Browsing by Author "Clay, Colin, advisor"
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Item Open Access Analysis of equine zygote development after intracytoplasmic sperm injection(Colorado State University. Libraries, 2016) Ruggeri, Elena, author; Carnevale, Elaine, advisor; Clay, Colin, advisor; Albertini, David, committee member; DeLuca, Jennifer, committee member; Seidel, George, committee memberIntracytoplasmic sperm injection (ICSI) is an established and widely used method to achieve oocyte fertilization in equine reproductive assisted technologies. However, not all the oocytes fertilized by ICSI undergo cleavage and develop into viable embryos. Limited knowledge on equine zygote development after ICSI is available, and reasons why developmental failure occurs after ICSI have been only partially studied and need further investigation. Fertility decline and early embryo loss is associated with maternal aging in the mare, and it is concomitant with reduced oocyte quality. Relatively little is known about the effect of maternal aging and zygote developmental failure or success in the mare. Effects of in vitro maturation of the oocyte or zygote development in the mare still need to be clarified and further studied. The overall objective of this dissertation was to study equine zygote development after ICSI using confocal microscopy. Objectives were to: (1) compare cytoskeletal and nuclear changes during progression of equine zygote development after ICSI for in vivo versus in vitro matured oocytes; (2) compare changes in cytoskeletal and chromosomal configurations after ICSI between oocytes from young and old mares to define maternal-aging related alterations; (3) determine cytoskeletal and nuclear alterations associated with fertilization failure in ICSI-produced presumptive zygotes in young and old mares; (4) determine cell-aging and cell donor-aging effects on cytoskeleton and chromatin configurations. Specifically, in our studies we evaluated the tubulin and actin cytoskeleton, chromatin, and kinetochores/centromeres. Immunostaining and confocal imaging of the equine zygotes was performed using a spinning disk confocal microscope. After ICSI, five distinct events of development were observed with no major differences over time whether oocytes matured in vivo or in vitro. Oocytes matured in vivo appeared to reach the pronucleus stage earlier after ICSI compared to in vitro matured oocytes. Abnormal phenotypes associated with fertilization failure were more significant in oocytes matured in vitro than in vivo. When ICSI was performed in oocytes from young and old mares, similar stages of zygote development were observed, and the number of zygotes reaching the pronucleus stage was similar between the two age groups. Nucleolus like bodies, sites of ribosomal RNA involved in embryonic genome activation, were observed only in zygotes at the pronucleus stage from young mares; no nucleolus-like bodies were observed in pronuclei of zygotes from old mares. Pronuclei morphology, based on CREST staining, and DNA localization, also differed between pronuclei of young and old mares. Actin vesicles were observed significantly more often within zygotes from old mares compared to young mares during all stages of zygote developmental progression. When potential zygotes were analyzed after failure of cleavage after ICSI, actin vesicles were greater in area, perimeter and number in oocytes from old mares than those from young mares. Tubulin cytoskeletal multiasters were associated with cell aging and with increased interval after ICSI for young mares but not old mares. In conclusion, zygotes produced from oocytes matured in vivo versus in vitro or collected from young and old mares went through similar stages of development, with pronuclei attainment appearing to be a crucial event in zygote development. Actin vesicles were a major cytoskeletal difference associated with oocyte origin and a potential factor involved in developmental failure of the oocyte. Confocal microscopy and image analysis were novel methods used to describe the equine zygote development and allowed us to elucidate the cytoskeletal and nuclear remodeling events that follow fertilization after ICSI in the mare.Item Open Access Androgen signaling in the placenta(Colorado State University. Libraries, 2014) Cleys, Ellane Rachael, author; Bouma, Gerrit, advisor; Clay, Colin, advisor; Tobet, Stuart, committee member; Di Pietro, Santiago, committee memberPlacental estrogen signaling is known to regulate placental trophoblast function and differentiation. However, the role of placental androgen signaling has never been investigated, despite the rise of maternal serum androgens throughout gestation. Recent findings have shown increased maternal serum androgen in patients with the placental induced disorder preeclampsia. Preeclampsia, a maternal hypertension and proteinuria condition instigated by insufficient trophoblast differentiation and invasion into maternal spiral arteries, is also associated with increased placental expression of androgen receptor and an increased risk of incidence in patients with polymorphisms in androgen receptor that decrease androgen signaling. These findings suggest a crucial role for placental androgen signaling. Moreover, research investigating androgen's role in cancer progression has shown that many androgen responsive genes regulate cell proliferation, differentiation to invasive phenotypes, and tissue vascularization, all processes necessary for normal placental development. Androgen signaling in tumor tissues is further regulated by androgen receptor complexes with histone lysine demethylases. These complexes are recruited to androgen response elements in DNA and dynamically regulate histone tail modifications for transcription initiation. This led us to the overall hypothesis that (1) androgen signaling in trophoblast cells is important for placental development, and (2) androgen receptor complexes with histone lysine demethylases in the placenta to regulate vascularization, growth and invasion factors in trophoblast cells. To test this hypothesis, we utilized a prenatal androgenization ewe model as well as human first trimester placental samples and immortalized human trophoblast cell lines. Using the prenatal androgenized ewe model, we report for the first time expression of histone lysine demethylases in the placenta. Furthermore, we showed androgen receptor complexes with histone lysine demethylases and is recruited to an androgen response elements in the 5'untranslated flanking sequence of vascular endothelial growth factor in the sheep placenta. We also report that histone lysine demethylase are present in human first trimester syncytiotrophoblast and complex with androgen receptor in immortalized trophoblasts. Additionally, we demonstrated that androgen receptor complexes with histone lysine demethylases are also present in choriocarcinoma ACH-3P and BeWo cells. Dihydrotestosterone treatment in these cells led to down-regulation of androgen responsive genes, specifically KDM3A and MMP2. Inhibition of androgen receptor through flutamide treatment altered mRNA levels for genes regulating vascularization, including HIF1α, PPARα, and PPARy. Hypoxia also decreased CYP19 levels, however, further investigation is needed to confirm dihydrotestosterone and flutamide effect on protein expression in trophoblast cells. These data suggest that histone lysine demethylases complex with androgen receptor to regulate androgen responsive genes, including those directing placental vascularization and development. However, further experiments are needed to confirm the necessity of histone lysine demethylases for targeted androgen signaling in trophoblast cells and to determine if androgen directly regulates trophoblast differentiation and invasion. These findings suggest androgen signaling may play a critical role in placental development.Item 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 memberThe 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.Item Open Access Regulation of local L-type calcium channel signaling in anterior pituitary gonadotropes(Colorado State University. Libraries, 2017) Dang, An Khanh, author; Amberg, Greg, advisor; Clay, Colin, advisor; Tamkun, Michael, committee member; Navratil, Amy, committee member; Duval, Dawn, committee memberThe binding of gonadotropin-releasing hormone (GnRH) to its receptor initiates signaling cascades in gonadotropes which result in enhanced luteinizing hormone (LH) and follicle stimulating hormone (FSH) biosynthesis and secretion. Most dramatic is the sharp rise in LH secretion ("LH surge") that precedes and is necessary for follicular maturation and ovulation. Ca2+ influx activates mitogen-activated protein kinases (MAPKs) which lead to increased transcription of LH and FSH genes. Interestingly, previous research suggests that two MAPK signaling pathways, ERK and JNK, are activated by either Ca2+ influx through L-type Ca2+ channels or by global Ca2+ signals originating from intracellular stores, respectively. These discrete Ca2+ sources for divergent signaling cascades provides a mechanism in which gonadotropes can decode different pathways for appropriate gonadotropin release during various stages of the ovulatory cycle. However, direct evidence supporting an underlying subplasmalemmal local Ca2+ signaling through L-type Ca2+ channels distinct from intracellular Ca2+ was lacking. Here we used a combination of electrophysiology and total internal reflection fluorescence (TIRF) microscopy to visualize discrete sites of Ca2+ influx (Ca2+ sparklets) in gonadotrope-derived αT3-1 cells in real time. These localized GnRH-induced Ca2+ influxes are mediated by L-type Ca2+ channels and important for downstream ERK activation. In addition, precise structural and molecular elements to create a microenvironment suitable for localized subplasmalemmal L-type Ca2+ channel signaling was necessary for gonadotrope function, in which GnRH-dependent stimulation of L-type Ca2+ channel influx was found to require PKC and a dynamic actin cytoskeleton. More recently, we have further elucidated molecular mechanisms modulating localized L-type Ca2+ channel influx. Reactive oxygen species (ROS) are cognate signaling molecules that mediate cell function, but their role in regulating Ca2+ in gonadotropes is unknown. We have explored GnRH regulation of both NADPH oxidase complexes and mitochondrial sources of ROS and assessed ROS modulation of L-type Ca2+ channel activity in gonadotropes. We identified GnRH-induced spatially localized ROS "puncta" in αT3-1 cells, and ROS increased local Ca2+ channel activity in both αT3-1 cells and primary mouse gonadotropes. In addition, GnRH increased mitochondrial oxidation activity at the subplasmalemmal surface and mitochondrial ROS increased localized L-type Ca2+ channel influx. Also, active L-type Ca2+ channels were associated with subplasmalemmal mitochondria. Taken together, this dissertation explored the first direct evidence for localized L-type Ca2+ channel signaling in αT3-1 cells and elucidated signaling mechanisms in gonadotropes. Specifically, cellular organization via an intact cytoskeletal platform and ROS regulated L-type Ca2+ channel sparklet activity that are important for the downstream ERK activation and gonadotropin gene expression that regulates reproduction.