Research Doctorate - Doctor of Philosophy / On leaving the testis, spermatozoa can neither swim nor fertilize the oocyte. These functional properties are acquired as spermatozoa engage in a process of post-testicular maturation in the epididymis. The studies described in this thesis were designed to elucidate some of the fundamental mechanisms associated with the regulation of epididymal maturation in mouse spermatozoa. The initial studies described in this thesis investigated the expression of a cAMP/PKAdependent, tyrosine phosphorylation signaling pathway that becomes activated during epididymal sperm maturation. It was demonstrated that the entry of spermatozoa into the epididymis was accompanied by the sudden stimulation of this pathway, initially in the principal piece of the cell and subsequently in the midpiece. The competence of these cells to phosphorylate the entire sperm tail, particularly the mitochondria, was accompanied by a capacity to exhibit hyperactivated motility on stimulation with cAMP. A distinctly different pattern of tyrosine phosphorylation involving the acrosomal domain of the sperm head was provoked as spermatozoa entered the caput epididymis and then remained high until these cells entered the distal corpus and cauda. However, tyrosine dephosphorylation of the sperm acrosomal domain during epididymal transit did not appear to be functionally involved in controlling the acrosome reaction. Research into the biochemical basis of sperm epididymal maturation revealed that this process was associated with the activation of sperm mitochondria, leading to the creation of a mitochondrial membrane potential (MMP) and activation of mitochondrial free radical generation. Immature caput spermatozoa displayed a low MMP whereas mature caudal spermatozoa actively maintained a high MMP. Moreover mitochondrial generation of reactive oxygen species (ROS) could be triggered by antimycin A in mature caudal spermatozoa but not in immature caput spermatozoa, suggesting a lack of electron flux in the latter. The molecular mechanisms responsible for regulating mitochondrial function were also found to be reversible, as washing the cells free of epididymal fluid allowed caput spermatozoa to acquire a high MMP and generate ROS while incubating caudal spermatozoa in caput epididymal fluid, suppressed MMP and their ability to generate ROS. Pharmacological suppression of mitochondrial activity was subsequently found to be associated with the inhibition of hyperactivated motility. These results strongly suggested that fluid from the caput epididymis contained a mitochondrial inhibitor and that activation of mitochondrial activity was due to the removal or inactivation of this inhibitor during epididymal transit. This causative factor was not species specific. Incubation of ejaculated human spermatozoa in murine epididymal fluid systematically suppressed their MMP. The characterization of caput epididymal fluid suggested that the putative mitochondrial inhibitor is a heat-resistant protein with a molecular weight larger than 30 kDa. The final results presented in this thesis demonstrate that a full-length Riken protein is a potential candidate for the putative mitochondrial inhibitor that switches off mitochondrial function in caput spermatozoa. Indeed, these results represent the first report suggesting that the epididymal maturation is associated with activation of sperm mitochondria and the first study of a testis specific protein that could be a regulator of mitochondrial function in the male germ line. Further characterization of the mechanisms by which epididymal spermatozoa control mitochondrial function may hold the key to our understanding of sperm maturation. It may also lead us to a clear exposition of the molecular basis of human male infertility, potentially serve as a target for infertility treatment and possibly contribute to the development of novel contraceptive agents.

Cystatin Related Epididymal Spermatogenic protein (CRES) is expressed in both the testis and epididymis and found associated with spermatozoa. It appears as non-glycosylated (14 and 12 kDa) and glycosylated isoforms (19 and 17 kDa). The role of CRES is enigmatic and dependent on localization of its isoforms, which is the objective of this study. The initial approach was to investigate testicular and epididymal origins of these isoforms by immunohistochemistry and immunogold cytochemistry. To further pinpoint CRES localization we then selectively extracted and fractionated epididymal spermatozoa in order to find by immunoblotting which sperm fractions contained CRES isoforms. Immunohistochemical analysis of mouse spermatogenesis showed that CRES was expressed in the tail cytoplasm of elongating spermatids from step 9-16, with a pattern reminiscent of outer dense fibre (ODF) proteins. Ultrastructural immunocytochemistry revealed that the immunogold label was concentrated over growing ODFs and mitochondrial sheath in the testes which persisted in spermatozoa through the epididymis. Sequential extractions of isolated sperm tails with Triton X-100-dithiothreitol (DTT) to remove the mitochondrial sheath, whose extract contained an unrelated 66 kDa immunoreactive band, followed by either sodium dodecyl sulfate (SDS)-DTT or urea-DTT to solubilise accessory fibres of the tail revealed a 14 kDa immunoreactive band associated with the ODF. In addition, Western blots revealed glycosylated and non-glycosylated CRES isoforms in nonyl phenoxylpolyethoxylethanol (NP40) extracts of the caput, but not cauda, sperm. Immunohistochemical analysis of the caput and cauda epithelium showed that CRES is secreted by the Golgi apparatus of the ii initial segment, fills the proximal caput lumen, and disappears by mid caput. Western blots of caput and cauda tissue and luminal fluid revealed 14 and 19 kDa immunoreactive bands in caput tissues and luminal fluid, but not in the cauda. This study concludes that there are two origins of CRES, one arising in the testis and the other in the epididymis. Testicular CRES is ionically and covalently associated with the ODF while epididymal CRES is detergent soluble and is most likely associated temporarily with the surface of caput epididymal sperm. / Thesis (Master, Anatomy & Cell Biology) -- Queen's University, 2010-09-03 14:22:01.913

Wie in anderen Zellen sind auch bei Säugerspermien spezifische Lipide und Proteine der Zellmembran aufgrund ihrer heterogenen lateralen Verteilung in speziellen Domänen angereichert, die in unterschiedlichen räumlichen und zeitlichen Dimensionen existieren und der Zelle funktionale Variabilität ermöglichen. Aufgrund der fehlenden aktiven Proteinbiosynthese bietet dies den Spermien eine Möglichkeit, auf unterschiedliche Anforderungen zu reagieren. In der vorliegenden Arbeit wurden daher sogenannte detergenzresistente Membrandomänen (DRMs) aus Eberspermien unterschiedlicher Reifestadien präpariert und untersucht. Dabei stieg bereits in den Dichtegradienten mit zunehmender Reife die Dichte, bei der die opaleszenten Banden auftraten. Eine Analyse dieser mittels 31P-NMR zeigte mit zunehmender Reife eine Anreicherung an Glycerophosphatidylethanolamin und Phosphatidylinositol bei den Glycerophospholipiden, der Gehalt an Sphingomyelin hingegen nahm während der Nebenhodenreifung und auch nach der Ejakulation ab. Diese Veränderungen könnten auf eine Destabilisierung von Membrandomänen hindeuten, um eine Zusammenlagerung zu größeren Domänenclustern zu erleichtern, möglicherweise in Vorbereitung auf Kapazitation und Akrosomenreaktion. Zunächst werden die destabilisierten Membrandomänen jedoch durch die Anlagerung von Seminalplasmaproteinen geschützt, was vermutlich für das verringerte Lipid- zu Proteinverhältnis der DRMs bei Ejakulatspermien sorgt. Aufgrund der generellen Kälteempfindlichkeit von Eberspermien findet ihre Lagerung üblicherweise bei 16°C statt. Dies ist aus mikrobiologischer Sicht nachteilig gegenüber einer kälteren Lagerungstemperatur. Eine Untersuchung der Spermien von 64 Ebern zeigte jedoch bei 10% der Ejakulate eine individuum-spezifische Resistenz gegenüber der Lagerung bei 4°C. Die DRMs der kälteresistenten Spermien hatten einen erhöhten Anteil an langkettigen, mehrfach ungesättigten Fettsäuren, wie 31P-NMR und MALDI-TOF MS Analysen zeigten. / The lateral distribution of lipids and proteins in the plasma membrane is heterogeneous. Therefore specific lipids and proteins in membranes of mammalian spermatozoa are enriched in special domains of varying size and different time scales enabling the cell’s membrane functional variability. Being transcriptional inactive this is especially relevant for spermatozoa in responding to multiple challenges on their way to fertilization. Therefore so called detergent resistant membrane domains (DRMs) from boar spermatozoa of different developmental stages were investigated. Already in the sucrose density gradients differences were visible, so the opalescent bands of more maturated sperm had a higher density. An analysis of these bands by 31P-NMR showed an enrichment of glycerophosphatidylethanolamine and phosphatidylinositol during maturation and a decrease of sphingomyelin during maturation in the epididymis and even after ejaculation. This suggests destabilization of DRMs and hence of putative membrane domains. This could enable clustering to bigger membrane domain platforms in preparation for capacitation and acrosome reaction. First, however, seminal fluid proteins cover the spermatozoa protecting the membrane with the destabilized membrane domains. This could have led to the detected decrease of the lipid to protein ratio in DRMs of ejaculated sperm. Boar spermatozoa are sensitive to storage at cold temperatures and are therefore usually stored at 16°C, which is especially disadvantageous with regard to growing of bacteria. A screening of sperm from 64 boars showed a ratio of 10% individuals with cold resistant sperm which could be stored at 4°C without quality loss. The DRMs of cold resistant sperm had a higher proportion of longchained, polyunsaturated fatty acids, as shown by analysis with 31P-NMR und MALDI-TOF MS.