Retinoblastoma-binding protein 4 (RBBP4) is a subunit of chromatin remodeling factor 1 (CAF-1) and is essential for mammalian oocyte maturation, embryo survival, and embryo implantation. RBBP4 also localizes to the chromatin and is a ubiquitously expressed nuclear protein. Previous methods used to study this protein include short interfacing RNAs (siRNAs) and CRISPR/Cas9. These techniques have limitations such as determining an indirect depletion of proteins, may trigger compensatory mechanisms, and may not be useful in non-dividing primary cells. A new, acute, and rapid endogenous protein depletion technique called Trim-Away, can overcome these limitations. Trim-Away is also widely applicable since it can be used with many off-the-shelf reagents. Trim-Away utilizes the TRIM21-antibody interaction within the cytosol and the ubiquitin-proteasome pathway (UPP) to target and degrade a protein of interest. Studying RBBP4 using Trim-Away can offer insight into possible new functions of RBBP4 and its maternal effect, and increase the knowledge on a new, acute, and endogenous protein depletion technique. Here we report that, RBBP4 is required for proper blastocyst development and RBBP4 is more abundant in MII oocytes than GVBD oocytes. We also report that the loss of RBBP4 hinders RNA synthesis and causes cell death in later stages of embryo development. While our Trim-Away methodology can deplete RBBP4 as early as the 2-cell stage in embryos, our oocyte Trim-Away protocol needs to be optimized.

Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Pharmacology & Toxicology Student: Zuzana Faustová Supervisor: Prof. Doutor Jorge Miguel de Ascenção Oliveira PharmDr. Lukáš Červený, Ph.D. Title of diploma thesis: The impact of SIRT1 inhibition on zebrafish morphology and behavior After discovery of connection between yeast Silent Information Regulator 2 (Sir2) and its ability to alter lifespan, Sir2 and its seven mammalian orthologs became very attractive therapeutic target. These so called sirtuins are members of a histone deacetylase family. They possess unique catalytic activity having nicotinamide adenine dinucleotide as a cofactor and their function can be influenced by environmental factors. The aim of this diploma thesis was to extend knowledge of Sirtuin 1 (SIRT1), which is from all mammalian sirtuins considered to have the closest relation to yeast Sir2. At first we tested the impact of SIRT1 inhibition on early developmental stages of zebrafish (Danio rerio) embryos and larvae, finding out that SIRT1 is important for normal development and SIRT1 inhibition or malfunction result in cardiovascular defects, delayed development, and death. Additionally, we tried to learn more about SIRT1 and its connection with Parkinson's disease by combining nontoxic doses...

<div> <p>Mammalian embryos undergo a dramatic amount of epigenetic remodeling during the first week of development to establish the correct epigenetic status to support the developmental program. SWI/SNF chromatin remodeling complexes are multi-subunits complexes and utilize energy from ATP hydrolysis to modify chromatin structure non-covalently. The collection of subunits determines the identity of a given SWI/SNF chromatin-remodeling complex, directs its activity, and dictate where that complex will act. The aims of this study were to 1) determine the requirement of SNF5, a SWI/SNF core subunit found in BAF and PBAF complexes during preimplantation porcine embryo development, 2) determine the requirement of BRD7, a PBAF complex-specific subunit during preimplantation porcine embryo development, and 3) investigate the role of <i>CDH1</i>, a downstream gene regulated by ARID1A, another subunit found exclusively in BAF complexes, in cleavage stage porcine embryos. Our results indicate that the differential requirement for each subunit during early embryo development. Depletion of different subunits results in embryo arrest at distinct developmental stage. Together, our data suggest the SWI/SNF chromatin remodeling complexes are necessary for proper porcine embryo development and this requirement is associated with the composition of the complex.</p> </div> <br>

L’anémie de Fanconi (FANC) est une maladie génétique humaine causant chez les patients une anémie sévère, une susceptibilité accrue à certains cancers, ainsi que des anomalies du développement dont un hypogonadisme. La voie FANC est impliquée dans la réponse au stress réplicatif et la réparation de l’ADN, et joue un rôle potentiel dans la régulation physiologique des cellules souches fœtales et adultes. Dans les modèles murins de la voie FANC, le phénotype majeur est une infertilité liée à un problème de développement du lignage germinal, qui est un paradigme pour l’étude des mécanismes contribuant à la pathologie. Notre étude a porté sur la caractérisation du défaut des cellules germinales primordiales (CGP) dans les embryons murins fancg-/-. Nous avons mis en évidence un défaut numérique des CGP très tôt dans le développement du lignage germinal dès les stades 9,5-10,5 jours post coïtum (jpc). Les CGP présentent un léger défaut de la prolifération à 10,5-11,5 jpc, mais aucun blocage de cycle. L’atteinte proliférative semble trop faible pour expliquer à elle seule la diminution drastique du nombre de CGP, ainsi que le profil mosaïque des gonades embryonnaires avec la présence de cordons dépourvus de CGP observé à 13,5 jpc. L’étude in vivo et ex vivo du comportement migratoire des CGP fancg-/- a permis de mettre en évidence des anomalies de la motilité des CGP, probablement liée à une activation anormale de la GTPase RAC1. Nous avons observé à 11,5 jpc une diminution du nombre de cellules au niveau du front de migration de la population de CGP. Ces anomalies entraîneraient ainsi un retard de migration et l’incapacité pour une fraction des GCP fancg-/- d’atteindre correctement les crêtes génitales à 11,5 jpc. La déplétion des CGP semble ainsi liée principalement à un défaut migratoire conduisant à une augmentation de la mort des CGP à 11,5 jpc, associé au défaut prolifératif intrinsèque des CPG fancg-/-. / Fanconi Anemia (FANC) is a genetic human disease, causing in patient a severe anemia, a higher risk to develop some cancers, and developmental anomalies including hypogonadism. The FANC pathway is involved in the replicative stress response and DNA repair, and has a potential role in the physiological regulation of fetal and adult stem cells. In mice models of the FANC pathway, the main phenotype is the sterility issue, associated to a developmental defect of the germ cell lineage, and is so a paradigm to study the pathology. Our study aimed to characterize the primordial germ cell (PGC) defect in fancg-/- mouse embryos. We showed a numerical defect of PGC early in the germ cell lineage development, as soon as 9.5–10.5 days post coitum (dpc). PGC show a mild defect of proliferation at 10.5–11.5 dpc, but no cell cycle arrest. This low proliferative defect can neither fully explain the drastic decrease of the PGC number, nor the mosaic profile of fetal gonads displaying cords without PGC at 13.5 dpc. In vitro and ex vivo studies of the migration behavior of fancg-/- PGC highlight abnormalities of the PGC’s motility, probably linked to abberant RAC1 GTPase activity. We also observed at 11.5 dpc a decrease of the cell number at the front of migration of the PGC population in 11.5 dpc fancg-/- embryos. Those motility defects could induce a migration delay, preventing a fraction of the fancg-/- PGC population to reach correctly the genital crests by 11.5 dpc. Hence, PGC depletion seems mainly linked to a migratory defect leading to increased cell death in PGC at 11.5 dpc, associated to an intrinsic proliferation defect of fancg-/- PGC.

In the modern poultry industry, intense genetic selection for meat production has negatively influenced the reproductive performance of commercial birds. Parthenogenesis, embryonic development in unfertilized eggs without any sperm-egg interactions, is known to hinder the normal fertilization process and could be one of the reasons for this reduced reproductive performance in the poultry industry. Therefore, the overall objective of this research was to gain a better understanding of the process of parthenogenesis using Chinese painted quail as the model. Studies on Chinese painted quail reproduction revealed that they are very inefficient in sustained sperm storage and that number of sperm penetrating the egg and subsequent embryonic development potentially alter egg transit time through the oviduct. This poor sperm storage capacity and high sperm-egg interaction requirement might be responsible for the occurrence of parthenogenesis in this species; and in fact, this makes Chinese painted quail an excellent choice for parthenogenesis research. Further, dams selected for parthenogenesis as well as embryonic development, including parthenogen size, alter egg components by possibly delaying the transit time of the egg through the oviduct. Also, both dams and sires selected for the parthenogenesis trait appear to influence their progenies performance, including 1st wk mortality and occurrence of parthenogenesis. Additionally, vaccination of virgin hens with live pigeon pox virus increases parthenogenesis as well as parthenogen size and livability by the direct action of the virus on the embryo. Moreover, live Newcastle disease virus under in vitro conditions was found to have similar effects on the embryo. Because parthenogenesis exists in the modern poultry industry, even the accidental selection of the trait in either males or females could have a negative impact on overall chick production and performance. Also, as vaccination is a routine practice in the industry, it is possible that vaccination of birds that carry the trait will reduce fertility and hatchability due to enhanced parthenogenesis. Overall, currently it appears that, parthenogenesis is adversely affecting the poultry industry; and therefore, additional research on the accurate determination of losses in the poultry industry due to parthenogenesis could further benefit the industry.

Lake and round whitefish are cold-adapted freshwater species with similar life histories and spawning behaviours. There have been several studies on the embryonic development of both species (particularly for lake whitefish), most utilizing constant temperatures. However, temperatures fluctuate in the field due to natural (e.g. seasonal changes) and anthropogenic (e.g. water discharged from once-through cooling processes) effects. Releases from once-through cooling processes may contain low levels of chemicals (e.g. morpholine) and radiation (e.g. tritium). This thesis examined and compared the impacts of thermal, morpholine, and radiation stressors on lake and round whitefish embryogenesis. To examine the effects of fluctuating incubation temperatures, lake and round whitefish were reared at constant temperatures, with seasonal temperature declines/inclines, transient temperature spikes, or seasonal temperature changes combined with temperature spikes. Round whitefish embryos had significantly higher mortality when reared at 8°C compared to lake whitefish, and seasonal temperature changes impacted development rate, growth, and hatch dynamics for both species. Temperature spikes had relatively little effect on development. The effects on embryonic development of chronic morpholine and low-dose radiation exposures were examined in round whitefish to compare with existing data in lake whitefish. Round whitefish embryos were more impacted by morpholine than lake whitefish (larger effects on growth and mortality at relatively lower concentrations) and v less impacted by low-dose radiation (little effect on growth or hatch dynamics). Post hatch, round whitefish embryos reared at 8°C, with rapid seasonal inclines, or with 500 mg L-1 morpholine had elevated mortality. All irradiated embryos had decreased mortality post-hatch compared to non-irradiated embryos. Thus, embryonic exposure to all stressors examined appears to alter post-hatch survival. This thesis better defines the effects of fluctuating incubation temperatures, chronic morpholine, and chronic radiation exposures on the embryonic development of lake and round whitefish. It also suggests that embryonic incubation conditions are important beyond hatching. / Thesis / Master of Science (MSc) / Lake and round whitefish are cold-adapted freshwater species. Both species play important ecological roles, with lake whitefish generally perceived as more economically and culturally important. Many studies have detailed lake whitefish embryonic development under constant stressors (e.g. temperature) but there are relatively few studies on round whitefish embryonic development. Both species experience seasonal temperature fluctuations in nature and may experience additional anthropogenic temperature, chemical, and radiation stress due to discharge from once-through cooling processes at thermal power plants, which may contain low levels of morpholine and radiation. Our study suggests that round whitefish embryos are more sensitive to elevated temperature and morpholine levels, but less impacted by chronic low-dose irradiation relative to lake whitefish embryos. The growth and development of both species are significantly affected by seasonal temperature changes.

The objectives of this study were (1) to determine the optimum in vitro culture conditions for in vitro fertilized bovine embryos among culture methods and (2) to investigate whether bovine fertilization and embryo development would occur in simulated microgravity conditions in vitro. In the first part of this study, the result showed that the microdrop method was the optimum culture method among groups; however, FBS supplementation did not significantly affect the bovine preimplantation embryo development in vitro. In the second part of this study, the result showed that bovine in vitro fertilization did not occur in simulated microgravity conditions. Moreover, none of the presumptive zygotes and 2-8 cell stage embryos were able to develop to further stages, while embryos cultured in standard microdrop method culture conditions developed normally.

No description available.

Extracellular matrix (ECM) modifications occur during plant growth, development, and in response to environmental stimuli. Key modulators of ECM modification in vertebrates, the extracellular matrix metalloproteinases (MMPs), have also been described in a few plants. Here, we report the identification of Loblolly pine (Pinus taeda) Pta1-MMP and its characterization during seed development and germination. The Pta1-MMP protein has the structural characteristics of other plant MMPs, and a recombinant protein (rPta-MMP) generated by using EST sequences for a seed-expressed MMP exhibits Zn2+-dependent protease activity, and is inhibited by the active site-binding hydroxamate inhibitor GM6001 and EDTA. The Pta1-MMP gene is expressed during embryo development, with transcript levels increasing from proembryo to early cotyledonary stage, then declining during late cotyledonary expansion and maturation drying. Protein extracts exhibited similar developmental-stage MMP-like activity. Seed imbibition in water facilited germination, which was stimulated by GA3 and inhibited by ABA. The timing of germination was mirrored by the presence of MMP-like protease activity in both water- and GA3-imbibed embryos. Pta1-MMP transcript levels increased in association with germination for both GA3- and water-treated embryos, in agreement with MMP-like activity. In contrast, by 10 days after imbibition, Pta1-MMP transcripts in ABA-treated embryos were at levels similar to the other treatments, although MMP-like activity was not observed. The application of GM6001 during Loblolly pine seed imbibition inhibited germination in a dose-dependent manner. Our results suggest that Pta1-MMP is required for ECM modification, facilitating the cell division and expansion required for both embryo development and germination. To our knowledge, this is the first report of an MMP in any gymnosperm and also its involvement in embryo development and subsequent germination. / Ph. D.

The objective of part one of this study was to determine if the presence of porcine growth hormone (pGH) during oocycte in vitro maturation (IVM) affected subsequent embryo development. Pig cumulus-oocyte complexes (COC) (n=987) were aspirated from slaughterhouse derived ovaries and cultured in BSA-free NCSU 23 medium containing porcine follicular fluid (10% v/v), cysteine (0.1 mg/ml) and hormonal supplements (eCG and hCG, 10 IU/ml each), 10 ng/ml EGF, and with or without pGH (100 ng/ml) for 22 h. The COC were then cultured in the same medium with or without 100 ng/ml pGH, but without hormonal supplements for an additional 22 h. After the completion of maturation culture, cumulus cells were removed and oocytes were co-incubated with frozen-thawed spermatozoa for 8 h. Putative embryos were transferred to NCSU 23 containing 0.4% BSA and cultured for 144 h. Embryo development was assessed on d 6 of culture. The treatment groups were as follows: treatment 1 = control group cultured in IVM medium alone; treatment 2 = 100 ng/ml pGH present of the first 22 h of maturation culture and absent for the second 22 h of maturation culture; treatment 3 = 100 ng/ml pGH absent for the first 22 h of maturation culture, but present for the second 22 h of maturation culture; and treatment 4 = 100 ng/ml pGH present throughout the entire IVM period. Embryos were visually scored for developmental stage at 144 h following fertilization. Each oocyte in the study received a developmental score, based on a scale of 1 = uncleaved, 2 = 2-cell embryo, 3 = 4- to 8-cell embryo, 4 = 9- to 16-cell embryo, 5 = morula, and 6 = blastocyst. The addition of pGH did not affect porcine embryo development as compared to the control (1.57 ± .08, 1.67 ± .08, 1.47 ± .08, and 1.60 ± .08, respectively; P > .10). Replicates within the study differed significantly from each other (P < .01) primarily because the development in replicate 6 was greater than for all others. There was a significant treatment by replicate interaction (P < .05); pGH added during the first 22 h of IVM and pGH added during the second 22 h of IVM in replicate 6 resulted in higher development scores than for controls and continuous pGH addition. However, in replicate 2, continuous pGH resulted in the greatest development. These results suggest that pGH may exert a stimulatory effect on embryo development when present in the IVM media; however, further studies using pGH in IVM culture are necessary. The objectives of the second part of the study were to examine aspects of intracytoplasmic sperm injection (ICSI) using membrane-disrupted spermatozoa, in vitro fertilization (IVF), and sperm-mediated gene transfer in the pig. Porcine oocytes were shipped overnight in maturation media at 39°C in a portable incubator. After 22 h of maturation culture, oocytes were washed in maturation medium without gonadotropins and cultured for an additional 22 h. Cumulus cells were removed and oocytes were divided into four treatment groups: treatment 1 = ICSI using membrane-damaged spermatozoa coincubated with linear green fluorescent protein (GFP) DNA; treatment 2 = ICSI using membrane damaged spermatozoa; treatment 3 = IVF with frozen-thawed spermatozoa coincubated with linear GFP DNA prior to IVF; treatment 4 = IVF with frozen-thawed spermatozoa with no DNA coincubation. Embryos were scored for developmental stage at 144 h following fertilization. Each oocyte in the study received a developmental score, based on a scale of 1 = uncleaved, 2 = 2-cell embryo, 3 = 4-cell embryo, 4 = 5- to 8-cell embryo, 5 = 9- to 16-cell embryo, 6 = morula, and 7 = blastocyst. Although no overall difference in development score was observed following the four different treatments, a treatment difference among cleaved oocytes was observed when comparing only the two ICSI treatments (P < .05); development scores were greater in the ICSI treatment in which sperm were not coincubated with linear GFP DNA prior to injection than when the coincubation was performed (3.76 ± .21 vs. 3.13 ± .17, respectively). No differences in development score were observed in the two IVF treatments. The percentage of embryos expressing the GFP transgene on d 6 of culture following fertilization was 7.3% in the ICSI+GFP group and 0% in all other treatment groups. Thus, sperm-mediated gene transfer using ICSI in the pig has been demonstrated, although success rates were low. / Master of Science