Graphene Oxide increases mammalian spermatozoa fertilizing ability by extracting cholesterol from their membranes and promoting capacitation

Abstract

Graphene Oxide (GO) is a widely used biomaterial with an amazing variety of applications in biology and medicine. Recently, we reported the ability of GO to improve the in vitro fertilization (IVF) outcomes in swine, a validated animal model with a high predictive value for human fertility. For that reason, here we characterized the mechanisms involved in this positive interaction by adopting an experimental approach combining biological methods (confocal microscopy analysis on single cell, flow cytometry on cell populations and co-incubation with epithelial oviductal cells), physical-chemical techniques (Differential Scanning Calorimetry and Thermogravimetric Analysis), and chemical methods (mass spectrometry and lipid measurement). As a result, we propose a model in which GO is able to extract cholesterol from the spermatozoa membrane without causing any detrimental effect. In this way, the cholesterol extraction promotes a change in membrane chemical-physical properties that could positively affect male gamete function, modulating sperm signalling function and increasing in this way the fertilizing potential, without losing the ability to physiologically interact with the female environment. In conclusion, these data seem to suggest new intriguing possibilities in engineering sperm membrane for improving assisted reproduction technologies outcomes, even in human medicine.

Graphene oxide: A glimmer of hope for Assisted Reproductive Technology

“…During capacitation, numerous membrane lipids are displaced, increasing the membrane fluidity and modulating the activity of several enzymes and proteins, leading to a deep remodeling of the membranes. Here, by using an already validated FRAP approach [42], we evidenced an increase in the membrane fluidity of some spermatozoa released from BOECs, probably caused by the extraction of cholesterol by GO [23], one of the events responsible for the beginning of capacitation. In order to appraise the results obtained, we compared the calculated diffusion coefficients (CDC) with those obtained when using P4, considered as a physiological stimulus.…”

“…However, recently our group has evaluated the dose-dependent effects of GO on boar sperm function, concretely in some events related to capacitation (i.e., [Escriba aquí] the ensemble of events that spermatozoa undergo to become fully fertile), revealing a low toxicity level and a positive effect on the IVF outcomes when spermatozoa were capacitated in the presence of GO at a specific concentration (1 µg/mL) [7]. Since our results stand out the potential use of GO to improve the fertilization rates in IVF assays, we adopted in our earlier study an experimental approach in which swine spermatozoa were incubated under capacitating conditions, evidencing the effect of GO on cholesterol extraction from sperm plasma membrane [23], a key event of sperm capacitation. After cholesterol extraction, the sperm membrane undergoes physicochemical modifications, increasing the lipid disorder and the fluidity and enabling the fusion between the plasma membrane (PM) and the outer acrosome membrane (OAM).…”

Graphene and Reproduction: A Love-Hate Relationship

“…Since it is well accepted that spermatozoa are transcriptionally silent (although with controversy [ 81 ]), most of the biochemical events take place or initiate within the sperm membrane. Our group was able to confirm, in a later work [ 82 ], the cholesterol extraction from the sperm membrane by the action of GO, thus increasing the sperm membrane fluidity and contributing to the acquisition of the fertilizing ability.…”

Graphene Oxide Improves in vitro Fertilization in Mice With No Impact on Embryo Development and Preserves the Membrane Microdomains Architecture

“…A MEF was used as a starting material for isolation of TI. In detail, based on a previously published model (Bernabò et al, 2019), we compared control samples (i.e., without cholesterol acceptors) with GO treatment and with samples treated with bovine serum albumin (BSA), which is a physiological extracellular acceptor of cholesterol, and MβCD, a stronger cholesterol extractor routinely used in mouse IVF, as mentioned above.…”

“…Recently, a significant improvement has been found in fertility when sperm were exposed to GO during the process that leads them to become fertile, the capacitation, as recently published in swine (Bernabò et al, 2018) and bovine models (Ramal-Sanchez et al, 2019), probably due to an extraction of cholesterol from the sperm membrane and thus an intense lipid membrane remodeling (Bernabò et al, 2019).…”