Expression of ZO-1 and occludin at mRNA and protein level during preimplantation development of the pig parthenogenetic diploids

Expression of mRNAs and proteins of ZO-1 and occludin was analyzed in pig oocytes and parthenogenetic diploid embryos during preimplantation development using real-time RT-PCR, western blotting and immunocytochemistry. All germinal vesicle (GV) and metaphase (M)II oocytes and preimplantation embryos expressed mRNAs and proteins of ZO-1 and occludin. mRNA levels of both ZO-1 and occludin decreased significantly from GV to MII, but increased at the 2-cell stage followed by temporal decrease during the early and late 4-cell stages. Then, both mRNAs increased after compaction. Relative concentration of zo1α- was highest in 2-cell embryos, while zo1α+ was expressed from the morula stage. Occludin expression greatly increased after the morula stage and was highest in expanded blastocysts. Western blotting analysis showed constant expression of ZO-1α- throughout preimplantation development and limited translation of ZO-1α+ from the blastocysts, and species-specific expression pattern of occludin. Immunocytochemistry analysis revealed homogeneous distribution of ZO-1 and occludin in the cytoplasm with moderately strong fluorescence in the vicinity of the contact region between blastomeres, around the nuclei in the 2-cell to late 4-cell embryos, and clear network localization along the cell-boundary region in embryos after the morula stage. Present results show that major TJ proteins, ZO-1 and occludin are expressed in oocytes and preimplantation embryos, and that ZO-1α+ is transcribed by zygotic gene activation and translated from early blastocysts with prominent increase of occludin at the blastocyst stage.     

Characteristics of preimplantational development of porcine parthenogenetic diploids relative to the existence of amino acids in vitro

The present study was designed to investigate the effects of amino acids on the in vitro development of porcine parthenogenetic diploids that were produced by electrostimulation (El-St) and cytochalasin B treatment of in vitro-matured oocytes. The culture medium for development, based on Whitten medium, contained 0.5 mg/ml of hyaluronic acid (mWM), and a two-step culture system in which 290 mOsmol before the 4-cell stage (48 or 72 h after El-St) and, subsequently, 256 mOsmol up to the blastocyst stage (mWMs) were used. In experiment 1, the diploids were cultured for 168 h in mWMs supplemented with 0.01-5 mg/ml of polyvinyl alcohol (PVA). In experiment 2, the diploids were cultured in mWMs containing 0.5 mg/ml of PVA (PVA-mWMs) for 0, 48, or 72 h and then cultured for 168 h after El-St in PVA-mWMs supplemented with essential amino acids for Eagle basal medium without glutamine (E-AA) and nonessential amino acids for minimum essential medium (NE-AA). The results showed that diploids can develop up to the blastocyst stage in mWMs including 0.05-5.0 mg/ml of PVA (49%-53% vs. 63%, P > 0.05), but the replacement of BSA with PVA alone could not support the expansion of blastocysts (11%-20% vs. 39%, P < 0.05) or their proliferation. The addition of both E-AA and NE-AA (E+NE-AA) to PVA-mWMs from the 1-cell stage resulted in severe inhibition of the development of diploids to the blastocyst stage. However, the addition of E+NE-AA to PVA-mWMs later than 48 or 72 h after El-St well supported the development of diploids to the blastocyst stage and supported the expansion of blastocysts. In experiments 3-5, which types of amino acids in E-AA inhibited the development of diploids during the first 48 h after El-St were determined. In experiment 6, the stimulatory effects of E-AA and/or NE-AA after the 4-cell stage were examined. The results of those experiments clearly showed that the presence of nonpolar E-AA, especially for valine, leucine, isoleucine, and methionine, during the first 48 h after El-St caused severe delay of the first division and inhibition of development beyond the 4-cell stage. The presence of NE-AA after the 4-cell stage produced a favorable condition for the expansion of blastocysts (33%), whereas the presence of E-AA increased the cleavage rates of the diploids after compaction and the total number of cells in the blastocysts (53.7 +/- 2.7) and inner cell mass (12 +/- 0.5). These findings indicate that the presence of nonpolar E-AA in a protein-free medium during the first 48 h causes the 4-cell block in porcine parthenogenetic diploids.     

Effects of hormones and osmolarity in the culture medium on germinal vesicle breakdown of porcine oocytes

The present study was conducted to examine effects of hormones and osmolarity on germinal vesicle breakdown (GVBD) and histone H1 kinase (H1K) activity in porcine oocytes cultured in vitro. The basic medium used for culture of oocytes was modified Tyrode's solution in which the osmolarity was adjusted to 134 to 495 mOsm by changing the concentration of sodium chloride (NaCl). When the hormones were present, osmolarity of medium that allows GVBD of oocytes was less than 400 mOsm. However, the range of osmolarity of medium that allows meiotic maturation of oocytes was 210 to 362 mOsm. On the other hand, without hormonal supplement, the incidence of GVBD in oocytes decreased as the osmolarity of the medium increased in the rage of 210 to 362 mOsm. By increasing the osmolarity of the medium from 210 to 362 mOsm by addition with sorbitol instead of NaCl, the incidence decreased from 89.1% to 13.3%. In oocytes cultured in medium of 210 mOsm without hormones, the percentage of oocytes that underwent GVBD and had increased H1K activity 20 h after culture was significantly higher (P < 0.05) than those of oocytes cultured in the same medium supplemented with hormones or medium of 362 mOsm. These results indicate that in vitro induction of GVBD in porcine oocytes is strongly affected by osmolarity of the medium in the absence of hormones. The results also suggest that, under low osmolarity (210 mOsm), GVBD is accelerated with rapid increase of H1K activity.     

The effects of the physical characteristics of the culture medium on the development of red seaweeds in tissue culture

Explants of Gelidium versicolor, Grateloupia doryphora and Laurencia sp. were cultivated in Provasoli enriched seawater culture medium (PES) adjusted to several osmolalities (0.5, 0.7, 1.0 and 1.5 Os kg^–1) and solidities (agar concentration = 3, 8 and 15 g L^–1). Osmolality was adjusted by dilution of seawater with distilled water (50, 70 and 100% seawater) and by NaCl addition. Explants of Laurencia sp. and Grateloupia doryphora showed bud regeneration and callus formation. Explants of Gelidium versicolor only showed bud regeneration. Osmolalities of 0.5 and 1.05 Os kg^–1. inhibited or drastically reduced bud regeneration and callus formation. The highest callus formation and bud regeneration were observed at 0.7 to 1.0 Os kg^–1. An increase in the agar concentration of the culture medium was positively correlated with callus formation and negatively correlated with bud regeneration. An increase in the percentage of seawater increased the solidity of the culture medium and was positively correlated with callus formation. Glycerol was an effective carbon source for the vegetative propagation of axenic explants of Grateloupia doryphora, promoting growth and bud regeneration. An increase in glycerol concentration in the culture medium increased its osmolality, inhibiting the growth of the explants and their morphogenetic development.     

Buoyant density of Escherichia coli is determined solely by the osmolarity of the culture medium

In previous studies, we had shown that the buoyant density ofEscherichia coli is determined by the osmolarity of the growth medium by varying the osmolarity of the medium with NaCl or sucrose. However, the buoyant density of the cells always exceeded that of the growth medium. Here we determined the effect of medium with a buoyant density greater than the expected buoyant density of cells by adding Nycodenz to Luria broth. Percoll gradients of cells were analyzed by laser light scattering. The buoyant density for 125- and 375-mOsM-grown cells was 0.002 g/ml and 0.003 g/ml more, respectively, for cells grown in the presence of Nycodenz than those grown without Nycodenz, while the buoyant density of 250-mOsM-grown cells was 0.005 g/ml less for cells grown in the presence of Nycodenz than those grown without Nycodenz. Cells grown in 500-mOsM medium with or without Nycodenz had the same buoyant density. the buoyant density of cultures grown in defined medium was the same as those grown in rich medium, with only the medium osmolarity correlating to buoyant density. We conclude from these experiments that neither buoyant density nor chemical make-up of the medium determines the buoyant density of cells grown in that medium. Only the medium osmolarity determines cell buoyant density, suggesting thatE. coli has no mechanisms to sense buoyant density.     

The effects of various antioxidants on the development of parthenogenetic porcine embryos

The major objective of this study was to improve the development rate of parthenogenetic porcine embryos. In this study, the anti-oxidative and anti-apoptotic effects of three antioxidants, β-mercaptoethanol (β-ME), α-tocopherol, and extracellular superoxide dismutase (EC-SOD), were examined on the development of parthenogenetic porcine embryos. The development rate of parthenogenetic porcine embryos to the blastocyst stage was 8.1% for control; 19.1%, 14.6%, and 5.0% for 1, 3, and 5 μM β-ME; 17.2% and 17.5% for 50 and 100 μM α-tocopherol and 12.0% and 4.0% for EC-SOD transgenic mouse embryonic fibroblast (Tg-MEF) and EC-SOD non-transgenic mouse embryonic fibroblast (NTg-MEF) conditioned medium at day 3, respectively. Here, β-ME, α-tocopherol, and EC-SOD Tg-MEF conditioned medium increased the development rate of parthenogenetic porcine embryos to the blastocyst stage (P?<?0.05). The average number of total cells and apoptotic cells at the blastocyst was analyzed at the optimal conditions of the three antioxidants. The three antioxidants increased the average number of total cells at the blastocyst, and they decreased apoptotic cells at the blastocyst as compared to control without supplementation (P?<?0.05). When the reactive oxygen species levels in two-cell embryos after 1 μM β-ME and 100 μM α-tocopherol treatment were examined, those were lower than control group (P?<?0.05). In conclusion, it was found that the three antioxidants, β-mercaptoethanol, α-tocopherol, and EC-SOD Tg-MEF, conditioned medium can play a role as a strong stimulator in the development of parthenogenetic porcine embryos.     

Effects of activation methods and culture conditions on development of parthenogenetic porcine embryos

The effects of different activation methods and culture conditions on early development of porcine parthenotes were examined. Three different activation methods were tested: (1) electroporation; (2) electroporation followed by incubation in the presence of butyrolactone I, an inhibitor of cdc2 and cdk2 kinases; and (3) electroporation followed by a treatment with cycloheximide, a blocker of protein synthesis. The activated oocytes were cultured in two different media, NCSU-23 and PZM-3 under 5% CO2 in air. In a separate experiment, the effects of high (approximately 20%) or low (5%) O2 tension on early embryo development were also evaluated. The average pronuclear formation was less (p<0.05) in the electroporated oocytes (83.9+/-1.7%) compared with those activated by electroporation and butyrolactone I or electroporation plus cycloheximide (92.8+/-0.8 and 93.0+/-1.0%). In PZM-3 medium, the average frequencies of blastocyst formation (59.7+/-3.6%) and hatching (10.6+/-1.3%) were greater than those in NCSU-23 medium (39.9+/-3.1% blastocyst formation, p<0.05; and 0.2+/-0.2% hatching; p<0.001). Furthermore, the average nuclear number was also greater (p<0.001) in blastocysts developed in PZM-3 (50.2+/-1.3) than in those developed in NCSU-23 (35.3+/-1.1). Blastocyst formation was similar (p>0.10) among the three activation procedures when parthenotes were cultured in NCSU-23, while in PZM-3 more (p<0.05) parthenotes produced by electroporation plus butyrolactone or electroporation plus cycloheximide developed into blastocysts compared to electroporation alone (64.9+/-5.2 and 68.6+/-3.5% compared with 45.6+/-4.7%). Incidences of apoptotic nuclei were similar (p>0.10) among all treatments. No difference in development was found between parthenotes that developed under high versus low O2 tension (p>0.10). These results demonstrate that activation methods targeting the calcium signaling pathway at several points trigger embryonic development more efficiently than electroporation alone. The data also imply that the PZM-3 medium provides for enhanced culture conditions for the early development of parthenogenetic porcine embryos than NCSU-23.     

Stage-specific excitation of cannabinoid receptor exhibits differential effects on mouse embryonic development

Anandamide (N-arachidonoylethanolamine), an arachidonic acid derivative, is an endogenous ligand for both the brain-type (CB1-R) and spleen-type (CB2-R) cannabinoid receptors. We have previously demonstrated that preimplantation mouse embryos express mRNA for these receptors and that the periimplantation uterus contains the highest level of anandamide yet discovered in a mammalian tissue. We further demonstrated that 2-cell mouse embryos exposed to low levels of anandamide (7 nM) or other known cannabinoid agonists in culture exhibit markedly compromised embryonic development to blastocysts and that this effect is mediated by CB1-R. In contrast, the present study demonstrates that blastocysts exposed in culture to the same low levels of cannabinoid agonists exhibited accelerated trophoblast differentiation with respect to fibronectin-binding activity and trophoblast outgrowth. Again, these effects resulted from activation of embryonic CB1-R. There was a differential concentration-dependent effect of cannabinoids on the trophoblast, with an observed inhibition of differentiation at higher doses. These results provide evidence for the first time that cannabinoid effects are differentially executed depending on the embryonic stage and cannabinoid levels in the environment. Since uterine anandamide levels are lowest at the sites of implantation and highest at the interimplantation sites, the new findings imply that site-specific levels of anandamide and/or other endogenous ligands in the uterus may regulate implantation spatially by promoting trophoblast differentiation at the sites of blastocyst implantation.     

Stage-specific effects of cAMP signaling during distal lung epithelial development

cAMP signaling is postulated to play a role in distal lung epithelial differentiation based on several observations. First, it enhances fibroblast growth factor-induced transdifferentiation of early tracheal epithelium into respiratory epithelium. Second, there are cAMP-responsive elements in the heterologous promoters of Sftpb and Sftpa genes. Third, cAMP augments the effect of dexamethasone in maintaining differentiation of human fetal type II pneumocyte culture. However, this concept has not been thoroughly tested in vivo. In the current study, we modulated cAMP signaling in developing distal lung epithelium in vivo using an inducible transgenic system that expressed a mutant form of Galpha(s) (Galpha(s)Q227L). We failed to demonstrate the ability of cAMP to promote distal epithelial maturation during embryonic stages. The results argue against its physiological role in this process. In addition, induction of cAMP signaling at the late pseudoglandular stage but not during the canalicular or saccular stage surprisingly delayed distal differentiation by suppressing the expression of Sftpc, Sftpa, and Aquaporin5 as well as the formation of lamellar bodies. This stage-specific inhibitory effect was observed in the absence of cellular toxicity or changes in branching. Transgenic lungs did not show significant changes in the known pathways that are important for distal differentiation. Therefore, we propose the existence of yet-to-be identified cAMP-sensitive novel regulators of early distal lung epithelial differentiation. Although the delay of differentiation seemed to be reversible at later stages, it still led to pronounced permanent postnatal airspace enlargement due to impaired paracrine function of distal epithelium in regulating alveolar myofibroblast development.     

Effects of media NaCl concentration and osmolarity on the culture of early-stage porcine embryos and the viability of embryos cultured in a selected superior medium

One- and 2-cell porcine embryos were obtained from oviductal flushes and cultured for 96 hours in media with varied osmolarity that resulted from alterations in NaCl and sorbitol content. The viability of experimental embryos cultured to advanced stages was determined by comparison with that of the controls, noncultured embryos transferred to recipient gilts. The data suggest that variation in embryonic development in the experimental media is related to the NaCl concentration rather than to osmolarity. Increased NaCl concentration impairs development of the embryos to the advanced morula/blastocyst stages (P<0.001). There was no difference in the pregnancy rate between the recipients of cultured (45%) and noncultured (57%) embryos on Day 25. There was, however, a higher embryonic survival rate (P<0.05) within the control gilts.     

Limited multiplication of Mycobacterium lepraemurium in parabiotic culture, as influenced by osmolarity of an alkaline-galactomannan medium

Kato, Laszlo (Institut de Microbiologie et d'Hygiène de l'Université de Montréal, Montreal, Quebec, Canada), and Bela Gozsy. Limited multiplication of Mycobacterium lepraemurium in parabiotic culture, as influenced by osmolarity of an alkaline-galactomannan medium. J. Bacteriol. 91:1859-1862. 1966.-Limited multiplication of Mycobacterium lepraemurium has been achieved in an alkaline (pH 8.4) galactomannan-containing medium, when cultivated alone or in parabiosis with a feeder strain, Torula minuta. Hyperosmolarity (NaCl, 2.0%) enhanced multiplication in both cases. With 2.0% NaCl in the medium, selective lysis of the feeder cells occurred, without damage to M. lepraemurium. Multiplication depends more on the physical properties (viscosity, hyperosmolarity, and alkaline pH) of the medium than on its chemical composition. The described conditions are proposed as a model for cultivation trials with other "apparently noncultivable" microorganisms.     

Stage-specific effects of Campoletis sonorensis parasitism on Heliothis virescens development and prothoracic glands

Abstract The ichneumonid endoparasitoid Campoletis sonorensis Cameron (Hymenoptera: Ichneumonidae) injects a polydnavirus when it oviposits into a host. We compared the development of Heliothis virescens (F.) (Lepidoptera: Noctuidae) larvae parasitized in the penultimate (fourth) stadium with those parasitized in the last (fifth) stadium by C.sonorensis and show that hosts stung in the fifth stadium exhibited arrested or delayed development compared to the controls. Parasitoids developed normally to the point of emergence in larvae stung in the fifth stadium but most did not successfully emerge from the host. The prothoracic glands in all successfully parasitized fifth stadium hosts and most unsuccessfully parasitized fifth stadium hosts showed some degree of virally-induced degeneration. Larvae stung in the fourth stadium developed more slowly than controls and either did not moult or developed to a fifth and sometimes a supernumerary sixth stadium before parasitoid emergence. Unsuccessfully parasitized hosts were delayed in their development but eventually moulted to the fifth and, in some cases, a supernumerary sixth stadium before pupating. Hosts stung in the fourth stadium showed no signs of prothoracic gland degeneration whether successfully parasitized or not. In addition, calyx fluid injections into early fourth stadium hosts did not cause prothoracic gland degeneration even after these hosts moulted to the fifth stadium, suggesting that degeneration induced by polydnavirus is specific to the last stadium of the host.     

A novel microtube culture system that enhances the in vitro development of parthenogenetic murine embryos

Oil is an indispensable material in micro-droplet culture; it prevents medium from evaporation, and its transparency facilitates monitoring. However, lipophilic factors in the medium can be absorbed into the oil overlay, and conversely, deleterious materials can diffuse into the medium. In the present study, we describe a novel oil-free microtube culture (MTC) system. Parthenogenetic mouse embryos were placed into 0.2-mL thin-wall flat cap PCR tubes and cultured to the blastocyst stage. Conventional drop culture was used as a control. Embryos in MTC had a higher blastocyst formation rate (89.2%) and larger population of cells in the blastocysts (92.0+/-6.9; mean+/-S.E.M.) compared with drop culture (78.3% and 74.7+/-8.1; P<0.05 for each). The large blastocyst cell population in MTC was due to higher numbers of trophectoderm (TE) cells (70.5+/-5.9 versus 53.8+/-7.4; P<0.05) rather than inner cell mass cells. The presence of more TE cells was attributed to faster development in MTC. Embryos cultured in oil-covered MTC had fewer TE cells (61.5+/-5.6) than oil-free cultures (70.5+/-5.9; P<0.05). In conclusion, oil-free MTC was an alternative to conventional micro-drops, without the deleterious effects of oil.     

Stage-specific effects of Lobophora on the recruitment success of a reef-building coral

Coral Reefs - Benthic marine organisms rely on the dispersal and recruitment of propagules to replenish depleted populations following disturbances. Yet, ecological interactions between colonizing...     

Basal medium development for serum-free culture: a historical perspective

The evolution of basal synthetic formulations to support mammalian cell culture applications has been facilitated by the contributions of many investigators. Definition of minimally-required nutrient categories by Harry Eagle in the 1950's spawned an iterative process of continuous modification and refinement of the exogenous environment to cultivate new cell types and to support emerging applications of cultured mammalian cells. Key historical elements are traced, leading to the development of high potency, basal nutrient formulations capable of sustaining serum-free proliferation and biological production. Emerging techniques for alimentation of fed batch and continuous perfusion bioreactors, using partial nutrient concentrates deduced from spent medium analysis, can enhance medium utilization and bioreactor productivity.     

High-throughput and real-time study of single cell electroporation using microfluidics: effects of medium osmolarity

Electroporation has been widely accepted as an important tool for the delivery of exogenous molecules into cells. Previous mechanistic studies have been carried out by observing either the average behavior from a large population of cells or the response from a small number of single cells. In this study, we demonstrated a novel microfluidic method with high throughput (up to 30 Hz) for real-time studies of single cell electroporation events. Electroporation occurred when cells flowed through a section of a microfluidic channel defined by special geometry. A CCD camera was used to monitor the response of cells starting from the onset of the electroporation. We studied the swelling of Chinese hamster ovary cells and the rupture of cell membrane during electroporation using this technique. We applied buffers with different osmolarities to investigate the effects of medium osmolarity, based on results from a population of single cells. We were able to establish the distributions of the rates of swelling and membrane rupture in the cell population. We also explored establishing the correlation between the property (the cell diameter) and the behavior (the swelling rate) of single cells. Our results indicated that the processes of swelling and rupture occurred more rapidly in the hypotonic or hypertonic buffers than in the isotonic buffer. Statistical analysis did not reveal strong linear correlation between the cell size and the swelling rate. These proof-of-concept studies reveal the potential of applying microfluidics to study electroporation of a cell population at single cell level in real time with high throughput. The limitations associated with this approach were also addressed.     

The influence of different carbon sources and medium osmolarity on the potassium requirements of Candida utilis NCYC 321, growing in continuous culture

In order to study the influence of different carbon sources on the K⁺-requirements of Candida utilis NCYC 321, this yeast was grown at several different dilution rates in potassium-limited continuous cultures with either glucose, glycerol, ethanol, citrate or lactate serving as the carbon and energy source.It was found that the nature of the carbon source profoundly influenced the cellular potassium content, especially at low dilution rates, but that these differences could not be correlated with any differences in relative growth rate (i.e., / _max. And although small amounts of potassium seemingly were needed to serve in osmoregulation and in the cotransport of some acidic carbon sources (lactate and citrate), these requirements were negligible.Independent of carbon source, a strong correlation existed between the intracellular potassium concentration and the yield value on oxygen (Y _O), and between cellular potassium concentration and growth rate. From these two correlations it was concluded that potassium probably was involved mainly in processes associated with ATP synthesis in this yeast.Finally the effect of the addition of NaCl to the medium was tested with glucose-containing cultures that were either carbon- or potassium-limited. Up to a concentration of 20 g/l, NaCl was without influence on Y _O, Y _glucose and q _{O 2}, but effected a slight increase in the cellular potassium content of the potassium-limited cells and a decrease in that of the glucose-limited cells.