CYSTATIN RELATED EPIDIDYMAL SPERMATOGENIC PROTEIN RESIDES IN THE OUTER DENSE FIBRES AND LIKELY TRANSIENTLY ASSOCIATES WITH THE SURFACE OF EPIDIDYMAL MOUSE SPERMATOZOA

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.

A FUNCTIONAL, COMPARATIVE AND CLINICAL ANALYSIS OF SPERM-BORNE OOCYTE ACTIVATING FACTOR, PAWP

Successful fertilization depends upon the activation of metaphase II arrested oocytes by sperm-borne oocyte activating factor (SOAF). Failure of oocyte activation is considered as the cause of treatment failure in a proportion of infertile couples. SOAF induces the release of intracellular calcium in oocyte which leads to meiotic resumption and pronuclear formation. Calcium release is either in the form of single calcium transient in echinoderm and amphibian oocytes or several calcium oscillations in ascidian and mammalian oocytes. Although the SOAF attributes are established, it is not clear which sperm protein(s) play such role. Sperm postacrosomal WW binding protein (PAWP) satisfies a developmental criteria set for a candidate SOAF. This study shows that recombinant human PAWP protein or its transcript acts upstream of calcium release and fully activates the amphibian and mammalian oocytes. Interference trials provided evidence for the first time that PAWP mediates sperm-induced intracellular calcium release through a PPXY/WWI domain module in Xenopus, mouse and human oocytes. Clinical applications of PAWP were further investigated by prospective study on the sperm samples from patients undergoing intracytoplasmic sperm injection (ICSI). PAWP expression level, analyzed by flow cytometry, was correlated to ICSI success rate and embryonic development. This study also explored the developmental expression of the other SOAF candidate, PLCζ in male reproductive system and its function during fertilization. Our findings showed for the first time that PLCζ most likely binds to the sperm head surface during epididymal passage and is expressed in epididymis. We demonstrated that PLCζ is also compartmentalized early in spermiogenesis and thus could play an important role during spermiogenesis. Detailed analysis of in vitro fertilization revealed that PLCζ disappears from sperm head during acrosome reaction and is not detectable during sperm incorporation into the oocyte cytoplasm. In conclusion, this dissertation provides evidence for the essential non-redundant role of sperm PAWP in amphibian and mammalian fertilization; recommends PAWP as a biomarker for prediction of ICSI outcomes in infertile couples; and proposes that sperm PLCζ may have functions other than inducing oocyte activation during fertilization.

Identification and Development of Enzymes Associated With the Sperm Inner Acrosomal Membrane and Their Function during Fertilization

In order for mammalian fertilization to transpire, spermatozoa must transit through the female reproductive tract and penetrate the outer investments of the oocyte: the cumulus oophorus and the zona pellucida. In order to penetrate the oocyte, spermatozoa must undergo the acrosome reaction. The acrosome reaction results in the exposure of the inner acrosomal membrane (IAM) and proteins that coat it to the extracellular environment. After the acrosome reaction, the IAM becomes the leading edge of spermatozoa undergoing progressive movement. Thus the enzymes which effect lysis of the oocyte investments ought to be located on the IAM. An objective of this study was to identify and characterize enzymatic activity detected on the IAM and provide evidence that they play a role in fertilization. This study also describes procedures for fractionating spermatozoa and isolating the IAM and proteins on its intra- and extra-vesicular surfaces, and describes their development during male gametogenesis. Since the IAM is exposed to the extracellular environment and oviductal milieu after the acrosome reaction, this study also sought to characterize interactions and relationships between factors in the oviductal environment and the enzymes identified on the IAM. The data presented provide evidence that MMP2 and acrosin are co-localized on the IAM, originate from the Golgi apparatus in gametogenesis, and suggest they cooperate in their function. Their localization and results of in vitro fertilization suggests they have a function in zona pellucida penetration. The data also provide evidence that plasminogen, originating from the oviductal epithelium and/or cumulus-oocyte complex, is present in the immediate environment of sperm-egg initial contact and penetration. Additionally, plasminogen interacts with MMP2 and enhances its enzymatic action on the IAM. The data also provide evidence that MMP2 has an important function in penetration of the cumulus oophorus. Holistically, this thesis provides evidence that enzymes on the IAM, originating from the Golgi apparatus in development, have an important function in penetration of the outer investments of the oocyte, and are aided in penetration by plasminogen in the female reproductive tract.