Browsing by Author "Carnevale, Elaine, advisor"

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Open Access
Analysis of equine zygote development after intracytoplasmic sperm injection
(Colorado State University. Libraries, 2016) Ruggeri, Elena, author; Carnevale, Elaine, advisor; Clay, Colin, advisor; Albertini, David, committee member; DeLuca, Jennifer, committee member; Seidel, George, committee member
Intracytoplasmic sperm injection (ICSI) is an established and widely used method to achieve oocyte fertilization in equine reproductive assisted technologies. However, not all the oocytes fertilized by ICSI undergo cleavage and develop into viable embryos. Limited knowledge on equine zygote development after ICSI is available, and reasons why developmental failure occurs after ICSI have been only partially studied and need further investigation. Fertility decline and early embryo loss is associated with maternal aging in the mare, and it is concomitant with reduced oocyte quality. Relatively little is known about the effect of maternal aging and zygote developmental failure or success in the mare. Effects of in vitro maturation of the oocyte or zygote development in the mare still need to be clarified and further studied. The overall objective of this dissertation was to study equine zygote development after ICSI using confocal microscopy. Objectives were to: (1) compare cytoskeletal and nuclear changes during progression of equine zygote development after ICSI for in vivo versus in vitro matured oocytes; (2) compare changes in cytoskeletal and chromosomal configurations after ICSI between oocytes from young and old mares to define maternal-aging related alterations; (3) determine cytoskeletal and nuclear alterations associated with fertilization failure in ICSI-produced presumptive zygotes in young and old mares; (4) determine cell-aging and cell donor-aging effects on cytoskeleton and chromatin configurations. Specifically, in our studies we evaluated the tubulin and actin cytoskeleton, chromatin, and kinetochores/centromeres. Immunostaining and confocal imaging of the equine zygotes was performed using a spinning disk confocal microscope. After ICSI, five distinct events of development were observed with no major differences over time whether oocytes matured in vivo or in vitro. Oocytes matured in vivo appeared to reach the pronucleus stage earlier after ICSI compared to in vitro matured oocytes. Abnormal phenotypes associated with fertilization failure were more significant in oocytes matured in vitro than in vivo. When ICSI was performed in oocytes from young and old mares, similar stages of zygote development were observed, and the number of zygotes reaching the pronucleus stage was similar between the two age groups. Nucleolus like bodies, sites of ribosomal RNA involved in embryonic genome activation, were observed only in zygotes at the pronucleus stage from young mares; no nucleolus-like bodies were observed in pronuclei of zygotes from old mares. Pronuclei morphology, based on CREST staining, and DNA localization, also differed between pronuclei of young and old mares. Actin vesicles were observed significantly more often within zygotes from old mares compared to young mares during all stages of zygote developmental progression. When potential zygotes were analyzed after failure of cleavage after ICSI, actin vesicles were greater in area, perimeter and number in oocytes from old mares than those from young mares. Tubulin cytoskeletal multiasters were associated with cell aging and with increased interval after ICSI for young mares but not old mares. In conclusion, zygotes produced from oocytes matured in vivo versus in vitro or collected from young and old mares went through similar stages of development, with pronuclei attainment appearing to be a crucial event in zygote development. Actin vesicles were a major cytoskeletal difference associated with oocyte origin and a potential factor involved in developmental failure of the oocyte. Confocal microscopy and image analysis were novel methods used to describe the equine zygote development and allowed us to elucidate the cytoskeletal and nuclear remodeling events that follow fertilization after ICSI in the mare.
Open Access
Association of oocyte and early embryo morphology with age and the establishment and maintenance of pregnancy after ICSI in mares
(Colorado State University. Libraries, 2010) Frank-Guest, Bethany Linda, author; Carnevale, Elaine, advisor; Seidel, George, Jr., committee member; Hendrickson, Dean, committee member
Increasing maternal age in humans, horses and lab animals has been associated with a decrease in fertility. Oocyte quality and morphology have been implicated as primary causes of reduced fertility in older mares. Selected oocyte morphological parameters have been correlated with pregnancy development in humans and horses. Objective measurements of morphology to assess oocyte quality would provide a critical evaluation and help identify zygotes with the highest developmental potential for transfer, to optimize recipient utilization and pregnancy rates. The hypotheses of the research were that oocyte and early embryo morphology differ with donor mare age and correspond with developmental potential. Objectives for the first study were to compare: 1) oocyte donor age with oocyte morphology and developmental competency after ICSI, and 2) oocyte morphology with developmental competency (cleavage, early pregnancy, late pregnancy and pregnancy loss) after ICSI. Objectives for the second study were to compare developmental potential of ICSI produced embryos with: 1) oocyte donor age, and 2) cleavage characteristics, and 3) rate of embryonic development. Oocytes were collected from donor mares in a clinical ICSI programs. The mares were divided into the following age groups and fertility categories: 1) 3-13 yr with Known fertility, 2) 2-13 yr with Unknown fertility, 14-19 yr, 20-23 yr and 24-27 yr. Approximately 24 h after induction of follicle maturation, and oocytes were collected and cultured approximately 18 h before being stripped of cumulus cells. Photographic images (200x) were captured before oocytes were injected with sperm. Images of oocytes were measured using digital calipers within a computer software program. Ooplasm volume was larger (p<0.05) for oocytes from mares 14-19 yr and 20-23 yr than mares 3-13 yr Known than for mares 24-27 yr. Perivitelline space volume was similar between mares 3-13 yr Unknown and mares 20-23 yr, but was smaller (p<0.05) between mares 3-13 Unknown and the other age groups. Oocyte diameter (OD) was smaller (p=0.05) between oocytes from donors 3-13 yr Known and donors 14 -19 yr, but similar among all other groups. Inner zona pellucida diameter (IZPD) differed (p=0.03) only between mares 14-19 yr and mares 3-13 yr Unknown, with oocytes from mares 14- 19 yr having the largest numerical IZPD and mares 3-13 yr Unknown having the smallest IZPD. Ooplasm diameter (OpD) was smaller (p≤0.02) for oocytes from mares 3-13 yr Known than from mares 14-19 or 20-23 yr. The diameter of the zona pellucida with the surrounding matrix (ZPTM) was greater (p<0.05) for mares 3-13 yr Unknown than for all other groups. The rate of embryo development (hours per cell) prior to oviductal embryo transfers was faster (P<0.05) for embryos that did versus did not produce an early pregnancy and tended (P≤0. l) to be faster for embryos that did versus did not produce a late pregnancy. Embryonic vesicles that had a more rapid increase in diameter were more often (p<0.05) maintained to the late pregnancy stage. Donor mare age exerted a large effect on the development and outcome of pregnancies. Oocyte morphology was not a reliable indicator of oocyte developmental potential, although speed of early embryonic development was associated with embryonic competency.
Open Access
Characterization of equine sperm attributes and selection for intracytoplasmic sperm injection
(Colorado State University. Libraries, 2018) Gonzalez-Castro, Raul A., author; Carnevale, Elaine, advisor; Graham, James, advisor; Seidel, George, committee member; Gerrit, Bouma, committee member; Ann, Hess, committee member
When performing intracytoplasmic sperm injection (ICSI), many in vivo mechanisms for sperm selection are bypassed; however, sperm must still be capable of activating the oocyte for successful fertilization. Limited information is available for horses on the effect of sperm preparation method and sperm characteristics that affect ICSI outcome. The overall objectives of this dissertation were to: 1) study the association between sperm sorting methods, sperm population characteristics, and equine ICSI outcome, and 2) characterize sperm oocyte activating factors in stallion sperm, such as phospholipase C zeta (PLCz) and postacrosomal WW binding protein (PAWP). In Experiment 1, a microfluidic device was used to sort frozen-thawed sperm from stallions (n=19), which resulted in a sperm subpopulation with improved motility, morphology, viability and DNA integrity (P<0.05) compared to the original sample. Then, microfluidic sorting was compared with the swim-up procedure and density gradient centrifugation. Swim-up was the least effective method to separate equine sperm. Microfluidic sorting and density gradient centrifugation sorted a sperm subpopulation with similar parameters, improving motility, viability and DNA integrity. After ICSI (n=45), no differences (P>0.3) were observed for cleavage and embryo development among sorting methods. In Experiment 2, sperm population parameters from which individual sperm were selected for injection were analyzed immediately after ICSI and correlated with the outcome. Sperm morphology, viability, membrane integrity measurement of hypoosmotic swelling and DNA integrity were evaluated in frozen-thawed sperm (n=114) used for ICSI in a program. Among sperm parameters, viability correlated positively with normal morphology and membrane integrity (P<0.05). Normal sperm morphology and DNA integrity were not predictive of ICSI outcome. Viability was predictive of cleavage and blastocyst formation, and membrane integrity was predictive of early pregnancy (P<0.05). In Experiment 3, PLCz and PAWP were identified, localized and quantified in stallion sperm, and the relationship with other sperm parameters was investigated. PLCz was identified as a 71 kDa protein and located in the acrosomal and postacrosomal region, midpiece and principal piece of the tail. PAWP was identified by two bands of ~28 and ~32 kDa, located in the postacrosomal region, midpiece and principal piece of the tail. The expression of PLCz and PAWP correlated positively (P=0.04) when analyzed for sperm of 14 stallions. Flow cytometric assessment was feasible for PLCz, but not for PAWP. Expression and percentages of positive labeled sperm for PLCz varied among stallions (n=21). Expression of PLCz was higher in live than dead sperm (P<0.005), and DNA fragmentation correlated negatively with PLCz expression (P<0.04). In conclusion, microfluidic sorting and density gradient centrifugation resulted in a subpopulation of sperm with high quality parameters for ICSI. The probability of sperm-injected oocytes to develop into an embryo and to establish pregnancy improved when sperm were selected from a sample population with higher viability and membrane integrity. This is the first report that describes PAWP in equine sperm, which displayed a novel localization in the midpiece and principal piece of sperm tail in addition to the expected postacrosomal region. Protein levels of PLCz and PAWP were correlated in sperm heads. The expression of PLCz in sperm varied widely among stallions and was associated with DNA integrity. Sperm membrane integrity is indicative of well-maintained plasma membrane architecture, conserving sperm quality and membrane components that are required for oocyte activation and early embryo development. Assessment of PLCz in stallion sperm represents a potent feature to investigate sperm quality for equine ICSI, and potentially can serve as a prognostic biomarker for oocyte activation ability and male infertility. Further studies are needed to determine the relationship between PLCz and PAWP with fertility in horses.
Open Access
Impact of equine sperm phospholipase C zeta content and sperm tail components on cleavage rates after intracytoplasmic sperm injections of equine and bovine oocytes
(Colorado State University. Libraries, 2020) Amoroso G. Sanches, Fabio, author; Carnevale, Elaine, advisor; Black, Jerry, advisor; Hatzel, Jennifer, committee member; Graham, James K., committee member; Veeramachaneni, Rao, committee member
Intracytoplasmic sperm injection (ICSI) is used in equine assisted reproductive medicine to generate offspring when other procedures fail. However, for some stallions, ICSI is not successful in producing embryos. This could be caused by multiple factors associated with stallion or mare gametes, which result in low cleavage and embryo development rates. Phospholipase C zeta (PLCz) has been identified as a sperm- associated factor that contributes to oocyte activation and is correlated to ICSI success in other species. We hypothesized that sperm population content of PLCz is associated with cleavage after ICSI and that components of the sperm tail, by virtue of containing oocyte activation factors can activate oocytes and induce cleavage. For the experiments, ICSI was performed using equine sperm with "High" or "Low" PLCz content on bovine oocytes (heterologous model) and equine oocytes to confirm results in the same species. More bovine oocytes (P=0.04) and equine oocytes (P=0.01) cleaved after injections with sperm having High PLCz content than Low PLCz content (bovine: High, 33/62, 53% and Low, 19/56, 34%; and equine: High, 9/10, 90% and Low 4/12, 33%). The addition of equine sperm tail components to the injection of sperm from a stallion with low PLCz population during ICSI improved (P<0.05) cleavage rates of bovine oocytes. In conclusion, PLCz content of equine sperm population was associated with cleavage rates following ICSI. Bovine oocytes provided a heterologous model to estimate the ICSI potential of an equine sperm population before use with equine oocytes, providing a more feasible and less costly system to evaluate sperm. Components of the equine sperm tail appear to assist activation for sperm from stallions with low PLCz content.
Embargo
Metabolic support of preimplantation embryo growth and viability
(Colorado State University. Libraries, 2024) Fresa, Kyle Joseph, author; Carnevale, Elaine, advisor; Chicco, Adam, advisor; Tesfaye, Dawit, committee member; Krisher, Rebecca, committee member; Gentile, Christopher, committee member
Early embryo metabolism involves essential and dynamic biological reactions that support viability, growth, and pregnancy establishment. Embryo metabolism not only serves to provide energy through ATP synthesis, but also facilitates the production of macromolecules such as proteins, nucleotides, and lipids. The ways in which embryos balance catabolic and anabolic activity during the preimplantation stage are not well understood; however, understanding these processes may lead to improved fertility treatments, embryo culture, and pregnancy outcomes. The studies described in this dissertation utilize innovative methods, such as stable isotope tracer analysis to track carbon and nitrogen flux through various pathways, oxygen microsensors to determine individual embryo respiration under various conditions, and proteomic analysis to determine the impacts of metabolic disturbances on embryo viability. The overarching hypothesis of this dissertation is that embryo viability is dependent on efficient and tightly regulated metabolic activity, and disturbances to metabolic function ultimately lead to reduced developmental potential. To test this hypothesis, a series of projects were conducted to 1) evaluate the importance of phosphoenolpyruvate carboxykinase (PEPCK) during early development, 2) uncover the function of PEPCK to support catabolic and anabolic activity in early embryos, and 3) determine the impacts of delayed embryo development on embryo metabolism and pathway regulation. These projects revealed important insights into the impact of embryo metabolism on development, including the discovery of a novel, PEPCK-mediated pathway that embryos utilize to balance energy production and biosynthesis. Furthermore, the impact of delayed embryo development was demonstrated to alter embryo metabolic activity and pathway regulation, including increased aerobic activity and altered protein expression. These findings improve our understanding of metabolic activity and regulation during preimplantation development, highlighting the impact of metabolic activity to promote ATP production, biosynthesis, developmental kinetics, and ultimately survival. The experimental outcomes presented in this dissertation provide a foundation for targeted approaches to improve embryo development and reproductive success.
Open Access
The effect of aging on gene expression and mitochondrial DNA in the equine oocyte and follicle
(Colorado State University. Libraries, 2009) Campos-Chillon, Lino Fernando, author; Carnevale, Elaine, advisor
The decline in fertility of aged mares is linked to declining oocyte quality. Oocyte viability is dependant on the ability of oocytes to remain in meiotic arrest until the initiation of maturation and adequate cumulus communication. We hypothesize that aging is associated with quantitative and temporal differences in meiotic arrest and resumption in oocytes, decreased oocyte secretion of paracrine factors and lower mitochondrial numbers, ultimately resulting in a dissociation of oocyte and follicular maturation. The objectives of this study were to clone and determine quantitative and temporal differences in mRNA content of the LH receptor (LHR), amphiregulin (AREG) and epiregulin (EREG) in granulosa cells; PDE4 in cumulus cells; and PDE3A, GPR3, GDF9, BMP15, and mitochondrial DNA (mtDNA) in oocytes during in vivo maturation in young (3-12 yr) and old (>20 yr) mares. Oocytes and follicular cells were collected by transvaginal follicular aspiration. Follicle maturation was induced in estrous mares with a follicle >30 mm by injection of 750 µg of recombinant equine LH. Aspirations were conducted at 0, 6, 9, and 12 h after LH administration. Total RNA was isolated from single denuded oocytes and associated lysed cumulus and granulosa cells. For each gene, mean mRNA copy number for each time point and age group were compared by ANOVA and Fisher's LSD. Regression coefficients were generated to compare oocyte mitochondrial numbers and correlations between gene expression within age groups. Expression of LHR mRNA in granulosa cells was different (p<0.05) between age groups. Young mares displayed a significant drop in LHR mRNA between 0 h and 6, 9, and 12 h; while the pattern of expression in old mares was similar (p>0.05) among times and higher (p<0.05) at 6 h than in young mares. Expression of AREG mRNA in granulosa cells peaked (p<0.05) at 9 h; however, the magnitude of expression at 6 and 9 h was higher (p<0.05) in old than young mares. Similarly, EREG expression peaked (p<0.05) at 9 h in young and old mares but was higher (p<0.05) for old mares. Expression of PDE4D peaked (p<0.05) at 6 and 12 h in old and young mares, respectively. The patterns of expression of GPR3 for oocytes of young and old mares were different and peaked (p<0.05) at 9 and 12 h, respectively. Magnitude of expression of PDE3A for oocytes of old mares at 6 and 9 h was higher (p<0.05) than in young mares. Expression of GDF9 and BMP15 was different (p<0.05) between ages. Mean expression of both genes in the old group was similar over time; however, in young mare oocytes maximum expression was at 6 h (p<0.05). Correlation coefficients between GDF9 and BMP15 for old and young mares were 0.94 and 0.99, respectively. Numbers of copies of oocyte mtDNA did not vary in young mares; however, there was a temporal decrease (p<0.05) of oocyte mitochondrial copy numbers in old mares. The main effect for age for mtDNA was similar for old and young mares.
Open Access
The role of cell-secreted vesicles in equine ovarian follicle development
(Colorado State University. Libraries, 2013) da Silveira, Juliano Coelho, author; Bouma, Gerrit, advisor; Carnevale, Elaine, advisor; Winger, Quinton, committee member; Veeramacheneni, DN Rao, committee member; Tjalkens, Ronald, committee member
Ovarian follicular development is a process responsible for generating a gamete and steroid hormones, which are important for reproduction and general health. Failure of intrafollicular cell communication is one of the causes behind infertility. Recently, cell-secreted vesicles (microvesicles and exosomes) were described as mediators of cell communication through the transfer of bioactive material such as protein, mRNA and miRNA. Cell-secreted vesicles are present in different body fluids. The overall hypothesis is that cell-secreted vesicles are present in ovarian follicular fluid and are involved in regulating TGF-β signaling members during follicular development. In order to test this hypothesis we utilized the mare as an animal model due to the well described follicular dynamics and the easy access to sufficient experimental material. Firstly, we described the presence of microvesicles and exosomes in ovarian follicular fluid from pre-ovulatory follicles. Further we demonstrated the presence of cell-secreted vesicles markers such as miRNA and proteins. We also demonstrated that microvesicles are taken up by granulosa cell in vitro and in vivo. Secondly, we demonstrated the role of exosomes mediating regulation of TGF-β signaling members during follicular development at mid-estrous and pre-ovulatory stages. Thirdly, we demonstrated association between relative levels of TGF-β signaling members and exosomal miRNAs during follicular development in young and old mares. The data indicates that cell-secreted vesicles play an important role mediating cell proliferation and differentiation through the regulation of TGF-β signaling members during follicular development.