Biomedical subjects

I Lagutina

Publications and source records attributed to I Lagutina.

7 recordsLinked to original sources

PubMed2006

Developmental competence of equine oocytes and embryos obtained by in vitro procedures ranging from in vitro maturation and ICSI to embryo culture, cryopreservation and somatic cell nuclear transfer.

Development of assisted reproductive technologies in horses has been relatively slow compared to other domestic species, namely ruminants and pigs. The scarce availability of abattoir ovaries and the lack of interest from horse breeders and breed associations have been the main reasons for this delay. Progressively though, the technology of oocyte maturation in vitro has been established followed by the application of ICSI to achieve fertilization in vitro. Embryo culture was initially performed in vivo, in the mare oviduct or in the surrogate sheep oviduct, to achieve the highest embryo development, in the range of 18-36% of the fertilised oocytes. Subsequently, the parallel improvement of in vitro oocyte maturation conditions and embryo culture media has permitted high rates of embryo development from in vitro matured and in vitro cultured ICSI embryos, ranging from 5 to 10% in the early studies to up to 38% in the latest ones. From 2003, with the birth of the first cloned equids, the technology of somatic cell nuclear transfer has also become established due to improvement of the basic steps of embryo production in vitro, including cryopreservation. Pregnancy and foaling rates are still estimated based on a small number of in vitro produced equine embryos transferred to recipients. The largest set of data on non-surgical embryo transfer of in vitro produced embryos, from ICSI of both abattoir and in vitro-matured Ovum Pick Up (OPU) oocytes, and from somatic cell nuclear transfer, has been obtained in our laboratory. The data demonstrate that equine embryos produced by OPU and then cryopreserved can achieve up to 69% pregnancy rate with a foaling rate of 83%. These percentages are reduced to 11 and 23%, respectively, for cloned embryos. In conclusion, extensive evidence exists that in vitro matured equine oocytes can efficiently develop into viable embryos and offspring.

Animals↗

PubMed2005

Establishment, differentiation, electroporation, viral transduction, and nuclear transfer of bovine and porcine mesenchymal stem cells.

Mesenchymal stem cells (MSCs) reside in the bone marrow and have the potential for multilineage differentiation, into bone, cartilage, and fat, for example. In this study, bovine and porcine MSCs were isolated, cultured to determine their replication ability, and differentiated with osteogenic medium and 5-azacytine. Both bovine and porcine undifferentiated MSCs were electroporated and virally transduced to test the efficiency of genetic modification and the maintainance of differentiation ability thereafter. Nuclear transfer experiments were carried out with bovine and porcine MSCs, both at the undifferentiated state and following differentiation. Our results indicate that bovine and porcine MSCs have limited lifespans in vitro--approximately 50 population doublings. They can be efficiently differentiated and characterized along the osteogenic lineage by morphology, alkaline phosphatase, Von Kossa, oil red stainings, and RT-PCR. Electroporation and selection induce high levels of EGFP expression in porcine but not in bovine MSCs. Following genetic modification, MSCs retain their pluridifferentiation ability as parental cells. Cloned embryos derived from bovine and porcine undifferentiated MSCs and their derivatives along the osteogenic lineage give rise to consistently high preimplantation development comparable to adult fibroblasts.

Animals↗

PubMed2004

Production and quality of bovine oocytes and embryos.

Many factors influence the efficiency of the in vitro embryo production technology in cattle but the most important are the physiological conditions of the donor and the culture protocols for oocyte maturation and fertilization and for embryo culture from zygote to blastocyst. Therefore, general factors such as age, body conditions and herd management play a pivotal role together with more specific factors such as reproductive soundness and ovarian cyclicity. Given that good quality and competent oocytes are available a complex series of processes, including oocyte maturation, fertilization and culture of the derived zygotes, must be completed to generate viable embryos.

Animals↗

PubMed2003

Bovine embryo technologies.

Embryo technologies are a combination of assisted reproduction, cellular and molecular biology and genomic techniques. Their classical use in animal breeding has been to increase the number of superior genotypes but with advancement in biotechnology and genomics they have become a tool for transgenesis and genotyping. Multiple ovulation and embryo transfer (MOET) has been well established for many years and still accounts for the majority of the embryos produced worldwide. However, no progress has been made in the last 20 years to increase the number of transferable embryos and to reduce the side effects on the reproductive performance of the donors. In vitro embryo production (IVP) is a newer and more flexible approach, although it is technically more demanding and requires specific laboratory expertise and equipment that are most important for the quality of the embryos produced. Somatic cell cloning is a rapidly developing area and a very valuable technique to copy superior genotypes and to produce or copy transgenic animals. More knowledge in oocyte and embryo biology is expected to shed new light on the early developmental events, including epigenetic changes and their long lasting effect on the newborn.Embryo technologies are here to stay and their use will increase as advances in the understanding of the mechanisms governing basic biological processes are made.

Animals↗

PubMed2001

Bovine oocytes treated prior to in vitro maturation with a combination of butyrolactone I and roscovitine at low doses maintain a normal developmental capacity.

Butyrolactone I (BL-I) and Roscovitine (ROS), two specific and potent inhibitors of M-phase promoting factor (MPF) kinase activity, were used to block germinal vesicle breakdown (GVBD) of cattle oocytes. A concentration 6.25 microM BL-I and 12.5 microM ROS blocked over 93.3 +/- 2.5% of oocytes in germinal vesicle (GV) stage during a 24-hr culture period. Following a second 24-hr culture step in maturation medium (IVM) almost all (91.5 +/- 3.0%) inhibited oocytes resumed meiosis and reached the metaphase II (MII) stage. The MII kinetics was different for inhibited and control oocytes. Fifty percent MII was reached at 13-14 hr in BL-I + ROS treated oocytes, compared to 18 hr in control oocytes. Therefore, control oocytes were fertilised (IVF) after 22 hr IVM and inhibited oocytes after 16 or 22 hr IVM. After IVF, percentage of grade 1 freezable embryos on day 7 (D + 7) as well as percentage of blastocyst formation on D + 8 in the group of BL-I + ROS treated oocytes fertilised after 16 hr IVM were higher (P < 0.05) compared with the other experimental group fertilised after 22 hr IVM but not different in comparison with the control. Survival to freezing and thawing of grade 1 embryos frozen on D + 7 was employed as viability criteria and was similar in all groups. Thus, the presence of BL-I + ROS in the prematuration medium of bovine oocytes determines a reversible meiotic block, without compromising their subsequent developmental competence.

4-Butyrolactone↗

PubMed2001

The ABC transporter Bcrp1/ABCG2 is expressed in a wide variety of stem cells and is a molecular determinant of the side-population phenotype.

Stem cells from bone marrow, skeletal muscle and possibly other tissues can be identified by the 'side-population' (SP) phenotype. Although it has been assumed that expression of ABC transporters is responsible for this phenotype, the specific molecules involved have not been defined. Here we show that expression of the Bcrp1 (also known as Abcg2 murine/ABCG2 human) gene is a conserved feature of stem cells from a wide variety of sources. Bcrp1 mRNA was expressed at high levels in primitive murine hematopoietic stem cells, and was sharply downregulated with differentiation. Enforced expression of the ABCG2 cDNA directly conferred the SP phenotype to bone-marrow cells and caused a reduction in maturing progeny both in vitro and in transplantation-based assays. These results show that expression of the Bcrp1/ABCG2 gene is an important determinant of the SP phenotype, and that it might serve as a marker for stem cells from various sources.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMedDate not supplied

Effect of donor cell age on the efficiency of nuclear transfer in rabbits.

The ability of rabbit fibroblasts of different ages to be reprogrammed following nuclear transfer (NT) to aged recipient oocytes was evaluated. The rate of NT blastocysts reconstructed with presumptive G1 stage morula cells or fetal fibroblasts was significantly higher (41.5% and 51.4%) than was those of cloned embryos reconstructed with fibroblasts from young (4-month-old) or aged (5-year-old) animals (16.7% and 7.1%, respectively, P < 0.025). Serum starvation significantly increased the development of NT embryos to the morula-blastocyst stage (67.6% versus 22.9%, P < 0.025). Transfer of 168 NT embryos derived from nuclei of morula cells and 106 control embryos into 21 recipients resulted in 10 pregnancies, 2 NT and 18 control pups, respectively. In the first experiment, transfer of 142 cleaved NT embryos reconstructed with fetal fibroblasts and 86 control embryos into eight recipient does resulted in five pregnancies and the birth of 20 control pups. In the second experiment, after transfer of 112 NT embryos derived from fetal fibroblasts into six recipients, 10 (8.9%) sites of implantation were revealed in two does (33.3%) on day 14 of gestation. This study provides evidence that nuclei of morula cells and fetal and adult fibroblasts differ in their ability to be reprogrammed by recipient cytoplasm following nuclear transfer.

Animals↗