Zoology - Theses
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ItemMale germ cell transplantation(University of Melbourne, 1995)This thesis has explored the success of male germ cell transplantation in rats. Busulfan-treated black and white Long Evans rats were used as the germ cell-recipients, and white Sprague Dawley fetal and neonatal rats were used to provide donor germ cells which were transplanted by 2 different routes. Adult Long Evans male rats were injected intraperitoneally once or twice with busulfan, an alkylating agent, at a dose of 10 mg/kg body weight. Forty four days after the first busulfan treatment, their testes were essentially devoid of spermatogenic cells and therefore suitable as germ cell recipients. Following exposure to busulfan during intrauterine life on which primordial germ cells (PGCs) were exclusively destroyed, the rats at days 4 - 5 of age were devoid of germ cells and therefore used as newborn recipients. Donor fetal and newborn testes were excised and dissociated by trypsin-EDTA (fetal tissues) or collagenase/trypsin-EDTA (neonatal tissues) treatment. PGCs and gonocytes were purified by equilibrium centrifugation on a discontinuous Percoll gradient column. Alkaline phosphatase activity was used as a marker for both PGCs and gonocytes. Trypan blue exclusion was used to determine cellular viability. The donor germ cells were injected into the testes of the anaesthetised recipients by retrograde injection into the rete testis, or random multiple testicular injections. A total of 74 recipient rats were transplanted with either primordial germ cells or gonocytes. Forty eight of them were paternity tested with 563 fertile Sprague Dawley females, and they produced 2,280 progeny. However, none of the progeny was derived from the donor germ cells, as judged by coat colour. However, a detailed histological analysis of the recipient testes showed that the donor germ cells had apparently differentiated into mini-tubules or irregular segments of seminiferous epithelium within the lumen of the host�s seminiferous tubules, and exhibited qualitatively normal spermatogenesis in 13 out of 19 animals following rete testis injections. The stage of spermatogenesis of the donor intraluminal seminiferous epithelium was closely synchronised with that of the host�s epithelium, suggesting that the spermatogenic cycle is regulated locally by the intraluminal microenvironment. None of 15 neonatal or adult recipients given random multiple intratesticular injections showed any evidence of intraluminal spermatogenesis, suggesting that the donor germ cells were unable to migrate through the basement membrane of the host seminiferous tubules. In summary, this thesis has demonstrated that it is possible to transplant purified PGCs and gonocytes from one rat to another. Male germ cell transplantation therefore provides an interesting new tool for investigating the control of spermatogenesis, and if the success rate of the procedure can be improved, so that the donated germ cells all integrated with the host�s Sertoli cells, it could become an invaluable technique for manipulating the male germ cell line.