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. 2023 Mar 1;24(5):4788.
doi: 10.3390/ijms24054788.

Catechin versus MoS2 Nanoflakes Functionalized with Catechin: Improving the Sperm Fertilizing Ability-An In Vitro Study in a Swine Model

Costanza Cimini  1 , Marina Ramal-Sanchez  2 , Angela Taraschi  1 , Flavio Della Pelle  1 , Annalisa Scroccarello  1 , Ramses Belda-Perez  1 , Luca Valbonetti  1   3 , Paola Lanuti  4   5 , Marco Marchisio  4   5 , Mario D'Atri  6   7 , Claudio Ortolani  6 , Stefano Papa  6 , Giulia Capacchietti  1 , Nicola Bernabò  1   3 , Dario Compagnone  1 , Barbara Barboni  1
Affiliations

Catechin versus MoS2 Nanoflakes Functionalized with Catechin: Improving the Sperm Fertilizing Ability-An In Vitro Study in a Swine Model

Costanza Cimini et al. Int J Mol Sci. .

Abstract

Nowadays, the adoption of In Vitro Fertilization (IVF) techniques is undergoing an impressive increase. In light of this, one of the most promising strategies is the novel use of non-physiological materials and naturally derived compounds for advanced sperm preparation methods. Here, sperm cells were exposed during capacitation to MoS2/Catechin nanoflakes and catechin (CT), a flavonoid with antioxidant properties, at concentrations of 10, 1, 0.1 ppm. The results showed no significant differences in terms of sperm membrane modifications or biochemical pathways among the groups, allowing the hypothesis that MoS2/CT nanoflakes do not induce any negative effect on the parameters evaluated related to sperm capacitation. Moreover, the addition of CT alone at a specific concentration (0.1 ppm) increased the spermatozoa fertilizing ability in an IVF assay by increasing the number of fertilized oocytes with respect to the control group. Our findings open interesting new perspectives regarding the use of catechins and new materials obtained using natural or bio compounds, which could be used to implement the current strategies for sperm capacitation.

Keywords: catechins; in vitro fertilization; molybdenum disulfide; sperm capacitation; spermatozoa.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
(A) Graphical sketch of the MoS2 sonochemical exfoliation assisted by catechin. (1) Bulk MoS2, (2) catechin structure, (3) sonochemical exfoliation process, (4) MoS2/CT nanoflakes. (B) SEM micrograph of the bulk-MoS2 (unexfoliated); (C) SEM micrograph of the exfoliated MoS2/CT (left); picture of the MoS2-CT colloidal water-dispersion (right). (D) SEM magnification of the MoS2/CT nanoflakes.
Figure 2
Figure 2
Acrosome integrity. The graph shows the percentage of non-reacted spermatozoa after 1.5 h of capacitation for the different experimental groups: CTRL, CT 10 ppm, CT 1 ppm, CT 0.1, MoS2/CT 10 ppm, MoS2/CT 1 ppm and MoS2/CT 0.1 ppm. A normal acrosome damage rate was obtained, similar to the control (CTRL) group (p > 0.05). Three independent technical and biological (from different boars) replicates were performed.
Figure 3
Figure 3
Flow cytometry analysis of intracellular calcium concentration. The graphs show the frequency of spermatozoa emitting a specific fluorescence intensity, which was subdivided into intervals ranging from 0 to 20,000 a.u. Capacitation was performed up to 1.5 h. Fluo 4-AM was used in combination with PI. Three independent technical and biological experiments were performed (n = 3).
Figure 4
Figure 4
Flow cytometry analysis of membrane disorder and fluidity. The graphs show the frequency of spermatozoa emitting a specific fluorescence intensity, which was subdivided into intervals ranging from 0 to 50,000 a.u. Capacitation was performed up to 1.5 h. DilC-12 was used as a probe. Three independent technical and biological experiments were performed (n = 3).
Figure 5
Figure 5
Flow cytometry analysis of mitochondrial activity. The graphs show the frequency of spermatozoa emitting a specific fluorescence intensity, which was subdivided into intervals ranging from 0 to 30,000 a.u. Capacitation was performed up to 1.5 h. Mitotracker Red was used in combination with a near-IR probe. Three independent technical and biological experiments were performed (n = 3).
Figure 6
Figure 6
Western blot analysis of PKA activity and tyrosine phosphorylation patterns. The image illustrates (A) the PKA activity and (B) the tyrosine phosphorylation patterns after 1.5 h of incubation under capacitating conditions. Antibodies were incubated on the same blot after membrane stripping and re-blotting. Blots were cut prior to hybridization. α-tubulin was used as a load control. Each of the eight lanes contains 1 × 107 spermatozoa from different animals. At least three independent experiments with different animals were performed.
Figure 7
Figure 7
IVF outcomes. Three different groups were subjected to IVF assay: CTRL, CT 0.1 and MoS2/CT 0.1 ppm. Data are expressed as percentages, showing the number of fertilized oocytes, the number of polyspermic oocytes and the number of spermatozoa per polyspermic oocyte, comparing the groups of spermatozoa capacitated in the presence of MoS2/CT and CT (0.1 ppm) to the control group. Data were analyzed using Dunnett’s test. * p < 0.05 versus control. Four independent experiments were performed.
Figure 8
Figure 8
Experimental design. Spermatozoa were exposed to MoS2 functionalized with catechin at different concentrations (10, 1, 0.1 ppm), catechin alone at the same concentrations (10, 1, 0.1 ppm) for 1.5 h in capacitation medium, a control group (CTRL) was maintained. Different sperm capacitation events were analyzed: acrosome damage, membrane fluidity, mitochondrial activity, intracellular calcium concentration, biochemical phosphorylation patterns and IVF assays.
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