Thesis (Ph. D.)--University of Washington, 2007. / Vita. Includes bibliographical references (leaves 109-141).

This study was conducted to investigate the requirement for sperm processing in microsurgical subzonal placement of sperm in rabbit oocytes. Fertilization rates with standard in vitro fertilization and microsurgical subzonal sperm placement were found to be similar (56 and 55%) when sperm treated with high-ionic strength Brackett's defined inedium to initiate capacitation were used. Statistically significant reductions in fertilization rates for both standard in vitro fertilization and subzonal placement were noted when twice-washed spermatozoa were used. Initiation of capacitation of spermatozoa results in higher fertilization results even when the zona pellucida is bypassed during fertilization.

Mammalian sperm are not able to fertilize immediately upon ejaculation; they become fertilization-competent after undergoing changes in the female reproductive tract collectively termed capacitation. Although it has been established that capacitation is associated with an increase in tyrosine phosphorylation, little is known about the role of this event in sperm function. In this work we used a combination of two dimensional gel electrophoresis and mass spectrometry to identify proteins that undergo tyrosine phosphorylation during capacitation. Some of the identified proteins are the mouse orthologues of human sperm proteins known to undergo tyrosine phosphorylation. Among them we identified VDAC, tubulin, PDH E1 β chain, glutathione S-transferase, NADH dehydrogenase (ubiquinone) Fe-S protein 6, acrosin binding protein precursor (sp32), proteasome subunit alpha type 6b and cytochrome b-c1 complex. In addition to previously described proteins, we identified two testis-specific aldolases as substrates for tyrosine phosphorylation. Genomic and EST analyses suggest that these aldolases are retroposons expressed exclusively in the testis, as has been reported elsewhere. Because of the importance of glycolysis for sperm function, we hypothesize that tyrosine phosphorylation of these proteins can play a role in the regulation of glycolysis during capacitation. However, neither the Km nor the Vmax of aldolase changed as a function of capacitation when its enzymatic activity was assayed in vitro, suggesting other levels of regulation for aldolase function. Looking upstream the kinase cascade, the identity of the kinase (s) that brings about the phosphorylation of the tyrosine residues remains to be elucidated. It has been suggested that the non receptor tyrosine kinase Src family is involved in the capacitation associated phosphorylation cascade. Using an immunological approach we show that the only Src family member present in mouse sperm extract is Src. The capacitation associated tyrosine phosphorylation is greatly reduced in the presence of Src specific inhibitors (SU6656 and SKI606) in vivo. As a means of control for the activity of Src inhibitors in our system, parallel experiments assaying the activity of PKA both in vivo and in vitro were realized. Surprisingly, Src inhibitors down regulates the phosphorylation of serine/threonine residues that correlate on earlier events in the capacitation, as assayed by western blot with PKA substrates antibody. However, in vitro kinase activity of PKA showed no effect of Src inhibitors in the phosphorylation of the PKA specific substrate, kemptide.

Les spermatozoïdes complètent l'acquisition de leur pouvoir fécondant lors d'un processus de maturation post-éjaculatoire nommé la capacitation. Ce processus nécessite un ensemble de modifications membranaires et intracellulaires, dont l'augmentation de la phosphorylation en tyrosine des protéines. Différentes enzymes à activité tyrosine kinase participent à cette augmentation mais aucune n'a été trouvée responsable de la totalité des événements de phosphorylation en tyrosine associés à la capacitation. L'expression de la Janus kinase (JAK) 1 fut récemment démontrée dans les spermatozoïdes. Toutefois, sa contribution à l'augmentation du contenu en phosphotyrosine des protéines n'est pas déterminée. Dans les cellules somatiques, les substrats des JAKs sont des facteurs de transcription de la famille des signal transducer and activator of transcription (STAT). Cette cascade de signalisation intervient dans une multitude de processus biologiques et est activée en réponse à la liaison des cytokines et des facteurs de croissance. Bien que l'expression du récepteur de l'interleukine-6 (IL-6) et de certains membres de la famille des STATs a préalablement été rapportée dans les spermatozoïdes, leur rôle lors du processus de capacitation n'est pas encore déterminé. Le but de ce projet de doctorat consistait donc à caractériser les intermédiaires de la voie JAK/STAT présents dans les spermatozoïdes humains. J'ai d'abord confirmé la présence du récepteur de 1TL-6 à la surface membranaire des spermatozoïdes sans qu'il soit possible, toutefois, de démontrer son activation et d'identifier des substrats potentiels de JAK1. De façon surprenante, les facteurs de transcription STAT1, 3, 4, 5 et 6 sont tous détectés dans les spermatozoïdes. À l'exception de STAT4, présent au niveau de la thèque périnucléaire, tous ces intermédiaires sont associés aux structures cytosquelettiques du flagelle. La localisation cellulaire des STATs ajoutée à l'absence d'activité de transcription dans les spermatozoïdes suggèrent fortement que ces protéines jouent un rôle moins classique dans les gamètes mâles matures. L'inhibition de STAT3 lors du processus de capacitation produit une baisse de la motilité et une altération de la fonction mitochondriale. Les résultats de cette étude sont en accord avec un rôle de STAT3 dans des activités indépendantes de son activité de facteur de transcription et reliées à la survie des spermatozoïdes lors des événements précédents la fécondation. Enfin, j'ai tenté d'explorer l'implication de STAT3 dans la voie dépendante de l'AMPc. Cette dernière étude suggère qu'une voie de signalisation commune mène à l'activation de la protéine kinase dépendante de l'AMPc et a permis d'identifier un nouveau substrat potentiel de cette enzyme dans les spermatozoïdes, soit la triosephosphate isomérase.

L'objectiu principal d'aquesta tesi és contribuir a un millor coneixement dels mecanismes moleculars implicats en el correcte assoliment de la capacitació "in vitro" (IVC) i posterior reacció acrosòmica "in vitro" (IVAR) dels espermatozoides porcins. Amb aquesta finalitat es va estudiar, per una banda, l'evolució temporal de l'estructura subpoblacional en els espermatozoides porcins durant aquests processos i es va observar canvis espeífics en el percentatge de les diferents subpoblacions mòbils sense que es perdés l'estructura de 4 subpoblacions descrita en l'ejaculat porcí. Per altra banda, la IVC i la IVAR indueixen no sols canvis en la fosforil·lació dels residus tirosina de les proteïnes sinó que s'ha demostrat que la fosforil·lació en els residus serina i treonina també està relacionada amb la IVC i IVAR. Així, s'ha descrit, no tan sols l'aparició de fosforil·lació específica en determinades proteïnes, sinò també la inducció de canvis conformacionals en altres proteïnes i l'aparició de senyal de fosforil·lació en serina i treonina en l'acrosoma de l'espermatozoide relacionada amb IVC i IVAR. En tercer lloc, s'observà un augment de l'activitat mitocondrial durant la IVC i els primers minuts de IVAR. Nogensmenys, en aquest treball es descriu un patró concret d'activació mitocondrial, des de nuclis concrets situats majoritàriament a la part més perifèrica de la peça intermitja de l'espermatozoide cap a la part central d'aquesta. Tanmateix l'actina i la mitofusina2 són dos proteïnes que podrien jugar un paper important en la modulació de la funció mitocondrial en l'espermatozoide porcí. / The first and principal aim of this thesis is to improve our knowledgement in molecular mechanisms and control of "in vitro" capacitation (IVC) and further acrosome reaction (IVAR) in boar spermatozoa. We have studied the structural motile subpopulation, the protein phosphorylation in concrete in tyrosine, serine and threonine residues and finally the mitochondrial activity during IVC and IVAR.Our results suggest that capacitation-induced motility changes are related to specific changes in the percentage of each motile-sperm subpopulation in the ejaculate without losing the overall, specific four-subpopulation structure. In this way, the maintenance of a four-subpopulation structure seems to be important in the control of the whole ejaculate physiology.Moreover, this study indicates that the changes in protein phosphorylation associated with IVC and subsequent IVAR affected not only pTyr, but also pSer and pThre, and they comprise not only the appearance of specific phosphorylated proteins, like pTyr 32-kDa, pSer-75 kDa and pSer 80-kDa proteins, but also structural changes that induce changes in pI and displacements in the sperm location of the phosphorylated proteins, such as the appearence of specific IVC- and IVAR-linked pSer and pThre signals in the acrosome. Finally, it have been shown that the increase of boar sperm mitochondria activity during IVC and the first minutes of IVAR is instrumental to perform the sperm function changes associated with these processes. Furthermore, the increase in mitochondria activity is originated in concrete nucleation points at the midpiece. Finally, actin and mitofusin-2 play important roles in the modulation of boar sperm mitochondria function, both by originating changes in the protein membrane environment and by changes in the mitochondrial structure by itself.

Thesis (M.Sc. in Physiology) -- Faculty of Graduate Studies, Mahidol University, 1979. / Financial support by National Research Council.

Thèse (M.Sc.)--Université Laval, 2002. / Bibliogr.: f. [87]-99. Publié aussi en version électronique.

Purpose - The purpose of the present study is to compare intrauterine msemination (IUI) pregnancy rates (PR) as a function of diagnosis and ovulation protocol utilizing an extended semen transport time. This allowed clients to conveniently collect IUI specimens in the comfort and privacy of their home. A single IUI per treatment cycle was performed. Basic Procedures - Three-hundred-ten consecutive infertilty couples having unexplained, male factor, ovulatory dysfunction, endometriosis, tubal factor or combined diagnostic factors receiving a total of 584 cycles of IUI were included. Ovulation protocols included LH surge, clomiphene citrate (CC)-hCG, CC-gonadotropins(Gn)-hCG, Gn-hCG or leuprolide acetate (L)-Gn-hCG followed 36-42 hours by a single IUI. Pregnancy rates per cycle (fecundity) and per couple (fertility) as a function of diagnosis, ovulation protocol and cycle number were evaluated. In each cycle the couples processed the specimen by adding sperm washing medium at room temperature to the specimen 30 min following collection and allowed it to incubate for two hours prior to IUI during transport. Main Findings - Overall, fecundity was 11.8% (69/584) and fertility was 22.3% (69/310); respectively by diagnosis was: unexplained 22.6%,38.8%; male factor 18.8%,42.9%; ovulatory dysfunction 12.4,22.6%; endometriosis 5.3%,11.1%; tubal factor 7.6%,13.3%; and combined factors 9.7%, 20.0%. Unexplained vs endometriosis (P < 0.0001, P < 0.005), tubal factor (fecundity P < 0.008) and ovulatory dysfunction (fecundity P < 0.027) was statistically different. Male factor vs endometriosis (P < 0.011, P < 0.036) was significantly different. Ovulatory dysfunction vs endometriosis was significantly different (fecundity P < 0.027). Pregnancies by ovulation protocol: LH surge 4.5%,10.5%; CC-hCG 9.4%,14.9%; CC-Gn-hCG 13.7%,23.7%; Gn-hCG 17.5%,45.3%; L-Gn-hCG 3.5%,6.7%. For Gn-hCG vs L-Gn-hCG (P < 0.009, P < 0.030) and LH surge (fecundity P < 0.033). CC-Gn-hCG vs CC-hCG (fertility P < 0.050) and L-Gn-hCG (P < 0.033, P < 0.034). Gn-hCG vs CC-hCG (fecundity P < 0.043). Conclusions - We conclude that IUI is effective when utilizing an extended transport time allowing most couples to collect the specimen at home and is most effective when utilizing Gn-hCG therapy. Based on our analysis, endometriosis, tubal factor and combined diagnostic categories should proceed earlier to higher level assisted reproductive technologies.

After ejaculation, mammalian spermatozoa must undergo a series of complex and poorly understood cellular events known as “capacitation” in order to be able to fertilize an oocyte. Among these, biochemical changes such as an increase in tyrosine phosphorylation of some sperm proteins have been correlated with the sperm capacity to fertilize an egg and found to be regulated by a cAMP dependent pathway. The influx of ions such as Ca2+ and HCO3- induce the activation of a soluble adenylyl cyclase (SACY) increasing the cAMP levels within the cell that leads to the activation of a protein kinase A (PKA), and a subsequent increase in protein tyrosine phosphorylation. This modification in sperm proteins seems to be essential for induction of a change in the motility pattern known as hyperactivation that enables the sperm to penetrate the zona pellucida of the oocyte and initiate fertilization. Since PKA is a serine/threonine kinase, it is not clear how it mediates protein tyrosine phosphorylation during sperm capacitation. Based on the finding that in somatic cells PKA activates c-Src, it has been proposed that the Src family of protein kinases (SFK) are the intermediate players involved in tyrosine phosphorylation induced by PKA activity. In order to better understand the molecular mechanisms involved in stallion sperm capacitation, the objectives of our study were: (1) To analyze PKA activity during stallion sperm capacitation (2) To evaluate the involvement of the Src family of protein kinases (SFK) on stallion sperm phosphorylation events associated with capacitation. Standard In Vitro Fertilization (IVF) has not been reproducibly successful in the horse. Recent data indicate that good fertilization rates may be achieved after treatment of sperm with procaine to induce hyperactivation. Our objectives were also to determine if drugs used in other species as well as procaine induce hyperactivation in stallion sperm and to evaluate biochemical changes such as protein tyrosine phophorylation.

Les spermatozoïdes sont des cellules haploïdes hautement spécialisées ayant pour but ultime la fécondation de l'ovocyte. Afin de devenir aptes à la fécondation, les cellules germinales mâles subissent plusieurs étapes de maturation, impliquant bien des modifications tant au niveau protéique, membranaire et intracellulaire. Toutefois, les spermatozoïdes de mammifères fraîchement éjaculés sont incapables de féconder et acquièrent leur plein pouvoir fécondant à l'intérieur du tractus génital femelle, lors de leur transit pour rencontrer l'ovule. Ces différentes étapes de maturation permettent aux spermatozoïdes d'obtenir, entre autres, leur caractéristique unique concernant leur composition protéique et lipidique. Cette complexité membranaire est un mécanisme permettant aux spermatozoïdes d'avoir une spécificité de signal qui rend possible la capacitaton et la réaction acrosomiale au moment et endroit opportuns. Enfin, ce mémoire traitera du fractionnement membranaire et des partenaires d'interaction de la tyrosine kinase YES1 chez les spermatozoïdes humains.