Ph.D. - Developmental and Reproductive Biology

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Selenoprotein I is essential for proper central nervous system development: A study on ethanolamine phospholipid deficiency in oligodendrocyte development and function
(University of Hawai'i at Manoa, 2024) Nunes, Lance Gregory A.; Hoffmann, Peter R.; Developmental & Reproductive Biology
Selenoprotein I (SELENOI; EPT1) is an endoplasmic reticulum resident ethanolamine phosphotransferase that catalyzes the final reaction of the ethanolamine branch of the Kennedy Pathway of phospholipid synthesis, in which phosphatidylethanolamine (PE) and plasmenyl-PE are produced. PE is a major component in mammalian cellular membranes and plays a key role in membrane architecture while also serving as a precursor for biologically active molecules. Plasmenyl-PE is enriched in nervous tissues, particularly in myelin, serving a critical role in a variety of biological functions including neuroprotection through scavenging reactive oxygen species (ROS). Rare mutations in SELENOI have been shown to be associated with a complex form of hereditary spastic paraplegia (HSP), a large group of neurodevelopmental and/or neurodegenerative disorders of multigenetic origin that results in spasticity and weakness in the lower limbs. Patients deficient of SELENOI suffer from a myriad of debilitating symptoms including severely delayed growth, cerebral and cerebellar atrophy, delayed motor development, lower limb spasticity, and hypomyelination. We developed a murine central nervous system-specific SELENOI conditional knockout (cKO) and observed profound deficits in tests of motor coordination accompanied by hypomyelination and reactive gliosis in regions of the corticospinal tract. Lipidomic and flow cytometric analyses of whole brains revealed altered lipid profile, elevated lipid peroxidation, and reduced percentage of myelinating oligodendrocytes. These findings suggested that cells of the oligodendroglial lineage are most vulnerable to the loss of SELENOI. During proliferation and differentiation, oligodendrocyte progenitor cells (OPCs) increase their uptake of iron which leads to elevated levels of ROS and, consequently, lipid peroxidation. The intracellular accumulation of iron and lipid peroxidation are two hallmarks of ferroptosis, a non-apoptotic cell death pathway. We, therefore, hypothesized that loss of SELENOI-derived plasmenyl-PE in OPCs results in increased vulnerability to lipid peroxidation and ferroptosis. We isolated SELENOI cKO OPCs and measured their sensitivity and response to ferroptosis induction. We then evaluated their differentiation and proliferation capacities ex vivo. We found that SELENOI cKO OPCs exhibited a smaller increase in iron uptake and greater increase in lipid peroxidation levels in response to ferroptosis induction compared to WT controls. Notably, we observed no effect on cell viability. Furthermore, we noted a decreased differentiation capacity and an increased rate of proliferation accompanied by downregulation of cell cycle exit and neuronal differentiation protein 1 (CEND1) in these OPCs. These data collectively suggest that SELENOI cKO OPCs evade ferroptosis by mitigating overwhelming levels of lipid peroxidation through restricting iron uptake, and that the resultant iron uptake restriction may negatively influence factors that drive differentiation and suppress proliferation, such as CEND1. Further investigation is needed to discern the precise molecular pathways through which SELENOI exerts its effects on these processes, as understanding these mechanisms could open new avenues for therapeutic interventions in myelination disorders.
IDENTIFYING THE MOLECULAR FUNCTION OF MOUSE ZFY1 AND ZFY2 IN MALE REPRODUCTION
(University of Hawai'i at Manoa, 2024) Holmlund, Hayden Robert; Ward, Monika A.; Developmental & Reproductive Biology
The mouse zinc finger genes Zfy1 and Zfy2 are essential for normal spermatogenesis. We recently produced Zfy1/2 double-knock-out (Zfy DKO) mice and observed severe defects in male fertility. The mechanism by which Zfy contributes to reproduction remains unknown but based on its predicted peptide sequence and the phenotype of Zfy DKO mice, we hypothesize that ZFY is a transcription factor that regulates genes essential for reproduction. To determine which genes are regulated by ZFY, we (1) performed transcriptome analysis on Zfy KO germ cells and (2) created tagged Zfy knock-in mice to identify ZFY co-interactors. Bulk RNA sequencing analysis was performed on XY, Zfy1 KO, Zfy2 KO, and Zfy DKO primary spermatocytes (SC1), secondary spermatocytes (SC2) and round spermatids (RS) cells. Ontology and gene set enrichment analysis revealed deregulation of apoptosis, chromatin re-organization, and spermatogenesis pathways in Zfy KO germ cells. Follow-up assays revealed that Zfy KO mice produce sperm with poorly reorganized chromatin and motility deficiencies, and an increase in apoptotic spermatocytes was observed in Zfy DKO testis cross sections. Next, CRISPR/Cas9 genome editing was used to create five distinct Zfy knock-in (KI) models (XYZfy2-FLAG, XYZfy2-3xFLAG, XYZfy1-HA, XYZfy2-HA, and XYZfy1-HA,Zfy2-MYC), and to generate knockouts of two other Y chromosome genes, Prssly and Teyorf1 KO mice to establish if these genes are responsible for similar spermatogenesis phenotypes as those of Zfy KO males. Tagged ZFY protein expression was detected in XYZfy1-HA and XYZfy2-HA mouse testis. A pilot immunoprecipitation followed by tandem mass spectrometry (IP-LC/MS) with a single XY and XYZfy2-HA testis confirmed enrichment of ZFY2 and identified H2AX as a potential binding partner. However, follow-up IP-LC/MS and chromatin immunoprecipitation (ChIP) assays have been inconclusive in determining the biochemical function of ZFY. Phenotypical analysis of Prssly and Teyorf1 KO males revealed that they are fertile with no deficiencies in spermatogenesis. In conclusion, this work described in this dissertation has revealed novel functions of Zfy, and the knock-in lines can be used for further study of Zfy function. Upon completion, this work will advance understanding of the function of Zfy in mice, which could in turn inform our knowledge on the homologous human ZFY homologue.
DEFINING THE ROLE OF IRON-DEPENDENT CELLULAR INJURY IN CARDIOMYOCYTES
(University of Hawai'i at Manoa, 2024) Kawasaki, Nicholas Kaulike; Matsui, Takashi; Developmental & Reproductive Biology
Heart failure remains one of the leading causes of death in America and while advancements in preserving patient outcomes after myocardial infarctions are improving the adverse effects after a myocardial infarction are still prevalent. To develop better therapeutic approaches, greater understanding of ischemia-reperfusion (I/R) injury and its mechanism will be required. In this dissertation, I aim to better understand cell death and scarring relating to I/R injury and determine if ferroptosis plays a significant role in that cell death cascade in cardiac I/R injury. To do this we used a handful of models to determine the unique patterning of scarring in I/R injury and discern if ferroptosis plays a major role in its formation. Initially we examined a set of human cadaveric hearts with and without percutaneous coronary intervention (PCI) and discerned that PCI resulted in scarring following myofibers instead of coronary arteries. We then used a murine in vivo model to determine that scarring along myofibers was consistent with I/R injury and occurred in the early phases of LV remodeling. Next, we developed a novel in vitro model of injury extending from cell-to-cell to determine the role of ferroptosis. Through this model we saw that ferroptosis was a major factor to cell death extending from cell-to-cell. Lastly, we used an ex vivo model to induce I/R conditions and saw that ferroptosis inhibitors were sufficient to prevent cell death along myofibers, solidifying that ferroptosis is a major factor in the fibrotic scarring that extends along myofibers. However, a clear mechanism of how ferroptosis extends cell death or what is communicated between cells still needs to be identified. Better understanding of ferroptosis in I/R injury could bring new therapeutic insights into combatting heart failure.
ENGINEERED RECOMBINASES: TOOLS FOR THERAPEUTIC HUMAN GENOME EDITING.
(University of Hawai'i at Manoa, 2024) Sato, Ryuei; Owens, Jesse B.; Developmental & Reproductive Biology
Numerous genetic human diseases will require precise and effective genome-targeting technologies to be treated in the future. Ideally, these technologies would be able to edit any genomic locus with high efficiency, high DNA sequence specificity, and few or no unintended side effects. Currently, a method for delivering a large DNA fragment to a single defined sequence in an efficient and safe manner does not exist. Current targeted approaches for gene editing that rely on CRISPR or other site-specific nucleases typically require double-strand DNA breaks (DSBs) that often generate unwanted byproducts or lead to chromosomal abnormalities. These technologies act passively; following the DNA break, rate-limiting host factors are needed to insert the donor DNA. Furthermore, because homologous directed repair (HDR) factors are only present during cell division, HDR is inefficient in non-dividing cells that make up most of the tissues of the body. Here, my studies will demonstrate effective methodologies to address these shortcomings in gene editing. I developed a system for actively inserting a large DNA sequence to a known target sequence. Rational mutations were incorporated into the endogenous DNA-binding domain of the piggyBac transposase to reduce non-specific binding and promote preferential binding and targeted insertion by catalytically dead Cas9 (dCas9). This strategy enabled us, for the first time, to direct transposition to the genome using RNA in human cells. Directed evolution could also improve proteins that are less characterized and are difficult to enhance using rational design. Using phage-assisted continuous evolution (PACE), we mutated the bacteriophage PhiC31 serine integrase to increase activity at the natural human pseudo site, Xq22.1 which meets the requirements to be considered a genetic safe harbor. Finally, we used PACE to increase the activity of two integrases, PhiC31 and Bxb1, on their native sites, resulting in mutant enzymes with a 10-fold increase in recombination. When combined with twin prime editing (PE), we were able to insert a 7kb donor into a single safe harbor loci in the human genome with efficiencies of more than 80% of available genomic targets. These technologies could be applicable to both preclinical research and potential gene replacement therapies for a multitude of genetic diseases.
The Effects of the FOXO3 Longevity Associated Variant on Biomarkers of Aging and Stress
(University of Hawaii at Manoa, 2022) Torigoe, Trevor; Allsopp, Richard; Developmental & Reproductive Biology
The Forkhead Box O3 (FOXO3) protein is one of four transcription factors that make up the FOXO family. The FOXOs are the mammalian homologues of the Caenorhabditis elegans daf-16 gene which has been associated with longevity. The four mammalian isoforms, FOXO1, -3, -4, and -6 are differentially expressed in various tissues. FOXO3 is expressed throughout the body, most significantly in the heart, brain, liver, muscle, spleen, testes, ovaries, and resident stem cells. Previous studies have shown that FOXO3 is positioned as a key regulator in multiple longevity regulatory pathways although the specific mechanisms are not well understood. We developed studies to explore the possible effect of the longevity-associated FOXO3 rs2802292 allele on age related change in telomere length, telomerase activity, FOXO3 gene expression, and plasma pro- and anti-inflammatory cytokine levels. The general hypothesis is that the FOXO3 longevity-variant is associated with increased FOXO3 expression, elevated levels of established healthy aging biomarkers, and reduction in biomarkers of stress response. In the initial study, Okinawa Study #1, telomere length and telomerase activity were assessed with respect to age as a function of both FOXO3 rs2802292 longevity associated variants and APOE variants in a population of Okinawan-Japanese. 121 subjects from Okinawa, Japan, ranging in age from 21-95 years, were recruited during annual physical appointments, where after consent, peripheral circulatory blood was collected and sent to the John A. Burns School of Medicine at the University of Mānoa for work-up and analysis. Telomere length was assessed in mononuclear cells using Southern blot analysis and telomerase activity was analyzed using the telomere repeat amplification protocol (TRAP). FOXO3 genotyping of the rs2802292 variant was performed using amplification-refractory mutation system allele-specific PCR while genotyping of the Apolipoprotein E variant done by standard PCR amplification and restriction enzyme digestion. We found that carriage of the FOXO3 G-allele was associated with protection against telomere loss with respect to age compared to those lacking the G-allele (TT-carriers). Importantly, this effect was only observed in subjects of middle age (arbitrary cut off of 50) and older. We did not detect an effect of carriage of the G-allele on telomerase activity in this preliminary study. In contrast, carriage of neither the ϵ4 -allele (risk allele) nor the ϵ2 -allele (protective allele) of APOE were associated with an effect on telomere attrition with respect to age. To further our analysis from our first study, we initiated a second larger Okinawa study (Okinawa Study #2), to assess telomere length, telomerase activity, FOXO3 gene expression, and circulatory plasma cytokines IL-1β, IL-2, IL-6, IL-10, and TNFα. We recruited 325 Okinawan Japanese subjects with roughly equal numbers of men and women (ages 19-104 years; 49:51 M:F ratio). FOXO3 genotyping, and telomerase activity was assessed using the same protocols as in the first study. Telomere length was analyzed using a monochrome multiplex quantitative PCR, FOXO3 gene expression was analyzed by quantitative PCR, and cytokine levels were assessed using a Milliplex MAP Human High Sensitivity T Cell Panel on a Luminex 200 system. In agreement with our previous findings, the FOXO3 longevity variant conferred protection against telomere shortening in peripheral blood mononuclear cells from adult aged 55 and older in both men and women (P<0.001). This was accompanied by higher levels of telomerase activity in carriers of the longevity-associated FOXO3 G-allele (P=0.015). Carriage of the FOXO3 G-allele was associated with a modest increase in FOXO3 gene expression in both young and old groups compared to decreased gene expression in TT-carriers, the difference in trend being most significant in the young population (P=0.02). We also observed two sex-specific protective effects of the FOXO3 rs2802292 G-allele in the older population with respect to inflammatory cytokine levels. In women, carriers of the G-allele displayed a modest decline in levels of pro-inflammatory cytokine IL-6 with respect to age in contrast to increasing levels with age in TT-carrier counterparts (P=0.07). This protective effect in maintaining lower levels of pro-inflammatory cytokines was highly significant in comparison to G-carrier men (P=0.0006). For the anti-inflammatory cytokine IL-10, G-carrier men in the old population retained IL-10 levels with age better than TT-carriers (P=0.04), and even more significantly compared to G-carrier women (P=0.007). Finally, in a collaborative study with Exeter University, we analyzed the expression of the FOXO3 isoform levels in peripheral blood using TaqMan PCR. Using a limited subset of RNA samples from the Okinawa Study#2 population, we were unable to find a significant effect of the longevity associated FOXO3 rs2802292 genotype on moderating levels of FOXO3-FL (full length) and FOXO3-TR (truncated) isoforms, although a trend towards increased expression in G-allele carriers was observed. Collaborators at the University of Exeter analyzed peripheral blood samples from the UK BioBank with respect to the FOXO3 rs13217795 longevity-associated SNP that is in linkage disequilibrium with the rs2802292 SNP. They found that carriage of the longevity-associated “C”-allele was associated with increased expression of the full-length isoform in peripheral blood as compared to TT-carriers. Truncated isoform expression was not observed in peripheral blood samples in either the Okinawan or UK BioBank cohorts. Through these findings, we believe that we have developed a foundation for FOXO3 and aging research that will provide an infrastructure to further life science and medical research in numerous specialties that will ultimately improve our understanding of healthy aging and further the development and technologies available to help patients in doing so.
Adaptative Functions Of Sensory And Behavior Laterality In Astyanax Mexicanus
(University of Hawaii at Manoa, 2021) Lima Fernandes, Vânia Filipa; Yoshizawa, Masato; Developmental & Reproductive Biology
Laterality consists of the asymmetrical use of the left or the right side in terms of morphology (the asymmetrical disposition of visceral organs), behavior (left or right-handedness), and neural processing. For example, regarding neural processing in humans, the left side of the brain is more often associated with analytical reasoning, while the right-side hemisphere is associated with emotion-processing tasks. Laterality is widely reported across vertebrates and several studies demonstrate that it confers a variety of advantages, such as performing intricate motor activities, spatial learning, cognitive and multitasking abilities, and escaping from predators. However, there is a major gap regarding how laterality arose through the course of evolution. Astyanax mexicanus, commonly known as the blind cavefish, has been presented as an excellent vertebrate model to study evolutionary processes. This teleost species is composed of two morphs: an eyed, river-dwelling surface morph (surface fish) and a blind cave-dwelling morph (cavefish). Sensory-behavior laterality has been described for cavefish regarding the detection of novel immotile objects. Cave-dwelling morphs have evolved different foraging strategies, such as attraction to a source of water disturbance/vibration, which is associated with the enhancement of non-visual sensory systems. This behavior is advantageous for capturing prey in the dark. Vibration Attraction Behavior (VAB) is a foraging behavior differently expressed in cave and surface morphs. Cavefish approaches the vibrating rod significantly more than surface fish, showing that cave individuals have a higher VAB level in comparison with the surface counterpart. Prey-capture assays have shown that cavefish (VAB positive) outcompeted surface fish (VAB negative) in the dark, which indicates that this foraging behavior may be crucial for survival in a cave environment. Further studies have demonstrated that VAB is mediated by the Lateral Line system, more precisely by the mechanosensory units present at the third infraorbital bone (IO3) region. The IO3 bone shows a higher degree of asymmetrical patterning and fragmentation across different cavefish populations. Furthermore, studies in zebrafish have reported an association between bone formation and neuromasts disposition during embryonic development. We will first investigate if the bone formation and consequently mechanosensory development at the IO3 region influence VAB output, and we will address if this sensory-behavior system exhibits laterality (Chapter 2). Additionally, we will discriminate both sensory and behavior laterality across different populations of Astyanax mexicanus, and we will functionally assess the asymmetric contribution of the mechanosensory system towards VAB (Chapter 3). Finally, we will address this foraging behavior lateral/asymmetrical modality in response to starvation (Chapter 3). Our results indicate that mechanosensory asymmetrical usage and behavior laterality vary according to food availability and across different cave populations over time. The most diversified cave population showed a combination of sensory and behavioral laterality, suggesting a novel advantage as a strategy to save energy in food-limited environments.
Three-dimensional Mouse Stem Cell-derived Gastruloids: An In Vitro Approach To Study The Impact Of Teratogens During Gastrulation
(University of Hawaii at Manoa, 2021) Li, Aileen S.; Marikawa, Yusuke; Developmental & Reproductive Biology
Teratogens are harmful chemical, physical or biological environmental exposure to childbearing and pregnant women that can increase the incidences of birth defects. To minimize the incidences of birth defects, it is important to identify teratogenic agents. However, there is no easy and reliable test to screen for teratogenic effects of substances. Epidemiologic studies in human are only retrospective, relying on hundreds of tragic incidences that had already happened to attain statistically sound conclusions, whereas experimentations using live animals are costly, laborious and associated with ethical issues. To compensate for these caveats of in vivo studies, in vitro teratogen tests using pluripotent stem cells are explored. Pluripotent stem cells can self-renew in culture indefinitely while retaining the developmental ability of early embryos. They can be induced to differentiate in vitro to mimic key aspects of embryogenesis, which may be used to evaluate the adverse effects of teratogenic exposures. The goal of my dissertation project is to validate one such stem cell-based in vitro embryogenesis model and apply it to elucidate the molecular mechanisms of some known teratogenic agents. First, we demonstrated that the morphogenesis and gene expression profile of the 3D stem cell model, known as the P19C5 gastruloid, are regulated by the same molecular machineries that function during early stages of normal embryogenesis, when body axis elongation and patterning take place. Second, we identified the molecular mechanisms, by which anti-epileptic medication, valproic acid, exerts its teratogenic effects through enhanced activation of retinoic acid signaling. Third, we revealed that methoxyacetic acid, the major metabolite of industrial products, including plasticizers and organic solvents, also enhance retinoic acid signaling to cause its adverse effects. Based on my dissertation project, we expect P19C5 gastruloid can serve as an effective in vitro model to assess the teratogenic potential of environmental exposures that may cause birth defects and explain the mechanisms of teratogenic agents to reduce the incidences of tragic birth defects
Evaluating Endogenous Carbon Monoxide (co) Production As An Indicator For Pulmonary Function Testing (PFT)
(University of Hawaii at Manoa, 2019) Kim, Jung Eun; Titchenal, Charles A.; Developmental & Reproductive Biology
Chronic lung diseases, such as chronic obstructive pulmonary disease (COPD) and asthma, are associated with considerable morbidity and mortality in the United States. COPD is currently the 4th leading cause of death in the United States and a major cause of morbidity. More than 26 million Americans have asthma, which accounts for 14.2 million doctor’s office visits each year, according to the Centers for Disease Control and Prevention (CDC). These lung diseases involve chronic inflammation and oxidative stress. However, the diseases are not diagnosed and treated efficiently in routine clinical practice because of the difficulties in monitoring inflammation. Consequently, it is often too late to alter respiratory dysfunction when patients’ referral for pulmonary function testing is delayed. The purpose of this descriptive-correlational study was to explore the possible associations between pulmonary function test (PFT) measurements and exhaled carbon monoxide (eCO) levels as an indicator of generalized inflammation. Data analysis was conducted to determine a potential level of eCO to use as an indicator for conducting PFT. The contributions of demographic factors, patient history of smoking habits and drug use, and the medical diagnosis to the prediction of principal variables were also investigated. A sample of 343 subjects, who were referred by their physicians for a routine care PFT, were recruited from the Queen’s Medical Center Pulmonary Lab in Honolulu, Hawaii. A study-specific information sheet was provided to the subjects as part of the informed consent process, and the subjects’ data were collected through a Demographic Data Questionnaire. Exhaled carbon monoxide (eCO) levels were measured with a portable carbon monoxide analyzer (MicroCO Meter), and the maximum values from three successive measurements were used in all calculations. Carbon monoxide was detectable and measured reproducibly in the exhaled air of all subjects. Forced vital capacity (FVC), forced expiratory volume in the first second (FEV1), FEV1/FVC, and forced expiratory flow 25-75% (FEF25-75%)) decreased with elevated concentrations of eCO. In females, decreased lung volumes (total lung capacity, functional residual capacity, and residual volume) were associated with increased eCO levels. In males, increased lung volumes were associated with increased eCO levels. Diffusion capacity of lungs for carbon monoxide (DLCO) and eCO levels also showed the opposite correlation between females and males. DLCO of female subjects markedly decreased with increased levels of eCO while DLCO of male subjects mildly increased. Appropriate cut-off points of eCO levels also were examined to determine the most efficient use of eCO as an indicator for PFT. The present study found that a cut-off point for eCO of 6 ppm provided the best relationship between sensitivity and specificity in predicting the need for PFT. In conclusion, eCO measurement, which is noninvasive, quick, inexpensive, and easily administered by primary care physicians, could serve as a useful biomarker for monitoring patients with pulmonary diseases. Therefore, eCO measurement may be clinically useful as a diagnostic tool to identify inflammation and to serve as an indicator of the need to conduct PFT.
In vitro Morphogenesis in Developmental Toxicity Testing: Assay Validation and the Search for Teratogenic Mechanisms
(University of Hawaii at Manoa, 2018-05) Warkus, Erica L. L.; Developmental & Reproductive Biology
Teratogens are chemicals that can cause birth defects in the developing embryo during pregnancy. Major structural birth defects affect 2-5% of children, but the etiology of this developmental error is often unknown. Exposure to teratogens (chemicals that cause birth defects) during gestation may play a causative role in many of these congenital defects. Therefore, it is important to identify teratogenic chemicals before pregnant women are exposed to them, but there is no easy, reliable test to determine potential teratogenicity of pharmaceutical drugs or industrial chemicals (pesticides, herbicides, solvents, etc.). Current developmental and reproductive toxicity (DART) testing uses millions of pregnant animals in outdated, inefficient regulatory studies. Embryonic stem cell-based in vitro tests (EST) produce faster, clearer results and have the potential to revolutionize DART testing, but they often lack the biologic complexity created during embryonic gastrulation and morphogenesis. The embryo is most sensitive to teratogenic exposures during the first three to eight weeks of fetal life. During this time, the process of morphogenesis shapes nearly all three-dimensional (3-D) organs and tissue structures in the embryo. Many of the common birth defects (e.g., neural tube closure defects and cardiac septal defects) are caused by disruptions in this vital process. Since morphogenesis drives the formation of fetal structures and is often affected by teratogenic drugs, a screen for potential teratogens should incorporate similar 3-D structural complexity, but this is a common deficit of in vitro teratogenicity tests. Aggregated P19C5 stem cells spontaneously form “embryoid bodies” (EBs) and recapitulate the germ layer differentiation and structural remodeling that occur during embryonic gastrulation and morphogenesis. Exposure to teratogens disrupts EB morphology, making P19C5 morphogenesis a sensitive indicator of developmental toxicity. In this research project, I validated the P19C5 system as a morphology-based teratogen-screening assay and demonstrate that the P19C5 assay can be used to investigate teratogenic mechanisms that cause structural defects. We expect that this test will allow us to accurately identify chemicals that may cause fetal malformations and help us understand the mechanisms of teratogenic chemicals in order to reduce the number of children born with preventable birth defects.
Sox9 Driven Sex Determination in Mice with Limited Y Chromosome Gene Contribution
(University of Hawaii at Manoa, 2018-05) Ortega, Egle A.; Developmental & Reproductive Biology
The window of sex determination is a short but critical time during which sex fate of the embryo is decided depending on the chromosomal predisposition inherited from the parents. This genetic information guides the direction of gonadal sex development towards acquisition of anatomical male or female sexual characteristics. In most mammals, male sex determination is initiated by expression of the Y chromosome encoded Sry. The duration of SRY signaling is transient - just long enough to ensure upregulation of autosomally encoded Sox9. This way, SRY acts as a molecular switch that initiates differentiation of the bipotential gonad into a testis. However, once SRY signaling ceases, Sox9 remains at the center of male-specific molecular signaling profile that ensures masculinization of the genital ridges. The focus of my dissertation is on the effects of Sry-to-Sox9 replacement on the development of genital ridges, as well as spermatogenesis and fertility in adult males. Work presented here explores the hypothesis that transgenic overexpression of Sox9 can replace the function of endogenous Sry at the time of sex determination and can sufficiently support spermatogenesis and fertility in adult males. The sex-determining function of transgenic Sox9 was tested using two genetic models: (1) mice that carried X chromosome and Y chromosome lacking the endogenous Sry (XYTdym1); (2) mice that carried a single X chromosome and lacked all endogenous Y chromosome genes (XO). The progression of spermatogenesis in XYTdym1 males transgenic for Sox9 (XYTdym1Sox9) was assessed through various sperm and testicular analyses such as sperm counts, motility, and morphology evaluation, as well testis weight, shape, histology, vasculature assessment and mRNA expression levels. The fertility of XYTdym1Sox9 males was tested through fecundity trials and in vitro (IVF). These investigations revealed that despite some testicular abnormalities transgenic overexpression of Sox9 transgene in the context of YTdym1 can replace the function endogenous Sry and allows for sufficient levels of spermatogenesis and fertility. In the mouse only two Y chromosome genes were shown to be necessary for successful assisted reproduction: Sry and spermatogonial proliferation factor Eif2s3y. When Sry and Eif2s3y were transgenically added to mice carrying a single X chromosome (XO), these mice (XEif2s3yOSry) developed as phenotypic males and produced haploid gametes. The second part of my dissertation, focuses on replacement of Sry and Eif2s3y with their non Y chromosome encoded homologues, Sox9 and Eif2s3x. We found that males with Y chromosome genes limited to two (XEif2s3yOSry), one (XOSry,Eif2s3x and XEif2s3yOSox9) or none (XOSox9,Eif2s3x) can father offspring with assisted reproduction. However, we also observed that these males had various abnormalities of the seminiferous epithelium and testicular interstitium. Moreover, sometimes the gonads of the transgenic males resembled ovotestes and had elevated expression of ovarian markers. These findings led to a hypothesis that pathologies observed in males with limited Y chromosome genes arise at the time of sex determination due to altered signaling in the genital ridges. To test this hypothesis, I compared gonadal development in males without Y chromosome and in which sex determination is initiated either by Sry or Sox9, and spermatogenesis is driven by either Eif2s3y or Eif2s3x. My findings support the initial hypothesis and suggest that all XO males with limited Y chromosome genes have altered gonadal development at the critical time of sex determination. The failure to completely antagonize female specific signaling and establish characteristic male-like gonadal morphology is especially prominent in genotypes that have sex determination driven by transgenic overexpression of Sox9.
The Licensing Protein ORC4 is Required for Polar Body Extrusion During Murine Meiosis
(University of Hawaii at Manoa, 2017-12) Nguyen, Hieu T.; Developmental & Reproductive Biology
Six proteins, ORC1-6, make up the origin recognition complex (ORC) that initiates licensing of the DNA replication origin. We have previously shown that subunit ORC1, ORC2, ORC3, and ORC5 are localized between the separating maternal chromosomes at anaphase II just after fertilization. During investigation, we identified ORC4 as having an unexpected localization in the polar body wherein ORC4 surrounds one set of chromosomes during both female meiotic divisions. The ORC4 structure, or ORC4 cage as we have termed it, eventually is discarded in the polar bodies while the chromosome set that does not interact with ORC4 segregate into the oocyte. Interestingly, none of the other five ORC proteins were found to be involved in this structure. In Zygotic G1, ORC4 surrounds the nuclei of the polar bodies, but was not detectable in the pronuclei. When the zygote entered mitosis, ORC4 was only detected in the polar body. At this point, the ORC4 that was in the polar body also migrated into the nuclei, suggesting that ORC4 or an associated protein is modified during the first embryonic cell cycle to allow it to bind DNA. We experimentally forced oocytes to extrude sperm chromatin as a pseudo-polar body and found that under these conditions, the sperm chromatin did become enclosed in an ORC4 cage. Next, we attempted to prevent the formation of the ORC4 cage by injecting peptides that contained sequences of different ORC4 protein domains into metaphase II oocytes just before typical cage formation. Our rationale was that the ORC4 peptides would block protein-protein interactions required for cage formation. Two out of six tested peptides prevented the ORC4 cage formation and simultaneously inhibited polar body extrusion, resulting in the formation of two pronuclei that were retained in the oocyte. Our previous results demonstrated that recombinantly expressed ORC4, which contained histidine tag at the C terminus, could be utilized by oocytes to form the ORC4 cage at one set of chromosomes. Using immunocytochemistry (ICC), we were able to show that ORC4-His tags incorporated with endogenous ORC4. However, we could not use these tags to study on live cells because they required cell fixation to identify by ICC. To test the localization of ORC4 in live cells, we generate a fusion ORC4-eGFP and ORC4 labeling with FlAsH. These constructs were then used to synthesize mRNA and microinjected into MII oocyte. Through confocal fluorescence fluctuation spectroscopy (FFS) measurements, we are able to record the movement of ORC4 signals moving forward to form a cage or PB. This work provides the first evidence that the ORC4 plays a necessary role for polar body extrusion.
Microscopic and Macroscopic Investigations of Male Development Anatomy and Fertility, and the Role of Y Chromosome Genes
(University of Hawaii at Manoa, 2016-08) Ruthig, Victor
There have been decades of work on the Y chromosome and how it relates to maleness and male reproduction. In light of the increasing decline of human male fertility and dependence on assisted reproduction more knowledge about the causes behind male infertility is needed to find more solutions for affected men. This dissertation details histological investigations aiming to elucidate function of Y chromosome genes in male development and fertility using a mouse model. The testis and the seminiferous tubules within were explored and spermatogenesis was quantitatively assessed. Males lacking Y chromosome and transgenic for either the key Y-derived transgenes (Sry and Eif2s3y) or transgenes of their non Y-derived homologues (Sox9 and Eif2s3x) were shown to produce haploid germ cells that could be used for assisted reproduction technologies (ART) and yield live offspring. Another Y chromosome gene, Zfy2, was identified as a gene allowing for transformation of round spermatids into sperm. While in males with only two Y chromosome genes, Sry and Eif2s3y, spermatogenesis progressed to round spermatid stage, addition of Zfy2 allowed for complete spermatogenesis and formation of sperm capable of generating offspring with ART. Investigations into the testicular abnormalities in mice with limited Y chromosome gene contribution showed relationship between the number of Y genes present and the severity and distribution of cellular abnormalities in the seminiferous epithelium and defects of the testis interstitium. Human spermatogenesis was also histologically investigated utilizing testis biopsies from normal and infertile men, some with Y chromosome azoospermic factor (AZF) deletions, validating mice with Y chromosome deficiencies as a model for human male Y-linked infertility. Gross anatomy investigations into human male urogenital anatomy were also undertaken using a novel dissection method in order to grow the repository of male urogenital teaching tools at the medical school. Both examined specimens had common male genital pathologies (direct inguinal hernia, varicocele) and so also showcase physical abnormalities that can affect male reproductive health.
The role of sperm DNA damage in the origin of infertility associated with Y chromosome long arm deletions in the mouse model
(University of Hawaii at Manoa, 2013-12) Riel, Jonathan M.
In mouse and man Y chromosome deletions are frequently associated with spermatogenic defects. Mice with severe non-pairing Y chromosome long arm (NPYq) deficiencies are infertile in vivo and in vitro. We have previously shown that sperm from these males, although having grossly malformed heads, were able to fertilize oocytes via intracytoplasmic sperm injections (ICSI) and yield live offspring. However, in continuing ICSI trials we noted a reduced efficiency when cryopreserved sperm were used and with epididymal sperm as compared to testicular sperm. Our initial study tested if NPYq deficiency is associated with sperm DNA damage-a known cause of poor ICSI results. We observed that epididymal sperm from mice with severe NPYq deficiency are impaired in oocyte activation ability, and have an increased incidence of oocyte arrest and paternal chromosome breaks. Comet assays revealed increased DNA damage in both epididymal and testicular sperm, and transmission electron microscopy showed sperm having impaired membrane integrity and abnormal chromatin condensation. We therefore concluded that the increased DNA damage associated with NPYq deficiency might be a consequence of disturbed chromatin remodeling taking place during spermiogenesis. There are four distinct multi-copy genes found in the long arm of the Y chromosome. One of them, Sly, is known to control the expression of sex chromosome genes after meiosis; Sly deficiency results in a remarkable upregulation of sex chromosome genes. Sly deficiency has also been shown to be the underlying cause of sperm head anomalies and infertility associated with NPYq gene loss. We therefore hypothesized that Sly is our target gene. To test this, we examined mice with transgenically (RNAi) silenced Sly. Our analysis of Sly-deficient mice demonstrated similar 'sperm DNA damage' phenotype. This confirmed that lack of Sly is responsible for the sperm DNA damage/chromatin packaging defects observed in mice with NPYq deletions. This project provides the first evidence of DNA damage in sperm from mice with NPYq deficiencies and that the multi-copy NPYq-encoded Sly gene plays a key role in processes regulating chromatin remodeling and thus maintaining DNA integrity in sperm.
The potential role of hypoxia inducible factor 1 alpha in multipotent germ cells and neural stem/progenitor cells
(University of Hawaii at Manoa, 2014-12) Takahashi, Natsumi
Hypoxia inducible factor 1 alpha (HIF1α) is a key transcription factor which transcriptionally activates an array of genes involved in adaptive responses to physiological hypoxia. HIF1α has been implicated in many disease conditions as well as normal development of many organs including the heart, the brain and the bones. In recent years, a number of studies have revealed that HIF1α also maintains undifferentiated state of many types of stem cells such as embryonic stem cells, hematopoietic stem cells and mesenchymal stem cells. In this study, we sought to investigate whether HIF1α is localized in intestinal stem cells, hair follicle stem cells and germline cells, in which HIF1α expression has not yet been reported. Although we did not detect any HIF1α signal in intestinal and hair follicle stem cell compartments, we observed a clear expression of HIF1α in both male and female germ cells. Furthermore, our results showed the presence of HIF1α throughout the fetal and neonatal development of both male and female germ cells, raising a possibility that HIF1α may play a role as a transactivator of glycolytic genes in highly proliferative cells such as primordial germ cells and spermatogonial cell as those cell types heavily depend on glycolysis for energy production. Lastly, we investigated whether HIF1α regulates telomerase activity in the cultured neural stem/progenitor cell (NSPCs) population, as our previous study revealed that HIF1α transactivates mTert and subsequently regulates telomerase activity in mouse embryonic stem cells. Our shRNA-mediated Hif1α knockdown assay resulted in the decreased levels of telomerase activity, strongly suggesting that HIF1α transactivates mTert in NSPCs. Regulation of telomerase activity may be important to ensure the long term survival of NSPCs and continuous neurogenesis throughout the life of an organism. Together, the results of this study illustrate the involvement of HIF1α in normal development and maintenance of types of stem/progenitor cells in which the localization and function of HIF1α was never extensively discussed, pointing out that further investigation may lead to better understanding of the roles of HIF1α in multipotent cells.

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