Theses and Dissertations

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Open Access
A retinal contribution to chronic opioid-induced sleep/wake dysfunction
(Colorado State University. Libraries, 2023) Bergum, Nikolas, author; Vigh, Jozsef, advisor; Myers, Brent, committee member; Hentges, Shane, committee member; Chanda, Soham, committee member
Light is among the most important environmental factors that regulate mammalian sleep/circadian behaviors. Melanopsin-expressing intrinsically photosensitive retinal ganglion cells (ipRGCs) transmit environmental light information to key sleep/circadian centers in the brain through a process known as photoentrainment. Interestingly, past studies have revealed that ipRGCs express µ-opioid receptors (MORs), the primary molecular target for opioid analgesics. Furthermore, MOR agonists can directly inhibit ipRGC firing. Therefore, we hypothesize that opioid drugs acting on MORs expressed by ipRGCs could disrupt ipRGC-mediated regulation sleep/wake rhythms in response to environmental light/dark cycles. To test this idea, we need to confirm that morphine reaches the mouse retina following systemic delivery. To accomplish this, tissue (retina, brain and serum) was collected from mice following intraperitoneal morphine administration. Importantly, results from this study show that systemically administered morphine selectively accumulates in the mouse retina, but not the serum or the brain. To test the role that MORs expressed by ipRGCs play in opioid-induced dysregulation of sleep/circadian behaviors, we used mini-telemetry devices to assess how chronic morphine alters their sleep/wake behavior in mice. Importantly, we performed these experiments in wildtype mice along with mice lacking MORs exclusively in ipRGCs (McKO). Results from these studies reveal that McKO animals exhibit decreased morphine-induced locomotion compared to controls, which implicates MORs expressed by ipRGCs as a mediator of opioid-induced sleep-wake alterations. Finally, we tested whether ipRGCs developed cellular tolerance to MOR agonists following chronic exposure to morphine. The lack of cellular tolerance development at the level of solitary ipRGCs provides a potential cellular correlate for the persistent sleep/wake dysfunction commonly attributed to chronic opioid exposure. Taken together, these findings support the idea that opioid accumulation in the eye persistently activate MORs on ipRGCs, continuously altering the ability of ipRGCs to transmit light information to the brain's sleep/wake circuitry. This alteration in photic input to the brain could underlie some of the sleep/wake problems associated with long-term opioid use.
Open Access
Age-dependent decline in Kv4 channels, underlying molecular mechanisms, and potential consequences for coordinated motor function
(Colorado State University. Libraries, 2019) Vallejos, Maximiliano Jose, author; Tsunoda, Susan, advisor; Amberg, Gregory C., committee member; Bouma, Gerrit, committee member; Mykles, Donald, committee member; Tamkun, Michael, committee member
The voltage-gated potassium channel, Kv4, is widely expressed in the central nervous system and it is responsible for a highly conserved rapidly inactivating A-type K+ current. Kv4 channels play a role in the regulation of membrane excitability, contributing to learning/memory and coordinated motor function. Indeed, recent genetic and electrophysiological studies in Drosophila have linked Kv4 A-type currents to repetitive rhythmic behaviors. Because a deterioration in locomotor performance is a hallmark of aging in all organisms, we were interested in examining the effects of age on Kv4/Shal channel protein. In this dissertation, I use Drosophila as a model organism to characterize an age-dependent decline in Kv4/Shal protein levels that contributes to the decline in coordinated motor performance in aging flies. Our findings suggest that accumulation of hydrogen peroxide (H2O2) is amongst the molecular mechanisms that contribute to the age-dependent decline of Kv4/Shal. We show that an acute in vivo H2O2 exposure to young flies leads to a decline of Kv4/Shal protein levels, and that expression of Catalase in older flies results in an increase in levels of Kv4/Shal and improved locomotor performance. We also found that the scaffolding protein SIDL plays a role in maintaining Kv4/Shal protein levels and that SIDL mRNA declines with age, suggesting that an age-dependent loss of SIDL may also lead to Kv4/Shal loss. In behavioral studies, we found that a knockdown of SIDL resulted in a lethal phenotype, leading to a large decline in Drosophila eclosion rates, an event that requires coordinated peristaltic motions. Expression of SIDL or Kv4/Shal in this SIDL knockdown genetic background resulted in a partial rescue; these results are consistent with a model in which SIDL and Kv4/Shal play a role in coordinated peristaltic motions and are required for successful eclosion. The results presented in this dissertation provide new insight into the possible molecular mechanisms that underlie an age-dependent decline in Kv4/Shal protein. We identify two contributing factors: 1) ROS accumulation, and 2) the interacting protein SIDL. Our data also suggests that this age-dependent decline in Kv4/Shal levels is likely to be conserved across species, at least in some brain regions. Because Kv4/Shal channels have been implicated in the regulation of long-term potentiation and in repetitive rhythmic behaviors, the loss of Kv4/Shal may contribute to the age-related decline in learning/memory and motor function.
Open Access
Amino acid transmitters and the neural control of feeding and energy homeostasis
(Colorado State University. Libraries, 2016) Dicken, Matthew S., author; Hentges, Shane T., advisor; Tamkun, Michael M., committee member; Amberg, Gregory C., committee member; Tjalkens, Ronald B., committee member
Consuming the correct number of calories to maintain a healthy bodyweight is a delicate balancing act between intake and energy expenditure, and humans in modern society seem to have a keen knack for throwing the balance off-center. In the U.S. alone, more than 1/3 of adults are obese based on the body mass index scale, and $147 billion is the estimated annual medical cost for obesity in the United States. On the other end of the feeding spectrum, anorexia in the U.S. has been steadily rising since the 1960s, and has the highest mortality rate of any mental illness. While great strides have been made in understanding the neuronal regulation of energy balance, there is a need to more fully understand the homeostatic systems within the hypothalamus that are so powerful that they are able to drive individuals to poor health or death, often even in the face of consciously fighting their urges. Two groups of functionally opposed neurons contained within the arcuate nucleus of the hypothalamus, Neuropeptide Y / Agouti-related peptide (NPY/AgRP) and proopiomelanocortin (POMC) cells (the so-called first order feeding neurons), have been extensively studied for their roles in energy homeostasis—mostly through research into the peptides they are named after. There is clear evidence that peptides play an essential role for the function of NPY/AgRP and POMC cells, but what about simple amino acid transmitters? While it is known that GABA is packaged and released by NPY/AgRP cells and that this release is relevant to feeding behavior, there is still a dearth of information about this aspect of the circuitry, very much an area waiting to be mined. This study focuses on better understanding the functional release and relevance of amino acid transmitters packaged in both NPY/AgRP and POMC cell populations. Evidence is presented here for the conclusive release of both GABA and glutamate from POMC cells within intact circuitry. For NPY/AgRP neurons, evidence is presented for a shift in functional release of GABA from these neurons onto POMC cells depending on feeding state, corroborated by concurrent in situ hybridization experiments. Using a combination of electrophysiology and in situ hybridization approaches, evidence is also provided that mRNA levels of glutamate decarboxylase can act as a general proxy for functional GABA release. Altogether, these results indicate that amino acid transmitters play a significant role in first order feeding neuron physiology. Not only does this warrant further study on the significance of each transmitter alone and their purpose in comparison with the peptides released, but also the interplay between POMC cell and NPY/AgRP cell amino acid transmitters and their many shared downstream targets. Imbalances in proper glutamatergic and GABAergic signaling may significantly contribute to obesity, and advancing this area of study could lead to correcting those imbalances to restore healthy energy homeostasis.
Embargo
An investigation of synaptic vesicle docking and priming and a proposed method for quantitatively measuring both in Drosophila using electron tomography
(Colorado State University. Libraries, 2023) Twiggs, Jasmin A., author; Reist, Noreen, advisor; Hoerndli, Frederic, committee member; Hoke, Kim, committee member; Tamkun, Michael, committee member
The nervous system, as the body's command center, plays a crucial role in cellular communication within the brain and between the brain and other body systems. Neurons, the individual cellular units, transmit electrical information and communicate with other cells through neurotransmitter release in response to electrical stimuli. Chapter 1 introduces the foundational concepts of neuronal structure and function and delves into the mechanisms underlying neurotransmitter release. Special attention is given to the neuromuscular junction (NMJ), a well-studied chemical synapse crucial for muscle movement. The synaptic vesicle cycle is introduced, with particular emphasis on docking and priming. The significance of active zones, specialized sites for efficient signal transmission, and their associated structural components are underscored. Synaptotagmin, a pivotal protein in calcium-triggered vesicle fusion, is discussed with emphasis on its C2B polylysine motif. Throughout the chapter, the utility of Drosophila as a model system for studying synaptic processes, particularly at the NMJ, is emphasized. In sum, Chapter 1 provides the foundational knowledge essential for comprehending the intricate cellular and molecular facets of synaptic communication within the nervous system, serving as a precursor to subsequent chapters' investigations. Chapter 2 examines synaptotagmin's C2B polylysine motif and its role in synaptic vesicle docking at the Drosophila NMJ. It explores the polylysine motif's potential involvement in endocytosis, demonstrates an unaffected interaction with AP-2, and uses electron microscopy to find no significant changes in vesicle distribution. The findings suggest that the reduced neurotransmitter release in the polylysine mutant is likely due to an impairment in vesicle priming. Chapter 3 introduces a method for studying synaptic vesicle docking and priming in Drosophila, using electron tomography. I address the limitations of conventional electron microscopy and underscore the need for higher-resolution techniques to assess molecular structures that mediate physiological processes. Chapter 3 also emphasizes the significance of the contact area between docked vesicles and the presynaptic membrane as a correlate of vesicle priming. The protocol, expected results, and key considerations are discussed. The methods presented in Chapter 3 offer a promising approach for understanding synaptic processes. In Chapter 4, I discuss key considerations for when standard electron microscopy can be used for assessing vesicle docking. Then, I discuss how the electron tomography method presented in Chapter 3 could not only confirm the results found in Chapter 2, that the synaptotagmin C2B polylysine motif is not implicated in vesicle docking but could also be used to directly test the mutant's role in priming. Specific aims for future studies on the synaptotagmin polylysine mutation in Drosophila are presented, potential results and interpretations are discussed. Finally, I showcase interesting, unpublished findings from electron tomograms I have taken at the Drosophila NMJ and discuss their potential significance.
Open Access
Analysis of equine zygote development after intracytoplasmic sperm injection
(Colorado State University. Libraries, 2016) Ruggeri, Elena, author; Carnevale, Elaine, advisor; Clay, Colin, advisor; Albertini, David, committee member; DeLuca, Jennifer, committee member; Seidel, George, committee member
Intracytoplasmic sperm injection (ICSI) is an established and widely used method to achieve oocyte fertilization in equine reproductive assisted technologies. However, not all the oocytes fertilized by ICSI undergo cleavage and develop into viable embryos. Limited knowledge on equine zygote development after ICSI is available, and reasons why developmental failure occurs after ICSI have been only partially studied and need further investigation. Fertility decline and early embryo loss is associated with maternal aging in the mare, and it is concomitant with reduced oocyte quality. Relatively little is known about the effect of maternal aging and zygote developmental failure or success in the mare. Effects of in vitro maturation of the oocyte or zygote development in the mare still need to be clarified and further studied. The overall objective of this dissertation was to study equine zygote development after ICSI using confocal microscopy. Objectives were to: (1) compare cytoskeletal and nuclear changes during progression of equine zygote development after ICSI for in vivo versus in vitro matured oocytes; (2) compare changes in cytoskeletal and chromosomal configurations after ICSI between oocytes from young and old mares to define maternal-aging related alterations; (3) determine cytoskeletal and nuclear alterations associated with fertilization failure in ICSI-produced presumptive zygotes in young and old mares; (4) determine cell-aging and cell donor-aging effects on cytoskeleton and chromatin configurations. Specifically, in our studies we evaluated the tubulin and actin cytoskeleton, chromatin, and kinetochores/centromeres. Immunostaining and confocal imaging of the equine zygotes was performed using a spinning disk confocal microscope. After ICSI, five distinct events of development were observed with no major differences over time whether oocytes matured in vivo or in vitro. Oocytes matured in vivo appeared to reach the pronucleus stage earlier after ICSI compared to in vitro matured oocytes. Abnormal phenotypes associated with fertilization failure were more significant in oocytes matured in vitro than in vivo. When ICSI was performed in oocytes from young and old mares, similar stages of zygote development were observed, and the number of zygotes reaching the pronucleus stage was similar between the two age groups. Nucleolus like bodies, sites of ribosomal RNA involved in embryonic genome activation, were observed only in zygotes at the pronucleus stage from young mares; no nucleolus-like bodies were observed in pronuclei of zygotes from old mares. Pronuclei morphology, based on CREST staining, and DNA localization, also differed between pronuclei of young and old mares. Actin vesicles were observed significantly more often within zygotes from old mares compared to young mares during all stages of zygote developmental progression. When potential zygotes were analyzed after failure of cleavage after ICSI, actin vesicles were greater in area, perimeter and number in oocytes from old mares than those from young mares. Tubulin cytoskeletal multiasters were associated with cell aging and with increased interval after ICSI for young mares but not old mares. In conclusion, zygotes produced from oocytes matured in vivo versus in vitro or collected from young and old mares went through similar stages of development, with pronuclei attainment appearing to be a crucial event in zygote development. Actin vesicles were a major cytoskeletal difference associated with oocyte origin and a potential factor involved in developmental failure of the oocyte. Confocal microscopy and image analysis were novel methods used to describe the equine zygote development and allowed us to elucidate the cytoskeletal and nuclear remodeling events that follow fertilization after ICSI in the mare.
Open Access
Anatomic plasticity and functional impacts of neural – immune and neural – epithelial signaling in the intestine
(Colorado State University. Libraries, 2021) Schwerdtfeger, Luke A., author; Tobet, Stuart A., advisor; Chicco, Adam, committee member; Myers, Brent, committee member; Ryan, Elizabeth, committee member
The intestinal wall is a multicompartmental barrier tissue composed of over 25 distinct cell types with integrated and complex signaling both within and between compartments. The gut wall is also a large endocrine organ comprised of cells capable of producing dozens of peptides used for hormonal and other signaling functions. However, the mechanistic roles that neural secretions play in regulating the gut epithelial barrier in health and disease are not well known. Additionally, frequently used models available for studying intestinal function outside of the body lack the complexity to investigate neural – epithelial and neural – immune signaling interactions. Using a bifurcated approach to method development, we created two culture systems for maintaining the full thickness of the intestinal wall ex vivo. One method allows for culture of mouse or human organotypic intestinal slices that maintain the gut wall for 6 or 4 days, respectively. This system does not however, maintain a true luminal – epithelial barrier as seen in the in vivo gut. The second method, a microfluidic organotypic device (MOD) enables maintenance of explanted mouse or pig intestinal tissue for up to 3 days ex vivo, with an intestinal barrier intact. These two methods allow for investigating and cross-validating of numerous biological questions now previously possible using traditional culture models. Neuronal fiber proximity to gut epithelia has been shown, with goblet, tuft and enteroendocrine cells being closely opposed by fibers. Goblet cells secrete mucopolysaccharides, a first line of defense separating luminal microbiota from host tissue. I have recently shown that vasoactive intestinal peptide (VIP) can regulate goblet cell production in organotypic slices of mouse ileum. This peptide is also in close proximity to Paneth cells in the base of the crypt, and enteric mast cells. There were sex differences in baseline mast cell neuronal proximity, quantities, and cell size in mouse ileum. Further, mast cells showed a sex difference in responses to lipopolysaccharide challenge. Further investigation of neurosecretory factor regulation of immune and epithelial function is needed, both in goblet cells and other secretory epithelia like anti-microbial producing Paneth cells, and in immune components like mast cells. Graphical illustration of the dissertation project is included below.
Open Access
Androgen signaling in the placenta
(Colorado State University. Libraries, 2014) Cleys, Ellane Rachael, author; Bouma, Gerrit, advisor; Clay, Colin, advisor; Tobet, Stuart, committee member; Di Pietro, Santiago, committee member
Placental estrogen signaling is known to regulate placental trophoblast function and differentiation. However, the role of placental androgen signaling has never been investigated, despite the rise of maternal serum androgens throughout gestation. Recent findings have shown increased maternal serum androgen in patients with the placental induced disorder preeclampsia. Preeclampsia, a maternal hypertension and proteinuria condition instigated by insufficient trophoblast differentiation and invasion into maternal spiral arteries, is also associated with increased placental expression of androgen receptor and an increased risk of incidence in patients with polymorphisms in androgen receptor that decrease androgen signaling. These findings suggest a crucial role for placental androgen signaling. Moreover, research investigating androgen's role in cancer progression has shown that many androgen responsive genes regulate cell proliferation, differentiation to invasive phenotypes, and tissue vascularization, all processes necessary for normal placental development. Androgen signaling in tumor tissues is further regulated by androgen receptor complexes with histone lysine demethylases. These complexes are recruited to androgen response elements in DNA and dynamically regulate histone tail modifications for transcription initiation. This led us to the overall hypothesis that (1) androgen signaling in trophoblast cells is important for placental development, and (2) androgen receptor complexes with histone lysine demethylases in the placenta to regulate vascularization, growth and invasion factors in trophoblast cells. To test this hypothesis, we utilized a prenatal androgenization ewe model as well as human first trimester placental samples and immortalized human trophoblast cell lines. Using the prenatal androgenized ewe model, we report for the first time expression of histone lysine demethylases in the placenta. Furthermore, we showed androgen receptor complexes with histone lysine demethylases and is recruited to an androgen response elements in the 5'untranslated flanking sequence of vascular endothelial growth factor in the sheep placenta. We also report that histone lysine demethylase are present in human first trimester syncytiotrophoblast and complex with androgen receptor in immortalized trophoblasts. Additionally, we demonstrated that androgen receptor complexes with histone lysine demethylases are also present in choriocarcinoma ACH-3P and BeWo cells. Dihydrotestosterone treatment in these cells led to down-regulation of androgen responsive genes, specifically KDM3A and MMP2. Inhibition of androgen receptor through flutamide treatment altered mRNA levels for genes regulating vascularization, including HIF1α, PPARα, and PPARy. Hypoxia also decreased CYP19 levels, however, further investigation is needed to confirm dihydrotestosterone and flutamide effect on protein expression in trophoblast cells. These data suggest that histone lysine demethylases complex with androgen receptor to regulate androgen responsive genes, including those directing placental vascularization and development. However, further experiments are needed to confirm the necessity of histone lysine demethylases for targeted androgen signaling in trophoblast cells and to determine if androgen directly regulates trophoblast differentiation and invasion. These findings suggest androgen signaling may play a critical role in placental development.
Open Access
Angiotensin-II signaling in the pars reticulata GABA-ergic neurons in the substantia nigra and its implications in nigral neurotransmission
(Colorado State University. Libraries, 2021) Singh, Maibam Ratan, author; Amberg, Gregory C., advisor; Vigh, Jozsef, committee member; Tsunoda, Susan, committee member; Tamkun, Michael, committee member; Garrity, Deborah, committee member
Renin-Angiotensin-system is one of the most widely studied hormonal systems in the peripheral system and is primarily associated with the essential function of regulating blood pressure, fluid and electrolyte balance in the body. Most of the drugs used to treat hypertension currently are targeted towards one or more components of the RAS system. However, increasing studies have presented evidence of local RAS in tissues completely independent of the humoral system. In the CNS, in addition to highly vascularized areas in the brain lacking the blood-brain-barrier (BBB) such as the circumventricular organs, all RAS components have also been found in the brain regions inside the BBB and are suspected to be involved in neuronal differentiation, neurotransmission, and learning and memory. Increasing studies have reported the interaction of brain RAS with pathophysiological mechanisms of many neurological and psychiatric illnesses. However, this extrarenal effect of RAS is only beginning to gain some scientific attention, and the underlying mechanisms are far from elucidated. All the RAS components are strongly expressed in the midbrain, especially the substantia nigra. Accumulating evidence in recent years has implicated Angiotensin-II (Ang-II), the primary effector peptide of RAS, in the selective degeneration of dopaminergic neurons in the substantia nigra compacta (SNc) in animal models of Parkinson's disease. Ang-II is believed to induce G-protein signaling through Ang-II type 1 receptor (AT1-R) and increase cellular oxidative stress, intracellular calcium load and activate apoptotic pathways in SNc dopaminergic neurons. Interestingly, studies have also shown Ang-II mediated striatal dopamine release in rats. These studies suggest that Ang-II signaling can induce both intracellular effects and influence dopaminergic neuronal output in the midbrain. However, if Ang-II signaling exists in other neuronal cell types in the substantia nigra is not known. Substantia nigra is comprised of two primary cell types: dopaminergic and GABAergic neurons. The majority of dopaminergic neurons are located in the SNc, and the SNr is comprised of GABAergic projection neurons with few interspersed dopaminergic neurons. Besides being one of the major output neurons of basal ganglia, SNr GABAergic projection neurons also provide significant inhibitory input to the neighboring SNc dopaminergic neurons, not through a direct axonal projection like its other target areas but via its extensive network of axon collaterals. Inhibitory input from the SNr GABAergic neurons contributes to the essential balance between afferent excitatory and inhibitory inputs to SNc dopaminergic neurons that tightly regulates their cellular activity and output. Indeed, SNr GABAergic neurons are necessary for the voluntary control of movement and are implicated in basal ganglia dysfunctions associated with movement disorders such as Parkinson's disease. RAS components are also expressed in the SNr GABAergic neurons, but it is not known if Ang-II signaling exists in these cells and what effects it may have on intranigral neurotransmission and dopaminergic cell activity. Here we used a combination of electrophysiology, imaging, and optogenetics to characterize and investigate the role of Ang-II in local neurotransmission in the substantia nigra. We found a heterogeneous effect of Ang-II in the nigral dopaminergic and GABAergic neurons. Ang-II suppressed both electrically and light-evoked activity of SNr GABAergic neurons through a combination of mechanisms: enhancement of postsynaptic GABAa receptors and increasing the action potential duration. On the contrary, Ang-II had no noticeable direct effect on the activity of SNc dopaminergic neurons and its GABAa receptors. This provides the first evidence of novel Ang-II signaling in SNr GABAergic neurons and its heterogeneous effect in the two nigral cell types. Interestingly, in contrast to observed suppression of SNr GABAergic neuronal activity by Ang-II, under phasic photoactivation of SNr GABAergic neurons, Ang-II enhanced the feedforward inhibitory input to SNc dopaminergic neurons. This shows a non-linear effect of Ang-II on population output of nigral GABAergic neurons and may indicate the involvement of an intricate intranigral network formed by the axon collaterals of SNr GABAergic neurons that can further modulate its effect on postsynaptic targets.
Open Access
Applying in vitro-produced embryos and sexed sperm to dairy cattle reproduction
(Colorado State University. Libraries, 2011) Rasmussen, Sara-Lesley, author; Seidel, George E., Jr., advisor; Graham, James K., committee member; McCue, Patrick M., committee member
This study compared the pregnancy rates between embryo transfer of bovine embryos produced in vitro with sexed vs control sperm and artificial insemination (AI) using sexed and unsexed sperm. Cleavage rates for oocytes fertilized with sexed vs control sperm were not different for two of the three bulls used, but were lower (p < 0.05) for the third bull sexed (44%) vs control sperm (70%). There were fewer transferable blastocysts produced per oocyte with sexed sperm (9-19%) than for unsexed sperm (18-26%); (p < 0.05). All cows were on an Ovsynch program to synchronize ovulation. Respective 60 d pregnancy rates at two Colorado dairies were as follows: control AI (43%, n=88; 43%, n=44); AI with X-sorted sperm (34%, n=82; 34%, n=62); embryo transfer (ET) with in vitro-produced (IVP) embryos using unsexed sperm (22%, n=68; 21%, n=39); and ET with IVP embryos using sexed sperm (7%, n=72; 37%, n=40). The pregnancy rate (day 60) for AI using sexed sperm was 78% of that of control sperm. ET pregnancy rates were generally lower than AI rates. At one dairy, abortions between days 32 and term were higher for X-sort ET pregnancies (79% n=14) than for AI control pregnancies (20% n=40); (P < 0.001). However, the other dairy experienced only a 12%, (n=17) abortion rate for transferred embryos produced from X-sorted sperm. The sex ratio of calves was similar to previous studies for AI with control sperm (52% bull calves, n=50), AI with X-sorted sperm (12% bull calves, n=40); ET with IVP embryos using unsexed sperm (50% bull calves, n=18); and ET with IVP embryos using sexed sperm (11% bull calves, n=18). Findings from this experiment indicate that embryo production with sexed sperm is not successful enough to be applied to large-scale dairies that already have successful breeding programs in place.
Open Access
Association of oocyte and early embryo morphology with age and the establishment and maintenance of pregnancy after ICSI in mares
(Colorado State University. Libraries, 2010) Frank-Guest, Bethany Linda, author; Carnevale, Elaine, advisor; Seidel, George, Jr., committee member; Hendrickson, Dean, committee member
Increasing maternal age in humans, horses and lab animals has been associated with a decrease in fertility. Oocyte quality and morphology have been implicated as primary causes of reduced fertility in older mares. Selected oocyte morphological parameters have been correlated with pregnancy development in humans and horses. Objective measurements of morphology to assess oocyte quality would provide a critical evaluation and help identify zygotes with the highest developmental potential for transfer, to optimize recipient utilization and pregnancy rates. The hypotheses of the research were that oocyte and early embryo morphology differ with donor mare age and correspond with developmental potential. Objectives for the first study were to compare: 1) oocyte donor age with oocyte morphology and developmental competency after ICSI, and 2) oocyte morphology with developmental competency (cleavage, early pregnancy, late pregnancy and pregnancy loss) after ICSI. Objectives for the second study were to compare developmental potential of ICSI produced embryos with: 1) oocyte donor age, and 2) cleavage characteristics, and 3) rate of embryonic development. Oocytes were collected from donor mares in a clinical ICSI programs. The mares were divided into the following age groups and fertility categories: 1) 3-13 yr with Known fertility, 2) 2-13 yr with Unknown fertility, 14-19 yr, 20-23 yr and 24-27 yr. Approximately 24 h after induction of follicle maturation, and oocytes were collected and cultured approximately 18 h before being stripped of cumulus cells. Photographic images (200x) were captured before oocytes were injected with sperm. Images of oocytes were measured using digital calipers within a computer software program. Ooplasm volume was larger (p<0.05) for oocytes from mares 14-19 yr and 20-23 yr than mares 3-13 yr Known than for mares 24-27 yr. Perivitelline space volume was similar between mares 3-13 yr Unknown and mares 20-23 yr, but was smaller (p<0.05) between mares 3-13 Unknown and the other age groups. Oocyte diameter (OD) was smaller (p=0.05) between oocytes from donors 3-13 yr Known and donors 14 -19 yr, but similar among all other groups. Inner zona pellucida diameter (IZPD) differed (p=0.03) only between mares 14-19 yr and mares 3-13 yr Unknown, with oocytes from mares 14- 19 yr having the largest numerical IZPD and mares 3-13 yr Unknown having the smallest IZPD. Ooplasm diameter (OpD) was smaller (p≤0.02) for oocytes from mares 3-13 yr Known than from mares 14-19 or 20-23 yr. The diameter of the zona pellucida with the surrounding matrix (ZPTM) was greater (p<0.05) for mares 3-13 yr Unknown than for all other groups. The rate of embryo development (hours per cell) prior to oviductal embryo transfers was faster (P<0.05) for embryos that did versus did not produce an early pregnancy and tended (P≤0. l) to be faster for embryos that did versus did not produce a late pregnancy. Embryonic vesicles that had a more rapid increase in diameter were more often (p<0.05) maintained to the late pregnancy stage. Donor mare age exerted a large effect on the development and outcome of pregnancies. Oocyte morphology was not a reliable indicator of oocyte developmental potential, although speed of early embryonic development was associated with embryonic competency.
Open Access
Behavioral effects of estrogen receptor beta acting locally to regulate the expression of tryptophan hydroxylase 2 (THP2) in serotonergic neurons of the dorsal raphe nuclei
(Colorado State University. Libraries, 2008) Donner, Nina Caroline, author; Handa, Robert J., advisor; Tjalkens, Ronald, committee member; Clay, Colin McKeown, committee member; Tobet, Stuart, committee member
Affective disorders often involve serotonin (5-HT)-related dysfunctions and are twice as common in women than men. Interactions between estrogen and the brain 5-HT system have long been proposed to contribute to sex differences in mood and anxiety disorders, but the mechanisms underlying this phenomenon have yet to be revealed. Estrogen signaling is mediated by two different receptors termed estrogen receptor alpha and estrogen receptor beta. While estrogen receptor alpha (ERalpha) has mainly reproductive responsibilities, in brain, estrogen receptor beta (ERbeta) has been shown to attenuate anxiety- and despair-like behaviors in rodent models. However, little is known about ERbeta regulation of function in the brainstem raphe nuclei. The raphe nuclei are the main 5-HT system of the brain, and projections from the dorsal raphe nuclei (DRN) innervate many important forebrain and limbic areas. The work presented in this thesis addressed the possibility that ERbeta may be involved in the regulation of 5-HT gene expression specifically in DRN neurons. My studies examined the effects of systemic versus local, intracerebral application of the selective ERbeta agonist diarylpropionitrile (DPN) and the nonselective ERligandestradiol (E) on tryptophan hydroxylase 2 (TPH2) mRNA expression within the DRN of female rats. TPH2 is the brain-specific, rate-limiting enzyme catalyzing 5-HT synthesis, and is expressed in every 5-HT neuron. Thus, it provides an excellent tool to assess the capacity for 5-HT production with the DRN. In these studies, TPH2 mRNA expression was assessed via in situ hybridization. In addition, relevant behavioral parameters were tested in all animals to evaluate each compound’s effect on two closely related, but yet different mental states, anxiety-like and despair-like behavior. Both, chronic systemic and chronic local DPN administration to ovariectomized (OVX) female rats significantly enhanced TPH2 mRNA expression in mid- and caudal subregions of the DRN after 8 days of treatment. Respective controls received systemic vehicle (27% hydroxypropyl-beta-cyclodextrin) or blank control pellets. Local application of DPN caused a stronger effect than systemic drug delivery. Chronic local delivery of E (0.5 μM) increased TPH2 mRNA expression in the same subregions of the DRN as did DPN, but its overall effect was weaker compared to the selective ERbeta agonist. Interestingly, while systemic DPN-administration confirmed the anxiolytic nature of ERbeta in two separate anxiety tests (elevated plus maze and open field test), the effect was lost when DPN was delivered locally. However, local DPN- as well as E-treatment both resulted in attenuated despair-like behavior, as measured in the forced-swim test. Chapter 3 describes the experimental design, results and interpretation of these studies in depth. Taken together, my data indicate that local actions of ERbeta agonist onto DRN neurons are sufficient to decrease despair-like behavior, whereas ERbeta stimulation of other brain regions is necessary to alter anxiety-like behaviors. Correspondingly, ERbeta acts locally to control TPH2 mRNA expression and presumably 5-HT synthesis in the certain subregions of the rat DRN. These results suggest an important role of ERbeta for regulating cellular events in the female DRN, and offer new opportunities for therapeutic treatments of depressive disorders.
Open Access
Characterization of equine sperm attributes and selection for intracytoplasmic sperm injection
(Colorado State University. Libraries, 2018) Gonzalez-Castro, Raul A., author; Carnevale, Elaine, advisor; Graham, James, advisor; Seidel, George, committee member; Gerrit, Bouma, committee member; Ann, Hess, committee member
When performing intracytoplasmic sperm injection (ICSI), many in vivo mechanisms for sperm selection are bypassed; however, sperm must still be capable of activating the oocyte for successful fertilization. Limited information is available for horses on the effect of sperm preparation method and sperm characteristics that affect ICSI outcome. The overall objectives of this dissertation were to: 1) study the association between sperm sorting methods, sperm population characteristics, and equine ICSI outcome, and 2) characterize sperm oocyte activating factors in stallion sperm, such as phospholipase C zeta (PLCz) and postacrosomal WW binding protein (PAWP). In Experiment 1, a microfluidic device was used to sort frozen-thawed sperm from stallions (n=19), which resulted in a sperm subpopulation with improved motility, morphology, viability and DNA integrity (P<0.05) compared to the original sample. Then, microfluidic sorting was compared with the swim-up procedure and density gradient centrifugation. Swim-up was the least effective method to separate equine sperm. Microfluidic sorting and density gradient centrifugation sorted a sperm subpopulation with similar parameters, improving motility, viability and DNA integrity. After ICSI (n=45), no differences (P>0.3) were observed for cleavage and embryo development among sorting methods. In Experiment 2, sperm population parameters from which individual sperm were selected for injection were analyzed immediately after ICSI and correlated with the outcome. Sperm morphology, viability, membrane integrity measurement of hypoosmotic swelling and DNA integrity were evaluated in frozen-thawed sperm (n=114) used for ICSI in a program. Among sperm parameters, viability correlated positively with normal morphology and membrane integrity (P<0.05). Normal sperm morphology and DNA integrity were not predictive of ICSI outcome. Viability was predictive of cleavage and blastocyst formation, and membrane integrity was predictive of early pregnancy (P<0.05). In Experiment 3, PLCz and PAWP were identified, localized and quantified in stallion sperm, and the relationship with other sperm parameters was investigated. PLCz was identified as a 71 kDa protein and located in the acrosomal and postacrosomal region, midpiece and principal piece of the tail. PAWP was identified by two bands of ~28 and ~32 kDa, located in the postacrosomal region, midpiece and principal piece of the tail. The expression of PLCz and PAWP correlated positively (P=0.04) when analyzed for sperm of 14 stallions. Flow cytometric assessment was feasible for PLCz, but not for PAWP. Expression and percentages of positive labeled sperm for PLCz varied among stallions (n=21). Expression of PLCz was higher in live than dead sperm (P<0.005), and DNA fragmentation correlated negatively with PLCz expression (P<0.04). In conclusion, microfluidic sorting and density gradient centrifugation resulted in a subpopulation of sperm with high quality parameters for ICSI. The probability of sperm-injected oocytes to develop into an embryo and to establish pregnancy improved when sperm were selected from a sample population with higher viability and membrane integrity. This is the first report that describes PAWP in equine sperm, which displayed a novel localization in the midpiece and principal piece of sperm tail in addition to the expected postacrosomal region. Protein levels of PLCz and PAWP were correlated in sperm heads. The expression of PLCz in sperm varied widely among stallions and was associated with DNA integrity. Sperm membrane integrity is indicative of well-maintained plasma membrane architecture, conserving sperm quality and membrane components that are required for oocyte activation and early embryo development. Assessment of PLCz in stallion sperm represents a potent feature to investigate sperm quality for equine ICSI, and potentially can serve as a prognostic biomarker for oocyte activation ability and male infertility. Further studies are needed to determine the relationship between PLCz and PAWP with fertility in horses.
Open Access
Characterization of mediators of cardiac and renal development in response to increased prenatal testosterone
(Colorado State University. Libraries, 2008) Maresh, Ryan W., author; Anthony, Russell, advisor
Previously reported differences in cardiac and kidney weights of offspring from ewes prenatally treated with testosterone suggested the development of systemic hypertension. To determine if prenatal androgen excess influences the expression of key mediators, angiopoietin 1 (Ang1), angiopoietin 2 (Ang2), tunica interna endothelial cell kinase-2 (Tie2), angiotensin II receptor subtypes 1 and 2 (AT1 and AT2), endothelial nitric oxide synthase (eNOS), vascular endothelial growth factor (VEGF), VEGF receptor 1 (VEGF R1), VEGF receptor 2 (VEGF R2), insulin receptor β (IRβ), glucose transporter 1 (GLUT1), and glucose transporter 4 (GLUT4) were evaluated from fetal hearts, fetal kidney, left ventricle (LV), right ventricle (RV), and kidney. We hypothesized that mRNA and protein concentrations were altered and that the changes persist into adulthood. At fetal Day 90 a significant increase (p = 0.021) in IRβ mRNA concentration of was seen in the heart, whereas VEGF mRNA concentration was significantly reduced (p = 0.044) in the kidney. In 9 month RV, eNOS mRNA concentration was significantly reduced in the treatment group (p = 0.019). In the 9 month kidney, significant increases in GLUT4 mRNA concentration (p = 0.047) and eNOS protein concentration (p = 0.027) were seen in the treatment group. At 21 months of age, Ang1 mRNA concentration in the treatment group was significantly reduced (p = 0.025) in the LV. AT1 mRNA concentration was significantly decreased (p = 0.004) in the RV of the treatment group. LV eNOS concentration was significantly reduced (p = 0.032), while RV eNOS mRNA concentration was significantly decreased (p < 0.001) in the treatment group. Significant decreases in GLUT1 mRNA concentration were detected in the LV (p = 0.013) and RV (p = 0.006), and RV GLUT4 mRNA concentration was significantly decreased (p = 0.003) in the treatment group. LV IRβ concentration was significantly reduced (p = 0.036) in the treatment group. In the kidney, VEGF R2 mRNA concentration was significantly reduced (p = 0.024) in the treatment group. Taken together, the data supports the development of systemic hypertension and insulin resistance with age following prenatal androgen exposure.
Open Access
Characterization of periattachment factor
(Colorado State University. Libraries, 2008) Purcell, Scott H., author; Anthony, Russell V., advisor; Seidel, George E., advisor
The purpose of these studies was to characterize expression of periattachment factor (PF) mRNA in the developing sheep conceptus, localize PF in the sheep conceptus, determine if degrading PF mRNA in the sheep conceptus was detrimental to development, and characterize PF in human tissues and cells. Sheep conceptuses were collected on d 11, 13, 15, 16, 17, 21, and 30 post-mating. oPF mRNA exhibited a quadratic expression pattern (P<0.05). No oPF mRNA was detected in d 11 conceptuses. From d 13, oPF mRNA increased to a peak at d 16 before declining. Peak expression of oPF mRNA in the conceptus occurs during rapid elongation and initial attachment of the conceptus to the endometrium. Immunolocalization of PF in a d 15 conceptus showed predominantly nuclear staining in trophectoderm and trophendoderm. Next, embryos collected from superovulated ewes on d 8 post-mating were infected with either a lentivirus expressing a short-hairpin (sh) RNA designed to target PF mRNA for degradation, a lentivirus expressing a shRNA containing 3 mismatched nucleotides, or a control lentivirus expressing no shRNA. Following infection, blastocysts were transferred into recipient ewes and collected back on d 15 of gestation. While 94 and 88% of the control and mismatched shRNA-treated conceptuses elongated by d 15, none of the embryos treated with the lentivirus expressing shRNA against PF mRNA elongated, and most died. This indicates that oPF is required for conceptus elongation and survival. Human PF mRNA was detected in human carcinomas and in the first trimester cytotrophoblast cell line, hTR8. Immunohistochemistry showed PF in the nuclei of carcinomas and in first and second trimester cytotrophoblasts. PF was also present in the invading cytotrophoblast columns. In an in vitro invasion assay of first trimester cytotrophoblasts, hPF mRNA increased from 0, 3, to 12 h as invasion occurred. To further characterize PF in human cells, a lentiviral construct expressing an shRNA targeting the hPF mRNA sequence was developed that resulted in 63% mRNA reduction in BeWo cells.
Open Access
Characterization of selected gene expression patterns as potential markers for oocyte quality in young versus old mares
(Colorado State University. Libraries, 2010) Rodrigues, Bernardo de Lima, author; Clay, Colin M., advisor; Carnevale, Elaine M., advisor; Tjalkens, Ronald B., committee member; Bouma, Gerrit J., committee member; Bruemmer, Jason E., committee member
As a female ages a series of alterations in normal physiology take place, during this process fecundity decreases. The reproductive system starts to shut down as a consequence of hormonal, histological and anatomical changes, and in the middle of this process, playing a critical role, is the oocyte. As in women, fertility decreases with aging in mares. Recently the mare has been suggested as a promising model to study age-related infertility in women due to similarities in the reproductive cycle and similar age-associated reproductive changes. During the last decades the use of assisted reproductive techniques (ART) in human and veterinary medicine to treat infertility has increased. Unfortunately, ART can only partially compensate for declining fertility- particularly the age related decline in fertility. Therefore, we need to understand the mechanisms involved in age-associated infertility and improve both the diagnostic tools and the techniques currently used in ART. In that regard the identification of reliable oocyte quality markers is of great interest, specifically of extrinsic markers in follicular cells and follicular fluid (FF). Follistatin (FS1) and anti-müllerian hormone (AMH) have been suggested as potential oocyte quality markers. In addition, the rate of apoptosis in follicular cells has also been suggested to be a good indicator of oocyte quality In this study, our goal was to use the young and old mare model to obtain competent and incompetent oocytes, respectively, to try and elucidate the involvement of apoptosis of follicular cells and/or of the oocyte in the determination of oocyte quality. Oocytes, follicular fluid, granulosa and cumulus cells were collected through transvaginal follicular aspirations from young ( 4-10 years) and old (>20 years) mares. Preovulatory follicles were aspirated 30-36 h post induction of follicular maturation, which was performed by administration (i.v) of a combination of deslorelin and hCG when the biggest follicle reached 35 mm. We used real time PCR to examine expression of pro-apoptotic ( CASP2, CASP 3) and pro-survival (XIAP) genes, as well as of FST and AMH expression in these cell types. In addition we measured androgens and estrogens in FF and calculated the androgens to estrogens ratio to assess follicular atresia. We also sought to determine FF concentrations of FST and AMH, and relate it to oocyte quality. There was no difference in CASP3 expression levels in granulosa and cumulus cells between the two age groups. In addition, there was no difference in CASP2 and CASP3 mRNA expression in oocytes from young and old mares. XIAP mRNA levels were expressed 3.3 fold higher in oocytes from young when compared to old mares, and there was a tendency for XIAP to be more highly expressed in granulosa cells of young mares. In contrast, the levels of XIAP mRNA in cumulus cells were 1.46 fold higher in old when compared to young mares. There was no difference in the expression levels of AMH in granulosa cells between young and old mares, but in cumulus cells there was a tendency for AMH to be higher expressed in cells from old vs. young mares. Unfortunately we were not able to analyze AMH FF concentrations. FST mRNA levels in oocytes were similar between the age groups, but FST concentrations in FF of preovulatory follicles from young mares (197 ± 16.7 ng/mL) were higher (p=0.02) than in FF from old (153.3 ± 22.7 ng/mL) mares. In both age groups FST FF concentrations in preovulatory follicles significantly decreased when compared to mid-estrus and post-deviation follicles. In conclusion, we believe that our data suggest that FST follicular fluid levels can be a non-invasive marker to assess oocyte quality in the horse, and that FST levels decrease in preovulatory follicles of the horse. In addition, expression levels of caspase-3 in follicular cells, and caspases 3 and 2 in the oocyte, does not seem to be involved in the mechanism of fertility loss in the old mare. Finally, XIAP mRNA levels may be important for oocyte quality in the horse.
Open Access
Characterizing the target of ivermectin, the glutamate-gated chloride channel, and other insecticide targets as candidate antigens for an anti-mosquito vaccine
(Colorado State University. Libraries, 2015) Meyers, Jacob, author; Partin, Kathryn, advisor; Foy, Brian, advisor; Vigh, Jozsef, committee member; Tsunoda, Susan, committee member
The latest WHO World Malaria Report estimates that, in 2013, there were 198 million cases worldwide causing 584,000 malaria-related deaths. Current malaria control programs primarily target malaria vectors through the use of long lasting insecticide treated bed nets and indoor residual spraying of pyrethroid-based insecticides. However, pyrethroid resistance is becoming widespread in many An. gambiae populations across Africa (Ranson et al., 2011; Trape et al., 2011). Out of recent efforts to find new vector-targeting interventions with novel modes of action, the endectocide ivermectin (IVM) has arisen as a new candidate to control malaria transmission. IVM, when imbibed by vectors from host-treated blood meals, has proven to efficiently kill or disable An. gambiae s.s. both in the lab and the field (Kobylinski et al., 2010; Sylla et al., 2010). More recently, IVM mass drug administrations in multiple locations across west Africa have been shown to temporarily reduce the proportion of P. falciparum-infected An. gambiae in IVM-treated villages (Kobylinski et al., 2011; Alout et al., 2014). The primary target of IVM is the invertebrate glutamate-gated chloride channel (GluCl) (Cully et al., 1994; Cully et al., 1996; Janssen et al., 2007; McCavera et al., 2009; Janssen et al., 2010; Moreno et al., 2010). The purpose of the first chapter of this thesis was to characterize GluCl from An. gambiae in order to understand the physiological role of GluCl and how IVM may be affecting mosquito physiology. Cloning of the An. gambiae GluCl (AgGluCl) revealed unique splicing sites and products not previously predicted. We expressed AgGluCl clones in Xenopus laevis oocytes to measure its electrophysiological activity in response to glutamate and IVM. We also examined AgGluCl isoform-specific transcript levels across different tissues, ages, blood feeding status and gender and GluCl tissue expression in adult An. gambiae. Given that GluCl can be targeted by drugs found in a blood meal and that GluCl is not expressed in mammals, we wanted to test the efficacy of AgGluCl as a candidate mosquitocidal vaccine antigen. We administered a polyclonal anti-AgGluCl immunoglobulin G (anti-AgGluCl IgG) to An. gambiae mosquitoes through a blood meal or directly into the hemocoel by intrathoracic injections and found it significantly reduced An. gambiae survivorship. By co-administering anti-AgGluCl IgG with a known GluCl agonist, IVM, we discovered anti-AgGluCl IgG reverses the mosquitocidal effects of IVM. Our results describing the mosquitocidal properties of anti-AgGluCl IgG suggest that other neuronal proteins could be used as candidate antigens for a mosquitocidal vaccine. The An. gambiae GABA-gated chloride channel (resistance to dieldrin; AgRDL) is another member of the cys-loop ligand-gated ion channels with a similar structure and physiological function to AgGluCl. The An. gambiae voltage-gated sodium channel (AgVGSC) is the target of dichlorodiphenyltrichloroethane (DDT) and the pyrethroid class of insecticides (Soderlund and Bloomquist, 1989). VGSCs are also the target of multiple classes of spider, scorpion and snail toxins, demonstrating that peptides binding to VGSC extracellular residues can affect channel function (Nicholson, 2007; King et al., 2008; Stevens et al., 2011; Klint et al., 2012). Preliminary results shows that IgG targeting AgRDL or AgVGSC similarly reduce An. gambiae survivorship. Finally we tested anti-AgGluCl IgG against A. aegypti and C. tarsalis to see if this strategy has broad potential across both Anopheline and Culicine mosquitoes. However, blood meals containing anti-AgGluCl IgG had no effect on A. aegypti or C. tarsalis survivorship. We determined that this was due to a barrier in antibody translocation from the blood meal to the hemolymph. Since the IgG target, AgGluCl, is only expressed in the hemocoel, antibody translocation was required for mosquito toxicity.
Open Access
Cholinergic synaptic homeostasis is regulated by Drosophila α7 nicotinic acetylcholine receptors and Kv4 potassium channels
(Colorado State University. Libraries, 2021) Eadaim, Abdunaser Omar, author; Tsunoda, Susan, advisor; Tamkun, Michael, committee member; Amberg, Gergory, committee member; Bouma, Gerrit, committee member; Clay, Colin, committee member; DeLuca, Jennifer, committee member
Homeostatic synaptic plasticity (HSP) is an important mechanism that stabilizes neural activity during changes that occur during development and learning and memory formation, and some pathological conditions. HSP in cholinergic neurons has been implicated in pathological conditions, such as Alzheimer's disease and nicotine addiction. In a previous study in primary Drosophila neuron culture, cholinergic activity was blocked using pharmacological tools and this induced a homeostatic response that was mediated by an increase in the Drosophila α7 (Dα7) nAChR, which was subsequently tuned by an increase in the voltage-dependent potassium channel, Kv4/Shal. In this study, we inhibit cholinergic activity in live flies using temperature-sensitive mutant alleles of the choline acetyltransferase gene (Chats2 mutants). We show that this in vivo activity inhibition induces HSP similarly mediated by Dα7 nAChRs followed by an up-regulation of Kv4/Shal. We show that the up-regulation of Dα7 nAChRs alone is sufficient to induce an increase in Kv4/Shal protein, as well as mRNA. Finally, we test the involvement of transcription factors, dCREB2 and nuclear factor of activated T cells (NFAT) in the up-regulation of Kv4/Shal. In particular, we find that NFAT is required for the inactivity-induced up-regulation of Kv4/Shal channels. Our studies reveal a novel receptor-ion channel system transcriptionally coupled to prevent over-excitation.
Open Access
Cloning and expression of a porcine zona pellucida gene: an approach to immunocontraception
(Colorado State University. Libraries, 1991) Fontenot, Gregory Kenneth, author; Bowen, R. A., advisor; Jonathan Carlson, committee member; Niswender, Gordon, committee member
Immunization with native porcine zona pellucida (ZP) proteins has been shown to induce infertility in females of several species and is thus a potentially valuable method of contraception. However, more extensive testing and commercialization of such a vaccine has been hampered by the limited availability of ZP proteins from natural sources. Availability of recombinant ZP proteins should simplify production of a practical ZP vaccine. The objective of this research was to clone a porcine ZP gene and use it to develop a recombinant ZP vaccine for use in pet animals. Polyadenylated RNA isolated from swine ovary was used to generate a cDNA library in the bacteriophage lambda gt11. This library was screened immunologically for ZP sequences using a polyclonal antiserum raised against solubilized porcine ZP. One immunoreactive clone, PZP, contained an insert of approximately 2.6 kb and was characterized further. The three Eco RI fragments constituting PZP were isolated and subcloned into a plasmid vector. The amino acid sequence deduced from the nucleotide sequence of PZP is considered to represent 305 residues from the carboxyterminal end of the ZP protein. A putative N-glycosylation site is present at residue 288 of this polypeptide. Comparison of the deduced amino acid sequence of PZP with deduced protein sequences from all of the other ZP proteins published to date failed to reveal significant homology. To confirm that PZP represented a ZP mRNA, a 418 bp fragment of the cDNA was expressed as a fusion protein in E. coli and used to hyperimmunize a rabbit. Antibodies to the PZP fusion protein bound to ZP surrounding porcine oocytes, stained ZP in sections of porcine ovary and immunoprecipitated a porcine ZP protein that was tentatively identified as ZP2. The PZP fusion protein was preliminarily evaluated as a vaccine in rabbits. Two groups of four adult rabbits were immunized three or four times with PZP2 fusion protein emulsified in either one of two adjuvants. Reproductive function was evaluated eight and sixteen weeks after initial immunization. In comparison to control rabbits, no effect of vaccination on reproductive function was observed.
Open Access
Connecting structural changes to cell transformation patterns in the canine degenerative mitral valve
(Colorado State University. Libraries, 2015) Abbott, Kaitlin Marie, author; Orton, E. Christopher, advisor; Frye, Melinda, committee member; Schenkel, Alan, committee member
Degenerative mitral valve disease (DMVD) is a significant problem in the canine population and also affects humans. Recent studies have provided insight into molecular and cellular mechanisms that likely contribute to disease progression. Better understanding of the cellular processes that mediate the degenerative process could lead to treatments that prevent or slow this degeneration benefiting both canine and human patients. Structural changes to degenerative valves such as nodules, leaflet thickening, increased opacity, loss of elasticity and loss of valve architecture have been well documented. Abnormal cell transformation patterns such as the transformation of valvular interstitial cells to activated myofibroblasts have been characterized in degenerative mitral valve tissue, as well as other irregular cell behavior such as the overproduction of glycosaminoglycan and matrix remodeling factors that have become hallmarks of the disease. Despite these important discoveries, much remains unknown about cell signaling in degenerative mitral valve disease and how cell activity changes a normal valve to the diseased phenotype. An overarching hypothesis of this study is that investigating signaling mechanisms active in degenerative valves could provide insight into cellular processes mediating the disease. A specific hypothesis that emerged from initial results is that endothelial to mesenchymal transition (EndMT), a process important in valvulogenesis, could be active in degenerative mitral valves. The first goal of this study was to compare protein abundance in degenerative and normal mitral valves to determine if there exists previously unidentified signaling molecules that could be initiating or perpetuating the cellular transformations and abnormalities present in DMVD. The second goal was to investigate these proteins using immunohistochemistry to characterize their activity in the tissue matrix and show evidence of their contribution to structural changes of the valve. The first goal was accomplished by doing a targeted microarray analysis of signaling proteins comparing their relative abundance in normal and degenerative mitral valves. This analysis yielded an increased abundance of signaling proteins that have been associated with EndMT. The second goal was accomplished by immunohistochemistry to determine the spatial distribution of selected proteins from the microarray analysis with markers of endothelial cells and mesenchymal cells (activated myofibroblasts). Targeted microarray analysis of signaling proteins revealed increased abundance of 18 proteins including the growth factor HB-EGF, its partner molecule ADAM17, and the cell adhesion molecule integrin β3, all possible mediators of EndMT (Chapter 4). Immunohistochemistry studies demonstrated the presence of cells positive for the endothelial marker CD31 within the valve interstitum. These CD31 positive cells co-localized with areas of myofibroblast transformation in degenerative valves identified by positive staining for α-smooth muscle actin (αSMA). Expression of signaling proteins including HB-EGF and ADAM17 also co-localized to these areas (Chapter 5). In conclusion, these results support active EndMT in canine degenerative mitral valves. EndMT could be contributing to the formation of high cellular density myofibroblast transformation which has been postulated to mediate mitral valve degeneration.
Open Access
Cryopreservation of cooled semen for equine intracytoplasmic sperm injection
(Colorado State University. Libraries, 2010) Daigneault, Bradford W., author; Carnevale, Elaine M., advisor; Denniston, David J., committee member; Graham, James K., committee member; Bruemmer, Jason E., committee member
The development of intracytoplasmic sperm injection (ICSI) for use in the horse has resulted in unique needs for semen. Modifications of ways to handle sperm for ICSI-related procedures were investigated through a series of three experiments. In Experiment 1, semen was cooled for 24 h and then frozen to provide a viable population of sperm for ISCI. The objectives of this study were to compare the efficacy of: (1) two extenders for cooling semen prior to freezing and (2) four extenders for freezing cooled semen. Stallion semen was extended in two commercial cooling extenders (CST and INRA96) and cooled for 24 h before freezing in four extenders. Freezing extenders were a modified- French (FR8), Lactose-EDT A (LAC 10), glycerol (G), and glycerol egg yolk (GEY). Extenders were added directly to cooled semen and diluted to a final concentration of 20 x 106 sperm/mL before freezing. After thawing, sperm were evaluated at 0, 45 and 75 min. Cooling extender did not affect sperm motility after freezing. Total motility and progressive motility was similar (p>0.05) for all freezing extenders at O and 45 min, but sperm frozen in glycerol egg yolk (GEY) yielded the highest total and progressively motile sperm at 75 min. Differences in motility over time were not detected except for sperm frozen in glycerol (G) in which total and progressive motility declined from Oto 75 min. For all extenders, some motile sperm were obtained from semen cooled for 24 h and then frozen. In Experiment 2, five media were evaluated for holding sperm to determine which best maintained the motility of stallion sperm over time. The experiment was designed to determine appropriate media in which to handle sperm for ICSI selection procedures. The media compared were: 1) GIVF, 2) FDCM, 3) TALP, 4) Emcare (EM) and 5) Mare Mojo (MM). The only media designed for holding sperm was T ALP; the remaining media were formulated as holding media for oocytes and embryos. Motility of sperm was analyzed every hour for 5 h. No differences in total or progressive motility were detected among groups until 5 h. At 5 h, sperm held in TALP had higher (p<0.05) total and progressive motility than all other groups. Sperm can be held in any of the five media evaluated and remain motile for ICSI. However, T ALP resulted in the best sperm motility over time. Experiment 3 was performed to evaluate a hyaluronic acid (HA) binding assay for the selection of sperm for ICSI. Sperm binding to hyaluronic acid has been characterized for many species. Sperm that bind to HA have been shown to exhibit more normal chromosomal structures and improve fertilization. The objective of Experiment 3 was to determine if fresh and frozen stallion sperm bind to HA when introduced to premanufactured hyaluronic acid binding kits (HBA, Biocoat Inc., Horsham, PA). Fresh semen pooled from two stallions exhibited approximately 15% of sperm bound to HA. Four frozen samples from Experiment 1 were thawed and pooled. Frozen sperm did not bind to the assays. For fresh and frozen samples, sperm displayed some hyperactivation, uncharacteristic circling, and tails bent at the mid-piece. Sperm were observed to agglutinate in congregations of fifty or more sperm. Hyaluronic acid seemed to have an effect on stallion sperm that has not been described for other species. Further investigation of HA binding for selection of sperm for ICSI is warranted.