Michael J. Dykstra

Ultrastructural Morphometry of Bovine Blastocysts Produced In Vivo or In Vitro

Abstract The objective of this study was to compare the ultrastructure of bovine blastocysts produced in vivo or in vitro by using morphometric analysis. Blastocysts produced in vivo (multiple ovulations, MO) were obtained from superovulated Holstein cows. For blastocysts produced in vitro, cumulus-oocyte complexes aspirated from ovaries of Holstein cows were matured and fertilized in vitro. At 20 h postinsemination (hpi), zygotes were distributed into one of three culture media: 1) IVPS (in vitro produced with serum): TCM-199 + 10% estrous cow serum (ECS); 2) IVPSR (in vitro produced with serum restriction): TCM-199 + 1% BSA until 72 hpi, followed by TCM-199 + 10% ECS from 72 to 168 hpi; and 3) mSOF (modified synthetic oviductal fluid): mSOF + 0.6% BSA. At 168 hpi, six or seven grade 1 blastocysts from each of the four treatments (MO, IVPS, IVPSR, and mSOF) were fixed and prepared for transmission electron microscopy. Random micrographs of each blastocyst were used to determine the volume density of cellular components. Overall, as blastocysts progressed in development, the volume densities of cytoplasm and intercellular space decreased (P < 0.05) and the volume densities of mature mitochondria, nuclei, blastocoele, and apoptotic bodies increased (P < 0.05). Across treatments, the proportional volumes of nuclei and inclusion bodies were increased in inner cell mass cells compared with trophectoderm cells for mid- and expanded blastocysts. For blastocysts produced in vitro, the volume density of mitochondria was decreased (P < 0.05) as compared with that of blastocycts produced in vivo. The proportional volume of vacuoles was increased (P < 0.05) in blastocysts from the mSOF treatment as compared with blastocysts produced in vivo. For mid- and expanded blastocysts from all three in vitro treatments, the volume density of lipid increased (P < 0.05) and the volume density of nuclei decreased (P < 0.05) compared with those of blastocysts produced in vivo. In conclusion, blastocysts produced in vitro possessed deviations in volume densities of organelles associated with cellular metabolism as well as deviations associated with altered embryonic differentiation. However, the specific nature of these deviations varied with the type of culture conditions used for in vitro embryo production.

FoxJ1-dependent gene expression is required for differentiation of radial glia into ependymal cells and a subset of astrocytes in the postnatal brain

Neuronal specification occurs at the periventricular surface of the embryonic central nervous system. During early postnatal periods, radial glial cells in various ventricular zones of the brain differentiate into ependymal cells and astrocytes. However, mechanisms that drive this time- and cell-specific differentiation remain largely unknown. Here, we show that expression of the forkhead transcription factor FoxJ1 in mice is required for differentiation into ependymal cells and a small subset of FoxJ1+ astrocytes in the lateral ventricles, where these cells form a postnatal neural stem cell niche. Moreover, we show that a subset of FoxJ1+ cells harvested from the stem cell niche can self-renew and possess neurogenic potential. Using a transcriptome comparison of FoxJ1-null and wild-type microdissected tissue, we identified candidate genes regulated by FoxJ1 during early postnatal development. The list includes a significant number of microtubule-associated proteins, some of which form a protein complex that could regulate the transport of basal bodies to the ventricular surface of differentiating ependymal cells during FoxJ1-dependent ciliogenesis. Our results suggest that time- and cell-specific expression of FoxJ1 in the brain acts on an array of target genes to regulate the differentiation of ependymal cells and a small subset of astrocytes in the adult stem cell niche.

TRITRICHOMONAS FOETUS AND NOT PENTATRICHOMONAS HOMINIS IS THE ETIOLOGIC AGENT OF FELINE TRICHOMONAL DIARRHEA

Recently, several investigators have reported large-bowel diarrhea in cats associated with intestinal trichomonad parasites. These reports have presumptively identified the flagellates as Pentatrichomonas hominis, an organism putatively capable of infecting the intestinal tracts of a number of mammalian hosts, including cats, dogs, and man. The purpose of the present study was to determine the identity of this recently recognized flagellate by means of rRNA gene sequence analysis; restriction enzyme digest mapping; and light, transmission, and scanning electron microscopy (SEM).

Ultrastructural morphometry of bovine compact morulae produced in vivo or in vitro.

Abstract The objective of this study was to compare the ultrastructure of bovine compact morulae produced in vivo or in vitro using morphometric analysis. Compact morulae produced in vivo were obtained from superovulated Holstein cows. Compact morulae produced in vitro were obtained from cumulus-oocyte complexes aspirated from ovaries of Holstein cows. The complexes were matured and fertilized in vitro. At 20 h postinsemination (hpi), zygotes were distributed into 1 of 3 culture media: 1) IVPS (in vitro produced with serum): TCM-199 + 10% estrous cow serum (ECS); 2) IVPSR (in vitro produced with serum restriction): TCM-199 + 1% BSA until 72 hpi followed by TCM-199 + 10% ECS from 72 to 144 hpi; 3) mSOF (modified synthetic oviductal fluid): SOF + 0.6% BSA. At 144 hpi, five grade 1 compact morulae from each of the four treatments were prepared for transmission electron microscopy. The volume density occupied by cellular components was determined by the point-count method using a sampling of seven to nine random micrographs from each compact morula. The volume density of lipid was greater (P < 0.05) in compact morulae from IVPS, IVPSR, and mSOF treatments compared with those produced in vivo. There was a reduced proportional volume of total mitochondria in compact morulae from the IVPS treatment compared with those produced in vivo (P < 0.05). For compact morulae from the IVPS culture treatment, the volume density of vacuoles was greater than that for compact morulae produced in vivo (P < 0.05). The cytoplasmic-to-nuclear ratio for compact morulae from the IVPS treatment was increased (P < 0.05) compared with the ratio for those produced in vivo. In conclusion, compact morulae produced in vitro differed ultrastructurally from those produced in vivo. Compact morulae produced in IVPS culture medium possessed the greatest deviations in cellular ultrastructure.

Hydrocarbon synthesis by enzymatically dissociated oenocytes of the abdominal integument of the German Cockroach, Blattella germanica

In insects, hydrocarbons waterproof the cuticle, protect the insect from the external environment, and serve as semiochemicals or their metabolic precursors. In the German cockroach, Blattella germanica, hydrocarbons are synthesized by the abdominal integument, but the precise site of biosynthesis is not known. We developed a method for separation of oenocytes from other cells in the abdominal integument using enzymatic dissociation followed by Percoll gradient centrifugation. Radiolabeled propionate was then used to monitor de novo synthesis of hydrocarbons by dissociated cells. Oenocyte-enriched cell suspensions of abdominal sternites synthesized hydrocarbons, whereas suspensions enriched with epidermal cells did not. Our results show conclusively that hydrocarbons are produced by oenocytes not only in insects whose oenocytes are localized within the hemocoel, but also in those insects whose oenocytes are within the abdominal integument. Furthermore, these data support a hemolymph pathway for transport and delivery of hydrocarbons to both external and internal tissues, including the epicuticle, fat body, and ovaries.

Characterization of the Aphanomyces species involved with ulcerative mycosis (UM) in Menhaden.

Aphanomyces and Saprolegnia were isolated from characteristic lesions of Ulcerative Mycosis (UM) on Atlantic menhaden and other fish. One of the Aphanomyces isolates was selected for cultural studies which revealed that the fungus was stimulated to increased vegetative growth and improved zoosporulation by low levels of NaCl. The salt tolerance exhibited by this fungus surpassed the known recorded limits for Oomycetes in regard to zoosporogenesis. The relationship of the fungal isolates to the lesions found on fish and the association of prevailing climatological conditions with the disease suggest a complex interaction between the fungus, possibly pre-stressed fish, and the salinity of the estuary systems of North Carolina.

Metals mimic airway epithelial injury induced by in vitro exposure to Utah Valley ambient particulate matter extracts.

Epid emiologic studies have shown positive associations between changes in ambient particulate matter (PM) levels in Utah Valley during 1986–1988, and the respiratory health of the local population. Ambient PM reductions coincided with closure of an open-hearth steel mill, the major industrial source of particulate emissions in the valley. In this report, water extracts of PM filters from steel mill ope rational (UE-86, UE-88) and closure (UE-87) periods were analyzed for their elemental composition. Their relative toxicity was determined by expos ing primary rodent airway epithelial cultures to equal masses of extracted material. To elucidate extract subcomponents mediating the effects observed, cells were also exposed to surrogate metal mixtures. Potential interactions between the two predominant metals in the UE-86/88 samples, zinc (Zn) and copper (Cu), were further investigated. Data indicated that, relative to the UE-87 (plant closed) sample, UE-86/88 samples contained more sulfate, calcium, potassium, magnesium and, although presentin much lower amounts, a variety of metals including Zn, Cu. iron, lead, strontium, nickel, manganese, and vanadium N). Cell expos ure to UE-86 and UE-88, but not UE-87, resulted in time- and concentration-dependent epithelial injury based on biochemical and light/electron microscopic changes. Cell injury induced by metal mixtures containing equivalent amounts of Zn + Cu + V was commensurate with that induced by the corresponding extract, although divergent antioxidant responses were observed. Expos ure to Zn + Cu resulted in significantly greater epithelial toxicity and stress (c-Jun N-terminal protein kinase activation) responses than did exposure to Zn or Cu individually. The parallel epithelial injury induced by the extracts and their surrogate Zn+Cu+V mixtures suggests that these metals are mediating the acute airwayep ithelial effects observed; however, metal interactions appear to play a critical role in the overall cellular effects induced by the PM-derived extracts. These experimental findings are in good accord with epidemiologic reports of adverse airway and respiratory health effects in Utah Valley residents.

Chronic Inflammatory Cells with Epithelial Cell Characteristics in Teleost Fishes

Certain cells that participate in the chronic inflammatory response of teleost fishes have many features typical of epithelioid cells of mammals. Such features include high metabolic activity, frequent phagolysosomes, and cytoplasmic interdigitations between adjacent cells; however, the epithelioid granulomas formed in response to certain diseases in teleost fishes also have several features associated with epithelial cells. Cases of ulcerative mycosis or acid-fast bacterial infection in Atlantic menhaden (Brevoortia tyrannus), fungal infection in silver perch (Bairdiella chrysoura), and mycobacteriosis in Mozambique tilapia (Oreochromis mossambicus) had epithelioid cells that were joined together by well-formed desmosomes with tonofilaments. “Mature granulomas” of the ulcerative mycosis-infected menhaden stained positively for cytokeratin, a cytoskeletal protein that is considered to be highly specific for epithelial cells. The consistent presence of these heretofore unrecognized epithelial features suggest that they may be characteristic of certain types of cells participating in piscine chronic inflammation.

Notexin-induced muscle injury in the dog

Notexin, a myotoxic phospholipase, was used to induce focal necrosis in the sartorius muscles of normal mixed-breed adult dogs and in 12-week-old beagles. Notexin injury caused pathologic changes similar to those of Duchenne muscular dystrophy (DMD) and its canine homologue, golden retriever muscular dystrophy (GRMD). All three conditions are characterized by increased serum creatine kinase (CK) levels, sarcolemmal defects, delta lesions, hyaline degeneration of myofibers, calcium-positive myofibers, and minimal effects on neurovascular structures. Four and 24 h after exposure to notexin, serum CK levels were elevated, and many myofibers were necrotic. In addition, by 24 h the necrotic areas were heavily invaded by mononuclear cells, and calcium-positive myofibers were prominent. Capillaries appeared intact even in areas of marked myonecrosis. Massive cellular infiltrate and myotube formation was evident at 3 days post injury. By 7 days, most affected fascicles were occupied by small immature myofibers. Regeneration was largely complete at 21 days. Our results suggest that notexin-induced muscle injury in dogs will be useful in the evaluation of potential therapies for DMD such as myoblast transplantation.

Capillary Pressure as Related to Water Holding in Polyacrylamide and Chicken Protein Gels

UNLABELLED The ability of food gels to hold water affects product yield and organoleptic quality. Most researchers believe that water is held by capillarity such that gels having smaller mean pore diameter and a more hydrophilic surface hold water more tightly. To date, however, only qualitative evidence relating pore size to water holding (WH) properties has been provided. The present study sought to provide quantitative confirmation of this hypothesis. Scanning electron microscopy coupled with image analysis was used to measure pore size, and water contact angle with the gel surface was measured by the captive bubble method, in both model polyacrylamide gels and heat-induced protein (minced chicken breast) gels. These were related to water lost during cooking of meat pastes to form gels (cooking loss (CL)), as well as water lost upon centrifugation (expressible water (EW)) or by capillary suction (CSL) of all prepared gels, as inverse measures of WH. As predicted by the Young-Laplace equation for calculating capillary pressure, the presumed mechanism of WH, gels with lower water losses exhibited a more hydrophilic surface (smaller contact angle). Yet, both lower CL and CSL correlated with larger mean pore diameter of gels, not smaller as had been expected. Polyacrylamide gels varied more in WH than did prepared meat gels, yet only the capillary suction method was sensitive enough to detect these differences. PRACTICAL APPLICATION  The ability of gels to hold water is important for economics of processing, food quality, and food safety. This study investigated the prevailing theory for how gels hold water, capillarity. Both the pore sizes of gel microstructures and the degree of hydrophilicity of the polymers comprising each gel were quantitatively assessed and related to water holding (WH) properties, and this was the first report using such methodologies. It appeared that the degree of hydrophilicity was much more important explaining WH properties than pore size, and that future research of this kind should be carried out.