Following reports by Shapira et al. that α₂Macro-globulin (α₂M) is abnormal in cystic fibrosis (CF), the author set out to examine the properties of α₂M isolated from the plasma of children with CF and from the plasma of age/sex matched controls. To do so, a technique capable of isolating pure, physiologically "active" α₂M from small plasma samples had to be developed. By a two-step chromatographic technique, involving Cibacron Blue Sepharose chromatography and immuno-adsorption, the author was able to isolate "active" CF and control α₂M of at least 98 percent purity from 5 ml of plasma, regardless of plasma haptoglobin type. Having accomplished this, comparative studies of CF and control α₂M were undertaken. Four parameters were investigated: (1) the molar protease binding of α₂M (2) the interaction of α₂M -bovine cationic trypsin (BCT) complexes with the low molecular weight substrate BAEE, (3) the stability of formed α₂M-BCT complexes, and (4) the subunit structure of α₂M. Contrary to the reports of Shapira and his colleagues, this author found no differences between the subunit structure of CF and control α₂M nor between the abilities of CF and control α₂M to interact with BCT. Based upon these findings, the author believes that no firm evidence exists to implicate an α₂M defect in the pathogenesis of cystic fibrosis. / Medicine, Faculty of / Pathology and Laboratory Medicine, Department of / Graduate

As CFTR plays an important role in HCO3- transport, and HCO3- sensitive soluble adenylyl cyclase (sAC) has been shown to be largely responsible for the cAMP production in spermatogenetic cells, we hypothesized that CFTR-mediated HCO3- transport was important to spermatogenesis via sAC pathway in spermatogenetic and Sertoli cells. Using intracellular pH measurement, we demonstrated that CFTR is involved in HCO3- transport in Sertoli cells. RT-PCR results showed that increased HCO3- concentrations in the culture medium resulted in upregulation of CFTR expression. The results also showed that the intracellular cAMP level in Sertoli cells increased as the extracellular HCO3- concentration increased. HCO3- also caused phosphorylation of the cAMP response element binding (pCREB) proteins transcription factor on serine 133, a modification known to be required by Sertoli cells to support spermatogenesis. This phosphorylation could be inhibited by CFTR inhibitor, further lending support to the notion that CFTR is important for HCO3- transport in Sertoli cells, leading to HCO3- dependent events that are important for spermatogenesis. / CFTR is known to be widely expressed in epithelial cells of male reproductive tracts, but its expression in spermatogenic cells is less well known. We first confirmed the expression of CFTR in spermatogenic cells and mature sperm in rodents. Our study thus focused on the important role of CFTR in the processes related to male fertility including spermatogenesis and sperm capacitation. / Cystic fibrosis transmembrane conductance regulator (CFTR) is an anion channel, mutations of which cause cystic fibrosis, a disease characterized by defective Cl- and HCO3- transport. While over 95% of CF male patients are infertile because of congenital bilateral absence of the vas deferens (CBAVD), the question whether CFTR mutations are involved in other forms of male infertility is under intense debates. / In conclusion, our study has demonstrated the role of CFTR in male reproductive system. We have further elucidated its possible physiological role and the underlying molecular mechanisms. These studies may pave the way for the development of method strategies for diagnosis and treatment of CFTR related infertility in male. / Our study also detected CFTR in both human and mouse sperm. CFTR inhibitor or antibody significantly reduced sperm capacitation, and the associated HCO 3--dependent events including increases in intracellular pH, cAMP production and membrane hyperpolarization. The fertilizing capacity of the sperm obtained from heterozygous CFTR mutant mice is also significantly lower as compared to that of the wild type. These results suggest that CFTR in sperm may be involved in the transport of HCO3- important for sperm capacitation and that CFTR mutations with impaired CFTR function may lead to reduced sperm fertilizing capacity and male infertility other than CBAVD. / We further demonstrated the physiological role of CFTR in spermatogenesis using CFTR knockout mice as an in vivo model. Although TUNNEL staining showed normal percentage of apoptotic cells in seminiferous tubules, Cftr -/- mice had spermatogenetic defects in histology section and fewer number of mature sperm compared with wild type (WT) mice. Consistent with the proposed role of CFTR in spermatogenesis, RT-PCR and Western blot results showed reduced expression of spennatids specific gene, Protamine 1, Protamine 2, and CREM, which have been known to be involved in the process of spermatogenesis, in Cftr-/- mice. / Xu, Wenming. / "January 2008." / Source: Dissertation Abstracts International, Volume: 69-08, Section: B, page: 4506. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2008. / Includes bibliographical references (p. 121-138). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract in English and Chinese. / School code: 1307.