which are candidates for the genetic control of spermatogenesis. Although it is known which genes are involved, a general principle of cause and effect cannot yet be deciphered and the deletion type has non-uniform histological phenotypes. METHODS AND RESULTS: We analysed morphological parameters of testicular biopsies from 17 patients diagnosed for Y chromosome microdeletions. As control groups we analysed testes from patients with idiopathic Sertoli cell-only (SCO) syndrome (n = 11), mixed atrophy (n = 10) and complete spermatogenesis (n = 11). A detailed genetic analysis on the extension of the observed microdeletions revealed similar breakpoints in the distal and proximal region of the AZFc region, indicating a common mechanism of homologous recombination for such deletions, as has been suggested before. Morphometric parameters such as the diameter of the tubules, lumen, thickness of the lamina propria and height of the tubule epithelia were investigated. The diameter of the tubules from patients with microdeletions was found to be significantly smaller compared with patients with mixed atrophy. Considering also the size of the tubules, lumen and epithelia, a Y-chromosomal microdeletion represents an intermediate state between an idiopathic SCO and normal spermatogenesis. The immunohistochemical analysis of six different Sertoli cell markers, cytokeratin 18, vimentin, inhibin alpha subunit, 14-3-3 theta, FSH receptor and androgen receptor, revealed no impact of AZF deletion on the specific expression pattern of these genes. CONCLUSIONS: Our results suggest that, notwithstanding the deletion of a common region in the AZFc region, microdeletions of the Y chromosome lead to an intermediate status between idiopathic SCO and complete spermatogenesis, resulting in a heterogeneous histological profile regardless of the seminiferous activity. The Sertoli cell function seems not to be altered.

Adult↗
PubMed2001

Dissipation of potassium and proton gradients inhibits mitochondrial hyperpolarization and cytochrome c release during neural apoptosis.

Exposure of rat hippocampal neurons or human D283 medulloblastoma cells to the apoptosis-inducing kinase inhibitor staurosporine induced rapid cytochrome c release from mitochondria and activation of the executioner caspase-3. Measurements of cellular tetramethylrhodamine ethyl ester fluorescence and subsequent simulation of fluorescence changes based on Nernst calculations of fluorescence in the extracellular, cytoplasmic, and mitochondrial compartments revealed that the release of cytochrome c was preceded by mitochondrial hyperpolarization. Overexpression of the anti-apoptotic protein Bcl-xL, but not pharmacological blockade of outward potassium currents, inhibited staurosporine-induced hyperpolarization and apoptosis. Dissipation of mitochondrial potassium and proton gradients by valinomycin or carbonyl cyanide p-trifluoromethoxy-phenylhydrazone also potently inhibited staurosporine-induced hyperpolarization, cytochrome c release, and caspase activation. This effect was not attributable to changes in cellular ATP levels. Prolonged exposure to valinomycin induced significant matrix swelling, and per se also caused release of cytochrome c from mitochondria. In contrast to staurosporine, however, valinomycin-induced cytochrome c release and cell death were not associated with caspase-3 activation and insensitive to Bcl-xL overexpression. Our data suggest two distinct mechanisms for mitochondrial cytochrome c release: (1) active cytochrome c release associated with early mitochondrial hyperpolarization, leading to neuronal apoptosis, and (2) passive cytochrome c release secondary to mitochondrial depolarization and matrix swelling.

Animals↗
PubMed2001

Maternal or fetal origin of rhesus monkey (Macaca mulatta) amniotic fluid leukocytes can be identified by polymerase chain reaction using the zinc finger Y gene.

Leukocytes can be found in substantial numbers within the intrauterine tissues and amniotic fluid of women, and play a central role in the pathophysiology of infection-related preterm labor by their production of proinflammatory mediators. It remains unclear whether these leukocytes represent a fetal immune response, a maternal response, or a combination of the two. The objective of this study was to develop a test in the rhesus monkey (Macaca mulatta) suitable for determining the percentage of male fetal cells present in a population of leukocytes recovered from blood or amniotic fluid. We found inadequate specificity for rhesus monkey cells using commercial human Y-chromosome paint kits (fluorescence in situ hybridization (FISH)). Human-specific primers for the repetitive Y chromosome DYZ-1 locus employed in the polymerase chain reaction (PCR) produced an unacceptable percentage of false positives. However, we successfully developed a PCR-based test using rhesus-specific primers for the zinc finger Y (ZFY) locus. Densitometry of PCR products from known ratios of male and female adult peripheral leukocytes generated a linear standard curve which provided quantitative results and required only 400 cells per sample. The rhesus beta globin (RBG) gene served as an internal control. The PCR test correctly discriminated the sex of peripheral leukocytes in 20 adult males, 20 adult females, two male fetuses, and one female fetus. Serial samples of amniotic fluid from four chronically catheterized rhesus monkeys bearing male fetuses were used to confirm the utility of this assay for quantifying fetal cells in amniotic fluid. In conclusion, we have developed a PCR test which is suitable for distinguishing male from female cells in adult and fetal blood and in amniotic fluid, which lends itself to a variety of diagnostic and biologic applications in the rhesus monkey and potentially in other nonhuman primates.

Amniotic Fluid↗
PubMed2001

Up-regulation of Bcl-xL in response to subtoxic beta-amyloid: role in neuronal resistance against apoptotic and oxidative injury.

Neuron death in Alzheimer's disease is believed to be triggered by an increased production of amyloidogenic beta-amyloid peptides, involving both increased oxidative stress and activation of a conserved death program. Bcl-xL, an anti-apoptotic protein of the Bcl-2 family, is expressed at high levels in the adult nervous system. Exposure of neuronal cultures to subtoxic concentrations of beta-amyloid peptide 1-40 (1-10microM) or the fragment 25-35 (1-10microM) up-regulated both bcl-xL mRNA and Bcl-xL protein levels, determined by reverse transcriptase-polymerase chain reaction and western blot analysis. Bcl-xL protein was also up-regulated during oxidative stress induced by exposure to hydrogen peroxide (3-100microM) or ferric ions (1-10microM). In contrast, apoptotic stimuli (exposure to staurosporine or serum withdrawal) actually decreased neuronal Bcl-xL expression. To investigate the role of Bcl-xL in cell death relevant to Alzheimer's disease, we stably overexpressed Bcl-xL in human SH-SY5Y neuroblastoma cells. Cells overexpressing Bcl-xL were significantly protected from beta-amyloid neurotoxicity and staurosporine-induced apoptosis compared to vector-transfected controls. In contrast, Bcl-xL overexpression only conferred a mild protection against oxidative injury induced by hydrogen peroxide. We conclude that up-regulation of Bcl-xL expression in response to subtoxic concentrations of beta-amyloid is a stress response that increases the resistance of neurons to beta-amyloid neurotoxicity primarily by inhibiting apoptotic processes.

Alzheimer Disease↗
PubMed2001

Multiple kinetics of mitochondrial cytochrome c release in drug-induced apoptosis.

We investigated cytochrome c release kinetics in response to three apoptosis-inducing agents (tumor necrosis factor-alpha, staurosporine, and valinomycin) in MCF-7/Casp-3 cells stably transfected with enhanced green fluorescent protein (EGFP)-tagged cytochrome c. All three agents induced significant caspase activation in the cultures determined by monitoring the cleavage of fluorigenic caspase substrates in extracts from drug-treated MCF-7/Casp-3 cells, albeit the valinomycin-induced activation was less pronounced. Time-lapse confocal microscopy showed that tumor necrosis factor-alpha and staurosporine caused rapid, one- or multiple-step release of cytochrome c-EGFP from mitochondria. In contrast, valinomycin-induced cytochrome c-EGFP release occurred slowly over several hours. Unlike staurosporine, the valinomycin-induced cytochrome c release was not associated with translocation of the proapoptotic Bax protein to the mitochondria, and was not accompanied by co-release of the proapoptotic Smac protein. Immunoprecipitation experiments revealed that cytochrome c was also released out of the cell into the extracellular space before loss of plasma membrane integrity. Our data indicate the existence of multiple kinetics of cytochrome c release in drug-induced apoptosis.

Apoptosis↗
PubMed2000

Delayed mitochondrial dysfunction in excitotoxic neuron death: cytochrome c release and a secondary increase in superoxide production.

An increased production of superoxide has been shown to mediate glutamate-induced neuron death. We monitored intracellular superoxide production of hippocampal neurons during and after exposure to the glutamate receptor agonist NMDA (300 microm). During a 30 min NMDA exposure, intracellular superoxide production increased significantly and remained elevated for several hours after wash-out of NMDA. After a 5 min exposure, superoxide production remained elevated for 10 min, but then rapidly returned to baseline. Mitochondrial membrane potential also recovered after wash-out of NMDA. However, recovery of mitochondria was transient and followed by delayed mitochondrial depolarization, loss of cytochrome c, and a secondary rise in superoxide production 4-8 hr after NMDA exposure. Treatment with a superoxide dismutase mimetic before the secondary rise conferred the same protection against cell death as a treatment before the first. The secondary rise could be inhibited by the complex I inhibitor rotenone (in combination with oligomycin) and mimicked by the complex III inhibitor antimycin A. To investigate the relationship between cytochrome c release and superoxide production, human D283 medulloblastoma cells deficient in mitochondrial respiration (rho(-) cells) were exposed to the apoptosis-inducing agent staurosporine. Treatment with staurosporine induced mitochondrial release of cytochrome c, caspase activation, and cell death in control and rho(-) cells. However, a delayed increase in superoxide production was only observed in control cells. Our data suggest that the delayed superoxide production in excitotoxicity and apoptosis occurs secondary to a defect in mitochondrial electron transport and that mitochondrial cytochrome c release occurs upstream of this defect.

Animals↗
PubMed2000

Clonal propagation of primate offspring by embryo splitting.

Primates that are identical in both nuclear and cytoplasmic components have not been produced by current cloning strategies, yet such identicals represent the ideal model for investigations of human diseases. Here, genetically identical nonhuman embryos were produced as twin and larger sets by separation and reaggregation of blastomeres of cleavage-stage embryos. A total of 368 multiples were created by the splitting of 107 rhesus embryos with four pregnancies established after 13 embryo transfers (31% versus 53% in vitro fertilization controls). The birth of Tetra, a healthy female cloned from a quarter of an embryo, proves that this approach can result in live offspring.

Animals↗
PubMed2000

TransgenICSI reviewed: foreign DNA transmission by intracytoplasmic sperm injection in rhesus monkey.

This brief review considers the status of transgenesis by intracytoplasmic sperm injection (ICSI) with nonhuman primates. GFP expressing rhesus macaques embryos (mean = 34.6%; N = 81) were produced by ICSI using rhodamine-tagged DNA encoding the green fluorescence protein (GFP) gene bound on sperm. Rhodamine signal was lost at the egg surface during in vitro fertilization (IVF) but could be traced by dynamic imaging during ICSI within the egg cytoplasm. GFP gene was expressed as early as the 4-cell stage in ICSI embryos but not in embryos produced by in vitro fertilization (IVF). The percentage of GFP expressing blastomeres increased during embryogenesis to the blastocyst stage. Three offspring resulted from seven embryo transfers-a set of anatomically normal twins (a male and a female) stillborn 35 days premature, and a healthy male born at term. Although transgene was not detected in the offspring, the successful production of live primates using DNA bound sperm by ICSI suggests an alternative route to creating transgenic animals. It also raises concern regarding transmission of infectious material during ICSI.

Animals↗
PubMed2000

Atypical decondensation of the sperm nucleus, delayed replication of the male genome, and sex chromosome positioning following intracytoplasmic human sperm injection (ICSI) into golden hamster eggs: does ICSI itself introduce chromosomal anomalies?

OBJECTIVE: To examine nuclear decondensation, positioning of sex chromosomes, and the S-phase in human sperm nuclei following intracytoplasmic sperm injection (ICSI) into hamster eggs. DESIGN: Prospective analysis of hamster eggs and human sperm following ICSI. SETTING: Division of Reproductive Sciences, Oregon Health Sciences University and Oregon Regional Primate Research Center. PATIENT(S): Fertile donor sperm from a commercial source. INTERVENTION(S): Human sperm were examined by immunofluorescence stain, bromodioxyuridine (BrdU) uptake assay and fluorescence in situ hybridization following ICSI into hamster eggs. MAIN OUTCOME MEASURE(S): Immunostaining and fluorescence in situ hybridization. RESULT(S): Decondensation of human sperm nuclei occurred initially in the basal region, and perinuclear theca of sperm persisted around the condensed apical region. In some sperm nuclei, following ICSI the sex chromosomes were in the apical region, remaining condensed for longer than in the basal region. S-phase entry of human sperm nuclei following ICSI was delayed compared to the zona-free hamster egg penetration assay. CONCLUSION(S): These results force questions about the mechanism of male pronuclear formation after ICSI and suggest new strategies for understanding the basis of chromosomal anomalies leading to birth defects as well as continuing improvements in the safety and efficacy of infertility therapies.

Animals↗
PubMed2000

Foreign DNA transmission by ICSI: injection of spermatozoa bound with exogenous DNA results in embryonic GFP expression and live rhesus monkey births.

Exogenous DNA transfer, mediated by intracytoplasmic sperm injection (ICSI) with plasmid-bound spermatozoa, results in the production of transgene expressing embryos in rhesus macaques (Macaca mulatta, mean = 34.6%; n = 81). Rhodamine-tagged DNA encoding the green fluorescent protein (GFP) gene binds avidly to spermatozoa. The rhodamine signal, while lost at the egg surface during in-vitro fertilization (IVF), is traced by dynamic imaging during ICSI and remains as a brilliant marker on the microinjected spermatozoa within the oocyte cytoplasm. The transgene is expressed in preimplantation embryos produced by ICSI, but not IVF, as early as the 4-cell stage with the number of expressing cells and the percentage of expressing embryos increasing during embryogenesis to the blastocyst stage. The three offspring that resulted from seven embryo transfers (a set of anatomically normal twins, one male and one female, stillborn 35 days premature, and a healthy male born at term) demonstrate that primate spermatozoa with exogenously bound DNA retain their full reproductive capacity in ICSI, but raise the concern that, theoretically, ICSI could transmit infectious material as well.

Animals↗
PubMed2000

Dynamic imaging of the metaphase II spindle and maternal chromosomesin bovine oocytes: implications for enucleation efficiency verification, avoidanceof parthenogenesis, and successful embryogenesis.

Manipulations of DNA and cellular structures are essential for the propagation of genetically identical animals by nuclear transfer. However, none of the steps have been optimized yet. This study reports a protocol that improves live dynamic imaging of the unfertilized bovine oocyte's meiotic spindle microtubules with microinjected polymerization-competent X-rhodamine-tubulin and/or with vital long-wavelength excited DNA fluorochrome Sybr14 so that the maternal chromosomes can be verifiably removed to make enucleated eggs the starting point for cloning. Suitability of the new fluorochromes was compared to the conventional UV excitable Hoechst 33342 fluorochrome. Enucleation removed the smallest amount of cytoplasm (4-7%) and was 100% efficient only when performed under continuous fluorescence, i.e., longer fluorescence exposure. This was in part due to the finding that the second metaphase spindle is frequently displaced (60.7 +/- 10%) from its previously assumed location subjacent to the first polar body. Removal of as much as 24 +/- 3% of the oocyte cytoplasm underneath the polar body, in the absence of fluorochromes, often resulted in enucleation failure (36 +/- 6%). When labeled oocytes were exposed to fluorescence and later activated, development to the blastocyst stage was lowest in the group labeled with Hoechst 33342 (3%), when compared to Sybr14 (19%), rhodamine-tubulin (23%), or unlabeled oocytes (37%). This suggests that longer wavelength fluorochromes can be employed for live visualization of metaphase spindle components, verification of their complete removal during enucleation, and avoidance of the confusion between artifactual parthenogenesis versus "cloning" success, without compromising the oocyte's developmental potential after activation.

Animals↗