Establishment of polarity is a critical process that occurs early during development. In Drosophila melanogaster, axis determination occurs by localization of determinants during oogenesis. Mutations in cappuccino (capu) lead to defects in polarity establishment of both the anterior/posterior (A/P) and dorsal/ventral (D/V) axes during oogenesis. In the oocytes laid by capu mutant females, determinants that define these axes are either mislocalized or are absent. Several lines of evidence suggest that the regulation of cytoskeleton by the gene product encoded by capu is involved in Drosophila oogenesis.Capu, a member of the formin family of proteins, known to be regulators of actin dynamics, interacts both genetically and physically with chickadee (chic) which encodes the actin binding protein Profilin. I show here that mutations in both capu and chic lead to defects in the endocytic uptake of yolk into developing oocytes. I show that mutations in these loci lead to accumulation of abnormally large yolk granules and that this is a post internalization defect in the oocyte of capu and chic females. I also present evidence which indicates that an interaction with capu is necessary for chic regulation of yolk granule biogenesis.This is the first evidence for a formin subfamily of formin proteins to have a role in endocytosis. While this new function identified for the actin associated proteins Capu and Profilin indicates that regulation of actin cytoskeleton plays a role in endocytosis during oogenesis, the mechanism of this regulation and possible actin independent roles played by Capu and Profilin in this process are yet to be determined.Like capu, mutations in spire (spir) also show defects in A/P and D/V axes during oogenesis. Spir is an actin binding protein and like capu, mutations in spir shows defects in cytoskeletal architecture and suggests that capu and spire alter microtubule distribution in the oocyte during oogenesis.To identify molecular partners of capu and spir and their roles during oogenesis, I performed a genome-wide deficiency screen to identify regions of the genome that interact with these genes. I identified regions in the genome that showed interaction with capu and spir. While I was able to narrow down the region of interaction to a smaller cytological interval, gaps in the deficiency coverage and lack of mutants in those regions prevented me from identifying interacting loci in those regions.

No description available.

Niveoscincus ocellatus is an important species in historical analyses of the evolution of viviparity because it is the species upon which the type II chorioallantoic placenta was based. Here we describe the net nutrient uptake across the placenta of N. ocellatus for comparison with other species of skinks with complex placentae. N. ocellatus is highly placentotrophic, with neonates being 1.68-times larger in dry matter than the fresh eggs. There is an increase of nitrogen from 6.3 ± 0.2 mg to 9.2 ± 0.6 mg, and ash from 3.8 ± 0.3 mg to 6.7 ± 0.6 mg. The increase in ash is made up by a more than two-fold increase in the amounts of calcium, potassium and sodium. There is no significant difference in lipids in the neonates compared to fresh eggs, so considerable lipid must have crossed the placenta to provide energy for embryonic development. N. ocellatus is significantly more placentotrophic than Niveoscincus metallicus, which also has a complex chorioallantoic placenta. Discovery of substantial placentotrophy in this genus confirms that two lineages of Australian lygosomine skinks (represented by the genera Pseudemoia and Niveoscincus) have evolved this pattern of embryonic nutrition and supports the hypothesis that the evolution of reptilian placentotrophy involves specialisations in addition to structural modifications of the chorioallantoic placenta.

This study tested different concentrations of egg yolk in canine freezing extender void of glycerol, a commonly used cryoprotectant, by examining the motility and morphology throughout the freezing process: initial (baseline after extender added), post-cool (after three hours at 5"C) and post-thaw (after freezing.)  Initial values of pH, osmolarity, motility and morphology were obtained for comparison of the samples.  Spermatozoa from six normal dogs as determined by progressive linear motility > 70% and normal morphology > 60% was used. Semen was collected and pooled for five freezing trials.  The concentrations of egg yolk used in the extender were: 0%, 10%, 20%, 30% and 40%. Assessment of each sample was blinded to the treatments until all results were obtained and statistics had been analyzed. Based on this study a 20% egg yolk concentration is slightly superior to a 30% egg yolk concentration when assessing post-thaw motility, morphology and longevity and significantly superior to a 0%, 10% or 40% egg yolk concentration. The study also showed motility and normal post-cool and post-thaw sperm morphology did not always correlate.  Utilization of 0% and 10% concentrations of egg yolk has negative effects on semen quality as measured by the motility and/or morphology.  Results confirm freezing does not affect secondary sperm abnormalities, abnormalities of the tail and distal section of the middle piece, during cooling or freezing.  Primary abnormalities, abnormalities of the head and midpiece, increased in the 0% extender during cooling and all extenders during freezing. The pH of the extenders before the addition of sperm was significantly different. Once sperm was added to the extenders, there was no longer a significant difference in pH.  There was a positive correlation for both motility and normal morphology percentages post-cool and post-thaw for the extenders with similar osmolarity to the semen. / Master of Science

No description available.

The chicken yolk sac and small intestine are connected through the yolk stalk and share many biological similarities. During the embryonic stage, the extra-embryonic yolk sac helps the embryo to absorb nutrients primarily in the last two weeks of incubation. The chicken yolk sac physically moves yolk contents from the yolk sac to the small intestine at the end of embryogenesis. This is the time when the small intestine replaces the yolk sac in assimilating nutrients for the embryo and later for the posthatch chicken. Additionally, both chicken small intestinal epithelia and the yolk sac secrete beta defensins for promoting intestinal health. Since there are heterogeneous cell types along the mammalian intestinal villus, which are derived from the intestinal stem cells in the crypts, we investigated if cells of the chicken yolk sac and small intestine have the same ontogeny as mammalian intestinal epithelial cells. In this dissertation, we mainly focused on the spatial expression of nutrient transporters (PepT1 and SGLT1), intestinal stem cell markers (Lgr5 and Olfm4), and avian beta defensins in the chicken yolk sac and small intestine during the embryonic and early posthatch stages. RNAscope in situ hybridization was used to identify the distribution of cells expressing PepT1 mRNA in both the chicken yolk sac and small intestine. PepT1 mRNA was found to be expressed by epithelial cells in both the yolk sac and small intestine. In the yolk sac, PepT1 mRNA was uniformly distributed in each endodermal epithelial cell along the villus-like structure. The pattern of PepT1 mRNA expression observed in the chicken yolk sac during the last 10 days of incubation revealed that PepT1 mRNA was increased from e11 to e13, and decreased from e15 to day of hatch. The peak of PepT1 mRNA expression was between e13 and e15, when the yolk sac reaches maximum absorptive area and the growth of the chicken embryo is at its fastest rate. However, the expression of PepT1 mRNA in the intestine was only detected in columnar enterocytes along the villus and not in goblet cells or cells in the crypts. The immunofluorescence assay confirmed that PepT1 protein was located at the brush border membrane of the enterocytes and that protein expression of PepT1 was restricted to the intestinal epithelial cells from approximately the middle to the tip of the villus. In order to identify intestinal stem cells, we used the known mammalian stem cell markers, Lgr5 and Olfm4. Both Lgr5 and Olfm4 are specifically expressed by cells in the chicken intestinal crypts, suggesting that they can be used as biomarkers for chicken intestinal stem cells. Dual labelling of PepT1 and Olfm4 mRNA on the same chicken intestinal sample revealed that there was a gap between PepT1-expressing enterocytes and Olfm4-expressing intestinal stem cells. The cells in this gap were presumably transit amplifying (TA) cells. Additionally, we also found that the TA cell zone along the intestinal villus was reduced during chicken growth. This TA cell population could be clearly detected at day of hatch and d1 posthatch but not later. The expression of SGLT1 mRNA was localized to yolk sac endodermal epithelial cells and showed a sharp increase at the end of incubation. This increase of SGLT1 mRNA coincided with the increase in glucose in the yolk, indicating that the chicken embryo needs glucose as energy for hatching. The mRNA expression profiles of various avian beta defensins have been examined by qPCR and in situ hybridization to investigate the immune function of the yolk sac and small intestine. We found that AvBD10 mRNA showed the highest expression level in the yolk sac and was expressed predominantly in the yolk sac endodermal epithelial cells. Additionally, the expression of AvBD10 mRNA showed a development-specific pattern, which increased from e9 to e11, and decreased from e13 towards day of hatch. The expression patterns of AvBD1, 2, and 7 mRNA were similar to each other. These three genes were found to be expressed by chicken heterophils distributed in the yolk sac blood islands and small intestinal blood vessels. Only a subset of heterophils, which might be activated, were able to express AvBD1, 2, and 7 mRNA. In the intestine, the expression of AvBD10 mRNA was localized to cells along the villus at e19 and day of hatch, but later to only a few cells located above the intestinal crypts. In summary, the endodermal epithelial cells are responsible for the absorptive and immune functions of the chicken yolk sac. The yolk sac mesoderm is critical for embryonic hematopoiesis and innate immunity. The chicken small intestinal epithelial cells are derived from the intestinal stem cells in the crypts. These epithelial cells have different cell types, which are functioning to absorb nutrients and secrete antimicrobial peptides. / Ph. D. / The chicken yolk sac and small intestine are connected to each other and share many biological similarities. Both chicken small intestinal and yolk sac epithelia play critical roles for nutrient absorption and immune defense. In this dissertation, the mRNA for nutrient transporters such as the peptide transporter, PepT1 and the sodium-glucose co-transporter, SGLT1 were found to be expressed by absorptive epithelial cells in both the yolk sac and small intestine. Additionally, both intestinal and yolk sac epithelial cells expressed avian beta defensins (AvBDs), which are important chicken host defense peptides. In the small intestine, there are a number of differentiated cell types that originate from stem cells in the crypt that express the known mammalian stem cell markers, Olfm4 and Lgr5 mRNA. However, in the chicken yolk sac, only the stem cell marker Lgr5 mRNA was expressed by endothelial cells. In summary, the yolk sac epithelial cells are responsible for the absorptive and immune functions for the embryonic stage. The chicken small intestinal epithelial cells are derived from the intestinal stem cells in the crypts. These epithelial cells have different cell types, which function to absorb nutrients and secrete antimicrobial peptides.

Egg yolk contamination of egg whites continues to be a serious problem in the egg industry. The ability of egg whites to form stable and voluminous foams is greatly inhibited by accidental yolk contamination, even at extremely small levels. Experiments were conducted to determine if lipase can regenerate the functional properties of yolk-contaminated egg whites. Treatments included control, 0.2% yolk-contamination, and 0.2% yolk-contamination that was treated with lipase and colipase and heated at 37°C for 1 hour. Lipase from Mucor meihei and colipase from porcine pancreas were added to yolk-contaminated egg white samples to target and hydrolyze the triglycerides from egg yolk. Enzymatic hydrolysis was confirmed using thin-layer chromatography. Treatment of yolk-contaminated samples with lipase, colipase and heat yielded a drastic improvement in a number of the functional properties, including the final foam volume, foam capacity, and foaming power. These functional properties showed complete restoration to control levels. However, foam stability and foam drainage levels were not statistically different from yolk-contaminated samples that had not been enzymatically treated. Enzyme treated yolk-contaminated egg whites were also tested in an angel food cake system. There were three treatments, including an uncontaminated control, a contaminated control, and a lipase and colipase treated yolk-contaminated sample. Comparison between treatments was performed by volume analysis. The enzyme treated yolk-contaminated egg whites performed similarly to uncontaminated control angel food cakes. / Master of Science

Cryopreservation of germplasm is widely used in agriculture, biotechnology, conservation of threatened species and human reproductive medicine. There is a need however to improve the reproductive efficiency of breeding with cryopreserved semen, which may involve increasing the post-thaw quality of sperm through improvements in cryopreservation extenders. Extenders including egg yolk from chickens are successfully used worldwide for cryopreservation of bovine semen, whereas the protective agent in the egg yolk is believed to be the low-density lipoprotein (LDL) fraction. Egg yolks of different avian species vary in their cholesterol, phospholipid and polyunsaturated fatty acid content which have been shown to have important effects on sperm’s freezing capability. The purpose of this study was to determine the cryoprotective effect of clarified egg yolk and LDLs extracted from different egg yolk sources (chicken, chicken omega-3, pigeon, quail and turkey) on bovine sperm. Semen from six bulls was collected four times each by electroejaculation, split and diluted with the 10 following extenders: chicken clarified (Ccl), chicken omega-3 clarified (O3cl), pigeon clarified (Pcl), quail clarified (Qcl), turkey clarified (Tcl), chicken LDL (CLDL), chicken omega-3 LDL (O3LDL), pigeon LDL (PLDL), quail LDL (QLDL) and turkey LDL (TLDL). The extended semen was evaluated, cryopreserved and examined directly after thawing (0h) and after two hours at 37 ˚C (2h). Computer assisted sperm analysis (CASA) was used to determine total sperm motility (TM), progressive motility (PM), straight line velocity (VSL), curvilinear velocity (VCL) and average path velocity (VAP). Intact plasma membrane (IPM) and intact acrosomes (IA) were measured by flow cytometry. The percentage change (loss; Δ%) of each sperm characteristic was calculated and used to compare the effect of the extenders. From extending to 0h post-thaw, the pigeon LDL extender lead to greater losses in sperm total and progressive motility, as well as of intact acrosomes, than the other nine extenders tested (P < 0.05). During 0h to 2h post-thaw, the sperm in PLDL extender experienced greater losses in total and progressive motility (P < 0.0001), as well as in curvilinear velocity (P < 0.05), than in all the other nine extenders. Sperm in turkey clarified extender had a greater loss in the velocity parameters (VSL, VAP, VCL) than sperm in several of the other extenders such as O3cl, CLDL, O3LDL, QLDL and TLDL from 0h to 2h (P < 0.05). Concomitantly, sperm in the Tcl extender had a greater loss in the velocity parameters and of intact acrosomes compared to sperm in its counterpart, the turkey LDL extender, from 0h to 2h post-thaw (P < 0.05). The differences produced in post-thaw quality of cryopreserved bovine sperm in the pigeon LDL and turkey clarified extenders were attributed to methodological differences in these egg yolk preparations compared with the other eight extenders. Importantly, the results demonstrate that with most egg yolk preparations derived from a variety of species, there are equivalent cryoprotective effects afforded by the use of omega-3 chicken, pigeon, quail, or conventional chicken egg yolk in a clarified form in freezing extenders for bovine semen. We further proved that the freezing capabilities of bovine semen extenders containing the low-density lipoprotein fraction of omega-3 chicken, quail, turkey and conventional chicken egg yolk were similar.

Development of the yolk sac of squamate reptiles (lizards and snakes) differs from other amniote lineages in the pattern of growth of extraembryonic mesoderm, which produces a cavity, the yolk cleft, within the yolk. The structure of the yolk cleft and the accompanying isolated yolk mass influence development of the allantois and chorioallantoic membrane. The yolk cleft of viviparous species of the Eugongylus group of scincid lizards is the foundation for an elaborate yolk sac placenta; development of the yolk cleft of oviparous species has not been studied. We used light microscopy to describe the yolk sac and chorioallantoic membrane in a developmental series of an oviparous member of this species group, Oligosoma lichenigerum. Topology of the extraembryonic membranes of late stage embryos differs from viviparous species as a result of differences in development of the yolk sac. The chorioallantoic membrane encircles the egg of O. lichenigerum but is confined to the embryonic hemisphere of the egg in viviparous species. Early development of the yolk cleft is similar for both modes of parity, but in contrast to viviparous species, the yolk cleft of O. lichenigerum is transformed into a tube-like structure, which fills with cells. The yolk cleft originates as extraembryonic mesoderm is diverted from the periphery of the egg into the yolk sac cavity. As a result, a bilaminar omphalopleure persists over the abembryonic surface of the yolk. The bilaminar omphalopleure is ultimately displaced by intrusion of allantoic mesoderm between ectodermal and endodermal layers. The resulting chorioallantoic membrane has a similar structure but different developmental history to the chorioallantoic membrane of the embryonic hemisphere of the egg.

Embryos of oviparous Reptilia (=turtles, lepidosaurs, crocodilians and birds) extract calcium for growth and development from reserves in the yolk and eggshell. Yolk provides most of the calcium to embryos of lizards and snakes. In contrast, the eggshell supplies most of the calcium for embryonic development of turtles, crocodilians and birds. The yolk sac and chorioallantoic membrane of birds recover and transport calcium from the yolk and eggshell and homologous membranes of squamates (lizards and snakes) probably transport calcium from these two sources as well. We studied calcium mobilization by embryos of the snake Pantherophis guttatus during the interval of greatest embryonic growth and found that the pattern of calcium transfer was similar to other snakes. Calcium recovery from the yolk is relatively low until the penultimate embryonic stage. Calcium removal from the eggshell begins during the same embryonic stage and total eggshell calcium drops in each of the final 2 weeks prior to hatching. The eggshell supplies 28% of the calcium of hatchlings. The timing of calcium transport from the yolk and eggshell is coincident with the timing of growth of the yolk sac and chorioallantoic membrane and expression of the calcium binding protein, calbindin-D28K, in these tissues as reported in previous studies. In the context of earlier work, our findings suggest that the timing and mechanism of calcium transport from the yolk sac of P. guttatus is similar to birds, but that both the timing and mechanism of calcium transport by the chorioallantoic membrane differs. Based on the coincident timing of eggshell calcium loss and embryonic calcium accumulation, we also conclude that recovery of eggshell calcium in P. guttatus is regulated by the embryo.