Measurement by quantitative PCR of changes in HPRT, PGK-1, PGK-2, APRT, MTase, and Zfy gene transcripts during mouse spermatogenesis

Singer-Sam, Judith; Robinson, Murray O.; Bellvé, Anthony R.; Simon, Mel I.; Riggs, Arthur D.

doi:10.1093/nar/18.5.1255

Cited by 231 publications

(109 citation statements)

References 36 publications

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“…It is believed that premeiotic mammalian sperm cells are hypomethylated and that high levels of DNA methyltransferase (14) result in the de novo methylation of sperm DNA during meiosis to give hypermethylated sperm nuclei (15). This change is exactly the opposite direction to that observed by us in tobacco pollen.…”

Section: Discussionmentioning

confidence: 60%

Developmental changes in DNA methylation of the two tobacco pollen nuclei during maturation

Oakeley

Podestà

Jost

1997

Proc. Natl. Acad. Sci. U.S.A.

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Changes in DNA methylation during tobacco pollen development have been studied by confocal f luorescence microscopy using a monoclonal anti-5-methylcytosine (anti-m 5 C) antibody and a polyclonal anti-histone H1 (antihistone) antibody as an internal standard. The specificity of the anti-m 5 C antibody was demonstrated by a titration series against both single-stranded DNA and double-stranded DNA substrates in either the methylated or unmethylated forms. The antibody was found to show similar kinetics against both double-and single-stranded DNA, and the f luorescence was proportional to the amount of DNA used. No signal was observed with unmethylated substrates. The extent of methylation of the two pollen nuclei remained approximately constant after the mitotic division that gave rise to the vegetative and generative nuclei. However, during the subsequent development of the pollen, the staining of the generative nucleus decreased until it reached a normalized value of 1 ⁄5 of that of the vegetative nucleus. The use of a confocal microscope makes these data independent of possible focusing artefacts. The anti-histone antibody was used as a control to show that, while the antibody staining directed against 5-methylcytosine changed dramatically during pollen maturation, the histone signal did not. We observed the existence of structural dimorphism amongst tobacco pollen grains, the majority having three pollen apertures and the rest with four. However, the methylation changes observed occurred to the same extent in both subclasses.Tobacco pollen undergoes a series of well defined stages during its development. Four amphihaploid sporocytes are produced by means of a meiotic division from an amphidiploid pollen mother cell. The cells then each form a highly resilient cell wall composed primarily of callose and cellulose. The nucleus then migrates to one of the poles of the pollen grain, where it undergoes a mitotic division to give two amphihaploid daughter cells. This division is not an equal one, and the cell closest to the pollen pole contains minimal cytoplasm and is known as the generative cell because it is destined to divide and form the gametes (1). The larger cell is called the vegetative cell, and its nucleus is responsible for controlling the development of the pollen tube. The generative cell rapidly detaches from the pole and floats freely within the cytoplasm of the vegetative cell (2). The two cells are separated only by plasma membranes (2). When mature pollen germinates, it produces a pollen tube that grows chemotropically toward the ovaries (3-5). The vegetative nucleus of tobacco always enters the pollen tube first and is then followed by the generative nucleus. Shortly after this, the vegetative nucleus disintegrates. The generative cell then undergoes a second mitotic division in the pollen tube to give two amphihaploid sperm cells that can fertilize the egg and the polar nuclei in the ovary (6), resulting in the embryo and endosperm, respectively.Previous studies, using genomic sequencing...

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Section: Discussionmentioning

confidence: 60%

Developmental changes in DNA methylation of the two tobacco pollen nuclei during maturation

Oakeley

Podestà

Jost

1997

Proc. Natl. Acad. Sci. U.S.A.

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“…62 RNA samples were purified from DNA contamination by digestion with DNaseI. 43 The RT-PCR assays were performed essentially according to Singer-Sam et al 63 At the reverse transcription step, the reaction mixture (final volume 30 ml) contained: 1.5 mM MgCl 2 , 0.2 mM each dNTPs, 0.4 mM primers (each), RNA (0.5-3 mg), MMLV reverse transcriptase (30 U, Gibco BRL), ribonuclease inhibitor (24 U, Fermentas) and Taq polymerase (1.2 U, Fermentas). Samples were incubated in a thermal cycler for 10 min at 501C.…”

Section: Rna Isolation Rt-pcr and Hybridizationmentioning

confidence: 99%

Spermatocyte-specific expression of constitutively active heat shock factor 1 induces HSP70i-resistant apoptosis in male germ cells

et al. 2005

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Spermatocytes, the most sensitive male germ cells to heatinduced apoptosis, do not respond to hyperthermia by inducing heat shock proteins (HSPs), including HSP70i, which has been previously shown to confer resistance to apoptosis in somatic cells. To dissect the mechanism of heat-induced apoptosis and to determine if we could protect spermatocytes by expressing HSP70i, we engineered transgenic mice that express in spermatocytes constitutively active heat shock transcription factor (HSF)1. Such HSF1 expression did not lead to transcription of inducible Hsp70 genes, but instead induced caspase-dependent apoptosis that mimicked heat shock-induced death of spermatogenic cells. Both mitochondria-dependent and death receptor-dependent pathways appear to be involved in such HSF1-induced apoptosis: the levels of Bcl-2 family proteins became increased, p53 protein accumulated and expression levels of caspase-8 and deathreceptor-interacting proteins (including Fas-associated death domain protein and TNF receptor associated death domain protein) became elevated. Surprisingly, the constitutive spermatocyte-specific expression of HSP70i in doubletransgenic males did not protect against such HSF1-induced apoptosis.

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“…The number of PCR cycles was initially determined to yield PCR products during the exponential phase of amplification. RT-PCR performed under these conditions provides reliable detection of greater than 2-fold differences in mRNA levels (24). All of the reagents used for RNA isolation and RT-PCR were from Invitrogen Canada, except for Taq DNA polymerase and PCR buffer, which were purchased from Amersham Biosciences (Baie d'Urfé, Quebec, Canada).…”

Section: Semiquantitative Rt-pcrmentioning

confidence: 99%

Thy-1 Signaling in the Context of Costimulation Provided by Dendritic Cells Provides Signal 1 for T Cell Proliferation and Cytotoxic Effector Molecule Expression, but Fails to Trigger Delivery of the Lethal Hit

Haeryfar

Al‐Alwan

Rowden

et al. 2003

The Journal of Immunology

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Cross-linking of the GPI-anchored protein Thy-1 results in T cell proliferation and IL-2 synthesis. However, the exact function of Thy-1 in the process of T cell activation remains unknown, as does the effect of costimulation on Thy-1-driven T cell responses. In this study, we have investigated the ability of Thy-1 to substitute for traditional signal 1 in the context of costimulation provided by dendritic cells. Dendritic cells dramatically enhanced T cell proliferation and IL-2 synthesis in response to Thy-1 triggering by anti-Thy-1 mAb. This effect was not dependent on dendritic cell Fcγ receptors, but was a result of B7-mediated costimulation (signal 2). T cells were also activated when microbeads coated with a combination of anti-Thy-1 and anti-CD28 mAbs were used to supply signals 1 and 2, respectively. Thy-1-stimulated T cells adhere to target cells and express perforin, granzyme B, and Fas ligand, but fail to kill target cells due to an inability to reorganize their secretion machinery. Moreover, in contrast to TCR signaling, Thy-1 triggering failed to induce cytotoxicity in redirected lysis assays. We conclude that Thy-1 triggering can partially substitute for signal 1, which, in combination with a strong signal 2, leads to robust T cell proliferation, IL-2 synthesis, and cytotoxic effector molecule expression, but does not induce cytolytic function. The block at the level of cytotoxic effector function that results when T cells are activated in the absence of a classical, Ag-specific signal 1 may constitute a mechanism to ensure the specificity of CTL responses and prevent potentially harmful promiscuous cytotoxicity.

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Measurement by quantitative PCR of changes in HPRT, PGK-1, PGK-2, APRT, MTase, and Zfy gene transcripts during mouse spermatogenesis

Cited by 231 publications

References 36 publications

Developmental changes in DNA methylation of the two tobacco pollen nuclei during maturation

Developmental changes in DNA methylation of the two tobacco pollen nuclei during maturation

Spermatocyte-specific expression of constitutively active heat shock factor 1 induces HSP70i-resistant apoptosis in male germ cells

Thy-1 Signaling in the Context of Costimulation Provided by Dendritic Cells Provides Signal 1 for T Cell Proliferation and Cytotoxic Effector Molecule Expression, but Fails to Trigger Delivery of the Lethal Hit

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