Apoptosis in mouse fetal and neonatal oocytes during meiotic prophase one

Background The 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. Results Microspreads 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. Conclusions Our 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

Physiological responses of cultured bovine granulosa cells to elevated temperatures under low and high oxygen in the presence of different concentrations of melatonin

Our understanding of the effects of temperature on granulosa cell (GC) physiology is primarily limited to in vitro studies conducted under atmospheric (approx 20% O2) conditions. In the current series of factorial experiments we identify important effects of O2 level (i.e. 5% vs 20% O2) on GC viability and steroidogenesis, and go onto report effects of standard (37.5°C) vs high (40.0°C) temperatures under more physiologically representative (i.e. 5%) O2 levels in the presence of different levels of melatonin (0, 20, 200 and 2000 pg/mL); a potent free-radical scavenger and abundant molecule within the ovarian follicle. Cells aspirated from antral (4 to 6 mm) follicles were cultured in fibronectin-coated wells using serum-free M199 for up to 144 h. At 37.5 C viable cell number was enhanced and luteinization reduced under 5 vs 20% O2. Oxygen level interacted (P<0.001) with time in culture to affect aromatase activity and cell estradiol (E2) production (pg/mL/105 cells). These decreased between 48 and 96 h for both O2 levels but increased again by 144 h for cells cultured under 5% but not 20% O2. Progesterone (P4) concentration (ng/mL/105 cells) was greater (P<0.001) under 20 vs 5% O2 at 96 and 144 h. Cell number increased (P<0.01) with time in culture under 5% O2 irrespective of temperature. However, higher doses of melatonin increased viable cell number at 40.0°C but reduced viable cell number at 37.5°C (P=0.004). Melatonin also reduced (P<0.001) ROS generation at both O2 levels across all concentrations. E2 increased with time in culture at both temperatures under 5% O2, however P4 declined between 96 to 144 h at 40.0 but not 37.5°C. Furthermore, melatonin interacted (P<0.001) with temperature in a dose dependent manner to increase P4 at 37.5°C but to reduce P4 at 40.0°C. Transcript expression for HSD3B1 paralleled temporal changes in P4 production, and those for HBA were greater at 5% than 20% O2, suggesting that hemoglobin synthesis is responsive to changes in O2 level. In conclusion, 5% O2 enhances GC proliferation and reduces luteinization. Elevated temperatures under 5% O2 reduce GC proliferation and P4 production. Melatonin reduces ROS generation irrespective of O2 level and temperature, but interacts with temperature in a dose dependent manner to influence GC proliferation and luteinization

A New Spin on Galactic Dust

We present a new puzzle involving Galactic microwave emission and attempt to resolve it. On one hand, a cross-correlation analysis of the WHAM H-alpha map with the Tenerife 10 and 15 GHz maps shows that the well-known DIRBE correlated microwave emission cannot be dominated by free-free emission. On the other hand, recent high resolution observations in the 8-10 GHz range with the Green Bank 140 ft telescope by Finkbeiner et al. failed to find the corresponding 8 sigma signal that would be expected in the simplest spinning dust models. So what physical mechanism is causing this ubiquitous dust-correlated emission? We argue for a model predicting that spinning dust is the culprit after all, but that the corresponding small grains are well correlated with the larger grains seen at 100 micron only on large angular scales. In support of this grain segregation model, we find the best spinning dust template to involve higher frequency maps in the range 12-60 micron, where emission from transiently heated small grains is important. Upcoming CMB experiments such as ground-based interferometers, MAP and Planck LFI with high resolution at low frequencies should allow a definitive test of this model.Comment: Minor revisions to match accepted ApJ version. 6 pages, 4 figs. Color figures and more foreground information at http://www.hep.upenn.edu/~angelica/foreground.html#spin or from [email protected]

Induction of Nod2 in Myelomonocytic and Intestinal Epithelial Cells via Nuclear Factor-kB Activation

Nod2, a member of the Apaf1/Nod protein family, confers responsiveness to bacterial products and activates NF-kB, a ranscription factor that plays a central role in innate immunity. Recently, genetic variation in Nod2 has been associated with susceptibility to Crohn’s disease. Here, we report that expression of Nod2 is induced upon differentiation of CD34+ hematopoietic progenitor cells into granulocyte or monocyte/macrophages. In peripheral blood cells, the highest levels of Nod2 were observed in CD14+ (monocytes), CD15+ (granulocytes), and CD40+/CD86+ (dendritic cells) cell populations. Notably, stimulation of myeloblastic and epithelial cells with bacterial lipopolysaccharide or TNF resulted in up-regulation of Nod2. A search for consensus sites within the Nod2 promoter revealed a NF-kB binding element that was required for transcriptional activity in response to TNF . Moreover, ectopic expression of p65 induced transactivation, whereas that of dominant-negative I B blocked the transcriptional activity of the Nod2 promoter. Upon stimulation with TNF or lipopolysaccharide, both p50 and p65 subunits of NF-kB were bound to the Nod2 promoter. Thus, Nod2 expression is enhanced by proinflammatory cytokines and bacterial components via NF-kB, a mechanism that may contribute to the amplification of the innate immune response and susceptibility to inflammatory disease

Transcriptional regulation of oncogenic protein kinase Cε (PKCε) by STAT1 and Sp1 proteins

Overexpression of PKCε, a kinase associated with tumor aggressiveness and widely implicated in malignant transformation and metastasis, is a hallmark of multiple cancers, including mammary, prostate, and lung cancer. To characterize the mechanisms that control PKCε expression and its up-regulation in cancer, we cloned an ∼1.6-kb promoter segment of the human PKCε gene (PRKCE) that displays elevated transcriptional activity in cancer cells. A comprehensive deletional analysis established two regions rich in Sp1 and STAT1 sites located between -777 and-105 bp (region A) and-921 and-796 bp (region B), respectively, as responsible for the high transcriptional activity observed in cancer cells. A more detailed mutagenesis analysis followed by EMSA and ChIP identified Sp1 sites in positions -668/-659 and-269/-247 as well as STAT1 sites in positions -880/-869 and- 793/-782 as the elements responsible for elevated promoter activity in breast cancer cells relative to normal mammary epithelial cells. RNAi silencing of Sp1 and STAT1 in breast cancer cells reduced PKCε mRNA and protein expression, as well as PRKCE promoter activity. Moreover, a strong correlation was found between PKCε and phospho-Ser-727 (active) STAT1 levels in breast cancer cells. Our results may have significant implications for the development of approaches to target PKCε and its effectors in cancer therapeutics.Centro de Investigaciones Inmunológicas Básicas y AplicadasFacultad de Ciencias Médica