Development, validation and in vitro applications of novel 3D models to study gamete interaction in mammals

Abstract

Despite gamete recognition being a widely studied process, not all the molecular mechanisms are known in detail and especially in livestock species such as porcine and bovine, the knowledge is scarce. New reproducible and scalable strategies to carry on studies in the reproductive biology field should be developed to elucidate the molecular mechanisms of gamete recognition, to improve the assisted reproductive technologies and to design new non-hormonal contraceptives. Ideally, these strategies should be easily transferable between species. The main objective of this Doctoral Thesis was to develop and validate a new in vitro tool to study gamete interaction and to predict the fertility in seminal samples. The three-dimensional (3D) model extensively studied during this thesis is based in the use of magnetic sepharose beads coated with recombinant proteins that are known to be involved in gamete interaction. In Chapter 1, the model based on magnetic sepharose beads (B) coated with single recombinant porcine zona pellucida (ZP) glycoproteins (BZP) that mimic the 3D oocyte's shape was generated, validated and characterized by protein SDS-PAGE, immunoblot and imaging with confocal and electron microscopy. BZP closely mimic native cumulus-oocyte complexes by maintaining a spherical shape, supporting cumulus cell adhesion and providing a glycoprotein-specific surface. From these observations, the conclusions of the first chapter are that the BZP are stable through the time, support cumulus cells adhesion, sperm binding and provide a valuable tool to explore the molecular basis of gamete recognition in pigs. In Chapter 2, the application of the model to study the role of single porcine ZP glycoproteins in gamete interaction was explored. Results after sperm-BZP incubation showed that over 60 % of beads had at least one sperm bound, being BZP2 model the one with the highest number of sperm bound per bead (8.82 ± 0.63, N=289) and higher percentage of acrosome reacted sperm bound to the beads. Meanwhile, BZP3 and BZP4 models presented a higher number of acrosomal shrouds (3.77 ± 0.17 N=156; and 3.48 ± 0.15 N=153 respectively) and a higher percentage of unbound acrosome reacted sperm. IVF output increased when porcine COCs were inseminated in presence of BZP2. The conclusion of this second chapter is that ZP3 and ZP4 glycoproteins mainly induce acrosome reaction whereas ZP2 is involved in sperm-ZP binding in porcine species. Besides, BZP2 model might be a useful tool to improve the final IVF efficiency in pigs. After studying the role of the single porcine ZP glycoproteins successfully in Chapter 2, using the 3D model developed and validated in Chapter 1, in Chapter 3, the 3D model was tested in the bovine species. A new model based on beads coated with recombinant bovine JUNO protein (BJUNO) was developed and validated. Results showed that BJUNO is stable through time and bull sperm bound specifically to BJUNO. Moreover, BJUNO model responded differently when incubated with sperm from different sources (fresh ejaculated vs epididymal) or different fertilizing capacity (high vs low fertility bulls). The number of sperm bound per bead was higher when incubated with fresh ejaculated and high fertility bull sperm respectively. These findings document an innovative and valid sperm-binding assay to predict mammalian fertility by responding in a different manner between ejaculates from different fertilizing capacity. In summary, the models can be a useful tool to study the gamete interaction in depth, sperm binding, the acrosome reaction induction, the improvement of IVF protocols and they might be implemented as a semen fertility predictor in the future. These models are easily translated between species, scalable and reproducible, being a potential tool to be transferred to the industry.