Mountain Scholar :: Browsing by Author "Bouma, Gerrit, committee member"

Browsing by Author "Bouma, Gerrit, committee member"

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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
Characterizing the cancer preventative properties of rosemary extract
(Colorado State University. Libraries, 2020) Van Steenberg, Julia Elizabeth, author; Kato, Takamitsu, advisor; Legare, Marie, committee member; Bouma, Gerrit, committee member
Flavonoids have been established as antioxidants, with some demonstrating tumor suppression abilities; however, there has been little investigation into their abilities as selectively toxic agents, particularly in cancer cell lines. This investigation was carried out on three different cell lines: V79 a Chinese hamster lung cell, V-C8 a Breast Cancer type 2 (BRCA2) susceptibility protein mutant of V79, and a gene corrected variant of V-C8 containing the human chromosomes with intact BRCA2 gene. The latter two were used as off target cell lines to ensure only the BRCA2 deficient cells were negatively impacted and that intact human BRCA2 would be spared as well. A rosemary extract provided by Gifu University in Japan was tested for any potential cancer prevention abilities in BRCA2 deficient cell lines of breast cancer, due to inhibition of poly (ADP-ribose) polymerase (PARP) in the aforementioned three cell lines. Additional qualities of the rosemary extract were done in vitro using both the extract as a whole and the four known compounds within the extract, including the primary compounds of carnosic acid and carnosol. The four compounds were tested in pure form to better understand what part of the extract was causing any selective toxicity observed. Using mammalian cell culture, we were able to prove selective cell toxicity of BRCA2 deficient cells in comparison to the two off target cell lines. Further investigation revealed that rosemary extract acted as a PARP inhibitor, a quality that is associated with synthetic cell lethality in BRCA2 deficient cells. Finally the antioxidant capability of this mixture was quantified against a known antioxidant, ascorbic acid. Although rosemary extract as a whole does possess some minor antioxidant capabilities, it is capable of some level of hydrogen donation at sub LD50 concentrations in healthy cells. Antioxidants, in particular ascorbic acid, has shown evidence of improving quality of life, survival rate, and decreasing the overall initial risk of developing breast cancer. While there is not presently data in living systems, there is promise that due to selective cell toxicity and antioxidant abilities our rosemary extract will be able to act preventively against cancer developing in BRCA2 deficient cells.
Open Access
Characterizing the microbiota and profiling small non-coding RNAs in the compartments of the equine hindgut
(Colorado State University. Libraries, 2018) Reed, Kailee Janelle, author; Coleman, Stephen J., advisor; Bruemmer, Jason, committee member; Turk, Phillp, committee member; Bouma, Gerrit, committee member
Gastrointestinal homeostasis is a complex relationship that encompasses the host's immune response, physiology, gut structure and the microbes residing within the host. Each one of these has pathways of communication in order to keep the host in a 'healthy state' or homeostasis. While each category has been extensively researched independently, interactions that occur between host and microbe are largely still unknown, especially within the equine species. Because horses are extremely prone to various gastrointestinal diseases, understanding the microbial populations and how the horse might communicate with those populations will provide more insight on equine gut homeostasis. The main objectives were to delineate the microbial structures residing within compartment of the hindgut and to begin to profile gene expression patterns of small RNAs within the same areas. Two different populations of animal subjects were used for the two projects in this thesis: a herd from the University of Kentucky (n=6) and a herd from Colorado State University (n=3). The herd from Kentucky was used for the microbiota data set in order to determine the microbial population structure within the cecum, right ventral colon, right dorsal colon and feces. First, we characterized microbial communities present in each of these anatomical sites and then completed a multivariate model to determine similarities of compartments and compared those to the fecal sample. The population of microorganisms observed in the proximal hindgut appeared similar between cecum and ventral colon, while the dorsal colon and fecal samples appeared to be more alike. Interestingly, there is an anatomical structure separating ventral and dorsal portions of the colon called the pelvic flexure. This could possibly be an indication of the host's contribution of determining the microbial communities in each anatomical region. We also demonstrated that while some microbial signatures from the proximal gut were identified in the feces, the distal gut seemed to be more represented in the fecal sample. The herd from Colorado was used to produce the gene expression data for the second project and the main focus was to profile microRNA (miRNA) expression along the hindgut. These small non-coding RNAs have been identified to be involved in gastrointestinal homeostasis within the intestinal epithelium and are host derived molecules. We demonstrated that each tissue (n=8 for each horse) had unique miRNA expression profiles and these miRNAs identified were used to complete a target pathway analysis which shows possible pathways that could be associated with the biological function of each intestinal site. While each project had different objectives, they are both key players of gastrointestinal homeostasis. For future research, we plan to combine these two areas of study by knowing which miRNA could target specific bacteria residing in the gut, which may further the knowledge of how the host contributes to the population structure of the microbes within their gastrointestinal tracts.
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
Comparison of dose-dependent outcomes in induction of cytogenotoxic responses by novel glucosyl flavonoids
(Colorado State University. Libraries, 2014) Engen, Anya, author; Kato, Takamitsu, advisor; Hanneman, William, advisor; Legare, Marie, committee member; Bouma, Gerrit, committee member
The flavonoids quercetin, and its glucosides isoquercetin and rutin, are phytochemicals commonly consumed in plant-derived foods. They are associated with potential health-promoting effects such as anti-inflammation, anti-viral, anti-carcinogenesis, neuro- and cardio-protection, etc. Semi-synthetic water soluble quercetin glucosides, maltooligosyl isoquercetin (MI), monoglucosyl rutin (MO) and maltooligosyl rutin (MA) were developed to overcome solubility challenges for improved incorporation in food and medicinal applications. Quercetin and its glucosides are known to induce genetic instability and decrease cell proliferation, which are possible mechanisms of anti-carcinogenesis in in vitro and animal studies. Using an in vitro system of Chinese hamster ovary (CHO) cells, this thesis project examined the differences in cytogenotoxic responses induced by natural and novel flavonoids. Treatments with flavonoids at a concentration range of 0.1 and 1,000 ppm induced sister chromatid exchanges (SCE) and micronuclei (MN) in CHO cells. Compared to spontaneous occurrences, significant increases in SCE and MN were observed in both natural and synthetic flavonoid-treated cells in a dose-dependent manner. The natural flavonoids exhibited greater potency than the synthetic compounds, where quercetin was most potent. An analysis of the effects of flavonoids on DNA repair via the poly(ADP ribose) polymerase (PARP) pathway using ELISA showed that all three natural flavonoids along with MI and MO were capable of inhibiting PARP activity by 50%. Quercetin was observed to be the strongest natural inhibitor of PARP. In growth studies using the same treatment dosages as the SCE-MN experiments, colony formation data corroborate those of the growth inhibition studies, in which all flavonoids exerted varying inhibitory effects on cell proliferation. These cytogenetic studies demonstrated that quercetin, isoquercetin and rutin generally exerted more potency than the synthetic compounds, requiring lower doses to achieve efficacy. The ability of both the natural and the synthetic flavonoids to cause genomic instability and impair cell growth may have human health implications for chemoprevention.
Open Access
Cross-kingdom microRNA detection and influence of diet on endogenous equine microRNAs
(Colorado State University. Libraries, 2014) Nulton, Lisa C., author; Bruemmer, Jason E., advisor; Bouma, Gerrit, committee member; Hess, Tanja, committee member
Every year the equine industry spends millions of dollars on research to enhance equine performance, whether it be reproductive or athletic. Equine nutrition is a leading contributor to equine performance and health, with poor nutrition and diet being associated with an ever-increasing incidence of metabolic related disorders. However, there is a lack of clinical markers to diagnose disease processes associated with poor nutrition or nutrient absorption. It has been well established that changes in nutrient intake can have a direct impact on health, but new evidence has emerged indicating that diet can influence endogenous miRNA and consequently gene expression. Also, recent research has identified the presence of exogenous plant miRNA in mammalian serum as a result of food consumption, but the role these miRNAs have on physiology is unclear. miRNAs are small, non-coding RNA molecules that post-transcriptionally regulate mRNA to silence gene expression via translational repression or degradation. We first hypothesized that diet-derived plant miRNAs can be detected in serum and tissue. For this study, twelve horses were randomly assigned to one of three groups (n=4/group) and fed alfalfa hay, extruded corn and alfalfa hay, or rice bran and alfalfa hay. Additionally, multiple tissues were harvested from four necropsied horses for detection of plant miRNAs in tissue. Our results reveal the presence of plant miRNAs in the serum and tissue of horses. Using an mRNA target prediction database, we were able to identify several potential mRNAs that could be regulated by exogenous plant miRNAs. These results suggest that diet-derived plant miRNAs enter into the circulation of the horse and are capable of being taken up by tissues. This data is important for understanding the role diet-derived plant miRNAs may have on equine biological processes. Furthermore, these results suggest a novel way in which plant miRNAs function as not only a nutrient in equine diet but also as a potential regulator of endogenous mRNA. Due to the large influence diet can have on the health of horses, we were then interested in investigating the role of diet on endogenous miRNAs in the horse. We hypothesized that either a rice bran (high-fat) or corn (high-non-structural carbohydrate) diet will alter the relative endogenous miRNA profile in horse serum exosomes. For this study we utilized the same serum samples collected from the feed trial horses in the previous study. Our results discovered 37 differentially expressed (P≤0.05) miRNAs in horses fed the corn diet high in NSC (starch and simple sugars) 23 days after feed treatment began. Pathway analysis of significantly different (P≤0.05) miRNAs indicated gastrointestinal disease, hepatic system disease, connective tissue disorders and inflammatory diseases as the primary predicted networks in which the differentially expressed miRNAs are involved. The horses fed rice bran (high-fat diet) exhibited 11 miRNAs that were differentially expressed on day 23. Cellular development, growth and proliferation were predicted to be the primary networks associated with these differentially expressed miRNAs. Finally, 11 miRNAs were different at day 23 in horses fed alfalfa. Ingenuity pathway analysis (IPA) revealed that these miRNAs are associated with connective tissue disorders and inflammatory response. This data indicates that dietary changes can result in altering endogenous miRNA. This information is important to understanding the influence nutrition has on gene expression and thus on health and disease.
Open Access
Effects of pregnancy status on organic anion transporters and prostaglandin receptors in equine endometrium during maternal recognition of pregnancy
(Colorado State University. Libraries, 2010) Cleys, Ellane R., author; Bruemmer, Jason, advisor; Hansen, Thomas, committee member; Denniston, David, committee member; Bouma, Gerrit, committee member
To view the abstract, please see the full text of the document.
Open Access
Evaluating the equine endometrial transcriptome during maternal recognition of pregnancy
(Colorado State University. Libraries, 2018) Klohonatz, Kristin, author; Bruemmer, Jason, advisor; Coleman, Stephen, committee member; Bouma, Gerrit, committee member; Hess, Ann, committee member; Thomas, Milton, committee member
To view the abstract, please see the full text of the document.
Open Access
Evaluating the genotoxic and cytotoxic effects of synthetic nucleosides in vitro
(Colorado State University. Libraries, 2019) Haskins, Jeremy S., author; Kato, Takamitsu A., advisor; Leary, Del, committee member; Bouma, Gerrit, committee member
The convoluted interplay between various cellular organelles has been a prominent area of study since humans have had the ability to research and explore the microscopic cellular world. Particularly, significant attention has been exercised in the effect that various compounds, pharmaceuticals, drugs, and therapies have on cellular division; particularly cancer cell division. Although documentation is scant, monitoring cell division has been of great interest for years. The utilization and administration of tritiated thymidine, to visualize cellular replication, was unarguably the first strategy to monitor cellular division. However, this method was deemed toxic and cumbersome. 5-bromo-2'-deoxyuridine (BrdU) soon took high notoriety. BrdU, a halogenated pyrimidine, and its structurally related analogs are known to mimic the deoxynucleoside, thymidine, during S-phase of cellular replication. BrdU is incorporated in place of thymidine during S-phase, and its rate of incorporation can be monitored via immunohistochemical antibody detection. However, current literature has demonstrated that BrdU presents a number of complications regarding long-term labeling, cell cycle progression, cellular mutagenicity and cytotoxicity, and unwanted photosensitivity. BrdU's shortcomings were bypassed by the advent of 5-ethynyl-2'-deoxyuridine (EdU) (25). EdU, an additional synthetic analog of thymidine, having a terminal 5'-ethynyl- substituent, instead of thymidine's or BrdU's terminal 5'-methyl- or 5'-bromo- substituent, respectively. EdU has gained popularity as the preferred method in detecting cellular division due to its inherent ability to readily incorporate into newly synthesized DNA. In order to detect and incorporate BrdU into DNA, the process requires the expenditure of an antibody. Binding of the BrdU-antibody tandem to DNA necessitates denaturation of DNA via volatile acid or heat treatment, which presents complications as unqualified and unfaithful base-paring and reannealing. Conversely, EdU incorporation and detection is a fast, simple, and effective method in labeling actively dividing cells. By way of "click" chemistry, EdU is readily introduced and synthesized into new DNA. The latter is accomplished, in part by a small-sized fluorescent azide, qualifying easy access to DNA without considerable steric hindrance. It is expected that successful incorporation of EdU, via "click" chemistry will result in high resolution microscopy analysis. However, current research suggests that implementation of EdU may result in unwanted biological effects. Using an in vitro system, the experimental basis described herein sought to determine the effects that BrdU or EdU had on cell cytotoxicity and genotoxicity when incorporated in DNA. Whilst a vast majority of research experiments use concentrations of said nucleosides' in the range of 10-50 µM, these conditions may induce strong genotoxic and cytotoxic effects inherently higher than the expected background frequency. By treating various DNA repair deficient cells with BrdU or EdU, at concentrations ranging from 1-100 µM, there was a significant increase in the induction of sister chromatid exchanges. Also, with identical concentrations as the latter, the doubling time of particular DNA repair deficient cell lines increased dramatically. To examine the effects of BrdU and EdU on DNA repair, a poly (ADP ribose) polymerase (PARP) ELISA assay was carried out. The PARP assay concluded that BrdU possessed the highest degree of PARP inhibition, with thymidine second, and EdU with the least PARP inhibition. One suggested mechanism by which BrdU is thought to implicate or hinder DNA repair is through its incorporation and modification of DNA repair thus, slower repair kinetics. Hypoxanthine-guanine phosphoribosyltransferase (HPRT) mutation analysis suggests that manufacturers recommended EdU concentration (10µM), result in a significantly higher HPRT mutation frequency, compared to control. In addition to BrdU's SCE-induction capability and HPRT-induction incidence, clinical and radiotherapeutic properties have been examined. CHO cells exposed to 2 and 8 µM BrdU and 4 or 15 Gy X-rays, increase DNA repair duration, increased chromosomal fragmentation, and induce radiosensitization. However, little or no evidence is available in regard to EdU's propensity to affect cell viability. To assess the induction of cellular radiosensitivity and chromosomal aberrations, we investigated CHO and A549 (human lung cancer) cells replicative ability in the presence of three external radiation. An in vitro clonogenic and chromosomal aberration assay, in the presence of UVC-, photon (fluorescent)-, and γ-irradiation and BrdU or EdU, was implemented. Our results support BrdU's ability to decrease cell viability. Although each synthetic analogue presented their own biological contribution, their mechanism is still not fully understood. This study aims to discern any cytotoxic and/or genotoxic effects that EdU or BrdU pose on cell cycle progression, clonogenicity and viability, mutation-induction, chromosomal aberrations, and induction of radiosensitization.
Open Access
Exosomal micrornas as a biological marker of maternal recognition of pregnancy in the mare
(Colorado State University. Libraries, 2012) Cameron, Ashley D., author; Bruemmer, Jason E., advisor; Bouma, Gerrit, committee member; Enns, Mark, committee member
Pregnancy maintenance mandates a signal from the embryo or by-product of the presence of the embryo in the uterus of the mare. This signal must occur between days 12 and 16 post-ovulation. A mobile conceptus prior to day 16 is obligatory for maintenance of pregnancy. However other communication between the embryo and suppression of endometrial prostaglandin F2α has been thoroughly researched yet remains enigmatic. Exosomes, cell secreted vesicles of endocytic origin, have been associated in a wide variety of important physiological cell-to-cell communication. Exosomes are secreted from numerous cell types and have been isolated from a variety of biological samples including; urine, breast milk, serum, plasma, and semen. Exosome presence in peripheral fluids makes them attractive, non-invasive biomarkers. Exosomes contain specific cargo including messengerRNA (mRNA), microRNA (miRNA), and proteins. Exosomes containing miRNA have been implicated in normal and complicated pregnancies as well as signature miRNAs associated with pregnancy status in women. We hypothesized that exosomal miRNA profiles between pregnant and non-pregnant mares would differ due to the changing uterine environment during maternal recognition of pregnancy. To test this hypothesis exosomes were isolated from serum samples in a simple cross-over design before (n=8) during (n=5) and after (n=3) maternal recognition of pregnancy. Pregnancy was confirmed using trans-rectal ultrasonography and embryo collection. Exosome isolation was performed using Exoquick™ (System Biosciences, Inc.) and total RNA was isolated using TriReagent BD (Molecular Research Center, Inc.). The first experiment profiled 380 human miRNAs to identify differences by quantitative Reverse Transcriptase Polymerase Chain Reaction (qRT-PCR) between pregnant and non-pregnant mares after maternal recognition of pregnancy (n=3) on day 16 post-ovulation. For the second experiment, we designed 340 equine miRNAs primers to assess differences with qRT-PCR between pregnancy status. Samples from day 9 and 11 (n=8) and day 13 (n=5) were profiled to represent time points before and during maternal recognition of pregnancy in the mare. Pathway analysis of significantly different (P<0.05) miRNAs was performed employing Diana mirPath software. qRT-PCR identified six miRNAs differentially expressed on day 16 post-ovulation; two only in pregnant, two only in non-pregnant, and two significantly up-regulated in non-pregnant mares. qRT-PCR analysis revealed one, four, and seven miRNAs differentially expressed on days 9, 11, and 13 respectively with only day 9 samples present at higher levels in samples from pregnant mares. miRNAs significantly different in day 16 samples were not found to be significant in earlier time points. Pathway analysis indicated focal adhesion molecules as the primary pathway of miRNAs that were expressed higher in non-pregnant samples on days 11 and 13. Therefore we conclude that exosomal miRNAs from serum do demonstrate differential expression, and can be used as biomarkers for pregnancy and maternal recognition of pregnancy. Predicted targets of these miRNAs may be biologically relevant for determining the signal for maternal recognition of pregnancy in the mare.
Open Access
FADS2 expression modulates effect of dietary polyunsaturated fatty acids on western diet-induced glucose intolerance
(Colorado State University. Libraries, 2017) Linde, Peter, author; Chicco, Adam, advisor; Melby, Christopher, committee member; Bouma, Gerrit, committee member
Fatty Acid Desaturase 2 (FADS2) haplotypes associated with hyperactivity of its gene product, delta-6-desaturase (D6D), are associated with obesity and type-2 diabetes in humans. D6D regulates long-chain polyunsaturated fatty acid (PUFA) biosynthesis and is upregulated in several rodent models of obesity/insulin resistance, but its direct influence on diabetes is unclear. D6D activity might favor pathogenic effects of omega-6 FA linoleic acid (LA) by enhancing production of its product arachidonic acid (AA). Conversely, D6D may promote protective effects of omega-3 FA α-linolenic acid (ALA) by enhancing production of ALA to long-chain PUFAs that displace AA in cell membranes. It is hypothesized that abundant LA found in the modern western diet will be converted to AA promoting an inflammatory phenotype. The present study is to determine the interaction of heterozygous knockout (HET) or transgenic overexpression (TG) of FADS2 in mice fed high fat diets (HFD), as well as the interaction of LA:ALA content in the HFD. Adult male mice with HET (low), wild type (WT; medium), and TG (high) expression of FADS2 were fed HFD (45% w/w) containing 8% PUFA supplied by a balanced mix of LA and ALA (1:1), LA-rich (41:1), or ALA-rich (1:4) for 16 weeks. Glucose intolerance developed in WT mice, with no difference between diets. In HET mice, glucose intolerance was attenuated but this protection was removed by ALA rich diet. TG mice developed more glucose intolerance than WT. TG mice fed high LA diets were more glucose tolerant than high ALA and mixed diets. In conclusion, FADS2 expression modulates metabolic responses to high fat feeding. HET provides some protection against glucose intolerance, except when given an ALA rich diet. Transgenic overexpression increases glucose intolerance while a high LA diet attenuates this effect. This is inconsistent with current hypotheses that AA production from LA increases metabolic risk.
Open Access
Hypothalamic concentration of kisspeptin and GnRH during breeding season (BS) and non breeding season (NBS) in sheep
(Colorado State University. Libraries, 2016) Urias Castro, Christian, author; Nett, Terry, advisor; Clay, Colin, advisor; Han, Hyng Chul, committee member; Bouma, Gerrit, committee member
The kisspeptin system has emerged as an important regulator of mammalian reproduction. In ewes, kisspeptin neurons are located in specific hypothalamic regions such as the preoptic area (POA) and medio-basal hypothalamus (MBH) which include the arcuate nucleus (ARC). A specific radioimmunoassay (RIA) for the quantification of hypothalamic kisspeptin was developed to test the hypothesis that estradiol decreases the production of kisspeptin during the NBS in the MBH in addition to other forebrain areas that harbor kisspeptin neurons and/or axons such as the POA, the anterior hypothalamic area (AHA), and the median eminence (ME). The kisspeptin RIA results indicated that the concentrations of kisspeptin per milligram of tissue were decreased during NBS in the MBH and the POA with a tendency for lower kisspeptin concentrations observed in the AHA. Likewise, the total content of kisspeptin was decreased in the MBH and POA during the NBS, with a similar tendency for lower content of kisspeptin observed in the AHA during the NBS. Supporting the notion that kisspeptin modulates secretion of GnRH at the level of the ME, a positive correlation between these neuropeptides was observed during the BS in this region. It may be, that kisspeptin neurons are relevant for the seasonal regulation of GnRH and LH secretion exerted by estradiol, since the GnRH neurons do not express estrogen receptor alpha (ERα) which is the relevant ER subtype for the regulation of the hypothalamic pituitary gonadal axis (GnRH/LH pulsatility). We investigated if the negative feedback exerted by estradiol during the NBS, promoting a decrease in the concentrations of kisspeptin in the ARC, can be blocked by the intracerebral ventricular (ICV) administration of an estradiol antagonist (ICI) to promote an increase in LH pulsatility. As expected, in ewes that received the ICI treatment an increase in the average number of LH pulses was observed. The increased frequency in LH pulsatility was probably a consequence of eliminating estradiol inhibitory actions over ARC kisspeptin neurons which send axonal projections to the ME and promote the release of GnRH, and thus LH. Interestingly, the ME is a circumventricular organ (CVO) located outside of the blood brain barrier (BBB). Ovariectomized ewes were immunized against kisspeptin and antiserum to kisspeptin generated. The antibodies to kisspeptin were intended to eliminate kisspeptin release from the ME and consequently block kisspeptin input to GnRH axon terminals. The blockade of kisspeptin input to GnRH axon terminals was intended to inhibit the release of GnRH and hence LH. When compared to controls, ewes immunized against kisspeptin tended to have lower average and basal secretion of LH. The lack of significant decrease observed in immunized ewes suggests that higher titers to kisspeptin could be needed to fully suppress GnRH and hence LH pulsatility. Still, the tendency for lower levels of LH observed in immunized ewes suggest that kisspeptin release from the ME is relevant for the modulation of the pulsatile secretion of GnRH and thus LH. Likewise, delayed onset of the preovulatory like surge of LH in immunized ewes suggests a partial inhibition of the massive release of kisspeptin/GnRH was obtained during the start of the surge. However the possibility that the preovulatory surge of kisspeptin/GnRH is also regulated inside the BBB or that kisspeptin independent or indirect mechanisms play an important role in the generation of the GnRH/LH surge in ewes cannot be ruled out.
Open Access
Immunoreactivity of anti pZP antibodies from the serum of SpayVac vaccinated mares to equine zona protein
(Colorado State University. Libraries, 2014) Mask, Tracy Ann, author; Bruemmer, Jason, advisor; Bouma, Gerrit, committee member; Kane, Albert, committee member; Ransom, Jason, committee member
Immunocontraception with the porcine zona pellucida (pZP) antigen is a well-published means of wild horse contraception. There are three pZP vaccines currently proposed for use in horses, Zonastat-H®, PZP-22, and SpayVac®. Despite abundant research concerning the safety and contraceptive efficacy of pZP vaccines in numerous species, the contraceptive mechanisms of the pZP antigen remain unclear and have not been investigated thoroughly for SpayVac. We investigated the immunoreactivity of anti pZP antibodies from the serum of SpayVac vaccinated mares to equine zona protein using Western blot and immunohistochemical techniques. These techniques were first applied using a bovine model because bovine oocytes are more easily obtained in large quantities relative to equine oocytes. Once the procedure was validated, equine samples were utilized. Western blot analysis revealed immunoreactivity of anti pZP antibodies that were produced in response to SpayVac vaccination to protein isolated from mature equine oocytes, equine zona pellucidae, equine follicular tissue, and equine ovarian stromal tissue. Immunohistochemical analysis identified the location of binding of anti pZP antibodies to the zona pellucida of mature oocytes isolated from Graafian follicles as well as the zona pellucida of immature oocytes in ovarian tissue. Western blot and immunohistochemical analyses also indicate high specificity of anti pZP antibodies for equine zona protein and predominant affinity for zona protein 3. Collectively, results suggest a model where anti pZP antibodies produced in response to SpayVac vaccination are immunoreactive to equine zona protein in vitro. If available in the follicular fluid and able to permeate the ovary following SpayVac vaccination, anti pZP antibodies may act on not only mature oocytes, but also oocytes of growing follicles in vivo. The results of this study lend insight into the infertility observed following SpayVac vaccination, and may also help explain the long-term ovarian effects following pZP vaccination reported by other studies.
Open Access
In vitro capacitation of stallion spermatozoa
(Colorado State University. Libraries, 2010) Spizziri, Beth Erin, author; Graham, James K., advisor; Bouma, Gerrit, committee member; Bruemmer, Jason, committee member; Seidel, George, committee member
Equine in vitro fertilization has resulted in limited success, and progress is hindered due to a lack of understanding the molecular and biochemical events involved in stallion sperm capacitation. As no single test exists to determine if a stallion sperm is capacitated, individual events of capacitation can be monitored to determine if treatments can induce in vitro changes involved in sperm capacitation. In addition, the limited availability of equine oocytes for experimentation has led to the use of heterologous oocyte assays to determine if various sperm treatments to induce sperm capacitation can result in these sperm fertilizing oocytes in vitro. In experiment 1, sperm plasma membrane cholesterol content of sperm was examined after treatment with capacitation inducing agents. Samples treated with methyl-~-cyclodextrin (MBC) exhibited lower (p<0.05) cholesterol content after 3 h incubation (16 μg/108 sperm) than control sperm at Oh (22 μg/108 sperm). Samples preloaded with cholesterol, after incubation with cholesterol-loaded-cyclodextrin (CLC), contained more cholesterol than control sperm (p<0.05). The second experiment was designed to determine if that protein tyrosine phosphorylation, a component of sperm capacitation, occurs under in vitro conditions. Sperm were capacitated in vitro in Modified Whitten's (MW) medium alone or with dilauroylphosphatidylcholine (PC 12; 40 μm), calcium ionophore A23187 (2 μm), or MBC ( 1 μm) for 0, 30, 90, and 180 min, and the amount of protein tyrosine phosphorylaton was assessed. PC 12-treated sperm exhibited the highest amount of protein tyrosine phosphorylation at time Oh. Control sperm exhibited the highest amount of protein tyrosine phosphorylation following a 3 h incubation. Tyrosine phosphorylation was negligible with MBC and calcium ionophore A23187 treatments. The third experiment was designed to adapt detection of protein tyrosine phosphorylation detection of stallion spermatozoa to flow cytometery. When sperm were incubated with nothing (control), PC12 (40 μm), MBC (1 μm), or calcium ionophore A23187 (2 μm) for 0, 10, 20, 30, 45, 60, 90, 120, or 180 min, and then fixed, permeabilized and incubated with a fluorescein isothiocyante (FITC)-labeled monoclonal antibody to phosphorylated protein, no consistent results were obtained using flow cytometry. Experiment 4 was designed to detect and classify sperm as hyperactive using novel software, minimum square binding ratio (MSBR). Control and CLC-treated stallion spermatozoa were incubated in MW or MW plus 5 mM procaine and then capacitated with PC12 (40 μm) or MBC (1 μm) for 15 min or 3 h. Sperm motility parameters were assessed using both the standard computer assisted sperm analysis (CASA) and the MSBR classification. Procaine treatment only, induced hyperactive motility in CLC-treated PC 12-capacitated sperm after 3 h incubation when using standard CASA analysis. MBC- treated spermatozoa exhibited the greatest changes in sperm motion parameters after 3 h. However, MSBR analysis indicated that neither PC 12 nor MBC-treated sperm were hyperactive at either time point, although all procaine supplemented samples had higher percentages of hyperactive sperm than control sperm (p < 0.05). Experiment 5 was designed to determine the effects of procaine supplementation on the acrosome reaction of stallion sperm treated with PC 12 or MBC. Stallion spermatozoa incubated in MW or MW plus 5 mM procaine were treated with nothing (control), PC12 (40 μm), or MBC (1 μm) for either 15 min or 3 h. The samples were then dual stained with FITC-PNA and propidium iodide (PI) and assessed by· flow cytometry. While PC 12 and MBC induced acrosome reactions in sperm, procaine had no effect on inducing acrosome reactions in stallion spermatozoa. Fertilization of bovine oocytes in vitro, with PC 12-(15 μM) treated stallion sperm resulted in higher cleavage rates (25% ± 3) than untreated sperm (9 % ± 4; p < 0.05). The ability of stallion spermatozoa to fertilize bovine oocytes following zona pellucida laser disruption was then addressed. Bovine oocytes given laser treatment exhibited lower cleavage rate when untreated or PC 12-treated sperm were co-incubated with them (3 and 4 % ± 2; p < 0.05) lhan zona intact oocytes inseminated with similarly treated sperm (9 vs. 30% ± 2; p < 0.05).
Open Access
Mechanism-based thresholds of toxicological concern (TTC) for developmental and reproductive toxicity of anticancer compounds
(Colorado State University. Libraries, 2015) Stanard, Brad, author; Hanneman, William, advisor; Legare, Marie, committee member; Bouma, Gerrit, committee member; Schaeffer, Joshua, committee member
To view the abstract, please see the full text of the document.
Open Access
Preimplantation genetic diagnosis of equine embryos
(Colorado State University. Libraries, 2010) Cullingford, Erika L., author; Seidel, George Jr., advisor; McCue, Patrick, advisor; Ahola, Jason K., committee member; Bouma, Gerrit, committee member
In horses, determination of certain genetic traits/alleles in embryos before embryo transfer would be advantageous due to the costs of resulting pregnancies. An attractive option is preimplantation genetic diagnosis (PGD), but to date few biopsied equine embryos have resulted in pregnancies. In the current experiment, 37 embryos ranging from 160 - 575 μm in diameter were biopsied. To obtain embryos, donor mares were monitored using transrectal ultrasonography. When a follicle > 35 mm in diameter was observed, 2,500 IU hCG or 1.5 mg deslorelin acetate was administered, and mares were inseminated daily until ovulation was detected. Embryos were recovered nonsurgically on days 6.5 – 7 (day 0 = ovulation). Trophoblast biopsies were collected in a 30 μl droplet of Syngro Holding Medium (Bioniche, Belleville, ON) using a piezo drill and beveled injection pipette. After removal of the embryo, the droplet containing the biopsied cells was moved into an Eppendorf tube and centrifuged. Supernatant was removed leaving ~5 μl sample, which was snap frozen for later genetic testing. Fifteen biopsied embryos were immediately transferred nonsurgically into uteri of synchronized recipients. Day 16 pregnancy rate for embryos ≤ 300 μm was 75.0% (6 of 8; 175 – 240 μm), which was not significantly different from control embryos of the same size (77.3%; 17 of 22). For embryos > 300 μm, day 16 pregnancy rate was 28.6% (2 of 7; 320 and 400 μm), which was not significantly different from control embryos of the same size (62.5%; 10 of 16). Additionally, 22 embryos (150 - 440 μm) were vitrified by standard procedures after biopsying and later warmed and transferred directly. No embryos > 300 μm (n = 3) became pregnancies after vitrification. Day 16 pregnancy rate for ≤ 300 μm was 47.4% (9 of 19; 150 – 225 μm), which was significantly different (p < 0.05) from direct transfer and control embryos of the same size (75.0% and 77.3%, respectively). Three of these pregnancies (150 - 200 μm) resulted in the formation of empty trophoblastic vesicles by 25 d. All pregnancies were terminated on or after 25 d to collect embryos for further genetic testing. For preimplantation genetic testing, a duplex nested polymerase chain reaction (PCR) was developed for amplification of the DNA from the biopsied cells using primers for sex chromosome-linked zinc finger protein genes (ZFx/ZFy; 445 bp), and 2 pairs of primers for equine-specific sex-determining region on the Y-chromosome (SRY; 217 bp, 121 bp). Experiments on XX and XY genomic DNA from white blood cells revealed accurate genetic testing on as little as ~9 pg DNA, which equals ~1 cell. Sex determination on biopsied material occurred for 30% of samples, one of which was confirmed from a placental sample. Low PGD results indicate either lack of sensitivity of the test, or more likely the loss of cells during the steps of transfering the biopsied cells to Eppendorf tubes. We concluded that biopsy collection, preimplantation genetic diagnosis, and direct transfer can be performed on equine embryos without compromising pregnancy rates when performed on embryos ≤ 300 μm. Vitrification lowered pregnancy rates of biopsied embryos (p < 0.05). Continued effort in improving genetic tests and in vitrifying equine embryos, especially those > 300 μm, is warranted.
Open Access
Profiling equine endometrial gene expression during maternal recognition of pregnancy
(Colorado State University. Libraries, 2013) Klohonatz, Kristin M., author; Bruemmer, Jason E., advisor; Bouma, Gerrit, committee member; Thomas, Milton, committee member
In order to maintain a pregnancy in the mare the presence of a conceptus in the uterus must be recognized by the endometrium. This is known as maternal recognition of pregnancy (MRP) and is required to prevent the secretion of prostaglandin F2α (PGF), starting on day 14 post-ovulation, from the endometrium into circulation. The secretion of PGF initiates luteolysis of the corpus luteum, which is secreting progesterone, the hormone needed to maintain a pregnancy. However, little is known about maternal recognition of pregnancy in the mare. It is critical that the embryo is mobile throughout the entire uterine lumen to signal maternal recognition of pregnancy between days 12-14. The embryo ceases mobility on day 16 by fixing at the base of one of the uterine horns, independent of the side of ovulation. Previously, an equine specific microarray analysis was performed on days 14, 16, and 18 post-ovulation comparing endometrial gene expression between pregnant and non-pregnant mares. From this analysis, ten genes: juxtaposed with another zinc finger protein 1-like (JAZF1), secretory phospholipase A2 (sPLA2), S100 calcium binding protein G (S100G), estrogen receptor 1 (ESR1), solute carrier family 36 (proton/amino acid symporter), member 2 (SLC36A2), methyltransferase-like protein 7A-like (METTL7A), retinaldehyde dehydrogenase 1-like (RALDH1), eukaryotic translation initiation factor 2-alpha kinase 3 (EIF2AK3), dickkopf 1 homolog (DKK1), and adrenomedullin (ADM), were identified as having consistently higher or lower expression levels in the endometrium of pregnant mares at all three time points. The goal of this study was to confirm and expand upon the results of the microarray on days 14, 16, and 18, by real time PCR (RT-PCR), and to evaluate differential gene expression on day 12. We hypothesized that the expression of the aforementioned ten genes will be the same on day 12 endometrium from pregnant mares as days 14, 16, and 18 endometrium from pregnant mares because day 12 is the start of maternal recognition of pregnancy. To test this hypothesis, 12 normally cycling mares were utilized in a crossover design. Each mare was assigned to a random collection day (day 12, 14, 16, or 18 post-ovulation) and provided endometrial samples from a pregnant cycle and then a non-pregnant (non-mated) cycle (n=3 per day). Endometrial biopsy samples were snap frozen and stored until total RNA was isolated for RT-PCR. This analysis was consistent with the microarray results for days 14, 16, and 18. On day 12, 6 of the 10 differentially expressed genes had the same pattern of expression as day 14, but 4 of the genes had opposite expression levels on day 12. Endometrial samples were then collected on day 13 post-ovulation (n=3) and processed for protein isolation and immunohistochemical analysis. The specificity of rabbit polyclonal antibodies for sPLA2 and DKK1 for equine endometrium were confirmed by Western Blot analysis. Upon conformation of antibody specificity, immunohistochemistry was used to determine the localization of sPLA2, DKK1, and ESR1. sPLA2 was localized to the endometrial epithelium and glandular epithelium in the endometrium from both pregnant and non-pregnant mares. DKK1 showed a localization difference between endometrial samples from pregnant and non-pregnant mares. In the endometrium from the pregnant mare, DKK1 was localized to the endometrial epithelium and the glandular cells, and in the endometrium from the non-pregnant mare DKK1 was localized throughout the glandular region, but not in the endometrial epithelium. ESR1 also showed differential localization based upon pregnancy status. In the endometrium from the pregnant mare, ESR1 was located in the basal glandular region and not close to the lumen or in the endometrial epithelium. In endometrium from non-pregnant mares it was located throughout the entire glandular region and in the endometrial epithelium. This experiment identified the expression patterns of ten genes, previously identified from a microarray analysis, on days 12, 14, 16, and 18 post-ovulation in the endometrium from pregnant and non-pregnant mares. The expression patterns on days 14, 16, and 18 were consistent across each day. Day 12 revealed mixed results for the expression patterns of these genes, indicating that they were undergoing transcriptional regulation based upon the presence or absence of a mobile conceptus. By determining what signal causes these genes to be higher or lower expressed in the endometrium of pregnant mares, it may lead to the identity of the signal for maternal recognition of pregnancy in the mare.
Open Access
Regulatory microRNA delivered to stallion spermatozoa during epididymal maturation
(Colorado State University. Libraries, 2016) Twenter, Hannah, author; Bruemmer, Jason, advisor; Bass, Luke, committee member; Bouma, Gerrit, committee member; Coleman, Stephen, committee member
Stallion spermatozoa are produced in the seminiferous tubules of the testis. After spermatogenesis, spermatozoa migrate through the seminiferous tubules to the rete testis then efferent ducts which converge to form a single duct within the caput of the epididymis. The epididymis is a convoluted tubule with region-specific luminal profiles. The epididymis consists of three commonly descried regions; caput, corpus, and cauda. Each region performs distinct functions in epididymal maturation of spermatozoa. The caput is responsible for concentration of spermatozoa by reabsorbing excess fluid from the epididymal lumen. The corpus is where majority of maturation occurs as spermatozoa gain motility and shed their cytoplasmic droplet. The cauda primarily serves as a storage site for the spermatozoa until ejaculation or spontaneous emission. All regions of the epididymis are lined with multiple epithelial cell types, each with different functions to provide the ideal luminal environment for the maturation. These epithelial cells also create apical blebs containing small, membranous vesicles named epididymosomes. The apical blebs are released from the apical surface via apocrine secretion and will disintegrate in the lumen, releasing epididymosomes. Epididymosomes transport proteins from the epithelium to the spermatozoa in the lumen. They also contain microRNAs (miRNAs). MicroRNAs are small, non-coding post-transcriptional regulators of mRNAs and can interfere with mRNA transcription through translational repression or degradation. We hypothesize that epididymosomes also transfer miRNA from the epididymal epithelium to spermatozoa. Quantitative real-time polymerase chain reaction was used to determine the miRNA profile of epididymal tissue from the caput and cauda, epididymal spermtozoa from the caput and cauda, and epididymosomes from the caput, proximal corpus, distal corpus, and cauda. Our focus turned to 33 newly-acquired miRNAs with expression specific to the spermatozoa located within the cauda as these are fully mature cells. Comparing the miRNAs present in each sample, 11 miRNAs had a distinct path from epididymal tissue to epididymosomes to spermatozoa, suggesting that miRNAs are transported to spermatozoa from the epididymal epithelium via epididymosomes. Pathway analysis was performed using DIANA tools on the 33 miRNA with expression on in caudal spermatozoa using an a posteriori method with predicted pathways considered significant with P≤0.05. Fifty-one predicted pathways were statistically significant. Some of those predicted pathways suggest a role in cell motility and viability, while others influenced factors in the oocyte or embryo. By developing a better understanding of the mechanisms behind the processes that regulate sperm maturation as well as the roles in embryogenesis better diagnostics for infertility in the horse may be generated.
Open Access
Repeated sequences encoding Cys2His2 zinc finger motifs influence mRNA polyadenylation and localization
(Colorado State University. Libraries, 2017) Jalkanen, Aimee L., author; Wilusz, Carol, advisor; Wilusz, Jeffrey, advisor; Bailey, Susan, committee member; Bouma, Gerrit, committee member; Thamm, Douglas, committee member
The Cysteine2 Histidine2 zinc finger (C2H2-ZNF) proteins are a vast family with over 700 members in primates, many of which are transcription factors with important roles in development, differentiation, cell cycle progression, and tumor suppression. Due to the sheer number of C2H2-ZNF proteins and their roles in modulating expression of other genes, any mechanism for coordinating their expression could have wide-ranging impacts on cell function and phenotype. Previously, a large subset of C2H2-ZNF transcripts were determined to have significant populations with short poly(A) tails. Here, we show that multiple C2H2-ZNF mRNAs accumulate with very short or undetectable poly(A) tails, even when newly transcribed. Furthermore, these C2H2-ZNF mRNAs are restricted to the nucleus. Reporter mRNAs with sequences from the ZNF12 open reading frame (ORF) and/or the 3' untranslated region (3' UTR) have short poly(A) tails and are retained in the nucleus. Deletion analysis suggests that repeated sequence elements in the ZNF12 mRNA that code for zinc finger protein motifs are important in controlling both poly(A) tail length and nuclear localization. Remnants of C2H2-ZNF motif sequences found in the ZNF12 3' UTR are also able to confer short poly(A) tails and nuclear retention. Finally, we use RNA-fluorescence in situ hybridization (RNA-FISH) to reveal that ZNF12 reporter transcripts are found in foci within the nucleus that could represent sites for storage or processing. Overall, our findings suggest repeated sequence elements encoding C2H2-ZNF protein motifs play a dual role as regulatory elements that may coordinate expression of the C2H2-ZNF protein family by controlling post-transcriptional events.
Open Access
Sc-26196, a delta-6 desaturase inhibitor, normalizes glucose tolerance in ob/ob mice
(Colorado State University. Libraries, 2012) Routh, Melissa Anne, author; Chicco, Adam J., advisor; Bouma, Gerrit, committee member; Frye, Melinda, committee member; Paul, Laybourn, committee member
The incidence of diabetes has reached epidemic levels worldwide, and while researchers know more about the phenomenon than ever, an effective treatment still remains elusive. Recently, chronic low-grade inflammation has been shown to play a key role in insulin resistance and diabetes. The purpose of this study was to demonstrate a role for the delta-6 desaturase (D6D) in diabetes, and furthermore to elucidate the potential mechanism by which inhibition of D6D by sc-26196 treatment could restore glucose tolerance in obese insulin resistant ob/ob mice. Treatment of 4-month-old male ob/ob mice with the selective D6D inhibitor sc-26196 (SC, 100 mg/kg/d for 4 weeks) improved response to an acute glucose challenge (1 mg/g BW i.p.), as indicated by lower peak (146% vs. 219% of fasting) and final (85% vs. 180% of fasting 2 hours-post bolus) blood glucose levels versus untreated ob/ob mice (p<0.01). Increased hepatic macrophage infiltration in addition to increased phosphorylation of JNK proteins and inhibitory IRS phosphorylation in untreated ob mice was attenuated by sc-26196 treatment, which suggests that D6D inhibition improves glucose tolerance by decreasing inflammation and restoring insulin signaling by way of the IRS/PI3K pathway. This study demonstrates the effectiveness of sc-26196 treatment for glucose intolerance in ob/ob mice and results warrant future studies for use of sc-26196 in the clinical treatment of insulin resistance and diabetes.