Thesis (M. Phil.)--University of Hong Kong, 1980. / Typescript.

published_or_final_version / Anatomy / Master / Master of Philosophy

The overall aim of this thesis was to determine if day old turkey gonads could be cryopreserved and transplanted into recipient poults. This would allow for grafts to develop and mature normally and potentially produce donor-derived offspring. In addition, the monitoring of folliculogenesis in chickens was studied to determine if ultrasonography would be a useful technique to study this biological process, with the intention of using this method in future studies on ovarian graft development. Three studies were conducted: cell and tissue viability of vitrified day old turkey gonads, transplantation of day old turkey gonads into recipient poults, and monitoring of follicle growth in chickens using ultrasonography. The objective of the first study was to determine if day old turkey gonads were viable after vitrification using a standard protocol or with variation in equilibrium solution (ES) and/or vitrification solution (VS) absorption times. Three different ES time points were tested 10, 15 and 20 min (10ES, 15ES and 20ES) and two different VS time points 2 and 3 min (2VS and 3 VS). The cell and tissue viability was determined by Trypan Blue Assay and light microscopy, respectively. Testicular cell viability was conducted using three vitrification protocols and fresh tissue. All vitrification protocols along with fresh tissue were assessed by light microscopy, to evaluate histological alterations, in ovarian and testicular tissue. Protocols with the highest cell viability and best morphological scores were selected as being the most suitable for cryopreservation. Testicular tissue vitrified using 15ES or 20ES with 3VS had the highest cell viability. Ovarian and testicular tissue vitrified using 15ES with (3VS or 2VS), showed the best morphological scores, out of all the protocols. The second study was broken down into two parts: Part A (Trial 1 & 2) was to determine the most suitable age group for poults pre-surgery to give the highest survivability; Part B (Trial 3) was to determine if previously vitrified day old turkey gonads could develop and mature normally, by retrieving grafts post-surgery, at- different time points. In Trial 1, large white turkeys (LWT) 1, 3, 4 and 7 days of age were used and for Trial 2, LWT’s aged 1, 3, 4, and 5 days of age were used. In Trial 3, bronze turkeys at 1 day of age were used, and graft tissue was used from day old LWT’s previously vitrified (10ES/2VS) or fresh. For all Trials, the survivability at each time point was analyzed, and for the third Trial, the grafts recovered were histologically analysed. From Trials 1 and 2, seven and three day old poults had the highest survivability ratios (3/5 and 6/8) respectively. For Trial 3, day old male poults (96%) had a higher survivability than the females (68%). From Trial 3, transplants were only recovered in females and males up until 4 days and 4 weeks post-surgery respectively, with no fresh tissue grafts recovered. The histological analysis of testicular transplants showed deteriorating structure, with a steady progression away from normal morphology, post-surgery. The third study’s objective was to determine the growth rates and patterns of folliculogenesis in Barred Plymouth Rock (BPR) hens by using ultrasonography. Two ultrasound Trials were performed: the first to determine the optimum time interval between serial ultrasound scans to accurately map follicles, and the second to tackle the main objective of the third part of this thesis. For the first Trial, BPR hens were scanned periodically over 24 hours, follicle diameter and position were recorded and mapped with respect to the ovary and neighbouring follicles. Proportional follicle growth, compared to the first scanning session showed that the 24hr time point had the only significant (P<0.001) proportional follicle growth. It is recommended here that scans occur twice a day (morning and afternoon) to capture a more precise growth rate of follicles. In the second Trial, BPR hens were scanned twice a day, over an 11-day period. Follicle diameters (height and width) were recorded to calculate follicle area. The growth of each follicle’s area was compared to the time before ovulation to determine the overall follicle growth rate. Additionally, it was determined if time (night, day) and type of preovulatory follicle (F1-F5) played a significant role in follicle growth rates. The overall follicle growth rate was best described by a cubic equation (R2=0.907, P<0.001). Follicle growth rates were influenced by both time (P=0.009) and type (P<0.001). With F2 and F3 follicles (P<0.05) having a higher growth rate than F1 and F5 types. In conclusion, modifications to the standard vitrification protocol used on quail gonads were necessary to increase cell viability and lower morphological alterations for turkey gonads left whole. For future work it has been shown that day old turkey poults can survive gonad transplantation. The lack of development of grafts is most likely due to a combination of tissue damage after vitrification-warming procedures and insufficient immunosuppression of the host. This work has paved the way for the poultry industry to be able to cryogenically store turkey gonads and revive lines when required. Additionally it was shown that serial ultrasound scans twice a day provided accurate monitoring of follicle growth in Barred Plymouth Rock hens. For BPR hen’s follicle growth rates and patterns were successfully measured. This gives the industry another tool to better select superior laying hens and to create a more homogeneous laying flock. Future application of ultrasonography on gonad monitoring has the potential to show growth and maturation of grafted tissue before the production of donor-derived offspring, enabling earlier detection of successful transplantation.

Thesis (Ph. D.)--University of Missouri-Columbia, 2000. / Typescript. Vita. Includes bibliographical references (leaves 137-162). Also available on the Internet.

Thesis (Ph. D.)--University of Hong Kong, 2005.

Thesis (Ph. D.)--University of Missouri-Columbia, 2000. / Typescript. Vita. Includes bibliographical references (leaves 137-162). Also available on the Internet.

Chez les mammifères, la détermination du sexe permet à un organe bipotentiel, la gonade, de se différencier en ovaire ou en testicule. Nrg1 (Neuregulin 1) est un facteur de croissance qui agit par phosphorylation de protéines cibles. Chez la souris, Nrg1 est nécessaire à la fertilité des adultes. Les mutants perte de fonction présente une hypoplasie testiculaire. Cependant, Nrg1 est exprimé dans la gonade embryonnaire, ce qui suggère son rôle précoce dans le développement de cet organe. Son rôle durant la gonadogenèse restait encore à établir. Mes travaux ont montré que Nrg1 est impliqué dans la prolifération des précurseurs des cellules de Sertoli chez le mâle, dans l’établissement de la vascularisation et par conséquent l’organisation des cordons testiculaires. Ce phénotype est aggravé en l’absence d’expression du gène Rspo1, un facteur qui stimule également la prolifération des précurseurs des cellules de Sertoli. Cela suggère une fonction additive entre les voies de signalisation contrôlées par Nrg1 et Rspo1 au cours du développement testiculaire. Chez les femelles, les mutants Nrg1 présentent une hypoplasie ovarienne et une réduction de la fertilité. Cela est associé à des cellules germinales atypiques. L’impact de ce phénotype sur la fertilité reste à déterminer. Mmd2 est spécifiquement exprimé dans le testicule mais son rôle est encore inconnu. Pour le déterminer durant la gonadogenèse, j’ai généré des modèles murins de perte de fonction par le système CRISPR-Cas9. Les premiers résultats montrent des cellules somatiques apoptotiques dans les testicules de ce modèle murin. Ces études restent cependant préliminaires et les causes de ce phénotype à élucider. / In mammals, sex determination allows a bipotential organ, the gonad, to differentiate into either an ovary or a testis. NRG1 (Neuregulin 1) is a growth factor that promotes phosphorylation of target proteins. In mice, Nrg1 is required for adult fertility. Loss-of-function mutants exhibit testicular hypoplasia. Nrg1 is expressed in embryonic gonads, suggesting an early role in the formation of this organ. However this role remained to be analysed. Here I show that Nrg1 is involved in the proliferation of precursors of Sertoli cells in male. It is also involved in the establishment of the vasculature and in turn partitioning of testis cords. This phenotype is more severe in the absence of Rspo1 expression. RSPO1 signalling also enhances proliferation of precursors of Sertoli cells. This suggests an additive function between the signalling pathways controlled by Nrg1 and Rspo1 during testicular development. In female, Nrg1 mutant ovaries exhibit hypoplasia and reduced fertility. This is associated with atypical germ cells. The relevance of this phenotype on fertility remains to be determined. Mmd2 is specifically expressed in testis but its role is still unknown. To investigate Mmd2 function, I have generated loss-of-function mouse models using the CRISPR-cas9 system. Preliminary results show apoptotic somatic cells in the testes of these mice. However, the causes of this phenotype remain to be clarified.

Thesis (M. Phil.)--University of Hong Kong, 1989.

published_or_final_version / Zoology / Doctoral / Doctor of Philosophy

The ability of an animal to respond and adapt to stimuli is necessary for its survival and involves plasticity and coordination of multiple levels of biological organization, including behavior, tissue organization, hormones, and gene expression. Each of these levels of response is complex, and none of them responds to stimuli in isolation. Thus, to understand how each system responds, it is necessary to consider its role in the context of the entire organism. Here, I have used the African cichlid fish Astatotilapia burtoni and its extraordinary phenotypic plasticity to investigate how animals respond to a change in social status from subordinate to dominant and attempted to integrate these multiple levels of biological response, as well as the roles of several candidate neuromodulators,. First, I have described how male A. burtoni become more aggressive and reproductive during their transition to dominance as well as increasing circulating levels of testosterone and estradiol and the histological organization of their testes. I then mapped the distribution of expression of two behaviorally relevant neuropeptides, arginine vasotocin and isotocin, and their respective receptors, throughout the A. burtoni brain, and found that they were highly expressed in several brain areas important for social behavior and decision-making. I then investigated the role of arginine vasotocin in social status and behavior via pharmacological manipulation and qPCR, showing the importance of arginine vasotocin in controlling the transition to dominance. Lastly, I investigated the role of aromatase, testosterone, and estradiol in male A. burtoni, both in stable dominant males and in males as they transition to dominance, using pharmacological manipulation and quantitative radioactive in situ hybridization, illustrating that estradiol synthesis during dominance is dependent on aromatase activity and necessary for aggressive behavior. / text