Sorting and reorganization of centrosomes during oocyte maturation in the mouse.

In animal oocytes, the centrosome exists as an acentriolar aggregate of centrosomal material that is regulated in a dynamic manner throughout the process of meiotic maturation. Recently, it has been demonstrated that in female meiotic systems spindle assembly is likely regulated by chromosomal and microtubule/microtubule-associated influences. The purpose of this study was to analyze the distribution of the integral centrosomal protein, pericentrin, during the course of meiotic maturation. The function of the centrosome during meiotic progression was evaluated by exposing oocytes to pharmacological agents that perturb cytoplasmic homeostasis (cycloheximide, nocodazole, cytochalasin D, taxol, and vanadate). Pericentrin was localized to the spindle poles during metaphase of meiosis-I as O- and C-shaped structures. At anaphase, these structures fragment, become displaced from the spindle poles, and associate with the lateral spindle margin. The metaphase spindle at meiosis-II had incomplete pericentrin rings at both spindle poles. Vanadate treatment, a known inhibitor of dynein-ATPase, resulted in meiotic arrest, constriction of the spindle pole, and an aggregation of pericentrin at the spindle poles. After taxol exposure, pericentrin incorporation into both spindle poles and cytoplasmic centrosomes was increased. Treatment of oocytes with cycloheximide, nocodazole, and cytochalasin D, influenced early events associated with chromosome capture and spindle assembly and altered the number and distribution of cytoplasmic centrosomes. Thus, although pericentrin incorporation is not required for meiotic spindle formation, the dynamic reorganization of pericentrin and changes in centrosome microtubule nucleating capacity are involved in critical cell cycle transitions during meiotic maturation.

Oviduct during early pregnancy: hormonal regulation and interactions with the fertilized ovum.

The cyclic fluctuations in circulating levels of 17 beta-estradiol and progesterone that occur during the menstrual or estrous cycle are responsible for dramatic, cyclic changes in the epithelial lining and secretory status of the mammalian oviduct. The timely transition in the synthesis and release of oviduct proteins, due to the ovarian steroids, and their interactions with oocytes, sperm, and the fertilized ovum underscore key biological events during gamete interactions and early embryonic cleavage. The regulation of these secretory alterations during the first few days of pregnancy is discussed with respect to the influence of the ovarian steroids, their interactions with the embryo microenvironment, and the possible ways in which they may mediate the critical reproductive events of fertilization and embryo development.

Early embryonic development in the Djungarian hamster (Phodopus sungorus) is accompanied by alterations in the distribution and intensity of an estrogen (E2)-dependent oviduct glycoprotein in the blastomere membrane and zona pellucida and in its association with F-actin.

The luminal environment of the estrogen (E2)-dominated mammalian oviduct generates and sustains the environment in which the first embryonic cleavages take place. The objective of this study was to determine, by use of an antiserum against an E2-dependent sheep oviduct secretory glycoprotein (M(r) 90,000-92,000), whether the E2-dominated and pregnant oviduct of the Djungarian hamster (Phodopus sungorus) releases an antigenically related protein. If the protein was present, a secondary objective was to define its fate and association with filamentous-actin (f-actin) and chromatin patterns in early cleavage-stage embryos. Oviduct flushings containing embryos (1-cell fertilized, 2-, 4-, and 8-cells), and uterine flushings (> 16 cell embryos) were obtained from pregnant hamsters. Embryos were removed from flushings, and oviduct secretions were analyzed by Western blotting. The zona pellucida was removed with acid Tyrode's solution from approximately half of the 2-, 4-, and 8-cell embryos. Zona-intact and zona-free embryos were then fixed and subjected to triple immunofluorescence staining with an antiserum to the sheep oviduct protein, rhodamine phalloidin, and Hoechst 33258. An antigenically related protein M(r) 200,000) was detected in oviduct secretions of E2-treated, ovariectomized, and pregnant hamsters, and not in secretions from ovariectomized controls. In the zona pellucida of 1- and 2-cell embryos, the oviduct protein displayed an intertwining, reticular organization that was replaced by a diffuse and more intense accumulation in 4-, 8-, and > 16-cell embryos. In 2-cell embryos, punctate foci of the oviduct protein were distributed unevenly over the apical blastomere plasma membrane, forming patches in regions of f-actin exclusion, which were absent at later development stages. At the 4- and 8-cell stage of development, as blastomeres lost their spherical form by minimizing intercellular spaces, the oviduct protein took on a polarized arrangement and was intensely concentrated on membrane areas involved in cell-cell contact that were also the focus of f-actin. These data show that in early cleavage-stage hamster embryos, the intensity and pattern of staining for an E2-dependent oviduct protein (M(r) 200,000) that is released into the oviduct lumen during embryo transport can be distinguished on the basis of membrane f-actin display and blastomere number and shape. These events may mediate cellular processes related to blastomere cleavage, shaping, and/or adhesion that occur in the oviduct.

Centrosome and microtubule dynamics during meiotic progression in the mouse oocyte.

The disposition, function and fate of centrosomes were analysed in mouse oocytes undergoing in vitro meiotic maturation, using multiple-label fluorescence microscopy. Oocytes fixed at various points during meiotic progression were double labeled with either human centrosome-specific antibody, 5051, and anti-tubulin antibodies or 5051 and MPM-2 antibodies in order to evaluate the microtubule nucleation capacity and phosphorylation status of centrosomes during this process. Double labeling with anti-tubulin antibodies revealed two populations of centrosomes that undergo stage-specific changes in number, location and microtubule nucleation capacity in relation to spindle assembly and cytoplasmic events. Specifically, one population was consistently associated with chromatin throughout meiotic maturation whereas a second population of cytoplasmic centrosomes exhibited maximal numbers and nucleation capacity at prometaphase and anaphase of meiosis-I. Quantitative evaluation of cytoplasmic centrosomes indicated increased numbers during the transition from diakinesis to prometaphase and metaphase to anaphase and total disappearance during telophase. Colocalization studies with MPM-2 revealed that centrosomes were always phosphorylated. However, at metaphase of meiosis I and II the microtubule nucleation capacity of centrosomes was diminished. These results suggest the existence of two discrete populations of centrosomes in the mouse oocyte that are coordinately regulated to subserve aspects of microtubule organization relative to both nuclear and cytoplasmic events.