Influencia del pH ambiental en la motilidad del esperma de la anguila europea (Anguilla anguilla)

[ES] A diferencia de lo que ocurre en mamíferos, los espermatozoides de la mayoría de los peces se encuentran inactivos en los testículos. Su activación se produce como consecuencia de un choque osmótico (hiper o hipoosmótico, según se trate de especies de agua salada o dulce), cuando son liberados al medio externo. Sin embargo, no existe un consenso en la comunidad científica sobre cuál es el mecanismo fisiológico que induce la activación espermática. En la anguila europea, objeto de estudio, trabajos previos han mostrado que la composición iónica del plasma seminal es más importante para la activación que la del medio activador; además, han presentado al pH como el factor más importante de este último. El principal objetivo de este trabajo es estudiar la influencia del pH del medio extracelular (pHe), tanto del plasma seminal como del agua de mar, en la activación de la motilidad espermática en la anguila europea. Un grupo de machos de anguila europea fue sometido semanalmente a inyecciones de gonadotropina coriónica humana recombinante (rec-hCG) para inducir en ellos la maduración sexual y, a partir de la sexta dosis suministrada, la espermiación. Veinticuatro horas después de cada inyección se extrajeron las muestras de esperma y se evaluó, en diferentes condiciones, la motilidad total y otros parámetros cinéticos de las mismas, mediante sistemas CASA (Computer Assisted Sperm Analysis). El pH intracelular (pHi) se evaluó por medio de citometría de flujo. Los ensayos que se llevaron a cabo en el seno de este proyecto, incluyen: estudio del efecto del pH del medio diluyente (plasma seminal artificial) y del medio activador (agua de mar) en la motilidad del esperma y en el pH intracelular; estudio de la interacción del pH con el potasio (K+ ) del medio diluyente en la motilidad espermática; y análisis del efecto del pH intracelular en la motilidad de los espermatozoides, mediante el uso de un intercambiador de iones K+ /H+ . Los resultados mostraron que el pH del medio activador tiene una mayor influencia sobre la motilidad espermática que el pH del medio diluyente. Asimismo, se observó la existencia de un gradiente de pH entre el interior y el exterior celular en estado quiescente, así como de variaciones en el pH intracelular tras el inicio de la motilidad. Por último, no se observó efecto alguno de la interacción del pH con el potasio del medio diluyente sobre la motilidad espermática, pero sí del pH intracelular fijado con nigericin sobre esta.[EN] Unlike what happens in mammals, most fish sperm is inactive in the testicles. Its activation occurs as a consequence of an osmotic shock (hyper or hypoosmotic, depending on whether it is marine or freshwater species), when they are released to the external environment. However, there is no consensus in the scientific community about what is the physiological mechanism that induces sperm activation. In the European eel, object of study, previous research projects have shown that the ionic composition of the seminal plasma is more important for the activation than that of the activating medium; in addition, pH was shown as the most important factor of the latter. The main objective of this paper is to study the influence of the pH of the extracellular medium (pHe), both seminal plasma and seawater, in the activation of sperm motility in European eel. A group of European eel males underwent weekly injections of recombinant human chorionic gonadotropin (rec-hCG) to induce sexual maturation and, from the sixth dose supplied, the spermiation. Twenty-four hours after each injection, the sperm samples were collected and, under different conditions, the total motility and other kinetic parameters of the same were evaluated by means of CASA (Computer Assisted Sperm Analysis) systems. The intracellular pH (pHi) was evaluated via flow cytometry. The tests that were carried out within this project include: study of the effect of the pH of the diluting medium (artificial seminal plasma) and the activating medium (seawater) on sperm motility and intracellular pH; study of the interaction of the pH and the potassium (K+ ) of the diluent in sperm motility; and analysis of the effect of intracellular pH on sperm motility, through the use of a K+ /H+ ion exchanger. The results showed that the pH of the activating medium has a greater influence on sperm motility than the pH of the diluting medium. Likewise, the existence of a pH gradient between cellular interior and exterior in a quiescent state, as well as variations in intracellular pH after the onset of motility were observed. Finally, no effect of the interaction of the pH and the potassium of the diluent medium was observed on sperm motility, but the intracellular pH fixed with nigericin had an effect on it.[CA] A diferència del que passa en mamífers, els espermatozoides de la majoria dels peixos es troben inactius als testicles. La seva activació es produeix com a conseqüència d'un xoc osmòtic (hiper o hipoosmòtic, segons es tracti de peixos d'aigua salada o dolça), quan són alliberats al medi extern. No obstant això, no existeix un consens en la comunitat científica sobre quin és el mecanisme fisiològic que indueix l'activació espermàtica. En l'anguila europea, objecte d'estudi, treballs previs han mostrat que la composició iònica del plasma seminal és més important per a l'activació que la del medi activador; a més, han presentat el pH com el factor més important d'aquest últim. El principal objectiu d'aquest treball és estudiar la influència del pH del medi extracel·lular (pHe), tant del plasma seminal com de l'aigua de mar, en l'activació de la motilitat espermàtica de l'anguila europea. Un grup de mascles d'anguila europea va ser sotmès setmanalment a injeccions de gonadotropina coriònica humana recombinant (rec-hCG) per induir la maduració sexual i, a partir de la sisena dosi subministrada, l’espermiació. Vint-i-quatre hores després de cada injecció es van extraure les mostres d'esperma i es va avaluar, en diferents condicions, la motilitat total i altres paràmetres cinètics de les mateixes mitjançant sistemes CASA (Computer Assisted Sperm Analysis). El pH intracel·lular (pHi) es va avaluar per mitjà de citometria de flux. Els assajos que es van dur a terme en el si d'aquest projecte, inclouen: estudi de l'efecte del pH del medi diluent (plasma seminal artificial) i del medi activador (aigua de mar) en la motilitat de l'esperma i en el pH intracel·lular; estudi de la interacció del pH amb el potassi (K+ ) del medi diluent en la motilitat espermàtica; i anàlisi de l'efecte del pH intracel·lular en la motilitat dels espermatozoides, fent ús d'un intercanviador d'ions K+ /H+ . Els resultats van mostrar que el pH del medi activador té una major influència sobre la motilitat espermàtica que el pH del medi diluent. Així mateix, es va observar l'existència d'un gradient de pH entre l'interior i l'exterior cel·lular en estat quiescent, així com de variacions en el pH intracel·lular després de l'inici de la motilitat. Per últim, no es va observar cap efecte de la interacció del pH amb el potassi del medi diluent sobre la motilitat espermàtica, però sí del pH intracel·lular fixat amb nigericin sobre aquesta.Francés Herrero, E. (2018). Influencia del pH ambiental en la motilidad del esperma de la anguila europea (Anguilla anguilla). http://hdl.handle.net/10251/107595TFG

Future bioengineering applications in female reproductive medicine. From basic science to animal models.

Tesis con Mención Internacional.Los hidrogeles de matriz extracelular (MEC) constituyen biomateriales con un alto potencial en el campo de la ingeniería tisular. Su objetivo es recrear el entorno nativo tridimensional que las células necesitan para diferenciarse y desarrollar funciones específicas. Los hidrogeles de MEC tienen aplicaciones emergentes en el campo de la ginecología. Entre ellas se incluye el desarrollo de plataformas in vitro de nueva generación, como sistemas de recubrimiento 2D o encapsulación 3D, así como aplicaciones in vivo que entre las que se encuentra su uso como portadores celulares o depósitos de fármacos. Los cinco objetivos de esta tesis fueron (1) diseñar hidrogeles de MEC oviductales (OviECM) a partir de tejido de conejo descelularizado (DC), (2) estudiar el efecto de un recubrimiento de hidrogel de OviECM durante el desarrollo embrionario in vitro, (3) diseñar hidrogeles de MEC ováricos (OvaECM) a partir de corteza ovárica bovina DC, (4) probar el potencial del hidrogel de OvaECM bovina para promover y sostener la foliculogénesis murina in vitro, y (5) evaluar la viabilidad y eficacia del uso de hidrogeles de OvaECM como portadores de factores de crecimiento (GFs) en el nicho ovárico murino para revertir el fallo ovárico prematuro (FOP). Para alcanzar estos objetivos, se aisló tejido oviductal y ovárico de especies de conejo y bovino respectivamente, se sometieron a diferentes protocolos de descelularización y se procesaron para crear los hidrogeles de OviECM y OvaECM, cuyas propiedades fisicoquímicas se caracterizaron. El efecto de un recubrimiento de hidrogel de OviECM durante el desarrollo embrionario in vitro se evaluó en términos de viabilidad, comportamiento metabólico y expresión génica, así como de salud y fenotipo de las crías. Posteriormente, se evaluó el desarrollo de folículos murinos sobre hidrogeles de OvaECM mediante análisis de viabilidad, crecimiento, producción hormonal y competencia ovocitaria. Por último, se evaluó la viabilidad y eficacia de estos hidrogeles como portadores de GFs en el nicho ovárico murino para revertir el FOP. Generamos y caracterizamos en profundidad hidrogeles acelulares de oviducto y ovario, preservando gran parte de la riqueza bioquímica de los tejidos nativos. Nuestros resultados mostraron que el cultivo in vitro de embriones de conejo preimplantatorios sobre recubrimientos de OviECM mejoró su rendimiento metabólico y mostró diferentes perfiles de expresión génica en comparación con los cultivados en condiciones estándar. Estos embriones también mostraron mayores tasas de implantación y nacimiento, y dieron lugar a crías con mayor peso. Los recubrimientos de hidrogel de OvaECM fueron capaces de favorecer la foliculogénesis murina in vitro, induciendo un crecimiento más fisiológico que las condiciones de cultivo 2D y mejorando las tasas de maduración de ovocitos y desarrollo embrionario. Además, el hidrogel de OvaECM demostró ser un excelente portador de GFs, permitiendo la liberación prolongada de GFs en el nicho ovárico de un modelo murino de FOP. Se observaron mejoras en las poblaciones foliculares, la proliferación celular y la vasculatura, al tiempo que se redujeron la apoptosis y la fibrosis. Además, se obtuvieron más ovocitos y embriones por estimulación ovárica, y aumentó la fertilidad natural de los ratones a largo plazo.Extracellular matrix (ECM) hydrogels are promising and remarkable tissue engineering biomaterials. They aim to recreate the 3D native environment that cells need to differentiate and develop specific functions. ECM hydrogels have emerging applications in the field of gynecology. These include the development of next generation in vitro platforms, such as 2D coating or 3D encapsulation systems, as well as in vivo applications involving ECM hydrogels as cell carriers or drug depots. The five objectives of this thesis were (1) to design oviductal ECM (OviECM) hydrogels from decellularized (DC) rabbit tissue, (2) to study the effect of an OviECM hydrogel coating during in vitro embryonic development, (3) to design ovarian ECM (OvaECM) hydrogels from DC bovine ovarian cortex (OC), (4) to test the potential of the bovine OvaECM hydrogel to promote and sustain murine folliculogenesis in vitro, and (5) to evaluate the feasibility and effectivity of using OvaECM hydrogels as carriers of growth factors (GFs) into the murine ovarian niche to reverse premature ovarian failure (POF). To achieve these objectives, oviductal and ovarian tissue were isolated from rabbit and bovine species respectively, subjected to different decellularization protocols, and processed to create OviECM and OvaECM hydrogels, whose physicochemical properties were studied. The effect of an OviECM hydrogel coating during in vitro embryonic development was assessed in terms of viability, metabolic behavior, and gene expression, as well as health and phenotype of the offspring. Later, development of murine follicles on OvaECM hydrogels was assessed through analysis of viability, growth, hormone production, and oocyte competence. Finally, the feasibility and effectivity of these hydrogels as carriers of GFs into the murine ovarian niche to reverse POF was evaluated. We generated and characterized in depth oviduct and ovarian acellular hydrogels, preserving a large part of the biochemical richness of the native tissues. Our results showed that culturing in vitro preimplantation rabbit embryos on OviECM coatings improved their metabolic performance and showed different gene expression profiles compared to those cultured under standard conditions. These embryos also exhibited higher implantation and birth rates, and gave rise to offspring with a higher weight. OvaECM hydrogel coatings were able to support murine folliculogenesis in vitro, inducing more physiological growth than 2D culture conditions, and improving rates of oocyte maturation and embryo development. Furthermore, the OvaECM hydrogel proved to be an excellent carrier of GFs, allowing prolonged release of GFs into the ovarian niche of a murine POF model. Improvements in follicular populations, cell proliferation and vasculature were observed, while apoptosis and fibrosis were reduced. In addition, more oocytes and embryos were obtained by ovarian stimulation, and the natural fertility of the mice was increased in the long term.Ministerio de UniversidadesUniversitat de ValènciaFundación IV

Human Umbilical Cord-Based Therapeutics: Stem Cells and Blood Derivatives for Female Reproductive Medicine

There are several conditions that lead to female infertility, where traditional or conventional treatments have limited efficacy. In these challenging scenarios, stem cell (SC) therapies have been investigated as alternative treatment strategies. Human umbilical cord (hUC) mesenchymal stem cells (hUC-MSC), along with their secreted paracrine factors, extracts, and biomolecules, have emerged as promising therapeutic alternatives in regenerative medicine, due to their remarkable potential to promote anti-inflammatory and regenerative processes more efficiently than other autologous treatments. Similarly, hUC blood derivatives, such as platelet-rich plasma (PRP), or isolated plasma elements, such as growth factors, have also demonstrated potential. This literature review aims to summarize the recent therapeutic advances based on hUC-MSCs, hUC blood, and/or other plasma derivatives (e.g., extracellular vesicles, hUC-PRP, and growth factors) in the context of female reproductive medicine. We present an in-depth analysis of the principal molecules mediating tissue regeneration, compiling the application of these therapies in preclinical and clinical studies, within the context of the human reproductive tract. Despite the recent advances in bioengineering strategies that sustain delivery and amplify the scope of the therapeutic benefits, further clinical trials are required prior to the wide implementation of these alternative therapies in reproductive medicine

Future Challenges and Opportunities of Extracellular Matrix Hydrogels in Female Reproductive Medicine

Bioengineering and reproductive medicine have progressed shoulder to shoulder for several decades. A key point of overlap is the development and clinical translation of technologies to support reproductive health, e.g., scaffold-free constructs, polymeric scaffolds, bioprinting or microfluidics, and hydrogels. Hydrogels are the focus of intense study, and those that are derived from the extracellular matrix (ECM) of reproductive tissues and organs are emerging as promising new players given their results in pre-clinical models. This literature review addresses the recent advances in the use of organ-specific ECM hydrogels in reproductive medicine, considering the entire female reproductive tract. We discuss in-depth papers describing the development of ECM hydrogels, their use in in vitro models, and their in vivo application in preclinical studies. We also summarize the functions of hydrogels, including as grafts, carriers for cell transplantation, or drug depots, and present the potential and possible scope for use of ECM hydrogels in the near future based on recent scientific advances

Future Challenges and Opportunities of Extracellular Matrix Hydrogels in Female Reproductive Medicine

Bioengineering and reproductive medicine have progressed shoulder to shoulder for several decades. A key point of overlap is the development and clinical translation of technologies to support reproductive health, e.g., scaffold-free constructs, polymeric scaffolds, bioprinting or microfluidics, and hydrogels. Hydrogels are the focus of intense study, and those that are derived from the extracellular matrix (ECM) of reproductive tissues and organs are emerging as promising new players given their results in pre-clinical models. This literature review addresses the recent advances in the use of organ-specific ECM hydrogels in reproductive medicine, considering the entire female reproductive tract. We discuss in-depth papers describing the development of ECM hydrogels, their use in in vitro models, and their in vivo application in preclinical studies. We also summarize the functions of hydrogels, including as grafts, carriers for cell transplantation, or drug depots, and present the potential and possible scope for use of ECM hydrogels in the near future based on recent scientific advances

Development of Decellularized Oviductal Hydrogels as a Support for Rabbit Embryo Culture

[EN] The oviducts (fallopian tubes in mammals) function as the site of fertilization and provide necessary support for early embryonic development, mainly via embryonic exposure to the tubal microenvironment. The main objective of this study was to create an oviduct-specific extracellular matrix (oviECM) hydrogel rich in bioactive components that mimics the native environment, thus optimizing the developmental trajectories of cultured embryos. Rabbit oviducts were decellularized through SDS treatment and enzymatic digestion, and the acellular tissue was converted into oviductal pre-gel extracellular matrix (ECM) solutions. Incubation of these solutions at 37 degrees C resulted in stable hydrogels with a fibrous structure based on scanning electron microscopy. Histological staining, DNA quantification and colorimetric assays confirmed that the decellularized tissue and hydrogels contained no cellular or nuclear components but retained important components of the ECM, e.g. hyaluronic acid, glycoproteins and collagens. To evaluate the ability of oviECM hydrogels to maintain early embryonic development, two-cell rabbit embryos were cultured on oviECM-coated surfaces and compared to those cultured with standard techniques. Embryo development was similar in both conditions, with 95.9% and 98% of the embryos reaching the late morula/early blastocyst stage by 48 h under standard culture and oviECM conditions, respectively. Metabolomic analysis of culture media in the presence or absence of embryos, however, revealed that the oviECM coating may include signalling molecules and release compounds beneficial to embryo metabolism.This study was supported by Instituto de Salud Carlos III (PI17/01039-CP19/00149 [Irene Cervello]), Ministry of Economy, Industry and Competitiveness (AGL2017-85162-C2-1-R [Francisco Marco], BES-2015-072429 [Ximo Garcia]), Ministry of Science, Innovation and Universities (FPU18/06327 [Emilio Frances]) and Generalitat Valenciana (PROMETEO/2018/137 [Irene Cervello, Lucia de Miguel], ACIF/2017/118 [Sara Lopez]). The proteomics laboratory is a member of ProteoRed, PRB3, and is supported by grant PT17/0019 of the State Plan I+D+i 2013-2016, funded by Instituto de Salud Carlos III and European Regional Development Fund.Francés-Herrero, E.; De Miguel-Gómez, L.; López-Martínez, S.; Campo, H.; Garcia-Dominguez, X.; Diretto, G.; Faus, A.... (2021). Development of Decellularized Oviductal Hydrogels as a Support for Rabbit Embryo Culture. Reproductive Sciences. 28(6):1644-1658. https://doi.org/10.1007/s43032-020-00446-6S1644165828