Cryopreservation of highly extended pig spermatozoa remodels its proteome and counteracts polyspermic fertilization in vitro

Inmaculada Parrilla; Josep M Cambra; Cristina Cuello; Heriberto Rodriguez-Martinez; Maria A Gil; Emilio A Martinez

doi:10.1111/andr.13575

Cryopreservation of highly extended pig spermatozoa remodels its proteome and counteracts polyspermic fertilization in vitro

Andrology. 2023 Dec 22. doi: 10.1111/andr.13575. Online ahead of print.

Authors

Inmaculada Parrilla^{1

2}, Josep M Cambra^{1

2

3}, Cristina Cuello^{1

2}, Heriberto Rodriguez-Martinez⁴, Maria A Gil^{1

2}, Emilio A Martinez^{1

2}

Affiliations

¹ Department of Medicine and Animal Surgery, International Excellence Campus for Higher Education and Research "Campus Mare Nostrum,", University of Murcia, Murcia, Spain.
² Department of Medicine and Animal Surgery, Institute for Biomedical Research of Murcia (IMIB-Pascual Parrilla), Murcia, Spain.
³ Large Animal Models in Cardiovascular Research, Internal Medical Department I, TU Munich, Technical University of Munich, Munich, Germany.
⁴ Department of Biomedical & Clinical Sciences (BKV), BKH/Obstetrics & Gynecology, Linköping University, Linköping, Sweden.

PMID: 38131448
DOI: 10.1111/andr.13575

Abstract

Background: Currently, high polyspermy remains a significant obstacle to achieving optimal efficiency in in vitro fertilization (IVF) and in vitro embryo production (IVP) systems in pigs. Developing strategies that would prevent polyspermy is essential in overcoming this challenge and maximizing the potential of this reproductive biotechnology. Previous results have demonstrated that using boar spermatozoa subjected to a high-extension and reconcentration procedure and then cryopreserved resulted in significant improvements in IVF/IVP systems with high rates of monospermy and penetration.

Objective: The aim of the present study was to unveil the molecular mechanisms that may underlie the changes in fertilization patterns exhibited by highly extended and cryopreserved boar spermatozoa.

Materials and methods: To achieve this goal, we used quantitative proteomic analysis (LC-ESI-MS/MS SWATH) to identify differentially abundant proteins (DAPs) between highly extended (HE) and conventionally (control; CT) cryopreserved boar spermatozoa. Prior to the analysis, we evaluated the in vitro post-thawing fertilizing ability of the sperm samples. The results demonstrated a remarkable improvement in monospermy and IVF efficiency when using HE spermatozoa in IVF compared with CT spermatozoa.

Results: At the proteomic level, the combination of high-extension and cryopreservation had a significant impact on the frozen-thawed sperm proteome. A total of 45 proteins (24 downregulated and 21 upregulated) were identified as DAPs (FC > 1 or ≤1; p < 0.05) when compared with CT spermatozoa. Some of these proteins were primarily linked to metabolic processes and the structural composition of sperm cells. The dysregulation of these proteins may have a direct or indirect effect on essential sperm functions and significantly affect spermatozoa-oocyte interaction and, therefore, the sperm fertilization profile under in vitro conditions. While these findings are promising, further research is necessary to comprehend how the disturbance of specific proteins affects sperm fertilization ability.

Keywords: IVF; cryopreservation; high dilution; monospermy; porcine; spermatozoa.

Abstract

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