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In animal development, how an animal builds its body has been a fundamental and central question. Spanning different fields, we have a good understanding of how animals build their body axes (anterior-posterior, dorsal-ventral) and this understanding is now a routine concept when learning about animal development. There are a number of mysteries left when considering early development, and the establishment of left-right (LR) asymmetry remains one of them. One key problem is how initial symmetry is broken early in animal development and how this translates into molecular asymmetry. Our lack of understanding is based on the difficulty of adapting available techniques to non-model species. This thesis uses an alternative approach to the current LR asymmetry models, and reveals that by adapting techniques to non-model organisms and by using newly available technologies, there is still much to be learned and discovered in embryology.

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The steroid hormone progesterone is essential for the maintenance of pregnancy in mammalian species, but its role at term and during labour is not fully understood. While the effects of progesterone be either genomic or non-genomic, this thesis focuses on the genomic effects of progesterone, and its withdrawal, on rat uterine tissues. Using laser Capture Microdissection, homogeneous cell populations from the the outer myometrium, inner myometrium, and decidua basalis were isolated from uterine horns obtained from timed-pregnant Sprague Dawley rats that were randomised to receive either progesterone, mifepristone, or vehicle treatment. These rats had a 22-day gestational period, with natural systemic withdrawal of progesterone occurring on the 19th day of gestation (GD19). RNA sequencing was employed in the examination of the transcriptomes of the uterine layers, as well as gene expression profiling between GD19 and term. Analysis of the spatial expression of mRNA and proteins in the uterine tissues revealed localisation of thyrotropin-releasing hormone mRNA, as well as the Trh protein, to the decidua in late pregnancy. In addition, tenascin-n mRNA and proteins localised to the inner myometrium but not the outer myometrium during late pregnancy. Analysis of the temporal expression of mRNA transcripts showed distinct patterns of expression within each of the analysed tissues. Following the withdrawal of progesterone on GD19, changes first occur in the outer myometrium, followed by the inner myometrium, then the decidua where mRNA expression increases between GD22 before labour and GD22 during labour. In conclusion, this thesis presents a novel approach in which rat uterine tissues were studied separately. Potential tissue-specific markers for the decidua and the inner myometrium in late pregnancy were identified. The increase in gene expression prior to labour in the decidua would suggest a signal for the onset of labour. Furthermore, changes in the myometrial tissues suggest the presence of an in-built mechanism through which the myometrium knows when to prepare for labour.

Maternal effects are important mechanisms by which mothers’ may influence the phenotype of their offspring. Females may vary in the resources they can provide during offspring development and understanding the factors responsible for this variation is key to understanding offspring success- in early life as well as later life. Differential allocation has been reported to occur, however how it impacts on offspring and mother’s future reproduction still remains unclear. This is also true for maternal transferred substances like maternally transferred immunity. Contributions to date have been limited to snapshots in time, mean level of transfer and/or limited information regarding other maternal traits. For my thesis, I aim to further the understanding of maternal allocation effects and explore the transfer of maternal antibodies over an immune response of a mother, across multiple breeding attempts and accounting for embryo, maternal and paternal traits. Furthermore, I determine the effect of key male traits on general egg traits along with maternal antibodies. I examine this at the individual level using Chinese painted quail (Coturnix chinensis) who are prolific layers and sexually dimorphic. To date the majority of differential allocation studies have not necessarily addressed the assumptions of differential allocation theory. In Chapter 2 of this thesis I attempt to address some of these assumptions and explore the impact of male characteristics across a number of clutches and find separate effects of initial pairing and subsequent pairings. I found that mothers can create, by differential allocation, clutches of varying size but egg components (egg mass) appears to be largely influenced by initial clutch pairing and not by paternal traits. Furthermore, the effect on egg mass appears to be a secondary effect mediated by females adjusting their condition based on their initial pairing. I demonstrate that unlike general clutch traits (clutch size, egg mass) maternal antibodies are not affected by male characteristics (Chapter 3) carry-over effects of egg size means antibody levels may be influenced throughout life by early experiences. However, maternal immune response may be detrimentally linked to viability of offspring. Whereas maternally transferred antibodies appear to have no relationship with maternal or paternal traits, oocyte yolk antibodies during development were found to correlate with female antibodies up to 48hr prior to lay. In Chapter 4, I examine a neglected area regarding maternal effect- exploring variation between female in their transfer of antibodies. Individual females were highly consistent in the relative level of specific blood antibodies transferred to eggs across different phases of their immune response, across challenge types (bacterial and viral) and that some females consistently transfer significantly more than others. The relative level of circulating antibody transferred was independent of the individual’s overall strength of antibody response and related to the female’s body condition (while the individual’s own antibody responses were not). We found no evidence for any trade-offs between the amount transferred and overall reproductive investment in this chapter. In Chapter 5, I discuss the wider implications of my findings and suggest future research directions.

The production of viable germ cells during human embryonic development determines adult reproductive success. This is particularly true for females, as development of germ cells (GCs) into primordial follicles before birth is imperative for future fertility. During fetal development GCs migrate to the genital ridge to form the gonad, after which several tightly regulated events, including proliferation, differentiation, and association with somatic cells, must occur to form a functional gonad. In the ovary these processes also include the initiation and subsequent arrest of meiosis. These developmental processes are orchestrated by local autocrine and paracrine factors, many of which remain to be identified in the human. In order to decipher further the pathways by which the gonad and GCs develop, potential regulators including prostaglandin (PG) E2, the interleukin (IL)6-type cytokines, and the prokinetecins (PROKs), were examined in the human fetal ovary and PROKs in the human fetal testis. Patterns of gene expression, protein localisation, function, and interaction of the potential mediators throughout human development (8-20 weeks gestation) were determined. Primary fetal tissue was investigated, in addition to immortalized GCs (T-Cam2 cells) and a murine model of fetal ovarian development. PGE2 interacts with known regulators of GC development in non-reproductive organs. It was postulated PGE2 may regulate GC progression by modulating these factors. Examination of PGE2 receptors and precursor enzymes in the fetal ovary revealed that all were present and some were developmentally regulated, with mRNA expression increasing with gestation. These developmentally regulated components were localised to the GCs. The PGE2 receptors were among those differentially expressed, with one localised solely to mature GCs. Culture of human fetal ovary confirmed that PGE2 regulates known regulators of GC development, increasing expression of survival and anti-apoptotic factors. To test the hypothesis that PGE2 is necessary for female GC development, paracetamol, an inhibitor of PGE2 precursor enzymes, was utilised in a murine model of fetal exposure. Fetal ovaries from this experiment displayed disruption of normal development. The IL6-type cytokines are also postulated to be involved in early gonad development, and are known to regulate proliferation and differentiation of mouse embryonic stem and GCs in vitro. A significant increase in transcript levels of the shared receptor components was determined in second trimester human ovaries, as well as developmental increases of several of the IL6-type ligands. Both common receptor components were located specifically in the GCs identifying them as the target of IL6 action in the human fetal ovary. The PROKs regulate cell migration, proliferation and differentiation, and modulate secretion of PGE2 and expression of some IL6-type cytokines. To-date, PROKs have not been examined in the human fetal gonad. Transcript levels were higher in the fetal testis compared to the ovary, with receptor and ligand components increasing with gestation. Most components also increased with gestation in the ovary. However, location of PROK components was strikingly different between the two tissues, with GCs being the primary target of PROK action in the fetal ovary, and Leydig and interstitial cells being the target in the testis. PROKs interaction with other regulators of gonad development was examined utilising a GC line in the case of the ovary and primary interstitial cell cultures in the case of the testis. These studies have identified new factors involved in human fetal gonad development, and how they interact with known regulatory pathways of development.