Dissertations / Theses on the topic 'Sperm maturation'

Beyond the haploid genome, mammalian sperm carry a payload of epigenetic information with the potential to modulate offspring phenotype. Morphologically mature sperm exit the testes, but cannot swim or interact with the oocyte without extensive remodeling during epididymal transit; this includes modifications to the lipid composition of the sperm membrane, gain of necessary proteins, and a dramatic shift in sperm RNA content. Epididymal maturation has also been linked to changes in the sperm methylome suggesting that the epididymis might play a broader role in shaping the sperm epigenome. First, we characterized the genome-wide methylation landscape in seven germ cell populations from throughout the male reproductive tract. Our data emphasize the stability of cytosine methylation in mammalian sperm, and identify a surprising, albeit transient, period during which sperm are associated with extracellular DNA. Second, given our interest in the small RNA repertoire of sperm we set out to address known bias in sequencing protocols by comparing several small RNA cloning protocols. We found a protocol recently developed by Kathleen Collins’ lab (OTTR) to be superior to commercially available kits in providing an accurate representation of tRNA fragment levels as compared to Northern blotting. These results not only provide a more accurate representation of tRNA fragments, but also more complexity than previously seen allowing us to reassess the true sperm small RNA content. Taken together, these results provide significant insight into the mechanisms and factors modulating sperm epigenomics during post-testicular sperm maturation.

Background: There is evidence to show that varicocele repair can improve conventional sperm parameters and sperm DNA integrity in infertile men with a clinical varicocele. Objective: To further examine the effect of varicocelectomy on sperm quality, specifically, sperm nuclear chromatin integrity, distribution of nuclear sulfhydryl groups and sperm maturation. Design, Setting and Participants: We prospectively evaluated a consecutive series of infertile men (n=29) presenting to Ovo clinic with one year or more history of infertility, a clinically palpable varicocele and abnormal semen parameters. Six sperm donors with normal sperm parameters served as controls. Surgical Procedure: Microsurgical sub-inguinal varicocelectomy. Outcome Measures: (1) Conventional sperm parameters, (2) aniline blue staining (AB is specific to histone lysines), (3) iodoacetamide fluorescein (IAF targets free protamine sulfhydryl groups) and (4) sperm chromatin structure assay (SCSA) with the results expressed as % DNA fragmentation index (%DFI) and percent high DNA stainability (%HDS) before and 4 months after microsurgical varicocelectomy. Results: The sperm %DFI, %HDS (a measure of chromatin compaction), % 5-IAF staining (diffuse head staining), % AB staining (dark blue) were all significantly lower in the control group compared to infertile men with varicocele (8 vs. 20%, 4.0 vs. 9.6%, 1.7 vs. 16.3%, and 2.5 vs. 13.5% respectively). The %5-IAF and %AB staining decreased significantly after surgery (from 16.3 to 5.4%, and from 13.5% to 5.4%, respectively). Similarly, the %HDS and %DFI also decreased significantly after surgery (from 10% to 6% and from 20% to 13%, respectively). The only notable relationships were between aniline blue staining and %HDS post varicocelectomy (r= 0.57, P <0.05), and both %IAF staining and %DFI were inversely correlated with motility (r=-0.44 and -0.43, respectively). Conclusion: The data show that varicocelectomy is associated with a consistent improvement in sperm DNA integrity and chromatin compaction using three different assays of sperm chromatin integrity (SCSA, IAF, Aniline Blue).
Contexte: Il y a la preuve(l'évidence) pour montrer que la réparation de varicocele peut améliorer des paramètres de sperme conventionnels et l'intégrité d'ADN de sperme dans des hommes infertiles avec varicocele clinique. Objectif: Examiner l'effet de varicocelectomy sur la qualité de sperme, spécifiquement, le sperme l'intégrité chromatin nucléaire, la distribution de groupes sulfhydryl nucléaires et la maturation de sperme. Schéma, environnement et participants : Nous avons éventuellement évalué une série consécutive d'hommes infertiles (n=29) présentant à la clinique Ovo avec un an ou plus d'histoire d'infertilité, varicocele cliniquement palpable et des paramètres de sperme anormaux. Six donneurs de sperme avec des paramètres de sperme normaux ont servi de contrôles. Intervention chirurgicale: Microchirurgie sous-inguinale varicocelectomie.Mesures des résultats: (1) Des paramètres de sperme conventionnels, (2) l'aniline bleu teintant(tachant) (d'AB est spécifique à histone lysines), (3) iodoacetamide fluorescein (libère protamine sulfhydryl des groupes) et (4) le sperme chromatin l'essai de structure (SCSA) avec les résultats (DFI) de fragmentation d'ADN de % et le pour cent haut ADN stainability (%HDS) auparavant et 4 mois après varicocelectomy microchirurgical Résultats: Le pourcentage de spermatozoïdes avec le sperme de DFI%, HDS%, positif de 5-IAF coloration, une coloration positive AB (bleu foncé) étaient significativement inférieure dans le groupe témoin par rapport aux hommes infertiles ayant une varicocèle (8 vs 20%, 4,0 vs 9,6%, 1,7 vs 16,3%, et 13,5 vs 2,5% respectivement). Le pourcentage de spermatozoïdes avec positifs 5-IAF coloration et AB nucléaire positif diminué de façon significative après la chirurgie (de 16,3 à 5,4%, et de 13,5% à 5,4%, respectivement). Les HDS% et DFI% également diminué de façon significative après la chirurgie (de 10% à 6% et de 20% à 13%, respectivement). Les seules relations entre les notables étaient coloration au bleu d'aniline et varicocélectomie après HDS (r = 0,57, P <0,05), et les deux taches IAF et DFI% ont été inversement corrélée avec la motilité (r = -0,44 et de -0,43, respectivement).Conclusion: Les données montrent que varicocélectomie est associée à une amélioration constante de l'intégrité d'ADN de sperme et compaction de la chromatine en utilisant trois différents dosages cytochimiques de l'intégrité chromatine des spermatozoïdes (SCSA, l'IAF, le bleu d'aniline).

Introduction: Mature spermatozoa recognise and bind hyaluronic acid (HA) in the extracellular matrix via Hyaluronic Acid Binding Proteins (HABPs). HA-binding may be important in supporting the sperms’ progress in the female genital tract. Information on sperm HABPs is limited, however and the current study investigated their expression in human and bovine sperm. Materials and methods: The ability of differential density gradient centrifugation (DDGC) processed human sperm to bind HA was evaluated. DDGC sperm populations (90% and 45% fractions) and sperm interacting with HA were assessed for DNA fragmentation and chromatin compaction using acridine orange (AO) and aniline blue (AB) staining, respectively. Additionally, the effect of capacitation on HA-binding was assessed. Expression of HABPs in bovine and human sperm was also investigated alongside changes in their distribution following capacitation and the acrosome reaction. Proteomics was used to monitor changes in the human sperm proteome (including HABPs) following capacitation and the acrosome reaction. Following a protein panning procedure, proteomics was also used to help identify HABPs in both HA-binding and non-binding fractions. The presence of HA-binding motifs (BX7B and Link module, common in several well-known HABPs) was also evaluated in both fractions. Increasing the sensitivity of HABP isolation and detection was attempted using biotinylation of sperm surface proteins alongside HA-affinity chromatography. Results: Sperm recovered from 90% fractions had significantly higher HA-binding ability, lower levels of DNA fragmentation and higher levels of chromatin compaction. The reverse was true for sperm from the 45% fractions. HA-binding showed higher efficiency at selecting sperm with higher DNA integrity and chromatin compaction than DDGC. Capacitation enhanced HA binding, which may reflect associated changes in sperm HABPs. Immunocytochemistry localised CD44 to the acrosome and equatorial segment of non-capacitated human sperm and on the anterior acrosome and post-acrosomal sheath of non-capacitated bovine sperm. CD44 was more confined to the equatorial segment following capacitation in human sperm and to the post-acrosomal sheath of bovine sperm. Acrosomal labelling of CD44 was highly reduced after the acrosome reaction. Hyaluronic acid-TRITC (tetramethylrhodamine isothiocyanate) labelled the sperm membrane and the neck region, particularly strongly after capacitation and in acrosome reacted bovine sperm. Qualitative and quantitative characteristics of 121 proteins altered by capacitation and the acrosome reaction is also reported. Differentially regulated proteins are involved in sperm metabolism, energy production, cell signaling, sperm-oocyte recognition and sperm motility. Following capacitation, mass spectrometry (MS) detected an increase in cilia and flagella associated protein 20, containing two potentially HA-binding BX7B motifs. Analysis of the proteomics data from the panning fractions resulted in the identification of 28 proteins in HA-binding fractions with PDBsum and SAS server highlighting structural similarities between ZPBP2 (found only in HA-binding fractions) and the HA-binding domain of CD44. Data also showed that 50% of binding (including ADAM32 and cilia-and flagella associated protein 20 amongst others) and 34.5% of non-binding proteins contained BX7B motifs, indicating a likely selective enrichment of putative HABPs by the panning technique. Western blot analysis confirmed these results. Isolation of HABPs from bovine sperm using HA affinity chromatography followed by SDS PAGE revealed distinct protein bands of molecular masses 58 and 78 kDa, possibly corresponding to CD44 and RHAMM, respectively, were detected. Other protein bands detected at 16, 27, 38, 42 and 47 may be novel HABPs, including ZPBP2 (molecular mass 38 kDa). Conclusion: In conclusion, ZPBP2, ADAM32, Midkine and cilia and flagella associated protein 20 may have HA binding properties. This is also the first study which has brought together compelling evidence for the relationship between excising methods of enriching good quality sperm (DDGC) and HA-binding. Sperm DNA fragmentation and chromatin compaction status, therefore, reflects this relationship and supports claims for a positive sperm quality benefit for HA-based sperm selection particularly for ICSI, where the choice of sperm is more critical. In this regard, the use of more specific, anti-HABP-based methods for sperm selection (initially targeting RHAMM, CD44 and SPAM1) should also be considered.

[ES] En las últimas décadas, la anguila europea Anguilla anguila ha sufrido una disminución drástica de su población lo que ha llevado su inclusión como especie en peligro crítico en la lista roja UICN. Esta situación, junto con la gran importancia comercial de esta especie, evidencia la necesidad de tomar acciones como el desarrollo de la reproducción en cautividad y el control de la pesca. Una de las herramientas más interesantes para su uso en la biología de la conservación es la criopreservación de espermatozoides, que presenta varias ventajas para esta especie, incluyendo la sincronización de gametos, la selección de líneas genéticas o su uso para la creación de un criobanco. Sin embargo, el desarrollo de protocolos de criopreservación necesariamente requieren esperma de buena calidad. Además, se necesita un método preciso para evaluar la calidad del esperma tanto antes como después de la criopreservación. Sobre esta última cuestión, la motilidad de los espermatozoides de los peces se considera uno de los mejores biomarcadores para la evaluación de la calidad de los espermatozoides en los peces, y se puede estudiar de forma subjetiva u objetiva utilizando sistemas "computer assisted sperm analysis" (CASA-Mot). Primero, se realizó un experimento para evaluar la precisión y la exactitud de ambos métodos para estudiar la motilidad del esperma: el método subjetivo y la técnica objetiva que utiliza el sistema CASA-Mot. Además, se probó si el grado de experiencia de los técnicos en el caso del método subjetivo tiene un efecto en la precisión de la estimación de la motilidad y, por lo tanto, hay una influencia del personal del laboratorio en la evaluación de la motilidad del esperma. Aquí concluimos que tanto el método como la experiencia técnica eran factores clave para evaluar con precisión la motilidad del esperma en la anguila europea, por lo que se requiere el uso de CASA-Mot junto con material calificado para obtener resultados fehacientes. En segundo lugar, se evaluaron métodos alternativos para la maduración de los machos de anguila europeos probando dos tratamientos hormonales diferentes: OVI, una gonadotropina recombinante; y VET, una gonadotropina purificada a partir de orina femenina. Después de elegir el mejor tratamiento hormonal de los dos, se evaluó el efecto de tres dosis diferentes con el objetivo de obtener el mayor rendimiento al menor coste. Los resultados de este experimento apuntaron a OVI como el mejor tratamiento hormonal en una dosis semanal de 1.5 UI/g de pez, que proporciona la mayor rentabilidad, obteniendo esperma de alta calidad a menor precio. En un tercer experimento, y utilizando los conocimientos adquiridos en los dos primeros experimentos, se realizaron una serie de experimentos para estandarizar los protocolos de criopreservación de esperma de anguila europea disponibles en ese momento (utilizando DMSO o metanol como crioprotector). Los resultados apuntaron al protocolo que utiliza el metanol como el mejor de ellos dos en términos de motilidad, velocidad y viabilidad de los espermatozoides y la preservación de la integridad del ADN. Siguiendo este último método estandarizado, se realizó un cuarto experimento con el objetivo de mejorar el protocolo en términos de volumen (volúmenes de esperma más grandes) y de calidad espermática. Además, se desarrolló un protocolo simple de almacenamiento a corto plazo para complementar las opciones de preservar el esperma durante diferentes períodos de tiempo. De todas las condiciones de almacenamiento probadas, las diluciones 1/50 a 4 ºC mostraron los mejores resultados, manteniendo la motilidad en comparación con el control durante 3 días, y manteniendo cierta motilidad espermática (12%) después de 7 días. A partir del experimento de criopreservación, fue posible aumentar los volúmenes a 2 y 5 mL sin perder la calidad del esperma en comparación con volúmenes más pequeños, y mejorando las mot
[CAT] En les últimes dècades, l'anguila europea Anguilla anguila ha sofert una disminució dràstica de la seva població el que ha portat la seva inclusió com a espècie en perill crític en la llista vermella UICN. Aquesta situació, juntament amb la gran importància comercial d'aquesta espècie, evidencia la necessitat de prendre accions com el desenvolupament de la reproducció en captivitat i el control de la pesca. Una de les eines més interessants per al seu ús en la biologia de la conservació és la criopreservació d'espermatozoides, que presenta diversos avantatges per a aquesta espècie, incloent la sincronització de gàmetes, la selecció de línies genètiques o el seu ús per a la creació d'un criobanc. No obstant això, el desenvolupament de protocols de criopreservació necessàriament requereixen esperma de bona qualitat. A més, es necessita un mètode precís per avaluar la qualitat de l'esperma tant abans com després de la criopreservació. Sobre aquesta última qüestió, la motilitat dels espermatozoides dels peixos es considera un dels millors biomarcadors per a l'avaluació de la qualitat dels espermatozoides en els peixos, i es pot estudiar de forma subjectiva o objectiva utilitzant sistemes "computer assisted sperm analysis" (CASA-Mot). Primer, es va realitzar un experiment per avaluar la precisió i l'exactitud de tots dos mètodes per estudiar la motilitat de l'esperma: el mètode subjectiu i la tècnica objectiva que utilitza el sistema CASA-Mot. A més, es va provar si el grau d'experiència dels tècnics en el cas del mètode subjectiu té un efecte en la precisió de l'estimació de la motilitat i, per tant, hi ha una influència del personal del laboratori en l'avaluació de la motilitat del esperma. Vam concloure que tant el mètode com l'experiència tècnica eren factors clau per avaluar amb precisió la motilitat de l'esperma en l'anguila europea, de manera que es requereix l'ús de CASA-Mot juntament amb material qualificat per obtenir resultats fefaents. En segon lloc, es van avaluar mètodes alternatius per a la maduració dels mascles d'anguila europeus provant dos tractaments hormonals diferents: OVI, un gonadotropina recombinant; i VET, un gonadotropina purificada a partir d'orina femenina. Després de triar el millor tractament hormonal dels dos, es va avaluar l'efecte de tres dosis diferents amb l'objectiu d'obtenir el major rendiment al menor cost. Els resultats d'aquest experiment van apuntar a OVI com el millor tractament hormonal en una dosi setmanal de 1.5 UI/g de peix, que proporciona la major rendibilitat, obtenint esperma d'alta qualitat a un preu millor. En un tercer experiment, i utilitzant els coneixements adquirits en els dos primers experiments, es van realitzar una sèrie d'experiments per estandarditzar els protocols de criopreservació d'esperma d'anguila europea disponibles en aquest moment (utilitzant DMSO o metanol com crioprotector). Els resultats van apuntar al protocol que utilitza el metanol com el millor d'ells dos en termes de motilitat, velocitat i viabilitat dels espermatozoides i la preservació de la integritat de l'ADN. Seguint aquest últim mètode estandarditzat, es va realitzar un quart experiment amb l'objectiu de millorar el protocol en termes de volum (volums d'esperma més grans) i de qualitat espermàtica. A més, es va desenvolupar un protocol simple d'emmagatzematge a curt termini per complementar les opcions de preservar l'esperma durant diferents períodes de temps. De totes les condicions d'emmagatzematge provades, les dilucions 1/50 a 4ºC van mostrar els millors resultats, mantenint la motilitat en comparació amb el control durant 3 dies, i mantenint certa motilitat espermàtica (12%) després de 7 dies. A partir de l'experiment de criopreservació, va ser possible augmentar els volums a 2 i 5 ml sense perdre la qualitat de l'esperma en comparació amb volums més petits.
[EN] In the last decades, the European eel Anguilla anguilla has suffered a drastic decrease in the recruitment in most areas of their distribution range, leading the species to be included as critically endangered in the IUCN list. This situation, together with the high commercial importance of the species, evidence the need of taking actions such as development of reproduction in captivity and control of fisheries based on the complexity of their life cycle. One of the most interesting tools for its use in conservation biology is the sperm cryopreservation, which presents several advantages for this species such as the synchronization of gametes, selection of genetic lines or cryobanking. However, the development of cryopreservation protocols necessarily requires good quality sperm, and it is also needed an accurate method to assess sperm quality both pre- and post-cryopreservation. On this last matter, fish sperm motility is considered one of the best quality biomarkers for sperm quality assessment in fish, and it can be evaluated subjectively or objectively using computer assisted sperm analysis (CASA-Mot) systems. First, an experiment was conducted to evaluate the precision and accuracy of both methods for assessing sperm motility: the subjective method and the objective technique using CASA-Mot system. Moreover, it was tested whether the degree of expertise of the technicians in the case of the subjective method, has an effect on the accuracy of the motility estimation, and therefore there is an influence of the laboratory staff on the sperm motility assessment. Here we concluded that both the method and the technician expertise were key factors in order to accurately assess sperm motility in European eel, so the use of CASA-Mot together with qualified stuff is required to obtain reliable results. Secondly, and alternative methods for European eel males maturation was evaluated by testing two different hormonal treatments: OVI, a recombinant ¿-choriogonadotropin; and VET, a human chorionic gonadotropin purified from female urine. After choosing the best hormonal treatment, the effect of three different doses was evaluated aiming for best performance and lowest cost on the treatment. The results of this experiment pointed at OVI as the best hormonal treatment in terms on sperm quantity and quality in most of the weeks of treatment, and at a weekly dose of 1.5 IU/g fish, which also provide the greatest profitability, obtaining high quality sperm at a lower price. In a third experiment, and using the knowledge acquired in the two first experiments (using the OVI hormonal treatment and CASA-Mot to assess sperm quality), a series of experiments were conducted to standardize the European eel sperm cryopreservation protocols available at the moment (using DMSO or methanol as cryoprotectant). The results indicated that the protocol using methanol was the best of them two in terms of sperm motility and velocity, sperm viability and preservation of DNA integrity. Following this last standardized method, a fourth experiment was conducted aiming for improvement of the protocol in terms of volume (larger volumes) and sperm quality outcome. Moreover, a simple protocol for short-term storage was developed to complement the options to preserve sperm for different time periods. Of all the tested storing conditions, 1/50 dilutions at 4 ºC showed the best results, maintaining the motility compared to control for 3 days, and some sperm motility (12%) was still observed after 7 days. From the cryopreservation experiment, it was possible to scale up the cryopreserved volumes to 2 and 5 mL without losing sperm quality compared to lower volumes. Moreover, the protocol was further improved by supplementing the protocol with egg yolk as an additive, obtaining the highest cryopreserved sperm motilities (over 50%) ever reported in European eel.
Herranz Jusdado, JG. (2019). Improvement of techniques for sperm evaluation and cryobanking in European eel [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/130846
TESIS

Mammalian spermatozoa acquire functionality during epididymal maturation and ability to penetrate and fertilize the oocyte during capacitation. Sperm quality results indicated that both epididymal maturation and ejaculation are key events for further capacitation, because only ejaculated spermatozoa are capable to undergo the set of changes leading to capacitation. Epididymal maturation is associated with a progressive loss of phosphotyrosine residues followed by a subtle increase after in vitro capacitation. Sperm glycocalix galactose, glucose/mannose and N-acetyl-D-glucosamine content increased distally in the epididymis. Fertilin analysis highlight that proteolytic processing occurrs mainly in testis for ADAM-1, and throughout epididymis for ADAM-2. Fertilin migrates from acrosomal region to acrosomal ridge during sperm transit from the distal corpus to the proximal cauda. All together we establish a basis for sperm assessment about the maturative status of the ejaculated sperm and, open new perspectives in the characterization of proteins markers of the sperm maturation and/or fertility.
Els espermatozoides de mamífers adquireixen la seva funcionalitat durant la maduració epididimària i l’habilitat de penetrar i fecundar l’oòcit durant la capacitació. L’anàlisi de la qualitat espermàtica de mostres epididimaries, ejaculades i capacitades in vitro, indica que tant la maduració epididimària com l’ejaculació són processos essencials per la posterior capacitació. La maduració epididimària està associada a una pèrdua progressiva de residus tirosina fosforilats, seguit d’un increment després de la capacitació in vitro. En el glicocàlix dels espermatozoides epididimaris augmenten significativament els residus galactosa, glucosa/mannosa i N-acetil-D-glucosamina al llarg del seu trànsit pel conducte epididimari. La fertilina és processada a nivell testicular (ADAM-1) i a nivell epididimari (ADAM-2) i relocalitza i concentra a la regió apical durant el seu pas del corpus distal al cauda proximal. En conjunt, en aquest projecte, s'ha establert una base per a l’avaluació espermàtica del grau de maduresa dels espermatozoides ejaculats que obre noves perspectives per la identificació de marcadors de maduració espermàtica i/o de fertilitat en els espermatozoides.

Les biotechnologies de la reproduction sont aujourd’hui largement utilisées dans le contrôle de la fertilité animale et humaine. Ces techniques présentent cependant des rendements faibles et font actuellement l’objet de nombreuses recherches. Ce travail de thèse s’inscrit dans ce contexte et se focalise sur la recherche de nouvelles molécules pro-fertilité dans l’espèce bovine, et s’articule autour de deux axes : le premier consiste à tester les propriétés pro-fertilité d’une enzyme du métabolisme lipidique sur la maturation ovocytaire, la fécondation et le développement embryonnaire préimplantatoire in vitro, et le deuxième à découvrir des molécules permettant d’améliorer la fécondance des spermatozoïdes. Nous démontrons ici que l’application de l’enzyme améliore de manière significative le nombre et la qualité des embryons au stade blastocyste. Un savoir-faire quant à l’utilisation de cette enzyme (fenêtre de traitement, concentration,…) a été développé. Cette thèse a également permit de caractériser différents composés avec des propriétés différentes dont une molécule originale permettant d’augmenter la vitesse des spermatozoïdes. Ce composé est prometteur car il est aussi actif sur des spermatozoïdes issus du testicule, de l’épididyme ou de l’éjaculat, avant ou après congélation
The reproductive biotechnologies are now widely used in control of animal and human fertility. However, these technics have low yields and are currently the subject of much research. In this context, the present thesis focuses on the search for new pro-fertility molecules in cattle, organized around two axis : the first one is to test the pro-fertility properties of an enzyme from lipid metabolism on maturation, fertilization and preimplantation embryo development in vitro, and the second one is to discover molecules to improve sperm fertilizing ability. Here, we show that application of the enzyme significantly improve the number and quality of embryos at the blastocyst stage. Expertise in the use of this enzyme (time of treatment, concentration, etc.) was developed. This thesis also allowed the characterization of different compounds with different properties. Among them, one original molecule increase sperm velocity. This compound is promising because it works on sperm from the testis, epididymis or ejaculate before or after freezing

A hipótese desta pesquisa foi que a maturação epididimária dos espermatozoides caninos envolve modificações nas proteínas e ácidos graxos do fluido sintetizado pelos distintos segmentos epididimários, além de alterações no perfil de ácidos graxos da membrana plasmática, organização celular e morfológica dos espermatozoides. Para tanto, este projeto foi realizado com 20 cães em idade reprodutiva e negativos para Brucella canis. Após a orquiectomia bilateral, os testículos e epidídimos foram acondicionados a 5°C por no máximo 24 horas, em seguida, as amostras foram colhidas por incisões na cauda, corpo e cabeça do epidídimo. As amostras foram imediatamente processadas por avaliação subjetiva e automática da motilidade e vigor espermático, concentração, morfologia espermática, integridade da membrana plasmática, acrossomal e atividade mitocondrial. Foi possível observar determinar maior número de espermatozoides dentro da normalidade na cauda do epidídimo, especialmente, referindo-se à motilidade, integridade de membrana e atividade mitocondrial. A avaliação das modificações ultraestruturais dos espermatozoides permitiu observar a migração da gota citoplasmática e alterações acrossomais. No perfil de ácidos graxos observaram-se variações na quantidade e presença dos ácidos durante o traje epididimário, destacando o acréscimo do DHA na região da cauda epididimária. Ainda, no perfil protéico do plasma epididimário canino, foi possível identificar um padrão regional de secreção de proteínas, maior nas regiões da cabeça e corpo em relação à cauda do epidídimo. Apesar das importantes informações geradas a partir do presente trabalho, mais estudos são necessários para a compreensão da fisiologia reprodutiva, especialmente da maturação espermática epididimária na espécie canina.
The hypothesis of this research is that the canine maturation of epididymal spermatozoa involves progressive changes in the protein and fatty acid content of the fluid synthesized by the different epididymal segments, as well as changes in the fatty acid profile of the sperm plasma membrane, cell organization and morphology of the canine sperm. Therefore, this experiment was conducted with 20 dogs in reproductive age and free of Brucella canis. After bilateral orchiectomy, testes and epididymis were stored at 5°C for up to 24 hours and then the samples were harvested through incisions of the tail, corpus and caput of the epididymis. The samples were immediately processed for subjective and automatic motility and sperm vigor, sperm count, morphology, plasma membrane integrity, acrosomal and mitochondrial activity. It was possible to observe a greater number of mature sperm in the epididymal tail compared to the corpus and caput, especially referring to motility, membrane integrity and mitochondrial activity. The evaluation of ultrastructural changes of the spermatozoa allowed to observed the migration of the cytoplasmic droplets and acrosomal changes. In the fatty acid profile was observed variations in the amount and presence of acids during epididymal maturation, highlighting the addition of DHA in the epididymal tail region. Moreover, in the protein profile of the canine epididymal plasma, it was possible to identify a regional pattern of protein secretion, higher in the caput and corpus in relation to the tail epididymis. In spite of the important data generated from this study, further studies are needed for understand the reproductive physiology, especially the epididymal sperm maturation in dogs.

The objective of part one of this study was to determine if the presence of porcine growth hormone (pGH) during oocycte in vitro maturation (IVM) affected subsequent embryo development. Pig cumulus-oocyte complexes (COC) (n=987) were aspirated from slaughterhouse derived ovaries and cultured in BSA-free NCSU 23 medium containing porcine follicular fluid (10% v/v), cysteine (0.1 mg/ml) and hormonal supplements (eCG and hCG, 10 IU/ml each), 10 ng/ml EGF, and with or without pGH (100 ng/ml) for 22 h. The COC were then cultured in the same medium with or without 100 ng/ml pGH, but without hormonal supplements for an additional 22 h. After the completion of maturation culture, cumulus cells were removed and oocytes were co-incubated with frozen-thawed spermatozoa for 8 h. Putative embryos were transferred to NCSU 23 containing 0.4% BSA and cultured for 144 h. Embryo development was assessed on d 6 of culture. The treatment groups were as follows: treatment 1 = control group cultured in IVM medium alone; treatment 2 = 100 ng/ml pGH present of the first 22 h of maturation culture and absent for the second 22 h of maturation culture; treatment 3 = 100 ng/ml pGH absent for the first 22 h of maturation culture, but present for the second 22 h of maturation culture; and treatment 4 = 100 ng/ml pGH present throughout the entire IVM period. Embryos were visually scored for developmental stage at 144 h following fertilization. Each oocyte in the study received a developmental score, based on a scale of 1 = uncleaved, 2 = 2-cell embryo, 3 = 4- to 8-cell embryo, 4 = 9- to 16-cell embryo, 5 = morula, and 6 = blastocyst. The addition of pGH did not affect porcine embryo development as compared to the control (1.57 ± .08, 1.67 ± .08, 1.47 ± .08, and 1.60 ± .08, respectively; P > .10). Replicates within the study differed significantly from each other (P < .01) primarily because the development in replicate 6 was greater than for all others. There was a significant treatment by replicate interaction (P < .05); pGH added during the first 22 h of IVM and pGH added during the second 22 h of IVM in replicate 6 resulted in higher development scores than for controls and continuous pGH addition. However, in replicate 2, continuous pGH resulted in the greatest development. These results suggest that pGH may exert a stimulatory effect on embryo development when present in the IVM media; however, further studies using pGH in IVM culture are necessary. The objectives of the second part of the study were to examine aspects of intracytoplasmic sperm injection (ICSI) using membrane-disrupted spermatozoa, in vitro fertilization (IVF), and sperm-mediated gene transfer in the pig. Porcine oocytes were shipped overnight in maturation media at 39°C in a portable incubator. After 22 h of maturation culture, oocytes were washed in maturation medium without gonadotropins and cultured for an additional 22 h. Cumulus cells were removed and oocytes were divided into four treatment groups: treatment 1 = ICSI using membrane-damaged spermatozoa coincubated with linear green fluorescent protein (GFP) DNA; treatment 2 = ICSI using membrane damaged spermatozoa; treatment 3 = IVF with frozen-thawed spermatozoa coincubated with linear GFP DNA prior to IVF; treatment 4 = IVF with frozen-thawed spermatozoa with no DNA coincubation. Embryos were scored for developmental stage at 144 h following fertilization. Each oocyte in the study received a developmental score, based on a scale of 1 = uncleaved, 2 = 2-cell embryo, 3 = 4-cell embryo, 4 = 5- to 8-cell embryo, 5 = 9- to 16-cell embryo, 6 = morula, and 7 = blastocyst. Although no overall difference in development score was observed following the four different treatments, a treatment difference among cleaved oocytes was observed when comparing only the two ICSI treatments (P < .05); development scores were greater in the ICSI treatment in which sperm were not coincubated with linear GFP DNA prior to injection than when the coincubation was performed (3.76 ± .21 vs. 3.13 ± .17, respectively). No differences in development score were observed in the two IVF treatments. The percentage of embryos expressing the GFP transgene on d 6 of culture following fertilization was 7.3% in the ICSI+GFP group and 0% in all other treatment groups. Thus, sperm-mediated gene transfer using ICSI in the pig has been demonstrated, although success rates were low.
Master of Science

Les techniques d’assistance médicale à la procréation particulièrement l’induction de l’ovulation, la maturation in vitro des ovocytes et la culture embryonnaire prolongée impliquent la manipulation des gamètes ainsi que les embryons à des moments critiques de leur maturation et développement qui sont également des étapes clé du remodelage épigénétique. Par conséquent, elles pourraient interférer avec la reprogrammation épigénétique, en particulier la mise en place de la méthylation des gènes soumis a empreinte au cours de l'ovogenèse, ou son maintien au cours du développement préimplantatoire. Afin d’évaluer ce risque nous avons analysé le profil de méthylation de KvDMR1, qui régule l’expression de KCNQ1OT1, dans des ovocytes humains mûris in vivo ou in vitro, provenant de patientes stimulées ou non. Nos résultats montrent que la mise en place de la méthylation au niveau de KvDMR1 se poursuit au cours de la maturation de l’ovocyte du stade VG au stade MII, in vivo et in vitro et que l’induction ovarienne des patientes génère des ovocytes épigénétiquement immatures. Par ailleurs, l’étude de la méthylation de H19 DMR qui régule l’expression d’Igf2 et H19 dans des embryons d’ICSI, atypiques bloqués en culture prolongée et dans les spermes correspondants met en évidence une hypométhylation de l'allèle paternel et une méthylation de l'allèle maternel dans certains embryons, sans que l'on puisse établir de lien entre les dérégulations de l’empreinte et l’arrêt du développement au stade blastocyste
Assisted reproductive technologies particularly the induction of ovulation, oocytes in vitro maturation, and prolonged embryo culture require in vitro manipulation of gamete and embryos at critical times of their maturation and development. In consequence, they may interfere with epigenetic reprogramming and affect particularly demethylation and remethylation of imprinted genes. To evaluate such a risk, we have determined the methylation profile of KvDMR1, the region that regulates KCNQ1OT1 imprinted gene, in human oocytes retrieved from stimulated or unstimulated cycles, at different phases of their maturation in vivo or in vitro. Our results show that the timing of establishment of the methylation profile of KvDMR1 covers the maturation phase of oocyte growth, in vivo and in vitro, and that hyperstimulation likely recruits young follicles epigenetically immature. Analysis of the methylation profile of H19DMR (DMR of IGF2/H19) in atypical ICSI embryos and corresponding sperm suggests that imprinting disorders are not responsible of embryo developmental failure prior the blastocyst stage

El present treball analitza la morfologia espermàtica de l'ejaculat de Sus domesticus, la histologia del conducte epididimari i la qualitat de l'esperma epididimari. El material d'estudi prové de mascles reproductors porcins de les races Landrace i Pietrain, sans i sexualment madurs. La metodologia emprada es basa en l'examen al microscopi òptic (camp dar, contrast de fases i contrast interferencial) i al microscopi electrònic (de rastreig i de transmissió). Per a l'anàlisi estadística de les dades s'ha utilitzat el test de la X2 de Pearson (p<0,01). L'estudi de la morfologia espermàtica de l'ejaculat permet distingir diversos tipus de gàmetes que s'han classificat en tres grups: espermatozoides madurs, espermatozoides immadurs i espermatozoides aberrants, així com algunes cèI.lules somàtiques. L'espermatozoide madur de Sus domesticus és un gàmeta típic de mamífer (format per tres parts: cap, peça de connexió i cua) en que destaquen: la forma oval i plana del cap, el desenvolupament d'una protuberància acrosòmica apical en una de les cares del cap i la presencia dels cossos laminars en la peça de connexió. L'espermatozoide immadur es caracteritza per la presencia de la gota citoplasmàtica, el major desenvolupament de la protuberància acrosòmica apical i per la flexibilitat del cap. Els espermatozoides aberrants es descriuen i classifiquen segons la morfologia externa i la morfologia interna, distingint-se una amplia gama de malformacions que afecten les diverses parts de l'espermatozoide. Les cèl·lules somàtiques presents en l'ejaculat ofereixen les característiques pròpies d'un macròfag i se les ha observat englobant espermatozoides immadurs.
L'estudi de l'estructura i la ultraestructura de les tres regions anatòmiques de l'epidídim (caput, corpus i cauda) revela que: a) l'epiteli epididimari és pseudoestratificat amb esterocilis, b) cada regió epididimària presenta uns valors característics en relació al diàmetre intern del conducte, a l'alçada de l'epiteli, a la longitud dels esterocilis i al nombre de cèl·lules somàtiques luminals, i c) l'epiteli epididimari esta format per cinc tipus cel·lulars: les cèl·lules principals, les cèl·lules basals, les cèl·lules dares, les cèl·lules estretes i les cèl·lules basòfiles. Dels resultats obtinguts es pot deduir que: a) aquests cinc tipus cel·lulars es distribueixen al llarg del conducte epididimari de forma no homogènia, b) les cèl·lules basals, les cèl·lules principals, les cèI.Iules dares i les cèl·lules estretes són diversos estadis del desenvolupament d'un mateix tipus cel·lular especialitzat en la secreció i reabsorció cel·lular, i c) les cèl·lules basòfiles són les precursores de les cèl·lules somàtiques luminals.
La qualitat de l'esperma procedent de les tres regions de l'epidídim ha estat analitzada a partir dels següents paràmetres espermàtics: vitalitat, resistència osmòtica dels acrosomes, estabilitat cefàlica, morfologia, malformacions i aglutinació.
La vitalitat espermàtica disminueix progressivament al llarg del conducte epididimari. La resistència osmòtica dels acrosomes s'assoleix en la regió corporal de l'epidídim. L'estabilitat cefàlica dels espermatozoides és més elevada en les dues primeres regions de l'epidídim que en la regió caudal. Cada regió de l'epidídim es caracteritza per una morfologia espermàtica específica: a) el caput es caracteritza per l'elevat percentatge d'espermatozoides immadurs amb gota citoplasmàtica proximal, b) el corpus es caracteritza per l'elevat percentatge d'espermatozoides immadurs amb gota citoplasmàtica distal, i c) el cauda es caracteritza per l'elevat percentatge d'espermatozoides madurs. S'han estudiat les següents malformacions d'origen epididimari: espermatozoides de cua doblegada per l'anell de Jensen (origen en el cauda), espermatozoides de cua enrotllada i espermatozoides de cues fusionades (origen en el corpus). Els espermatozoides perden la capacitat de doblegar la cua per la peça intermèdia a mesura que avancen pel conducte epididimari. L'aglutinació espermàtica tendeix a augmentar progressivament al llarg del conducte epididimari, si bé, no s'han observat variacions significatives en els diversos tipus d'aglutinació.
La maduració epididimaria dels espermatozoides de Sus domesticus és un procés lent i complex, i la qualitat de l'ejaculat depèn de que aquesta maduració hagi estat completa. La presencia en l'esperma ejaculat de formes gamètiques pròpies de l'esperma epididimari és un signe d'una incompleta maduració dels espermatozoides; i, pot considerar-se com un paràmetre indicador d'estrés del mascle reproductor, tant més quant més s'assembli a la morfologia espermàtica de la regió cefàlica de l'epidídim.
This work analyzes the sperm morphology of the ejaculate of Sus domesticus, the histology of the epididymal duct and the quality of the epididymal sperm. The material of research comes from healthy and sexually mature boars (Landrace and Pietrain breeds). The methodology used is based on the examination by light microscopy, phase-contrast and interferential contrast microscopy and by electron microscopy (scanning and transmission). Comparisons of data of the seminal parameters observed in the three epididymal regions were made by the Pearson's X2 test at the 0.01 level of significance.
The study of the sperm morphology of the ejaculate allows distinguishing different gamete forms classified into three groups: mature, immature and aberrant spermatozoa, as well as some somatic cells. The mature spermatozoon of Sus domesticus is a typical gamete of a mammal (it can be divided into three regions: head, connecting piece and tail) characterized by the following features: the oval and plain shape of the head, the development of an apical semi lunar protuberance in one of the head's sides and the presence of the laminar bodies in the connecting piece. The immature spermatozoon is characterized by the presence of the cytoplasmic droplet, by the greater development of the apical acrosome protuberance and by the head's flexibility. The aberrant spermatozoa are described and classified according to the external and internal morphology, being observed a large range of malformations which affect to different regions of the spermatozoon. The somatic cells present in the ejaculate show the characteristics of a macrophage and have been observed including immature spermatozoa.
The study of the structure and ultra structure of the three anatomic regions of the epididymis (caput, corpus and caudal) reveals that: a) the epididymal epithelium is pseudo stratified with sterocilia, b) each epididymal region presents characteristic values in relation to the internal diameter of the duct, to the height of the epithelium, to the length of the stereocilia and to the number of luminal somatic cells, and c) the epididymal epithelium contains five cellular types: principal cells, basal cells, clear cells, narrow cells and basophilic cells. From the results obtained it can be deduced that: a) these five cellular types are not distributed homogeneously along the epididyrnal duct, b) the basal cells, clear cells and narrow cells are different stadia of the development of a same cellular type specialized in the cellular type specialized in the cellular secretion and resorption, and c) the basophilic cells are the precursors of the luminal somatic cells.
principal eells, c1ear eells and narrow cells are different stadia of the development of a same eellular type specialized in the eellular seeretion and resorption, and el the basophilie eells are the preeursors of the luminal somatie eells.
The sperm quality from the three epididymal regions has been analyzed starting from the following sperm parameters: vitality, osmotic resistance of the acrosomes, cephalic stability, morphology, malformations and agglutination. The sperm vitality decreases progressively along the epididymal duet. The osmotic resistance of the acrosomes occurs in the corporal region of the epididymis. The cephalic stability of the spermatozoa is higher in the first two epididymal regions than in the caudal region. Each epididymal region is characterized by specific sperm morphology: a) the caput is characterized by the high percent of immature spermatozoa with proximal cytoplasmic droplet, b) the corpus is characterized by the high percent of immature spermatozoa with distal cytoplasmic droplet, and c) the cauda is characterized by the high percent of mature spermatozoa. There have been studied the following malformations of epididymal origin: spermatozoa with tail folded at the Jensen's ring (cauda origin), coiled tail spermatozoa and spermatozoa with fused tails (corpus origin). The sperm agglutination tends to increase progressively along the epididymal duet, although it has not been observed significative variations in the different types of agglutination.
The epididymal maturation of the spermatozoa of Sus domesticus is a slow and complex process, and the quality of the ejaculate depends on a complete maturation of the spermatozoa. The presence of gametic forms characteristic of the epididymal sperm in the ejaculated is a sign of an incomplete maturation of the spermatozoa; and, it can be considered as a stress indicative parameter of the boar, the more when they look more similar to the sperm morphology of the cephalic region of the epididymis.

Many primate populations face the threat of extinction due to habitat loss, intensive agriculture, hunting for meat, the pet trade and/or use in traditional medicines. An alternative approach to in situ conservation includes gene banking and the use of assisted reproductive technologies (ART), such as oocyte in vitro maturation (IVM) and in vitro fertilization (IVF). Although many of these 'high-tech' solutions have not yet been proven viable for pragmatic wildlife conservation, basic research and development of these emerging tools can provide necessary information needed to optimize these techniques and institute ART as a routine practice in conservation efforts. A severely limiting factor in the successful application of ARTs is the availability of mature developmentally competent oocytes. Oocyte maturation involves many nuclear and cytoplasmic factors, which can be affected by maturation conditions and female age. In vitro maturation does not have the same success rate across species studied. In primates especially, IVM oocytes exhibit reduced developmental capacity upon fertilization when compared to in vivo matured (IVO) oocytes. This study aimed to investigate possible causes of reduced developmental capacity of primate IVM oocytes using the rhesus macaque (Macaca mulatta) as a model. Research efforts included investigation of ovarian senescence, oocyte karyotype and spindle morphology, and establishment of an optimal sperm cryopreservation protocol for use in IVF. Histological examination of the rhesus ovary demonstrated an age-related pattern of follicle depletion similar to that described in the human ovary. Oocyte karyotype analysis revealed a significant effect of IVM on the frequency of hyperhaploidy. In addition, immunostaining and confocal microscopy demonstrated a significant increase of anomalous chromosome congression on the oocyte metaphase II spindle equator in relation to IVM and donor female age. These results indicate that IVM can produce serious, if not lethal consequences for embryo development. This study presents baseline data on ovarian aging in the rhesus macaque and aspects of nuclear maturation during macaque IVM that may contribute to the design of primate oocyte recovery plans. Implementation of either of two sperm cryopreservation methods originally developed for rhesus and vervet monkeys will aid future investigation of the developmental capacity of IVM oocytes.

[ES] Resumen Como pez de gran valor económico, procedente de una de las líneas de teleósteos más antiguas, con un ciclo de vida misterioso, un potencial de acuicultura excepcional, y con importancia cultural y actividades de pesca en casi todos los países de Europa, la anguila europea posee un enorme valor socioeconómico. Este valor se suma a la desgraciada situación actual en peligro crítico de población natural de anguilas europeas. Como el ciclo de vida de la anguila aún no se ha conseguido cerrar en cautiverio, si la especie se extingue en la naturaleza, no seremos capaces de recuperarla. El cierre del ciclo de vida de la anguila europea ha sido, por lo tanto, el objetivo final de varios estudios. Sin embargo, a pesar de una investigación científica sustancial, desde la década de 1930, varios aspectos de la maduración de la anguila, como el mecanismo que bloquea la maduración de la anguila en la etapa prepúber en cautiverio, aún no se conocen bien. Por lo tanto, es necesario ampliar nuestro conocimiento sobre la reproducción de la anguila para inducir mejores hipótesis y lograr un progreso sustancial. Para profundizar en este campo, esta tesis se realizó con el objetivo específico de desarrollar métodos innovadores para la inducción de la maduración de la anguila y aumentar el conjunto de conocimientos sobre los procesos europeos de maduración de la anguila. Los procedimientos hormonales utilizados actualmente para la maduración sexual de la anguila artificial probablemente no induzcan el proceso natural de maduración. Por lo tanto, esta tesis ha evaluado el potencial de las hormonas recombinantes específicas de la anguila para inducir un proceso de maduración más natural. Este estudio específico mostró que la espermatogénesis completa y la espermiación se pueden inducir con gonadotropinas específicas de anguila recombinante; sin embargo, la calidad del gameto resultante es aún inferior a los resultados de los protocolos establecidos. Sin embargo, la utilización de hormonas recombinantes tiene un gran potencial para futuras implementaciones. Además, el experimento de gonadotropina recombinante ha generado nuevos detalles sobre el efecto de las gonadotropinas homólogas en el eje BPG de las anguilas europeas. Trabajos previos han llevado a la hipótesis de que un tratamiento térmico adecuado puede reducir o reemplazar parcialmente los tratamientos hormonales estándar para la maduración sexual de la anguila europea, o puede mejorar la calidad y / o cantidad de gametos. En esta tesis, se probó el efecto de varios regímenes térmicos en el eje BPG de machos de anguila europeos prepúberes, sin administración de hormonas. Los resultados muestran claramente que un tratamiento de agua de mar fría durante 2 semanas (10 ° C) afecta el eje BPG de los machos de anguila europeas. Los resultados específicos incluyeron un aumento en la sincronización de espermatogonias, niveles elevados de testosterona y 11-ketotestosterona en plasma, agrupamiento de muestras de transcriptomas del eje BPG del grupo tratado con agua de mar fría y posiblemente niveles aumentados de la proteína subunidad ß de la hormona luteinizante de la hipófisis. Los genes transcritos diferencialmente incluyeron varios genes, procesos y vías interesantes, que parecen estar involucrados en la maduración "natural" temprana de la anguila y que pueden ser biomarcadores adecuados para las distintas etapas de este proceso. Para un análisis adecuado de los datos transcriptómicos, se creó un transcriptoma de anguila europea de novo. Se demostró que este transcriptoma de novo posee una superior integridad al genoma de anguila europea disponible y, por lo tanto, es una herramienta útil para el análisis adicional de genes específicos. Un análisis de este transcriptoma reveló un gran número de pares de genes parálogos, que mostraron una baja divergencia entre secuencias sinónimas. Entre las hipótesis potenciales sobre e
[CAT] Com a espècie de renom culinari que pertany a un dels llinatges teleostis més antics, amb un cicle vital misteriós, un potencial d'aqüicultura excepcional, i una tradició pesquera a gairebé tots els països d'Europa, l'anguila europea posseeix un enorme valor socioeconòmic. No obstant això, aquest valor només fa que augmentar la preocupació de la seva població, que actualment es troba catalogada com "en perill crític d'extinció". Atès que el cicle de vida de les anguiles encara no ha estat tancat en captivitat, l'espècie no serà salvable en el cas que s'extingeixi en estat natural, per la qual cosa tancar el cicle de vida d'aquesta espècie ha estat l'objectiu final de diversos grups d'investigació durant els últims anys.. No obstant això, i malgrat la investigació científica de qualitat duta a terme des de la dècada de 1930, encara hi han diversos aspectes de la maduració de les anguiles -com el mecanisme que bloqueja la maduració sexual de l'anguila a l'etapa pre-puberal en captivitat- que son poc coneguts en l'actualitat. Per tal d'ampliar els coneixements sobre la reproducció de les anguiles i aconseguir un progrés substancial, aquesta tesi es va dur a terme amb l'objectiu específic de desenvolupar mètodes innovadors per a la inducció de la maduració de l'anguila europea, a més de afegir-hi el coneixement en els processos de maduració bàsics d'aquesta espècie. Els procediments hormonals utilitzats actualment per a la maduració artificial de l'anguila europea no acaben d'induir el procés de maduració natural tal i com probablement es dóna a la natura. Doncs, en primer lloc, aquesta tesi va avaluar el potencial d'hormones recombinants específiques d'anguila europea per induir un procés de maduració molt més natural. Aquest estudi específic va mostrar que mitjançant estes gonadotropines específiques d'anguila europea és possible induir l'espermatogènesi i l'espermiació completes. Tot i que els resultats van mostrar que la qualitat dels gamets va ser inferior als resultats que generen els protocols establerts fins ara amb un altre tipus d'hormones (generalment d'origen humà), la utilització d'hormones recombinants específiques es presenta amb un gran potencial per a la seva implementació futura en la inducció de la maduració sexual de l'anguila europea, ja que l'estudi va generar noves idees sobre l'efecte de les gonadotropines l'eix BPG de l'anguila europea. En segon lloc, i treballant amb la hipòtesi que un tractament tèrmic adequat pot reduir o substituir parcialment els tractaments hormonals estàndards per a la maduració sexual de l'anguila europea, en aquesta tesi es va provar l'efecte de diversos règims tèrmics (sense administració d'hormones) en l'eix BPG dels mascles europeus pre-puberals amb l'objectiu de millorar la qualitat i / o quantitat dels gamets. Els resultats mostraren clarament que un tractament d'aigua de mar de 2 setmanes a baixa temperatura (10 °C) va afectar l'eix BPG dels mascles europeus d'anguila. Resultats més específics van mostrar un augment de la sincronització de les espermatogonies, elevats nivells plasmàtics de testosterona i 11-ketotestosterona, una agrupació de mostres de transcriptoma de l'eix BPG del grup tractat amb aigua de mar freda i, possiblement, un augment dels nivells de la proteïna de la subunitat ß de la hormona luteinitzant de la hipofisi. Els gens transcrits diferencials van al·ludir a diversos gens, processos i vies interessants, que semblen estar implicats en la maduració inicial de l'anguila "natural" i podrien resultar biomarcadors adequats per a les etapes d'aquest procés. No obstant això, es necessiten estudis addicionals per avaluar el potencial dels biomarcadors d'aquests gens i, de manera complementària, comprovar si un pre-tractament d'aigua de mar freda pot millorar la resposta de les anguiles europees a un tractament hormonal artificial, com suggereixen els resultats. Finalment, amb l'objectiu
[EN] As an expensive fish from one of the most ancient teleost lineages, with a mysterious life cycle, exceptional aquaculture potential, and cultural associations and fishing activity in almost every country in Europe, the European eel possess huge socioeconomic value. This value only adds to the misfortune of the current critically endangered state of the wild European eel population. As the eel lifecycle has not yet been closed in captivity, the species will not be salvable if it went extinct in the wild. Closing the life-cycle of the European eel has thus been the ultimate objective of several studies. However, despite the substantial scientific investigation, since the 1930s, several aspects of eel maturation, such as the mechanism which blocks eel sexual maturation at the pre-pubertal stage in captivity, is still poorly understood. Therefore, it is necessary to broaden our knowledge of eel reproduction to induce better hypotheses and therethrough achieve substantial progress. In order to further this field, this thesis was conducted with the specific objective of developing innovative methods for induction of eel maturation and add to the pool of knowledge of European eel maturation processes. The hormonal procedures currently used for artificial eel sexual maturation are probably not inducing the natural maturation process. Therefore, this thesis has evaluated the potential of eel specific recombinant hormones to induce a more natural maturation process. This specific study showed that full spermatogenesis and spermiation can be induced with recombinant eel specific gonadotropins; however, the resulting gamete quality is still inferior to the results of established protocols. Nevertheless, the utilization of recombinant hormones holds a large potential for future implementation. Furthermore, the recombinant gonadotropin experiment has generated novel insights into the effect of homologous gonadotropins on the BPG axis of European eels. Previous work has led to the hypothesis that the right thermal environmental treatment may reduce or partially replace the standard hormonal treatments for sexual maturation of European eel, or may improve gamete quality and/or quantity. In this thesis, the effect of various thermal regimes was tested on the BPG axis of pre-pubertal European eel males, without administration of hormones. The results clearly show that a 2 week cold (10 °C) seawater treatment effects the BPG-axis of European eel males. Specific results included an increase in the synchronization of spermatogonial cells, elevated testosterone and 11-ketotestosterone plasma levels, clustering of BPG-axis transcriptome samples from the cold seawater treated group and possibly increased levels of pituitary luteinizing hormone ß-subunit protein. Differentially transcribed genes alluded to several interesting genes, processes, and pathways, which appears to be involved in early "natural" eel maturation and may prove to be suitable biomarkers for the stages of this process. In order for proper analysis of the transcriptomic data, a de novo European eel transcriptome was assembled. This de novo transcriptome was proven to have superior completeness to the available European eel genome and is thus a useful tool for further analysis of specific genes. An analysis of this transcriptome revealed a large number of paralog gene pairs, which showed low synonymous sequence divergence. Among the potential hypothesis regarding the origin of these paralog gene pairs, the hypothesis of a 4R whole genome duplication is among the most parsimonious. Several of these duplicated genes were involved in reproduction and the onset of puberty. Regardless of the origin, further analysis of these genes may reveal eel specific adaptations, which could help to better understand the exceptional reproductive system of eels.
Rozenfeld, C. (2019). Development of innovative methods for induction of European eel (Anguilla anguilla) spermatogenesis [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/125697
TESIS

Les techniques d'assistance médicale à la procréation particulièrement l'induction de l'ovulation, la maturation in vitro des ovocytes et la culture embryonnaire prolongée impliquent la manipulation des gamètes ainsi que les embryons à des moments critiques de leur maturation et développement qui sont également des étapes clé du remodelage épigénétique. Par conséquent, elles pourraient interférer avec la reprogrammation épigénétique, en particulier la mise en place de la méthylation des gènes soumis a empreinte au cours de l'ovogenèse, ou son maintien au cours du développement préimplantatoire. Afin d'évaluer ce risque nous avons analysé le profil de méthylation de KvDMR1, qui régule l'expression de KCNQ1OT1, dans des ovocytes humains mûris in vivo ou in vitro, provenant de patientes stimulées ou non. Nos résultats montrent que la mise en place de la méthylation au niveau de KvDMR1 se poursuit au cours de la maturation de l'ovocyte du stade VG au stade MII, in vivo et in vitro et que l'induction ovarienne des patientes génère des ovocytes épigénétiquement immatures. Par ailleurs, l'étude de la méthylation de H19 DMR qui régule l'expression d'Igf2 et H19 dans des embryons d'ICSI, atypiques bloqués en culture prolongée et dans les spermes correspondants met en évidence une hypométhylation de l'allèle paternel et une méthylation de l'allèle maternel dans certains embryons, sans que l'on puisse établir de lien entre les dérégulations de l'empreinte et l'arrêt du développement au stade blastocyste.

Research Doctorate - Doctor of Philosophy (PhD)
The mechanisms and molecules mediating marsupial fertilisation and reproduction will only be elucidated with a detailed understanding of the molecular nature of marsupial gametes and their post-testicular maturation. This thesis presents the first pivotal steps in defining the antigenic nature and maturation of marsupial spermatozoa. Numerous monoclonal antibodies produced against tammar wallaby (Macropus eugenii) and brushtail possum (Trichosurus vulpecula) sperm antigens provided specific tools for characterising the cellular, biochemical and molecular nature of these gametes. Marsupial spermatozoa, like those of their eutherian counterparts, are comprised of a complex array of antigens. Many of these antigens were restricted to specific cellular regions and surface domains, whilst others were distributed widely in the cell. The epitopes recognised by the monoclonal antibodies also displayed differential characteristics. Some were species-specific, whilst others were shared only by other marsupial spermatozoa or a wide variety of species. Similarly some antibodies bound sperm-specific epitopes, whilst others were common to somatic tissues. Most sperm antigens arose in the marsupial testis, however others were added or modified during post-testicular maturation in the epididymis. The characterisation of sperm antigens with potential roles in sperm maturation, fertilisation events were facilitated by the WSA- 1 monoclonal antibody. This antibody recognised a species and tissue-specific epitope shared by an acrosomal matrix antigen and an epididymal maturation antigen on wallaby spermatozoa. The acrosomal antigen arose in the wallaby testis, persisting unaltered during epididymal maturation and shared some sequence homology with proacrosin. The proacrosin/acrosin zymogen mediates sperm binding to ZP2 and zona penetration in eutherian species. This antibody might therefore be used to investigate the importance of proacrosin in marsupial fertilisation. The WSA- 1 maturation antigen was added to the whole surface of wallaby spermatozoa during epididymal transit. Secretion by the epididymal epithelium commenced in the proximal head of the epididymis and persisted distally in the tract whereas the antigen first associated with the sperm surface in the proximal body of the epididymis. Cross-linking of the WSA-1 carbohydrate epitope on the sperm surface resulted in potent midpiece-midpiece agglutination of wallaby ejaculated spermatozoa. Investigations in eutherian species suggest that the WSA-1 maturation antigen may function in sperm storage or the acquisition of sperm motility and fertility in the wallaby epididymis. As such the WSA-1 antigen is likely to be involved capacitation and/or fertilisation events in the female tract and has great promise as an immunocontraceptive target antigen. Intracellular antigenic maturation accompanies the morphological maturation of marsupial spermatozoa during epididymal transit. The PSA-10 monoclonal antibody recognised midpiece fibre network antigen(s) that arose concomitantly with the epididymal development of this structure in both possum and wallaby spermatozoa. Thus the PSA-lO monoclonal antibody provides an important tool for following the development and fate of a cytoskeletal structure which is unique to marsupial spermatozoa. The PSA-10 antibody also recognised an antigen associated with the outer acrosomal membrane of possum spermatozoa. Initial detection of PSA-10 acrosomal immunoreactivity also accompanied major morphological folding and consolidation of the possum acrosome during epididymal transit. Evidence of the antigenic modification of the marsupial sperm nucleus during spermiogenesis was provided by the differential binding patterns of two monoclonal antibodies. The WSA-3 and PSA-1 monoclonal antibodies each bound sperm-specific nucleoproteins in the wallaby testis. The WSA - 3 antigen was first detected in stage 10 spermatids and accumulated in spermatids in stages of spermiogenesis characterised by considerable nuclear condensation and elongation. However the PSA- 1 nucleoprotein was first detected quite late in spermiogenesis on stage 13 spermatids. These marsupial sperm nucleoproteins may play a role in the protein transitions thought to mediate chromatin binding and condensation in the mammalian spermatid nucleus. The construction of a wallaby testis cDNA library as part of this investiagation provides an invaluable tool for the identification of of the nucleotide sequences encoding marsupial sperm proteins. Screening the cDNA library resulted in the cloning of a partial sequence for a marsupial heat shock protein. The highly conserved heat shock proteins have important roles in the cellular processing and cytoprotection of testicular polypeptides. The significant contributions to our fundamental knowledge of marsupial sperm antigens and their epididymal maturation in this study provide a strong foundation for future examinations of marsupial capacitation and fertilisation events. Some of the antigens identified may also have applications to the regulation of fertility in pest marsupial species.

abstract: Infertility has become an increasing problem in developed countries and in many cases can be attributed to compromised sperm quality. Assessment of male fertility typically utilizes semen analysis which mainly examines sperm morphology, however many males whose sperm appear normal are sub- or infertile, suggesting that sperm from these males may be deficient in a protein or suite of proteins. To date, very little is known about the composition of sperm or the complex maturation process that confers motility and fertilization competency to sperm. Chapter 1 discusses the use of whole cell mass spectrometry to identify 1247 proteins comprising the Rhesus macaque (Macaca mulatta) sperm proteome, a commonly used model of human reproduction. This study provides a more robust proxy of human sperm composition than was previously available and facilitates studies of sperm using the rhesus macaque as a model. Chapters 2 & 3 provide a systems level overview of changes in sperm proteome composition that occurs during epididymal transit. Chapter 2 reports the proteomes of sperm collected from the caput, corpus and cauda segments of the mouse epididymis, identifying 1536, 1720 and 1234 proteins respectively. Chapter 3 reports the sperm proteome from four distinct segments of the Rhesus macaque epididymis, including the caput, proximal corpus, distal corpus and cauda, identifying 1951, 2014, 1764 and 1423 proteins respectively. These studies identify a number of proteins that are added and removed from sperm during epididymal transit which likely play an important role in the sperm maturation process. To date no comparative evolutionary studies of sperm proteomes have been undertaken. Chapter 4 compares four mammalian sperm proteomes including the human, macaque, mouse and rat. This study identified 98 proteins common to all four sperm proteomes, 82 primate and 90 rodent lineage-specific proteins and 494, 467, 566, and 193 species specific proteins in the human, macaque, mouse and rat sperm proteomes respectively and discusses how differences in sperm composition may ultimately lead to functional differences across species. Finally, chapter 5 uses sperm proteome data to inform the preliminary design of a rodent contraceptive vaccine delivered orally using recombinant attenuated Salmonella vaccine vectors.
Dissertation/Thesis
Ph.D. Biological Design 2013

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.

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.

博士
國立臺灣大學
生物科技研究所
102
SERPINE2, one of the potent serine protease inhibitors that modulate the activity of plasminogen activator and thrombin, is implicated in many biological processes. In the first study, we purified SERPINE2 from mouse seminal vesicle secretion based on its potent inhibitory activity against the urokinase-type plasminogen activator. A prominent amount of SERPINE2 was detected on ejaculated and oviductal spermatozoa, predominantly on uncapacitated sperm, suggesting the need to remove SERPINE2 before initiation of the capacitation process. Moreover, SERPINE2 could inhibit in vitro bovine serum albumin-induced sperm capacitation and prevent sperm binding to the egg, thus blocking fertilization. It acts through preventing cholesterol efflux, one of the initiation events of capacitation, from the sperm. These findings suggest that SERPINE2 protein may play a role as a sperm decapacitation factor. Plasminogen activators play a crucial role in follicle wall rupture during ovulation; however, their function in oocyte maturation during pre-ovulation remained unclear. Our second study provides the first evidence that PLAU (urokinase plasminogen activator) and its inhibitor SERPINE2 are involved in murine cumulus expansion and oocyte maturation. High SERPINE2 levels bound to the extracellular matrix of cumulus cells could reduce PLAU activity, and ultimately suppressing cumulus expansion and oocyte maturation. PLAU supplementation to culture medium may assist the final maturation of the immature human oocytes collected during assisted reproductive technology procedures, thus providing a potential therapeutic strategy. Based on these results, SERPINE2 protein possibly influencese on sperm to prevent precocious capacitation and the acrosome reaction before sperm reach the oviduct. In addition, we suppose that aberrantly high SERPINE2 protein levels in cumulus cells may be one of the etiologies for patients with defects in cumulus expansion and subsequent oocyte maturation.

碩士
國防醫學院
解剖學研究所
82
Sperm maturation during epididymal transit has been extenively studied in numerous mammalian species.Sperm maturation-related molecules secreted by epididymis are found in rat,hamster, mouse, monkey and human.Our recent studies identify two sperm maturation -related glycoproteins,GP-83 and GP-39 in human epididymis. GP-83 and GP-39 are found in tissue extract, epididymal fluid and sperm surface of corpus and cauda epididymis,not in caput epididymis. In this proposal,GP-83 was purified from human seminal fluid by DEAE-ion exchange,gel filtration chromatography and gel elution. Purified GP-83 was a glycoprotein which exhibited strong affinity to Con-A,PNA and WGA. The pI of GP-83 was 6.57.Purified GP-83 was further separated on SDS-polyacrylamide gel electrophoresis and eluted to immunize male New Zealand rabbit.Titer of the antisera was determined on immunoblot,on which GP-83 was recognized at the dilution of 1 to 10,000.Immunohistochemical localization with antisera revealed the presence of GP-83 in principal cells and sperm of corpus and cauda epididymis.In the principal cells, GP-83 was found in supranuclear region and on cell membrane.In primary culture of principal cells recovered from human corpus epididymis ,GP-83 was found in Golgi apparatus.Purified GP-83 was digested with Lys-C and separated on reverse phase HPLC. Digested polypep- tides were analyzed by amino acid micro- sequencing which revealed SLSQDQEHG and SQNQV?IPSQDQEHG.The partial sequence of GP-83 exhibited homology to semenogelin protein of seminal fluid.

Research Doctorate - Doctor of Philosophy (PhD)
Human infertility is now a major clinical problem affecting approximately one in six couples; with a male factor contributing to nearly 50% of these cases. Clinical analysis has shown that a majority of male infertile patients are still able to produce enough spermatozoa to achieve fertilization. However, for reasons that remain poorly defined, the functionality of these cells has become compromised. Improved understanding of how sperm acquire functional maturity would not only beneficial in terms of uncovering the causative basis of male gamete dysfunction, but also for the provision of urgently needed biomarkers of sperm quality to reliably predict the outcome of assisted reproductive technology treatments. In this context, it is generally accepted that spermatozoa released from the testes require additional phases of post-testicular development that occur during their transit through the epididymis and female reproductive tract before acquiring functional competence. Both biophysical and biochemical changes occur along this journey, eventually culminating in the ability of sperm to undergo an acrosome reaction and recognize the ocyte. Notably, due to spermatozoa being both transcriptionally and translationally silent, the acquisition of functional maturity is reliant on communication between the spermatozoa and the extrinsic factors that they encounter within the male and female reproductive tracts. Our recent work has shown that the dynamin family of enzymes may regulate several key steps in these communication pathways. The dynamin family comprises a group of large GTPases responsible for the regulation of membrane trafficking events such as endocytosis, exocytosis and intracellular trafficking. Such diverse functionality relies on the ability of dynamin to polymerize into a helix structure around the template lipid membrane, whereupon GTP hydrolysis drives the lengthwise constriction of the helix structure and leads to scission of the connection between the two membrane templates. Dynamin has three canonical isoforms, namely dynamin 1, dynamin 2 and dynamin 3. Although sharing over 80% sequence homology, recent studies have shown that each isoform may play distinct roles in regulating membrane trafficking events, depending on their localization and ability to interact with other protein targets. This is especially the case in male reproduction with our previously published studies having shown that dynamin 1 and 2 putatively regulate acrosomal exocytosis whilst dynamin 2 plays an essential role in regulating spermatogenesis in the mouse model. Herein, we have provided further evidence that dynamin is involved in sperm maturation through the regulation of epididymal epithelium secretion. Our detailed characterization of the three canonical dynamin isoforms have revealed that each are highly expressed during the early development phases of epididymal differentiation. Interestingly however, the widespread localization of these isoforms in the juvenile epididymis is replaced by segment and cell specific patterns coinciding with the arrival of testicular sperm into the tract. Notably, the expression of dynamin 2 in the Golgi apparatus of caput epithelial cells ideally positions the enzyme to regulate the classical merocrine pathway of protein secretion. This hypothesis was tested through the use of an in vitro caput epithelial cell line; i.e. mECap18 cells. Accordingly, pharmacological inhibition of dynamin selectively inhibited the secretion of a subset of proteins, such as CCT3 and CCT8, from the mECap18 cells. Having demonstrated that dynamin influences the secretion of epididymal proteins, we elected to explore if members of this family also participate in downstream communication between epididymal soma and sperm via the control of extracellular vesicle uptake. For this purpose, we elected to focus on epididymosomes, small membrane encapsulated vesicles that have been implicated in establishing the sperm proteomic and epigenetic landscape. Through the establishment of an in vitro co-culture model, we have documented the kinetics of epididymosome-mediated transfer of proteins to spermatozoa and identified the post-acrosomal sheath as the domain responsible for initial epididymosome – sperm adhesion. Such adhesion appears to be followed by the uptake of epididymosome cargo into the cell, a process that is reliant on both dynamin 1 and lipid rafts. In continuing our investigation of dynamin, we also elected to study the role of this family of mechanoenzymes in regulating the acrosome reaction in human spermatozoa. Based on previous data generated in a mouse model, we hypothesized that dynamin 1 and 2 play a conserved role in facilitating acrosomal exocytosis in human spermatozoa, and that this activity is linked to the phosphorylation status of the dynamin proteins. Consistent with this hypothesis, dynamin 1 and 2 were localized to the acrosomal domain of human spermatozoa and their pharmacological inhibition significantly compromised the ability of human spermatozoa to complete an acrosome reaction. This activity appears to be tied to the phosphorylation of dynamin, with our data identifying CDK1 as an important targeting kinase for dynamin 2. Accordingly, we recorded a significant loss of dynamin 2 expression in the acrosomal domain of poor quality human spermatozoa; a loss that was accompanied by a significant reduction in the ability of these cells to complete an acrosome reaction. Collectively these data support a conserved role for dynamin in regulating the acrosome reaction in both mouse and human spermatozoa. In summary, the data summarized in this thesis implicates dynamin as a key regulatory enzyme in both epididymal sperm maturation and downstream acrosomal exocytosis. In contrast to the overlapping role of the dynamin family members in somatic cells, our findings raise the prospect that different dynamin members fulfill distinct functions in the male reproductive system. Such distinctions raise the intriguing possibility of being able to target specific dynamin members for the purpose of male fertility regulation. Moreover, the functional conservation we observed between human and mouse models supports the utility of conditional knockout mouse models as an important tool with which to further dissect the role of dynamin in human male (in)fertility.

"April 2004."
Thesis (Ph.D.)--Chinese University of Hong Kong, 2004.
Includes bibliographical references (p. 158-181).
Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web.
Mode of access: World Wide Web.
Abstracts in English and Chinese.

碩士
國立中山大學
海洋資源研究所
86
The studies forcus on spermiogenesis and capacitation of sperm of tiger prawn, ~u2;Penaeus~u1; ~u2;monodon~u1;. After spermatogenesis of the sperm has finished in the testis, spermiogenesis are in progess in proximal vas deferens. The major change in the sperm during spermiogenesis is the extension of the spike. Sperm progessively extend its single spike from the front of the round main body. The growth of the spike finish when spermatids move into medial vas deferens ascending limb. There is no difference in spike length between the sperm in medial vas deferens limb and spermatophore. We studies sperm in male vas deferens (VD), spermatophore (SPH) and female seminal receptacle (SR) of the tiger prawn for understanding the capacitation of the sperm in this shrimp specues. During mating, spermatophores of male tiger prawn are transfered by petasma at the base of first pair of pleopods to female thelycum between fourth and fifth pair of pereipods. The sperm mass mix with the secretion of the thelycum and store in exoskeletal culticle seminal receptacles. Using egg water (EW) to induce sperm acitvation to examine the degree od sperm capacitation in different region of the reproduction tract. The results reveal that tiger prawn do proceed capacitation in the thelycum since the sperm from SR reacted to EW while the sperm from VD and SPH do not reacted to EW. We use spermatophore transplatation to artificially transfer the SPH to female thelycum to examine ~u2;in~u1; ~u2;vivo~u1; capacitation process in the tiger prawn. The results showes that more than 10% sperm are able to exocytosis induced by EW after 6 hr transplantation; more than 40% sperm are capable to be induced exocytosis by EW after 24 hr transplantation. Sperm isolated from medial vas deferens ascending limb and spermatophore were incubated in different pH artificial sea water (ASW), seminal fluid, male hemolymph and female hemolymph to process ~u2;in~u1; ~u2;vitro~u1; capacitation. The percentage of sperm exocytosis induced by EW were much higher in sperm being incubated in higher pH (pH8.0-9.0) than in lower pH (pH5.0-7.0) ASW. Sperm incubated in high pH ASW with the addition of seminal fluid had higher sperm exocytosis rate than incubated in same pH ASW. The results indicated that seminal fluid can promote the efficiency of inducing sperm capacitation in high pH ASW ~u2;in~u1; ~u2;vitro~u1;.-1 -aThe studies of morphology and physiology of sperm maturation in tiger prawn, ~u2;Penaeus~u1; ~u2;monodon~u1

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

Die Transkriptionsfaktoren der p53 Familie besitzen diverse Aufgaben sowohl während der Tumorentstehung als auch während der Entwicklung. In seiner ursprünglichen, evolutionär konservierten Rolle hat p63, ein Mitglied der p53 Familie, die Aufgabe die genetische Stabilität von Keimzellen zu gewährleisten und beeinflusst zudem die Keimbahnentwicklung. Mit Hilfe von „knockout“ (KO) Mausmodellen wurde bereits der Einfluss der 3 Transkriptionsfaktoren p53, p63 und p73 auf die weibliche und männliche Keimbahn untersucht. Während p53KO und p63KO Mäuse fruchtbar sind, führt der gemeinsame Verlust aller p73 Isoformen oder der Verlust der transkriptionell aktiven Isoform TAp73 hingegen zur Infertilität der Tiere. Weibliche TAp73KO Tiere sind deshalb infertil, da es bei ihnen zu einer Inhibition der Ovulation kommt, die Blastozystenentwicklung gestört ist und Spindeldefekte in den Oozyten auftreten. Eine Erklärung für die Infertilität von männlichen TAp73KO Mäusen wurde hingegen noch nicht gegeben. In der vorliegenden wissenschaftlichen Studie beschreiben wir eine bisher unbekannte Funktion des Transkriptionsfaktors TAp73: seinen Einfluss auf die Entwicklung und den Erhalt der adulten männlichen Keimbahn. Untersuchungen an Mausmodellen zeigen, dass „total“ p73KO und Isoform-spezifische TAp73KO Mäuse im Alter von 6 Wochen oder älter einen starken Verlust von sich entwickelnden Keimzellen in den testikulären Tubuli aufweisen. ΔNp73KO Tiere zeigen diesen Phänotyp nicht und ihre Testis Morphologie entspricht der der Wildtyp (WT) Tiere. Der Phänotyp der p73KO und TAp73KO Testes lässt sich durch eine starke Reduktion in der Zahl der späten Stadien der Spermatozyten und Spermatiden charakterisieren, wohingegen die Zahl der basalen Spermatogonien und pachytänen Spermatozyten des Keimepithels nicht betroffen ist (reguläre Zellzahl und Proliferation). Die Depletion von p73 oder TAp73 führt zu einer Anhäufung von apoptotischen und unreifen Keimzellen im Lumen des Epididymis, was auf eine atypische, verfrühte Ablösung der Keimzellen aus dem Keimepithel schließen lässt. Diese Beobachtung wird dadurch bestätigt, dass die Sertoli-Zellen, welche die Keimzellen stützen und ernähren, im Elektronenmikroskop verkürzte cytoplasmatische Arme sowie eine verstärkte Vakuolisierung aufweisen. Außerdem zeigt das Keimepithel große strukturelle Veränderungen, die dadurch gekennzeichnet sind, dass die Keimzellen nicht mehr dicht zu einem Epithel gepackt sind und die Sertoli-Keimzell-Verbindung gestört ist. Der Verlust der Epithelstruktur begleitet von der Reduktion der Keimzellen scheint das Ergebnis eines Defekts der Blut-Testis-Schranke (BTS) zu sein. Dies wird durch einen in vivo BTS Permeabilitätstest und die gestörte Morphologie der Sertoli-Sertoli „tight junctions“ deutlich. Der funktionelle Verlust der BTS führt zur Zerstörung der epithelialen Polarität und der Umgebung der sich entwickelnden Keimzellen. Somit wird auch die basal-luminal gerichtete Migration der Keimzellen entlang der Keimzelltaschen der Sertoli-Zellen verhindert. Der molekulare Hintergrund für das Ungleichgewicht in der Reorganisation der Zell-Zell Verbindungen wurde mit Hilfe der quantitativen Expressionsanalyse des Gesamtgenoms untersucht. Dabei wurde Testis Gewebe von TAp73KO mit WT Mäusen verglichen, um TAp73-Zielgene zu finden. Die Depletion von TAp73 führt zur Induktion von Genen, die an der Adhäsion und Zellmigration beteiligt sind; Integrine und Proteaseinhibitoren wie Timp1 und die Serpine zeigen eine Hochregulation in der Expression. Es ist bekannt, dass diese Proteine bei der Reorganisation von Zell-Zell Verbindungen im Testis involviert sind. Die Studie zeigt, dass TAp73 vorwiegend in den Keimzellen exprimiert wird und dass diese parakrin auf die Sertoli-Zellen zu wirken scheinen, da die Überexpression von TAp73 Zielgenen in isolierten Sertoli-Zellen in der Langzeitkultur zurückgeht. Zusammenfassend konnte zum ersten Mal gezeigt werden, dass die Funktion von TAp73 für die adulte Spermatogenese unerlässlich ist. TAp73 reguliert im Testis ein Transkriptionsprogramm von Genen, die an Adhäsion und Migration beteiligt sind, sichert den Zusammenhalt des Keimepithels und verhindert den frühzeitigen Verlust von Keimzellen. Somit wird die ungestörte Keimzellentwicklung ermöglicht. Umgekehrt führt die Depletion von TAp73 zu starken Defekten in Zell-Zell Verbindungen von sich entwickelnden Keimzellen im Keimepithel, was die Infertilität von p73KO und TAp73KO Mäusen erklärt.