Rabbit seminal plasma proteome: The importance of the genetic origin

[EN] The present study was conducted to characterise rabbit seminal plasma proteins (SP proteins) focusing on the influence of the genetic origin and seasonality. In addition, ß-NGF protein quantity in SP was determined. Semen samples were recovered from January to December 2014 using 6 males belonging to genotype A and six from genotype R. For each genotype, one pooled sample at the beginning, middle and end of each season was selected to develop the experiment. A total of 24 pools (3 for each season and genetic line) were analysed. SP proteins of the two experimental groups were recovered and subjected to in-solution digestion nano LC¿MS/MS and bioinformatics analysis. The resulting library included 402 identified proteins validated with ¿95% Confidence (unused Score¿1.3). These data are available via ProteomeXchange with identifier PXD006308. Only 6 proteins were specifically implicated in reproductive processes according to Gene Ontology annotation. Twenty-three proteins were differentially expressed between genotypes, 11 over-expressed in genotype A and 12 in genotype R. Regarding the effect of season on rabbit SP proteome, results showed that there is no clear pattern of protein variation throughout the year. Similar ß-NGF relative quantity was observed between seasons and genotypes. In conclusion, this study generates the largest library of SP proteins reported to date in rabbits and provides evidence that genotype is related to a specific abundance of SP proteins.This research was supported in part by the RTA2013-00058-00-00 from INIA, the European Social Fund and the European FEDER Funds. L. Casares-Crespo is supported by a scholarship from Instituto Valenciano de Investigaciones Agrarias (IVIA) and the European Social Fund. P. Fernandez-Serrano is supported by Spanish funds from IVIA and Ministerio de Empleo y Seguridad Social (Youth Guarantee Program). The authors are grateful to M. Luz Valero for her excellent technical assistance.Casares-Crespo, L.; Fernández-Serrano, P.; Vicente Antón, JS.; Marco-Jiménez, F.; Viudes De Castro, MP. (2018). Rabbit seminal plasma proteome: The importance of the genetic origin. Animal Reproduction Science. 189:30-42. https://doi.org/10.1016/j.anireprosci.2017.12.004S304218

Insemination extender supplementation with bestatin and EDTA has no effect on rabbit reproductive performance

[EN] The addition of aminopeptidase inhibitors (AMIs) to rabbit semen extenders could be a solution to decrease the hormone degradation (GnRH) by the aminopeptidases existing in the seminal plasma. Therefore, the quantity of GnRH needed to induce ovulation in doe would be comparable with the amount administered intramuscularly (i.m.). This study was conducted to evaluate the effects of two AMIs (bestatin and EDTA) on rabbit semen quality parameters, beta nerve growth factor ((beta-NGF) degradation and reproductive performance after artificial insemination. Results showed that seminal quality was not affected by the incubation with AMIs; the values of motility, acrosome integrity and sperm viability were not significantly different between the AMIs and the control groups (positive i.m. and negative intravaginally without AMIs). In addition, the aminopeptidase activity of seminal plasma was inhibited in a 55.5% by the AMIs as well as beta-NGF degradation. On the other hand, regarding the effect of AMIs on reproductive performance, our results showed that the presence of bestatin and EDTA did neither affect fertility (85.3 vs. 88.6%), nor the prolificacy rate (10.12 vs. 10.51 kits per delivery), comparing AMIs group to positive control group, respectively. We conclude that the addition of specific AMIs in the rabbit semen extender has no effect on reproductive performance. Therefore, due to the fact that AMIs inhibit part of the aminopeptidase activity that degrades the GnRH analogue and beta-NGF, they could be used to develop new extenders with less hormone concentration. (C) 2017 Elsevier Inc. All rights reserved.This research was supported in part by the RTA2013-00058-00-00 from INIA, the European Social Fund and the European FEDER Funds. L. Casares-Crespo is supported by a scholarship from Instituto Valenciano de Investigaciones Agrarias (IVIA) and the European Social Fund. P. Fernandez-Serrano is supported by funds from Instituto Valenciano de Investigaciones Agrarias (IVIA) and Ministerio de Empleo y Seguridad Social (Programa de Garantia Juvenil).Casares-Crespo, L.; Fernández-Serrano, P.; Vicente Antón, JS.; Moce Cervera, ET.; Castellini, C.; Stabile, A.; Viudes De Castro, MP. (2018). Insemination extender supplementation with bestatin and EDTA has no effect on rabbit reproductive performance. Theriogenology. 105:61-65. https://doi.org/10.1016/j.theriogenology.2017.09.009S616510

Development of new artificial insemination extenders supplemented with GnRH analogues to induce ovulation and proteomic characterization of rabbit semen

Tesis por compendioLos objetivos generales de esta tesis fueron desarrollar nuevos diluyentes de inseminación artificial (IA) suplementados con un análogo de GnRH y caracterizar el proteoma del semen de conejo. En el capítulo I, se evaluó la inclusión de un cóctel de inhibidores de proteasas en el diluyente de inseminación (DI) para evitar parte de la actividad proteasa del plasma seminal de conejo. La calidad seminal y la fertilidad no se vieron afectadas por el cóctel. Sin embargo, la prolificidad fue significativamente menor en el grupo experimental en comparación con los grupos de control positivo y negativo (8,2±0,22 vs. 9,3±0,23 y 9,2±0,26 gazapos por parto, respectivamente). De este capítulo, se puede concluir que la adición de una amplia variedad de inhibidores de proteasas en el diluyente de semen de conejo afecta negativamente la tasa de prolificidad y que en el futuro sería aconsejable probar inhibidores específicos de aminopeptidasas (AMIs). En el capítulo II, suplementamos el DI con AMIs (bestatina y EDTA), y estudiamos su efecto sobre la calidad seminal y el rendimiento reproductivo. La inclusión de AMIs no afectó la calidad seminal, ni la fertilidad (85,3 vs. 88,6%), ni la prolificidad (10,12 vs. 10,51 gazapos por parto) en comparación con el grupo control. Por lo tanto, concluimos que los AMIs se pueden utilizar en los DIs de conejo para inhibir parte de la actividad aminopeptidasa del plasma seminal (PS). En el capítulo III, probamos nuevos DIs de conejo que contenían AMIs con o sin nanopartículas de quitosano (CS)-sulfato de dextrano (DS) que atrapan el análogo de GnRH. Se estudiaron los siguientes diluyentes: C4 (4 µg de buserelina/coneja en medio control (MC): tris-ácido cítrico-glucosa suplementado con AMIs), C5 (5 µg de buserelina/coneja en MC), Q4 (4 µg de buserelina/coneja en nanopartículas CS-DS en MC) y grupo Q5 (5 µg de buserelina/coneja en nanopartículas CS-DS en MC). La fertilidad fue significativamente menor en el grupo C4 en comparación con los grupos C5, Q5 y Q4 (0,7 frente a 0,85, 0,85 y 0,82, respectivamente). Por el contrario, la prolificidad fue similar en los cuatro grupos experimentales. Por lo tanto, las nanopartículas de CS-DS como transportador de acetato de buserelina permiten reducir la concentración de la hormona en diluyentes con AMIs sin afectar la fertilidad ni la prolificidad. Por ello, la nanoencapsulación parece ser un sistema prometedor para proteger el análogo de GnRH en los diluyentes de IA de conejos. Por otro lado, el objetivo de los últimos tres capítulos fue caracterizar las proteínas del semen de conejo. En los capítulos IV y V, se estudiaron las proteínas del PS de conejo. Las muestras de semen se recuperaron utilizando 6 machos de cada línea genética (A y R) y seleccionando una muestra heteroespérmica del comienzo, del medio y del final de cada estación y de cada línea (24 muestras en total). En el capítulo IV, utilizamos la técnica de electroforesis en gel de poliacrilamida 1D. Siete bandas proteicas fueron significativamente diferentes entre las líneas genéticas y tres bandas entre las estaciones. En el capítulo V, se sometió el PS a nano LC-MS/MS y se identificaron y cuantificaron 402 proteínas. 23 proteínas se expresaron diferencialmente entre genotipos. Con respecto al efecto de la estación en el proteoma del PS de conejo, los resultados mostraron que no hubo un patrón claro de variación de proteína a lo largo del año. En el capítulo VI, se caracterizaron las proteínas del espermatozoide de conejo. Se recuperaron 6 muestras espermáticas utilizando 5 machos de cada genotipo y se sometieron a nano LC-MS/MS, identificándose y cuantificándose 487 proteínas. 40 proteínas se expresaron diferencialmente entre genotipos. En conclusión, los resultados de los tres últimos capítulos evidencian que el genotipo está relacionado con una abundancia específica de proteínas del PS y del espermatozoide. Finalmente, se creó laThe general objectives of this thesis were to develop new artificial insemination (AI) extenders supplemented with a GnRH analogue and to characterise the proteomic profile of rabbit semen. In chapter I, the inclusion of a protease inhibitors cocktail in the insemination extender (IE) to avoid part of the rabbit seminal plasma protease activity was evaluated. Seminal quality and fertility rate were not affected by the cocktail, having similar values between experimental and control groups. However, prolificacy rate was significantly lower in experimental group compared to positive and negative control groups (8.2 ±0.22 vs. 9.3 ±0.23 and 9.2 ±0.26 total born per litter, respectively). From this chapter, it may be concluded that the addition of a wide variety of protease inhibitors in the rabbit semen extender negatively affects prolificacy rate and it in the future it would be advisable to test specific aminopeptidase inhibitors (AMIs). Therefore, in chapter II, we supplemented the IE with AMIs (bestatin and EDTA), and we studied their effect on rabbit seminal quality and reproductive performance. Seminal quality was not affected by AMIs. Regarding reproductive performance, the inclusion of AMIs, did not affect fertility (85.3 vs. 88.6%), nor the prolificacy rate (10.12 vs. 10.51 kits per delivery) in comparison with control group. Thus, we concluded that AMIs can be used in rabbit IEs to inhibit part of the seminal plasma aminopeptidase activity. In chapter III, we test new rabbit IEs containing AMIs with or without chitosan (CS)-dextran sulfate (DS) nanoparticles entrapping the GnRH analogue. The following experimental extenders were studied: C4 (4 µg buserelin/doe in control medium (CM): Tris-citric acid-glucose supplemented with AMIs), C5 (5 µg of buserelin/doe in CM), Q4 (4 µg of buserelin/doe into CS-DS nanoparticles in CM) and Q5 group (5 µg of busereline/doe into CS-DS nanoparticles in CM). Results showed that fertility was significantly lower in C4 group compared to C5, Q5 and Q4 groups (0.7 vs. 0.85, 0.85 and 0.82, respectively). On the contrary, prolificacy was similar in the four experimental groups studied. Thus, CS-DS nanoparticles as carrier for buserelin acetate allow reducing the hormone's concentration in extenders supplemented with AMIs without affecting the fertility and prolificacy of rabbit females. Therefore, nanoencapsulation seems to be a promising system to protect the GnRH analogue in rabbit AI extenders. On the other hand, the aim of the last three chapters was to characterize rabbit semen proteins. In chapters IV and V, rabbit seminal plasma (SP) proteins were studied. Semen samples were recovered using 6 males from each genetic line (A and R). For each genotype, one pooled sample at the beginning, middle and end of each season was selected to develop the experiment (24 pools in total). In chapter IV, we used a 1D polyacrylamide gel electrophoresis approach. Seven protein bands were significantly different between genetic lines and three protein bands were significantly different between seasons. In chapter V, SP was subjected nano LC-MS/MS and 402 proteins were identified and quantified. Twenty-three proteins were differentially expressed between genotypes. Regarding the effect of season on rabbit SP proteome, results showed that there was no clear pattern of protein variation throughout the year. The results obtained in both chapters evidence that genotype is related to a specific abundance of SP proteins. In chapter VI, rabbit sperm proteins were characterised. Six samples were recovered during two months using 5 males from each genotype. Sperm proteins were subjected to nano LC-MS/MS and 487 proteins were identified and quantified. Forty proteins were differentially expressed between genotypes. In conclusion, rabbit sperm proteins showed that genotype has also a huge impact on their abundance. Finally, with these data, the first publicly accessible database of rabbit semen proteome was cEls objectius generals d'aquesta tesi van ser desenvolupar nous diluents d'inseminació artificial (IA) suplementats amb un anàleg de GnRH i caracteritzar el proteoma del semen de conill. En el capítol I, es va avaluar la inclusió d'un còctel d'inhibidors de proteases en el diluent d'inseminació (DI) per evitar part de l'activitat proteasa del plasma seminal de conill. La qualitat seminal i la fertilitat no es van veure afectades pel còctel. No obstant això, la prolificitat va ser significativament menor en el grup experimental en comparació amb els grups de control positiu i negatiu (8,2 ± 0,22 vs. 9,3 ± 0,23 i 9,2 ± 0,26 catxaps per part, respectivament). D'aquest capítol, es pot concloure que l'addició d'una àmplia varietat d'inhibidors de proteases en el diluent de semen de conill afecta negativament la taxa de prolificitat i que en el futur seria aconsellable provar inhibidors específics de aminopeptidasas (AMIS). En el capítol II, suplementàrem el DI amb AMIs (bestatina i EDTA), i vam estudiar el seu efecte sobre la qualitat seminal i el rendiment reproductiu. La inclusió de AMIs no va afectar la qualitat seminal, ni la fertilitat (85,3 vs. 88,6%), ni la prolificitat (10,12 vs. 10,51 catxaps per part) en comparació amb el grup control. Per tant, concloem que els AMIs es poden utilitzar en els DIs de conill per inhibir part de l'activitat aminopeptidasa del plasma seminal (PS). En el capítol III, vam provar nous DIs de conill que contenien AMIs amb o sense nanopartícules de quitosà (CS)-sulfat de dextrà (DS) que atrapen l'anàleg de GnRH. Es van estudiar els següents diluents: C4 (4 µg de buserelina/conilla en medi control (MC): tris-àcid cítric-glucosa suplementat amb AMIs), C5 (5 µg de buserelina/conilla en MC), Q4 (4 µg de buserelina/conilla en nanopartícules CS-DS en MC) i grup Q5 (5 µg de buserelina/conilla en nanopartícules CS-DS en MC). La fertilitat va ser significativament menor en el grup C4 en comparació amb els grups C5, Q5 i Q4 (0,7 enfront de 0,85, 0,85 i 0,82, respectivament). Per contra, la prolificitat va ser similar en els quatre grups experimentals. Per tant, les nanopartícules de CS-DS com a transportador d'acetat de buserelina permeten reduir la concentració de l'hormona en diluents amb AMIs sense afectar la fertilitat ni la prolificitat. Per això, la nanoencapsulació sembla ser un sistema prometedor per protegir l'anàleg de GnRH en els diluents d'IA de conills. D'altra banda, l'objectiu dels últims tres capítols va ser caracteritzar les proteïnes del semen de conill. En els capítols IV i V, es van estudiar les proteïnes del PS de conill. Les mostres de semen es van recuperar utilitzant 6 mascles de cada línia genètica (A i R) i seleccionant una mostra heteroespérmica del començament, del mitjan i del final de cada estació i de cada línia (24 mostres en total). En el capítol IV, utilitzàrem la tècnica d'electroforesi en gel de poliacrilamida 1D. Set bandes proteiques van ser significativament diferents entre les línies genètiques i tres bandes entre les estacions. En el capítol V, es va sotmetre el PS a nano LC-MS / MS i es van identificar i quantificar 402 proteïnes. 23 proteïnes es van expressar diferencialment entre genotips. Pel que fa a l'efecte de l'estació en el proteoma del PS de conill, els resultats van mostrar que no hi havia un patró clar de variació proteica al llarg de l'any. En el capítol VI, es van caracteritzar les proteïnes de l'espermatozoide de conill. Es van recuperar 6 mostres espermàtiques utilitzant 5 mascles de cada genotip i es van sotmetre a nano LC-MS / MS, identificant i quantificant 487 proteïnes. 40 proteïnes es van expressar diferencialment entre genotips. En conclusió, els resultats dels tres últims capítols evidencien que el genotip està relacionat amb una abundància específica de proteïnes del PS i de l'espermatozoide. Finalment, es va crear la primera base de dades d'accés públic del proteomaCasares Crespo, L. (2018). Development of new artificial insemination extenders supplemented with GnRH analogues to induce ovulation and proteomic characterization of rabbit semen [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/100854TESISCompendi

Can the Genetic Origin Affect Rabbit Seminal Plasma Protein Profile along the Year?

Contents The study was designed to evaluate the influence of genetic origin on rabbit seminal plasma protein profile variation along the year. Seminal plasma of rabbits from line A (maternal line) and R (paternal line) collected during a natural year was subjected to polyacrylamide gel electrophoresis (SDS-PAGE). The electrophoretic profile of rabbit seminal plasma resulted in multiple protein bands of different intensity ranging from 9 to 240kDa. Results showed that seven protein bands were significantly different between genetic lines, and among these, three protein bands were significantly different between seasons. The differentially expressed proteins were identified by MALDI-TOF/TOF or LC-MS/MS analysis and were the following ones: FAM115E-like (220, 113 and 59kDa), ectonucleoside triphosphate diphosphohydrolase 3 isoform X2 (72kDa), annexin A5 (32kDa), lipocalin allergen Ory c 4 precursor (19kDa), and haemoglobin subunit zetalike (13kDa) between genetic lines and FAM115E-like (113kDa), haemoglobin subunit zetalike (13kDa) and -nerve growth factor (12kDa) between seasons. These results indicate that proteins from rabbit seminal plasma are under both seasonal control and genetic control. Furthermore, the differential presence of these proteins could be one of the causes explaining the differences observed in fertility and seminal parameters between these two lines in earlier studies

Porcine oocyte vitrification in optimized low toxicity solution with open pulled straws

[EN] One of the greatest challenges for reproductive cryobiologists today is to develop an efficient cryopreservation method for human and domestic animal oocytes. The objective of the present study was to optimize a low toxicity solution called VM3 to vitrify porcine oocytes using an open pulled straw (OPS) device and to evaluate the effects on viability, chromosomal organization and cortical granules distribution. Two experiments were conducted in this study. Firstly, we determined the minimum concentration of cryoprotectant present in the VM3 solution required (7.6 M) for vitrification using an OPS device. The appearance of opacity was observed when using a cooling solution at -196 degrees C; no observable opacity was noted as vitrification. In addition, the ultrastructure of oocytes in VM3 or VM3 optimized solution was examined using cryo-scanning electron microscopy. The minimum total cryoprotectant concentration present in VM3 solution necessary for apparent vitrification was 5.6 M when combined with use of an OPS device. Use of both vitrification solutions showed a characteristic plasticized surface. In the second experiment, the relative cytotoxicity of vitrification solutions (VM3 and VM3 optimized) was studied. Oocyte viability, chromosomal organization and the cortical granules distribution were assessed by fluorescent stain. After warming, oocyte survival rate was similar to that of fresh oocytes. The vitrification process significantly reduced correct chromosomal organization and cortical granules distribution rates compared with the fresh oocytes group. However, correct chromosomal organization and cortical granules distribution rates did not differ among oocytes placed in different vitrification solutions. In conclusion, our data demonstrated that the VM3 solution can be optimized and that reduction in concentration to 5.6 M enabled vitrification of oocytes with an OPS device, however use of the VM3 optimised solution had no beneficial effect on vitrification of porcine oocytes.This work was supported by funds from the Generalitat Valenciana research programme (Prometeo 2009/125) and Ajudes per a la realitzacio de projectes precompetitius d'I+D per a equips [Grants for pre-competitive R&D projects for research teams] (GVPRE/2008/206). English text version was revised by the N. Macowan English language service.Marco Jiménez, F.; Casares-Crespo, L.; Vicente Antón, JS. (2014). Porcine oocyte vitrification in optimized low toxicity solution with open pulled straws. Zygote. 22(2):204-212. https://doi.org/10.1017/S0967199412000524S20421222

On the Role of Basal Autophagy in Adult Neural Stem Cells and Neurogenesis

9 páginas, 1 figura, 1 tabla.Adult neurogenesis persists in the adult mammalian brain due to the existence of neural stem cell (NSC) reservoirs in defined niches, where they give rise to new neurons throughout life. Recent research has begun to address the implication of constitutive (basal) autophagy in the regulation of neurogenesis in the mature brain. This review summarizes the current knowledge on the role of autophagy-related genes in modulating adult NSCs, progenitor cells and their differentiation into neurons. The general function of autophagy in neurogenesis in several areas of the embryonic forebrain is also revisited. During development, basal autophagy regulates Wnt and Notch signaling and is mainly required for adequate neuronal differentiation. The available data in the adult indicate that the autophagy-lysosomal pathway regulates adult NSC maintenance, the activation of quiescent NSCs, the survival of the newly born neurons and the timing of their maturation. Future research is warranted to validate the results of these pioneering studies, refine the molecular mechanisms underlying the regulation of NSCs and newborn neurons by autophagy throughout the life-span of mammals and provide significance to the autophagic process in adult neurogenesis-dependent behavioral tasks, in physiological and pathological conditions. These lines of research may have important consequences for our understanding of stem cell dysfunction and neurogenic decline during healthy aging and neurodegeneration.This work was supported by the Spanish Ministerio de Economía y Competitividad (Grant No. SAF2015-70433-R).Peer reviewe

On the Role of Basal Autophagy in Adult Neural Stem Cells and Neurogenesis.

Capítulo de libro online, fruto de una recopilación de distintos artículos sobre el tema "Glial and Neural Stem Cells as New Therapeutic Targets for Neurodegenerative Disorders"This work was supported by the Spanish Ministerio de Economía y Competitividad (Grant No. SAF2015-70433-R).Peer reviewe

Autophagy drives the conversion of developmental neural stem cells to the adult quiescent state

12 páginas, 6 figurasNeurogenesis in the adult mammalian brain relies on the lifelong persistence of quiescent neural stem cell (NSC) reservoirs. Little is known about the mechanisms that lead to the initial establishment of quiescence, the main hallmark of adult stem cells, during development. Here we show that protein aggregates and autophagy machinery components accumulate in developmental radial glia-like NSCs as they enter quiescence and that pharmacological or genetic blockade of autophagy disrupts quiescence acquisition and maintenance. Conversely, increasing autophagy through AMPK/ULK1 activation instructs the acquisition of the quiescent state without affecting BMP signaling, a gatekeeper of NSC quiescence during adulthood. Selective ablation of Atg7, a critical gene for autophagosome formation, in radial glia-like NSCs at early and late postnatal stages compromises the initial acquisition and maintenance of quiescence during the formation of the hippocampal dentate gyrus NSC niche. Therefore, we demonstrate that autophagy is cell-intrinsically required to establish NSC quiescence during hippocampal development. Our results uncover an important role of autophagy in the transition of developmental NSCs into their dormant adult form, paving the way for studies directed at further understanding the mechanisms of stem cell niche formation and maintenance in the mammalian brain.This work was supported by the Spanish MICINN (grant no. PID2019- 111225RB-I00) and PROMETEO/2018/055 grant from Generalitat Valenciana for H.M.; FPI fellowship BES-2016-077156 to I.C.-B.; APOSTD2019/058 from Generalitat Valenciana to L.C.-C.; and INVEST/ 2022/242 from Generalitat Valenciana to H.M. and C.F.-I.Peer reviewe