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This thesis is an evaluation of the hypothesis that the spennathecae of spiders affects the sperm precedence patterns in a predictable way (Austad 1984). Spermathecae come in two varieties: cul-de-sac and conduit. Cul-de-sac spennathecae, according to the hypothesis, are supposed to lead to second male sperm priority and conduit to first male sperm priority . The hypothesis was evaluated both directly and indirectly. Direct measurements were made of paternity in two species, Pholcus phalangioides and Tetragnatha montana, both of which are cul-de-sac species. It was found that P. phalangioides complies with the predicted precedence pattern and thus does not disprove the hypothesis. This second male priority pattern was despite a much shorter mating time by second mating males. In T. montana no precedence pattern was found, with equal likelihood of first or second mating males of gaining paternity. There was in T. montana a possible influence of the duration of mating affecting the precedence pattern, with longer mating males gaining a higher paternity no matter what order they mated in. It is discussed whether or not this is due to sperm loading or genitalic stimulation (Eberhard 1985). Indirect evaluation of the hypothesis included an analysis of mating behaviour in Zygiella x-notata which is a conduit species and was chosen as a comparison to the two cul-de-sac species. In Z. x-notata it was found that there was no difference between mating duration in first and second mating males. Mating persistence is thus the same in first and second mating males, suggesting that the males cannot detect that the female is a denuded resource to second mating males. Hence first male priority may not be a factor in this species. Other indirect methods of evaluating the hypothesis involved charting the incidence of mate-guarding and mating-plugs. The expected pattern of mate-guarding was for conduit species to pre-mate guard and for cul-de-sac species to post-mate guard, because of the predicted sperm precedence patterns associated with the spermathecae. The predicted pattern was not found. In the case of mating-plugs it was predicted that these should be deployed by cul-de-sac species because it is in these species that second males are able to usurp paternity to a large extent. The opposite pattern was found with mating-plugs of various design being utilized by conduit species. It is postulated that mating-plugs are the mechanism by which first male priorities are established in conduit species, where this pattern is found. The absence of plugs in cul-de-sac species is possibly the reason that second males can cuckold. The additional data collected since 1984 reveal that patterns of paternity found in spiders seem to be more complex than was originally assumed by Austad (1984). Spermathecae are species-specific in character and this may reflect a species specificity in sperm precedence patterns. Thus the conduit I cul-de-sac dichotomy may not reflect a useful prediction of paternity patterns.

A thesis submitted to the Faculty of Science, University of the Witwatersrand, Johannesburg, in fulfilment of the requirements for the degree of Doctor of Philosophy. Johanneburg, 2010. / Unable to load abstract

Mammalian females form their germ cells (oocytes) before or shortly after birth. The oocytes interact with somatic cells to form primordial follicles, creating the quiescent population from which oocytes will be recruited to grow throughout life. A female’s fertility life span is therefore, dependant on the size of this pool and the rate at which primordial follicle are activated to grow. However, there is still much we do not know about the quiescent follicle population and the mechanisms that control their recruitment into the growing follicle population are still unclear. There is evidence that the phosphoinositide-3-kinase (PI3K) pathway is key to activation of follicle growth. The role of the PI3K pathway has been primarily explored in the rodent model and has highlighted this pathway’s importance both in the activation of quiescent follicle growth and maintaining dormancy of the quiescent follicle population. This thesis aimed to explore if the PI3K pathway played a similar role in a large mono-ovulatory species as it does in the small polyovulatory rodent species. Bovine is a mono-ovulate species, which has similar attributes in its reproduction and folliculogenesis to the human in vivo; therefore using an in vitro bovine model might be a valuable indication of the role of the PI3K pathway in the human. Initial experiments tested if the bovine was a good model for human primordial follicle activation in an in vitro environment. It was observed that the bovine and human had comparative levels of activation and subsequent increases in both the primary and secondary follicle populations within an in vitro culture system. These similarities indicate that the bovine is a relevant model for the human in vitro. It is not possible to culture the entire bovine ovary. Therefore knowing the location of the primordial follicles is important to establishing what region(s) of the ovary to use. The overall concentration of ovarian follicles was higher in the cortex and gradually declined through the consecutive inner layers of the ovary. The distribution of the ovarian follicle populations were different in each distinctive region of the ovary with the quiescent follicles representing a much larger proportion of the ovarian follicle population in the cortex compared to the inner regions of the ovary. The location an ovarian follicle in the ovary was seen to influence its health in both the quiescent and growing follicle populations, with reduced health seen in the IV inner layers of the ovary compared to the cortex. This resulted in very few healthy quiescent follicles outside of the cortex region making it the more favourable region to culture in functional studies. The role of the PI3K pathway was therefore explored using an in vitro bovine model using the pharmacological compounds bpV (HOpic) and 740Y-P, both of which caused an up-regulation of the PI3K-pathway. It was observed that up-regulation of the PI3K pathway caused an increase in the activation of the quiescent follicle population, and the resulting primary follicles were larger in size. However, there was reduced health in both the growing and quiescent follicle populations. The ill health appears to be due to a disruption in the co-ordination of growth between oocyte and granulosa cells in the ovarian follicles, leading to enlarged oocytes in both the primary follicles and quiescent follicles. Although the PI3K pathway caused an increase in quiescent follicle activation and larger primary follicles there was no increase in the number of viable large secondary follicles obtained. The growth of the secondary follicles was unaltered by the initial activation of the quiescent follicles via the PI3K pathway. These experiments show that the PI3K pathway plays a role in primordial follicle activation in large mono-ovulate species. However, up-regulating the PI3K pathway results in a decrease in health of the quiescent and primary follicle populations, thus limiting its immediate value as a therapeutic target. This study has improved our understanding of the role of the PI3K pathway in primordial follicle activation in a large mono-ovulate species. It has highlighted that the up-regulation of the PI3K pathway using both bpV (HOpic) and 740Y-P increases the activation of the bovine ovarian follicles in vitro. However, up-regulating the PI3K pathway disrupts the development of the ovarian follicles resulting in retarded growth and thereby a decrease in the survival of both the quiescent and growing follicle populations. The similarities in activation, growth and development between the bovine and the human in vitro indicate that the results observed in the bovine are a good indication of what would occur in the human. This study has also improved our understanding of the location, distribution and viability of the ovarian follicle population within the ovary.

Mak Wai Yan. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (leaves 95-115). / Abstracts in English and Chinese. / Declaration --- p.i / Abstract --- p.ii / Acknowledgements --- p.viii / Table of Contents --- p.ix / List of Tables --- p.xiii / List of Figures --- p.xiv / List of Abbreviations --- p.xvi / Chapter Chapter 1 --- Introduction and Literature Review / Chapter 1.1 --- Introduction --- p.1 / Chapter 1.2 --- Endocrine control of reproduction in crustaceans --- p.2 / Chapter 1.3 --- The X-organ sinus gland (XOSG) complex --- p.3 / Chapter 1.3.1 --- CHH/MIH/GIH family of neuropeptides --- p.4 / Chapter 1.3.2 --- Gonad inhibiting hormone (GIH) --- p.6 / Chapter 1.3.3 --- Molt inhibiting hormone (MIH) --- p.7 / Chapter 1.3.4 --- Crustaceans hyperglycemic hormone (CHH) --- p.9 / Chapter 1.3.5 --- The chormatophorotropins (RPCH and PDH neuropeptide family) --- p.10 / Chapter 1.4 --- Other mechanisms of reproduction control --- p.11 / Chapter 1.4.1 --- Methyl farnesoate (MF) and mandibular organ inhibiting hormone (MOIH) --- p.11 / Chapter 1.4.2 --- Gonad stimulating hormone (GSH) --- p.13 / Chapter 1.4.3 --- Serotonin (5HT) --- p.13 / Chapter 1.4.4 --- Dopamine --- p.14 / Chapter 1.4.5 --- Enkephalin (ENK) --- p.15 / Chapter 1.4.6 --- 17β-Estradiol --- p.16 / Chapter 1.4.7 --- Progesterone --- p.17 / Chapter 1.5 --- Androgenic hormone (AH) --- p.18 / Chapter 1.6 --- Objective and methodology of present research --- p.19 / Chapter 1.7 --- Reproductive biology of the shrimp Metapenaeus ensis --- p.20 / Chapter Chapter 2 --- Materials and Methods / Chapter 2.1 --- Introduction --- p.26 / Chapter 2.2 --- Extraction of eyestalk RNA from the shrimp --- p.27 / Chapter 2.3 --- Construction of the eyestalk cDNA library --- p.28 / Chapter 2.3.1 --- First-strand cDNA synthesis --- p.28 / Chapter 2.3.2 --- cDNA amplification by LD PCR --- p.29 / Chapter 2.3.3 --- Proteinase K digestion --- p.29 / Chapter 2.3.4 --- Sfi I digestion --- p.30 / Chapter 2.3.5 --- cDNA size fractionation --- p.30 / Chapter 2.3.6 --- Ligation of cDNA to λ TriplEx2 Vector --- p.31 / Chapter 2.3.7 --- Packaging --- p.31 / Chapter 2.3.8 --- Titering the unamplified library --- p.32 / Chapter 2.3.9 --- Library amplification --- p.33 / Chapter 2.3.10 --- Titering the amplified library --- p.34 / Chapter 2.4 --- Mass Excision of the eyestalk cDNA library --- p.34 / Chapter 2.5 --- PCR screening of inserted sequence --- p.35 / Chapter 2.6 --- RNA arbitrarily primed polymerase chain reaction (RAP-PCR) --- p.35 / Chapter 2.7 --- Dot blot hybridization --- p.37 / Chapter 2.7.1 --- Probe Labelling --- p.37 / Chapter 2.7.2 --- Dotting of membrane --- p.38 / Chapter 2.7.3 --- Hybridization --- p.38 / Chapter 2.7.4 --- Washing and chemiluminescent detection --- p.39 / Chapter 2.7.5 --- Probe stripping for re-hybridization --- p.40 / Chapter 2.8 --- Sequencing --- p.40 / Chapter 2.9 --- BLAST search --- p.41 / Chapter 2.10 --- Northern blot analysis --- p.41 / Chapter 2.10.1 --- Probe labelling --- p.41 / Chapter 2.10.2 --- RNA formaldehyde denaturing gel electrophoresis --- p.41 / Chapter 2.10.3 --- Northern blot --- p.42 / Chapter 2.10.4 --- Pre-hybridization --- p.43 / Chapter 2.10.5 --- Hybridization --- p.43 / Chapter 2.10.6 --- Washing and chemiluminescent detection --- p.43 / Chapter 2.11 --- Real-time RT-PCR --- p.44 / Chapter 2.11.1 --- DNaseI treatment --- p.44 / Chapter 2.11.2 --- First strand synthesis --- p.44 / Chapter 2.11.3 --- Primer design and verification --- p.45 / Chapter 2.11.4 --- Real-time PCR --- p.45 / Chapter 2.11.5 --- Statistical analysis --- p.45 / Chapter Chapter 3 --- Results / Chapter 3.1 --- Experimental animals --- p.50 / Chapter 3.2 --- Total RNA extraction --- p.50 / Chapter 3.3 --- Library construction --- p.50 / Chapter 3.4 --- PCR screening of inserted sequences --- p.50 / Chapter 3.5 --- RNA arbitrarily primed polymerase chain reaction (RAP-PCR) --- p.51 / Chapter 3.6 --- Dot blot hybridization --- p.51 / Chapter 3.7 --- Sequencing and BLAST search --- p.52 / Chapter 3.8 --- Northern blot analysis --- p.53 / Chapter 3.8.1 --- Housekeeping gene - elongation factor la --- p.53 / Chapter 3.8.2 --- Insulin-like growth factor binding protein (IGFBP) --- p.54 / Chapter 3.8.3 --- Arrestin --- p.54 / Chapter 3.8.4 --- Opsin --- p.54 / Chapter 3.9 --- Real-time RT-PCR --- p.55 / Chapter 3.9.1 --- β-Actin --- p.55 / Chapter 3.9.2 --- Farnesoic acid O-methyltranferase (FAMeT) --- p.56 / Chapter Chapter 4 --- Discussion / Chapter 4.1 --- Application of RAP-PCR and dot blot analysis in identifying differential expressed genes --- p.79 / Chapter 4.1.1 --- Abundant genes --- p.79 / Chapter 4.1.2 --- Appearance of 16S rRNA in eyestalk cDNA library of M. ensis --- p.80 / Chapter 4.1.3 --- False positive --- p.80 / Chapter 4.2 --- Investigation on common housekeeping genes --- p.81 / Chapter 4.3 --- Potential functions of identified differential expressed genes in reproduction of shrimp --- p.82 / Chapter 4.3.1 --- Arrestin --- p.84 / Chapter 4.3.2 --- Opsin --- p.86 / Chapter 4.3.3 --- Insulin-like growth factor binding protein (IGFBP) --- p.88 / Chapter 4.3.4 --- β-Actin --- p.89 / Chapter 4.4 --- Investigation for farnesoic acid O-methyltransferase (FAMeT) in shrimp --- p.89 / Chapter Chapter 5 --- General Conclusion --- p.91 / References --- p.95 / Appendix --- p.116

Made available in DSpace on 2014-06-11T19:22:22Z (GMT). No. of bitstreams: 0 Previous issue date: 2012-02-10Bitstream added on 2014-06-13T19:27:36Z : No. of bitstreams: 1 urbinati_ec_me_jabo.pdf: 438124 bytes, checksum: ec3f06e377e44a119086563d92e6f440 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Os relatos de produtores e pesquisadores sobre problemas na fase de ovulação do pacu Piaractus mesopotamicus durante a reprodução artificial nos conduziu a avaliar o uso da prostaglandina F2α. O estudo foi realizado em duas estações reprodutivas (2009/2010 e 2010/2011), com dois experimentos na estação 2009/2010 (T1 e T2) um experimento na estação 2010/2011 (T3). Foi amostrado um total de 45 fêmeas. As fêmeas do grupo controle foram induzidas apenas com extrato bruto de hipófise de carpa (EC, 6mg/kg), enquanto as do grupo tratado receberam prostaglandina F2α (PGF - 2ml/fêmea na estação 2009/2010 e 5ml/fêmea na estação 2010/2011), além da dose de EC. Nas duas estações observou-se 100% de fêmeas desovadas no grupo tratado com PGF enquanto no grupo controle a desova ocorreu em 52,94% e 83,33% no primeiro e segundo experimentos da estação 2009/2010, respectivamente. Em 2010/2011, somente 25% das fêmeas controle desovaram. As taxas de fecundidade, fertilização e eclosão não diferiram (p>0,05) entre os grupos de fêmeas. A análise da freqüência de volume de ovócitos nas diferentes fases de desenvolvimento mostrou, nos ovários analisados pós-desova, valores significativamente maiores (p<0,05) de ovócitos vitelogênicos com quebra de vesícula germinativa, mas não ovulados no grupo controle e maior ocorrência de ovócitos pré-vitelogênicos no grupo tratado com PGF. Independente do tratamento, as maiores taxas de fertilidade ocorreram ao redor de 275 UTA, enquanto a maior parte das fêmeas desovou entre 276 e 323 UTA. Do mesmo modo, independente do tratamento, houve um decréscimo significativo (p<0,05) nas taxas de fertilização e eclosão na comparação das duas coletas de 2009/2010. Os dados obtidos neste estudo sugerem que a prostaglandina pode aumentar as taxas de desova em fêmeas de pacu induzidas à reprodução, com efeito evidente na liberação dos ovócitos dos folículos / Due to reports of fish farmers and researchers about the problems in the ovulation phase of P. mesopotamicus during its artificial reproduction we evaluated the use of prostaglandin F. The study was conducted in two spawning seasons (2009/2010 and 2010/2011), with two sampling in season 2009/2010 and one sampling in 2010/2011. Forty five females broodstock were sampled. Control group was injected with carp pituitary extract (CE, 6mg/kg), while treated group received prostaglandin F (PGF – 2ml/ 2009/2010 season and 5ml/2010/2011 season) in addiction to CE. In the two seasons 100% of treated with PGF groups spawned while in control groups 52,94% and 83,33% in first and second sampling of season 2009/2010 spawned, respectively. In 2010/2011 only 25% of control group spawned. Fecundity, fertility and hatching rates did not differ (p>0.05) between groups. The oocytes volume frequency analysis showed that in ovaries after spawning, in control group, there was a significant higher number (p<0.05) of vitelogenic oocytes with GVBD but anovulated, while in PGF treated group, a higher number (p<0.05) of pre-vitelogenic oocytes was observed. Independent on the treatment, the highest fertility rate occurred around 275 ATU, while most of the females spawned in a range of 276 and 323 ATU. The same way, independent on the treatment, there was a significant decrease (p<0.05) in fertility and hatching rates comparing samplings from 2009/2010. Data obtained in this study suggest that prostaglandin F might enhance spawning rate in hormonal induced females of pacu, with a notable effect on the release of oocytes from the follicles

鄭淑宜. / "2009年8月". / "2009 nian 8 yue". / Thesis (M.Phil.)--Chinese University of Hong Kong, 2009. / Includes bibliographical references (leaves 45-47). / Abstracts in Chinese and English. / Zheng Shuyi. / Chapter (一） --- 前言：物品的定 義 --- p.1 / Chapter (二） --- 看的習慣與物品意義的關 係 --- p.3 / Chapter 2.1. --- 個人化的注視 / Chapter 2.2. --- 多角度的視覺總合 / Chapter 2.3. --- 看得見的緣份 / Chapter (三） --- 觀物篇：物品的 美 --- p.9 / Chapter 3.1. --- 造型的美 / Chapter 3.2. --- 功能的美 / Chapter 3.3. --- 機器的美 / Chapter 3.4. --- 使用的美 / Chapter 3.5. --- 戀物情意結 / Chapter (四） --- 造物篇：藝術物品呈現的社會現象及作 用 --- p.18 / Chapter 4.1. --- 挪用現成物 / Chapter 4.1.1. --- 媚俗文化的出現 / Chapter 4.1.2. --- 商品雕塑 / Chapter 4.1.3. --- 價値取向的轉變 / Chapter 4.1.4. --- 品牌效應 / Chapter 4.2. --- 模擬物品 / Chapter 4.2.1. --- 模擬世界符號 / Chapter 4.2.2. --- 藝術商品 / Chapter 4.2.3. --- 生產技術和工人協作 / Chapter 4.3. --- 重新創造物品 / Chapter 4.3.1. --- 藝術設計品 / Chapter 4.3.2. --- 親身體驗的世界 / Chapter (五） --- 總結一創作超越真實的物品 --- p.38 / Chapter 5.1. --- 物品是記錄儀 / Chapter 5.2. --- 物品是自身和社會的觀照 / Chapter 5.3. --- 創作物品的成效 / 參考書目 --- p.45 / 附錄 --- p.48