Cellular source and mechanisms of high transcriptome complexity in the Mammalian testis.

Understanding the extent of genomic transcription and its functional relevance is a central goal in genomics research. However, detailed genome-wide investigations of transcriptome complexity in major mammalian organs have been scarce. Here, using extensive RNA-seq data, we show that transcription of the genome is substantially more widespread in the testis than in other organs across representative mammals. Furthermore, we reveal that meiotic spermatocytes and especially postmeiotic round spermatids have remarkably diverse transcriptomes, which explains the high transcriptome complexity of the testis as a whole. The widespread transcriptional activity in spermatocytes and spermatids encompasses protein-coding and long noncoding RNA genes but also poorly conserves intergenic sequences, suggesting that it may not be of immediate functional relevance. Rather, our analyses of genome-wide epigenetic data suggest that this prevalent transcription, which most likely promoted the birth of new genes during evolution, is facilitated by an overall permissive chromatin in these germ cells that results from extensive chromatin remodeling.

The epidthelial sodium channel ENaC and its regulators in the epidermal permeability barrier function

The highly amiloride-sensitive epithelial sodium channel ENaC is well known to be involved in controlling whole body sodium homeostasis and lung liquid clearance. ENaC expression has also been detected in the skin of amphibians and mammals. Mice lacking ENaC expression lose rapidly weight associated with an epidermal barrier defect that develops following birth. This dehydration is accompanied with a highly abnormal lipid matrix composition and an impaired skin surface acidification. This strongly suggests a role of ENaC in the maturation of barrier function rather than in the prenatal generation of the barrier, and may be as such an important modulator for skin hydration. In parallel, gene targeting experiments of regulators of ENaC activity, membrane serine proteases, also termed channel activating proteases, like CAP1/Prss8 and matriptase/MT-SP1 by themselves have been shown to be crucial for the epidermal barrier function. In our review, we mainly focus on the role of ENaC and its regulators in the skin and discuss their importance in the epidermal permeability barrier function.

Problems of allometric scaling analysis: examples from mammalian reproductive biology.

Biological scaling analyses employing the widely used bivariate allometric model are beset by at least four interacting problems: (1) choice of an appropriate best-fit line with due attention to the influence of outliers; (2) objective recognition of divergent subsets in the data (allometric grades); (3) potential restrictions on statistical independence resulting from phylogenetic inertia; and (4) the need for extreme caution in inferring causation from correlation. A new non-parametric line-fitting technique has been developed that eliminates requirements for normality of distribution, greatly reduces the influence of outliers and permits objective recognition of grade shifts in substantial datasets. This technique is applied in scaling analyses of mammalian gestation periods and of neonatal body mass in primates. These analyses feed into a re-examination, conducted with partial correlation analysis, of the maternal energy hypothesis relating to mammalian brain evolution, which suggests links between body size and brain size in neonates and adults, gestation period and basal metabolic rate. Much has been made of the potential problem of phylogenetic inertia as a confounding factor in scaling analyses. However, this problem may be less severe than suspected earlier because nested analyses of variance conducted on residual variation (rather than on raw values) reveals that there is considerable variance at low taxonomic levels. In fact, limited divergence in body size between closely related species is one of the prime examples of phylogenetic inertia. One common approach to eliminating perceived problems of phylogenetic inertia in allometric analyses has been calculation of 'independent contrast values'. It is demonstrated that the reasoning behind this approach is flawed in several ways. Calculation of contrast values for closely related species of similar body size is, in fact, highly questionable, particularly when there are major deviations from the best-fit line for the scaling relationship under scrutiny.

Permissivity of insect cells to Waddlia chondrophila, Estrella lausannensis and Parachlamydia acanthamoebae.

Recent large scale studies questioning the presence of intracellular bacteria of the Chlamydiales order in ticks and fleas revealed that arthropods, similarly to mammals, reptiles, birds or fishes, can be colonized by Chlamydia-related bacteria with a predominant representation of the Rhabdochlamydiaceae and Parachlamydiaceae families. We thus investigated the permissivity of two insect cell lines towards Waddlia chondrophila, Estrella lausannensis and Parachlamydia acanthamoebae, three bacteria representative of three distinct families within the Chlamydiales order, all documented in ticks and/or in other arthropods. We demonstrated that W. chondrophila and E. lausannensis are able to very efficiently multiply in these insect cell lines. E. lausannensis however induced a rapid cytopathic effect, which somehow restricted its replication. P. acanthamoebae was not able to grow in these cell lines even if inclusions containing a few replicating bacteria could occasionally be observed.

Habitat selection of three rodents in a frequently burned boreal environment : productivity and habitat diversity

How does fire affect the plant and animal community of the boreal forest? This study attempted to examine the changes in plant composition and productivity, and small mammal demography brought about by fire in the northern boreal environment at Chick Lake, N.W.T. (65053fN, 128°14,W). Two 5*6 ha plots measuring 375m x 150m were selected for study during the summers of 1973 and 197^. One had been unburned for 120 years, the other was part of a fire which burned in the spring of 1969. Grids of 15m x 15m were established in each plot and meter square quadrats taken at each of the 250 grid intersections in order to determine plant composition and density. Aerial primary production was assessed by clipping and drying 80 samples of terminal new production for each species under investigation. Small mammal populations were sampled by placing a Sherman live trap at each grid intersection for ten days in every month. The two plots were similar in plant species composition which suggested that most regrowth in the burned area was from rootstocks which survived the fire. The plant data were submitted to a cluster analysis that revealed nine separate species associations, six of which occured in the burned area and eight of which occured in the control. These were subsequently treated as habitats for purposes of comparison with small mammal distributions. The burned area showed a greater productivity in flowers and fruits although total productivity in the control area was higher due to a large contribution from the non-vascular component. Maximum aerial productivity as dry wieght was measured at 157.1 g/m and 207.8 g/m for the burn and control respectively. Microtus pennsylvanicus and Clethrionomys rutilus were the two most common small mammals encountered; Microtus xanthognathus, Synaptomys borealis, and Phenacomys intermedius also occured in the area. Populations of M. pennsylvanicus and C. rutilus were high during the summer of 1973; however, M. pennsylvanicus was rare on the control but abundant on the burn, while C. rutilus was rare on the burn but abundant in the control. During the summer of 197^ populations declined, with the result that few voles of any species were caught in the burn while equal numbers of the two species were caught in the control. During the summer of 1973 M. pennsylvanicus showed a positive association to the most productive habitat type in the burn which was avoided by C. rutilus. In the control £• rutilus showed a similar positive association to the most productive habitat type which was avoided by M. pennsylvanicus. In all cases for the high population year of 1973# the two species never overlapped in habitat preference. When populations declined in 197^f "both species showed a strong association for the most productive habitat in the control. This would suggest that during a high population year, an abundant species can exclude competitors from a chosen habitat, but that this dominance decreases as population levels decrease. It is possible that M. pennsylvanicus is a more efficient competitor in a recently burned environment, while C. rutilus assumes this role once non-vascular regrowth becomes extensive.

Interactions of diploid embryonal carcinoma cells and normal cell mass cells with 2.5 day mouse embryos following aggregation

Spontaneous teratocarcinomas are ovarian or testicular tumors which have their origins in germ cells. The tumors contain a disorganized array of benign differentiated cells as well as an undifferentiated population of malignant stem cells, the embryonal carcinoma or EC cells. These pluripotent stem cells in tissue culture share many properties with the transient pluripotent cells of the early embryo, and might therefore serve as models for the investigation of developmental events ill vitro. The property of EC cells of prime interest in this study is an in vivo phenomenon. Certain EC cell lines are known to be regulated ill vivo and to differentiate normally in association with normal embryonic cells, resulting in chimeric mice. These mice have two genetically distinct cell populations, one of which is derived from the originally malignant EC cells. This has usually been accomplished by injection of the EC cells into the Day 3 blastocyst. In this study, the interactions between earlier stage embryos and EC cells have been tested by aggregating clumps of EC cells with Day 2 embryos. The few previous aggregation studies produced a high degree of abnormality in chimeric embryos, but the EC cells employed had known chromosomal abnormalities. In this study, two diploid EC cell lines (P19 and Pi0) were aggregated with 2.5 day mouse embryos, and were found to behave quite differently in the embryonic environment. P19 containing aggregates generally resorbed early, and the few embryos recovered at midgestation were normal and non-chimeric. Pi0 containing aggregates survived in high numbers to midgestation, and the Pi0 cells were very successful in colonizing the embryo. All these embryos were chimeric, and the contribution by the EC cells to each chimera was very high. However, these heavily chimeric embryos were all abnormal. Blastocyst injection had previously produced some abnormal embryos with high Pl0 contributions in addition to the live born mice, which had lower EC contributions. This study now adds more support to the hypothesis that high EC contributions may be incompatible with normal development. The possibility that the abnormalities were due to the mixing of temporally asynchronous embryonic cell types in the aggregates was tested by aggregating normal pluripotent cells taken from 3.5 day embryos with 2.5 day embryos. Early embryo loss was very high, and histological studies showed that the majority of these embryos died by 6.5 days development. Some embryos escaped this early death such that some healthy chimeras were recovered, in contrast to recovery of abnormal chimeric embryos following Pl0-morula aggregations, and non-chimeric embryos following P19-morula aggregations. This somewhat surprising adverse effect on development following aggregation of normal cell types suggests that there are developmental difficulties associated with the mixing of asynchronous cell types in aggregates. However, the greater magnitude of the adverse effects when the aggregates contained tumor derived cells suggests that EC cells should not be considered the complete equivalent of the pluripotent cells of the early embryo.

Size regulation of double and half embryos in the mice musculus

Genetic chimeras made by aggregating early mouse embryos have many uses in developmental biology and have also provided insights into embryonic growth regulation. There is an indication that the embryo can regulate for an increase in size because although aggregation chimeras are twice as big as normal embryos when made, they are born of normal size. Upward regula..... tion of size reduced embryos is also possible. Half embryos made by the isolation or destruction of one of the blastomeres of a 2-cell embryo are also born of normal size. Little is known about the timing or the mechanism of this size regulation. In this study, the timing of size regulation in double and half embryos was clearly established by comparison of cell numbers derived from serial reconstruction of light microscope sections of control and experimental embryos. It was shown that size regulation in double embryos occurred around 6dl6h and in half embryos by 7dOh. Size regulation occurred in all tissues at the same time indicating a single control mechanism for the entire embryo. More detailed examination of the growth of double embryos revealed that size regulation occurred by alteration in cell cycle length~ No excessive cell death was found in double embryos compared to the controls and continuous labelling with [3H] thymidine showed no large non-dividing cell population in double embryos. However, a comparison of the mitotic index of double and control embryos after colcemid treatment, revealed a large difference between the two around 5dl6h to 6d16h. During this period, control embryos underwent a proliferative burst not shown by the double embryos. The mechanism for cell cycle control is not clear; it may be intrinsic to the embryo or determined by the uterine environment. Evidence was found suggesting that differentiation in the postimplantation embryo was cell number dependent. The timing of differentiative events was examined in half, double and control embryos. Proamnion formation, which occurs prior to size regulation, occurs at the same cell number but at different times in the three groups of embryos. However mesoderm which appears after size regulation was seen at the same time in all grollps of embryos.

Vegetational history and geochronology of several sites in South Southwestern Ontario with discussion on mastodon extinction in Southern Ontario /

This study has three purposes: to establish a chronologically controlled vegetational history for a number of sites in south Southwestern Ontario; to utilize the resulting data to support and/or add to the current understanding of Quaternary geology and stratigraphy, and the glacial and postglacial history of the Great Lakes in south Southwestern Ontario; and to attempt to propose a possible explanation for the extinction of the mastodon in Southern Ontario. Palynological and geochronological analyses were conducted on material collected from eleven sites (east to west): Verbeke Mastodon Site, Woloshko Mastodon Site, Walker Pond II, Pond Mills I, Lake Hunger Bog, Bouckaert Site. Mabee Site, Cornell Bog. Colles Lake I, Folden Mastodon Site and Forest Pond. Individual geochronologically controlled (where possible) vegetational histories were reconstructed for each of the sites investigated. The results of the individual studies, when considered in overview. indicated the existance of an established closed boreal forest throughout south Southwestern Ontario by 10,000 years B.P. This evidence for a significant climatic change coincident throughout south Southwestern Ontario supports the proposed age of 10,000 years B.P. for the Pleistocene/Holocene Boundary (Terasmae, 1972). Remnant patches of 'open spruce parkland' persisted in small local 'wet' areas. It was in these areas that the mastodon was restricted during early Holocene time. With continued encroachment by the surrounding boreal forest, possibly speeded up by this browser's destructive feeding habits, the spruce enclaves shrank and the mastodon became extinct in south Southwestern Ontario. The results of this thesis basically support Dreimanis' (1967, 1968) proposed 'Environmental-Climatic' theory for mastodon extinction. It is suggested that increased dryness during the present interglacial compared to the climate of earlier interglacials may be the key to unravelling the problem of mastodon extinction in eastern North America.

Demographic and genetic attributes of dispersing and resident individuals of an enclosed Microtus pennsylvanicus population

A dispersal polymorphism may exist in emigrants from cyclic populations of Microtus '~nnsylvanicus biasing trap-revealed movements of unenclosed animals in favour of sedentary or colonizing individuals. The dispersal tendency of emigrants from an enclosed population was investigated by releasing animals via tubes into one of two adjacent enclosures, one vacant and one inhabited. Individuals from the enclosed population were monitored for age, sex, weight and electrophoretically detectable serum transferrin genotype in an intensive live-trapping program. In 1973 the minimum number alive in the introduced enclosed study population reached approximately l67/ha when breeding stopped in October. In 1974 intensive breeding increased the population density to 333/ha by mid-July when a long decline in numbers and breeding intensity began without an intervening plateau. An adjacent unenclosed area had a much lower density and longer breeding season in 1974. The growth rate of young males in the enclosed population tended to be lowest during the decline period in 1974. Survival of the enclosed population was high throughout but was lowest during the decline phase in both sexes, especially males. Low transferrin heterozygote survival during the decline coincided with a significant heterozygote deficiency in females whereas in males genotype frequencies did not depart from Hardy-Weinberg equilibrium values throughout th.e study. Twenty-nine suitable ani.mals were released during the decline in five periods from July to November 1974. The proportions of males and transferrin heterozygotes in the released graun were generally greater than in the source population~ In the test enclosures 21% of the released animals continued their movement through the vacant area while 41% (no significant difference) moved through the inhabited enclosure. In the vacant test area, females had a greater tendency than males to continue dispersal whereas no difference was noted in the inhabited area. Low frequency of captures in the tubes, predator disturbances and cold weather forced the termination of the study. The role of dispersal as a population regulating mechanism was further substantiated. The genetic differences between emigrant and resident animals lend support to Howard's hypothesis that a genetic polymorphism influences the tendency to disperse. Support is also given to Myers' and Krebs' contention that among dispersers an additional density dependent polymorphism influences the distance dispersed.

Language shifting among the Hodenosaunee of Southern Ontario : Edwaenagé: tsgó -- Shogwaya> dihs> oh nidwawenó :de: shogwá:wi: tsáhohwejáda:t

Through aggressive legislative and educational policies Indigenous languages globally have been shifted to the language of the dominant society. Globalization has brought previously geo-politically and/or geo-linguistically isolated people and language . groups into close proximity that necessitated interaction and at times intense power struggles. There are currently approximately 6,000 spoken languages in the world, more than half are either endangered, dying or disappearing altogether. Canadian statistics reveal an overall 3 % decline in the intergenerational transmission of language. Of the original 60 Indigenous languages spoken in Canada, 8 are extinct, 13 are nearly extinct, and 23 are critical. The remaining languages have a slim chance of survival. Within the next 100 years only 4 Indigenous languages will remain. The Hodenosaunee languages of Southern Ontario are not incl~ded among the list of languages that will survive the next 100 years. There are, without a doubt, complex challenges in the maintenance of Indigenous languages within a dominant-culture influenced environment. Given the increasing awareness of the social impact of linguistic integrity and preservation of languages on Indigenous people as a whole, this study considers how language is currently being used; the social, economic, and political implications of language shifting; the need to shift our social consciousness in order to understand the urgency in privileging our Hodenosaunee languages; as well as ways in which we might achieve those goals as individuals, as families, and as a community.

Adenovirus-based exogenous gene expression in mammalian cells

Thesis (Ph.D.)--Brock University, 2010.

A possible role for chemokine signalling through CXCR4 as a downstream effector of retinoic acid signalling in the regenerating tail and spinal cord of Notophthalmus viridescens

The newt, Notopthalmus viridescens is one of the few tet rapod vertebrates capable of extensive regeneration of the central nervous system, however, the factors involved in this process are still unknown. Chemokine signalling through the receptor CXCR4, has been found to be involved in the development of the central nervous system of mammals and more recently in epimorphic fin regeneration in zebrafish. We have hypothesized that the CXCR4 signalling pathway is involved in spinal cord and tail regeneration in the adul t newt , possibly as a downstream target of retinoic acid signalling. We found that CXCR4 mRNA expression was observed in the brain, spinal cord, heart, gut, liver and regenerating tail blastemas. CXCR4 expression increased over the f i rst 12 days of tail regeneration and returned to basal expression levels at day 21 of regeneration. Inhibition of CXCR4 wi th AMD3100, a specific receptor antagonist, led to a decrease in CXCR4 mRNA in the regenerating tail 14 days post amputation. Histological analysis suggests a delay in the early stages of tail and spinal cord regeneration. Spinal cord explants t reated wi th CXCL12, the ligand to CXCR4, displayed enhanced neurite outgrowth in vitro. Explants t reated wi th AMD3100 abolished any retinoic acid enhanced neurite outgrowth effects suggesting a link between these signalling pathways.

The Role of Pyruvate Dehydrogenase Kinase-4 in Post-Exercise Glycogen Recovery

The pyruvate dehydrogenase (PDH) complex regulates the oxidation of carbohydrates in mammals. Decreased activation of PDH following exhaustive exercise may aid the resynthesis of glycogen through increased activity of PDH kinase-4 (PDK4), one of four kinases that decrease the activity of the PDH complex. The purpose of this study was to examine the role of PDK4 in post-exercise glycogen resynthesis. Wild-type (WT) and PDK4-knockout (PDK4-KO mice) were exercised to exhaustion and were sampled at rest (Rest), at exercise exhaustion (Exh), and after two-hours post-exercise (Rec). Differences in feeding post-exercise led to the addition of a PDK4-KO group, pair-fed (PF) with WT mice. Glycogen fully recovered in all Rec groups in muscle however remained low in the PF group in liver. Flux through PDH was elevated in PDK4-KO muscle with feeding and low in the PF group in both tissues. This suggests PDK4 may fine-tune flux through PDH during exercise recovery.

Alternative splicing of DNA polymerase beta in vertebrates

Alternative splicing (AS) is the predominant mechanism responsible for increasing eukaryotic transcriptome and proteome complexity. In this phenomenon, numerous mRNA transcripts are produced from a single pre-mRNA sequence. AS is reported to occur in 95% of human multi-exon genes; one specific gene that undergoes AS is DNA polymerase beta (POLB). POLB is the main DNA repair gene which performs short patch base excision repair (BER). In primate untransformed primary fibroblast cell lines, it was determined that the splice variant (SV) frequency of POLB correlates positively with species lifespan. To date, AS patterns of POLB have only been examined in mammals primarily through the use of cell lines. However, little attention has been devoted to investigating if such a relationship exists in non-mammals and whether cell lines reflect what is observed in vertebrate tissues. This idea was explored through cloning and characterization of 1,214 POLB transcripts from four non-mammalian species (Gallus gallus domesticus, Larus glaucescens, Xenopus laevis, and Pogona vitticeps) and two mammalian species (Sylvilagus floridanus and Homo sapiens) in two tissue types, liver and brain. POLB SV frequency occurred at low frequencies, < 3.2%, in non-mammalian tissues relative to mammalian (>20%). The highest POLB SV frequency was found in H. sapiens liver and brain tissues, occurring at 65.4% and 91.7%, respectively. Tissue specific AS of POLB was observed in L. glaucescens, P. vitticeps, and H. sapiens, but not G. gallus domesticus, X. laevis and S. floridanus.The AS patterns of a second gene, transient receptor potential cation channel subfamily V member 1 (TRPV1), were compared to those of POLB in liver and brain tissues of G. gallus domesticus, X. laevis and H. sapiens. This comparison was performed to investigate if any changes (either increase or decrease) observed in the AS of POLB were gene specific or if they were tissue specific, in which case similar changes in AS would be seen in POLB and TRPV1. Analysis did not reveal an increase or decrease in both the AS of POLB and TRPV1 in either the liver or brain tissues of G. gallus domesticus and H. sapiens. This result suggested that the AS patterns of POLB were not influenced by tissue specific rates of AS. Interestingly, an increase in the AS of both genes was only observed in X. laevis brain tissue. This result suggests that AS in general may be increased in the X. laevis brain as compared to liver tissue. No positive correlation between POLB SV frequency and species lifespan was found in non-mammalian tissues. The AS patterns of POLB in human primary untransformed fibroblast cell lines were representative of those seen in human liver tissue but not in brain tissue. Altogether, the AS patterns of POLB from vertebrate tissues and primate cell lines revealed a positive correlation between POLB SV frequency and lifespan in mammals, but not in non-mammals. It appears that this positive correlation does not exist in vertebrate species as a whole.

Seasonal reproductive endothermy in tegu lizards

With some notable exceptions, small ectothermic vertebrates are incapable of endogenously sustaining a body temperature substantially above ambient temperature. This view was challenged by our observations of nighttime body temperatures sustained well above ambient (up to 10°C) during the reproductive season in tegu lizards (~2 kg). This led us to hypothesize that tegus have an enhanced capacity to augment heat production and heat conservation. Increased metabolic rates and decreased thermal conductance are the same mechanisms involved in body temperature regulation in those vertebrates traditionally acknowledged as “true endotherms” : the birds and mammals. The appreciation that a modern ectotherm the size of the earliest mammals can sustain an elevated body temperature through metabolic rates approaching that of endotherms enlightens the debate over endothermy origins, providing support for the parental care model of endothermy, but not for the assimilation capacity model of endothermy. It also indicates that, contrary to prevailing notions, ectotherms can engage in facultative endothermy, providing a physiological analog in the evolutionary transition to true endothermy.