Rediscovering Boveri's centrosome in Ascaris (1888): its impact on human fertility and development.

We rediscover and review the brilliant work of Theodore Boveri, over a 100 years ago, on the centrosome of the round worm Ascaris and show how it impacts on our understanding of human fertilization and embryogenesis. Boveri was able to make fundamental predictions on the mechanics of fertilization and the dominant role of the sperm centrosome (Boveri's rule), which is now applicable to most animals. Using advanced digital imaging by light and electron microscopy, we explore centrosomal dynamics during Ascaris fertilization and the first cell cycle during cleavage.

Nuclear lamin antigen and messenger RNA expression in bovine in vitro produced and nuclear transfer embryos.

The nuclear lamina is a complex meshwork of nuclear lamin filaments that lies on the interface of the nuclear envelope and chromatin and is important for cell maintenance, nucleoskeleton support, chromatin remodeling, and protein recruitment to the inner nucleolus. Protein and mRNA patterns for the major nuclear lamins were investigated in bovine in vitro fertilized (IVF) and nuclear transfer embryos. Expression of lamins A/C and B were examined in IVF bovine germinal vesicle (GV) oocytes, metaphase II oocytes, zygotes, 2-cell, 8-cell, 16-32-cell embryos, morulae, and blastocysts (n = 10).

Production of a cloned calf using zona-free serial nuclear transfer.

The efficiency of generating cloned animals following somatic cell nuclear transfer appears to have reached a plateau, despite ongoing research to improve developmental outcomes. A major limitation appears in the restricted nature of the adult/donor cell to de-differentiate to form a totipotent nucleus. Serial nuclear transfer, a modified cloning technique, has increased the developmental competence of amphibian, murine and porcine cloned embryos.

Comparison of two approaches to nuclear transfer in the bovine: hand-made cloning with modifications and the conventional nuclear transfer technique.

The aim of the present study was to compare the in vitro and in vivo developmental competence of hand-made cloning (HMC) embryos with the conventional nuclear transfer (NT) method using five somatic cell lines and in vitro-fertilised (IVF; control) embryos. Modifications to the HMC procedure included fusion efficiency optimisation, effect of cytoplasmic volume and cloned embryo aggregation. The developmental competence of blastocysts from each of the treatment groups and cell lines used was assessed following transfer to 345 recipients.

Expression profiling of genes crucial for placental and preimplantation development in bovine in vivo, in vitro, and nuclear transfer blastocysts.

Placental abnormalities and failed implantation are characterized phenotypes that occur in many species as a result of somatic cell cloning. This study examines a number of genes, critical for early placental development and reports aberrant expression patterns in a number of cloned bovine blastocysts, thus implicating a role of these genes in failed implantation. Messenger RNA (mRNA) expression of eight genes critical for early placental and preimplantation development including Acrogranin, Cdx2, Eomes, ErbB3, ERR2, Hand1, MRJ, and Rex1 were analyzed in single, in vivo, in vitro, and cloned bovine blastocysts (produced by hand-made cloning (HMC) and serial hand-made cloning (SHMC)) following complementary DNA (cDNA) amplification with a SMART cDNA synthesis kit.

Influence of hormone environment and donor age on cryopreserved common wombat (Vombatus ursinus) ovarian tissue xenografted into nude mice.

Developmentally competent oocytes can be collected from xenografted ovarian tissues; however, optimal xenograft conditions need to be established for this technique to be of use in assisted reproduction. In the present study, common wombat ovarian tissue was xenografted under the kidney capsule of nude mice to clarify the role of recipient gonadal status and donor tissue age on graft establishment, follicle development and oocyte recovery. Eighty-nine per cent of all grafts were recovered; of these, 78% contained growing follicles.

A QF-PCR system to detect chromosome 13 aneuploidy from as few as ten cells.

Complete and partial trisomies of chromosome 13 are characterized by abnormal fetal development and birth defects. Despite the severe abnormalities associated with trisomy 13, some couples elect not to undergo invasive prenatal diagnosis (PND) due to the 0.5%-1.

Retinoic acid induces Pdx1-positive endoderm in differentiating mouse embryonic stem cells.

We have generated an embryonic stem (ES) cell line in which sequences encoding green fluorescent protein (GFP) were targeted to the locus of the pancreatic-duodenal homeobox gene (Pdx1). Analysis of chimeric embryos derived from blastocyst injection of Pdx1(GFP/w) ES cells demonstrated that the pattern of GFP expression was consistent with that reported for the endogenous Pdx1 gene. By monitoring GFP expression during the course of ES cell differentiation, we have shown that retinoic acid (RA) can regulate the commitment of ES cells to form Pdx1(+) pancreatic endoderm.

Human embryonic stem cells form embryoid bodies containing visceral endoderm-like derivatives.

Objective: The objective of this study was to determine the potential of human embryonic stem (hES) cells to provide an in vitro model of human extraembryonic endoderm development.

Methods: The hES cell line HES-2 was propagated in Dulbecco's modification of Eagle's medium (DMEM) in the presence of 20% fetal calf serum (FCS) on a mouse embryonic fibroblast feeder layer. Clumps of approximately 50-100 cells were transferred to fresh DMEM and FCS and grown as embryoid bodies (EBs) in suspension culture.

Derivation, propagation and differentiation of human embryonic stem cells.

Embryonic stem (ES) cells are in vitro cultivated pluripotent cells derived from the inner cell mass (ICM) of the embryonic blastocyst. Attesting to their pluripotency, ES cells can be differentiated into representative derivatives of all three embryonic germ layers (endoderm, ectoderm and mesoderm) both in vitro and in vivo. Although mouse ES cells have been studied for many years, human ES cells have only more recently been derived and successfully propagated.

Birth of a cloned calf derived from a vitrified hand-made cloned embryo.

The hand-made cloning (HMC) technique describes a simplified nuclear transfer process without the need for micromanipulators. The technique involves manual bisection of zona-free oocytes, selection of cytoplasts by Hoechst staining and fusion of a single somatic cell and two cytoplasts. In this proof-of-principle experiment, the objective was to examine the developmental competence of HMC embryos following embryo transfer.

Effect of ovariectomy and graft position on cryopreserved common wombat (Vombatus ursinus) ovarian tissue following xenografting to nude mice.

Ovarian tissue xenografting may be applied to increase the population size of rare or endangered animals. However, optimal grafting conditions, such as graft position and recipient hormonal status, are yet to be established. The present study, using common wombat ovarian tissue, showed that development of xenografted ovarian tissue to the antral follicle stage can be achieved irrespective of graft position.

In vitro maturation of oocytes from non-stimulated common wombats.

Assisted reproductive techniques, such as in vitro oocyte maturation in conjunction with in vitro fertilisation, may be used as a tool to manipulate reproduction. Using the common wombat as a model for the critically endangered northern hairy-nosed wombat, the present study examined whether oocyte maturation could be achieved under field conditions. At the time of collection, no oocytes were at the metaphase II (MII) stage (0/42).

Effects of electrical stimulation and seminal plasma on the motility of mouse sperm.

The effects of electric current (in vivo and in vitro) and seminal plasma on epididymal and ejaculated sperm obtained from C57BL x CBA and C57BL/6J mice were investigated by studying motility parameters, fertilization and embryo development. Electroejaculates were obtained by applying a series of computer-generated sinusoidal alternating currents (0.25-3.

Simplified technique for differential staining of inner cell mass and trophectoderm cells of mouse and bovine blastocysts.

Histological staining and counting of blastocyst inner cell mass (ICM) and trophectoderm (TE) cells differentially with chromatin-specific dyes is a more accurate indicator of cultured blastocyst quality and normality than total cell number assessment. The aim of this study was to test the effectiveness of a simplified method of chemically-defined differential blastocyst staining. The TE of cultured mouse and bovine blastocysts of different developmental stages was stained when blastocysts were treated with a permeabilizing solution containing the ionic detergent Triton X-100 and the fluorochrome propidium iodide.

Glucose metabolism of human morula and blastocyst-stage embryos and its relationship to viability after transfer.

The pregnancy rate and implantation rate following blastocyst transfer in the human have been reported to be high; however, it has remained necessary to transfer 2-3 blastocysts to achieve these rates. Morphological criteria are currently used to select blastocysts for transfer and have some limited correlation with ongoing viability. Glucose metabolism of 189 human morula to blastocyst stage embryos was analysed using a non-invasive ultramicrofluorescence technique to determine if this could be used to predict viability.

Fertilization of mouse oocytes using somatic cells as male germ cells.

Female and male mouse somatic cells were injected into mouse F(1) oocytes. The cells used included cumulus cells (female) and muscle derived fibroblasts (male). The ability of the cells to fertilize oocytes and support embryonic development was examined.

Semen collection from mice: electroejaculation.

The effects of device type (electrostimulator, function generator or computer-generated waveforms), waveform (square, triangle or sine wave), probe type (ring or strip) and anaesthetic compound (ketamine/xylazine combination or pentobarbitone sodium) were investigated on electroejaculation (EEJ) responses of C57B1 x CBA and C57Bl/6J mice. Ejaculates were analysed for total sperm count and motility variables using computer-assisted sperm analyses. Automated computer-generated waveforms delivered through a sound card were more effective and reproducible compared with waveforms generated by function generator and electrostimulator.

Transgenic strategy for demonstrating nuclear reprogramming in the mouse.

Totipotency can be restored to the nuclei of somatic cells by reprogramming the nucleus via the technique of nuclear transfer. As genes expressed in somatic cells differ from those expressed in early embryos, a change in gene expression must accompany nuclear reprogramming. In this study, nuclear reprogramming of somatic cell nuclei following nuclear transfer (NT) was demonstrated by the reactivation of developmentally regulated lacZ reporter genes.

Selected genetic factors associated with male infertility.

Studies into the mechanisms underlying spermatogenesis, the process by which spermatogonia undergo meiosis to become spermatozoa, have identified a number of genetic determinants of male infertility. Indeed, a more comprehensive knowledge of the genetic regulation of spermatogenesis has alleviated the dependence on the use of idiopathic infertility as a classification for sterile men for whom a cause for their infertility is unknown, as genetic factors become more accountable for this phenotype. This review focuses on selected areas implicated in male infertility including: (i) autosomal and sex chromosomal abnormalities; (ii) genetic disorders associated with impaired gonadotrophin secretion or action; (iii) microdeletions within regions of the Y-chromosome containing candidate gene families for spermatogenesis; (iv) the genetic nexus between cystic fibrosis and congenital bilateral absence of the vas deferens; and (v) insights into human infertility as gleaned from animal studies into mechanisms involving the Bcl-2 family of apoptosis regulators and the interaction between the c-kit encoded tyrosine kinase receptor and its ligand, stem cell factor.

Technical advances and pitfalls on the way to human cloning.

There exists a widespread consensus that the cloning of human beings to term would be detrimental to both the mother and child and of little value to society. However, the ambition of a few organisations and the recent advances in cellular and molecular technologies that led to the cloning of Dolly the sheep, for example, have meant that such a procedure will be possible if not illegal in the near future. The science associated with the cloning technologies practiced in other mammalian species reported to date provide important advances in our understanding of how cells function during early developmental processes and commit themselves to specific developmental pathways.

Cell synchronization for the purposes of nuclear transfer in the bovine.

We have examined the reprogramming ability of donor fibroblast nuclei in various phases of the cell cycle, upon transfer to cytoplasts, using a bovine nuclear transfer (NT) model. Bovine fetal fibroblasts were cultured in reduced serum and conditioned medium to induce quiescence (G0) and treated with nocodazole to induce M phase arrest. Unsynchronized actively dividing cells (control) were mainly in G1.

Experimental models for ovarian tissue and immature follicles.

Primordial and growing follicles are abundant within the ovaries of healthy young female mammals. While our understanding of follicular dynamics is based mainly on studies of normal ovaries in intact animals, techniques such as ovarian grafting and in vitro culture, in particular when used in combination with cryopreservation, have provided both significant additional insights and a source of mature oocytes. Primordial follicles are small, quiescent, and most commonly located within the collagen-rich outer portion of the ovary.

Identification and characterisation of known and novel transcripts expressed during the final stages of human oocyte maturation.

The final stages of oocyte maturation, from the germinal vesicle (GV) stage to metaphase II (MII) oocytes, are characterised by a series of dynamic events. These include germinal vesicle break down (GVBD), resumption of meiosis, and nuclear and cytoplasmic maturation to produce MII oocytes ready for fertilisation. To investigate the specific genes transcribed during these stages of oogenesis, we have prepared and analysed amplified cDNA representing the transcribed genes in a series of GV and MII oocytes.

Ooplasmic donation in humans: the potential for epigenic modifications.

Ooplasm donation, wherein ooplasm is transferred from a donor oocyte to a recipient oocyte in an effort to increase embryo viability, has been applied in the human, with resulting pregnancies and births. Neither the safety nor efficacy of this method has been adequately investigated. Mitochondrial heteroplasmy in the blood of children conceived using ooplasm donation has recently been described.