Des facteurs masculins sont identifiés dans près de la moitié des cas d’infertilité. À ce jour, les tests évaluant la fertilité masculine demeurent peu prédictifs de la survenue d’une grossesse. Dans le but de pallier cette lacune, nous avons mis au point deux nouveaux tests mesurant l’intégrité de l’ADN et le temps de survie des spermatozoïdes. Nous avons effectué une étude prospective portant sur 42 couples infertiles suivis en fécondation in vitro (FIV). Le spermogramme a été effectué selon les critères de l’Organisation Mondiale de la Santé (OMS) et le temps de survie des spermatozoïdes exposés à un détergent cationique a été mesuré en observant la mobilité sous microscope. L’intégrité de l’ADN des spermatozoïdes a été vérifiée par la nouvelle méthode de marquage radioenzymatique et par analyse de la structure de la chromatine (SCSA). Tous les tests ont été réalisés sur la partie des échantillons de sperme non utilisée par la clinique de fertilité. Le projet a été approuvé par le comité d’éthique du Centre Hospitalier Universitaire de Montréal (CHUM) et les patients ont préalablement signé un formulaire de consentement éclairé. L’analyse des paramètres du spermogramme et de l’intégrité de l’ADN n’a montré aucune différence statistiquement significative entre les données chez les couples avec ou sans grossesse. Cependant, le taux de grossesse biochimique était statistiquement plus élevé chez les couples dont le temps de survie des spermatozoïdes était long (>250 s) comparativement à ceux dont ce temps était court (≤250 s): 66% vs 27% respectivement (p<0,05). Les taux de grossesse clinique et d’implantation étaient aussi plus élevés, mais les différences n’atteignaient pas le seuil de signification statistique. Nos résultats confirment que le spermogramme et la mesure de la fragmentation de l’ADN des spermatozoïdes ne sont pas de bons facteurs prédictifs des résultats de la FIV. Par contre, le test de survie des spermatozoïdes serait un meilleur indicateur de la possibilité d’une grossesse en FIV. L’amélioration de sa spécificité et un plus grand nombre de sujets sont nécessaires avant de proposer son application en clinique de fertilité. / Male factors are known to be involved in almost half of the couples consulting for infertility. To date, the tests for evaluating male fertility are poor predictors of pregnancy. We developed two new tests to evaluate sperm function: a sperm survival test and a new method to measure sperm DNA integrity. This prospective study was conducted on 42 infertile couples undergoing in vitro fertilization (IVF). Assessment of sperm parameters was done according to the World Health Organization (WHO) criteria, and sperm survival upon exposure to a cationic detergent was measured by observing motility under the microscope. Sperm DNA integrity was verified by our new radioenzymatic method as well as by the sperm chromatin structure analysis (SCSA) method. All testing was performed on a remainder aliquot of the semen samples. The study was approved by the ethics committee of the Centre Hospitalier Universitaire de Montréal (CHUM), and informed consent was obtained before inclusion. Neither conventional semen analysis, nor sperm DNA fragmentation showed statistically significant difference between conception and non-conception cycles. However, the biochemical pregnancy rate was statistically higher in couples where the sperm survival time was long (>250 s) compared to short (≤250 s): 66% vs. 27% respectively, (p < 0.05). The clinical pregnancy rate and implantation rate were also higher but the differences did not reach statistical significance. Our study confirms that conventional semen analysis and the assay for sperm DNA integrity are not reliable indicators of IVF outcome. In contrast, our new sperm survival test seems to be a better predictor of the pregnancy rate after IVF. Improvement of its specificity and a larger cohort of patients are necessary before proposing its regular application in IVF clinics.

Ιατρικές έρευνες έχουν δείξει ότι η ανδρική υπογονιμότητα σχετίζεται άμεσα με την ύπαρξη κατατμημένου DNA στον πυρήνα των σπερματοζωαρίων. Οι διαταραχές στις τιμές της συγκέντρωσης σπερματοζωαρίων, της κινητικότητάς τους, του όγκου της εκσπερμάτισης και στη μορφολογία τους που παρατηρούνται σε ένα σπερμοδιάγραμμα έχουν σα βαθύτερο αίτιο την ύπαρξη κατατμημένου DNA. Το εργαστήριο πειραματικής εμβρυολογίας και ιστολογίας της Ιατρικής Αθηνών χρησιμοποιεί τη μέθοδο TUNEL (deoxynucleotidyl transferase-mediated dUTP nick end labeling) για να σηματοδοτήσει τα άκρα κάθε τμήματος του DNA με χρώμα διαφορετικό από αυτό που χρησιμοποιεί για το υπόλοιπο τμήμα του DNA. Αποτέλεσμα της επεξεργασίας που υφίστανται τα σπερματοζωάρια σε μια αντικειμενοφόρο πλάκα είναι ένα σύνολο από μπλε φθορίζοντα σπερματοζωάρια με πιθανό κόκκινο στο πυρήνα τους, στην περίπτωση που υπάρχει κατατμημένο DNA. Όσο μεγαλύτερος είναι ο βαθμός κατάτμησης, τόσο περισσότερο είναι το κόκκινο και τόσο περισσότερο παθολογικό το σπερματοζωάριο και άρα λιγότερο ικανό να γονιμοποιήσει. Τη διαδικασία της TUNEL ακολουθεί η φωτογράφηση της αντικειμενοφόρου πλάκας με κάμερα υψηλής ανάλυσης και μεγάλης ευαισθησίας, ειδική για εφαρμογές φθορισμού. Στη συνέχεια, οι εικόνες επεξεργάζονται με ειδικό λογισμικό, όπως έχει προταθεί στο «Automatic Analysis of TUNEL assay Microscope Images» από τους Kontaxakis et al. στο 2007 IEEE International Symposium on Signal Processing and Information Technology. Το αποτέλεσμα της επεξεργασίας των εικόνων είναι η ταξινόμηση των αντικειμένων που απεικονίζονται σε ομάδες από α) σπερματοζωάρια μονήρη β) επικαλυπτόμενα και γ) «σκουπίδια» όπως λευκοκύτταρα ή θραύσματα σπερματοζωαρίων. Στη συνέχεια για κάθε μονήρες σπερματοζωάριο γίνεται ο υπολογισμός των κόκκινων και μπλε pixels. Κατ’ αυτό τον τρόπο έχουμε ποσοτικοποιημένη την έκταση του κερματισμού κάθε σπερματοζωαρίου. Στόχος της διπλωματικής εργασίας είναι αρχικά η μελέτη και στη συνέχεια η σχεδίαση και υλοποίηση ενός συστήματος, το οποίο λαμβάνοντας υπόψη τα δεδομένα από την επεξεργασία εικόνας καθώς και δεδομένα που είναι γνωστά από το σπερμοδιάγραμμα, όπως η κινητικότητα και η συγκέντρωση των σπερματοζωαριών, χρησιμοποιώντας τεχνικές της υπολογιστικής νοημοσύνης θα εκπαιδεύεται και θα ταξινομεί αυτόματα ασθενείς ανάλογα με το συνολικό βαθμό κερματισμού του DNA τους. Τέλος, θα υπολογίζει και ένα κατώφλι ή μία περιοχή τιμών άνω της οποίας ένας ασθενής θα χαρακτηρίζεται ως στείρος. Απώτερος στόχος είναι να γίνει όλη η παραπάνω διαδικασία ένας έλεγχος ρουτίνας για τα εργαστήρια που ασχολούνται με την ανδρική υπογονιμότητα και την τεχνητή γονιμοποίηση, προφυλάσσοντας ζευγάρια από άσκοπες και επιβλαβείς για την υγεία της γυναίκας προσπάθειες τεχνητής γονιμοποίησης. / Studies have proven that male infertility is directly connected with the existence of fragmented DNA in sperm nucleus Structural disorders and functional abnormalities are often present in spermatozoa from infertile men, as they are the impact of DNA fragmentation. The histology and embryology laboratory in Medical School in Athens uses the TUNEL assay to mark the edges of DNA helix with color different from the rest of the helix. The result of this procedure is that the human spermatozoa are blue and in the interior of every cell, an area proportional to the degree of the cell DNA fragmentation has been stained in reddish color. The more reddish the area is, the more fragmented the DNA is and the more infertile the patient is. The TUNEL assay is followed by image collection using a camera of high sensitivity appropriate for fluorescence applications. Afterwards, the obtained images are processed as described in “Automatic Analysis of TUNEL assay Microscope Images” at IEEE International Symposium on Signal Processing and Information Technology in 2007. The results of the processing above is image segmentation, shapes classification in 3 groups, solitary spermatozoa, overlapped spermatozoa and debris and at last the area measurement of red pixel for each solitary spermatozoon. This way, we have in numbers how much fragmented the DNA is. This master thesis aims at the study and the design of a system, that taking into consideration the data from the image analysis accompanied by the data from the basic sperm analysis, like sperm concentration and motility, and using computational intelligence techniques, it will be trained and will automatically classify the patients according their DNA fragmentation degree. In the end, it will estimate a threshold or an area of values above which a patient will be considered as infertile. Our ultimate goal is the above procedure to be a routine for the labs that are dealing with male infertility and artificial insemination, so that couples are protected against pointless and prejudicial artificial insemination attempts.

Magister Scientiae (Medical Bioscience) - MSc(MBS) / Introduction: Eurycoma longifolia (Tongkat Ali; TA) is a Malaysian shrub used to treat various illnesses including male infertility. Considering that TA is also used to improve male fertility and no report regarding its safety has been published, this study investigated the effects of a patented, aqueous TA extract on various sperm and testicular functions. Materials and Methods: This study encompasses two parts (part 1: on spermatozoa; part 2: on TM3-Leydig and TM4-Sertoli cells). Part 1: Semen samples of 27 patients and 13 fertile donors were divided into two groups, washed and swim-up prepared spermatozoa, and incubated with different concentrations of TA (1, 10, 20, 100, 2000 μg/ml) for 1 hour at 37°C. A sample without addition of TA served as control. After incubation with TA, the following parameters were evaluated: viability (Eosin-Nigrosin test), total and progressive motility (CASA), acrosome reaction (triple stain technique), sperm production of reactive oxygen species (ROS; dihydroethidium test; DHE), sperm DNA fragmentation (TUNEL assay) and mitochondrial membrane potential (Δψm) (Depsipher kit). Part 2: TM3-Leydig and TM4-Sertoli cells incubated with different concentrations of TA (0.4, 0.8, 1.6, 3.125, 6.25, 12.5, 25, 50 μg/ml) and control (without extract) for 48 and 96 hours. After incubation with TA, the following parameters were evaluated: viability (XTT), cell proliferation (protein assay), testosterone (testosterone ELISA test) and pyruvate (pyruvate assay). Results Part 1: For washed spermatozoa, significant dose-dependent trends were found for viability, total motility, acrosome reaction and sperm ROS production. However, these trends were only significant if the highest concentrations were included in the calculation. In the swim-up spermatozoa, ROS production of spermatozoa showed a biphasic relationship with its lowest percentage at 10 μg/ml, yet, no significance could be observed (P=0.9505). No influence of TA could be observed for sperm DNA fragmentation nor Δψm. Part 2: The viability rates and protein production of TM3-Leydig and TM4-Sertoli cells at 48-hour exposure to TA showed increases whereas at 96-hour incubation periods viability and protein production declined especially as from concentration 25 μg/ml TA. Similar results could be seen for TM4-Sertoli cells pyruvate production. The testosterone production at 48-hour exposure marginally increased (P=0.0580) at the highest (50 μg/ml) concentration of TA. However, at 96-hour exposure to TA the testosterone production significantly (P=0.0065) increased. It is also apparent that after 96 hours the concentration of testosterone has increased [12 x 10-4 ng/ml] when compared to 48-hour exposure [6 x 10-7ng/ml] of Tongkat Ali. Conclusion: Part 1: Results indicate that the Tongkat Ali extract has no deleterious effects on sperm functions at therapeutically used concentrations (<2.5 μg/ml). Part 2: The cytotoxic effect of TA are only presented at higher concentration from 25 μg/ml. TM3-Leydig cells appears to be more resilient than TM4-Sertoli cells in viability and protein production yet at prolonged periods of exposure it is detrimental. Testosterone production only increases after 96 hours exposure to TA.

In mammals, freshly ejaculated spermatozoa do not possess the ability to fertilize a mature oocyte. They acquire fertilization competence upon residing for a period of time in the female reproductive tract. The physiological changes that bring about these time-dependent changes in motility pattern and acquisition of fertilizing ability of spermatozoa are collectively referred to as capacitation, culminating in sperm hyperactivation. Capacitation-associated increase in sperm protein tyrosine phosphorylation (PYP), exhibited by mammalian sperm, is one of the major downstream events, regulating hyperactivated motility. However, it is still unclear which are the tyrosine kinases and phosphatases involved in modulating the capacitation-associated increase in global PYP. In order to determine this, our laboratory earlier showed the role of PYP in hamster sperm capacitation using a specific EGFR protein tyrosine kinase (PTK) inhibitor, tyrphostin A47 (TP-47). Interestingly, inhibition of capacitation by 0.5 mM TP-47 was associated with induction of a slow circular motility pattern, accompanied by inhibition of PYP of certain proteins (Mr. 45,000-52,000), localized to the principle piece of the sperm flagellum. Two such proteins, hypo-tyrosine phosphorylated, were found to be tektin-2 and ODF-2, using 2D-PAGE followed by MS/MS analysis. Interestingly, a global phosphoproteome analysis of human spermatozoa showed that PYP changes are associated with capacitation and asthenospermic condition in infertile men is attributed to the failure of capacitation-associated increase in PYP. Such individuals exhibited impaired sperm motility. There is a need to understand the exact mechanism of phosphorylation of sperm flagellar proteins, which is necessary to assess sperm’s ability to fertilize the mature oocyte. Therefore, the focus of the present work was to elucidate the role of receptor tyrosine kinases (RTKs) and the non-receptor tyrosine kinases (NRTKs) in mammalian (hamster) sperm capacitation. Recent studies have shown that apart from EGFR other RTKs like IGF1R, FGFR, VEGFR, MuSK, TrkA are expressed in mammalian spermatozoa and actively involved in sperm capacitation. However, there is very little information available in the context of sperm capacitation and associated PYP. Therefore, attempts were made to understand the role of various RTKs (IGF1R, FGFR and VEGFR) in hamster sperm capacitation and associated PYP. Initially, the role of IGF1R tyrosine kinase during sperm capacitation was studied. Immunolocalization of IGF1R in spermatozoa showed a strong signal in the sperm acrosome and the principal piece of the sperm flagellum. Inhibition of IGF1R kinase with an IGF1R-specific inhibitor TP-1-O-Me-AG538 (TP-538) showed inhibitory effect on sperm capacitation and the associated hyperactivation. But, inhibitors of FGFR and VEGFR tyrosine kinases did not show such an effect. Interestingly, inhibition of IGF1R by TP538 was associated with inhibition of PYP of certain proteins (Mr. 45,000-120,000), localized to head, mid piece and principle piece regions of the sperm flagellum. Phosphoproteomic analysis using 2D-PAGE-western blot with anti-phosphotyrosine antibodies identified 17 differentially phosphorylated protein spots. Out of the 17 spots, 12 were identified by MALDI-MS/MS analysis. The proteins identified to be differentially phosphorylated, upon inhibition of IGF1R, were PDHE1, ODF-2, Tubulin β 2C chain, PDHE2 and ATP synthase β subunit. The RTKs being present in the membrane level may not be directly involved in the phopshorylation of downstream target proteins associated with the mitochondrial membrane, sperm axonemal structures and outer dense fibers. Therefore, the RTKs may interact directly or indirectly with the downstream NRTKs, which may be involved in the phosphorylation of target sperm proteins. Till date, six different families of NRTKs are shown to be expressed in mammalian spermatozoa. The major family of NRTKs involved in sperm function is the Src family of kinases. However, there is very little information available in the context of sperm capacitation and the associated PYP. Therefore, studies were carried out to understand the role of Src family of NRTKs in sperm capacitation and associated PYP. Presence of active Src signaling was observed by the immunolocalization of activated Src (pY416) in the acrosome, mid piece and the principal piece regions of the sperm flagellum. Inhibition of Src family of kinase with a specific Src family kinase inhibitor PP2, showed inhibition of sperm capacitation and the associated hyperactivation. Inhibition of Src family of kinases with PP2 was associated with decrease in PYP of several proteins (Mr. 45,000-120,000), localized mainly to the mid piece region, followed by the principle piece region of the sperm flagellum. Phosphoproteomic analysis using 2D-PAGE-western blot with anti-phosphotyrosine antibodies identified 38 differentially phosphorylated protein spots. Out of the 38 spots, 16 were identified by MALDIMS/MS analysis and these corresponded to seven proteins which included PDHE1, ODF-2, Tubulin β 2C chain, Tektin-2, GAPDS, PDHE2 and ATP synthase β subunit. Additionally, the biochemical and molecular characteristics of the identified proteins were also studied. Bioinformatic analysis predicted the presence of phosphorylation motifs for several kinases and interestingly, all the proteins identified had a Src kinase motif. Comparing the current observations and the previous work in the laboratory, two proteins ODF-2 and Tektin-2 were found to be regulated by EGFR, IGF1R and Src family of kinases. Therefore, characterization of the capacitation-associated tyrosine phoposphorylated proteins ODF-2 and Tektin-2 was performed. By employing PCR and Northern blotting techniques, the presence of the transcripts of both the proteins was shown. Additionally, the ontogeny of expression of ODF2 and Tektin-2 in hamster testis development was studied and the results indicated that the expression of both the proteins started from week 3 onwards till week 8. To confirm the meiotic stage-associated expression of ODF-2 and Tektin-2, germ cells were sorted based on their DNA content. ODF-2 and Tektin-2 transcripts were first expressed in the meiotic germ cells (pachytene spermatocytes) and their expression was upregulated in the post-meiotic germ cells (round spermatids). Sequential extraction of sperm proteins showed that, Tektin-2 was majorly extracted out in the Triton X-100 and DTT fraction, whereas, ODF-2 was maximally extracted in the presence of urea and DTT. In conclusion, these observations indicate that IGF1R and Src family of tyrosine kinases are critical for mammalian sperm capacitation and associated global PYP. Inhibition of sperm capacitation was associated with hypo-tyrosine-phospohorylation of certain proteins associated with mitochondrial membrane, axonemal structures and outer dense fibers of the sperm flagellum. Future work can be directed towards understanding the role of other RTKs and NRTKs involved in sperm capacitation and the molecular characterization of hypophosphorylated proteins critical for sperm function and its fertilization competence.

The following work demonstrates that paternal diet controls medically important metabolic phenotypes in offspring. We observe transmission of dietary information to the zygote via sperm, and this information evades reprogramming that typically occurs after fertilization. Cytosine methylation is implicated as a major contributor to meiotic epigenetic inheritance in several transgenerational phenomena. Our extensive characterization of the sperm methylome reveals that diet does not significantly affect methylation patterns. However, we find that extensive epivariability in the sperm epigenome makes important contributions to offspring variation. Importantly, coordinate cytosine methylation and copy number changes over the ribosomal DNA locus contributes to variation in offspring metabolism. Thus, rDNA variability acts independently of postadolescent paternal diet to influence offspring metabolism. Therefore, at least two mechanisms exist for epigenetically controlling offspring metabolism: stochastic epivariation and diet acting by an unknown mechanism to further modulate metabolism. This work argues that an offspring's phenotype can no longer be viewed solely as the result of genetic interactions with the developmental environment - the additional influences of paternal environment and inherited epigenetic variability must also be considered. These findings reveal novel contributions to metabolism that could revolutionize how we think about the risk factors for human health and disease.

Effective inhibition of sperm motility using a spermicide can be a promising approach in developing non-invasive male contraceptive agents. Copper is known to have contraceptive properties and has been used clinically for decades as intrauterine contraceptive devices (IUDs) for contraception in females. Beyond that, the spermicidal use of copper is not explored much further, even though its use can also subdue the harmful effects caused by the hormonal female contraceptive agents on the environment. Herein, the size, concentration, and timedependent in vitro inhibition of bovine spermatozoa by copper microparticles are studied. The effectivity in inhibiting sperm motility is correlated with the amount of Cu²⁺ ions released by the particles during incubation. The copper particles cause direct suppression of sperm motility and viability upon incubation and thereby show potential as sperm-inhibiting, hormone-free candidate for male contraception. In addition, biocompatibility tests using a cervical cell line help optimizing the size and concentration of the copper particles for the best spermicidal action while avoiding toxicity to the surrounding tissue.

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

Cilia and flagella are hair-like appendages of eukaryotic cells. They are actively bending structures that exhibit regular beat patterns and thereby play an important role in many different circumstances where motion on a cellular level is required. Most dramatic is the effect of nodal cilia whose vortical motion leads to a fluid flow that is directly responsible for establishing the left-right axis during embryological development in many vertebrate species, but examples range from the propulsion of single cells, such as the swimming of sperm, to the transport of mucus along epithelial cells, e.g. in the ciliated trachea. Cilia and flagella contain an evolutionary highly conserved structure called the axoneme, whose characteristic architecture is based on a cylindrical arrangement of elastic filaments (microtubules). In the presence of a chemical fuel (ATP), molecular motors (dynein) exert shear forces between neighbouring microtubules, leading to a bending of the axoneme through structural constraints. We address the following two questions: How can these organelles generate regular oscillatory beat patterns in the absence of a biochemical signal regulating the activity of the force generating elements? And how can the beat patterns be so different for apparently very similar structures? We present a theoretical description of the axonemal structure as an actively bending elastic cylinder, and show that in such a system bending waves emerge from a non-oscillatory state via a dynamic instability. The corresponding beat patterns are solutions to a set of coupled partial differential equations presented herein.

Cilia and flagella are hair-like appendages of eukaryotic cells. They are actively bending structures that exhibit regular beat patterns and thereby play an important role in many different circumstances where motion on a cellular level is required. Most dramatic is the effect of nodal cilia whose vortical motion leads to a fluid flow that is directly responsible for establishing the left-right axis during embryological development in many vertebrate species, but examples range from the propulsion of single cells, such as the swimming of sperm, to the transport of mucus along epithelial cells, e.g. in the ciliated trachea. Cilia and flagella contain an evolutionary highly conserved structure called the axoneme, whose characteristic architecture is based on a cylindrical arrangement of elastic filaments (microtubules). In the presence of a chemical fuel (ATP), molecular motors (dynein) exert shear forces between neighbouring microtubules, leading to a bending of the axoneme through structural constraints. We address the following two questions: How can these organelles generate regular oscillatory beat patterns in the absence of a biochemical signal regulating the activity of the force generating elements? And how can the beat patterns be so different for apparently very similar structures? We present a theoretical description of the axonemal structure as an actively bending elastic cylinder, and show that in such a system bending waves emerge from a non-oscillatory state via a dynamic instability. The corresponding beat patterns are solutions to a set of coupled partial differential equations presented herein.

[ES] La familia Tephritidae constituye un grupo de dípteros de gran interés económico debido a que incluye especies consideradas plagas en casi todas las áreas frutícolas del mundo. Entre las especies de tefrítidos citadas en España, se encuentra Ceratitis capitata, la mosca de la fruta del Mediterráneo, plaga de gran relevancia económica en el país. La gestión de esta plaga requiera de actuaciones en grandes áreas frutícolas y con integración de diversas tecnologías y herramientas de control, siendo las actuaciones principales los tratamientos químicos, el trampeo masivo y la aplicación de la técnica del insecto estéril (TIE). Conocer la biología reproductiva de C. capitata es de gran importancia para desplegar nuevos métodos de control y mejorar los ya existentes, especialmente la TIE. En este sentido, y haciendo uso de los avances biotecnológicos, se desarrolló un método molecular de cuantificación, basado en la aplicación de una PCR en tiempo real mediante el uso de marcadores del cromosoma Y, para estimar la cantidad de espermatozoides contenidos en las espermatecas (capítulo 2). Este método fue validado satisfactoriamente como medio de cuantificación de la cantidad de esperma almacenado en las espermatecas después del apareamiento para dos cepas de C. capitata, la cepa silvestre y la cepa Vienna-8, empleada internacionalmente en la producción masiva de machos estériles para el programa TIE. Además, los resultados obtenidos mostraron detecciones positivas de esperma en apareamientos de corta duración (10 minutos) y se confirmó la transferencia efectiva de esperma en el 78 % de los casos evaluados. Una vez obtenido un método más eficaz para cuantificar el esperma almacenado en las espermatecas, éste se empleó para estudiar (Capítulo 3) la influencia de la edad del macho y la duración de la cópula en la cantidad de esperma trasferido durante la misma, en ambas cepas. Estos aspectos que se han relacionado con el rendimiento de los machos estériles empleados en los programas TIE. Además de observarse un efecto de la cepa, se determinó una influencia positiva de la duración de la cópula en la cantidad de esperma recibido por las hembras. Los resultados mostraron un patrón gradual de transferencia no lineal que aumenta con la duración del apareamiento en ambas cepas. En cuanto a la edad de los machos no se observó una influencia en la cantidad de esperma transferido dentro del rango de edad evaluado. Por último, se estudió la capacidad de los machos Vienna-8 estériles para volver a aparearse e inseminar a las hembras (Capítulo 4). El fenómeno del apareamiento múltiple, relacionado con la eficacia reproductiva de C. capitata, ha sido observado en las poblaciones naturales, tanto en hembras (poliandria) como en machos (poliginia), pero ha sido poco estudiada en machos irradiados. Los resultados mostraron que el 57 % de los machos estériles recopularon hasta en tres ocasiones y el 73 % al menos en una ocasión y que en estos reapareamientos hubo transferencia efectiva de esperma en el 99 % de los casos. En cuanto a la cantidad de espermatozoides transferidos en las recópulas producidas, se observó una tendencia a disminuir respecto a la primera cópula, pero no se detectó una situación de aspermia. En conclusión, esta tesis desarrolla un nuevo método molecular basado en la detección del cromosoma Y para evaluar el esperma almacenado en las espermatecas de hembras en C. capitata. Este método ha sido validado para estimar los espermatozoides de las cepas silvestre y Vienna-8 de esta especie, y podría servir como modelo base para la cuantificación de gametos en otras especies de tefrítidos. Se han mejorado los patrones de transferencia del esperma con relación al tiempo de apareamiento para ambas cepas, y se ha determinado, por primera vez, el potencial de recópula de los machos estériles Vienna-8 y su capacidad para transferir esperma no viable a lo largo de múltiples apareamientos. / [CA] La família Tephritidae constituïx un grup de dípters de gran interés econòmic pel fet que inclou espècies considerades plagues en quasi totes les explotacions frutícoles del món, especialment les conegudes com a mosques de la fruita. Entre les espècies de tefrítids citades a Espanya, es troba Ceratitis capitata, la mosca de la fruita del Mediterrani, plaga de gran rellevància econòmica al país. La gestió d'aquesta plaga requerisca d'actuacions en grans àrees frutícoles i amb integració de diverses tecnologies i ferramentes de control, sent les actuacions principals els tractaments químics, el trampeig massiu i l'aplicació de la tècnica de l'insecte estèril (TIE). Conéixer la biologia reproductiva de C. capitata és de gran importància per a desplegar nous mètodes de control i millorar els ja existents, especialment la TIE. En este sentit, i fent ús dels avanços biotecnològics, en primer lloc (capítol 2) es va desenvolupar un mètode molecular de quantificació, basat en l'aplicació d'una PCR en temps real mitjançant l'ús de marcadors del cromosoma Y, per a estimar la quantitat d'espermatozoides continguts en les espermateques. Este mètode va ser validat satisfactòriament com a mitjà de quantificació dels espermatozoides emmagatzemats en les espermateques després de l'aparellament per a dos soques de C. capitata, la soca silvestre i la soca Vienna-8, empleada internacionalment en la producció massiva de mascles estèrils per al programa TIE. A més, els resultats obtinguts van mostrar deteccions positives d'esperma en aparellaments de curta duració (10 minuts) i es va confirmar la transferència efectiva d'esperma en el 78% dels casos avaluats. Una vegada obtingut un mètode més eficaç per a quantificar l'esperma emmagatzemat en les espermateques, aquest es va emprar per a estudiar (capítol 3) la influència de l'edat del mascle i la duració de la còpula en la quantitat d'esperma transferit durant esta, en ambdues soques. Estos aspectes s'han relacionat amb el rendiment dels mascles estèrils empleats en els programes TIE. A més d'observar-se un efecte de la soca, es va determinar una influència positiva de la durada de l'acoblament en la quantitat d'esperma rebut per les femelles. Els resultats van mostrar un patró gradual de transferència no lineal que augmenta amb la duració de l'aparellament en ambdues soques. Quant a l'edat dels mascles, no es va observar una influència en la quantitat d'esperma transferit dins del rang d'edat avaluat. Finalment, es va estudiar la capacitat dels mascles Vienna-8 estèrils per a tornar a emparellar-se i inseminar a les femelles (capítol 4). El fenomen de l'aparellament múltiple, relacionat amb l'eficàcia reproductiva de C. capitata, ha sigut observat en les poblacions naturals, tant en femelles (poliàndria) com en mascles (poligínia), però ha sigut poc estudiada en mascles irradiats. Els resultats van mostrar que el 57% dels mascles estèrils van recopular fins a tres ocasions i el 73% almenys en una ocasió i que en estos reaparellaments va haver-hi transferència efectiva d'esperma en el 99% dels casos. Quant a la quantitat d'espermatozoides transferits en les récopulas produïdes, es va observar una tendència a disminuir respecte a la primera còpula, però no es va detectar una situació d'aspèrmia. En conclusió, esta tesi desenvolupa un nou mètode molecular basat en la detecció del cromosoma Y per a avaluar l'esperma emmagatzemat en les espermateques de femelles en C. capitata. Este mètode ha sigut validat per a estimar els espermatozoides de les soques silvestre i Vienna-8 d'esta espècie, i podria servir com a model base per a la quantificació de gàmetes en altres espècies de tefrítids. S'han millorat els patrons de transferència de l'esperma en relació amb el temps d'aparellament per ambdues coques, i s'ha determinat, per primera vegada, el potencial de recòpula dels mascles estèrils Vienna-8 i la seua capacitat per a transferir esperma no viable al llarg de múltiples aparellaments. / [EN] Tephritidae family constitutes a great interest group of dipterans as it includes species considered pests in almost all fruit-growing areas around the world, especially those known as fruit flies. The Mediterranean fruit fly, Ceratitis capitata, is among the recorded tephritids species in Spain and it is recognized as a pest of great economic importance by Spanish government. The ability to adapt to different environmental conditions of each of its development stages and its polyphagy are two of the key aspects to become such a harmful pest. C. capitata has the largest host range known among tephritids. These biological and adaptative traits make management of this pest require actions in large fruit-growing areas and the integration of various technologies and control tools. The integrated pest management programme includes chemical treatments, mass trapping and the application of the sterile insect technique (SIT). The study of the reproductive biology of C. capitata is relevant for deploying new control methods and improving existing ones, especially SIT. Taking it into consideration, and making use of biotechnological advances, in chapter 2, we develop a molecular quantification method, based on the application of real-time PCR using Y-chromosome markers, to estimate the amount of sperm contained in spermathecae. This method was successfully validated to quantify the amount of spermatozoa stored in the spermathecae after mating for two strains of C. capitata, wild-type strain and Vienna-8 strain, used internationally in the mass production of sterile males in TIE programmes. Positive sperm detections were obtained in short-term matings (10 minutes) and effective sperm transfer was confirmed in 78% of the cases evaluated. Once obtained this effective method to quantify the sperm stored in the spermathecae, it was used to study in chapter 3 the influence of male age and copula duration on the amount of sperm transferred during mating in both strains. These aspects have been related to the performance of sterile males used in TIE programmes. In addition to observing a strain effect, it was determined a positive influence of copula duration on the amount of sperm received by females. The results showed a gradual pattern of non-linear transfer that increases with mating duration in both strains. Regarding the age of the males, no influence was observed on the amount of sperm transferred within the evaluated age range. Finally, the ability of sterile Vienna-8 males to remate and inseminate females was studied in chapter 4. The phenomenon of multiple mating, related to the reproductive efficiency of C. capitata, has been observed in natural populations, both in females (polyandry) and males (polygyny), but it has been barely studied in irradiated males. The results of this chapter showed that 57% of the sterile males recopulated up to three times and 73% at least once, and there was effective sperm transfer in 99% of these rematings. Regarding the amount of sperm transferred along the remating occasions, a tendency to decrease was observed with respect to the first copulation, but a situation of aspermia was not detected. In conclusion, this thesis develops a new molecular method based on the detection of the Y-chromosome to estimate the sperm stored in the spermathecae of females in C. capitata. This method has been validated to estimate spermatozoa from wild-type and Vienna-8 strains of this tephritid and it could be used as a model for the quantification of gametes in other fruit fly species. Sperm transfer patterns have been improved in relation to mating time for both strains, and the remating potential of sterile Vienna-8 males and their ability to transfer non-viable sperm over multiple matings have been determined for the first time in a period of their lifetime. / Català Oltra, M. (2024). Cuantificación y análisis de la transferencia de espermatozoides en Ceratitis capitata (Diptera: Tephritidae): Aplicaciones en programas de la Técnica del Insecto Estéril [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/213442