6 Development of a new method to label pig oocytes with nanoparticles to be applied in assisted reproductive techniques
G. Garrappa A B , G. Yarza-Muñoz C D , C. Luongo A , F. A. García-Vázquez A C and M. Jiménez-Movilla C DA Department of Physiology, Faculty of Veterinary Medicine, Campus Mare Nostrum, University of Murcia, Murcia, Spain
B Institute of Animal Research of the Semi-Arid Chaco, National Institute of Agricultural Technology, Tucumán, Argentina
C Murcian Institute of Biosanitary Research, Arrixaca, Murcia, Spain
D Department of Cell Biology and Histology, Faculty of Medicine, Campus Mare Nostrum, University of Murcia, Murcia, Spain
Reproduction, Fertility and Development 35(2) 127-128 https://doi.org/10.1071/RDv35n2Ab6
Published: 5 December 2022
© 2023 The Author(s) (or their employer(s)). Published by CSIRO Publishing on behalf of the IETS
Similar to other cellular systems, the capability to label oocytes with nanoparticles (NPs) could generate a breakthrough in assisted-reproduction techniques (ARTs), such as new strategies for the manipulation of oocytes or molecule/drug-carrier systems. We have developed a new method of NP binding to the zona pellucida (ZP) of mature oocytes using the intrinsic and specific ability of oviductin (OVGP1 [Ov]) as a link. The aims of this work were (1) to analyse the successful binding of NPs and Ov over time, (2) to evaluate the NPOv distribution around the ZP surface, and (3) to evaluate the harmlessness of the method in ARTs. Carboxyl-modified NPs (Estapor®) (diameter of ∼0.365 μm) were used in this study. Recombinant truncated porcine Ov was expressed and purified as described by Algarra et al. (2016). For conjugation, 10 µL (concentration/solid content [%] = 0.1) of NPs were incubated with Ov (6 µg; ∼75 kDa). Sequential electrophoresis and western blots (WBs) with a polyclonal anti-Ov antibody (Abcam) were performed to test the conjugation stability over time. WB results indicated that Ov was successfully conjugated to NPs, showing a stable binding of NPOv up to 31 days. Further, NPOv were co-incubated with mature porcine oocytes, showing a binding efficiency higher than 80%. The distribution of NPOv around ZP surface (%, QWin software, Leica Microsystems Ltd.) was evaluated at 0.5 h, 1 h, and 6 h of incubation, with a distribution ranging from ∼20% to ∼40% of the surface ZP area from the beginning of incubation (0.5 h) (n = 300). Finally, the harmlessness of NPOv bound to the ZP was tested by in vitro fertilisation (IVF) and embryo culture (EC). Three experimental groups were established: (1) control: oocytes not incubated with NPOv (n = 154 IVF; n = 493 EC); (2) NPOv (+): oocytes incubated with NPOv (n = 200 IVF; n = 401 EC); (3) NPOv (−): oocytes incubated with NPOv but removed after mechanical separation before IVF (n = 177 IVF; n = 404 EC). Statistical analysis was performed using IBM SPSS v23 (SPSS Inc.). A Pearson chi-squared test was used to analyse percentage data. For EC parameters, Kruskal-Wallis tests were used after a normality test (Kolmogorov-Smirnov). The IVF results showed a similar output (penetration, monospermy, efficiency; %) between groups. Also, EC parameters (cleavage [%], blastocyst rate [%], and blastocyst quality [diameter-µm and number of blastocyst cells]) had no differences between groups. In conclusion, the feasibility to label oocytes with NPOv was successfully demonstrated without no impact on ART output.
Research was supported by the Fundación Seneca-Agencia de Ciencia y Tecnología de la Región de Murcia “Ayudas a la realización de proyectos para el desarrollo de investigación científica y técnica por grupos competitivos 2018” (20887/PI/18) and “Fundación Séneca-Prueba de concepto” (21637/PDC/21) Spain, and by projects PID 2020-114109GB-I00 and PID2019-106380RB-I00, both funded by MCIN/AEI/10.13039/501100011033.
