M.S. - Developmental and Reproductive Biology

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https://hdl.handle.net/10125/36909

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Using in vitro gastrulation models to assess the teratogenicity of antiviral drugs: dolutegravir and remdesivir
(2021) Kirkwood-Johnson, Lauren; Marikawa, Yusuke; Developmental & Reproductive Biology
An assessment of the reproductive toxicity of the anti-COVID-19 drug molnupiravir using stem cell-based embryo models
(University of Hawaii at Manoa, 2023) Huntsman, Margaret Carrell; Marikawa, Yusuke; Developmental & Reproductive Biology
It is important that women of reproductive potential know what risks the medications they are consuming pose to reproductive health. However, reproductive risk information is particularly limited for new drugs, such as those to treat COVID-19. Because the FDA no longer requires in vivo animal testing during preclinical tests, the information obtained through suitable in vitro models are particularly crucial. The overall objective of this study was to evaluate the embryotoxic effects of molnupiravir, an anti-COVID-19 drug, using in vitro embryo models made of pluripotent stem cells. In Specific Aim 1, the efficacy of morphological assay using mouse P19C5 stem cell model, or embryoid bodies (EBs), was assessed in reference to known embryotoxic drugs. The ICH recently released a list of drugs with their in vivo concentrations known to cause embryotoxic effects. This ICH list can be used to validate the sensitivity and specificity of an in vitro assay to identify embryotoxicity. Adverse effects of drugs on the P19C5 EB model were determined from disruptions in morphology. The in vitro results with P19C5 EBs closely reflected those of the ICH’s in vivo results for many of the drugs examined, validating the efficacy of the assay. In Specific Aim 2, the adverse effects of molnupiravir, and its metabolite N-hydroxycytidine (NHC) were examined using in vitro embryo models made of P19C5 cells and also of human embryonic stem cells. Morphology-based analyses indicated that molnupiravir had an adverse effect at concentrations much higher than the therapeutic level, whereas NHC had a dose-dependent adverse effects at therapeutically relevant concentrations. Molecular studies using P19C5 EBs further demonstrated that NHC altered the expression patterns of several embryo-patterning genes, providing mechanistic insights into the embryotoxic action of molnupiravir. These results emphasize the need for further studies into the effectiveness of the in vitro embryo models and the embryotoxic effects of the COVID-19 antiviral drugs.
Optimizing The Targeting Of The Piggybac And T2022 Transposon System
(University of Hawaii at Manoa, 2022) Xie, Kristal; Owens, Jesse B.; Developmental & Reproductive Biology
Gene therapy, which involves correcting pre-existing genetic interruptions, could provide a long-term solution for those suffering from genetic diseases such as cystic fibrosis (CF), a monogenic, autosomal recessive disease. Typical methods, however, for introducing DNA into cells have limitations such as random insertion, limited cargo capacity, and immune response. Thus, this thesis aims to optimize DNA binding domain (DBD)-directed transposition to develop a novel approach that would result in a reliable and effective non-viral method of transporting a gene of interest to a particular location in the genome in non-dividing cells. We hypothesize that the piggyBac (pB) and t2022 transposon systems can be optimized to target and integrate at a specific TTAA site through (a) introducing mutations in the pB and the t2022 transposases to reduce integration at off-target TTAA sites and (b) pairing the pB and the t2022 transposon systems to programmable targetable nucleases that contain a DNA binding domain capable of binding to a DNA sequence near a TTAA site of interest. In Chapter 2, we generate mutations in the dimerization interface and the DNA binding domain of the pB and the t2022 transposases to reduce their natural abilities to bind and integrate at various TTAA sites in the genome. In Chapter 3, we fuse the modified pB and the t2022 transposases to a CRISPR-associated protein 9 (Cas9) and synthesize user-defined sgRNAs. that bind to our region of interest. We also fuse the modified pB and t2022 transposases to custom-built transcription activator-like effectors (TALEs) that can direct integration to specific TTAA sites in the genome. The sgRNAs and TALEs are designed to target a specific TTAA site in the cystic fibrosis transmembrane conductance regulator (CFTR) gene as well as a specific TTAA site in the genomic safe harbors: CCR5, hROSA26, and AAVS1. Overall, the optimization of the pB and t2022 transposon systems will lead to a safer and more efficient approach to delivering therapeutic genes into the genome that have the potential to reverse various genetic diseases.
Investigations Of The Effects Of Selected Y-chromosome Encoded Gene Deletion And Viral Infection On Testicular Function In A Mouse Model
(University of Hawaii at Manoa, 2022) Bakse, Jackson Elias; Ward, Monika A.; Developmental & Reproductive Biology
Murine and human testes are affected by both Y-chromosome encoded gene loss and the direct or indirect effects of some viral infections. Primary focus into the impact of Y-chromosome gene loss and viral infection on male fertility has been primarily focused on the Y-chromosome encoded testis determining factor, Sry, and viruses that transmit vertically, like ZIKA virus (ZIKV). Zinc finger Y encoded genes (Zfy), termed Zfy1 and Zfy2 in mouse and ZFY in humans, were once suspected to be the testes determining factor (TDF). This was proven incorrect when the true TDF was identified as another Y-chromosome gene, SRY (sex-determining region Y encoded). Subsequent research using mouse models with Zfy deficiencies and Zfy transgenic additions have indicated significant contributions from these genes to other aspects of male fertility in the mouse. However, complete knockout (KO) of Zfy genes has not been conducted. Existing data from testicular biopsies obtained from men who succumbed to SARS-CoV2 (COVID-19) infection, many with underlying conditions, revealed extensive testicular damage in the absence of active viral infection of the testes, despite the presence of the viral cellular receptor, angiotensin converting enzyme 2 (ACE2). However, the effect of SARS-CoV-2 infection on testicular function has not been researched in a well-controlled mouse model. The projects herein are based on the hypothesis that complete Zfy gene loss and SARSCoV-2 viral infection have profound effects on testicular function in the mouse. To assess effect of Zfy gene loss, select spermiogenic phenotypes were assessed in mice with complete Zfy1 (Zfy1 KO), Zfy2 (Zfy2 KO), and both Zfy homologs (Zfy DKO) knockout developed with CRISPR/Cas9 technology. Assessment of the testicular effect of COVID-19 disease was performed using ii transgenic K18-hACE mice expressing human ACE in epithelia, infected with SARS-CoV-2, and assessed 3-, 5-, 8-, and 14-days post infection (DPI). The analyses of sperm and testes Zfy KO mice using silver-stained sperm spreads and light microscopy and transmission electron microscopy (TEM) revealed defects in sperm morphogenesis in Zfy2 KO and Zfy DKO males, but not Zfy1 KO males. The analyses of testicular PAS-stained testicular sections from Zfy DKO males revealed seminiferous tubule abnormalities. Histopathological assessment of testes from K18-hACE mice at 5 different DPI revealed significant increase in seminiferous tubule abnormalities and increase in germ cell apoptosis males with DPI of at least 5. This data shows that the testes are highly susceptible to loss of Zfy1 and Zfy2, and to SARSCoV-2 infection, illustrating the fragility of male fertility. The novel mouse models utilized to examine the effect of Zfy deletion and COVID-19 disease allow for the assessment of attributed solely to intended manipulations (gene knockout or viral infection). This provides insight for further investigations to elucidate the mechanisms of Zfy gene contribution and COVID-19’s lasting impact on mice and by extension possibly mankind.
Three-dimensional (3-D) ovarian tissue culture system supported by synthetic polyvinyl alcohol (PVA) hydrogel
(University of Hawaii at Manoa, 2022) Miyagi, Marissa; Yamazaki, Yukiko; Developmental & Reproductive Biology
Cancer therapies such as radiation and chemotherapy may damage ovarian function and result in infertility. The development of assisted reproductive technologies (ARTs) supports fertility preservation and has become a significant area of study to improve the quality of life for cancer patients undergoing chemoradiotherapy. In vitro follicle culture is a promising ART to preserve the fertility of cancer patients. Generally, conventional two-dimensional (2-D) ovarian tissue culture is unable to maintain the normal architecture of the ovarian tissues, and suppresses the transition from secondary to antral follicle development. To overcome this problem, the Yamazaki lab previously developed a three-dimensional (3-D) ovarian tissue culture system supported by Matrigel. As a scaffold, Matrigel significantly enhanced antral follicle development and oocyte quality. However, Matrigel is an animal-derived natural hydrogel and is therefore not clinically applicable.In this study, we tried to develop a clinically applicable 3-D ovarian tissue culture system using the mouse ovary as a model. For this purpose, we replaced animal-derived Matrigel with synthetic polyvinyl alcohol (PVA) hydrogel. PVA hydrogel is non-animal derived and synthetically engineered. PVA hydrogel has a defined composition, no batch-to-batch variability, and can be applied clinically. To establish a 3-D ovarian tissue culture system supported by PVA hydrogel, we first set the appropriate mechanical condition of PVA hydrogel to support follicle development (Specific Aim 1: To determine whether PVA hydrogel supports follicle growth in the 3-D culture system). Next, we examined ECM-mimetic bioactive modification in PVA hydrogel (Specific Aim 2: To determine the effect of extracellular matrix (ECM)-derived cell-adhesive peptides on follicle growth and oocyte development). To examine the effect of three ECM-derived cell-adhesive peptides (collagen-derived Gly-Phe-Hyp-Gly-Glu-Arg (GFOGER), laminin-derived Tyr-Ile-Gly-Ser-Arg (YIGSR), and fibronectin-derived Arg-Gly-Asp (RGD)) on follicle growth and oocyte development, the ovarian tissues were cultured in peptide-modified PVA hydrogels. We found that (1) PVA hydrogel successfully supported follicle growth as a scaffold, (2) RGD-modified PVA hydrogel significantly promoted antral follicle development and oocyte maturation, and (3) the combination of three peptides in PVA hydrogel synergistically enhanced folliculogenesis in vitro. In conclusion, our 3-D system supported by synthetic PVA hydrogel can be a promising technology to preserve the fertility of cancer patients, as well as an excellent model to define bioactive molecules essential for in vitro folliculogenesis.
The Role of Deptor in Intrauterine Growth Restriction
(University of Hawaii at Manoa, 2021) Nunes, Lance Gregory A.; Urschitz, Johann; Developmental & Reproductive Biology
Maternal obesity or insufficient nutrient supply during pregnancy can result in fetal overgrowth and fetal growth restriction, respectively, and cause serious health problems for both the mother and the fetus. Dysregulated placental nutrient transport is believed to be a mediator for both conditions. The mammalian target of rapamycin (mTOR) is thought to be the nutrient sensor of the placenta. mTOR is comprised of two complexes, mTORC1 and mTORC2. Both mTORC1 and mTORC2 have downstream targets that regulate cell growth, proliferation, ion transport, cytoskeletal remodeling, and more. Intrauterine Growth Restriction (IUGR) is associated with decreased nutrient transport and restricted nutrient availability for the fetus. It has been shown that mTOR is downregulated in placentas of pregnancies complicated by IUGR. The DEP-domain containing mTOR interacting protein (DEPTOR) has recently been shown to be an endogenous inhibitor of mTOR. We hypothesized that placenta-specific DEPTOR knockdown (KD) results in restoration of mTOTRC1/2 signaling, normalizing placental amino acid transport capacity and preventing IUGR. In the first part of this thesis, we generated a transgenic mouse model that expressed a placenta-specific DEPTOR KD cassette to study the in vivo effects of placental DEPTOR KD. Results showed that trophoblastic DEPTOR inhibition leads to increased expression of mTORC1/2 signaling, increased activity of the two main placental amino acid transport systems, and a marked increase in birth weight. In the second part of this thesis, we aimed to optimize parameters for placental sonoporation, a minimally invasive in vivo gene delivery technique that utilizes ultrasound and DNA conjugated lipid microbubbles. We observed successful placental transgene expression after sonoporation following intravenous injection of DNA conjugated lipid microbubbles. Taken together, this thesis has elucidated the role of placental DEPTOR in the regulation of mTORC1/2 signaling and fetal nutrient transport, providing insight into the molecular mechanisms that govern fetal growth and, ultimately, health. Furthermore, the successful sonoporation trials illustrate the possibility for this technique to be used as an alternative to transgenics for studying in vivo gene modulation, as well as a potential adaptable technique for the treatment of complications due to placental dysregulation.
The Role of N-WASP in ORC4 Mediated Polar Body Extrusion
(University of Hawaii at Manoa, 2020) Elento, Dominique Chanel Olita; Ward, W. Steve; Developmental & Reproductive Biology
Oocyte meiosis is completed though two asymmetric cellular divisions, where the oocyte extrudes half of its DNA two times, into two smaller daughter cells called polar bodies. N-WASP is an important factor in the actin polarization pathway. We investigated whether N-WASP is required for the extrusion of both polar bodies during mouse female meiosis. Previous work in our laboratory demonstrated that the DNA replication protein origin recognition complex 4 (ORC4) forms a cage around the chromosomes that will be extruded during polar body extrusion (PBE), but not the chromosomes that remain in the oocyte. My hypothesis is that N-WASP’s involvement in action polarization may be important for PBE, and related to ORC4 cage formation. We first localized N-WASP during oocyte progression through meiosis and found that it co-localizes with ORC4, except that N-WASP was not present in the initial stage, GV oocytes, while ORC4 was. This finding suggested the possibility that N-WASP was synthesized during meiosis. We tested this by injecting siRNA into GV oocytes upon collection, then allowing them to mature. We found that only 23.2% + 2.5% (MEAN+SD) of oocytes injected with siRNA directed towards N-WASP progressed to MII (the normal end point for in vitro GV maturation), while 92.9% + 10.1% of oocytes injected with control siRNA progressed to MII. These data suggest that (1) N-WASP may function in the same process that regulates PBE that ORC4 does, (2) that N-WASP is synthesized during meiosis, and (3) that N-WASP is required for progression to MII.
Identification of Quantitative Trait Loci (QTL) for Vibration Attraction, Locomotor, and Sleep Behaviors in Astyanax mexicanus
(University of Hawaii at Manoa, 2020) Lactaoen, Kimberly Diego; Yoshizawa, Masato; Developmental & Reproductive Biology
Rapid evolutionary shifts of multiple behavioral traits are frequently observed in the animal kingdom. Certain genetic mechanisms of these behavioral traits are thought to parallel psychiatric conditions. What puzzles geneticists and evolutionary biologists is that many behavioral traits are multigenic, which conflicts with rapid evolution. One possible explanation for this was shown through human genetics studies on Autism Spectrum Disorder (ASD), in which a high-burden of common risk variants induce many behavioral shifts in an individual. A core assumption is that many risk variants should be pleiotropic—one gene influences multiple behaviors. To test this assumption, we used Astyanax mexicanus, a teleost species constituting two morphs, a surface morph and a cave morph. The cave morph, evolved from its conspecific, exhibits many distinct behavioral phenotypes derived from cave-type mutations in the genome. In this thesis, F2 progeny generated from a pair of a cavefish and a surface fish (N=580), addressed the hypothesis that the evolution of a set of polygenic behaviors are influenced by pleiotropic loci. In Chapter 1, four diversified behaviors (vibration attraction, locomotor activity, sleep, and turning) were measured in cavefish (N=8), surface fish (N=8), and F2 hybrids (N=580). Behavioral correlation analyses among F2 hybrids indicated a tight link between night activity and night sleep, suggesting shared (pleiotropic) genetic factors among these behaviors. In Chapter 2, a genetic linkage map comprising of 27 linkage groups with 1,445 markers (40,268 cM) was generated through the genotyping-by-sequencing data of genomic DNA extracted from the F2 individuals in Chapter 1. On this linkage map, 16 quantitative trait loci (QTL) associated with vibration attraction, locomotor, and sleep behaviors were detected. Also, 11 of 16 QTLs were associated with two or more behavioral measurements, supporting the hypothesis that rapid multi-behavioral shifts are underlain by pleiotropic genetic factors. Interestingly, newly detected QTLs for daytime and nighttime sleep were distinct, suggesting separate pathways for regulating diurnality. In summary, shared genetic factors can provide the framework for studying rapid evolution and can inform potential target pathways in the study of psychiatric conditions.
Juvenile Shank3b Deficient Mice Present With Behavioral Phenotype Consistent With Autism Spectrum Disorder
(University of Hawaii at Manoa, 2017-12) Balaan, Chantell; Developmental & Reproductive Biology
Autism spectrum disorder (ASD) is a pervasive, multifactorial neurodevelopmental disorder diagnosed according to deficits in three behavioral domains: communication, social interaction, and stereotyped/repetitive behaviors. Mutations in Shank genes account for ~1% of clinical ASD cases with Shank3 being the most common gene variant. In addition to maintaining synapses and facilitating dendritic maturation, Shank genes encode master scaffolding proteins that build core complexes in the postsynaptic densities of glutamatergic synapses. Male mice with a deletion of the PDZ domain of Shank3 (Shank3B KO) were previously shown to display ASD-like behavioral phenotypes with reported self-injurious repetitive grooming and aberrant social interactions. Our goal was to extend these previous findings and use a comprehensive battery of highly detailed ASD-relevant behavioral assays including an assessment of mouse ultrasonic communication carried out on key developmental days in male and female Shank3B KO mice. We demonstrate that ASD-related behaviors, atypical reciprocal social interaction and indiscriminate repetitive grooming, are apparent in juvenile stages of development of Shank3B KO mice. Our findings underscore the importance of utilizing Shank mutant models to understand the impact of this gene in ASD etiology, which may enable future studies focusing on etiological gene-environment interactions in ASD.
Exocyst Dysfunction Impairs Urothelial Development Resulting in Congenital Ureter Obstruction
(University of Hawaii at Manoa, 2016-08) Napoli, Josephine
Hydronephrosis is the most commonly detected abnormality in prenatal ultrasounds. The most Prevalent cause of prenatal hydronephrosis is congenital obstructive nephropathy, with obstructions at the ureteropelvic junction (UPJ) region as the most common site for obstruction. The cause and developmental process of obstruction formation at the UPJ is poorly understood. We developed a Sec10 conditional knockout (Sec10-CKO) mouse model where Sec10, a critical subunit of the exocyst complex, has been knocked out in the urothelium that lines the ureter. Sec10-CKO embryos develop complete bilateral UPJ obstructions and hydronephrosis by day 18.5 of gestation (E18.5). Using this model, we were able to study the development of prenatal hydronephrosis. We determined that the urothelium lining the ureters of Sec10-CKO mice fails to differentiate, with the earliest evidence of this starting at E16.5. Loss of key terminal differentiation markers, including uroplakins, weakens the urothelial barrier that prevents urine from permeating into ureter tissues. Our data suggests that a luminal wound healing response at the UPJ starts at E17.5, which would correspond with the time point urine first starts to flow. We measured three characteristics consistent with a fibrotic wound healing response: (1) increased expression of TGFβ1, a key regulator of wound healing and fibrosis, (2) gene expression profiles consistent with myofibroblast activation, with increased proliferation at the site of obstruction, and (3) evidence of stromal remodeling. Our findings support a model where prenatal UPJ obstructions may be caused by an impaired urothelial barrier that allows urine to permeate and damage tissues lining the lumen, inducing a fibrotic wound healing response.

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