Maternally-derived transcripts and proteins support early bovine and ovine embryo development until the 8- to 16-cell stage, at which time embryonic transcripts become essential for continued development. One purported mechanism for the switch from maternal to zygotic control of development (maternal to zygotic genome activation; MZGA) is the appearance of transcription factors that activate specific genes in the embryonic genome. Members of the E26 transformation specific (Ets) family are unique transcription factors involved in development, differentiation, and protease regulation. This study was undertaken to evaluate expression and function of the Ets transcription factors, Ets-1, Ets-2, and Elf-1, in early bovine and ovine embryos from the one-cell stage to Day 15 of pregnancy (Day 0 onset of estrus). In the first experiment, bovine embryos from the one- to 16-cell stages were derived by in vitro maturation, fertilization, and culture. Days 6, 8, 10, 12, and 14 embryos were collected nonsurgically from estrous synchronized and superovulated cows. RNA was extracted at the appropriate time interval and reverse transcribed. The resultant cDNA was amplified by PCR using primers designed for Ets-1, Elf-1, and Ets-2. Ets-1 transcripts were present in both primary and matured oocytes, cleavage stage embryos, and Days 10, 12, and 14 embryos, as well as in the positive control, bovine ovary. Elf-1 transcripts were detected in the matured oocyte, cleavage stage embryos, and Days 6, 10, and 14 embryos. Ets-2 transcripts were not observed in the embryonic stages investigated or the bovine ovary. Ovine embryos were surgically collected from synchronized and superovulated ewes and similarly analyzed for Ets-1 and Elf-1 expression using the same RNA extraction and RT-PCR technique. Embryos expressed both transcripts at Days 13 and 15, but did not show expression at any of the earlier stages evaluated. The second experiment was designed to determine if inhibition of ETS-1 translation would interfere with development and plasminogen activator (PA) production in bovine embryos. Plasminogen activator production was evaluated in Days 5 and 6 embryos nonsurgically collected from superovulated cows and cultured in 1, 2.5, 5, or 10 ��M concentrations of sense or antisense Ets-1 oligonucleotides. In preliminary experiments, 1 ��M antisense was ineffective in suppressing PA production, and 10 ��M oligonucleotides were detrimental to development. Day 5 embryos treated with 2.5 ��M oligonucleotides inhibited developmental effect and total PA production was (P<0.05) lower in antisense treatments when compared to either control or sense treatments. No difference (P>0.10) in PA production was observed between Day 6 embryos treated with 2.5 or 5 ��M sense and antisense oligonucleotides. A significant time effect on PA production was observed in both Day 5 and Day 6 embryos cultured in either 2.5 or 5 ��M concentrations of oligonucleotides. Based on these results, it is unlikely that Elf-1 and Ets-2 are involved in MZGA because the former is constituitively expressed throughout development, and the latter was not observed. There is some uncertainty regarding the expression of Ets-1 during MZGA. This factor may be expressed after MZGA for controlling PA production and other proteases involved in extracellular matrix turnover and early germ layer formation. / Graduation date: 2003

In the first experiment, estradiol production was measured in cultured embryos collected on Days 7, 9, 11, 13 and 15 post-mating. Estradiol was detected in Day 7 embryos and production increased approximately 50-fold (P<0.05) in medium recovered from Day 13 compared to Day 11 embryos and was greatest (P<0.05) from Day 15 embryos. The second experiment evaluated the luteotrophic effect of estradiol in the llama. Daily injections of vehicle only, 5 or 10 mg estradiol benzoate (EB) in isopropylmyristate were administered on Days 7-15 (Day 0=hCG ovulation induction). Mean progesterone levels were greater (P<0.05) on Days 14, 15, 16, and 17 in llamas injected with 10 mg EB compared to llamas injected with vehicle or 5 mg EB. The final experiment analyzed differences in estrogen receptor expression in the corpus luteum (CL), ovary, and uterus using Reverse Transcription-Polymerase Chain Reaction. A significant linear regression (P<0.05) was detected for both ER�� (increased from Days 7-11), and ER�� (decreased from Days 7-11) in llama CL. ER�� expression in ovary was higher (P<0.05) compared to Days 7 and 9 CL. ER�� expression in CL was lower on Day 9 (P<0.05) than on Days 7 and 11 in pregnant animals. ER�� expression decreased (P<0.01) from Days 7-11 in uterus and expression was lower in pregnant versus non-pregnant uterus (P<0.10). ER�� expression was higher in pregnant versus non-pregnant uterus (P<0.10). No differences (P>0.10) in ER�� or ER�� expression were detected in endometrium due to reproductive status or days post-mating. A decrease (P<0.01) in ER�� expression was detected in pregnant uterus from Days 7-13. ER�� expression was lower (P=0.12) in juvenile uterus versus pubertal females. ER�� expression in non-pregnant uterus was different (P<0.05) by days post-mating and reproductive status. These data suggest that estradiol produced by the embryo may be involved in embryo migration and maternal recognition of pregnancy in the llama, and these events may be mediated by differential expression of ER subtypes / Graduation date: 2000

published_or_final_version / Anatomy / Master / Master of Philosophy

Graduation date: 1996

As bacteria are becoming resistant to conventional antibiotics, researchers are looking for new ways to combat microbial infection. We have begun to adopt genetic and functional genomic approaches to define the molecular determinants of pathogen resistance in the dung beetle, Euoniticellus intermedius. This dung beetle survives microbe-rich environments such as dung. This ability makes it a potential model for the study of infectious agents and ecological damage. To date, E. intermedius has not been studied at the molecular level. In this study, a range of complimentary analytical techniques were used to characterize the E. intermedius embryonic cell line established in our laboratory. These techniques characterize morphology, growth characteristics, karyotype, isoenzyme patterns and embryonic development. Complete characterization of the E. intermedius cell line is essential for the cell banks and for the regulatory requirements in biopharmaceutical production. This study followed gene sequences and their comparisons for both adult and cell line to confirm that the E. intermedius (EISA08) cell line is originated from the embryonic E. intermedius dung beetle. cDNA was synthesized from mRNA isolated from E. intermedius adult beetles and cell line (EISA08) was sequenced using GS (FLX) technology by a commercial facility, Inqaba Biotechnical Industries (Pty) Ltd, South Africa. In addition to characterization of the cell line, two genes, namely hopscotch and ribosomal protein S9 (RpS9), were selected from the Flylab genome data base. The E. intermedius database is a web-based system for the genome and transcriptome of the dung beetle to evaluate the immune system of the dung beetle (http://Flylab.wits.ac.za/). hopscotch was selected because it is believed to be involved in the JAK-STAT signalling pathway for anti-viral response, embryonic development and cell growth. Rsp9 was chosen as a loading control because it is expected to be a housekeeping gene. The conserved molecular signalling pathway JAK-STAT is used by E. intermedius (as in other insects and humans) for immune defence and early embryonic development. The project followed hopscotch and Rsp9 gene expression in all the E. intermedius life cycle developing stages; adult, pupae, larvae, embryo, and cell line cell growth, life cycle developing stages and embryonic development has was monitored. E. intermedius embryonic development is described as short germ-band. E. intermedius embryogenesis is regarded as basal and is observed in most arthropods. The study revealed that E. intermedius hopscotch is over expressed in the early developing stages, embryo, larvae, and pupae and in the newly established cell line EISA08. The results from this study lead to the suggestion that E. intermedius JAK-STAT pathway is activated early and has an important role in embryonic development, cell proliferation and immune defence. Studies of E. intermedius could provide more insight into the properties and evolution of innate immunity and embryonic development.

No description available.

During early embryonic development, endodermal cells leave the inner cell mass (ICM) and migrate over an extracellular matrix (ECM), located on the blastocoelic side of the trophectoderm, to form a continuous layer of extraembryonic endoderm. Cell migration events depend on a family of cell surface proteins known as integrins that bind specific ECM proteins. In an effort to understand the mechanisms involved in bovine endodermal cell migration, two experiments were conducted. In the first experiment, expression of the ECM proteins fibronectin, laminin and vitronectin was evaluated by immunofluorescent staining in in vivo and in vitro developing embryos during Day 6-10 and Day 7-10, respectively (Day 0=onset of estrus). Fibronectin was detected in all stages of in vivo and in vitro embryos, however no difference (P>0.10) was observed due to day or developmental stage. Laminin staining was moderately expressed in all stages of in vivo embryos, with an increase (P<0.05) in Day 10 in vivo embryos. Laminin staining in Day 9 in vitro embryos was less intense (P<0.05) than Day 7 and 8 in vitro embryos. Higher (P<0.05) expression of laminin was observed in Day l0 in vivo embryos as compared to Day 10 in vitro. Vitronectin staining was expressed throughout all stages of development. Day 6 in vivo embryos exhibited more intense (P<0.05) staining compared to Day 8 in vivo embryos. Day 10 in vivo embryos expressed more (P<0.05) vitronectin than Day 10 in vitro embryos. In the second experiment, the effects of ECM-type and inhibitors of integrin binding on bovine endodermal cell outgrowth from the ICM were evaluated. Day 7 embryos were nonsurgically collected and cultured for 96 h on either fibronectin-layered microdrops containing 0 (control), 0.5 or 1.0 mg/ml RGD and/or EILDV peptides or vitronectin-layered microdrops containing 0, 0.5 or 1.0 mg/ml RGD peptides. At 24-h intervals, ICM were photographed and the numbers of cells leaving the ICM were counted. Areas of cellular outgrowth were calculated from the photomicrographs. Compared to the control, addition of 0.5 or 1.0 mg/ml RGD, EILDV or RGD and EILDV did not (P>0.10) reduce the areas of cellular outgrowth from the ICM on matrices of fibronectin. Numbers of cells in outgrowths were greater (P<0.05) in control ICM compared to 0.5 mg/ml RGD, but this effect was eliminated (P>0.10) when the inhibitor concentration was increased to 1.0 mg/ml. Addition of 0.5 or 1.0 mg/ml RGD did not reduce (P>0.10) the area of cellular outgrowth from the ICM on vitronectin and had no effect (P>0.10) on numbers of cells in the outgrowths. Detection of fibronectin, laminin and vitronectin by immunofluorescence suggests these proteins are present in the developing bovine embryo to support endodermal cell migration and stabilization in extraembryonic endoderm formation. Because cell migration over fibronectin and vitronectin was not inhibited by the RGD and EILDV peptides, endodermal cells must use either an integrin that recognizes alternative binding sites in fibronectin and vitronectin or an alternative cell adhesion system. / Graduation date: 2003

Glutamine serves as a major energy source for all stages of preimplantation mouse embryo development, whether the embryos are raised in vivo or in vitro from the one-cell stage. Glutamate dehydrogenase (GDH) and glutamine synthetase (GS) are enzymes that are involved in the metabolism of glutamine. GDH catalyzes the conversion of glutamate into a-ketoglutarate, a primary component of the tricarboxylic acid cycle. GS catalyzes the conversion of glutamate to glutamine. The expression of GDH RNA and GS RNA were analyzed in preimplantation mouse embryos using reverse transcription (RT) with an oligo dT primer followed by Polymerase Chain Reaction (PCR) amplification of GDH and GS cDNAs using gene specific primers. Data show that GDH RNA is expressed in mouse embryos grown in vivo at the one-cell, two-cell, eight-cell, and blastocyst stages of development. GS RNA is not expressed at the one-cell stage, but first appears at the two-cell stage and is expressed at the eight-cell and blastocyst stages. Semiquantitative PCR analysis using a globin internal standard demonstrated that GS RNA is present at high levels at the two-cell stage and declines by 51 % by the blastocyst stage. These results suggest that, within the preimplantation mouse embryo, GDH RNA is expressed by both the maternal genome as well as the embryonic genome, while GS RNA is only expressed by the embryonic genome. This study provides an explanation for why glutamine is utilized as an energy source during preimplantation development, which allows for a better understanding of glutamine metabolism and its role during early mouse development. / Department of Biology

The cell cycle is a highly regulated process driven by endogenous factors that have regulatory functions. Certain proteins such as cyclins and cyclin-dependent kinases (cdks) are needed to progress through the four phases of the cell cycle. Cell cycle regulatory proteins have been characterized in somatic cells and exhibit phase specific expression patterns. However, the changes in expression of these proteins have not been characterized in early cleavage stage mouse embryos. This study utilized indirect immunofluorescence microscopy to determine the expression pattern of cell cycle regulators cyclin B 1, cyclin E, and CDK2 during the first three cell cycles of preimplantation mouse embryo development. Results suggest unique and specific patterns of expression for all three cell cycle regulators at different stages of the cell cycle. In G1 of the first cell cycle, cyclin E is expressed at high levels, whereas cyclin B 1 and CDK2 are expressed at moderate levels. During DNA synthesis (S phase), CDK2 levels slightly increase. However, cyclin B 1 and cyclin E levels begin to decline in S and continue to decrease to minimal levels in G2. CDK2 expression follows a similar trend during G2, decreasing considerably. During the second cell cycle, cyclin B 1 and CDK2 show staining patterns similar to the first cell cycle. The expression of cyclin E is maintained at a moderate level throughout the entire second cell cycle. Cyclin B 1, cyclin E, and CDK2 are all expressed at moderate levels during GI of the third cell cycle. During S phase, cyclin B 1 and CDK2 are maintained at moderate levels, but cyclin E is decreased to minimal levels. The expression of all three proteins was minimal during G2. This study provides baseline information on the unique expression patterns of cell cycle regulators in early mouse embryos. The determination of cell cycle protein expression will allow for a better understanding of the complex mechanisms in the division process during preimplantation mouse embryo development. / Department of Biology

A dissertation submitted to the Faculty of Science, University of the Witwatersrand, Johannesburg, in fulfilment of the requirements for the degree of Master of Science. Johannesburg, 2014. / Insects are confronted by a large variety of potentially harmful microorganisms to which they are resistant as they are able to build up an efficient innate defense system that relies on three tightly interconnected reactions. One of the reactions is the transient and rapid synthesis of a battery of antimicrobial peptides (AMPs). Development of antimicrobial therapeutic drugs and vaccines is very crucial due to factors such as the emergence of multiple-drug resistance. AMPs have been termed natural antibiotics because of their large spectrum of activity. The current study focused on the isolation and identification of cationic antimicrobial peptides and the analysis of immune response in the South African Euoniticellus intermedius embryonic (SAEIE08) cell line upon exposure to pathogens. E. intermedius is of the Coleopteran order in the Scarabaeoidea superfamily. Liquid growth inhibition assay showed higher antimicrobial activity in SAEIE08 that was treated with heat-killed E. coli compared to untreated. Further evidence for antimicrobial activity was seen as a clear zone of inhibition in solid growth inhibition assay when a gel run with protein extracts was plated and overlayed with live E. coli. Changes in protein expression patterns that were analysed in SDS-PAGE and 2-D PAGE indicated the most intense bands and spots at low molecular weight sizes around 10 kDa and/or 16 kDa which implicated increased induction of AMP expression upon exposure to pathogen. Homologues of Saccharomyces cerevisiae proteins were found in some of the 5′/3′ RACE sequences. Possible explanation for matches to these homologues could be that short sequences were used for database searches. The proteins were identified as flavin-containing monooxygenase, long-chain fatty acyl-CoA synthetase, severe depolymerization of actin protein and serine/threonine protein kinase. Interestingly, these proteins play roles in metabolism, cell proliferation and/or molecular pathways which do occur when cells are exposed to stress. There was also an insect peptide allatotropin from Spodoptera frugiperda. The results show that there is inducible antimicrobial activity in embryonic E. intermedius cell line.