David G. Capco

Activation of protein kinase C after fertilization is required for remodeling the mouse egg into the zygote

Fertilization of the mammalian egg initiates numerous biochemical and structural changes which remodel the egg into a single‐celled zygote. To date, the most extensively studied phenomenon of fertilization in virtually all species has been the relationship between sperm penetration and the induction of the initial rise in intracellular‐free calcium ([Ca2+]i) concentration within the egg. In contrast, relatively few studies have focused on the biochemical events following this rise in calcium, and even fewer studies have directly linked the biochemical events to the structural changes which must ensue for proper development of the embryo. In this study, we exploited recently developed technologies to investigate the action of protein kinase C (PKC), a presumed downstream transducer of the initial rise in [Ca2+]i, during fertilization and artificial activation with calcium ionophore or phorbol 12‐myristate 13‐acetate (PMA). The newly developed myristoylated PKC pseudosubstrate (myrPKCΨ) was used to specifically inhibit PKC, thereby averting the trauma of injecting the egg with nonmyristoylated PKCΨ. Following fertilization, eggs which were pretreated with myrPKCΨ were not capable of forming a second polar body and pronuclear formation was significantly inhibited. Spatial and temporal localization of PKC using confocal microscopy to visualize the PKC reporter dye, Rim‐1, demonstrated localization of PKC to the lateral aspects of the forming second polar body after fertilization, or after artificial activation with calcium ionophore or PMA. In vivo biochemical analysis of eggs which were fertilized or artificially activated demonstrated that PKC activity rose at the same time (40 min) as the second polar body formed and then subsided over the next 5 hr post activation. From these data, we conclude that PKC plays an integral role in directing the transformation from egg to embryo. Mol. Reprod. Dev. 46:587–601, 1997.

Regulation of cell adhesion during embryonic compaction of mammalian embryos: Roles for PKC and β‐catenin

Beta‐catenin has a number of roles in early development including involvement in cell adhesion, cell signaling, and developmental fate specification. This study investigates the mechanisms that regulate embryonic compaction, the first cell adhesion event in early mammalian development. Mammalian embryos can be induced to compact at an earlier developmental stage than normal by treatment with agonists that activate protein kinase C (PKC), and this treatment is used to identify and analyze the minimum essential changes required for embryonic compaction. It was predicted that: (1) since activation of PKC can induce compaction prematurely in mouse embryos, phosphorylation of the protein components of the adherens complex would occur during induced compaction and that these components would be required for the cell adhesive event; (2) these same proteins should be phosphorylated during compaction in normal development; (3) new, highly‐specific inhibitors of PKC activity would inhibit compaction during normal development and induced compaction; and (4) some PKC isotypes would become localized to the junctional membranes during the process of compaction. In agreement with these predictionst, β‐catenin became phosphorylated on serine/threonine residues both during induced compaction and normal development. Inhibitors to PKC, but not inhibitors to other kinases, blocked compaction. Furthermore, the alpha isotype of PKC is recruited to the membranes of the apposing blastomeres both during induced compaction and during normal development immediately before compaction begins and before β‐catenin becomes part of the detergent‐resistant cytoskeleton at the junction. Mol. Reprod. Dev. 54:135–144, 1999.

Transient localizations of messenger RNA in Xenopus laevis oocytes

Abstract The distribution of poly(A)-containing RNA [poly(A) + RNA] was examined in sections of oocytes, mature eggs, and early embryos of Xenopus laevis by in situ hybridization with [ 3 H]poly(U). Poly(A) + RNA was uniformly distributed in the cytoplasm of previtellogenic oocytes. In contrast, distinct cytoplasmic regions exhibiting high concentrations of poly(A) + RNA were detected in vitellogenic and full-grown oocytes. These regions include a perinuclear area located between the germinal vesicle (GV) and the cortex of stage 3 oocytes, a cortical area in stage 4 oocytes, a vegetal subcortical area and a subnuclear zone in stage 5 oocytes, and a vegetal subcortical area in stage 6 oocytes. No selective accumulations of poly(A) + RNA were observed in the mitochondrial mass of previtellogenic oocytes or in the GV at any time during oogenesis. The poly(A) + RNA localizations in the peripheral regions of full-grown oocytes disappeared during the first maturation division leading to a uniform poly(A) + RNA distribution in the mature egg and did not reappear during early embryogenesis. The results suggest that transient localizations of poly(A) + RNA are present in the oocyte cytoplasm during oogenesis of Xenopus laevis .

Differential accumulation and localization of maternal poly(A)-containing RNA during early development of the ascidian, Styela.

Abstract The regional distribution of poly(A) + RNA was examined in sections of Styela oocytes and fertilized eggs by in situ hybridization with [ 3 H]poly(U). The nucleus and cytoplasm of previtellogenic oocytes contain equivalent densities of [ 3 H]poly(U) binding sites. The concentration of these sites is reduced in the cytoplasm, but not the nucleus, during vitellogenesis. Consequently, the germinal vesicle (GV) plasm of mature oocytes is characterized by an eightfold elevation in [ 3 H]poly(U) binding activity relative to the surrounding cytoplasm. The distinctive cytoplasmic regions of the mature oocyte do not exhibit differential concentrations of [ 3 H]poly(U) binding sites. Following fertilization which triggers GV breakdown, meiosis, and ooplasmic segregation, the high density of [ 3 H]poly(U) binding sites characteristic of the GV plasm is conserved in the basophilic cytoplasm during its extensive migration and eventual accumulation in the animal hemisphere of the egg. The insensitivity of the [ 3 H]poly(U) binding sites of the basophilic cytoplasm to actinomycin D suggests that they are of maternal origin. It is concluded that maternal poly(A) + RNA is subject to differential accumulation in the GV plasm and its derivative ooplasm during the early development of Styela .

Differential distribution of poly(A)-containing RNA in the embryonic cells of Oncopeltus fasciatus: Analysis by in situ hybridization with a [3H]poly(U) probe☆

Abstract The regional distribution of poly(A)+ RNA was examined in the embryonic cells of the milkweed bug, Oncopeltus fasciatus, by in situ hybridization of histological sections with a [3H]poly(U) probe. As shown by a number of control experiments, this probe interacts specifically with poly(A) sequences preserved in the sections. Using this method, it was shown that labeling of periplasmic and vitellophage nuclei increases markedly early during syncytial blastoderm formation. At this time, label also increases in the vitellophage cytoplasm but not in the cytoplasm surrounding the blastodermal nuclei. Labeling continues to increase in the blastodermal nuclei during cellularization and germ band differentiation without a concomitant accumulation in the blastodermal cell cytoplasm. At the time of germ band invagination, the region of the most intense subcellular labeing shifts from the nucleus to the cytoplasm of the invaginated cells. This shift is not evident in the blastodermal cells which remain at the surface of the egg to become the serosa. In the serosa and the vitellophage energids, labeling then decreases as histogenesis proceeds. Significant labeling of the nuclei and cytoplasm of the invaginated germ band cells continues through germ layer formation. It is concluded that poly(A)+ RNA, probably synthesized de novo following oviposition, is subject to differential intracellular distribution in three types of Oncopeltus embryonic cells which may reflect cell-specific patterns of mRNA or poly(A) metabolism.

Cytoskeletal reorganization during early mammalian development: Analysis using embedment-free sections

We have examined cytoskeletal reorganization during early embryonic development in the hamster by employing detergent extraction to remove soluble components of the embryos and reveal the underlying structural network. This procedure allows examination of both the cortical cytoskeleton and the cytoskeleton of the egg interior. Sections of eggs and embryos were prepared for transmission electron microscopy with the removable embedding medium, diethylene glycol disterate which allows thicker sections than conventional embedment procedures thereby providing more spatial cues for studying organization. The cytoskeleton reorganizes after fertilization, at the time of compaction and again at the blastocyst stage. These cytoskeletal reorganizations are considered in terms of the blastomere polarity hypothesis and the involvement of the cytoskeleton with early embryonic development.

Progesterone acts through protein kinase C to remodel the cytoplasm as the amphibian oocyte becomes the fertilization-competent egg

The fertilization-competent Xenopus egg undergoes a contraction of its cortex towards the apex of the pigmented animal hemisphere within 10 min of fertilization. Evidence suggests that protein kinase C (PKC) is involved in the assembly of this contractile network and we show that PKC is rapidly activated as a result of exposure of oocytes to progesterone. Xenopus oocytes contain at least five different isotypes of PKC. Three actin-binding proteins (i.e. vinculin, talin and ankyrin) appear to play an early role in the assembly of the contractile network and one of the proteins (vinculin) becomes phosphorylated shortly after progesterone treatment as the contractile network is assembling. Our results indicated that progesterone acts through a phospholipase to activate PKC and that PKC participates in the remodeling of the cytoplasmic compartment as the oocyte becomes the egg.

Colocalization of CaM KII and MAP kinase on architectural elements of the mouse egg: Potentiation of MAP kinase activity by CaM KII

The conversion of the egg to a zygote requires the initiation of several signaling pathways that act in an orchestrated fashion to rapidly remodel the egg. Architectural elements within the egg can serve to localize components of these signaling pathways and colocalization of such components provides the opportunity for interaction between different signaling pathways. This study examines the localization as well as the state of activation of two different kinases, MAP kinase and calcium/calmodulin‐dependent protein kinase II (CaM KII). The meiotic spindle serves as a site for enrichment of these kinases. However, activated MAP kinase and activated CaM KII exhibit a developmental stage–specific pattern of localization that represents a subset of the area occupied by the distribution of the total mass of MAP kinase and CaM KII. Suppression of CaM KII activity results in reduction in the amount of MAP kinase as well as a decreased level of activity of MAP kinase. Since CaM KII becomes active as a result of fertilization, the former kinase could serve to potentiate MAP kinase activity and the colocalization of these two kinases may facilitate such an interaction. Mol. Reprod. Dev. 58:69–77, 2001.

Adsorption of hematite nanoparticles onto Caco-2 cells and the cellular impairments: effect of particle size

The increasing applications of engineered nanomaterials nowadays have elevated the potential of human exposure through various routes including inhalation, skin penetration and digestion. To date there is scarce information on a quantitative description of the interactions between nanoparticles (NPs) and cell surfaces and the detrimental effects from the exposure. The purpose of this work was to study in vitro exposure of Caco-2 cells to hematite (alpha-Fe(2)O(3)) NPs and to determine the particle size effects on the adsorption behaviors. Cellular impairment was also investigated and compared. Hematite NPs were synthesized as part of this study with a discrete size distribution and uniform morphology examined by dynamic light scattering (DLS) and confirmed by transmission electron microscopy (TEM). Caco-2 cells were cultured as a model epithelium to mirror human intestinal cells and used to evaluate the impacts of the exposure to NPs by measuring transepithelial electrical resistance (TEER). Cell surface disruption, localization and translocation of NPs through the cells were analyzed with immunocytochemical staining and confocal microscopy. Results showed that hematite NPs had mean diameters of 26, 53, 76 and 98 nm and were positively charged with minor aggregation in the buffer solution. Adsorption of the four sizes of NPs on cells reached equilibrium within approximately 5 min but adsorption kinetics were found to be size-dependent. The adsorption rates expressed as mg m(-2) min(-1) were greater for large NPs (76 and 98 nm) than those for small NPs (26 and 53 nm). However, adsorption rates, expressed in units of m(-2) min(-1), were much greater for small NPs than large ones. After the adsorption equilibrium was reached, the adsorbed mass of NPs on a unit area of cells was calculated and showed no significant size dependence. Longer exposure time (>3 h) induced adverse cellular effects as indicated by the drop in TEER compared to the control cells without the exposure to NPs. NPs initially triggered a dynamic reorganization and detachment of microvilli structures on Caco-2 cell surfaces. Following this impact, the drop in TEER occurred more significantly, particularly for the exposure to 26 nm NPs, which was consistent with the observations with confocal microscopy that the junctions were more severely disrupted by 26 nm NPs than other sizes. In conclusion, this paper demonstrates the interactions at the ultrastructural level from initial surface adsorption of NPs upon cells, to the subsequent microvilli reorganization, membrane penetration and the disruption of adherens junction and provides the fundamental information on size effects on NP behavior which is often poorly addressed for in vitro cytotoxicity studies of NPs.

Involvement of the PKC family in regulation of early development

Protein kinase C (PKC) isotypes have been implicated in a number of key steps during gametogenesis, fertilization, and early development. The 11‐member family of PKC isotypes, many with different cofactor requirements for activation, can provide for differential activation of the specific kinases. In addition the enrichment of particular PKC isotypes to unique locations within gametes, zygotes, and early embryos likely promotes specific substrate interactions. Evidence exists to indicate involvement of PKC isotypes during sperm capacitation and the acrosome reaction, during resumption of meiosis in the oocytes, regulating the spindle organization in meiosis I and II, at fertilization, in the pronuclei, in the mitotically dividing blastomeres of the embryo, and at the plasma membranes of blastomeres at the time of embryonic compaction. Evidence also exists for crosstalk with other signaling pathways and one or more isotypes of PKC appear to be active at each major developmental transition. Mol. Reprod. Dev. 77: 95–104, 2010.