Geraldine M. Hartshorne

Non-conservation of mammalian preimplantation methylation dynamics

In mammals, active demethylation of sperm pronuclear DNA shortly after fertilisation is thought to be important for reprogramming subsequent embryonic development [1–3]. A further passive loss of methylation has been observed as DNA replicates between 2-cell and morula stages, with somatic cell levels being re-established at or after the blastocyst stage when differentiated lineages are first formed [1,3,4]. We now demonstrate non-conservation in the DNA methylation dynamics of the sheep embryo and suggest this challenges the perceived role of DNA methylation in mammalian preimplantation development. A dramatic loss of cytosine methylation from the male pronucleus has previously been observed in mouse, pig and cow with a 5-methylcytosine antibody [1]. By contrast, we did not observe loss of methylation from either pronucleus in the in vivo-derived ovine zygote (Figure 1A). With the same immunostaining technique, we observed asymmetry in pronuclear methylation in mouse embryos as reported previously, ruling out procedural differences (Figure 1B). Examination of pronuclear stages revealed demethylation in the human zygote (Figure 1C) and no demethylation in the rabbit zygote (Figure 1D). In contrast with a previous study [1], we observed only partial asymmetric demethylation in the bovine zygote (Figure 1E). Collectively, this suggests that demethylation of the paternal genome is not an obligate requirement for early mammalian development. Also in contrast to the mouse and cow [1], we observed no passive demethylation throughout sheep preimplantation development upon purely visual inspection, but rather an apparent increase between the 8-cell and morula stages (Figure 2A–E). Only at the blastocyst stage is demethylation visible in the sheep trophectoderm, whereas the cells of the inner cell mass (ICM) remain methylated (Figure 2F). This might be important as trophectoderm cells are the first differentiated cell type to form during development and trophoblast-specific gene expression is essential for embryonic nutrition and implantation. Quantification of confocal images (Figure 2G), demonstrates a significant decrease in methylation intensity from the 2-cell to the 8-cell stage (43%, p < 0.05). However, nuclear size also decreases between these stages (41%, p < 0.01), thus the ratio of mean methylation intensity to nuclear size was not different. Moreover, quantification revealed that the apparent increase observed between the 8-cell and morula stages is also a visual artefact. A simple explanation is that methylation levels do not increase but nuclear intensities appear higher due to the increased nuclear compaction (55% size reduction from 8-cell to morula (p < 0.001, Figure 2G); we …

Patterns of Meiotic Recombination in Human Fetal Oocytes

Abnormal patterns of meiotic recombination (i.e., crossing-over) are believed to increase the risk of chromosome nondisjunction in human oocytes. To date, information on recombination has been obtained using indirect, genetic methods. Here we use an immunocytological approach, based on detection of foci of a DNA mismatch-repair protein, MLH1, on synaptonemal complexes at prophase I of meiosis, to provide the first direct estimate of the frequency of meiotic recombination in human oocytes. At pachytene, the stage of maximum homologous chromosome pairing, we found a mean of 70.3 foci (i.e., crossovers) per oocyte, with considerable intercell variability (range 48-102 foci). This mean equates to a genetic-map length of 3,515 cM. The numbers and positions of foci were determined for chromosomes 21, 18, 13, and X. These chromosomes yielded means of 1.23 foci (61.5 cM), 2.36 foci (118 cM), 2.5 foci (125 cM), and 3.22 foci (161 cM), respectively. The foci were almost invariably located interstitially and were only occasionally located close to chromosome ends. These data confirm the large difference, in recombination frequency, between human oocytes and spermatocytes and demonstrate a clear intersex variation in distribution of crossovers. In a few cells, chromosomes 21 and 18 did not have any foci (i.e., were presumptively noncrossover); however, configurations that lacked foci were not observed for chromosomes 13 and X. For the latter two chromosome pairs, the only instances of absence of foci were observed in abnormal cells that showed chromosome-pairing errors affecting these chromosomes. We speculate that these abnormal fetal oocytes may be the source of the nonrecombinant chromosomes 13 and X suggested, by genetic studies, to be associated with maternally derived chromosome nondisjunction.

Uterine Selection of Human Embryos at Implantation

Human embryos frequently harbor large-scale complex chromosomal errors that impede normal development. Affected embryos may fail to implant although many first breach the endometrial epithelium and embed in the decidualizing stroma before being rejected via mechanisms that are poorly understood. Here we show that developmentally impaired human embryos elicit an endoplasmic stress response in human decidual cells. A stress response was also evident upon in vivo exposure of mouse uteri to culture medium conditioned by low-quality human embryos. By contrast, signals emanating from developmentally competent embryos activated a focused gene network enriched in metabolic enzymes and implantation factors. We further show that trypsin, a serine protease released by pre-implantation embryos, elicits Ca2+ signaling in endometrial epithelial cells. Competent human embryos triggered short-lived oscillatory Ca2+ fluxes whereas low-quality embryos caused a heightened and prolonged Ca2+ response. Thus, distinct positive and negative mechanisms contribute to active selection of human embryos at implantation.

Apoptosis in mouse fetal and neonatal oocytes during meiotic prophase one

BackgroundThe vast majority of oocytes formed in the fetal ovary do not survive beyond birth. Possible reasons for their loss include the elimination of non-viable genetic constitutions arising through meiosis, however, the precise relationship between meiotic stages and prenatal apoptosis of oocytes remains elusive. We studied oocytes in mouse fetal and neonatal ovaries, 14.5–21 days post coitum, to examine the relationship between oocyte development and programmed cell death during meiotic prophase I.ResultsMicrospreads of fetal and neonatal ovarian cells underwent immunocytochemistry for meiosis- and apoptosis-related markers. COR-1 (meiosis-specific) highlighted axial elements of the synaptonemal complex and allowed definitive identification of the stages of meiotic prophase I. Labelling for cleaved poly-(ADP-ribose) polymerase (PARP-1), an inactivated DNA repair protein, indicated apoptosis. The same oocytes were then labelled for DNA double strand breaks (DSBs) using TUNEL. 1960 oocytes produced analysable results.Oocytes at all stages of meiotic prophase I stained for cleaved PARP-1 and/or TUNEL, or neither. Oocytes with fragmented (19.8%) or compressed (21.2%) axial elements showed slight but significant differences in staining for cleaved PARP-1 and TUNEL to those with intact elements. However, fragmentation of axial elements alone was not a good indicator of cell demise. Cleaved PARP-1 and TUNEL staining were not necessarily coincident, showing that TUNEL is not a reliable marker of apoptosis in oocytes.ConclusionOur data indicate that apoptosis can occur throughout meiotic prophase I in mouse fetal and early postnatal oocytes, with greatest incidence at the diplotene stage. Careful selection of appropriate markers for oocyte apoptosis is essential.

Oogenesis and cell death in human prenatal ovaries: what are the criteria for oocyte selection?

Prenatal oogenesis produces hundreds of thousands of oocytes, most of which are discarded through apoptosis before birth. Despite this large-scale selection, the survivors do not constitute a perfect population, and the factors at the cellular level that result in apoptosis or survival of any individual oocyte are largely unknown. What then are the selection criteria that determine the size and quality of the ovarian reserve in women? This review focuses on new data at the cellular level, on human prenatal oogenesis, offering clues about the importance of the timing of entry to meiotic prophase I by linking the stages and progress through MPI with the presence or absence of apoptotic markers. The characteristics and responsiveness of cultured human fetal ovarian tissue at different gestational ages to growth factor supplementation and the impact of meiotic abnormalities upon apoptotic markers are discussed. Future work will require the use of a tissue culture model of prenatal oogenesis in order to investigate the fate of individual live oocytes at different stages of development.

Telomere lengths in human oocytes, cleavage stage embryos and blastocysts.

Telomeres are repeated sequences that protect the ends of chromosomes and harbour DNA repair proteins. Telomeres shorten during each cell division in the absence of telomerase. When telomere length becomes critically short, cell senescence occurs. Telomere length therefore reflects both cellular ageing and capacity for division. We have measured telomere length in human germinal vesicle (GV) oocytes and preimplantation embryos, by quantitative fluorescence in situ hybridization (Q-FISH), providing baseline data towards our hypothesis that telomere length is a marker of embryo quality. The numbers of fluorescent foci suggest that extensive clustering of telomeres occurs in mature GV stage oocytes, and in preimplantation embryos. When calculating average telomere length by assuming that each signal presents one telomere, the calculated telomere length decreased from the oocyte to the cleavage stages, and increased between the cleavage stages and the blastocyst (11.12 versus 8.43 versus 12.22 kb, respectively, P < 0.001). Other methods of calculation, based upon expected maximum and minimum numbers of telomeres, confirm that telomere length in blastocysts is significantly longer than cleavage stages. Individual blastomeres within an embryo showed substantial variation in calculated average telomere length. This study implies that telomere length changes according to the stage of preimplantation embryo development.

Telomere lengths in human pronuclei, oocytes and spermatozoa

Telomeres are chromosome ends that control functions related to cell division. Short telomeres are proposed to underlie infertility, female reproductive ageing and abnormal embryogenesis, but there is little direct evidence on telomere length in gametes and embryos. The aim of this study was to measure telomere lengths in individual human oocytes, spermatozoa, male and female pronuclei, in order to compare parental contributions to telomere lengths in the human zygote. Quantitative fluorescence in situ hybridization was used to measure average telomere length in pronuclei of oocytes fertilized for research using a known fertile sperm sample. Pronuclei derived from male and female gametes were distinguished by 5-methylcytosine staining. Results were compared with those for unfertilized mature and immature oocytes and individual spermatozoa decondensed in vitro. Fifty unselected men and one sperm donor provided semen samples and 32 women donated oocytes surplus to IVF treatment. Telomeres in mature oocytes and female pronuclei were significantly longer than those in individual spermatozoa and male pronuclei (P < 0.0001). Telomeres were longer in immature oocytes than in mature oocytes (P < 0.04). Sperm telomere length increased with male age (P < 0.05). Neither sperm nor oocyte telomere lengths were significantly associated with clinical parameters or outcome of treatment. In conclusion, telomere length measurements directly comparing human pronuclei under identical conditions show that male-derived telomeres are shorter on average than female-derived telomeres at fertilization. We propose that from this starting point, telomere lengths are probably modified by recombination events in the oocyte until telomerase increases at the blastocyst stage. Our findings do not support the use of gamete telomere lengths as a fertility diagnostic tool.

Altered patterns of meiotic recombination in human fetal oocytes with asynapsis and/or synaptonemal complex fragmentation at pachytene.

Meiotic recombination was analysed in human fetal oocytes to determine whether recombination errors are associated with abnormal chromosome synapsis. Immunostaining was used to identify the synaptonemal complex (SC, the meiosis-specific proteinaceous structure that binds homologous chromosomes) and the DNA mismatch repair protein, MLH1, that locates recombination foci. It was found that 57.1-74.2% of zygotene oocytes showed fragmentation and/or defective chromosome synapsis. Fewer such abnormal cells occurred at pachytene (15.8-28.9%). MLH1 foci were present from zygotene to diplotene in both normal and abnormal oocytes. However, the proportions of oocytes having MLH1 foci, and mean numbers of foci per oocyte, were both lower in abnormal oocytes. Oocytes with fragmented SC had more foci than those with synaptic anomalies. Analysis of chromosomes 13, 18, 21 and X by fluorescence in-situ hybridization (FISH) did not implicate particular chromosomes in recombination deficiency. These observations indicate that recombination is disturbed in oocytes with SC fragmentation and/or synaptic abnormalities during meiotic prophase I. Such disturbances might be a risk factor for selection of fetal oocytes for atresia, as occurs for homologous chromosome pairing. Recombination errors may potentially increase the risk of abnormal chromosome segregation in oocytes that survive and contribute to the reserve in the mature ovary.

Unique geometry of sister kinetochores in human oocytes during meiosis I may explain maternal age-associated increases in chromosomal abnormalities

ABSTRACT The first meiotic division in human oocytes is highly error-prone and contributes to the uniquely high incidence of aneuploidy observed in human pregnancies. A successful meiosis I (MI) division entails separation of homologous chromosome pairs and co-segregation of sister chromatids. For this to happen, sister kinetochores must form attachments to spindle kinetochore-fibres emanating from the same pole. In mouse and budding yeast, sister kinetochores remain closely associated with each other during MI, enabling them to act as a single unified structure. However, whether this arrangement also applies in human meiosis I oocytes was unclear. In this study, we perform high-resolution imaging of over 1900 kinetochores in human oocytes, to examine the geometry and architecture of the human meiotic kinetochore. We reveal that sister kinetochores in MI are not physically fused, and instead individual kinetochores within a pair are capable of forming independent attachments to spindle k-fibres. Notably, with increasing female age, the separation between kinetochores increases, suggesting a degradation of centromeric cohesion and/or changes in kinetochore architecture. Our data suggest that the differential arrangement of sister kinetochores and dual k-fibre attachments may explain the high proportion of unstable attachments that form in MI and thus indicate why human oocytes are prone to aneuploidy, particularly with increasing maternal age. Summary: Sister kinetochores in meiosis I human oocytes are not physically fused, with the degree of separation increasing with maternal age. This may have implications for the high incidence of aneuploidy in human oocytes.

Influence of p53 and genetic background on prenatal oogenesis and oocyte attrition in mice

BACKGROUND Meiotic progression, and the number of oocytes surviving to birth, determine the ovarian reserve, yet the control of prenatal oogenesis is poorly understood. We investigated the effects of genetic background and p53 upon oogenesis in mice. METHODS Fetal and neonatal ovaries were analysed in B6CBf1 and B6CBf2 mice from 15.5 to 21 days post-coitum (dpc) and p53 (a tumour suppressor gene) knockout, heterozygous and wild-type mice from 15.5 to 16 dpc. Oocytes in meiotic prophase I (MPI) were identified by labelling synaptonemal complex protein 3, and the specific stage of MPI was classified by the appearance of axial elements. Apoptosis and DNA breaks were assessed by cleaved poly-(ADP-ribose) polymerase (PARP-1) and terminal deoxynucleotidyltransferase-mediated dUTP nick-end labelling (TUNEL), respectively. RESULTS The leptotene, zygotene and pachytene stages were earlier in f1 than f2 generations with significant differences at all stages (P< or =0.002). The p53(-/-) oocyte populations and those with the presence of p53 gene differed significantly in terms of: (i) the proportion of oocytes reaching specific stages of MPI on 15.5 and 16 dpc and (ii) the proportion of oocytes having observed abnormalities in synaptonemal complexes (P < 0.001). The absence of p53 resulted in faster progression of oocytes and more with compressed and abnormal axial elements. We observed significant differences between p53(-/-), p53(+/-) and p53(+/+) mice in terms of cleaved PARP-1 staining and TUNEL. CONCLUSION Genotype has an important impact on prenatal meiosis and oocyte apoptosis. p53 affects the speed of oocyte development and may influence the oocyte selection through apoptosis during MPI.