Effects of prolonged exposure of mouse embryos to elevated temperatures on embryonic developmental competence.

To investigate effects of heat stress on developmental competence, in-vitro fertilized zygotes were incubated at different temperatures until 96 h post human chorionic gonadotrophin (HCG). Under severe and moderate conditions (41°C and 40°C), most embryos did not overcome the 2-cell block. In long-term mild heat stress (39°C until 96 h post HCG), cleavage and blastocyst formation were comparable to non-heat-stress control, but the number of live pups per transferred embryo and mean litter size were significantly affected (P < 0.

Actin depolymerization is associated with meiotic acceleration in cycloheximide-treated ovine oocytes.

Oocytes treated with the protein synthesis inhibitor cycloheximide (CHX) arrest at the germinal vesicle (GV) stage and undergo accelerated GV breakdown (GVBD) after CHX is removed. However, little is known about the underlying mechanism of accelerated meiotic maturation. Here, we investigated this mechanism and found that oocytes released from CHX arrest have higher amounts of cyclin B1 (CCNB1) and phosphorylated mitogen-activated protein kinase (pMAPK) proteins.

Ovine induced pluripotent stem cells are resistant to reprogramming after nuclear transfer.

Induced pluripotent stem cells (iPSCs) share similar characteristics of indefinite in vitro growth with embryonic stem cells (ESCs) and may therefore serve as a useful tool for the targeted genetic modification of farm animals via nuclear transfer (NT). Derivation of stable ESC lines from farm animals has not been possible, therefore, it is important to determine whether iPSCs can be used as substitutes for ESCs in generating genetically modified cloned farm animals. We generated ovine iPSCs by conventional retroviral transduction using the four Yamanaka factors.

Interactions between FGF18 and retinoic acid regulate differentiation of chick embryo limb myoblasts.

During limb development Pax3 positive myoblasts delaminate from the hypaxial dermomyotome of limb level somites and migrate into the limb bud where they form the dorsal and ventral muscle masses. Only then do they begin to differentiate and express markers of myogenic commitment and determination such as Myf5 and MyoD. However the signals regulating this process remain poorly characterised.

Siberian Sturgeon Oocyte Extract Induces Epigenetic Modifications of Porcine Somatic Cells and Improves Developmental Competence of SCNT Embryos.

Somatic cell nuclear transfer (SCNT) has generally demonstrated that a differentiated cell can convert into a undifferentiated or pluripotent state. In the SCNT experiment, nuclear reprogramming is induced by exposure of introduced donor nuclei to the recipient cytoplasm of matured oocytes. However, because the efficiency of SCNT still remains low, a combination of SCNT technique with the ex-ovo method may improve the normal development of SCNT embryos.

Proteomic analysis of early reprogramming events in murine somatic cells incubated with Xenopus laevis oocyte extracts demonstrates network associations with induced pluripotency markers.

The reprogramming of somatic cells into a pluripotent/embryonic-like state holds great potential for regenerative medicine, bypassing ethical issues associated with embryonic stem cells (ESCs). Numerous methods, including somatic cell nuclear transfer (SCNT), fusion to pluripotent cells, the use of cell extracts, and expression of transcription factors, have been used to reprogram cells into ES-like cells [termed induced pluripotent stem cells (iPSCs)]. This study investigated early events in the nuclei of permeabilized murine somatic cells incubated in cytoplasmic extract prepared from Xenopus laevis germinal vesicle-stage oocytes by identifying proteins that showed significant quantitative changes using proteomic techniques.

Production of good-quality blastocyst embryos following IVF of ovine oocytes vitrified at the germinal vesicle stage using a cryoloop.

The cryopreservation of immature oocytes at the germinal vesicle (GV) stage would create an easily accessible, non-seasonal source of female gametes for research and reproduction. The present study investigated the ability of ovine oocytes vitrified at the GV stage using a cryoloop to be subsequently matured, fertilised and cultured in vitro to blastocyst-stage embryos. Selected cumulus-oocyte complexes obtained from mature ewes at the time of death were randomly divided into vitrified, toxicity and control groups.

The combined treatment of calcium ionophore with strontium improves the quality of ovine SCNT embryo development.

Summary Poor embryo quality is a major problem that contributes to the failure of pregnancy in somatic cell nuclear transfer (SCNT). The aims of this study were to improve the quality of ovine SCNT embryos by modifying the conventional activation protocol with the addition of SrCl2. In order to achieve this objective we conducted a series of experiments with in vitro-matured oocytes to optimize conditions for oocyte activation with strontium, and subsequently applied the protocol to SCNT embryos.

In vitro fertilization of ovine oocytes vitrified by solid surface vitrification at germinal vesicle stage.

Cryopreservation of immature oocytes at germinal vesicle (GV) stage would provide a readily available source of oocytes for use in research and allow experiments to be performed irrespective of seasonality or other constraints. This study was designed to evaluate the recovery, viability, maturation status, fertilization events and subsequent development of ovine oocytes vitrified at GV stage using solid surface vitrification (SSV). Cumulus oocyte complexes (COCs) obtained from mature ewes were randomly divided into three groups (1) SSV (oocytes were vitrified using SSV), (2) EXP (oocytes were exposed to vitrification and warming solutions without vitrification) or (3) Untreated (control).

The novel use of modified pig zygotic medium for the efficient culture of the preimplantation mouse embryos.

A high potassium concentration in culture media is considered detrimental to in vitro culture of mouse embryos. Here we show that pig zygotic medium (PZM) containing a higher concentration of potassium, and modified to contain 0.2 mM glucose and 0.

Ovine oocytes vitrified at germinal vesicle stage as cytoplast recipients for somatic cell nuclear transfer (SCNT).

The development of embryos produced by somatic cell nuclear transfer (SCNT) using vitrified oocytes as cytoplast recipients has been reported in cattle but not in sheep. This study investigated the parthenogenetic development of ovine oocytes vitrified and thawed at the germinal vesicle (GV) stage, matured in vitro, and then activated using two activation protocols. The optimal activation protocol was then used to assess development when vitrified oocytes were used as cytoplast recipients for SCNT.

Nuclear-cytoplasmic incompatibility and inefficient development of pig-mouse cytoplasmic hybrid embryos.

Inter-species somatic cell nuclear transfer (iSCNT) embryos usually fail to develop to the blastocyst stage and beyond due to incomplete reprogramming of donor cell. We evaluated whether using a karyoplast that would require less extensive reprogramming such as an embryonic blastomere or the meiotic spindle from metaphase II oocytes would provide additional insight into the development of iSCNT embryos. Our results showed that karyoplasts of embryonic or oocyte origin are no different from somatic cells; all iSCNT embryos, irrespective of karyoplast origin, were arrested during early development.

Reprogramming of ovine somatic cells with Xenopus laevis oocyte extract prior to SCNT improves live birth rate.

The birth of live animals following somatic cell nuclear transfer (SCNT) has demonstrated that oocytes can reprogram the genome of differentiated cells. However, in all species the frequency of development of healthy offspring is low; for example, in sheep, approximately only 5% of blastocysts transferred develop to term, and less than 3% develop to adulthood. Such low efficiencies, coupled with the occurrence of developmental abnormalities, have been attributed to incomplete or incorrect reprogramming.

Caffeine treatment of ovine cytoplasts regulates gene expression and foetal development of embryos produced by somatic cell nuclear transfer.

Treatment of ovine oocytes during the latter stages of maturation in vitro with caffeine, a phosphodiesterase inhibitor, can increase the activities of maturation promoting factor and mitogen-activated protein kinases at metaphase II. When used as cytoplast recipients for somatic cell nuclear transfer (NT), caffeine-treated oocytes produced blastocysts with increased cell numbers. The objectives of these studies were to determine the effects of caffeine treatment on the expression profile of genes involved in early embryonic development and whether induction or maintenance of pregnancy was subsequently altered.

Generation of mtDNA homoplasmic cloned lambs.

Generally in mammals, individual animals contain only maternally inherited mitochondrial DNA (mtDNA), as paternal (sperm)-derived mitochondria are usually eliminated during early development. Somatic cell nuclear transfer (SCNT) bypasses the normal routes of mtDNA inheritance and introduces not only a different nuclear genome into the recipient cytoplast (in general an enucleated oocyte) but also somatic mitochondria. Differences in mtDNA genotype between recipient oocytes and potential mtDNA heteroplasmy due to persistence and replication of somatic mtDNA means that offspring generated by SCNT are not true clones.

Treatment of ovine oocytes with caffeine increases the accessibility of DNase I to the donor chromatin and reduces apoptosis in somatic cell nuclear transfer embryos.

Caffeine treatment of ovine oocytes increases the activity of maturation-promoting factor (MPF) and mitogen-activated protein kinases (MAPKs) and, in somatic cell nuclear transfer (SCNT) embryos, increases the frequency of nuclear envelope breakdown (NEBD) and premature chromosome condensation (PCC). At the blastocyst stage, caffeine-treated SCNT embryos have increased cell numbers. One explanation for this is that NEBD and PCC release chromatin-bound somatic factors, allowing greater access of oocyte factors involved in DNA synthesis and nuclear reprogramming to donor chromatin.

A-type lamin dynamics in bovine somatic cell nuclear transfer embryos.

The persistence of A-type nuclear lamin in somatic cell nuclear transfer (SCNT) embryos has been proposed as a marker for incomplete nuclear reprogramming. Using monoclonal antibodies to A/C- (A/C-346 and A/C-131C3) and B-type lamin, we compared distribution during early development of bovine IVF, parthenogenetic and SCNT embryos. A/C-346 staining was observed in the pronuclei of IVF embryos and in nuclei at the two-cell stage, but was not detected in subsequent cleavage stages up to and including hatched blastocysts.

Epigenetic marks in somatic chromatin are remodelled to resemble pluripotent nuclei by amphibian oocyte extracts.

Reprogramming pluripotency after nuclear transplantation shows that molecules in oocytes can remodel somatic chromatin to a stem cell state. Here we report on an ex-ovo system using axolotl oocyte extracts to remodel epigenetic marks of somatic chromatin. Molecules present in axolotl oocyte extracts induce the reduction of the overall levels of H3K9me3, HP1alpha and DNA methylation of somatic cells, and they increase the levels of H3K9ac.

Differential acetylation of histone H4 lysine during development of in vitro fertilized, cloned and parthenogenetically activated bovine embryos.

The oocyte is remarkable in its ability to remodel parental genomes following fertilization and to reprogram somatic nuclei after nuclear transfer (NT). To characterize the patterns of histone H4 acetylation and DNA methylation during development of bovine gametogenesis and embryogenesis, specific antibodies for histone H4 acetylated at lysine 5 (K5), K8, K12 and K16 residues and for methylated cytosine of CpG dinucleotides were used. Oocytes and sperm lacked the staining for histone acetylation, when DNA methylation staining was intense.

Caffeine treatment prevents age-related changes in ovine oocytes and increases cell numbers in blastocysts produced by somatic cell nuclear transfer.

Maturation-promoting factor (MPF) and mitogen-activated protein kinase (MAPK) are key regulators of both meiotic and mitotic cycles. Oocytes arrested at metaphase of the second meiotic division (MII) contain high levels of both kinases; however, these activities decline with age. Caffeine (an inhibitor of Myt1/Wee1 activity) can increase MPF and MAPK activities in ovine oocytes; however, the effects of caffeine treatment on the activation, nuclear configuration and developmental potential of ovine SC nuclear transfer (SCNT) embryos were unknown.

Spindle position assessment prior to ICSI does not benefit fertilization or early embryo quality.

Intracytoplasmic sperm injection (ICSI) is traditionally performed with the first polar body at 6 or 12 o'clock, and the injection pipette inserted at 3 or 9 o'clock. This positioning aims to direct the path of the injection pipette at a distance from the presumed metaphase II spindle position. Since spindles can now be imaged directly in living oocytes using computer-assisted polarized light microscopy, the effectiveness of this positioning precaution was studied.

A comparison of headfirst and tailfirst microinjection of sperm at intracytoplasmic sperm injection.

Objective: To investigate whether the direction of sperm loading and exit from the injection pipette during intracytoplasmic sperm injection (ICSI) had any bearing on ability to cause fertilization or affect subsequent embryonic development.

Design: Prospective randomized trial.

Setting: Hospital-based IVF center.

Contrasting effects of in vitro fertilization and nuclear transfer on the expression of mtDNA replication factors.

Mitochondrial DNA (mtDNA) is normally only inherited through the oocyte. However, nuclear transfer (NT), the fusion of a donor cell with an enucleated oocyte, can transmit both donor cell and recipient oocyte mtDNA. mtDNA replication is under the control of nuclear-encoded replication factors, such as polymerase gamma (POLG) and mitochondrial transcription factor A (TFAM).