The Red Balloon; Words, Words, Words; The Actor\u27s Nightmare

The Red Balloon by Damian Trasler, Words, Words, Words by David Ives, and The Actor\u27s Nightmare by Christopher Durang are the Marinello One-Acts featured at John Carroll University in March of 2010.https://collected.jcu.edu/plays/1030/thumbnail.jp

Coordinate regulation of DNA methyltransferase expression during oogenesis

<p>Abstract</p> <p>Background</p> <p>Normal mammalian development requires the action of DNA methyltransferases (DNMTs) for the establishment and maintenance of DNA methylation within repeat elements and imprinted genes. Here we report the expression dynamics of <it>Dnmt3a </it>and <it>Dnmt3b</it>, as well as a regulator of DNA methylation, <it>Dnmt3L</it>, in isolated female germ cells.</p> <p>Results</p> <p>Our results indicate that these enzymes are coordinately regulated and that their expression peaks during the stage of postnatal oocyte development when maternal methylation imprints are established. We find that Dnmt3a, Dnmt3b, Dnmt3L and Dnmt1o transcript accumulation is related to oocyte diameter. Furthermore, DNMT3L deficient 15 dpp oocytes have aberrantly methylated <it>Snrpn</it>, <it>Peg3 </it>and <it>Igf2r </it>DMRs, but normal IAP and LINE-1 methylation levels, thereby highlighting a male germ cell specific role for DNMT3L in the establishment of DNA methylation at repeat elements. Finally, real-time RT-PCR analysis indicates that the depletion of either DNMT3L or DNMT1o in growing oocytes results in the increased expression of the <it>de novo </it>methyltransferase <it>Dnmt3b</it>, suggesting a potential compensation mechanism by this enzyme for the loss of one of the other DNA methyltransferases.</p> <p>Conclusion</p> <p>Together these results provide a better understanding of the developmental regulation of <it>Dnmt3a</it>, <it>Dnmt3b </it>and <it>Dnmt3L </it>at the time of <it>de novo </it>methylation during oogenesis and demonstrate that the involvement of DNMT3L in retrotransposon silencing is restricted to the male germ line. This in turn suggests the existence of other factors in the oocyte that direct DNA methylation to transposons.</p

Loss of spermatogonia and wide-spread DNA methylation defects in newborn male mice deficient in DNMT3L

<p>Abstract</p> <p>Background</p> <p>Formation of haploid spermatozoa capable of fertilization requires proper programming of epigenetic information. Exactly how DNMT3L (DNA methyltransferase 3-Like), a postulated regulator of DNA methyltransferase activity, contributes to DNA methylation pattern acquisition during gametogenesis remains unclear. Here we report on the role of DNMT3L in male germ cell development.</p> <p>Results</p> <p>A developmental study covering the first 12 days following birth was conducted on a <it>Dnmt3L </it>mutant mouse model; lower germ cell numbers and delayed entry into meiosis were observed in <it>Dnmt3L</it>^-/-males, pointing to a mitotic defect. A temporal expression study showed that expression of <it>Dnmt3L </it>is highest in prenatal gonocytes but is also detected and developmentally regulated during spermatogenesis. Using a restriction enzyme qPCR assay (qAMP), DNA methylation analyses were conducted on postnatal primitive type A spermatogonia lacking DNMT3L. Methylation levels along 61 sites across chromosomes 4 and X decreased significantly by approximately 50% compared to the levels observed in <it>Dnmt3L</it>^+/+germ cells, suggesting that many loci throughout the genome are marked for methylation by DNMT3L. More so, hypomethylation was more pronounced in regions of lower GC content than in regions of higher GC content.</p> <p>Conclusion</p> <p>Taken together, these data suggest that DNMT3L plays a more global role in genomic methylation patterning than previously believed.</p

Sperm DNA methylome abnormalities occur both pre- and post-treatment in men with Hodgkin disease and testicular cancer

Combination chemotherapy has contributed to increased survival from Hodgkin disease (HD) and testicular cancer (TC). However, questions concerning the quality of spermatozoa after treatment have arisen. While studies have shown evidence of DNA damage and aneuploidy in spermatozoa years following anticancer treatment, the sperm epigenome has received little attention. Our objectives here were to determine the impact of HD and TC, as well as their treatments, on sperm DNA methylation. Semen samples were collected from community controls (CC) and from men undergoing treatment for HD or TC, both before initiation of chemotherapy and at multiple times post-treatment. Sperm DNA methylation was assessed using genome-wide and locus-specific approaches. Imprinted gene methylation was not affected in the sperm of HD or TC men, before or after treatment. Prior to treatment, using Illumina HumanMethylation450 BeadChip (450 K) arrays, a subset of 500 probes was able to distinguish sperm samples from TC, HD and CC subjects; differences between groups persisted post-treatment. Comparing altered sperm methylation between HD or TC patients versus CC men, twice as many sites were affected in TC versus HD men; for both groups, the most affected CpGs were hypomethylated. For TC patients, the promoter region of GDF2 contained the largest region of differential methylation. To assess alterations in DNA methylation over time/post-chemotherapy, serial samples from individual patients were compared. With restriction landmark genome scanning and 450 K array analyses, some patients who underwent chemotherapy showed increased alterations in DNA methylation, up to 2 to 3 years post-treatment, when compared to the CC cohort. Similarly, a higher-resolution human sperm-specific assay that includes assessment of environmentally sensitive regions, or "dynamic sites," also demonstrated persistently altered sperm DNA methylation in cancer patients post-treatment and suggested preferential susceptibility of "dynamic" CpG sites. Distinct sperm DNA methylation signatures were present pre-treatment in men with HD and TC and may help explain increases in birth defects reported in recent clinical studies. Epigenetic defects in spermatozoa of some cancer survivors were evident even up to 2 years post-treatment. Abnormalities in the sperm epigenome both pre- and post-chemotherapy may contribute to detrimental effects on future reproductive health. The online version contains supplementary material available at 10.1186/s13148-022-01417-1

Restriction landmark genomic scanning (RLGS) spot identification by second generation virtual RLGS in multiple genomes with multiple enzyme combinations.

BackgroundRestriction landmark genomic scanning (RLGS) is one of the most successfully applied methods for the identification of aberrant CpG island hypermethylation in cancer, as well as the identification of tissue specific methylation of CpG islands. However, a limitation to the utility of this method has been the ability to assign specific genomic sequences to RLGS spots, a process commonly referred to as "RLGS spot cloning."ResultsWe report the development of a virtual RLGS method (vRLGS) that allows for RLGS spot identification in any sequenced genome and with any enzyme combination. We report significant improvements in predicting DNA fragment migration patterns by incorporating sequence information into the migration models, and demonstrate a median Euclidian distance between actual and predicted spot migration of 0.18 centimeters for the most complex human RLGS pattern. We report the confirmed identification of 795 human and 530 mouse RLGS spots for the most commonly used enzyme combinations. We also developed a method to filter the virtual spots to reduce the number of extra spots seen on a virtual profile for both the mouse and human genomes. We demonstrate use of this filter to simplify spot cloning and to assist in the identification of spots exhibiting tissue-specific methylation.ConclusionThe new vRLGS system reported here is highly robust for the identification of novel RLGS spots. The migration models developed are not specific to the genome being studied or the enzyme combination being used, making this tool broadly applicable. The identification of hundreds of mouse and human RLGS spot loci confirms the strong bias of RLGS studies to focus on CpG islands and provides a valuable resource to rapidly study their methylation

Bmp4 gene is expressed at the putative site of fusion in the midfacial region

The molecular mechanisms by which the primordia of the midface grow and fuse to form the primary palate portion of the craniofacial region are not well characterized. This is in spite of the fact that failure of growth and/or fusion of these primordia leads to the most common craniofacial birth defect in humans (i.e. clefts of the lip and/or palate). Bmp4 plays a critical role during early embryonic development and has previously been shown to play a role in epithelial-mesenchymal interactions in the craniofacial region of chicks. We analyze the expression of bmp4 in mouse as the midfacial processes undergo fusion to form the primary palate. We show that bmp4 is expressed in a very distinct manner in the three midfacial processes (lateral nasal, LNP, medial nasal, MNP, and maxillary processes, MxP) that ultimately fuse to form the midface. Prior to fusion of the midfacial processes, bmp4 is expressed in the ectoderm of the LNP, MNP, and MxP in a distinct spatial and temporal manner near and at the site of fusion of the midface. Bmp4 appears to demarcate the cells in the LNP and MNP that will eventually contact and fuse with each other. As fusion of the three prominences proceeds, some bmp4 expressing cells are trapped in the fusion line. Later, the expression of bmp4 switches to the mesenchyme of the midface underlying its initial expression in the ectoderm. The switch occurs soon after fusion of the three processes. The pattern of expression in the midfacial region implicates the important role of bmp4 in mediating the fusion process, possibly through apoptosis of cells in the putative site of fusion, during midfacial morphogenesis.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/72184/1/j.1432-0436.2003.710304.x.pd

Betaine is accumulated via transient choline dehydrogenase activation during mouse oocyte meiotic maturation

Betaine (N,N,N-trimethylglycine) plays key roles in mouse eggs and preimplantation embryos first in a novel mechanism of cell volume regulation and second as a major methyl donor in blastocysts, but its origin is unknown. Here, we determined that endogenous betaine was present at low levels in germinal vesicle (GV) stage mouse oocytes before ovulation and reached high levels in the mature, ovulated egg. However, no betaine transport into oocytes was detected during meiotic maturation. Because betaine can be synthesized in mammalian cells via choline dehydrogenase (CHDH; EC 1.1.99.1), we assessed whether this enzyme was expressed and active. Chdh transcripts and CHDH protein were expressed in oocytes. No CHDH enzyme activity was detected in GV oocyte lysate, but CHDH became highly active during oocyte meiotic maturation. It was again inactive after fertilization. We then determined whether oocytes synthesized betaine and whether CHDH was required. Isolated maturing oocytes autonomously synthesized betaine in vitro in the presence of choline, whereas this failed to occur in Chdh/ oocytes, directly demonstrating a requirement for CHDH for betaine accumulation in oocytes. Overall, betaine accumulation is a previously unsuspected physiological process during mouse oocyte meiotic maturation whose underlying mechanism is the transient activation of CHDH

The Activation of DNA Damage Detection and Repair Responses in Cleavage-Stage Rat Embryos by a Damaged Paternal Genome

Male germ cell DNA damage, after exposure to radiation, exogenous chemicals, or chemotherapeutic agents, is a major cause of male infertility. DNA-damaged spermatozoa can fertilize oocytes; this is of concern because there is limited information on the capacity of early embryos to repair a damaged male genome or on the fate of these embryos if repair is inadequate. We hypothesized that the early activation of DNA damage response in the early embryo is a critical determinant of its fate. The objective of this study was to assess the DNA damage response and mitochondrial function as a measure of the energy supply for DNA repair and general health in cleavage-stage embryos sired by males chronically exposed to an anticancer alkylating agent, cyclophosphamide. Male rats were treated with saline or cyclophosphamide (6 mg/kg/day) for 4 weeks and mated to naturally cycling females. Pronuclear two- and eight-cell embryos were collected for immunofluorescence analysis of mitochondrial function and biomarkers of the DNA damage response: γH2AX foci, 53BP1 reactivity, and poly(ADP-ribose) polymer formation. Mitochondrial activities did not differ between embryos sired by control- and cyclophosphamide-exposed males. At the two-cell stage, there was no treatment-related increase in DNA double-strand breaks; by the eight-cell stage, a significant increase was noted, as indicated by increased medium and large γH2AX foci. This was accompanied by a dampened DNA repair response, detected as a decrease in the nuclear intensity of poly(ADP-ribose) polymers. The micronuclei formed in cyclophosphamide-sired embryos contained large γH2AX foci and enhanced poly(ADP-ribose) polymer and 53BP1 reactivity compared with their nuclear counterparts. Thus, paternal cyclophosphamide exposure activated a DNA damage response in cleavage-stage embryos. Furthermore, this damage response may be useful in assessing embryo quality and developmental competence

3D Cohort Study : The Integrated Research Network in Perinatology of Quebec and Eastern Ontario

Background: The 3D Cohort Study (Design, Develop, Discover) was established to help bridge knowledge gaps about the links between various adverse exposures during pregnancy with birth outcomes and later health outcomes in children. Methods: Pregnant women and their partners were recruited during the first trimester from nine sites in Quebec and followed along with their children through to 2 years of age. Questionnaires were administered during pregnancy and post-delivery to collect information on demographics, mental health and life style, medical history, psychosocial measures, diet, infant growth, and neurodevelopment. Information on the delivery and newborn outcomes were abstracted from medical charts. Biological specimens were collected from mothers during each trimester, fathers (once during the pregnancy), and infants (at delivery and 2 years of age) for storage in a biological specimen bank. Results: Of the 9864 women screened, 6348 met the eligibility criteria and 2366 women participated in the study (37% of eligible women). Among women in the 3D cohort, 1721 of their partners (1704 biological fathers) agreed to participate (73%). Two thousand two hundred and nineteen participants had a live singleton birth (94%). Prenatal blood and urine samples as well as vaginal secretions were collected for ≥98% of participants, cord blood for 81% of livebirths, and placental tissue for 89% of livebirths. Conclusions: The 3D Cohort Study combines a rich bank of multiple biological specimens with extensive clinical, life style, and psychosocial data. This data set is a valuable resource for studying the developmental etiology of birth and early childhood neurodevelopmental outcomes

Loss of DNMT1o Disrupts Imprinted X Chromosome Inactivation and Accentuates Placental Defects in Females

The maintenance of key germline derived DNA methylation patterns during preimplantation development depends on stores of DNA cytosine methyltransferase-1o (DNMT1o) provided by the oocyte. Dnmt1omat-/- mouse embryos born to Dnmt1Δ1o/Δ1o female mice lack DNMT1o protein and have disrupted genomic imprinting and associated phenotypic abnormalities. Here, we describe additional female-specific morphological abnormalities and DNA hypomethylation defects outside imprinted loci, restricted to extraembryonic tissue. Compared to male offspring, the placentae of female offspring of Dnmt1Δ1o/Δ1o mothers displayed a higher incidence of genic and intergenic hypomethylation and more frequent and extreme placental dysmorphology. The majority of the affected loci were concentrated on the X chromosome and associated with aberrant biallelic expression, indicating that imprinted X-inactivation was perturbed. Hypomethylation of a key regulatory region of Xite within the X-inactivation center was present in female blastocysts shortly after the absence of methylation maintenance by DNMT1o at the 8-cell stage. The female preponderance of placental DNA hypomethylation associated with maternal DNMT1o deficiency provides evidence of additional roles beyond the maintenance of genomic imprints for DNA methylation events in the preimplantation embryo, including a role in imprinted X chromosome inactivation. © 2013 McGraw et al