Mammalian embryo quality is often assessed by morphology, the timing of mitotic events, and aneuploidy. These parameters are more commonly investigated in non-equid mammalian species, therefore, the parameters which indicate embryo quality in the horse remains to be fully elucidated. The overall objective of this dissertation was to optimize the in vitro production (IVP) of equine embryos by understanding the consequences of errors that can arise during pre-mitotic and post-mitotic events. This was accomplished in 3 chapters. Chapter 1 is an introduction to the dissertation. Chapter 2: Review of relevant literature provides a review of the literature pertaining to early mammalian embryo development, knowledge gaps, and implications for equine embryo quality assessment. The specific aims of the experiments conducted in Chapter 3: Pronuclear formation and cytoplasmic extrusion and Chapter 4: Aneuploidy in the early cleavage stage equine embryo were to determine the pre- and post-mitotic events in the zygote using fluorescence microscopy, and the exact percentage of aneuploid embryos in the cleavage stage equine embryo. Currently, the general timing of pronuclear (PN) apposition and fusion is unknown, and this information is critical to bridge the gap in understanding the transition from the PN phase to first cleavage. We found that in vitro PN formation is delayed by more than 6 hours compared to previous observations of PN in the in vivo equine model. The sperm head is still intact at 12 hours post intracytoplasmic sperm injection (ICSI). Additionally, cytoplasmic extrusion (CE) is a pre-cleavage event unique to the horse, and previous studies have shown that CE is a critical event in equine embryo development and occurs as it is associated with developmental success. While CE has also been described as a fragmentation event, there was little previous evidence that the extruded material contains DNA. Using fluorescence microscopy, we confirmed that no DNA was present in the extruded CE material and at this moment, the zygote was at the metaphase of mitosis. In Chapter 4, euploid and aneuploid blastomeres were determined by single-cell sequencing for the first time in the horse. To accomplish this, embryos were removed from culture between days 2 and 3 of embryo culture following ICSI. Because embryonic endpoints were unknown, a prediction model using machine learning using time-lapse data from Chapter 3 was used to determine if the embryos would have failed or survived to blastocyst stage and whether the early developmental stages were predictive of aneuploidy. According to the results from the sequencing data and prediction model, the blastocyst prediction model could not distinguish embryos with a higher percentage of euploid blastomeres in the horse. The percentage of aneuploid embryos was alarmingly high (81.25%), which could be explained by the frequent multipolar divisions observed during the early cleavage divisions. The findings from this work inform future research efforts to improve IVP practices in the horse with potential applications for improving embryo outcome predictions, selection methods as well as pregnancy outcomes.

Appropriate timing of reproduction is crucial to animals’ reproductive success and fitness. Animals living in unpredictable environments do not constrain their breeding to one season, but rather breed any time of the year in which good conditions occur. These animals, termed “opportunistic” breeders, cannot predict when good conditions will occur based on the seasons, and must instead respond quickly to proximate environmental factors to successfully breed and raise young. Animals may respond to a number of different cues in the environment to decide when is the best time to breed, and thus the brain must integrate these varying sources of information to produce an appropriate decision.

Cue integration in the brain takes place in the hypothalamus located at the base of the brain and, if conditions are favorable, will result in a physiological reproductive cascade. In stimulatory conditions gonadotropin-releasing hormone (GnRH) is released from the hypothalamus, which stimulates the production and release of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) from the pituitary gland. These two hormones act on the gonads to increase production of testosterone, estradiol and gametes, leading to an increase in fertility. These changes in physiology are associated with an increase in reproductive behavior, including courtship and song behavior. Release of GnRH from the hypothalamus is considered the gateway for reproduction, so research on the mechanisms of reproductive timing often focus on how information is transmitted to the GnRH system and hormones that influence GnRH release.

The goal of this dissertation is to increase our understanding of the mechanisms underlying reproductive timing in response to social cues, stress, and food availability in an avian model of opportunistic breeding, the zebra finch (Taeniopygia guttata). Social cues and food availability stimulate and stress inhibits reproduction in zebra finches, but the endocrine underpinnings of these phenomena are undefined. Chapter 1 provides an overview of the current scientific knowledge regarding the effects of social cues, stress, and food availability on reproductive physiology. The remaining chapters detail experiments I conducted to further elucidate the mechanisms responsible for the effects of these cues on reproductive physiology and behavior, and the dissertation concludes with a discussion chapter.

Chapter 2, “Social stimulation, reproductive behavior and the thyroid hormone deiodinase system in an opportunistically-breeding songbird”, examines the role the presence of an available mate plays in activating reproduction. Isolated males were compared to those receiving a female stimulus or a male stimulus animal. I measured key genes in various tissues of the reproductive axis, activation and protein content of the hypothalamus, and reproductive behaviors. My findings suggest that the hypothalamus is indeed active in response to an available mate and appropriate reproductive behaviors increase, but more work needs to be done to determine how GnRH release might be governed by social cues.

Chapter 3, “Differential response of GnIH in the brain and gonads following acute stress

in a songbird”, looks at stress as a potential mechanism by which to time reproduction. Stress is a known inhibitor of reproduction across vertebrate taxa, and thus can be used to time breeding by increasing the stress hormone corticosterone (resulting in decreased reproductive axis function) during times when environmental conditions are poor. This chapter focuses on gonadotropin-inhibitory hormone (GnIH), a hormone known to inhibit reproduction at all levels of the reproductive axis, in the hypothalamus and gonads. While previous research has suggested a role for GnIH in stressed-induced reproductive inhibition, this is the first experiment to show a differential response of the GnIH system in the hypothalamus and the gonads in response to stress. This study provides more information about the role of GnIH, particularly that the GnIH response to stress may not be as conserved throughout vertebrate evolution as previously thought.

Chapter 4, “Corticosterone measurements differ in plasma sampled from a wing vein and

trunk blood”, is concerned with the methodology used to measure hormones in laboratory and field settings and the potential sources of intra-individual hormone variability. When comparing corticosterone levels between wing blood, a source commonly used in field studies, and trunk blood, a source commonly used in terminal studies, we found differences in corticosterone levels within an individual. This finding illustrates that the source of blood collection for hormone assay is an important source of variability and must be considered in experimental design and when comparing results across studies.

Chapter 5, “Perception of food availability determines reproductive response to food restriction in the zebra finch Taeniopygia guttata”, investigates the role of perception in food-induced reproductive suppression and activation. Animals that were food restricted decreased reproductive parameters (GnRH, general activity), while animals that were food restricted but could see food during restriction maintained higher levels of GnRH and activity similar to controls. This suggests that the visual cue of food is enough to maintain reproductive activity even in conditions of starvation, and that perceptual information as well as metabolic information from food is important in these reproductive activity considerations. Additionally, measurement of expression of the orexigenic peptide neuropeptide Y (NPY) and its correlation with GnRH across groups indicates a potential novel role for this peptide in governing reproductive activation in response to the perception of food. Taken together, these studies indicate the importance of brain integration of social cues, stress, and food availability to initiate breeding during favorable conditions in opportunistic breeders.

Retroviral replicating vectors (RRV) are capable of tumor-selective replication and stable integration into the cancer cell genome, and are currently under investigation for prodrug activator gene therapy of recurrent high grade glioma in phase II/III clinical trials. In preclinical studies evaluating RRV gene therapy in intracranial glioma models, we showed effective long-term tumor control in human glioma xenograft models as long as prodrug administration was continued. In contrast, tumor eradication was achieved even after cessation of prodrug administration in immunocompetent syngeneic glioma models. Accordingly, my research has focused on investigating the immunological mechanisms involved in RRV gene therapy, and evaluating combined viro-immunotherapy approaches to improve RRV-mediated tumor transduction and therapeutic efficacy, as well as to confer a dominant foreign antigen to be targeted by adoptively transferred T cells.

RRV replication may be delayed at lower inoculation doses, if initial distribution is confined to the area surrounding an intratumoral injection site. More rapid tumor transduction may be achieved if the inoculum is distributed more evenly within the tumor. Alloreactive cytotoxic T lymphocytes (alloCTL) are migratory cells trained to react to major histocompatibility complex (MHC) that are selectively displayed on gliomas. Combining alloCTL and RRV gene therapy as individual therapies showed added benefit compared to either modality alone. We further tested the use of alloCTL as vector producer cells to deliver marker and suicide genes to tumors after their transduction with RRV (alloCTL/RRV). When compared against either therapy alone in a subcutaneous glioma model, the greatest benefit was achieved by alloCTL/RRV. This suggests that alloCTL/RRV can serve as anti-tumor effector cells targeting endogenous antigens, as well as motile ‘carriers’ that facilitate spread of RRV to tumor cells.

Aside from MHC, there are few well-characterized / immunogenic tumor-associated antigens with defined epitopes in glioblastoma. The efficient spread and permanent genomic integration of RRV can be utilized to induce stable expression of viral antigens that may represent useful immunotherapeutic targets. We developed an RRV displaying the lymphocytic choriomeningitis virus glycoprotein gp33-43 (gp33) epitope fused to the viral env gene. Target cell-specific cytotoxicity by transgenic lymphocytes engineered with a T cell receptor recognizing the gp33 epitope (P14 cells) was observed in vitro and in vivo. P14 adoptive transfer in immunocompetent hosts increased survival. Thus, the use of RRV to deliver highly immunogenic viral antigens to glioma could enable more effective immunotherapy for this disease.