Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2020 Jun 16:8:629.
doi: 10.3389/fbioe.2020.00629. eCollection 2020.

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

Nicola Bernabò  1   2 , Luca Valbonetti  1   2 , Marcello Raspa  2 , Antonella Fontana  3 , Paola Palestini  4 , Laura Botto  4 , Renata Paoletti  5 , Martin Fray  6 , Susan Allen  6 , Juliana Machado-Simoes  1 , Marina Ramal-Sanchez  1 , Serena Pilato  3 , Ferdinando Scavizzi  2 , Barbara Barboni  1
Affiliations

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

Nicola Bernabò et al. Front Bioeng Biotechnol. .

Abstract

During the latest years, human infertility worsened all over the world and is nowadays reputed as a global public health issue. As a consequence, the adoption of Assisted Reproductive Technologies (ARTs) such as In Vitro Fertilization (IVF) is undergoing an impressive increase. In this context, one of the most promising strategies is the innovative adoption of extra-physiological materials for advanced sperm preparation methods. Here, by using a murine model, the addition of Graphene Oxide (GO) at a specific concentration has demonstrated to increase the spermatozoa fertilizing ability in an IVF assay, finding that 0.5 μg/ml GO addition to sperm suspensions before IVF is able to increase both the number of fertilized oocytes and embryos created with a healthy offspring given by Embryo Transplantation (ET). In addition, GO treatment has been found more effective than that carried out with methyl-β-cyclodextrin, which represents the gold standard in promoting in vitro fertility of mice spermatozoa. Subsequent biochemical characterization of its interaction with male gametes has been additionally performed. As a result, it was found that GO exerts its positive effect by extracting cholesterol from membranes, without affecting the integrity of microdomains and thus preserving the sperm functions. In conclusion, GO improves IVF outcomes in vitro and in vivo, defining new perspectives for innovative strategies in the treatment of human infertility.

Keywords: cholesterol; detergent resistant membrane; graphene oxide; in vitro fertilization; mouse spermatozoa; rafts; sperm capacitation; sperm membrane.

PubMed Disclaimer

Figures

FIGURE 1
FIGURE 1
Graph showing the effects of increasing GO concentrations on sperm acrosome integrity when incubating under capacitating conditions.
FIGURE 2
FIGURE 2
Graph representing the relationship between GO concentration and IVF outcomes, expressed as difference in percentage (Δ IVF%) respect to the CRTL.
FIGURE 3
FIGURE 3
Confocal microscopy image showing an example of an embryo generated using spermatozoa exposed to 0.5 μg/ml GO. The (upper panel) shows an embryo with (from left to right) an overlay of the membranes in red (DilC12) and the nuclei in blue (DAPI), only the nuclei, only the membranes. The (lower panel) illustrates, from left to right an overlay of nuclei and membrane showing the Depth Coded maxIP (Intensity Projection) gradient visualization (to evaluate the 3D structure).
FIGURE 4
FIGURE 4
An example picture of pups derived by transferred embryos obtained after IVF with GO treated spermatozoa.
FIGURE 5
FIGURE 5
Distribution of Cav-1 between insoluble (TI) and soluble (TS) membrane fractions, isolated from MEF prepared from sperm incubated in a capacitating medium (BSA, MB CD, GO treatments), or from sperm collected in a non-capacitating medium (CTRL). The data reported were expressed as cav-1 abundance in each group. Values are means ± SE for three separate experiments. *P < 0.05 versus control.
FIGURE 6
FIGURE 6
Percentage distribution of proteins in TI and TS fractions, isolated from a membrane-enriched fraction (MEF) prepared from sperm incubated in a capacitating medium (BSA, MβCD, GO treatments) or from sperm collected in a non-capacitating medium (CTRL). The data reported were expressed as percentage change relative to control value. Values are the mean of three protein determinations ± SE obtained from three independent experiments. *P < 0.05 versus control.
FIGURE 7
FIGURE 7
Cholesterol/Phospholipids ratio in (A) insoluble (TI) and (B) soluble (TS) membrane fractions, isolated from MEF prepared from sperm incubated in a capacitating medium (BSA, MβCD, GO treatments) or from sperm collected in a non-capacitating medium (CTRL). Values are means ± SE for three separate experiments. ***p < 0.001 versus control.
FIGURE 8
FIGURE 8
Cholesterol normalized to total protein content in (A) insoluble (TI) and (B) soluble (TS) membrane fractions, isolated from MEF prepared from sperm incubated in a capacitating medium (BSA, MβCD, GO treatments), or from sperm collected in a non-capacitating medium (CTRL). Values were expressed as percentage change relative to control value. Values are means ± SE for three separate experiments. **p < 0.01 versus control, ***p < 0.001 versus control.
See this image and copyright information in PMC

References

    1. Asghar W., Shafiee H., Velasco V., Sah V. R., Guo S., El Assal R., et al. (2016). Toxicology study of single-walled carbon nanotubes and reduced graphene Oxide in human sperm. Sci. Rep. 6:30270. 10.1038/srep30270 - DOI - PMC - PubMed
    1. Barratt C. L. R., Björndahl L., De Jonge C. J., Lamb D. J., Osorio Martini F., McLachlan R., et al. (2017). The diagnosis of male infertility: an analysis of the evidence to support the development of global WHO guidance—challenges and future research opportunities. Hum. Reprod. Update 23 660–680. 10.1093/humupd/dmx021 - DOI - PMC - PubMed
    1. Barré-Sinoussi F., Montagutelli X. (2015). Animal models are essential to biological research: issues and perspectives. Futur. Sci. OA 1:FSO63. 10.4155/fso.15.63 - DOI - PMC - PubMed
    1. Behringer R., Gertsenstein M., Vintersten Nagy K., Nagy A. (2014). Manipulating the Mouse Embryo A Laboratory Manual, 4th #dn Edn Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press.
    1. Bernabò N., Fontana A., Sanchez M. R., Valbonetti L., Capacchietti G., Zappacosta R., et al. (2018). Graphene Oxide affects in vitro fertilization outcome by interacting with sperm membrane in an animal model. Carbon N. Y. 129 428–437. 10.1016/j.carbon.2017.12.042 - DOI