Search PubMed⌕ Search

Biomedical subjects

Alan O Trounson

Publications and source records attributed to Alan O Trounson.

At least 19 recordsLinked to original sources
PubMed2007

Optimization of a microarray based approach for deriving representative gene expression profiles from human oocytes.

The purpose of the present study was to optimize a protocol for deriving reproducible and representative gene expression profiles from very rare research samples of human oocytes using microarrays. Immature oocytes produced as a result of administration of gonadotrophins for the treatment of infertility were donated to research. Linear amplification (L-amp) and exponential amplification (E-amp) were both capable of generating sufficient product for hybridization to the microarrays even from the low amount of template mRNA present in a single human oocyte. Slightly higher numbers of transcripts were detected by microarray following linear rather than E-amp but both techniques generated a product with reliably reproducible sensitivity and fidelity providing oocytes were pooled in minimum numbers of three. The majority of the variance associated with amplification and hybridization to arrays comes from the molecular processing. Slightly greater additional variance is associated with biological differences in immature oocytes from the same or different donors. The findings suggest that representative gene expression profiles can be generated from human oocytes for comparative purposes following L-amp and hybridization to microarray. However, oocytes must be pooled for the starting template for each array and sufficient independent microarray experiments performed to minimize the variance associated with molecular processing.

Female↗
PubMed2006

Developmental effects of sublethal mitochondrial injury in mouse oocytes.

Mitochondrial dysfunction may be acquired or inherited by oocytes without detectable morphological abnormalities. This pathology may account for some examples of unexplained pregnancy loss in women following transfer of morphologically normal in vitro fertilization (IVF) embryos. The present study was intended to determine whether sublethal mitochondrial injury in mouse oocytes before IVF negatively affects pre- and postimplantation development, and to further define the latency of developmental compromise in relation to aberrant mitochondrial metabolism. Mature mouse oocytes were loaded with the mitochondrial fluorophore rhodamine-123 and photosensitized for 20 sec, a duration previously found to permit preimplantation embryo development to the blastocyst stage and so deemed "sublethal." This treatment resulted in some aberrations in cytoplasmic patterning of organelles, but did not inhibit zygote mitochondrial metabolism. Blastocyst development following IVF was not significantly inhibited following sublethal oocyte photosensitization; however, a decrease in trophectoderm cell numbers was observed relative to untreated controls. Following intrauterine transfer, blastocysts derived from sublethally photosensitized oocytes implanted but later aborted at a higher rate, formed fetuses with lower average weights, and, in rare cases, formed abnormal fetuses relative to controls. Photosensitization for more prolonged durations resulted in failed fertilization (2 min) and rapid oocyte degeneration (10 min). Therefore, photosensitization duration and the consequent degree of mitochondrial dysfunction are negatively related to the onset of developmental compromise. Acquired low-level mitochondrial injury is heritable by the resultant embryos and can cause postimplantation developmental compromise that may be relevant to some clinically observed outcomes following human assisted reproduction strategies, including reduced birth weights for gestational age. Future strategies for the detection and prevention of mitochondrial dysfunction may assist in improving outcomes for some clinically infertile women.

Animals↗
PubMed2006

From oogonia to mature oocytes: inactivation of the maternal centrosome in humans.

The fine structure of human oogonia and growing oocytes has been reviewed in fetal and adult ovaries. Preovulatory maturation and the ultrastructure of stimulated oocytes from the germinal vesicle (GV) stage to metaphase II (MII) stage are also documented. Oogonia have large nuclei, scanty cytoplasm with complex mitochondria. During folliculogenesis, follicle cell processes establish desmosomes and deep gap junctions at the surface of growing oocytes, which are retracted during the final stages of maturation. The zona pellucida is secreted in secondary follicles. Growing oocytes have mitochondria, Golgi, rough endoplasmic reticulum (RER), ribosomes, lysosomes, and lipofuscin bodies, often associated with Balbiani bodies and have nuclei with reticulated nucleoli. Oocytes from antral follicles show numerous surface microvilli and cortical granules (CGs) separated from the oolemma by a band of microfilaments. The CGs are evidently secreted by Golgi membranes. The GV oocytes have peripheral Golgi complexes associated with a single layer of CGs close to the oolemma. They have many lysosomes, and nuclei with dense compact nucleoli. GV breakdown occurs by disorganization of the nuclear envelope and the oocyte enters a transient metaphase I followed by MII, when it is arrested and ovulated. Maturation of oocytes in vitro follows the same pattern of meiosis seen in preovulatory oocytes. The general organization of the human oocyte conforms to that of most other mammals but has some unique features. The MII oocyte has the basic cellular organelles such as mitochondria, smooth endoplasmic reticulum, microfilaments, and microtubules, while Golgi, RER, lysosomes, multivesicular, residual and lipofuscin bodies are very rare. It neither has yolk nor lipid inclusions. Its surface has few microvilli, and 1-3 layers of CGs, aligned beneath the oolemma. Special reference has been made to the reduction and inactivation of the maternal centrosome during oogenesis. The MII spindle, often oriented perpendicular to the oocyte surface, is barrel-shaped, anastral and lacks centrioles. Osmiophilic centrosomes are not demonstrable in human eggs, since the maternal centrosome is nonfunctional. However, oogonia and growing oocytes have typical centrioles, similar to those of somatic cells. The sperm centrosome activates the egg and organizes the sperm aster and mitotic spindles of the embryo, after fertilization.

Centrosome↗
PubMed2006

Future and applications of cloning.

The birth of viable offspring from somatic cell nuclear transfer (SCNT) in mammals caused a major re-examination of the understanding of the commitment of cells to specific tissue lineages during differentiation. The questions of whether cells undergo dedifferentiation or transdifferentiation during the development of offspring and how these changes are controlled is a source of ongoing debate that is yet to be resolved. Irrespective of the outcome of this debate, it is clear that cloning using SCNT has a place and purpose in the future of research and animal breeding. The future uses of SCNT could include the production of transgenic mice, the production of transgenic livestock and assisting with the re-establishment of endangered species. Human medicine also would benefit from future use of SCNT because it would allow the production of patient-specific embryonic stem cells.

Animals↗
PubMed2006

Formation of human prostate tissue from embryonic stem cells.

Rodent models and immortalized or genetically modified cell lines are frequently used-but have limited utility-for studying human prostate development and maturation. Using rodent mesenchyme to establish reciprocal mesenchymal-epithelial cell interactions with human embryonic stem cells (hESCs), we generated human prostate tissue expressing prostate-specific antigen (PSA) within 8-12 weeks. This human prostate model shows species-conserved signalling mechanisms that could extend to integumental, gastrointestinal and genital tissues.

Animals↗
PubMed2005

Mouse embryonic stem cell colonisation of carbonated apatite surfaces.

Apatites play a crucial role in the body and have been used extensively in biomedical implants. The influence on stem cell behaviour is not known and so this study will explore whether sintered carbonated apatites are favourable for propagation of stem cells. Different weight substitutions of carbonated apatite, specifically 2.5 wt% (2.5 wt%CAP) and 5 wt% (5 wt%CAP), were sintered and characterised prior to the investigation of their potential as a matrix for the support of mouse embryonic stem (ES) cells. Characterisation of the apatites included elemental analysis, X-ray diffraction, surface roughness, specific surface area, density, and solubility. The ability of carbonated apatite to support mouse ES cell colonisation and maintenance in the presence of leukaemia inhibitory factor was determined by an enumeration of live versus dead cells within a population, and immunoreactivity to Oct4, a transcription factor and stem cell marker, following growth on each matrix. It was found that while both compositions allowed for the colonisation of mouse ES cells, the cells were not maintained in an undifferentiated state, as evidenced by a reduction in the number of cells staining positive for Oct4 expression. This study shows that an increase in carbonate content within sintered apatites leads to a higher cell number, a desired aspect for stem cells to populate scaffolds intended for tissue engineering. This study presents carbonated apatites as a suitable matrix for the initial colonisation and differentiation of ES cells for tissue engineering applications.

Animals↗
PubMed2005

Transcripts from a human primordial follicle cDNA library.

BACKGROUND: Human primordial follicles (PFs) or the oocyte-pre-granulosa complex, constitute the earliest and most immature stage of human oogenesis. The factors, signalling networks and the precise role of the oocyte and the pre-granulosa cells in initiating growth and recruitment from this finite resting pool remain largely unknown at present. METHODS: To obtain a gene resource of this oogenesis stage and thereby determine a molecular blueprint of the human PF, a cDNA library was constructed from 50 isolated human PFs using the phagemid vector pTriplEx2. RESULTS: Sequence analysis showed that 46.67% of these clones corresponded to known genes while 29.48% were uncharacterized genes that included hypothetical proteins, human cDNA clones and novel genes. Bioinformatics analysis revealed a preponderance of mitochondrial genes and repeat elements followed by ribosomal proteins, transcription and translation genes. Transcripts for heat shock proteins, cell cycle, embryogenesis genes and apoptosis genes were identified. Members of the ubiquitin-proteasome pathway, MAPK, p38/JNK, GPCR, Wnt, NF-kappaB and notch signalling pathways were identified. A mitochondrial pathway and a transcription factor pathway in the human PF were generated. The gene networks in the transcription factor pathway provided a first glimpse of the balance between proliferation and cell death/apoptosis in this earliest stage of oogenesis. CONCLUSIONS: The abundance and diversity of retroviral elements and transcriptional repressor genes in the human PF suggest these could contribute to the maintainance of this oogenesis stage. The role of these genes in initial recruitment and in subsequent oogenesis stages will be greatly facilitated and elucidated by printing a human PF cDNA array of the sequenced clones and using it for gene profiling.

Cell Differentiation↗
PubMed2005

Effect of female age on mouse oocyte developmental competence following mitochondrial injury.

Oocytes from aging ovaries contain mitochondria with morphological and genetic flaws. How these flaws relate to phenotypes of oocyte developmental compromise associated with clinical infertility is not well understood. This study was conducted to investigate the role of mitochondria in the developmental compromises observed with female aging using a mouse model of mitochondrial dysfunction. Oocytes obtained from aging (30-40 wk) (C57BL/6J x CBACaH)F1 (B6CBAF1) hybrid female mice were photosensitized with mitochondrial fluorophore rhodamine-123 for variable durations and compared to similarly treated oocytes derived from pubertal mice (4-6 wk). Blastocyst development of normally fertilized oocytes from both age-groups correlated negatively in mathematically unique profiles with irradiation time, with a more sudden decline in development for oocytes from aging mice. Complete inhibition of blastocyst development occurred following a shorter duration of photosensitization for oocytes from aging compared to pubertal animals (60 vs. 90 sec). Prolonged photosensitization resulted in mitochondrial uncoupling and promoted localized generation of reactive oxygen species, mitochondrial permeabilization, and apoptotic phenotypes. Thus, aging oocytes are more developmentally sensitive to mitochondrial damage than pubertal oocytes but undergo similar metabolic and apoptotic responses. These and future findings may encourage further optimization of laboratory-based strategies to minimize mitochondrial injury to oocytes, particularly those from older women, and improve clinical outcomes for women with age-related etiologies of infertility.

Age Factors↗
PubMed2005

Chromosome 21 mosaic human preimplantation embryos predominantly arise from diploid conceptions.

OBJECTIVE: High rates of chromosomal mosaicism in human IVF embryos question the accuracy of preimplantation genetic diagnosis, and, with the majority of embryo transfers still resulting in no pregnancy, chromosomal mosaicism is likely to be a contributing factor to human IVF failure. The aim of this study was to investigate the origin and nature of chromosome 21 (Ch21) cell division errors in human IVF embryos. DESIGN: Perform single cell Ch21 allelic profiling on human IVF embryos. SETTING: Academic research environment. PATIENT(S): Women of advanced maternal age (> 35 yrs) (n = 65) undergoing infertility treatment; and amniocytes/chorionic cells from trisomy 21 pregnancies (n = 28). INTERVENTION(S): Cells were analyzed by single cell allelic profiling, MAIN OUTCOME MEASURE(S): The origin and nature of cell division errors. RESULT(S): The vast majority of Ch21 mosaic embryos (approximately 80%) originated from diploid conceptions. In contrast, all fetal trisomy 21 originated from aneuploid conceptions. Increasing maternal age was significantly associated with aneuploid conceptions, meiotic cell division error, and adverse pregnancy outcome (P < .05). The mean daily FSH dose that produced embryos with normal Ch21 cell division was significantly lower than the mean daily FSH dose that produced embryos with mitotic Ch21 cell division errors (P < .01) and embryos with meiotic cell division errors (P < .05). CONCLUSION(S): Chromosomal mosaicism of Ch21 in human IVF embryos predominantly originate from diploid conceptions. Further understanding of chromosomal mosaicism with respect to IVF parameters, such as daily FSH dose, may eventually lead to improvements in IVF outcomes.

Adult↗
PubMed2005

Stereomicroscopic and histological examination of bovine embryos following extended in vitro culture.

Attempts to support survival of mammalian embryos after hatching have met with limited success, although some mouse studies have reported growth at the post-implantation stage. The aim of the present research was to establish and characterise an in vitro culture system that could support extended growth and differentiation of bovine embryos. Abattoir-derived oocytes were matured and fertilised in vitro. Presumptive zygotes were cultured in modified synthetic oviduct fluid (SOFaaci) medium supplemented with 5% cow serum (CS). On Day 9, single hatched blastocysts (n = 160) were randomly allocated to SOFaaci supplemented with either 5% bovine serum albumin, 5% CS, 5% fetal calf serum (FCS) or SOF only and cultured on a collagen gel substrate for up to 45 days. Embryos were evaluated at various time-points until complete disaggregation or the total disappearance of embryonic cells. Blastocyst viability post hatching was severely compromised in protein-free SOFaaci medium. Addition of FCS generated increased embryonic growth for the longest time period (Day 45) when compared to the other groups. Long-term survival of embryonic cells was observed stereomicroscopically by the proliferation and development of three-dimensional tubular structures to 85% confluence in culture. Haematoxylin and eosin staining of morphological structures obtained from all treatment groups revealed embryos displaying trophoblast, inner cell mass and hypoblast development to varying degrees. Regardless of treatment, extended in vitro culture did not result in development comparable with that described for in vivo embryos. In the present work, however, there was evidence of extended culture of bovine embryos beyond that achieved previously. However, further research is required to identify the exact requirements for extended in vitro culture for bovine embryos.

Animals↗
PubMed2005

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. Vitrification was also used to enable management of recipient resources and to assess the susceptibility of membranes to cryopreservation following zona removal. Increasing cytoplasmic volume to 150% or aggregating two embryos improved the blastocyst development rate and increased the total cell number. Although HMC embryo transfers established a significantly higher pregnancy rate on Day 30 than fresh IVF or NT embryo transfers, the overall outcome in terms of cloned live births derived from either fresh or vitrified/thawed HMC or NT embryo transfers across the five cell lines did not differ. The birth and continued survival of clones produced with HMC technology with equivalent efficiency to NT shows that it can be used as an alternative method for the generation of cloned offspring in the bovine.

Animals↗
PubMed2004

Mitochondrial dysfunction in mouse oocytes results in preimplantation embryo arrest in vitro.

Oocyte mitochondrial dysfunction has been proposed as a cause of high levels of developmental retardation and arrest that occur in human preimplantation embryos generated using assisted reproductive technology in the treatment of some causes of female infertility. To investigate this, a model of mitochondrial dysfunction was developed in mouse oocytes using a method of photosensitization of the mitochondrion-specific dye, rhodamine-123. After in vitro fertilization, dye-loaded and photosensitized oocytes showed developmental arrest in proportion to irradiation time. Morphological and metabolic assessments of zygotes indicated an increase in mitochondrial permeability that subsequently resulted in apoptotic degeneration. Development was partially restored by inhibition of mitochondrial permeability transition pore formation by oocyte pretreatment with cyclosporin A. Oocyte mitochondria are therefore physiological regulators of early embryo development and potential sites of pathological insult that may perturb oocyte and subsequent preimplantation embryo viability. These findings have important implications for the treatment of clinically infertile women using assisted reproductive technologies.

Animals↗
PubMed2004

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. Many biochemical differences and culture requirements between mouse and human ES cells have been described, yet despite these differences the study of murine ES cells has provided important insights into methodologies aimed at generating a greater and more in depth understanding of human ES cell biology. One common feature of both mouse and human ES cells is their capacity to undergo controlled differentiation into spheroid structures termed embryoid bodies (EBs). EBs recapitulate several aspects of early development, displaying regional-specific differentiation programs into derivatives of all three embryonic germ layers. For this reason, EB formation has been utilised as an initial step in a wide range of studies aimed at differentiating both mouse and human ES cells into a specific and desired cell type. Recent reports utilising specific growth factor combinations and cell-cell induction systems have provided alternative strategies for the directed differentiation of cells into a desired lineage. According to each one of these strategies, however, a relatively high cell lineage heterogeneity remains, necessitating subsequent purification steps including mechanical dissection, selective media or fluorescent or magnetic activated cell sorting (FACS and MACS, respectively). In the future, the ability to specifically direct differentiation of human ES cells at 100% efficiency into a desired lineage will allow us to fully explore the potential of these cells in the analysis of early human development, drug discovery, drug testing and repair of damaged or diseased tissues via transplantation.

Animals↗
PubMed2004

Human embryonic stem cells: prospects for development.

It is widely anticipated that human embryonic stem (ES) cells will serve as an experimental model for studying early development in our species, and, conversely, that studies of development in model systems, the mouse in particular, will inform our efforts to manipulate human stem cells in vitro. A comparison of primate and mouse ES cells suggests that a common underlying blueprint for the pluripotent state has undergone significant species-specific modification. As we discuss here, technical advances in the propagation and manipulation of human ES cells have improved our understanding of their growth and differentiation, providing the potential to investigate early human development and to develop new clinical therapies.

Animals↗
PubMed2003

Birth of rats following nuclear exchange at the 2-cell stage.

We report full-term development of nuclear transfer embryos following nuclear exchange at the 2-cell stage. Nuclei from 2-cell rat embryos were transferred into enucleated 2-cell embryos and developed to term after transfer to recipients (NT2). Pronuclear exchange in zygotes was used for comparison (NT1). Zygotes and 2-cell embryos were harvested from 4-week-old female Sprague-Dawley rats. Nuclear transfer was performed by transferring the pronuclei or karyoplasts into the perivitelline space of recipient embryos followed by electrofusion to reconstruct embryos. Fused couplets were cultured for 4 or 24 h before being transferred into day 1 pseudopregnant recipients (Hooded Wistar) at the 1- or 2-cell stage. In vitro culture was also carried out to check the developmental competence of the embryos. In vitro development to the blastocyst stage was not significantly different between the two groups (NT1, 34.3%; NT2, 45.0%). Two of three recipients from NT1 and two of five recipients from NT2 became pregnant. Six pups (3 from NT1, 3 from NT2) were delivered from the four foster mothers. Three female pups survived; 2 from NT1 and 1 from NT2. At 2 months of age these pups appeared healthy, and were mated with Sprague-Dawley males. One rat derived from NT1 delivered 15 pups (5 males, 10 females) as did the rat from NT2 (7 males, 8 females). Our results show that by using karyoplasts from 2-cell stage embryos as nuclear donors and reconstructing them with enucleated 2-cell embryos, healthy rats can be produced.

Animals↗
PubMed2003

Handmade somatic cell cloning in cattle: analysis of factors contributing to high efficiency in vitro.

Widespread application of somatic cell cloning has been hampered by biological and technical problems, which include complicated and time-consuming procedures requiring skilled labor. Recently, zona-free techniques have been published with limited or no requirement for micromanipulators. The purpose of the present work was to optimize certain steps of the micromanipulator-free (i.e., handmade) procedure, to analyze the morphology of the developing blastocysts, and to explain factors involved in the high efficiencies observed. Optimization of the procedure included selection of the appropriate medium for enucleation, orientation of pairs at fusion, timing of fusion, and culture conditions. As a result of these improved steps, in vitro efficiency as measured by blastocysts per reconstructed embryo and blastocysts per working hour was among the highest described so far. The cattle serum used in our experiments was superior to other protein sources for in vitro embryo development. One possible explanation of this effect is the considerable mitogenic activity of the cattle serum compared with that of commercially available fetal calf serum. Morphological analysis of blastocysts by inverted microscopy, inner cell mass-trophoblast differential staining, and transmission electron microscopy revealed high average quality. A high initial pregnancy rate was achieved after the transfer of single blastocysts derived by aggregation of two nuclear transfer embryos into recipients. The improved handmade somatic cell nuclear transfer method may become a useful technology as a simple, inexpensive, and efficient alternative to traditional somatic cell nuclear transfer.

Animals↗
PubMed2002

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. Differential display analysis disclosed that the overall gene expression profiles between different samples of GV oocytes are very similar, regardless of their source, while those between the MII oocytes are markedly variable. A comparison of expression profiles in oocytes and somatic (cumulus) cells identified several known genes preferentially-expressed in oocytes (e.g., a zona pellucida gene), as well as five novel sequences. Two of the five novel sequences are homologous to retrotransposon sequences, long terminal repeat (LTR) and long interspersed nuclear element (LINE) 1, and two other sequences show partial homology to known ESTs and genomic sequences. The remaining sequence, which is identical to shorter ESTs isolated from germ cell tumor cDNA libraries, was extended towards its 5' end by PCR, using the original cDNA preparation from which it was isolated as a template. Expression of the resultant 1.1-kb transcript is restricted to the testis and ovary, and its expression correlates with cell pluripotency in that it is expressed in embryonal carcinoma cells, but not in their differentiated derivative cells.

Amino Acid Sequence↗