Physiological role of mitochondrial and plasma membrane channels in sperm
- Autores: Ariadna Delgado-Bermúdez
- Directores de la Tesis: Marc Yeste Oliveras (dir. tes.)
- Lectura: En la Universitat de Girona ( España ) en 2022
- Idioma: inglés
- Tribunal Calificador de la Tesis: Christine Aurich (presid.), Ana Josefa Soler Valls (secret.), Robert Pedley (voc.)
- Programa de doctorado: Programa de Doctorado en Tecnología por la Universidad de Girona
- Materias:
- Enlaces
- Tesis en acceso abierto en: TDX
- Resumen
The spermatozoon is a highly specialised cell that travels along the female reproductive tract to deliver its genome into the oocyte, and encounters varying extracellular conditions during its journey. The physiological interaction with this varying environment is crucial for sperm to undergo capacitation, which is a process they must go through to acquire effective fertilising ability and trigger the acrosome reaction. Cryopreservation, which is the most efficient procedure for long-term storage of sperm, also challenges the male gamete because of the drastic changes in extracellular medium composition. The adaptation and physiological response to these variations in extracellular medium are related to the exchange of water and other molecules across the plasma membrane, but also across intracellular membranes. Considering their hydrophobicity, additional mechanisms to simple diffusion are needed to allow the flow of water and solutes through the aforementioned membranes. Water permeability is facilitated by aquaporins (AQPs), a family of transmembrane water channels. Different groups of AQPs present distinct structure and permeability: orthodox AQPs, aquaglyceroporins (GLPs) and superAQPs. While the presence and subcellular distribution of AQPs in sperm varies between different species, their main function is linked to osmoadaptation and is highly conserved between species. Voltage-gated proton channel (HV1 or HVCN1) has also been identified in sperm from different species of mammals. Because it regulates proton flow through the plasma membrane, its permeability is linked to intracellular pH, which regulates capacitation-associated events. Finally, different types of potassium channels have been found in mammalian sperm. Although many different types of K+ channels have been identified in mammalian cells, the SLO subfamily of channels seems to be highly relevant in sperm. Essentially, K+ channels are crucial in sperm physiology through the regulation of plasma membrane potential, which activates different signalling pathways, including those driving capacitation-related events. Acknowledging the relevance of these three types of channels (AQPs, HVCN1 and K+ channels) in sperm function, their involvement in mammalian sperm capacitation and cryopreservation was investigated in this Dissertation. For this purpose, in the first study, the role of AQPs during in vitro capacitation of pig sperm was explored through the use of three different inhibitors. Cooper sulphate, a specific inhibitor of AQP3, caused a drastic increase in reactive oxygen species (ROS) levels, which could be related to the intracellular accumulation of these molecules, as they can permeate certain members of the AQPs family. The presence of mercury chloride, an unspecific inhibitor of all AQPs but AQP7, impaired membrane lipid disorder and sperm motility. Finally, the inhibition of all AQPs using silver sulfadiazine (AgSDZ) caused a drastic decrease in intracellular pH and tyrosine phosphorylation levels after the induction of acrosomal reaction. Three additional studies aimed to explore the role of AQPs during pig and horse sperm cryopreservation. For this purpose, three different inhibitors were added to cryopreservation media, and their effects were also evaluated separately in ejaculates with good (GFE) and poor cryotolerance (PFE). On the one hand, 1,3-propanediol (PDO), an efficient inhibitor of orthodox AQPs with a mild inhibitory effect on GLPs, maintained better post-thaw sperm quality compared to control samples, suggesting that this molecule has a cryoprotective role in both species. Acetazolamide (AC), an inhibitor of AQP1 and AQP4, had some detrimental effects that could occur as a collateral impact on other sperm proteins. Finally, phloretin (PHL), which inhibits AQP3 and AQP7, impaired post-thaw sperm quality drastically. In addition, when GFE and PFE samples were analysed separately, GFE were more sensitive to positive effects, whereas PFE were less resilient to detrimental effects on their post-thawing quality. The last study aimed to explore the relevance of HVCN1 and K+ channels, with a special focus on SLO1, during pig sperm cryopreservation; to this end, three different inhibitors were added to the cryopreservation medium. Paxilline (PAX), a specific inhibitor of SLO1, did not alter sperm quality and function after thawing. Similarly, the unspecific inhibition of 6TM K+ channels through tetraethyl ammonium (TEA) did not cause significant effects on sperm cryotolerance. Yet, when 2-guanidino benzimidazole (2-GBI) was used as a specific inhibitor of HVCN1, sperm experienced a dramatic impairment in their function and survival after cryopreservation, including alterations in membrane integrity, including mitochondrial, plasma and acrosomal membranes; and ROS levels. In conclusion, the results of this Dissertation support that membrane channels have a crucial role during both capacitation and cryopreservation processes, which might be in relation to osmoadaptability and intracellular pH regulation. Alterations in the permeability of membrane channels have drastic consequences for sperm quality and function, the most relevant alterations being an impairment of membrane structure and an increase in ROS levels.
