Magnetic-Assisted Control of Eggs and Embryos via Zona Pellucida-Linked Nanoparticles.

Eggs and embryo manipulation is an important biotechnological challenge to enable positioning, entrapment, and selection of reproductive cells to advance into a new era of nature-like assisted reproductive technologies. Oviductin (OVGP1) is an abundant protein in the oviduct that binds reversibly to the zona pellucida, an extracellular matrix that surrounds eggs and embryos. Here, the study reports a new method coupling OVGP1 to magnetic nanoparticles (NP) forming a complex (NPOv). NPOv specifically surrounds eggs and embryos in a reversible manner. Eggs/embryos bound to NPOv can be moved or retained when subjected to a magnetic force, and interestingly only mature-competent eggs are attracted. This procedure is compatible with normal development following gametes function, in vitro fertilization, embryo development and resulting in the birth of healthy offspring. The results provide in vitro proof-of-concept that eggs and embryos can be precisely guided in the absence of physical contact by the use of magnets.

Sperm-Binding Assay Using an In Vitro 3D Model of the Mammalian Cumulus-Oocyte Complex.

We have recently described a new model to study gamete interaction in mammalian species. The model recreates the spherical surface of the oocyte by using magnetic Sepharose beads coated with a layer of a recombinant protein involved in gamete interaction (such as ZP2, or the IZUMO1 receptor JUNO) and an external layer of cumulus oophorus cells, thus mimicking, to some extent, a native cumulus-oocyte complex. Once generated, this 3D model can be used in a sperm-binding assay to obtain valuable information about the molecular basis of gamete interaction, since different recombinant proteins can be used to coat the bead surface, thus generating a variety of models to be used for several species. Furthermore, thanks to the ability of the model to decoy sperm, the physiological status of the bound sperm can be studied, making this a powerful tool to select sperm with high fertilizing capacity, to unmask subfertile animals in livestock breeding centers, or for toxicological studies. Here, we describe how to generate and use this model for sperm-binding assays, using porcine sperm as an example, and ZP2, a protein from zona pellucida, as the recombinant protein of interest. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Generation of the in vitro 3D model Alternate Protocol 1: Binding cumulus oophorus cells to the model Basic Protocol 2: Quality control of the model by SDS-PAGE electrophoresis and western blot Support Protocol 1: Immunochemistry to confirm proper protein distribution on surface of beads Support Protocol 2: Elution of recombinant conjugated proteins Basic Protocol 3: Sperm-binding assay Alternate Protocol 2: Sperm preparation by the swim-up method.

JUNO protein coated beads: A potential tool to predict bovine sperm fertilizing ability.

Considerable variation in fertility exists between bulls in AI centres, despite passing minimum post-thaw quality control checks. The development of a reliable in vitro test to predict bull fertility could enable the identification and selection of high fertility bulls, without the need to resort to test inseminations. An in-depth knowledge of the molecular basis of fertilization is a prerequisite to the development of such a test or combination of tests. To date, JUNO is the only oocyte plasma membrane receptor described to be involved in gamete binding for which the partner in the sperm, IZUMO1, is known. Despite the fact that this interaction appears to be conserved among mammals, it has not been confirmed yet in some species including cattle. Furthermore, an association between binding and fertility has not been tested. Here, we propose a sperm-binding assay based on magnetic sepharose beads coated with bovine recombinant JUNO protein (BJUNO) to study sperm binding. Bull sperm bound specifically to BJUNO demonstrating that the JUNO-IZUMO1 interaction is conserved in cattle. Moreover, the assay was able to distinguish between epididymal and ejaculated sperm. Lastly, the number of sperm cells bound to BJUNO was significantly lower for frozen-thawed sperm from bulls of low vs high field fertility. In conclusion, our findings document a novel valid sperm-binding assay to predict mammalian sperm function and to investigate the role of specific proteins involved in gamete recognition and fusion.

Mammalian spermatozoa and cumulus cells bind to a 3D model generated by recombinant zona pellucida protein-coated beads.

The egg is a spherical cell encapsulated by the zona pellucida (ZP) which forms a filamentous matrix composed of several glycoproteins that mediate gamete recognition at fertilization. Studies on molecular mechanisms of sperm-egg binding are limited in many mammalian species by the scarcity of eggs, by ethical concerns in harvesting eggs, and by the high cost of producing genetically modified animals. To address these limitations, we have reproduced a three-dimensional (3D) model mimicking the oocyte's shape, by means of magnetic sepharose beads coated with recombinant ZP glycoproteins (BZP) and cumulus cells. Three preparations composed of either ZP2 (C and N-termini; BZP2), ZP3 (BZP3) or ZP4 (BZP4) were obtained and characterized by protein SDS-PAGE, immunoblot and imaging with confocal and electron microscopy. The functionality of the model was validated by adhesion of cumulus cells, the ability of the glycoprotein-beads to support spermatozoa binding and induce acrosome exocytosis. Thus, our findings document that ZP-beads provide a novel 3D tool to investigate the role of specific proteins on egg-sperm interactions becoming a relevant tool as a diagnostic predictor of mammalian sperm function once transferred to the industry.