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Keith E Latham

Publications and source records attributed to Keith E Latham.

At least 19 recordsLinked to original sources

Somatic cell nuclei in cloning: strangers traveling in a foreign land.

The recent successes in producing cloned offspring by somatic cell nuclear transfer are nothing short of remarkable. This process requires the somatic cell chromatin to substitute functionally for both the egg and the sperm genomes, and indeed the processing of the transferred nuclei shares aspects in common with processing of both parental genomes in normal fertilized embryos. Recent studies have yielded new information about the degree to which this substitution is accomplished. Overall, it has become evident that multiple aspects of genome processing and function are aberrant, indicating that the somatic cell chromatin only infrequently manages the successful transition to a competent surrogate for gamete genomes. This review focuses on recent results revealing these limitations and how they might be overcome.

Animals↗

Analysis of polysomal mRNA populations of mouse oocytes and zygotes: dynamic changes in maternal mRNA utilization and function.

Transcriptional activation in mammalian embryos occurs in a stepwise manner. In mice, it begins at the late one-cell stage, followed by a minor wave of activation at the early two-cell stage, and then the major genome activation event (MGA) at the late two-cell stage. Cellular homeostasis, metabolism, cell cycle, and developmental events are orchestrated before MGA by time-dependent changes in the array of maternal transcripts being translated. Many elegant studies have documented the importance of maternal mRNA (MmRNA) and its correct recruitment for development. Many other studies have illuminated some of the molecular mechanisms regulating MmRNA utilization. However, neither the complete array of recruited mRNAs nor the regulatory mechanisms responsible for temporally different patterns of recruitment have been well characterized. We present a comprehensive analysis of changes in the maternal component of the zygotic polysomal mRNA population during the transition from oocyte to late one-cell stage embryo. We observe global transitions in the functional classes of translated MmRNAs and apparent changes in the underlying cis-regulatory mechanisms.

Animals↗

Optimization of procedures for cloning by somatic cell nuclear transfer in mice.

Cloning by somatic cell nuclear transfer is a complex procedure that is dependent on correct interactions between oocyte and donor cell genome. These interactions require minimal insult to either the oocyte or the transplanted nucleus. Available data also indicate that reprogramming the donor cell genome may be slow, so that the cloned embryo expresses genes typical of the donor cell, and thus has different characteristics from normal embryos. Procedures that minimize damage to the donor genome and that address the unique characteristics of the cloned construct should enhance the efficacy of the method.

Animals↗

The Primate Embryo Gene Expression Resource in embryology and stem cell biology.

The analysis of temporal patterns of gene expression in embryos is an essential component of any research program seeking to understand molecular mechanisms that control development. Little is known of early regulatory mechanisms that operate in primate oocytes and preimplantation-stage embryos. Such studies have been hindered by the cost of obtaining, and limited availability of, non-human primate oocytes and embryos, and by ethical and legal constraints on studies of human embryos. Over the past 4 years we have established the Primate Embryo Gene Expression Resource (PREGER) to circumvent these limitations. A set of over 200 samples of rhesus monkey oocytes and embryos has been converted to cDNA libraries, which are, in turn, used for a variety of molecular analyses. Both the libraries and cDNA dot blots can be distributed free of charge to anyone wishing to study gene expression at these stages. This includes providing an inexpensive and rapid method for confirming and extending results of gene discovery approaches such as microarray analysis. PREGER includes an on-line resource with a database and other useful tools for embryologists. The resource is being expanded to incorporate samples from other species and from embryonic stem cells.

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Non-equivalence of embryonic and somatic cell nuclei affecting spindle composition in clones.

Cloning by nuclear transfer remains inefficient but is more efficient when nuclei from embryonic cells or embryonic stem cells (ECNT) are employed as compared with somatic cells (SCNT). The factors determining efficiency have not been elucidated. We find that somatic and embryonic nuclei differ in their ability to organize meiotic and mitotic spindles of normal molecular composition. Calmodulin, a component of meiotic and mitotic spindle chromosome complexes (SCCs), displays sharply reduced association with the SCC forming after SCNT but not ECNT. This defect persists in mitotic spindles at least through the second mitosis, despite abundant calmodulin expression in the cell, and correlates with slow chromosome congression. We propose that somatic cell nuclei lack factors needed to direct normal SCC formation in oocytes and early embryos. These results reveal a striking control of SCC formation by the transplanted nucleus and provide the first identified molecular correlate of donor stage-dependent restriction in nuclear potency.

Animals↗

X chromosome reactivation and regulation in cloned embryos.

Somatic cell nuclear transfer embryos exhibit extensive epigenetic abnormalities, including aberrant methylation and abnormal imprinted gene expression. In this study, a thorough analysis of X chromosome inactivation (XCI) was performed in both preimplantation and postimplantation nuclear transfer embryos. Cloned blastocysts reactivated the inactive somatic X chromosome, possibly in a gradient fashion. Analysis of XCI by Xist RNA and Eed protein localization revealed heterogeneity within cloned embryos, with some cells successfully inactivating an X chromosome and others failing to do so. Additionally, a significant proportion of cells contained more than two X chromosomes, which correlated with an increased incidence of tetraploidy. Imprinted XCI, normally found in preimplantation embryos and extraembryonic tissues, was not observed in blastocysts or placentae from later stage clones, although fetuses recapitulated the Xce effect. We conclude that, although SCNT embryos can reactivate, count, and inactivate X chromosomes, they are not able to regulate XCI consistently. These results illustrate the heterogeneity of epigenetic changes found in cloned embryos.

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Maternal transmission ratio distortion at the mouse Om locus results from meiotic drive at the second meiotic division.

We have observed maternal transmission ratio distortion (TRD) in favor of DDK alleles at the Ovum mutant (Om) locus on mouse chromosome 11 among the offspring of (C57BL/6 x DDK) F(1) females and C57BL/6 males. Although significant lethality occurs in this backcross ( approximately 50%), differences in the level of TRD found in recombinant vs. nonrecombinant chromosomes among offspring argue that TRD is due to nonrandom segregation of chromatids at the second meiotic division, i.e., true meiotic drive. We tested this hypothesis directly, by determining the centromere and Om genotypes of individual chromatids in zygote stage embryos. We found similar levels of TRD in favor of DDK alleles at Om in the female pronucleus and TRD in favor of C57BL/6 alleles at Om in the second polar body. In those embryos for which complete dyads have been reconstructed, TRD was present only in those inheriting heteromorphic dyads. These results demonstrate that meiotic drive occurs at MII and that preferential death of one genotypic class of embryo does not play a large role in the TRD.

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Developmental regulation and in vitro culture effects on expression of DNA repair and cell cycle checkpoint control genes in rhesus monkey oocytes and embryos.

DNA repair is essential for maintaining genomic integrity, and may be required in the early embryo to correct damage inherited via the gametes, damage that arises during DNA replication, or damage that arises in response to exposure to genotoxic agents. The capacity of preimplantation stage mammalian embryos to repair damaged DNA has not been well characterized, particularly in primate embryos. In this study, we examined the expression of 48 mRNAs related to sensing different kinds of DNA damage, repairing that DNA damage, and controlling the cell cycle to provide an opportunity for DNA repair. The expression data reveal dynamic temporal changes, indicating a changing ability of the rhesus embryo to detect and repair different kinds of DNA damage. Low expression or overexpression of specific DNA repair genes may limit the ability of the embryo to respond to DNA damage at certain stages. Additionally, our data reveal that in vitro culture may lead to dysregulation of many such genes and a potentially impaired ability to repair DNA damage, thus affecting cellular viability and long-term embryo viability via effects on genome integrity. This effect of in vitro culture on nonhuman primate embryos may be relevant to assessing the potential advantages and disadvantages of prolonged in vitro culture of human embryos.

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Species-dependent expression patterns of DNA methyltransferase genes in mammalian oocytes and preimplantation embryos.

DNA methyltransferases (DNMTs) comprise a family of proteins involved in the establishment and maintenance of DNA methylation patterns in the mammalian genome. DNA methylation involves the transfer of the methyl group of the coenzyme S-adenosyl-L-methionine to the 5 position of cytosine residues within CpG dinucleotides. DNA methylation is implicated in the control of imprinted genes expression, X chromosome silencing, development of certain types of cancer, and embryonic development. DNA methylation is also believed to protect the genome from parasitic elements such as transposons, retrotransposons, and viruses. The aim of this study was to analyze the expression patterns of DNMT1, DNMT2, DNMT3A, DNMT3B, and DNMT3L genes in rhesus macaque (Macaca mulatta) oocytes and preimplantation stage embryos from fertilization to the hatched blastocyst stage, and to compare these results with the expression profiles in the mouse and other mammalian species. We describe species-dependent differences as well as similarities in expression patterns of DNMT genes among mammals.

Animals↗

X chromosome imprinting and inactivation in preimplantation mammalian embryos.

Dosage compensation for the mammalian X chromosome involves the silencing of one X chromosome to achieve equal X-linked gene expression between males and females. X chromosome inactivation (XCI) is controlled by a complex set of genetic elements located in a region known as the X chromosome inactivation center, and is regulated by a combination of genomic imprinting, cell lineage-dependent erasure of imprinting, an unidentified mechanism of X chromosome counting, an incompletely understood means of selection of one X chromosome for inactivation and developmentally regulated changes in X chromosome chromatin. A detailed understanding of when and how these components of XCI occur is essential for elucidating the operative mechanisms. A model accounting for early events related to XCI, including observations in uniparental and aneuploid embryos, is presented.

Aneuploidy↗

Early and delayed aspects of nuclear reprogramming during cloning.

The successful production of viable progeny following adult somatic cell nuclear transfer (cloning) provides exciting new opportunities for basic research for investigating early embryogenesis, for the propagation of valuable or endangered animals, for the production of genetically engineered animals, and possibly for developing therapeutically valuable stem cells. Successful cloning requires efficient reprogramming of gene expression to silence donor cell gene expression and activate an embryonic pattern of gene expression. Recent observations indicate that reprogramming may be initiated by early events that occur soon after nuclear transfer, but then continues as development progresses through cleavage and probably to gastrulation. Because reprogramming is slow and progressive, cloned embryos have dramatically altered characteristics in comparison with fertilized embryos. Events that occur early following nuclear transfer may be essential prerequisites for the later events. Additionally, the later reprogramming events may be inhibited by sub-optimum culture environments that exist because of the altered characteristics of cloned embryos. By addressing the unique requirements of cloned embryos, the entire process of reprogramming may be accelerated, thus increasing cloning efficiency.

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Effects of follicle size and oocyte maturation conditions on maternal messenger RNA regulation and gene expression in rhesus monkey oocytes and embryos.

The relationship between alterations in gene expression and differences in developmental potential in primate oocytes and embryos was examined. Oocytes from 3 sources were used for these studies: 1) in vivo-matured oocytes from monkeys stimulated with FSH and hCG, 2) in vitro-matured oocytes from large follicles of monkeys primed with FSH, and 3) in vitro-matured oocytes from small follicles from nonstimulated (NS) monkeys. Following in vitro fertilization, embryos from these oocytes displayed high, moderate, and low developmental competence, respectively. Oocytes from NS females displayed aberrant accumulation of a number of maternal mRNAs, followed by precocious loss of many maternal mRNAs by the 2-cell stage. Embryos from NS oocytes displayed alterations in expression of key transcription factors after the 8-cell stage. Oocytes and embryos from FSH-stimulated females also displayed alterations in gene expression relative to hCG-stimulated females, but these alterations were much less severe than those observed for NS oocytes and embryos. Our data are consistent with the hypothesis that continued development and maturation of the oocyte within the ovarian follicle in vivo facilitates the production of oocytes of the highest developmental potential, and that in vitro conditions may not support this process as effectively due to differences in the extracellular milieu. These observations are relevant to understanding the role of the in vivo environment on oocyte maturation, and the potential effects of in vitro maturation on human assisted reproduction methods.

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Maternal factors controlling blastomere fragmentation in early mouse embryos.

Interactions between sperm and egg are required to maintain embryo viability and cellular integrity. Differential transcriptional activities and epigenetic differences that include genomic imprinting provide mechanisms by which complementary parental genome functions support early embryogenesis. We previously showed that cytofragmentation can be influenced by the specific combination of maternal and paternal genotypes. Using maternal pronuclear transfer in mouse embryos, we examined the cellular basis for the maternal genotype effect. We found that the maternal genotype effect is predominantly controlled by the maternal pronucleus, with a lesser role played by the ooplasm. This effect of the maternal pronucleus is sensitive to alpha-amanitin treatment. The effect of the maternal component of the embryonic genome on cytofragmentation constitutes the earliest known effect of the embryonic genome on mammalian embryo phenotype. The results also indicate that clinical procedures seeking to define or manipulate oocyte quality in humans should take into account early effects of the embryonic genome, particularly the maternal genome.

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Recapitulation of the ovum mutant (Om) phenotype and loss of Om locus polarity in cloned mouse embryos.

The ovum mutant (Om) locus in mice affects early interactions between sperm and egg that in turn affect viability of embryos beyond the morula stage. Crosses of DDK females to males of many other inbred strains are 95% lethal around the morula stage, whereas reciprocal crosses are fully viable. Available data indicate that the early lethality is the result of an interaction between a factor in the ooplasm and the paternal genome. In this study, we examined whether this lethal interaction would likewise occur in cloned embryos produced by somatic cell nuclear transfer. We find that the Om effect is recapitulated but that the parental origin effect at the Om locus is no longer evident in cloned embryos.

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Rapid H1 linker histone transitions following fertilization or somatic cell nuclear transfer: evidence for a uniform developmental program in mice.

H1 linker histones (H1s) are key regulators of chromatin structure and function. The functions of different H1s during early embryogenesis, and mechanisms regulating their associations with chromatin are largely unknown. The developmental transitions of H1s during oocyte growth and maturation, fertilization and early embryogenesis, and in cloned embryos were examined. Oocyte-specific H1FOO, but not somatic H1s, associated with chromatin in oocytes (growing, GV-stage, and MII-arrested), pronuclei, and polar bodies. H1FOO associated with sperm or somatic cell chromatin within 5 min of intracytoplasmic sperm injection (ICSI) or somatic cell nuclear transfer (SCNT), and completely replaced somatic H1s by 60 min. The switching from somatic H1s to H1FOO following SCNT was developmentally regulated. H1FOO was replaced by somatic H1s during the late two- and four-cell stages. H1FOO association with chromatin can occur in the presence of a nuclear envelope and independently of pronucleus formation, is regulated by factors associated with the spindle, and is likely an active process. All SCNT constructs recapitulated the normal sequence of H1 transitions, indicating that this alone does not signify a high developmental potential. A paucity of all known H1s in two-cell embryos may contribute to precocious gene transcription in fertilized embryos, and the elaboration of somatic cell characteristics in cloned embryos.

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The primate embryo gene expression resource: a novel resource to facilitate rapid analysis of gene expression patterns in non-human primate oocytes and preimplantation stage embryos.

Detailed molecular studies of preimplantation stage development in a suitable nonhuman primate model organism have been inhibited due to the cost and scarcity of embryos. To circumvent these limitations, we have created a new resource for the research community, designated as the Primate Embryo Gene Expression Resource (PREGER). The PREGER sample collection currently contains over 160 informative samples of oocytes, obtained from various sized antral follicles, and embryos obtained through a variety of different protocols. The PREGER makes it possible to undertake quantitative gene-expression studies in rhesus monkey oocytes and embryos through simple and cost-effective hybridization-based methods. The PREGER also makes available other molecular tools to facilitate nonhuman primate embryology. We used PREGER here to compare the temporal expression patterns of five housekeeping mRNAs and three transcription factor mRNAs between mouse and rhesus monkey. We observed noticeable differences in temporal expression patterns between species for some mRNAs, but clear similarities for others. Our results also provide new information related to genome activation and the effects of embryo culture conditions on gene expression in primate embryos. These results provide one illustration of how the PREGER can be employed to obtain novel insight into primate embryogenesis.

Animals↗

Expression of genes encoding chromatin regulatory factors in developing rhesus monkey oocytes and preimplantation stage embryos: possible roles in genome activation.

One of the most critical events of preimplantation development is the successful activation of gene transcription. Both the timing and the array of genes activated must be controlled. The ability to regulate gene transcription appears to be reduced just prior to the time of the major genome activation event, and changes in chromatin structure appear essential for establishing this ability. Major molecules that modulate chromatin structure are the linker and core histones, enzymes that modify histones, and a wide variety of other factors that associate with DNA and mediate either repressive or activating changes. Among the latter are chromatin accessibility complexes, SWI/SNF complexes, and the YY1 protein and its associated factors. Detailed information about the expression and regulation of these factors in preimplantation stage embryos has not been published for any species. In order to ascertain which of these factors may participate in chromatin remodeling, genome activation, and DNA replication during early primate embryogenesis, we determined the temporal expression patterns of mRNA encoding these factors. Our data identify the predominant members of these different functional classes of factors expressed in oocytes and embryos, and reveal patterns of expression distinct from those patterns seen in somatic cells. Among each of four classes of mRNAs examined, some mRNAs were expressed predominantly in the oocyte, with these largely giving way to others expressed stage specifically in the embryo. This transition may be part of a global mechanism underlying the transition from maternal to embryonic control of development, wherein the oocyte program is silenced and an embryonic pattern of gene expression becomes established. Possible roles for these mRNAs in chromatin remodeling, genome activation, DNA replication, cell lineage determination, and nuclear reprogramming are discussed.

Acetylation↗