Biomedical subjects

V Bordignon

Publications and source records attributed to V Bordignon.

At least 19 recordsLinked to original sources

PubMed2006

Telophase-stage host ooplasts support complete reprogramming of roscovitine-treated somatic cell nuclei in cattle.

Nuclear-cytoplasmic incompatibilities are known to play a significant role in the developmental outcome of embryos produced by nuclear transfer, particularly when metaphase arrested oocytes are used as hosts for interphase donor nuclei. To further our understanding of how cell cycle coordination affects somatic cell cloning, somatic cells at different stages of the cell cycle were fused to host oocytes either before (metaphase II, M-II) or after (telophase II, T-II) activation. To obtain cells at different stages of the cell cycle, fetal fibroblast (FF) and granulosa cells (GC) were treated with roscovitine, an inhibitor of cyclin-dependent kinases (CDKs) resulting in a large percentage of cells in S/G(2)-phase. In contrast to the M-II group, which did better with confluent cells, embryos reconstructed with T-II cytoplasts resulted in higher rates of blastocyst formation when fused to cells recovered at 16-24 h after passage. Embryos reconstructed with FF treated with roscovitine and T-II cytoplasts (Rosc/T-II) resulted in similar blastocyst rate compared to those produced with confluent cells and M-II cytoplasts (Conf/M-II). Transfer of blastocysts to surrogate heifers resulted pregnancies and birth of healthy calves from Rosc/T-II and Conf/M-II reconstructed embryos. These results indicate that, when combined with nuclear donor cells at specific cell cycle stages, M-II and T-II bovine oocytes are similarly effective in supporting the reprogramming of somatic cell nuclei.

Animals↗

PubMed2006

Vascular endothelial growth factor gene polymorphisms increase the risk to develop psoriasis.

We investigated the relationship between eight polymorphisms in the gene encoding for vascular endothelial growth factor (VEGF) (-1540C > A, -1512Ins18, -1451C > T, -460T > C, -160C > T, -152G > A, -116G > A and +405G > C) and plaque-type psoriasis stratified for age at onset, gender and family history of dermatosis. For this purpose, 117 patients with chronic plaque-type psoriasis and 215 healthy subjects were enrolled. We found that being homozygous -1540AA, -1512InsIns, -1451TT, -460CC and -152AA conferred a significant risk in developing psoriasis compared with heterozygous (-1540CA, -1512 + Ins, -1451CT, -460CT and -152AG) and homozygous genotypes (-1540CC, -1512 + +-1451CC, -460TT and -152GG) grouped together [odds ratio (ORs) = 1.73, 1.73, 1.73, 1.77 and 1.87, respectively]. Conversely, having the -116AA or +405GG genotype did not significantly increase the risk of disease expression compared with other genotypes of the same loci. Interestingly, we found that -1540AA, -1512InsIns, -1451TT, -460CC and -152AA homozygous genotypes have a significant two-fold increased risk in developing psoriasis after the age of 40 years (late-onset psoriasis) (ORs = 2.19, 2.19, 2.19, 2.05 and 2.26; P = 0.02, 0.02, 0.02, 0.04 and 0.02, respectively) as compared with controls. On the contrary, we found no phenotype-genotype association of the same magnitude among the patients in whom psoriasis developed at or before the age of 40 years (early-onset psoriasis) compared with controls. Genotype distributions were not significantly different when cases and controls were stratified either by gender or family history of psoriasis. Finally, VEGF plasma concentration was not significantly different between patients and controls and was not correlated with the severity of the disease.

5' Untranslated Regions↗

PubMed2006

Age, gender and reactivity to allergens independently influence skin reactivity to histamine.

BACKGROUND: The ability to mount an IgE response to allergens is a prerequisite for the development of positive allergen skin tests. Histamine is commonly used as a positive control in skin prick testing and provides a measure of nonspecific skin reactivity, similar to bronchial hyper-responsiveness. METHODS: To determine whether allergen responsiveness, age, gender and season of the year contribute to histamine sensitivity, 620 subjects (502 of them with at least one known sensitizing allergen and the remaining 118 non-allergic controls) were prick-tested with a panel of allergens common in the Northern Italy semi-rural area where the patients lived, and with 10 mg/ml histamine dihydrochloride. RESULTS: We found higher histamine reactivity in allergic versus control individuals (median value 23.7 versus 19.8 mm2; p=0.0497). Likewise, we found in allergic subjects a correlation between allergen responsiveness in terms of number of positive allergens at skin prick test and sensitivity to histamine (mono- sensitized versus poly-sensitized subjects: p=0.0015). Moreover older age and male sex were associated with a higher response to histamine, also when separately considering allergic subjects (p<0.0001 in both cases: correlation coefficient for age versus histamine reactivity: r=0.3408). The correlation between allergen responsiveness and sensitivity to histamine was maintained also when statistically balanced for age and sex. CONCLUSION: Allergen responsiveness, gender and age allow more accurate prediction of histamine sensitivity than either parameter alone.

Adult↗

PubMed2005

Cytokine profiles during infliximab monotherapy in psoriatic arthritis.

BACKGROUND: Biological therapies are a new breakthrough in the treatment of psoriasis and psoriatic arthritis (PsA). Among these, tumour necrosis factor (TNF)-alpha antagonists such as infliximab and etanercept are the most promising as TNF is considered to be essential in driving cytokine cascade at sites of cutaneous and synovial inflammation in this disease. OBJECTIVES: To evaluate the time-related response of serum cytokine release during infliximab monotherapy and assess serum cytokine levels in order to provide a fast, minimally invasive tool to monitor and/or predict efficacy of anti-TNF-alpha therapy. METHODS: Twenty patients affected by PsA with Psoriasis Area and Severity Index (PASI) score between 0.4 and 42.8 were treated with infliximab for 30-42 weeks. The assessment of arthritis severity was performed using the American College of Rheumatology (ACR) criteria and ultrasonography evaluation. The treatment schedule consisted of infliximab (5 mg kg(-1) intravenously) at 0, 2 and 6 weeks and every 12 weeks on an individual basis determined by therapeutic results and adverse events reported. At baseline and before every infusion blood samples were taken to assess serum cytokine levels [TNF-alpha, interleukin (IL-6), E-selectin, vascular endothelial cell growth factor (VEGF), fibroblast growth factor (FGF), matrix metalloproteinase (MMP-2)]. RESULTS: Eighteen of 20 psoriatic patients achieved > 50% improvement and 14 of 20 patients attained > 75% improvement in the PASI score at 10 weeks. All arthritic patients achieved > 50% improvement (ACR-50) and 16 of 20 patients attained > 75% improvement (ACR-75) at 10 weeks. TNF-alpha did not decrease immediately during the first part of the study. A significant decrease was detected at week 12 (P < 0.01). In contrast, IL-6, VEGF, FGF and E-selectin showed significant decreases after early infliximab infusions. PASI was not correlated with TNF-alpha in the serum but was significantly correlated with FGF, VEGF and MMP-2. Treatment was well tolerated and there were no significant adverse events in most patients, other than an urticarial reaction and an autoimmune hepatitis. CONCLUSIONS: Monotherapy with infliximab has to be considered an efficacious and safe treatment for PsA in comparison with traditional disease-modifying antirheumatic drugs. The resolution of cutaneous and synovial symptoms is not related to TNF-alpha serum levels in the initial phases. Apoptosis may play an important role in the modulation of the inflammatory response.

Adult↗

PubMed2005

Prevalence of human papilloma virus type 5 DNA in lesional and non-lesional skin scales of Italian plaque-type psoriatic patients: association with disease severity.

Human papilloma virus type 5 (HPV-5) has been associated closely with psoriatic skin in Polish patients, while findings from other countries have indicated a more limited prevalence. The results of the present study, in which a type-specific nested PCR was used, indicated that scales of plaque-type psoriatic skin from 54 Italian patients had a high prevalence (74.1%) of HPV-5 DNA in lesional areas, and a reduced prevalence (33.3%) in non-lesional skin (33.3%), compared to 0% of 20 healthy subjects and 3.6% in the lesional areas of 28 patients with various other dermatological diseases. Individuals negative for HPV-5 DNA had a less severe disease. No correlation was found between the presence of HPV DNA and a patient's age or sex. The data demonstrated a statistically significant association between psoriasis and HPV-5, although results in other geographical areas suggest variable virus spread or ethnic variation in virus colonisation.

Adult↗

PubMed2003

Assessment of chromosomal abnormalities in bovine nuclear transfer embryos and in their donor cells.

Chromosomal anomalies were assessed in nuclear transfer (NT) embryos (n = 148) at 1-4-cell stage (n = 88), and morula (n = 60), as well as in donor cells (n = 97) derived from two different cell lines. Two different cytogenetic approaches were used: conventional karyotyping and fluorescent in situ hybridization (FISH) with painting probes, specific for bovine X and Y chromosomes. The total rate of NT embryos with abnormal nuclei was 43%. These anomalies were mainly nuclear fragmentation (30%), hypoploidy/hypoploidy-mixoploidy (9%, n = 14) and hyperploidy/hyperploidy-mixoploidy (3%, n = 5). The incidence at which these anomalies occurred in NT embryos varied according to the donor cell culture and paralleled the frequency of anomalies in donor cells. A higher frequency of total anomalies was observed in NT embryos (55%) derived from the donor cell cultures with the highest incidence of anomalies (23%). An increase in the rate of total anomalies of the cell, after transfer to recipient cytoplasm, was also observed. These results suggest that proper screening of donor cells for chromosomal anomalies must be performed prior to NT procedure. They also suggest that the NT procedure itself might have a detrimental effect on some mechanism of chromosome segregation and distribution during cell division.

Animals↗

PubMed2003

Variation of the skin end-point in patients treated with sublingual specific immunotherapy.

The reduction in skin reactivity in sensitized subjects is a common finding after injective specific immunotherapy. Few data are, on the contrary, available for sublingual immunotherapy. We assessed the skin reactivity by the end-point titration method in 90 monosensitized subjects (age range 3-50; mean age 19.63) at the baseline and for four consecutive years during the SLIT treatment with two different maintenance regimens. The yearly duration of drug intake was monitored throughout the observation period. Age and skin reactivity to histamine (p < 0.0001; r = 0.871), and age and skin prick end-point (p < 0.0001; r = 0.523) turned out to be statistically positively correlated at baseline. No correlation was on the contrary found between age and the ratio allergen wheal/histamine wheal (p = 0.857; r = -0.019). After 4 years all patients showed at least a significant 4-fold decrease of the skin end-point, whereas 46/90 (51.1%) showed a 16-fold decrease and 29/90 (32.2%) a 64-fold decrease (p < 0.001 in all cases). At the end of the SLIT treatment, 21/90 patients (23.3%) were not using any drug, whereas 16/16 (100%) had given up the use of eye drops, and 34/35 (97%) had given up the use of bronchodilators and bronchial steroids (p < 0.001). After SLIT, 52/90 patients showed a reduction by at least 50% of the yearly duration of drug intake (p < 0.0001). An apparent relationship between the progressively decreasing rate of use of drugs and the decrease in skin reactivity was found, but it did not reach the point of statistical significance (p = 0.081). Patients treated with the daily allergen administration schedule, in spite of a lower cumulative dosage, showed a significantly greater decrease in skin reactivity (p < 0.001) and a higher rate of both no use of any drug (p = 0.013) and of at least a 50% reduction of the yearly duration of drug intake (p = 0.001) as compared to patients treated with three allergen administrations per week.

Administration, Sublingual↗

PubMed2002

Differential diagnosis by the endpoint method in patients skin-reactive to more than one inhalant allergen.

In recent years there has been an increasing number of patients suffering from respiratory symptoms and skin test positive to more than one allergen, and in many cases neither the clinical history nor the specific IgE determination has proved useful in establishing a ranking among two or more positive allergens. This study was designed to test the usefulness of the skin-prick test endpoint technique for the identification of the most important allergen(s) among those skin test positive. To this end, 118 consecutive patients (age range 4-60 years) skin test positive to between 2 and 6 different allergens were selected and submitted to the skin-prick test endpoint technique with serial 1:4 dilutions of standardized commercial allergens. The skin-prick test endpoint technique was unable to establish a ranking in only 7 patients sensitized to two allergens, whereas a difference in the range 2-10 between the highest and the lowest endpoint dilution in the same patient could be established in all other cases. Some allergens known to be widely crossreactive (D. farinae and D. pteronyssinus; cultivated and common grasses; birch and hazel) showed a similar skin reactivity only in few cases when tested according to the skin-prick test endpoint technique. This finding suggests a specific sensitization only for one allergen (or mixture) of each crossreacting couple. Moreover, in 60/118 patients the allergen able to evoke the largest skin reaction with the standard diagnostic did not have the highest skin-prick endpoint, suggesting that the ranking of allergens on the basis of the dimension of the wheal obtained with the standard diagnostic is a concept that should be revised and updated.

Administration, Inhalation↗

PubMed2001

Factors controlling the loss of immunoreactive somatic histone H1 from blastomere nuclei in oocyte cytoplasm: a potential marker of nuclear reprogramming.

Nuclei of differentiated cells can acquire totipotency following transfer into the cytoplasm of oocytes. While the molecular basis of this nuclear reprogramming remains unknown, the developmental potential of nuclear-transfer embryos is influenced by the cell-cycle stage of both donor and recipient. As somatic H1 becomes immunologically undetectable on bovine embryonic nuclei following transfer into ooplasm and reappears during development of the reconstructed embryo, suggesting that it may act as a marker of nuclear reprogramming, we investigated the link between cell-cycle state and depletion of immunoreactive H1 following nuclear transplantation. Blastomere nuclei at M-, G1-, or G2-phase were introduced into ooplasts at metaphase II, telophase II, or interphase, and the reconstructed embryos were processed for immunofluorescent detection of somatic histone H1. Immunoreactivity was lost more quickly from donor nuclei at metaphase than at G1 or G2. Regardless of the stage of the donor nucleus, immunoreactivity was lost most rapidly when the recipient cytoplast was at metaphase and most slowly when the recipient was at interphase. When the recipient oocyte was not enucleated, however, immunoreactive H1 remained in the donor nucleus. The phosphorylation inhibitors 6-DMAP, roscovitine, and H89 inhibited the depletion of immunoreactive H1 from G2, but not G1, donor nuclei. In addition, immunoreactive H1 was depleted from mouse blastomere nuclei following transfer into bovine oocytes. Finally, expression of the developmentally regulated gene, eIF-1A, but not of Gapdh, was extinguished in metaphase recipients but not in interphase recipients. These results indicate that evolutionarily conserved cell-cycle-regulated activities, nuclear elements, and phosphorylation-linked events participate in the depletion of immunoreactive histone H1 from blastomere nuclei transferred in oocyte cytoplasm and that this is linked to changes in gene expression in the transferred nucleus.

Animals↗

PubMed2001

Reprogramming of telomerase activity and rebuilding of telomere length in cloned cattle.

Nuclear reprogramming requires the removal of epigenetic modifications imposed on the chromatin during cellular differentiation and division. The mammalian oocyte can reverse these alterations to a state of totipotency, allowing the production of viable cloned offspring from somatic cell nuclei. To determine whether nuclear reprogramming is complete in cloned animals, we assessed the telomerase activity and telomere length status in cloned embryos, fetuses, and newborn offspring derived from somatic cell nuclear transfer. In this report, we show that telomerase activity was significantly (P < 0.05) diminished in bovine fibroblast donor cells compared with embryonic stem-like cells, and surprisingly was 16-fold higher in fetal fibroblasts compared with adult fibroblasts (P < 0.05). Cell passaging and culture periods under serum starvation conditions significantly decreased telomerase activity by approximately 30-50% compared with nontreated early passage cells (P < 0.05). Telomere shortening was observed during in vitro culture of bovine fetal fibroblasts and in very late passages of embryonic stem-like cells. Reprogramming of telomerase activity was apparent by the blastocyst stage of postcloning embryonic development, and telomere lengths were longer (15-23 kb) in cloned fetuses and offspring than the relatively short mean terminal restriction fragment lengths (14-18 kb) observed in adult donor cells. Overall, telomere lengths of cloned fetuses and newborn calves ( approximately 20 kb) were not significantly different from those of age-matched control animals (P > 0.05). These results demonstrate that cloned embryos inherit genomic modifications acquired during the donor nuclei's in vivo and in vitro period but are subsequently reversed during development of the cloned animal.

Animals↗

PubMed2001

Complete replacement of the mitochondrial genotype in a Bos indicus calf reconstructed by nuclear transfer to a Bos taurus oocyte.

Due to the exclusively maternal inheritance of mitochondria, mitochondrial genotypes can be coupled to a particular nuclear genotype by continuous mating of founder females and their female offspring to males of the desired nuclear genotype. However, backcrossing is a gradual procedure that, apart from being lengthy, cannot ascertain that genetic and epigenetic changes will modify the original nuclear genotype. Animal cloning by nuclear transfer using host ooplasm carrying polymorphic mitochondrial genomes allows, among other biotechnology applications, the coupling of nuclear and mitochondrial genotypes of diverse origin within a single generation. Previous attempts to use Bos taurus oocytes as hosts to transfer nuclei from unrelated species led to the development to the blastocyst stage but none supported gestation to term. Our aim in this study was to determine whether B. taurus oocytes support development of nuclei from the closely related B. indicus cattle and to examine the fate of their mitochondrial genotypes throughout development. We show that indicus:taurus reconstructed oocytes develop to the blastocyst stage and produce live offspring after transfer to surrogate cows. We also demonstrate that, in reconstructed embryos, donor cell-derived mitochondria undergo a stringent genetic drift during early development leading, in most cases, to a reduction or complete elimination of B. indicus mtDNA. These results demonstrate that cross-subspecies animal cloning is a viable approach both for matching diverse nuclear and cytoplasmic genes to create novel breeds of cattle and for rescuing closely related endangered cattle.

Animals↗

PubMed2001

Variation in skin reactivity inhalant allergens estimated by the end-point technique.

Skin prick testing is commonly used to diagnose IgE-mediated sensitization to allergens, whereas the longitudinal variability in positive/negative results can be used for clinical and epidemiological purposes. Few publications have investigated the longitudinal variability in skin reactivity, and most studies have concluded that seasonal changes in exposure to environmental allergens are paralleled by a change in skin reactivity. Our trial investigated the relationship between variations in skin reactivity and seasonal exposure to allergens in sensitized subjects, by use of a sensitive method-the end-point technique. Forty-three patients monosensitized to inhalant allergens were selected, and the skin prick end-point technique with serial 1:4 dilutions of standardized commercial allergens was used to assess skin reactivity. Twenty-two patients sensitized to pollens with a short pollination period were tested before and after the pollen season, whereas the other 21 patients sensitized to house dust mites or to a species with a long pollination period such as Parietaria were tested in the same month 12 months later. Patients tested with the end-point technique showed a large interindividual variability in skin reactivity. None of the patients in either group showed a decrease in skin sensitivity with time whereas 9 out of 43 showed no change and 34 out of 43 showed an increase (p < .0001). According to our data, skin reactivity increases in sensitized subjects over time, and this increase seems not to have a circannual rhythm related to the seasonal exposure to environmental allergens, since it can be detected both in pollen- and mite-sensitized patients after a 12-month interval.

Administration, Inhalation↗

PubMed2000

Mitochondrial genotype segregation and effects during mammalian development: applications to biotechnology.

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.

Animals↗

PubMed2000

Benefits and problems with cloning animals.

Animal cloning is becoming a useful technique for producing transgenic farm animals and is likely to be used to produce clones from valuable adults. Other applications will also undoubtedly be discovered in the near future, such as for preserving endangered breeds and species. Although cloning promises great advantages for commerce and research alike, its outcome is not always certain due to high pregnancy losses and high morbidity and mortality during the neonatal period. Research into the mechanisms involved in the reprogramming of the nucleus is being conducted throughout the world in an attempt to better understand the molecular and cellular mechanisms involved in correcting these problems. Although the cause of these anomalies remains mostly unknown, similar phenotypes have been observed in calves derived through in vitro fertilization, suggesting that culture conditions are involved in these phenomena. In the meantime, veterinarians and theriogenologists have an important role to play in improving the efficiency of cloning by finding treatments to assure normal gestation to term and to develop preventative and curative care for cloned neonates.

Animal Husbandry↗

PubMed1999

Effect of equine chorionic gonadotropin on weaning-to-first service interval and litter size of female swine.

We evaluated the effect of PMSG on the weaning-to-first service interval, total litter size and born alive litter size in swine. Four doses of PMSG (0, 500, 750 and 1,000 IU) were administered intramuscularly after weaning to sows at 3 different farms, grouped by parities (1, 2 and 3 or higher) and 2 distinct time periods. The associations among main effects and response variables were assessed by analysis of variance. Polynomial orthogonal terms were used to adjust the estimates of weaning-to-first service interval, total litter size and born alive litter size for the interaction effect of parity and PMSG treatment. The weaning-to-first service interval did not differ across periods and farms (P>0.05), although the interval was shorter (P<0.05) for Parity 3+ sows (4.97 d) than for Parity 1 sows (5.29 d), with no other differences in intervals observed across parities (P>0.05). Time period did not influence litter size (P>0.05), but there were differences in litter size across farms (P<0.05). Both litter size traits were lower for Parity 1 sows than for higher parity sows (P<0.05), but there were no differences in litter size between Parity 2 and 3+ sows (P>0.05). Litter size increased with PMSG dose in both Parities 1 and 2 (P<0.05), but not in Parity 3+ (P>0.05). A significant quadratic effect (P<0.05) of PMSG treatment in weaning-to-first service interval was observed for both Parity 1 and 2 sows, with the shortest intervals occurring with the 750 IU dose for Parity 1 sows. Administration of PMSG after weaning was associated with a shortened weaning-to-first service interval in Parity 1 sows and increased litter size in Parity 1 and 2 sows.

Animals↗

PubMed1999

Developmentally regulated loss and reappearance of immunoreactive somatic histone H1 on chromatin of bovine morula-stage nuclei following transplantation into oocytes.

One difference between chromatin of bovine oocytes and blastomeres is that somatic subtypes of histone H1 are undetectable in oocytes and are assembled onto embryonic chromatin during the fourth cell cycle. We investigated whether this chromatin modification is reversed when nuclei containing somatic H1 are transplanted into ooplasts. Donor nuclei obtained from morula-stage bovine embryos were fused to ooplasts at different times before and after parthenogenetic activation of the ooplasts. After fusion, immunoreactive H1 became undetectable, and the loss occurred more rapidly when fusion was performed near the time of ooplast activation compared with several hours after activation, when the host oocytes were at a stage corresponding to interphase. Although the loss of immunoreactive H1 occurred independently of DNA replication and transcription, exposure of reconstructed oocytes to cycloheximide or 6-dymethylaminopurine (6-DMAP) delayed the loss of immunoreactive H1 from transplanted nuclei. During further development of nuclear-transplant embryos, somatic H1 remained undetectable at the 2- and 4-cell stages, and it reappeared on the chromatin at the 8- to 16-cell stage, as previously observed in unmanipulated embryos. We conclude that factors in oocyte cytoplasm are able to modify morula chromatin so that somatic H1 becomes undetectable, and that the amount or activity of these factors declines over time in activated ooplasts.

Adenine↗

PubMed1999

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

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

Animals↗

PubMed1998

Telophase enucleation: an improved method to prepare recipient cytoplasts for use in bovine nuclear transfer.

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.

Age Factors↗