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P. 520-528 The performance of cryopreserved semen in ovine artificial insemination still needs improvement. Some antioxidants have been tested, with variable success. We cryopreserved semen from Churra rams using TES-Tris-fructose with 4% glycerol and 10% egg yolk (EY) or 3.5% soybean lecithin (SL), with 1 mM of reduced glutathione (GSH), Trolox, crocin, or cysteamine. Samples were analyzed after thawing and incubation (6 hours, 38 °C) for motility (computer-assisted sperm analysis [CASA]), viability, acrosomal integrity, apoptosis, mitochondrial activity, chromatin status, and lipoperoxidation (malondialdehyde production). Interactions (antioxidant/extender/incubation) were significant for most variables. Extenders yielded similar results, although SL depressed mitochondrial activity and linearity (P < 0.001), it improved motility (P < 0.05), DNA fragmentation (P < 0.05), and acrosomal damage (P < 0.001). The control, GSH, and Trolox showed greater viability with SL (P < 0.01). Cysteamine depressed motility (0 hours: 51.6 ± 2.0% vs. 32.3 ± 4.3%; 6 hours: no motility vs. 32.5 ± 1.9%; P < 0.001), but improved viability when using EY (P = 0.004). Crocin increased acrosomal damage (P = 0.022) but improved linearity-related parameters after thawing (P = 0.014). Trolox considerably reduced malondialdehyde production in both extenders (8.6 ± 0.4 nmol per 108 cells vs. 14.2 ± 0.3 in EY and 20 ± 0.6 in SL; P < 0.001). Interestingly, thiol antioxidants (cysteamine and GSH) increased DNA fragmentation (percentage of DNA fragmentation index), whereas crocin reduced it (P < 0.05). Interactions between extender and antioxidant must be taken into account for improving sperm cryopreservation. Soybean lecithin seems to be a suitable replacement for EY, but its effect on mitochondria must be investigated. Trolox and crocin might be useful for ram semen freezing. SI

P. 541-550 The Cantabrian brown bear survives as a small remnant population in northern Spain and semen cryopreservation for future artificial insemination is one of the measures being implemented for conservation of this species. As part of this program we investigated the value of adding heat shock protein A8 (HSPA8) to media (N-[Tris(hydroxymethyl)methyl]-2-aminoethanesulfonic acid-TRIS-fructose with 20% egg yolk) used for chilling and cryopreserving the spermatozoa. Semen samples from eight brown bears were obtained by electroejaculation during the breeding season. In experiment 1, we tested three concentrations of HSPA8 (0.5, 1, and 5 μg/mL) to determine whether sperm motility (computer assisted sperm analysis system) and sperm survival could be improved during refrigeration (5 °C) up to 48 hours. Results showed that sperm viability (test with propidium iodide) was improved by the addition of 0.5 and 5 μg/mL HSPA8. In experiment 2, HSPA8 was added to the cryopreservation media (6% final glycerol concentration) before the freezing process. Though there were no differences in sperm viability immediately after thawing (analyses to 0 hours), plasma membrane permeability (test with YO-PRO-1) was significantly lower by the presence of HSPA8 (1 μg/mL) and acrosomal damage (test with peanut agglutinin-fluorescein isothiocyanate conjugate) was reduced by higher concentrations of HSPA8 (1 and 5 μg/mL) (analyses after thermal stress test incubating over 2 hours to 37 °C). In experiment 3, results of a simple progression test carried out through artificial mucus (hyaluronic acid 4 mg/mL) showed a significant decrease in the total number of sperm able to swim a distance of 0.5 to 2 cm through a capillary tube for all HSPA8-based extenders. Nevertheless, the distance traveled by the vanguard spermatozoa, which represent a highly motile subpopulation, was restored by the inclusion of 1 and 5 μg/mL HSPA8 in the cryopreservation media. Thus, the HSPA8 addition to extender improves the quality of brown bear (Ursus arctos) sperm during chilling (viability) and after cryopreservation (number of sperm with damaged acrosomes and “apoptotic-like” changes) SI

P. 1119-1128 Centrifugation is a crucial procedure in sperm cryopreservation protocols of brown bear (Ursus arctos), because the semen must be processed to increase sperm concentration and/or clean urine-contaminated samples. The efficacy of three media for centrifugation (Maxifreeze [IMV technologies, L'Aigle, France], Cushion Fluid Equine (Minitübe, Tiefenbach, Germany), and PureSperm [Nidacon, Gothenburg, Sweden]) on the quality of bear spermatozoa was evaluated. In experiment one, two cushioned media used for protecting against mechanical stress during centrifugation were analyzed. In experiment two, a density gradient based on PureSperm was assessed in relation to the maximum retrieval and the quality of fresh spermatozoa, and the freezability of the spermatozoa selected in this density gradient was studied in experiment three. Finally, the selection of frozen-thawed sperm using PureSperm was analyzed in experiment four. Our results indicate that the use of dense isotonic cushion solutions (Maxifreeze, Cushion Fluid Equine) in centrifugation did not improve the quality of recovered spermatozoa compared with standard centrifugation. However, a density gradient prepared with PureSperm improved the quality of spermatozoa in fresh semen and frozen-thawed semen, but the spermatozoa selected from the fresh sample with this density gradient did not show a better resistance to freezing with this density gradient in comparison with the control sample. SI

P. 105-112 In order to establish a semen bank for the endangered Cantabrian brown bear, we tested five glycerol concentrations and three freezing rates for electroejaculated semen. Electroejaculation was performed on nine males. Semen was diluted in TES–Tris–Fructose (20% egg yolk, 2% EDTA, 1% Equex) with 2%, 4%, 6%, 8% or 10% glycerol and frozen at −10, −20 or −40°C/min. Before and after cryopreservation, samples were analysed for motility (CASA), viability and acrosomal status (flow cytometry). Pre‐freezing results showed that glycerol concentration had no significant effect on total motility or progressive motility, but it significantly decreased VCL, ALH, viability and acrosomal status (p

P. 907-914 Thawed ram spermatozoa were incubated at 37°C in the presence of dehydroascorbic acid (DHA), TEMPOL (TPL), N‐acetyl‐cysteine (NAC) and rutin (RUT), at 0.1 and 1 mm, in order to test their effects on sperm physiology. Cryopreserved spermatozoa from four rams were thawed, pooled, washed and incubated in TALP‐Hepes with 1 mm or 0.1 mm of each antioxidant, performing a replicate with induced oxidative stress (Fe2+/ascorbate). Motility (CASA), viability and mitochondrial membrane potential (flow cytometry) were analysed at 2 and 4 h. Lipoperoxidation (MDA production), intracellular reactive oxygen species (ROS) and DNA status (TUNEL) were analysed at 4 h. Antioxidants, except DHA 0.1 mm, decreased motility and kinematic parameters, but had little effect on viability or mitochondrial activity. Except 1 mm DHA, the antioxidants reduced ROS at 4 h. Moreover, NAC 1 mm, rutin and TEMPOL reduced ROS and DNA damage in the presence of oxidative stress. N‐acetyl‐cysteine, rutin 1 mm and TEMPOL reduced lipoperoxidation in the presence of oxidative stress. However, DHA did not affect lipoperoxidation. At 1 mm, DHA increased DNA damage in the absence of oxidative stress. Dehydroascorbic acid effects could arise from spermatozoa having a low capacity for reducing it to ascorbic acid, and it may be tested in the presence of other antioxidants or reducing power. Future research should focus in testing whether the inhibition of motility observed for NAC, rutin and TEMPOL is reversible. These antioxidants might be useful at lower temperatures (refrigerated storage or cryopreservation) when their protective effects could be advantageous. SI

Thawed ram spermatozoa were incubated at 37°C in the presence of dehydroascorbic acid (DHA), TEMPOL (TPL), N-acetyl-cysteine (NAC) and rutin (RUT), at 0.1 and 1mm, in order to test their effects on sperm physiology. Cryopreserved spermatozoa from four rams were thawed, pooled, washed and incubated in TALP-Hepes with 1mm or 0.1mm of each antioxidant, performing a replicate with induced oxidative stress (Fe 2+/ascorbate). Motility (CASA), viability and mitochondrial membrane potential (flow cytometry) were analysed at 2 and 4h. Lipoperoxidation (MDA production), intracellular reactive oxygen species (ROS) and DNA status (TUNEL) were analysed at 4h. Antioxidants, except DHA 0.1mm, decreased motility and kinematic parameters, but had little effect on viability or mitochondrial activity. Except 1mm DHA, the antioxidants reduced ROS at 4h. Moreover, NAC 1mm, rutin and TEMPOL reduced ROS and DNA damage in the presence of oxidative stress. N-acetyl-cysteine, rutin 1mm and TEMPOL reduced lipoperoxidation in the presence of oxidative stress. However, DHA did not affect lipoperoxidation. At 1mm, DHA increased DNA damage in the absence of oxidative stress. Dehydroascorbic acid effects could arise from spermatozoa having a low capacity for reducing it to ascorbic acid, and it may be tested in the presence of other antioxidants or reducing power. Future research should focus in testing whether the inhibition of motility observed for NAC, rutin and TEMPOL is reversible. These antioxidants might be useful at lower temperatures (refrigerated storage or cryopreservation) when their protective effects could be advantageous., This work has been supported by Junta de Castilla y León (LE019A10-2), by the Spanish Ministry of Science and Innovation (AGL2010-15758 ⁄ GAN) and by INIA (RZ2010-00005-00-00). We thank ANCHE (National Association of Churra Breeders) for supporting this study with animals and personnel. M. Mata-Campuzano and Manuel Álvarez-Rodríguez were supported by FPI grants (Spanish Ministryof Science and Innovation). F. Martínez-Pastor was supported by the Juan de la Cierva programme and by the Ramón y Cajal programme (Ministry of Science and Innovation, Spain) was supported by the Juan de la Cierva programme (Ministry of Science and Innovation, Spain).

La vieira común (Pecten jacobaeus), molusco bivalvo pectínido, está vinculada, hasta en su nombre científico, al Camino de Santiago. Llamada también concha del peregrino, constituye uno de los símbolos por excelencia del Camino jacobeo, bien como insignia portada en el equipamento de los peregrinos bien como representación continua en fachadas de edificios emblemáticos situados a lo largo del Camino. La simbología existente alrededor de este bivalvo es muy rica, con sentidos muy diversos, y está asociada a la realización de obras buenas -por su parecido a los dedos de una mano-, al renacimiento personal -en torno al símbolo de Venus-, a la iniciación de un camino -por su similitud con una pata palmeada de oca- o, más claramente, en la ruta jacobea, a la culminación de la peregrinación. Todas estos sentidos confluyen para explicar la consolidación de este símbolo hasta nuestros días, de forma que la concha significa para muchos la espiritualidad-religión presente a lo largo del Camino

P. 1561-1565 We have tested several freezing protocols for brown bear semen, modifying the time when glycerol was added (before and after cooling to 5 °C). No differences were found among protocols, indicating a good tolerance of brown bear semen to glycerol. This finding indicates that freezing protocols for brown bear semen could be modified to fit practical solutions which would facilitate preparation of the seminal samples in the field with the addition of glycerol at ambient temperature. SI

La Hierba de Santiago es una de las plantas silvestres cuyas propiedades curativas han sido aprovechadas durante siglos por peregrinos e instituciones para intentar sanar las posibles dolencias de los caminantes. Se encuentra ampliamente distribuida por toda Europa, creciendo en los márgenes de los caminos, a disposición de quienquiera que pase por su lado. Curiosamente se trata de una planta tóxica, debido a su alta concentración de alcaloides pirrolicidínicos, y en la actualidad está prohibida su comercialización. Tanto su nombre vulgar como científico hacen alusión directa al Camino de Santiago, por lo que forma parte de la simbología imprescindible en la peregrinación hacia el Apóstol.

P. 67-78 Flow cytometry has become an important technique in sperm evaluation and is increasingly used both for routine assessment and for research in veterinary science. We have revised the literature, describing fluorescent probes that have been used for analysing spermatozoa by flow cytometry, regarding: viability, acrosomal status, capacitation, mitochondrial status, apoptotic markers, oxidative stress markers, DNA damage, sperm counting and sperm sizing. Details and problems of some techniques are reviewed, with special attention to the occurrence of non‐sperm particles in the samples (‘debris’). New and promising aspects of flow cytometry, such as sperm sorting using viability markers as selection criteria, are highlighted. The relationship between flow cytometry analyses and fertility and their future improvements are considered. SI

4 Pág., Artificial insemination (AI) for pigs relies on liquid storage of extended semen at 17°C, which preserves sperm quality and ensures its fertilizing capacity. Routine quality controls include the evaluation of sperm motility, viability and capacitation status. The physiological functions of all these features depend on transmembrane aquaporins (AQPs), proteins playing key roles in osmoadaptation. In this study, we made a relative quantification, using RT-qPCR, of the mRNA of several sperm AQPs in AI-liquid semen doses before and after a 48-hr incubation period, aiming to determine possible quantitative compromising expression changes during the process that could serve as a diagnostic tool. Our results showed a decrease in classical sperm motility variables (total and progressive motility and velocity) and sperm viability after 48-hr storage, whereas capacitation status increased overtime. mRNA expression increased in the orthodox AQP4 and AQP6 after 48-hr incubation, relative to control (0 hr) and 24-hr time-points. Moreover, mRNA expression of aquaglyceroporins AQP3, AQP7 and AQP10 was higher after 48-hr incubation, confirmed by AQP7-protein validation using Western blot. Our results indicate that expression levels of AQPs-mRNA can change in ejaculated pig spermatozoa under conditions of ex-vivo incubation that could modify sperm homeostasis, suggesting it could eventually become a relevant molecular biomarker to assess the efficiency of liquid storage of pig semen., This research was funded by the Research Council FORMAS, Stockholm (Projects 2017- 00946 and 2019- 00288), and by the Grant PID2019- 108320RJ- I00 and IJCI- 2015- 24380 funded by MCIN/AEI/10.13039/501100011033 (Spain) and FEDER funds (EU).

7 Pág. Departamento de Reproducción Animal., The expression of aquaglyceroporin 3 (AQP-3) has been demonstrated in the spermatozoa of several mammalian species and its role has been associated with cryotolerance. Post-thaw sperm quality from individual dromedary males with different response to freezing-thawing process was evaluated through sperm head morphometry. In order to understand the cellular mechanisms affected by cryoinjury we have explored the presence and distribution of sperm AQP-3 using western blotting and immunocytochemistry. WB showed different intensity of the specific signal bands at 28 kDa. Immunofluorescence assessments allowed us to identify five different and clear AQP-3 distribution patterns of labelling in the sperm plasma membrane; acrosome, post-acrosome, mid-piece, and principal and final tail. Although expression of AQP-3 varied among male ejaculates, the individual sperm response to freeze-thawing was not associated with AQP-3 expression. Thus, AQP3 expressions do not seem like a reliable predictor of sperm response to freeze-thawing process in this species. This work is the first to describe the morphometric characteristics of the heads of dromedary spermatozoa. No correlation was found between sperm head dimensions and sperm quality variables after freeze-thawing suggesting that dromedary camel sperm head morphometry is also not a reliable predictor of cryosurvival., This work was supported by Zoitechlab S.L. (Arquimea Group) - INIA contract CON18-141, MCINN/AEI/FEDER and EU grant AGL2017-85753-R and PID2020-113288RB-100/AEI/10.13039/501100011033 grant.

11 Pág. Departamento de Reproducción Animal​, Oocyte in vitro maturation (IVM) and vitrification procedures lead to detrimental effects on the overall oocyte quality. The addition of antioxidants during IVM, such as the coenzyme Q10 (Q10), has been demonstrated to positively impact on the cumulus-oocyte complexes due to its role in protection from oxidative damage and modulating gene transcription. Furthermore, glucocorticoids (GC) regulate gene transcription, energy metabolism and apoptosis during the early steps of reproduction. In this sense, most GC actions are mediated by the glucocorticoid receptor (NR3C1), a transcription factor. However, the specific roles of GC in ovarian physiology and oocyte maturation are still unknown. In this regard, a better knowledge on the expression of GC-related and apoptosis-related genes during IVM and cryopreservation procedures could potentially benefit the refinement of assisted reproductive techniques in the bovine species. The present study aims to explore the expression of NR3C1 mRNA in fresh and vitrified bovine oocytes and cumulus cells in response to Q10 (50 μM), and the effect of cortisol addition (0.25 μM, 0.5 μM) on the expression of NR3C1. We also studied the mRNA expression of NR3C1-related genes belonging to the GC regulation pathway, such as hydroxysteroid dehydrogenases (HSD11B1; HSD11B2), immunophilins (FKBP4; FKBP5), signal transducers and activators of transcription (STAT3; STAT5A), the mineralocorticoid receptor (NR3C2), and to the apoptosis pathway, such as the anti- (BCL2) and pro-apoptotic (BAX) mRNA transcripts in oocytes and cumulus cells 1) after IVM, and 2) after vitrification, both in presence or absence of Q10 supplementation during IVM. Our results show that there is an increase in the NR3C1 receptor expression after vitrification of oocytes, but not after exogenous cortisol supplementation during IVM. In addition, Q10 reduces the mRNA expression of HSD11B1 and FKBP5 in oocytes at levels of immature oocytes (HSD11B1 mRNA expression also in cumulus cells), and the BAX:BCL2 ratio mRNA expression. After vitrification in the presence of Q10, HSD11B2 mRNA expression increases in cumulus cells, while HSD11B1 and BAX:BCL2 mRNA expression decreases significantly both in oocytes and cumulus cells. In conclusion, our results show for the first time the effect of IVM, vitrification and Q10 supplementation on the mRNA relative expression of GC-related and apoptosis genes, and the effect of vitrification in the protein expression of NR3C1., This research was supported by the projects PID2019-108320RJ-I00, IJCI-2015-24380, PID2020-116531RB-I00 and RYC2020- 028615-I, funded by MCIN/AEI/10.13039/501100011033 (Spain) and FEDER funds (EU). MRC is supported by the Government of Spain, Ministry of Education, Culture and Sports (Training Programme for Academic Staff FPU15/06029). JG was supported by the Govern ment of Catalonia (Generalitat de Catalunya, AGAUR), co-financed with the European Social Found (2018FI_00236).

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Les tècniques de reproducció assistida han millorat enormement durant les darreres dècades. No obstant això, encara hi ha un llarg camí a recórrer per tal d'assolir taxes d'èxit que siguin acceptables. El desenvolupament d'estratègies per mitigar l'estrès cel·lular in vitro, juntament amb una millor comprensió de rutes de senyalització implicades en la fisiologia reproductiva i la fertilitat, pot proporcionar noves valuoses perspectives per aquest àmbit. La regulació dels glucocorticoides (GCs) pot ser una ruta de senyalització candidata en aquest sentit. Els GCs són hormones esteroidals que poden inhibir la reproducció durant les respostes al estrès, però que són essencials a nivell basal per a importants esdeveniments de la fisiologia reproductiva a la femella, incloent l'ovulació, la maduració del oòcit o la implantació. El rol dels GCs a la reproducció és complex i sembla variar en funció de l'espècie. El mecanisme involucrat en la seva regulació no és clar actualment, però moltes de les seves accions s'atribueixen al receptor de GCs, NR3C1, i també als enzims que controlen la disponibilitat de GC, així com a les immunofilines FKBP4 i FKBP5, implicades en la regulació de les accions de NR3C1 al nucli. En conseqüència, en aquesta tesi s'estudia el rol de l'expressió gènica i proteica del receptor (i altres molècules implicades en la regulació de GCs) en la funció reproductiva, mitjançant la determinació de la modulació en el tracte reproductiu de la femella durant diferents esdeveniments reproductius in vivo i in vitro. En aquest sentit, es va estudiar l'expressió de la regulació de GCs en cèl·lules del cúmulus i oòcits de boví sotmesos a maduració in vitro, vitrificació i suplementació amb coenzim Q10 (Q10), una molècula endògena amb importants propietats a nivell mitocondrial. Així, es va demostrar que la Q10 és beneficiosa per a la preservació de la integritat de l'oòcit post-vitrificació, que exerceix un efecte positiu contra l'apoptosi, i que pot influir en la regulació dels GCs en el gàmeta femení. A més, es van determinar els canvis en l'expressió basal relacionada amb els GCs a l'endometri i l'ampolla del boví, al llarg de les diferents fases del cicle estral. Aquests canvis van mostrar un patró espai-temporal de coincidència amb els esdeveniments reproductius, suggerint una funció rellevant dels GCs a l'oviducte durant la fase postovulatòria. Després d'aquesta aproximació, l'objectiu va ser entendre la influència de la interacció mascle-femella en l'expressió de la regulació de GCs, avaluant els efectes de la munta natural, la inseminació artificial i la infusió amb plasma seminal al tracte reproductiu de truges en fase preovulatòria. D'acord amb els nostres resultats, la munta natural indueix una regulació molt controlada cap a la restricció de l'acció dels GCs al reservori espermàtic, la qual cossa no succeeix després de la inseminació artificial. Finalment, en el conill, espècie d'ovulació induïda, es va avaluar la modulació de NR3C1 al tracte reproductiu causada per la influència de la interacció mascle-femella, així com durant diferents punts temporals, que es corresponen amb esdeveniments reproductius, com l'ovulació, la fecundació i els estadis embrionaris. Es va concloure que el plasma seminal pot induir l'expressió de NR3C1 a l'infundíbul, i que la munta natural augmenta NR3C1 en un patró espai-temporal coincident amb el dels embrions. En conjunt, aquesta tesi aporta nova informació sobre la complexa regulació dels GCs a la reproducció, i la seva modulació durant els esdeveniments reproductius en diferents espècies. Una millor comprensió d'aquesta ruta pot ajudar a desxifrar els mecanismes subjacents a la influència de l'estrès en la reproducció, trobar nous biomarcadors de fertilitat, així com desenvolupar noves estratègies amb finalitats reproductives. Las técnicas de reproducción asistida han mejorado enormemente durante las últimas décadas. Sin embargo, todavía existe un largo que camino que recorrer hasta alcanzar tasas de éxito que sean aceptables. El desarrollo de estrategias para mitigar el estrés celular in vitro, junto con una mayor comprensión de rutas de señalización implicadas en la fisiología reproductiva y la fertilidad, puede proporcionar nuevas valiosas perspectivas para el ámbito. La regulación de los glucocorticoides (GCs) puede ser una ruta de señalización candidata en este sentido. Los GCs son hormonas esteroideas que pueden inhibir la reproducción durante las respuestas a estrés, pero que son esenciales a nivel basal para importantes eventos de la fisiología reproductiva en la hembra, incluida la ovulación, la maduración ovocitaria o la implantación. El papel de los GCs en la reproducción es complejo y parece variar en función de la especie. El mecanismo involucrado en su regulación no está claro por el momento, pero muchas de sus acciones se atribuyen al receptor de GCs, NR3C1, y también a las enzimas que controlan la disponibilidad de GCs, así como a las inmunofilinas FKBP4 y FKBP5, implicadas en la regulación de las acciones de NR3C1 en el núcleo. En consecuencia, en la presente tesis se estudia el papel de la expresión génica y proteica del receptor (y otras moléculas implicadas en la regulación de GCs) en la función reproductiva, mediante la determinación de su modulación en el tracto reproductivo de la hembra durante diferentes eventos reproductivos in vivo e in vitro. En este sentido, se estudió la expresión de la regulación de GCs en células del cúmulus y ovocitos de bovino sujetos a maduración in vitro, vitrificación y suplementación con coenzima Q10 (Q10), una molécula endógena con importantes propiedades a nivel mitocondrial. Así, se demostró que la Q10 es beneficiosa para la preservación de la integridad del ovocito post-vitrificación, que ejerce un efecto positivo contra la apoptosis, y que puede influir en la regulación de los GCs en el gameto femenino. Además, se determinaron los cambios en la expresión basal relacionada con los GCs en el endometrio y la ampolla del bovino, a largo de las diferentes fases del ciclo estral. Estos cambios mostraron un patrón espaciotemporal de coincidencia con los eventos reproductivos, sugiriendo una función relevante de los GCs en el oviducto durante la fase postovulatoria. Tras este enfoque, el objetivo fue entender la influencia de la interacción macho-hembra en la expresión de la regulación de GCs, evaluando los efectos de la monta natural, la inseminación artificial y la infusión con plasma seminal en el tracto reproductivo de cerdas en fase preovulatoria. De acuerdo a nuestros resultados, la monta natural induce una regulación muy controlada para la restricción de la acción de los GCs en el reservorio espermático, la cual no sucede tras la inseminación artificial. Finalmente, en conejo, especie de ovulación inducida, se evaluó la modulación de NR3C1 en el tracto reproductivo después de la interacción macho-hembra, y durante diferentes puntos temporales, que se corresponden con eventos reproductivos como la ovulación, la fecundación y los estadios embrionarios. Se concluyó que el plasma seminal puede inducir la expresión de NR3C1 en el infundíbulo, y que la monta natural aumenta NR3C1 en un patrón espaciotemporal coincidente con el de los embriones. En conjunto, esta tesis aporta nueva información sobre la compleja regulación de los GCs en la reproducción, y su modulación durante los eventos reproductivos en diferentes especies. Una mayor comprensión de esta ruta puede ayudar a descifrar los mecanismos subyacentes a la influencia del estrés en la reproducción, encontrar nuevos biomarcadores de fertilidad, así como desarrollar nuevas estrategias con fines reproductivos. The assisted reproduction techniques have greatly improved over the last decades. However, there is still a long way to go until acceptable success rates are achieved. Developing strategies for reducing the cellular stress in vitro, together with a better understanding of signaling pathways involved in reproductive physiology and fertility, can provide new valuable insights in the field. The regulation of glucocorticoids (GCs) can be a candidate signaling pathway in this regard. GCs are steroid hormones that can inhibit reproduction during stress responses, but are also essential at baseline levels for important events of the female reproductive physiology, including ovulation, oocyte maturation or implantation. The role of GCs in reproduction is complex and seems to be different among species. The mechanisms involved in their regulation remain unclear, but most of their actions are attributed to the GC receptor, NR3C1, to the enzymes controlling GC availability, and immunophilins FKBP4 and FKBP5, involved in the regulation of the NR3C1 actions in the nucleus. Accordingly, the present thesis studies the role of the gene and protein expression of the GC receptor -and molecules involved in GC regulation- in the reproductive function, by determining its modulation in the female reproductive tract during reproductive events in vivo and in vitro. In this regard, we studied the GC regulatory expression in bovine oocytes and cumulus during in vitro maturation, vitrification, and supplementation with coenzyme Q10 (Q10), an endogenous molecule with important mitochondrial properties. Thus, we demonstrated that Q10 is beneficial for preserving the oocyte integrity after vitrification, exerts positive effects against apoptosis, and can influence the regulation of GCs in the female gamete. Moreover, we determined the changes in GC-related expression occurring at baseline level in the bovine endometrium and ampulla across different stages of the estrous cycle. These changes showed a spatiotemporal pattern that matched with reproductive events, suggesting a relevant role of GCs in the oviduct during the postovulatory phase. After this approach, we aimed to understand the influence of the male-female interaction in the expression of the GC regulation, by assessing the effects of natural mating, artificial insemination and seminal plasma infusion in the reproductive tract of preovulatory sows. According to our results, the natural mating induces a tight regulation for the restriction of the GC actions in the sperm reservoir which is not mimicked by artificial insemination. Finally, in the rabbit, an induced-ovulation species, we evaluated the modulation of NR3C1 in the female reproductive tract caused by the influence of the male-female interactions, and during different time points, corresponding to reproductive events such as ovulation, fertilization and presumed embryo developmental stages. We concluded that seminal plasma could trigger NR3C1 expression in the infundibulum, and mating increased NR3C1 in a spatiotemporal sequence corresponding to the assumed location of rabbit embryos. Overall, this thesis provides new knowledge about the complex GC regulation in reproduction and its modulation during reproductive events in different species. A better comprehension of this pathway may help to unravel the underlying mechanisms behind the stress influence on reproduction, find novel fertility biomarkers, and develop new potential strategies with reproductive purposes.

Posttranslational modifications of nuclear and nonnuclear proteins in spermatozoa Summary The number of couples who need the help of assisted reproductive technology (ART) has increased over the years. Approximately half of the cases are caused by male infertility, which is often diagnosed as idiopathic infertility. Therefore, the search for male fertility markers will improve male infertility diagnosis, thereby facilitating advanced sperm treatment and selection via ART. Posttranslational modifications (PTMs) of sperm nuclear and nonnuclear proteins are suitable candidates for such markers. The PTMs of protamines and histones reflect sperm chromatin maturity and its readiness for fertilization, and accordingly, they can predict the outcome of ART. However, the PTMs of nonnuclear proteins, including cytoplasmic, cytoskeletal, and membrane proteins, reflect the ability of sperm to undergo hyperactivation, capacitation, or acrosome reactions, which are processes essential for fertilization. We hypothesize that the PTMs of nuclear and nonnuclear proteins can reflect sperm quality and, thus, serve as a valuable marker in ART. Additionally, we suggest that the in vitro addition of hydrogen sulfide into the sperm-manipulating media improves sperm motility and viability via persulfidation. We used Western blot...

La vitrificació ha reemplaçat la congelació convencional per a la criopreservació d'ovòcits en diverses espècies. No obstant això, encara no s'ha establert el protocol òptim per a la criopreservació d'ovòcits bovins, encara que s'han desenvolupat diverses estratègies per a millorar els seus resultats. En aquest sentit, s'ha prestat poca atenció a l'estrès subletal per fred com a inductor de criotolerància en ovòcits bovins. La cold-inducible RNA-binding protein (CIRBP) pertany a les proteïnes induïbles per fred (CIPs), un grup de pèptids induïts per hipotèrmia i altres factors d'estrès. CIRBP és un candidat potencial per a millorar la criotolerància dels ovòcits bovins a causa de la seva funció en la supervivència cel·lular. A més, les CIPs exerceixen funcions crucials en el control del ARNm, la qual cosa planteja interrogants sobre la relació entre aquestes proteïnes i els canvis fisiològics que es produeixen en l'aparell reproductor femení. En conseqüència, la present tesi es va dur a terme amb l'objectiu general d'estudiar CIRBP com a molècula potencial per a millorar la vitrificació d'ovòcits bovins i la seva modulació durant els canvis fisiològics en el tracte reproductiu femení. En primer lloc, es va avaluar els efectes de la hipotèrmia en la maduració dels complexos cumulus-ovòcit. A més, es va determinar l'expressió proteica de CIRBP mitjançant Western blot. Es va demostrar que l'estrès per fred augmenta l'expressió de CIRBP. No obstant això, com s'esperava, es van detectar efectes perjudicials induïts pel fred. Després d'aquesta primera aproximació, es van buscar noves estratègies per a augmentar l'expressió de CIRBP evitant els efectes perjudicials del fred. Per a això, es van testar diferents concentracions de CIRBP exògena i una petita molècula hipotèrmico-mimètica ja establerta (zr17-2) com a suplement dels mitjans de maduració in vitro. Es van trobar pocs canvis en el ARNm de CIRBP i altres CIPs relacionats amb aquesta suplementació, però si es van observar increments d'expressió del ARNm de CIRBP i altres CIPs a causa de la hipotèrmia i vitrificació. Finalment, els estudis en el tracte reproductiu femení van revelar que CIRBP està modulada pels canvis hormonals fisiològics i la interacció mascle-femella. Aquesta tesi ofereix una nova visió de la fisiologia reproductiva de CIRBP, tant per a l'increment de coneixement bàsic com per al seu ús potencial en la millora de la vitrificació d'ovòcits bovins. Una millor comprensió del procés reproductiu pot servir com a nous biomarcadors, potencials eines d'avaluació reproductiva i millora de la producció animal. La vitrificación ha reemplazado la congelación convencional para la criopreservación de ovocitos en varias especies. Sin embargo, aún no se ha establecido el protocolo óptimo para la criopreservación de ovocitos bovinos, aunque se han desarrollado varias estrategias para mejorar sus resultados. En este sentido, se ha prestado poca atención al estrés subletal por frío como inductor de criotolerancia en ovocitos bovinos. La cold-inducible RNA-binding protein (CIRBP) pertenece a las proteínas inducibles por frío (CIPs), un grupo de péptidos inducidos por hipotermia y otros factores de estrés. CIRBP es un candidato potencial para mejorar la criotolerancia de los ovocitos bovinos debido a su función en la supervivencia celular. Además, las CIPs desempeñan funciones cruciales en el control del ARNm, lo que plantea interrogantes sobre la relación entre estas proteínas y los cambios fisiológicos que se producen en el aparato reproductor femenino. En consecuencia, la presente tesis se llevó a cabo con el objetivo general de estudiar CIRBP como molécula potencial para mejorar la vitrificación de ovocitos bovinos y su modulación durante los cambios fisiológicos en el tracto reproductivo femenino. En primer lugar, evaluamos los efectos de la hipotermia en la maduración de los complejos cumulus-ovocito. Además, determinamos la expresión proteica de CIRBP mediante Western blot. Se demostró que el estrés por frío aumenta la expresión de CIRBP. Sin embargo, como se esperaba, se detectaron efectos perjudiciales inducidos por el frío. Tras esta primera aproximación, se buscaron nuevas estrategias para aumentar la expresión de CIRBP evitando los efectos perjudiciales del frío. Para ello, testeamos diferentes concentraciones de CIRBP exógena y una pequeña molécula hipotérmico-mimética ya establecida (zr17-2) como suplemento de los medios de maduración in vitro. Se encontraron pocos cambios en el ARNm de CIRBP y otras CIPs relacionados con esta suplementación, pero si se observaron incrementos de expresión del ARNm de CIRBP y otras CIPs debido a la hipotermia y vitrificación. Finalmente, los estudios en el tracto reproductivo femenino revelaron que CIRBP está modulada por los cambios hormonales fisiológicos y la interacción macho-hembra. Esta tesis ofrece una nueva visión de la fisiología reproductiva de CIRBP, tanto para el incremento de conocimiento básico como para su uso potencial en la mejora de la vitrificación de ovocitos bovinos. Una mejor comprensión del proceso reproductivo puede servir como nuevos biomarcadores, potenciales herramientas de evaluación reproductiva y mejora de la producción animal. Vitrification has replaced the slow freezing method for cryopreservation of oocytes in several species. However, the optimal protocol for bovine oocyte cryopreservation remains to be established. Several strategies for improving bovine oocyte vitrification have been developed. In this regard, little attention has been given to sublethal mild hypothermia as an inductor of cryotolerance in bovine oocytes. The cold-inducible RNA-binding protein (CIRBP) belongs to the cold-inducible proteins (CIPs), a group of peptides induced by mild hypothermia and other stressors. CIRBP is a potential candidate to improve bovine oocyte cryotolerance due to its function in cell survival. In addition, CIPs play crucial roles in RNA transcript control, raising questions about the link between these proteins and the physiological changes in the female reproductive tract. Accordingly, the present thesis was conducted with the general objective to study CIRBP as a potential molecule to improve bovine oocyte vitrification and its modulation during physiological changes in the female reproductive tract. We first evaluated the effects of cold temperatures on the maturation of bovine cumulus-oocyte complexes. Additionally, we determined CIRBP protein expression by Western blot. Sublethal mild hypothermia was demonstrated to increase the expression of CIRBP. However, as expected, we reported detrimental effects induced by the sublethal stress. After this first approach, we aimed to find other strategies to increase the expression of CIRBP, avoiding the damaging effects of sub-physiological temperature variations. For this purpose, we tested different concentrations of exogenous CIRBP and a well-defined hypothermia mimetic small molecule (zr17-2), to supplement the in vitro maturation media of bovine cumulus-oocyte complexes. Minor changes in mRNA CIRBP expression and other CIPs were linked to this supplementation. Additionally, our results revealed that mild hypothermia and vitrification increased the mRNA expression of CIRBP and other CIPs. Finally, the female reproductive tract studies revealed that CIRBP is physiologically modulated by the cyclic hormonal changes and by the male-female interaction. Overall, this thesis offers new insight into CIRBP reproductive physiology, increasing the knowledge between CIRBP and the female reproductive tract and its potential use in improving bovine oocyte vitrification. A better understanding of the reproductive complex process may serve as novel clinical biomarkers, potential diagnostic tools for reproductive evaluation, and the improvement of animal production.