The centrosome plays a vital role in mitotic fidelity, ensuring establishment of bipolar spindles and balanced chromosome segregation. Centrosome duplication occurs only once during the cell cycle and is therefore highly regulated. Here, it is shown that in mouse embryonic fibroblasts (MEFs) lacking the p53 tumor suppressor protein, multiple copies of functionally competent centrosomes are generated during a single cell cycle. In contrast, MEFs prepared from normal mice or mice deficient in the retinoblastoma tumor suppressor gene product do not display these abnormalities. The abnormally amplified centrosomes profoundly affect mitotic fidelity, resulting in unequal segregation of chromosomes. These observations implicate p53 in the regulation of centrosome duplication and suggest one possible mechanism by which the loss of p53 may cause genetic instability.

Mos is an upstream activator of mitogenactivated protein kinase (MAPK) and, in mouse oocytes, is responsible for metaphase II arrest. This activity has been likened to its function in Xenopus oocytes as a component of cytostatic factor. Thus, Mos-deficient female mice are less fertile and oocytes derived from these animals fail to arrest at metaphase II Here we show that maturing MOS-'-oocytes fail to activate MAPK throughout meiosis, while p34edc2 kinase activity is normal until late in metaphase II when it decreases prematurely. Phenotypically, the first meiotic division of MOS-/-oocytes frequently resembles mitotic cleavage or produces an abnormally large polar body. In these oocytes, the spindle shape is altered and the spindle fails to translocate to the cortex, leading to the establishment of an altered cleavage plane. Moreover, the first polar body persists instead of degrading and sometimes undergoes an additional cleavage, thereby providing conditions for parthenogenesis. These studies identify meiotic spindle formation and programmed degradation of the first polar body as new and important roles for the Mos/MAPK pathway.The mos protooncogene encodes a protein serine/threonine kinase (1) and Mos is expressed at high levels in oocytes undergoing meiotic maturation (2,3). InXenopus oocytes, Mos has been shown to function as a meiotic initiator (4, 5) and an active component of cytostatic factor (CSF) (6), an activity that is responsible for the arrest of an unfertilized egg at metaphase II of meiosis (7). More recently, mitogen-activated protein kinase (MAPK), which is highly activated throughout oocyte maturation (8-12), has been identified as one of the major downstream targets of Mos (13)(14)(15)(16)(17). Mos has been implicated in the activation and stabilization of p34cdc2 kinase as maturation promoting factor (MPF) (4,(18)(19)(20), and evidence for the mutual dependency between MPF and the Mos/MAPK pathway in Xenopus oocytes has been reported (8,21,22).More recently, it has been shown that oocytes from mice homozygously deficient in mos (MOS-/-) fail to arrest at metaphase II and undergo parthenogenetic activation (23,24). These observations clearly demonstrated that Mos is an active component of CSF, but left unclear whether Mos functions prior to metaphase II arrest as it does in Xenopus.We generated MOS-'-mice and confirmed previous reports (23,24) that oocytes from these animals fail to arrest at metaphase II and instead undergo parthenogenetic activation.Also we show that maturing oocytes from MOS-1-mice fail to activate MAPK, while p34cdc2 kinase activation is normal until metaphase, when it decreases prematurely. Moreover, in MOS-'-oocytes, we observe that the first polar bodies can be abnormally large and sometimes undergo an additional cleavage instead of undergoing rapid degeneration. Thus, in addition to CSF activity, Mos/MAPK are required for regulating the size and degradation of the first polar body. MATERIALS AND METHODSGeneration of MOS-1-Knockout Mice. A replacement-type ...

Little is known about whether bone marrow-derived circulating progenitor cells (BMDCPCs) can transdifferentiate into adipocytes in adipose tissues or play a role in expanding adipocyte number during adipose tissue growth. Using a mouse bone marrow transplantation model, we addressed whether BMDCPCs can transdifferentiate into adipocytes under standard conditions as well as in the settings of diet-induced obesity, rosiglitazone treatment, and exposure to G-CSF. We also addressed the possibility of transdifferentiation to adipocytes in a murine parabiosis model. In each of these settings, our findings indicated that BMDCPCs did not transdifferentiate into either unilocular or multilocular adipocytes in adipose tissues. Most BMDCPCs became resident and phagocytic macrophages in adipose tissues--which resembled transdifferentiated multilocular adipocytes by appearance, but displayed cell surface markers characteristic for macrophages - in the absence of adipocyte marker expression. When exposed to adipogenic medium in vitro, bone marrow cells differentiated into multilocular, but not unilocular, adipocytes, but transdifferentiation was not observed in vivo, even in the contexts of adipose tissue regrowth or dermal wound healing. Our results suggest that BMDCPCs do not transdifferentiate into adipocytes in vivo and play little, if any, role in expanding the number of adipocytes during the growth of adipose tissues.

No abstract

Mitogen-activated protein kinase (MAPK) is selectively activated by injecting either mos or MAPK kinase (mek) RNA into immature mouse oocytes maintained in the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX). IBMX arrests oocyte maturation, but Mos (or MEK) overexpression overrides this block Under these conditions, meiosis I is significantly prolonged, and MAPK becomes fully activated in the absence of p34cdc2 kinase or maturationpromoting factor. In these oocytes, large openings form in the germinal vesicle adjacent to condensing chromatin, and microtubule arrays, which stain for both MAPK and centrosomal proteins, nucleate from these regions. Maturationpromoting factor activation occurs later, concomitant with germinal vesicle breakdown, the contraction of the microtubule arrays into a precursor of the spindle, and the redistribution of the centrosomal proteins into the newly forming spindle poles. These MATERIALS AND METHODSOocyte Collection, Culture, and Microinjection. B6C3 F1 female mice were injected intraperitoneally with 5 units of pregnant mare serum gonadotropin. Cumulus-enclosed oocytes were isolated 45-48 hr later and cultured in modified Whitten's medium (21) containing 0.4% BSA and 100 ,uM IBMX. For microinjection, oocytes were transferred to modified Whitten's media with Hepes (PGC Scientific, Gaithersburg, MD; specialty media) containing 5% fetal calf serum and 100 ,tM IBMX. Oocytes were injected in the cytoplasm with '10 pl of RNA (1 gg/,ul) in Dulbecco's PBS. Injected or uninjected oocytes were washed in modified Whitten's medium containing IBMX and incubated at 38.5°C in humidified 5% CO2 in air.RNA was synthesized in vitro from the mos-containing plasmid, PHTX, with T7 RNA polymerase and the RNA cap analog, 7-methyl-G(5')ppp(5')G (22). Gain-of-function mek* RNA (17) was synthesized in vitro using the same method. The RNA was precipitated twice in ethanol, then resuspended in Dulbecco's PBS.Immunofluorescence and Confocal Microscopic Analysis.Oocytes were stripped of their zonae pellucidae with acidic Tyrode's solution (pH 2.5), fixed with 1.8% paraformaldehyde in PBS (fixing solution) for 40 min at room temperature, and made permeable with 1% Triton X-100 in fixing solution for 40 min. Oocytes were washed with 0.1% Tween-20 in PBS for 20 min and incubated with 3% BSA, 10% goat serum, andAbbreviations: MAPK, mitogen-activated protein kinase; MT, microtubule; MEK, MAPK kinase; MPF, maturation-promoting factor;IBMX, 3-isobutyl-1-methylxanthine; GV, germinal vesicle; DAPI, 4',6-diamino-2-phenylindol; GVBD, GV breakdown. 4730The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S C. §1734 solely to indicate this fact.