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- ItemOpen AccessCloning 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 memberImmunization 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.
- ItemOpen AccessBehavioral 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 memberAffective 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.
- ItemOpen AccessMembrane organization of luteinizing hormone receptors during signal transduction(Colorado State University. Libraries, 2010) Wolf-Ringwall, Amber L., author; Roess, Deborah, advisor; Miller, Charles, committee member; Graham, James, committee member; Barisas, B. George, committee memberMechanisms involved in signal transduction by luteinizing hormone (LH) receptors are important for regulating key events in mammalian reproduction, such as ovulation, sex hormone production and maintenance of pregnancy. Studying the organization of LH receptors in the plasma membrane during hormone-mediated signaling provides insights into the protein interactions needed for important physiological responses. We used biochemical and biophysical methods to examine the role of the plasma membrane in contributing to LH receptor desensitization. Using single particle tracking and sucrose gradient ultracentrifugation, we determined that individual human LH receptors are confined in small membrane compartments and localize in membrane rafts for several hours following desensitization. These receptors do not demonstrate signaling via cyclic adenosine monophosphate (cAMP) while they are confined, suggesting that the microenvironment within these compartments may be different for desensitized versus actively signaling receptors. We also investigated self-association of human LH receptors using homotransfer fluorescence resonance energy transfer (homo-FRET) and fluorescence correlation spectroscopy. We determined that human LH receptors self-associate following desensitization and in response to increasing concentrations of human chorionic gonadotropin (hCG). LH receptors demonstrated the highest degree of aggregation in response to saturating concentrations of 100 nM hCG. Using single particle tracking, we examined whether native LH receptors expressed on KGN human granulosa-like tumor cells, or M l7 human neuroblastoma cells, become confined in small membrane compartments in response to hormone binding. We found that confinement of native LH receptors in small plasma membrane compartments depended on hCG concentration. With increasing concentrations of hCG, more LH receptors became confined in small membrane compartments with an average diameter of less than 100 nm. These receptors also exhibited slower rates of lateral diffusion. We reported the movement of non-functional hormone-receptors, labeled with deglycosylated hCG, into small membrane compartments in response to hCG treatment that saturated other available LH receptors on the membrane. This finding suggests that interactions between functional and non-functional LH receptors may occur in membrane microdomains during signal transduction.
- ItemOpen AccessEvaluation of kisspeptin in the mare(Colorado State University. Libraries, 2010) Magee, Christianne, author; Clay, Colin M., advisor; Tobet, Stuart A., committee member; Nett, Torrance M., committee member; Goodrich, Laurie R., committee member; Duval, Dawn L., committee memberIdentified in 2003 for their role in reproductive physiology, kisspeptins have become major players in the field of reproductive neuroendocrinology. With the ability to act as a central regulator for the onset of reproductive function in prepubertal and seasonal animals, the possibility that kisspeptin signaling could be used to modify seasonal reproductive function in the horse held great promise. My hypothesis was that kisspeptin, acting via a hypothalamic signaling mechanism to stimulate the GnRH neuron, could initiate reproductive function in the horse. The initial objectives of these studies were to (1) establish biological and physiological evidence for kisspeptin signaling in the hypothalamus of the mare, (2) demonstrate peripheral administration of kisspeptin could elicit a rise in serum luteinizing hormone (LH) concentrations in the diestrous mare, and (3) demonstrate that kisspeptin, acting via LH, could induce ovulation in the estrous mare. The diestrous mare has kisspeptin immunoreactive neurons in the hypothalamus that are in close proximity to Gonadotropin Releasing Hormone (GnRH) neurons. At the time of these initial studies, the equine sequence for the kisspeptin decapeptide (Kp-10) was not yet available; therefore, I utilized the rodent Kp-10 (rKp-10, YNWNSFGLRY-NH2). Even though I was using a heterologous ligand, the diestrous mare was responsive to administration of rKp-10 (0.5 and 1.0 mg) such that there was a short (< 1 hour), but significant (2-fold) rise in circulating levels of LH and follicle stimulating hormone (FSH) after kisspeptin administration. I was also able to establish a threshold dose for kisspeptin responsiveness in the diestrous mare as there was no change in serum gonadotropin levels following a 1.0 μg dose of rKp-10. In the estrous mare, a single injection of 1.0 mg rKp-10 IV was unable to induce ovulation (173), presumably due to the short duration of the kisspeptin induced LH surge as compared to the 3-5 day endogenous peri-ovulatory LH surge (306). To understand the dynamic of kisspeptin signaling to the hypothalamus and the anterior pituitary gland, I sought to determine the effect of treating mares with repeated injection of kisspeptide in diestrus and estrus. If the future of kisspeptin in the horse involves the use of modified agonists or antagonists, it will be necessary to understand how the mare responds to repeated stimulation with kisspeptin. Before beginning these studies, the equine sequence for Kp-I0 (eKp-10, YRWNSFGLRY-NH2) had become available. Therefore, I used the homologous peptide for these studies. By treating mares with eKp-10 (0.5 mg IV every 4 hours), the hypothalamus and pituitary gland were repeatedly stimulated to elicit a GnRH and gonadotropin response. Repeated administration of kisspeptin in the diestrous mare is not able to sustain a 2-fold increase in LH concentration for 48 hours following the initial injection. Interestingly, kisspeptin caused a decrease in basal LH, but not FSH levels, indicating a decrease in LH synthesis or secretion via a pituitary effect. Although the mare does not exhibit a change in peripheral LH levels following eKp-10 if a GnRH antagonist (e.g. Antide) has been administered, I sought some evidence for kisspeptin signaling directly to the anterior pituitary. To support the idea of a direct pituitary effect of kisspeptin, I challenged primary pituitary cells in culture with 100 nM GnRH and 100 nM of eKp-10. Surprisingly, I identified three populations of cells that respond with a change in intracellular calcium concentration and grouped them as follows: cells that responded to (1) both GnRH and eKp-l0, (2) only GnRH, or (3) only eKp-10. The identification of gonadotrope and non-gonadotrope kisspeptin responsive pituitary cells is the first evidence for a direct mechanism for kisspeptin signaling at the level of the equine pituitary gland. In the estrous mare, repeated administration of eKp-10 is not able to shorten the interval to ovulation whether it is administered before or after the development of a dominant follicle. Another surprising finding was a significant decrease in sexual receptivity in mares within 48 hours of beginning treatment with kisspeptin, which is likely due to a decrease in estradiol synthesis by the maturing follicle. Given the lack of ovulation induction in the estrous mare and the changes in behavioral receptivity, I do not recommend the use of kisspeptin as an ovulation inducing agent at this time. However, there was no decrease in basal LH levels in the estrous mares. Thus, kisspeptin may be signaling via different mechanisms in the estrous vs. diestrous mare. In summary, these studies do provide evidence for kisspeptin signaling in the mare, but they reveal that the signaling mechanism in the horse may be more complex than my original hypothesis of a simple, linear process that is working only through the GnRH neuron.
- ItemOpen AccessCharacterization 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 memberAs 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.
- ItemOpen AccessPreimplantation 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 memberIn 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.
- ItemOpen AccessInteraction of [VO₂(MA)₂]⁻ with model membranes: relevance to insulin enhancing effects of BMOV and its oxidized form(Colorado State University. Libraries, 2010) Schoeberl, Samantha Kay, author; Roess, Deborah, advisor; Graham, James, committee member; Crans, Debbie, committee memberAnti-diabetic vanadium-containing compounds and salts reportedly have effects on the overall organization of the cytoskeleton and the plasma membrane of cells. For ligand-mediated signaling, appropriate cytoskeletal and membrane lipid organization is essential for down-stream signaling to occur. A number of vanadium-containing compounds and salts are of interest because of their effects on these important cellular structures. Promising results in regulating diabetic symptoms such as glucose and lipid metabolism have been shown to result from the use of various anti-diabetic vanadium drugs. Their effects on the cytoskeleton and plasma membrane are reviewed Chapter I. Due to the importance of membrane interactions of vanadium-containing compounds with insulin-enhancing activity in ligand-mediated signaling, two simple membrane model systems were used to investigate the interactions of an oxidized metabolite of bis(maltolato)oxovanadium(IV) with model lipid interfaces. Studies were carried out using multinuclear NMR spectroscopy with a focus on 1H NMR techniques. In Chapter II we demonstrate that there were slight changes in 1H NMR spectra indicating that this BMOV metabolite was able to penetrate the lipid interface. These findings are important in understanding the pharmacologic mechanism of action of this anti-diabetic compound in cells and intact animals.
- ItemOpen AccessCryopreservation 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 memberThe 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.
- ItemOpen AccessAssociation 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 memberIncreasing 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.
- ItemOpen AccessRegulation of trophoblast stem cell maintenance and differentiation by LIN28 and AP-2γ(Colorado State University. Libraries, 2010) Fromme, Brittany A., author; Winger, Quinton A., advisor; Bouma, Gerrit J., advisor; Bailey, Susan M., committee member; Anthony, Russell V., committee memberThe placenta is a unique organ essential for survival of the fetus in all eutherian mammals. Failure to develop a normal placenta in humans can lead to diseases, such as pre-eclampsia, with high morbidity and mortality for both the mother and the fetus. These diseases are thought to be caused by abnormal proliferation and differentiation of cells in the placenta. A mouse trophoblast stem (TS) cell culture system is a useful tool in studying TS cell proliferation and differentiation into trophoblast giant cells (TGCs). TS cells cultured in proliferative media (70% conditioned media, 30% TS media, FGF4, and heparin sulfate) will remain proliferative, and TS cells cultured under differentiation media (100% TS media) will differentiate into TGCs. LIN28 is a protein that regulates mRNAs and miRNAs, and is abundantly expressed in many undifferentiated tissues. AP- 2y has been shown to be essential for TS cell maintenance and TGC formation. AP-2y null mutants display embryonic lethality at E7.5 due to a severely disrupted extraembryonic portion of the embryo. In TS cells, AP-2y has been shown to bind to the promoter region of Lin28. This study investigates the hypothesis that Liti28 and Ap-2y are necessary regulators of trophoblast stem cell maintenance and differentiation into TGCs. This study shows the pluripotency genes, Lin28, Sox2, and NrObl, to be differentially expressed in proliferating TS cells and differentiated TGCs. MiRNAs can be used as markers for proliferation or differentiation. 28 significantly different miRNAs were detected between TS cells and TGCs, 18 up-regulated in TGCs and 9 downregulated in TGCs. Expression of the miR-290 family, initially thought to be ES cell specific, was detected in proliferating TS cells suggesting TS cells have similar miRNA mediated regulation of proliferation compared to ES cells. The Let-7 family of miRNAs was found to be up-regulated in differentiated TGCs. The Let-7 family has been shown to be regulated by LIN28, where LIN28 prevents accumulation of mature Let-7 miRNAs. In this study Lin28 was highly expressed in proliferating cells and the Let-7’s are upregulated in differentiated TGCs. Lin28 function in TS cells was assessed by knocking down Lin28 using shRNA lentiviral technology. Lin28 knockdown TS cells were used to observe results of knockdown. We obtained a 78% reduction of Lin28 mRNA, but found that loss of Lin28 in TS cells did not affect morphology, proliferation or differentiation. AP-2y null TS cells grown in culture fail to differentiate morphologically into TGCs. Lin28, Sox2, and NrObl show no difference in expression when grown in conditions to differentiate the cells, indicating a failure of AP-2y null TS cells to differentiate into TGCs. RO3306 is a compound used to block Cyclin-dependent Protein Kinase 1 and force endoreduplication, causing TS cells to differentiate into TGCs. AP-2y null TS cells cannot be forced to differentiate into TGCs, and instead undergo cell death, when cultured with RO3306. Additionally, AP-2y null TS cells express the pluripotency markers Oct4, Stella, and Nanog which only are expressed in ES cells and germ cells. MiRNA profiling of AP-2y null TS cells indicates that cells in proliferative conditions resemble wild type counterparts, but when proliferative conditions are removed we observe an increase in expression of the ES cell specific miR-302 cluster. While there was no effect of proliferation in wild type cells, loss of Lin28 in AP-2y null TS cells via lentiviral knockdown leads to a partial rescue of TGC formation. This suggests that Liii28 must be down-regulated in order for TGC formation, and that AP-2y regulates Lin28 in TS cells. Taken together these data suggest a role for Lin28 in mouse TS cell proliferation and differentiation, where Lin28 must become down regulated in order for differentiation into TGCs. AP-2y has been shown to bind to the Lin28 promoter in TS cells; this regulation enables TS cell differentiation into TGCs. This study also shows the necessity of AP-2y for TS cell differentiation into TGCs; loss of AP-2y leads to a more pluripotent state rather than allowing for differentiation. Loss of AP-2y leads to expression of pluripotency markers Oct4, Nanog, and Stella, and the ES cell specific miR-302 cluster, indicating an increase in pluripotency. We conclude that AP-2y and LIN28 are essential molecular regulators of TS cell proliferation and differentiation.
- ItemOpen AccessTranslocation of insulin receptors into plasma membrane microdomains in response to insulin and to insulin-enhancing vanadium and chromium compounds(Colorado State University. Libraries, 2010) Al-Qatati, Abeer S. A., author; Roess, Deborah, advisor; Crans, Debbie, committee member; Graham, James, committee member; Anthony, Russ, committee memberWe have examined the translocation of insulin receptors into specialized, cholesterol-enriched membrane microdomains called lipid rafts following treatment of RBL-2H3 cells with insulin, bis-maltolatooxovanadium (BMOV) and tris(pyridinecarbxylato) chromium(III) (Cr(pic)3). Isopycnic sucrose gradient ultracentrifugation was used to subfractionate membrane fragments and insulin receptors were identified within low or high buoyant density membrane fractions using insulin receptor-specific antibodies and western blotting. Single particle tracking methods were used to confirm the confinement of individual insulin receptors within small membrane compartments on intact, viable RBL-2H3 cells. We demonstrated that insulin receptors translocate into lipid rafts upon binding insulin or following exposure to BMOV or Cr(pic)3 Phosphorylated insulin receptors also appeared in membrane raft fragments in response to insulin and/or insulin-mimicking compounds. Extraction of cholesterol from lipid rafts disrupted these microdomains and caused a decrease in the number of unphosphorylated and phosphorylated insulin receptors within these compartments. In addition to their ability to induce translocation of insulin receptors into lipid rafts, BMOV and Cr(pic)3 caused an increase in the number of phosphorylated IRS-1 molecules within these membrane fragments. To determine why Cr(pic)3 and BMOV might affect the distribution of insulin receptors in non-raft and raft compartments, membrane fluidity was evaluated in Cr(pic)3 and BMOV treated cells. Fluidity, as suggested by a decrease in lipid packing, was increased following treating 2H3 cells with either BMOV or Cr(pic)3 These results suggest that changes in lipid packing resulting from exposure of cells to either Cr(pic)3 and BMOV may affect the distribution of receptors in non-raft and raft compartments. Increased receptor localization in rafts or small membrane compartments evaluated by single particle tracking studies, would result in increased likelihood of insulin receptor phosphorylation within these signaling platforms. Thus rafts may be an important membrane structures involved in cell signaling events mediated by insulin receptors.
- ItemOpen AccessLuteinizing hormone induced oocyte maturation initiates mRNA decay in cattle(Colorado State University. Libraries, 2010) Walker, David Joshua, author; Seidel, George E., Jr., advisor; Clay, Colin M., committee member; Bruemmer, Jason E., committee member; Anthony, Russell V., committee memberOocyte maturation is a complex process consisting of signal transduction, ultrastructural changes, and mRNA transcription, translation, storage, and degradation. In vitro-matured oocytes initiate maturation in response to removal from an inhibitory follicular environment while in vivo-matured oocytes mature in response to the LH surge. Oocytes matured in vivo lead to more successful embryo production than those matured in vitro. This research concerned study of expression levels and action of selected transcripts involved in RNA processing that occur in in vivo oocyte maturation. The first experiment focused on the inability of GnRH to induce oocyte maturation in superstimulated cows during the luteal stage of the estrous cycle. Superstimulated cows were treated with PGF2a and GnRH to induce in vivo maturation or were treated with GnRH without PGF2a to induce an LH surge at 0, 3, 12, or 24 h before aspiration. While treatment with GnRH caused an increase in LH over no GnRH treatment, it was a smaller increase than that observed in cows treated with PGF2a before GnRH treatment (P<0.001) (No GnRH; 0.84 ng/mL, GnRH: 9.45 ng/mL, PGF2a: 93.86 ng/mL). Thus, increases in LH were sufficient to initiate epiregulin mRNA transcription in granulosa cells (P<0.06) with the greatest expression levels after 6h. However, germinal vesicle breakdown did not occur as reliably in cows with an intact corpus luteum 23 h after GnRH (treated with PGF2a 36 h prior to GnRH injection) (GV stage oocytes; Oh; 79%, 6h: 58%, 12h: 83%, 24h: 60%, vs. controls 6%)(P<0.001). In addition, cAMP levels remained stable in oocytes from cows treated with GnRH in the presence of progesterone regardless of post injection time, while control oocytes had a slightly elevated level of cAMP (Oh: 4.95, 6h: 3.98, 24h: 4.12, control: 7.41 fmol) (P>0.1). Phosphodiesterase 3A mRNA levels were unaffected by any treatment (P>0.10). These data suggest that although the stimulatory signaling of LH and epiregulin occur, cAMP levels are unaffected by GnRH treatment in the presence of progesterone. The second experiment evaluated mRNA concentrations in bovine oocytes of four transcripts involved in RNA regulation in mammalian cells; CUG-BP, PARK, eIF-4E, and PAP-1. In vivo- and in vitro-matured oocytes were collected 0, 3, or 6 hours after initiation of maturation via GnRH injection for in vivo-maturation and aspiration from follicles for in vitro-maturation. eIF-4E and PARN mRNA concentrations increased over time in both in vitro- and in vivo-matured bovine oocytes (P<0.05). In vivo-matured oocytes contained more eIF-4E mRNA molecules than in vitro-matured bovine oocytes (P<0.10). CUG-BP and PAP-1 concentrations remained stable over the first 6 h of maturation and were similar in the in vivo- and in vitro-matured oocytes. The final experiment concerned deadenylation patterns for the cyclin B1 3’ untranslated region (UTR) and GDF-9 3’UTR with a poly(A) tail of 60 adenosines. Bovine oocytes were injected with radiolabeled constructs after 0, 5, or 19 h of maturation and then cultured for 0, 1, or 3 h. Radiolabeled RNA was recovered from oocytes after culture and analyzed for changes in construct size, reflective of deadenylation or general degradation. Cyclin B1 underwent deadenylation before being degraded regardless of the stage of oocyte maturation. Analysis of gels showed an intermediate with 0 adenosines (AO) in the Cyclin B1 injected oocytes, while those injected with GDF-9 displayed no such intermediate. These findings indicate that injected GDF-9 transcript remains stable with a poly(A) tail of 60 adenosines or is simply degraded randomly in bovine oocytes matured for 0, 5, or 19 h. Deadenylation of Cyclin B1 mRNA begins immediately in bovine oocytes resulting in degradation of the Cyclin Bl.
- ItemOpen AccessIn 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 memberEquine 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).
- ItemOpen AccessOptimizing storage of bovine sperm between semen collection and sexing(Colorado State University. Libraries, 2010) Anema, Jennifer Lea, author; Seidel, George, Jr., advisor; Graham, James, committee member; Peel, Richard Kraig, committee memberTo view the abstract, please see the full text of the document.
- ItemOpen AccessEstradiol exposure alters gonadothropin-releasing hormone (GNRH) induced gonadotrope plasticity(Colorado State University. Libraries, 2010) Hartshorn, Cheryl, author; Tobet, Stuart, advisor; Clay, Colin, committee member; Hentges, Shane, committee member; Tjalkens, Ron, committee memberThe reproductive axis is dependent upon communication among the hypothalamus, pituitary and gonads. For successful ovulation, a large increase in circulating estradiol provides positive feedback at both the hypothalamic and pituitary levels to promote an luteinizing hormone (LH) surge. An LH surge is necessary for the final maturation of the pre-ovulatory follicle and ovulation. The cellular and molecular events underlying estradiol’s action(s) upon the anterior pituitary gland, specifically gonadotropes, remain elusive. Recent video microscopy experiments showed that pituitary cells in vitro in slice culture move in response to GnRH [Navratil, et al., 2007]; presumably these cells were gonadotropes. The current study utilized a novel transgenic animal model that has gonadotrope specific fluorescence provided by yellow fluorescent protein (YFP) [Wen et al., 2008]. I sought to determine if 17(3-estradiol (E2) working through either a genomic or non-genomic mechanism affected gonadotrope specific movements in response to GnRH. Consistent with earlier studies [Navratil et al., 2007], application of GnRH [100nM] altered the cytoarchitecture of gonadotropes with observable cell process extensions. Using live video- microscopy, exposure to 10nM E2 for fourteen hours significantly enhanced the ability of gonadotropes to extend processes in response to GnRH compared to short-term exposure of E2 (1.5 hours) or vehicle. There was no demonstrable effect of 1.5 hours of E2 exposure on GnRH-induced process extensions. I hypothesize that the differential effect of short-term versus long-term E2 exposure is due to a genomic mechanism that may underlie the ability of E2 to enhance GnRH induced cellular plasticity. Thus, E2 and GnRH may cooperate to maximize the secretory interface between gonadotropes and the adjacent vasculature during the pre-ovulatory LH surge.
- ItemOpen AccessTcfap2c regulation of primordial germ cell development(Colorado State University. Libraries, 2011) Guttormsen, Jillian Bosick, author; Winger, Quinton A., advisor; Bouma, Gerrit J., committee member; Anthony, Russell V., committee member; Garrity, Deborah, committee memberThe development of germ cells during embryonic development is driven by a complex expression pattern of genes. The transcription factor Tcfap2c is expressed in germ cells throughout development from specification to adult sperm and oocytes. Tcfap2c expression is first seen in primordial germ cells around embryonic day (E)6.75 and has been classified as a germ cell specification gene. This study implicates Tcfap2c as a potential key factor in germ cells during specification, proliferation, migration and differentiation. In order to investigate the role of Tcfap2c in germ cells, we utilized the Cre/loxP conditional gene mutation strategy. Cre/loxP allows us to overcome the early embryonic lethality that arises from loss of Tcfap2c in traditional knock-out mice by creating Tcfap2c null mutation in specifically-targeted tissues. We created Tcfap2c mutant mice using the epiblast-specific Sox2-Cre model. Mutant ovaries from this model failed to express both germ cell specific markers and meiotic markers at E12.5. Immunohistochemistry at E18.5 failed to detect the germ cell specific marker NOBOX or the meiotic protein SYCP3, which confirmed that Sox2-Cre, Tcfap2c mutant mice lacked germ cells at late embryonic stages. However, Sox2-Cre, Tcfap2c mutant mice die prior to or at birth preventing us from studying adult gonads from these mice. To this end we used tamoxifen inducible ERTM-Cre mice to create Tcfap2c mutation in adult animals. We assessed ERTM-Cre, Tcfap2c mutant animals for fertility and gametogenesis; surprisingly, fertility, spermatogenesis and oogenesis were not affected in Tcfap2c mutant gonads. These results show that Tcfap2c is not necessary for adult maturation of gonocytes to produce mature sperm and oocytes. However, Sox2-Cre, Tcfap2c mutants lack germ cells indicating that Tcfap2c is necessary during fetal germ cell differentiation. The Sox2-Cre model was limited because Tcfap2c was deleted in the entire embryo and the mutants died at birth. Prdm1-Cre was used to produce a mouse where Tcfap2c is only deleted in germ cells beginning around specification. Prdm1-Cre, Tcfap2c mutants initially specified germ cell-like cells at E7.5; however, by E8.5 the germ cell numbers were decreased and they had not initiated migration towards the genital ridges. By E9.5 few if any germ cells were observed in Prdm1-Cre, Tcfap2c mutants. At E12.5 no germ cells were seen in Prdm1-Cre, Tcfap2c mutant XX or XY gonads. Adult ovaries and testes from Prdm1-Cre, Tcfap2c mutant mice were noticeably smaller than littermate controls and showed no oogenesis or spermatogenesis. The Prdm1-Cre model showed that mutation of Tcfap2c results in loss of germ cells in embryos by E9.5 suggesting that Tcfap2c plays a role during germ cell specification, proliferation and migration. We identified Tcfap2c as an important factor during early germ cell development; however, Tcfap2c expression is observed in germ cells well past specification. We believe that Tcfap2c is present in germ cells during during fetal gonad differentiation because it plays a role in regulating the gene expression pathways necessary for this event. We show that Tcfap2c is expressed in germ cells during the period of fetal gonad differentiation. Gene expression analysis of gonads from E11.5-13.5 reveals Tcfap2c as the most highly expressed member of the Tcfap2 family member. Tcfap2c is a member of a transcription factor family that regulates gene expression by binding consensus sequences within target gene promoters. TCFAP2 binding sites are present in promoter regions of germ cell specific genes Cadherin1 (Cdh1) and Kit oncogene (Kit), as well as in the promoter regions of genes involved in regulating pluripotency High mobility group AT-hook 2 (Hmga2), Nanog homeobox (Nanog) and Lin28. Using chromatin immunoprecipitation we demonstrate that TCFAP2C binds the promoter regions of Cdh1, Kit, Hmga2, Nanog and Lin28. The interaction between TCFAP2C and the promoter regions of Cdh1, Kit, Hmga2, Nanog and Lin28 indicates that Tcfap2c likely plays a functional role in the regulation of these genes. These genes are necessary for germ cell survival, migration and pluripotency. In conclusion, our results provide a new understanding of the role of Tcfap2c during different stages of germ cell development from specification to differentiation.
- ItemOpen AccessApplying 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 memberThis 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.
- ItemOpen AccessDihydrotestosterone attenuates endotoxin, cytokine, and hypoxia-induced vascular inflammation(Colorado State University. Libraries, 2011) Osterlund, Kristen Leanne, author; Handa, Robert, advisor; Gonzales, Rayna, committee member; Amberg, Gregory, committee member; Garrity, Deborah, committee member; Tobet, Stuart, committee memberVascular inflammation plays a key role in the etiology of cardiovascular disease, particularly stoke. Vascular inflammation is under the control of several transcription factors, including nuclear factor kappa B and hypoxia inducible factor-1 alpha (HIF-1α). Activation of these transcription factors can lead to the production of inflammatory mediators such as cyclooxygenase-2 (COX-2). COX-2 plays a role in vascular inflammation, cerebral ischemia-induced injury, and has been implicated as a source of reactive oxygen species (ROS). Inflammatory mediators, such as endotoxin or cellular breakdown products released following injury, are known to signal through the Toll-like receptor 4 (TLR4). TLR4 activation leads to NFκB activation and subsequent production of COX-2. Like COX-2, TLR4 has also been implicated in injury-induced oxidative stress and cerebral ischemia damage. Previous studies have demonstrated that gonadal steroid hormones can also modulate vascular inflammation. Both protective and detrimental effects of androgens on the cardiovascular system have been reported. Since the potent androgen receptor (AR) agonist dihydrotestosterone (DHT) can be converted to 3β-diol, an estrogen receptor (ER) β-selective agonist, I hypothesized that ERβ may mediate some of the protective effects of androgens, while the AR may mediate some of the detrimental effects. The overall goal of this dissertation was to determine the mechanisms by which androgens can influence the vascular inflammatory response under both physiological and pathophysiological conditions. The hypothesis to be tested was that DHT influences vascular inflammation under both physiological and pathophysiological conditions. In my first set of experiments, using Western blot, I found that DHT increases expression of the vascular inflammatory mediator COX-2 under physiological conditions in human coronary artery vascular smooth muscle (VSM) cells and human brain VSM cells. This effect of DHT was attenuated in the presence of the AR antagonist bicalutamide. This data indicates that the pro-inflammatory effect of DHT under normal physiological conditions is AR mediated. In my second set of experiments, I examined the effects of DHT on vascular inflammation under a variety of pathophysiological conditions. Surprisingly, I found that DHT decreased cytokine-induced COX-2 expression and oxidative stress, endotoxin-induced COX-2 and TLR4 expression in human VSM cells. Furthermore, DHT also decreased hypoxia induced HIF-1α and COX-2 expression in human brain VSM cells and rat pial arteries. Finally, I found that DHT decreased hypoxia with glucose deprivation (HGD)-induced HIF-1α, COX-2 and TLR4 expression in human brain VSM cells. DHT`s anti-inflammatory effects during cytokine or HGD-induced inflammation in human brain VSM cells were not blocked by the AR antagonist bicalutamide, indicating that they were not AR mediated. These results led me to my second hypothesis, that DHT's anti-inflammatory effects are ERβ-mediated. In my third set of experiments, I found that the DHT metabolite/ERβ selective agonist 3β-diol also decreased cytokine-induced COX-2 expression in human brain VSM cells. Furthermore, DHT's ability to reduce cytokine-induced COX-2 expression in human brain VSM cells was inhibited by the non-selective estrogen receptor antagonist ICI 182,780 and the selective ERβ antagonist PHTPP. The mRNAs for steroid metabolizing enzymes in the pathway necessary to convert DHT to 3β-diol were detected in human brain VSM cells, as were AR and ERβ mRNAs. Therefore, DHT appears to be protective against cerebrovascular inflammation via conversion to 3β-diol and subsequent activation of ERβ in human brain VSM cells. The results of these studies indicate that: 1) DHT increases COX-2 expression under unstimulated/physiological conditions via an AR-dependent mechanism. 2) DHT decreases cytokine-, endotoxin,-hypoxia, and HGD-induced COX-2 expression via an AR-independent mechanism. 3) DHT decreases cytokine-induced reactive oxygen species. 4) DHT decreases hypoxia-induced HIF-1α expression. 5) DHT decreases HIF-1α and TLR4 expression during HGD via an AR-independent mechanism. 6) DHT's effect to attenuate cytokine-induced COX-2 expression is ERβ-mediated.
- ItemOpen AccessRemoving seminal plasma improves sex-sorting of bovine sperm(Colorado State University. Libraries, 2011) Burroughs, Chelsie Ann, author; Seidel, George E., Jr., advisor; Graham, James K., advisor; Curthoys, Norman P., committee memberTo view the abstract, please see the full text of the document.
- ItemOpen AccessMucosal immunization of mice with a recombinant Salmonella choleraesuis that expresses a multimeric gonadotropin releasing hormone fusion protein(Colorado State University. Libraries, 2011) Kemp, Jeffrey M., author; Graham, James, advisor; Huyvaert, Kathryn P., committee member; Bowen, Richard, committee member; Miller, Lowell, committee member; Rhyan, Jack, committee memberTo view the abstract, please see the full text of the document.