N,N,N’,N’-tetrakis-(2-Pyridylmethyl) ethylenediamine (TPEN) is a heavy metal chelator with high affinity for zinc. TPEN causes important responses in mammalian eggs. For example, these eggs are arrested at the MII stage by the Endogenous Mitotic Inhibitor 2 (Emi2), which prevents activation of the Anaphase Promoting Complex (APC) and degradation of Cyclin B. By chelating zinc, TPEN inactivates Emi2 and eggs undergo spontaneous exit of meiosis and egg activation. TPEN chelates Ca2+ with lower affinity, although in the Endoplasmic Reticulum (ER), where Ca2+ concentrations are high, TPEN may sequester Ca2+ preventing release into the cytoplasm. Initial exposure of TPEN to MII eggs failed to cause spontaneous intracellular Ca2+ release from the ER. Interestingly, in the case of GV oocytes, addition of TPEN caused Ca2+ influx. This influx could be blocked via the addition of 2-APB, a plasma membrane Ca2+ channel blocker. To determine the possible role of TPEN on chelation of ER Ca2+, MII and GV cells were incubated in TPEN and ER Ca2+ released was by exposure to Cyclopiazonic Acid (CPA), a sarco/endoplasmic reticulum (SERCA) pump inhibitor, or Ionomycin (IO), a Ca2+ ionophore. In MII oocytes, the amplitude of the rises caused by CPA and IO, in TPEN-treated oocytes, was smaller than controls and experienced a delay in return to baseline. In GV oocytes, TPEN enhanced rather than reduced Ca2+ responses to CPA and IO. Given its inability to fully chelate ER Ca2+, the use of TPEN as a tool to study Ca2+ homeostasis in mouse oocytes needs additional investigation.

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<p> Social learning, defined as learning from other individuals has been well studied in vertebrates and social insect species. I used fruit fly egg laying site selecting as a paradigm to test for social learning in a non-social insect. Focal females that experienced novel food together with mated females (models), who had laid eggs on that food, subsequently exhibited a stronger preference for laying eggs on that food over another novel food than focal females that experienced the same food alone. This socially influenced learning was also observed when focal females experienced both foods, one with mated models and their eggs and one alone. In contrast to the strong effect of a food with mated models and their eggs, neither a food with virgin models nor the aggregation pheromone (cVA) alone generated socially influenced learning. These results provide the first evidence for social learning about egg laying substrate in fruit flies. Further research utilizing fruit flies as a model system may help us gain a better understanding of the evolution and neurogenetics of social learning.</p> / Thesis / Master of Science (MSc)

Profiles of plasma estradiol and testosterone concentrations, cathepsin D, L, and B activities, and quantitative and qualitative protein content were developed and evaluated in four commercial strains of channel catfish, Gold Kist (2), Thompson and NWAC-103 for one year (age 2 to age 3). Great variation between individuals of the same strain precluded the identification of any significant, strain-specific differences for the variables under investigation. When variables from fish of all strains were collectively evaluated over time, both estradiol and testosterone concentrations significantly increased in July and then later from February to April. The increase in hormone concentration was accompanied by oocyte growth and increases in proteolytic activity of cathepsins D, L, and B, supporting the role of estradiol in regulating vitellogenesis. Vitellogenin was enzymatically broken down into smaller protein units by cathepsins L, D, and B that were separately predominant at different stages of oocyte development. During oocyte development, there were sequential relationships among hormone concentration, cathepsin activity, protein content, and predominant oocyte proteins. This observation was associated with high levels of activity of cathepsin L in February, suggesting an important role in protein degradation during that time, while high activity of cathepsin B occurred, stimulating during November to January. Cathepsin B is more important in oogenesis or early vitellogenesis, and cathepsin L assumes a principal role during middle vitellogenesis. Twenty hours subsequent to the injection of fish with either carp pituitary hormone or luteinizing hormone releasing hormone, increases in the concentration of plasma estradiol and testosterone, activities of cathepsins L, D, and B, egg size, and egg protein content occurred, stimulating the process of oocyte maturation. The percentages of spawning obtained were 18.8% of LHRH injected fish, 12.4% of CPE injected fish, 9.4% of fish not injected, and 0% of saline injected fish. Injection of females with LHRH can potentially serve as a tool to increase spawning success in appropriate commercial settings, particularly for improving three year old catfish spawning success early in the spawning season. Low estradiol levels in all three-year-old fish suggest that insufficient stimulation of vitellogenin production by estradiol may underlie the lack of vitellogenin incorporation into developing oocytes. In the present study, the measurement of the activities of the cathepsins and their relationships to other parameters were evaluated for the first time. This is also the first study to report plasma estradiol and testosterone concentrations, protein content, and egg size in 2 to 3-year old channel catfish. All of the parameters collectively evaluated may serve to assist in the selection of the best 2- year old channel catfish female broodstock, and to determine the optimal timing of treatments of hormone injection to increase reproductive performance.

Vaccination programs using live whole cell vaccines are presently being used to control outbreaks and to help protect flocks from field strains of Mycoplasma gallisepticum. The primary goal of the present study was to investigate the effects of a ts11-strain M. gallisepticum inoculation alone, and in conjunction with F-strain M. gallisepticum overlays at 22 or 45 weeks of age during lay. In this study, it was shown that a pre-lay ts11 vaccination had a broad range effect on the physiology of the commercial layer without affecting overall performance. The timing of an overlay vaccination using F-strain M. gallisepticum during lay also had varied effects on physiological parameters without affecting overall performance. This study indicates that using ts-11-strain M. gallisepticum in conjunction with F-strain M. gallisepticum does not negatively affect laying hen performance. Furthermore, this inoculation combination offers benefits above the use of those achieved through either inoculation alone

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This thesis addresses two hypotheses: 1) ooplasmic ion activity is regulated during water uptake by locust eggs, and, 2) activities of Ca⁺⁺ and H⁺ are maintained at levels appropriate for their use as signals for developmental processes, including activation, in insect eggs. Hypothesis 1 is based on the perturbing effect of large changes in intracellular Na and/or K activity on enzyme function in eukaryotic cells. Hypothesis 2 is based on the ionic hypothesis of activation developed from studies of eggs of marine invertebrates. Electrical potential difference across egg membranes (PD_egg), and ooplasmic activities of Na⁺, K⁺, Cl⁻, Ca⁺⁺ and H⁺ were measured with double-barrelled ion-selective microelectrodes. Locust eggs maintained a measurable potential difference across egg membranes throughout development. Input resistance (Rᵢₙ) decreased by approximately 5-fold in eggs after fertilization suggesting that the chorion is not the major barrier to ion movements into and out from the egg. Chilling and anoxia decreased the contribution of a metabolic component to PD_egg. The effects of hypercapnia on PD_egg and ooplasmic pH suggest that the metabolic component may be a H⁺-pump. Chloride diffusion contributes to PD_egg in eggs after fertilization; contributions of other ions were relatively small. Potential differences measured in internally perfused locust eggs after osmotic lysis of the serosal epithelium indicated that the chloride-dependent component of PD_egg is developed across the chorion and/or serosal cuticle. In vivo values of PD_egg are probably less negative than values measured in control saline because chloride content of ground water is low. Ooplasmic sodium and potassium activities remained at typically intracellular levels during water uptake, possibly due to release from internal stores. Chloride activities were typical of extracellular fluids and were not regulated during water uptake. Measurements of pH and pea in locust eggs are consistent with the increases in these parameters predicted by the ionic hypothesis of activation. calcium activity in the ooplasm of unfertilized locust eggs (pCa 6.4 -4.9) appeared to be at a level appropriate for the use of calcium as a signal or second messenger. Calcium activity increased 100fold within 1 day of fertilization, and 1000-fold by day 3. Calcium entry from external sources at fertilization and release from internal stores later in development may contribute to the progressive increase in ooplasmic Ca⁺⁺ activity. The ooplasm likely alkalinizes after oviposition as ambient pCo₂ declines. Available data suggest a metabolically-dependent proton pump may control ooplasmic pH in locust eggs, in contrast to the Na⁺/H⁺ exchanger implicated in alkalinization of marine invertebrate eggs. / Thesis / Master of Science (MS)

I investigated the maternal effects that take place in a joint-nesting bird: the smooth-billed ani. Female anis were shown to respond to increasing group size by increasing the number of eggs produced per capita, by tossing and burying more eggs per capita, and by taking longer to reach the dedicated incubation phase. These results support the hypothesis that females respond to increased egg laying competition by trying to skew the contents of the final incubated clutch of eggs in their own favor. I showed that in ani groups, yolk testosterone and estradiol deposited by females in eggs increased from early- to late-laid eggs. Increases in yolk steroid levels over the laying sequence may function to mitigate the disadvantage of being a later-hatched chick. This maternal influence may not be a mere reflection of a female's hormonal status as female plasma circulating levels of testosterone and estradiol did not vary in the same direction as yolk hormone profiles. I showed that yolk corticosterone levels, an indicator of maternal physiological stress, increased with laying order in multi-female groups, but not in single-female groups. Results suggest that laying females experience higher levels of stress in multifemale groups. The above results suggest that communal life in anis generates competition and egg production waste that likely reduces short-term per capita reproductive benefits. Female anis can vary egg quality via deposition of hormones in eggs, and also lay eggs of different sizes. I showed that circulating plasma testosterone levels were higher in nestlings with better begging abilities. Furthermore, nestlings hatched from eggs laid late in the laying sequence had better begging abilities. These results suggest that testosterone is an important controlling mechanism of begging behaviour, and that female testosterone depositions in eggs rave long lasting effects on offspring development and behavior. / Thesis / Doctor of Philosophy (PhD)