Epigenetic Control of Early Mouse Development.

Although the genes sequentially transcribed in the mammalian embryo prior to implantation have been identified, understanding of the molecular processes ensuring this transcription is still in development. The genomes of the sperm and egg are hypermethylated, hence transcriptionally silent. Their union, in the prepared environment of the egg, initiates their epigenetic genomic reprogramming into a totipotent zygote, in which the genome gradually becomes transcriptionally activated. During gametogenesis, sex-specific processes result in sperm and eggs with disparate epigenomes, both of which require drastic reprogramming to establish the totipotent genome of the zygote and the pluripotent inner cell mass of the blastocyst. Herein, we describe the factors, DNA and histone modifications, activation and repression of retrotransposons, and cytoplasmic localizations, known to influence the activation of the mammalian genome at the initiation of new life.

Benefit of combined triiodothyronine (LT(3)) and thyroxine (LT(4)) treatment in athyreotic patients unresponsive to LT(4) alone.

Despite some reports, the usefulness of levothyroxine (LT(4)) and levotriiodothyronine (LT(3)) combination therapy in hypothyroidism remains controversial. The objective of this paper is to study a benefit of additional LT(3) in athyreotic patients who failed to normalize TSH on LT(4) alone even with hyperthyroid serum T(4) values. In a survey of 200 athyreotic patients treated between 2006 and 2009, about 7% failed to normalize serum TSH levels following treatment with LT(4), though serum T(4) values in the hyperthyroid range were achieved. These patients (characterized by serum T(4)≥160 nmol/L and TSH≥5.0 mIU/L), were additionally treated with 10 µg b. i. d LT(3). LT(3) and LT(4) combination therapy resulted in decreased serum TSH levels into the normal range (12.8 vs. 1.22 mIU/L; p<0.01) and reduced LT(4) dose (153.3 vs. 117.5 µg; p<0.01) required for normalization of serum T(4) values (170.6 vs. 123.3 nmol/L; p<0.01). Serum T(3) values were higher (1.3 vs. 2.26 nmol/L; p<0.01) than those during monotherapy with LT(4). Our results indicate a subpopulation of athyreotic patients that could significantly benefit from combined LT(4) + LT(3) therapy in restoring normal TSH and thyroid hormone patterns. Further research should be undertaken to provide a genetic basis for these findings.

Increased thyroidal T4 to T3 conversion in autonomously functioning thyroid adenoma: from euthyroidism to thyrotoxicosis.

AIM: The aim was to investigate whether the intrathyroid conversion of T4 to T3 in autonomously functioning thyroid adenoma (AFTA) tissue could influence serum T3 levels and suppression of TSH, especially in patients with borderline thyroid function. PATIENTS AND METHODS: In ten patients with AFTA, thyroidal conversion of T4 to T3 was investigated in nodular and paranodular, TSH-suppressed tissue. All patients had normal serum T4 and suppressed TSH. Serum T3 was normal in six, and borderline or slightly increased in four. AFTA and paranodular tissues were surgically removed and frozen at -70°C, then homogenized in a glass homogenizer, centrifuged at 100,000×g, and particulate fraction collected as a pellet. Analysis mixture consisted of thyroid enzyme suspension in 50 µmol/L TRIS buffer with 5 µmol DTT and 200 µL 1.3 µmol T4. Incubation was performed at 37°C and the generation of T3 measured after 5, 10, 20 and 40 minutes respectively. RESULTS: T3 production (pmol/mg protein) was significantly higher in AFTA than in paranodular tissues (8.8 1.2/Mean ± SE/vs. 1.8 ± 0.2; p<0.01), and excessively high (9.8, 14.1, 14.2 and 15.0) in four patients with borderline or slightly supranormal serum T3. A significant correlation was found between serum T3 concentrations and T3 generation (T4 conversion) in AFTA tissues. CONCLUSION: Results suggest that increased thyroidal T4 to T3 conversion in AFTA tissue could be involved in an increased delivery of T3, increased serum T3 and suppressed serum TSH, particularly in patients with the disease evolving from euthyroid to an early hyperthyroid phase.

Unintended changes in cognition, mood, and behavior arising from cell-based interventions for neurological conditions: ethical challenges.

The prospect of using cell-based interventions (CBIs) to treat neurological conditions raises several important ethical and policy questions. In this target article, we focus on issues related to the unique constellation of traits that characterize CBIs targeted at the central nervous system. In particular, there is at least a theoretical prospect that these cells will alter the recipients' cognition, mood, and behavior-brain functions that are central to our concept of the self. The potential for such changes, although perhaps remote, is cause for concern and careful ethical analysis. Both to enable better informed consent in the future and as an end in itself, we argue that early human trials of CBIs for neurological conditions must monitor subjects for changes in cognition, mood, and behavior; further, we recommend concrete steps for that monitoring. Such steps will help better characterize the potential risks and benefits of CBIs as they are tested and potentially used for treatment.

Reprogramming and differentiation in mammals: motifs and mechanisms.

The natural reprogramming of the mammalian egg and sperm genomes is an efficient process that takes place in less than 24 hours and gives rise to a totipotent zygote. Transfer of somatic nuclei to mammalian oocytes also leads to their reprogramming and formation of totipotent embryos, albeit very inefficiently and requiring an activation step. Reprogramming of differentiated cells to induced pluripotent stem (iPS) cells takes place during a period of time substantially longer than reprogramming of the egg and sperm nuclei and is significantly less efficient. The stochastic expression of endogenous proteins during this process would imply that controlled expression of specific proteins is crucial for reprogramming to take place. The fact that OCT4, NANOG, and SOX2 form the core components of the pluripotency circuitry would imply that control at the transcriptional level is important for reprogramming to iPS cells. In contradistinction, the much more efficient reprogramming of the mammalian egg and sperm genomes implies that other levels of control are necessary, such as chromatin remodeling, translational regulation, and efficient degradation of no longer needed proteins and RNAs.

Cell-based interventions for neurologic conditions: ethical challenges for early human trials.

BACKGROUND: Attempts to translate basic stem cell research into treatments for neurologic diseases and injury are well under way. With a clinical trial for one such treatment approved and in progress in the United States, and additional proposals under review, we must begin to address the ethical issues raised by such early forays into human clinical trials for cell-based interventions for neurologic conditions. METHODS: An interdisciplinary working group composed of experts in neuroscience, cell biology, bioethics, law, and transplantation, along with leading disease researchers, was convened twice over 2 years to identify and deliberate on the scientific and ethical issues raised by the transition from preclinical to clinical research of cell-based interventions for neurologic conditions. RESULTS: While the relevant ethical issues are in many respects standard challenges of human subjects research, they are heightened in complexity by the novelty of the science, the focus on the CNS, and the political climate in which the science is proceeding. CONCLUSIONS: Distinctive challenges confronting US scientists, administrators, institutional review boards, stem cell research oversight committees, and others who will need to make decisions about work involving stem cells and their derivatives and evaluate the ethics of early human trials include evaluating the risks, safety, and benefits of these trials, determining and evaluating cell line provenance, and determining inclusion criteria, informed consent, and the ethics of conducting early human trials in the public spotlight. Further study and deliberation by stakeholders is required to move toward professional and institutional policies and practices governing this research.

Systems biology of the 2-cell mouse embryo.

The transcriptome of the 2-cell mouse embryo was analyzed to provide insight into the molecular networks at play during nuclear reprogramming and embryonic genome activation. Analysis of ESTs from a 2-cell cDNA library identified nearly 4,000 genes, over half of which have not been previously studied. Transcripts of mobile elements, especially those of LTR retrotransposons, are abundantly represented in 2-cell embryos, suggesting their possible role in introducing genomic variation, and epigenetic restructuring of the embryonic genome. Analysis of Gene Ontology of the 2-cell-stage expressed genes outlines the major biological processes that guide the oocyte-to-embryo transition. These results provide a foundation for understanding molecular control at the onset of mammalian development.

Expression of genes involved in mammalian meiosis during the transition from egg to embryo.

The ooplasm of higher eukaryotes provides substances necessary for completing the last stages of meiosis and initiating the first mitotic division. These processes are firmly attuned to other events in the egg and newly formed embryo, such as switching from the use of maternal transcripts to the onset of zygotic transcription. In mammals little is known about the molecular mechanisms guiding this transition, largely due to the lack of information about genes expressed in the egg and early embryos. Studies of yeast mitosis have contributed much of what is known about the vertebrate cell cycle, and recent reports indicate that homologs of yeast DNA repair genes also function during mammalian gametogenesis. To examine whether this conservation can be expanded to include genes operative in oocyte meiosis, we performed a computer-based search for homologs of yeast genes that are induced during sporulation in C. elegans, Drosophila, and mammals. Results from this study suggest that yeast and higher eukaryotes share genes that coordinate the overall process of meiosis. However intriguing differences exist, reflecting the distinctive mechanisms governing the progression of meiosis in each organism. ESTs representing more than half of the mammalian homologs are present in mouse cDNA libraries that contains genes controlling the meiosis/mitosis transition. About 50% of these genes contain potential cis-elements for cytoplasmic polyadenylation in their 3'-UTR, suggesting the importance of controlled translation in the egg and zygote.

Timely translation during the mouse oocyte-to-embryo transition.

In the mouse, completion of oocyte maturation and the initiation of preimplantation development occur during transcriptional silence and depend on the presence and translation of stored mRNAs transcribed in the growing oocyte. The Spin gene has three transcripts, each with an identical open reading frame and a different 3' untranslated region (UTR). (Beta)-galactosidase-tagged reporter transcripts containing each of the different Spin 3'UTRs were injected into oocytes and zygotes and (beta)-galactosidase activity was monitored. Results from these experiments suggest that differential polyadenylation and translation occurs at two critical points in the oocyte-to-embryo transition - upon oocyte maturation and fertilization - and is dependent on sequences in the 3'UTR. The stability and mobility shifts of ten other maternal transcripts were monitored by reprobing a northern blot of oocytes and embryos collected at 12 hour intervals after fertilization. Some are more stable than others and the upward mobility shift associated with polyadenylation correlates with the presence of cytoplasmic polyadenylation elements (CPEs) within about 120 nucleotides of the nuclear polyadenylation signal. A survey of the 3' UTRs of expressed sequence tag clusters from a mouse 2-cell stage cDNA library indicates that about one third contain CPEs. We suggest that differential transcript stability and a translational control program can supply the diversity of protein products necessary for oocyte maturation and the initiation of development.

Mammalian cloning: advances and limitations.

For many years, researchers cloning mammals experienced little success, but recent advances have led to the successful cloning of several mammalian species. However, cloning by the transfer of nuclei from adult cells is still a hit-and-miss procedure, and it is not clear what technical and biological factors underlie this. Our understanding of the molecular basis of reprogramming remains extremely limited and affects experimental approaches towards increasing the success rate of cloning. Given the future practical benefits that cloning can offer, the time has come to address what should be done to resolve this problem.

Cloning and embryonic stem cells: a new era in human biology and medicine.

The cloning of mammals using adult cells as nuclear donors has been achieved and the same procedure can be, at least theoretically, used to clone humans. Another recent technological advance, the derivation of human embryonic stem cells, opens up new possibilities in cell and tissue replacement therapy and heralds significant improvements in gene therapy. Besides suggesting new and potentially valuable medical applications, the insights gained through the use of these techniques could significantly enrich our understanding of basic mechanisms regulating human development. On the other hand, these preliminary results are viewed by many as the opening of the Pandora's box and there are loud voices clamoring that research in these areas be forbidden in perpetuity. I suggest in the following article that at present we do not know enough to make anything but an entirely emotional decision about future applications of these techniques. I try to summarize the current state of the kn owledge in the field and indicate how much further research is necessary if benefits and drawbacks are to be properly understood.

Expression of Melk, a new protein kinase, during early mouse development.

A new gene named maternal embryonic leucine zipper kinase, Melk, has been recently identified (Heyer et al. [1997] Mol. Reprod. Dev. 47:148-156). As a basis for further study of the function of the gene, we have examined the expression of Melk across a wide range of embryonic stages, from the ovulated egg and 2-cell embryo through the gastrulation and early organogenesis stages, by in situ hybridization and immunohistochemistry. Melk is expressed in a spatially and temporally specific pattern during mammalian embryogenesis. The strongest expression was detected during maturation of oocytes and preimplantation development. Given its expression pattern, Melk may play an important role during preimplantation embryonic development. Dev Dyn 1999;215:344-351.

The mouse cornichon gene family.

As part of a large scale mouse Expressed Sequence Tag (EST) project to identify molecules involved in the initiation of mammalian development, a homolog of the Drosophila cornichon gene was detected as a mouse maternal transcript present in the two-cell embryo. Cornichon is a multigene family in the mouse: the new gene, Cnih, maps to mouse chromosome 10, another cornichon homolog, Cnil, maps to chromosome 14 and two additional cornichon-related loci, possibly pseudogenes, localize to chromosomes 3 and 10, respectively. Cnih encodes an open reading frame (ORF) of 144 amino acids that is 93% homologous (68% identical) to the Drosophila protein, whereas the ORF of Cnil contains two extra polypeptide regions not found in these other proteins. Transcripts of Cnih are highly abundant in the full grown oocyte and the ovulated unfertilized egg, while Cnil message is only detectable after activation of the embryonic genome at the eight-cell stage. In situ hybridization shows specific localization of Cnih transcripts to ovarian oocytes. The lack of cytoplasmic polyadenylation of the maternally inherited Cnih transcript suggests that Cnih mRNA is translated in the full grown oocyte before, but not after, ovulation. In Drosophila, cornichon is involved in the establishment of both anterior-posterior and dorso-ventral polarity via the epidermal growth factor (EGF)-receptor signaling pathway. Finding Cnih in the mammalian oocyte opens a new perspective on the investigation of EGF-signaling in the oocyte.

Imprinting.

Imprinting provides a fascinating mechanism of control of gene expression so that the maternal and paternal alleles of some genes are unequally expressed. Imprinting is most likely established during gametogenesis by a mechanism not completely clear, though DNA methylation probably plays a certain role. Expression of imprinted gene significantly affects mammalian development so that only the maternal or only the paternal diploid genomes cannot support normal development. Since imprinting results in functional hemizygocity, mutation of the expressed allele can have the drastic consequences of a null mutation. For this reason identification of imprinted genes and further understanding of the imprinting mechanism represent an important change for human medical genetics.