||Carrell, Douglas T; Moskovtsev Surgery I.; Willis Jennifer; Liu Lihua; Mullen Brendan M.; Emery, Benjamin R.; Atkins, John F.; Christensen, Greg L.; Jones, Kirtly P.; Hatasaka Harry H.; Gibson, Mark; Peterson, C. Matthew
||During spermiogenesis, sperm chromatin undergoes dramatic remodeling. The testis-specific protamine proteins facilitate these nuclear changes by replacing the somatic cell histones, a process that produces highly condense, transcriptionally silent chromatin. In humans there are two forms of sperm protamine, protamine-1 (P1) and protamine-2 (P2), which occur in a strictly regulated one-to-one ratio. Sperm protamine-deficiency and P1/P2 ratio deregulation have been implicated in male infertility. The studies comprising this dissertation aimed to investigate the hypothesis that human sperm protamine deficiency: (1) occurs due to abnormal expression of the P1 and P2 proteins, (2) is associated with diminished semen quality, compromised sperm functional ability, and reduced sperm DNA integrity, (3) arises due to genetic aberrations in the protamine genes and unfaithful translation of the protamine transcripts, and (4) is negatively associated with fertility potential. In order to evaluate the hypothesis, studies were focused on three specific objectives: (1) to better characterize protamine-deficiency in infertile males, (2) to evaluate the molecular basis of protamine-deficiency, and (3) to evaluate the clinical significance and fertility implications of human sperm protamine deficiency. Quantitative analysis of sperm nuclear protein extracts revealed a large population of infertile men with abnormal sperm P1/P2 stoichiometry. Included in this group, were patients with abnormally elevated P1/P2 ratios, as well as a newly identified infertile population with significantly reduced P1/P2 ratios. Abnormal expression of the P1 and P2 proteins were shown to underlie these abnormally reduced and elevated ratios, respectively. Nuclear protein gene sequencing and protamine mRNA quantification subsequently revealed deregulated protamine expression is not a primary result of nuclear protein gene mutations, but instead may arise due to novel mechanisms of aberrant translation regulation or post-translational processing defects. Semen analysis, sperm penetration evaluation, and DNA fragmentation testing revealed aberrations in P1/P2 stoichiometry are associated with diminished semen quality, compromised sperm functional ability, and reduced sperm DNA integrity. Finally, an analysis of in vitro fertilization (IVF) outcomes revealed sperm protamine-deficiency is related to reduced fertilization ability and diminished IVF pregnancy rates. Taken together, these studies provide a comprehensive characterization of sperm protamine deficiency, elucidate the biochemical and molecular basis of abnormal protamine expression, and establish the clinical significance of the protamines in human male infertility.