Freezing-Thawing Procedures Remodel the Proteome of Ram Sperm before and after In Vitro Capacitation

Abstract

Mammalian sperm must undergo a set of structural and functional changes collectively termed as capacitation to ensure a successful oocyte fertilization. However, capacitation can be compromised by cryopreservation procedures, which alter the proteome and longevity of sperm. To date, how the protein changes induced by cryopreservation could affect the acquisition of sperm fertilizing potential remains unexplored. The present study investigated the protein profile of ram sperm during in vitro capacitation before and after cryopreservation to elucidate the impact of cryopreservation on sperm capacitation at a molecular level. Fresh and cryopreserved ram sperm were incubated under capacitating (CAP) and non-capacitating (NC) conditions for 240 min. The sperm proteome of these four treatments was analyzed and compared at different incubation times using reverse phase liquid chromatography coupled to mass spectrometry (RP-LC-MS/MS). The comparison between fresh and cryopreserved sperm suggested that cryopreservation facilitated an apoptosis-stress response and redox process, while the comparison between sperm incubated in CAP and NC conditions showed that capacitation increased those biological processes associated with signaling, metabolism, motility, and reproductive processes. In addition, 14 proteins related to mitochondrial activity, sperm motility, oocyte recognition, signaling, spermatogenesis, and the apoptosis-stress response underwent significant changes in abundance over time when fresh and cryopreserved sperm incubated in CAP and NC conditions were compared. Our results indicate that disturbances in a ram sperm proteome after cryopreservation may alter the quality of sperm and its specific machinery to sustain capacitation under in vitro conditions.

Keywords: capacitation; cryopreservation; proteome; ram sperm.

Figure 1

Venn diagram showing the distribution of ram sperm proteins between different treatments. F CAP and F NC represents the proteins detected in fresh sperm incubated under capacitating and non-capacitating conditions for all times, respectively; C CAP and C NC represent the proteins detected in cryopreserved sperm incubated under the same conditions for all times.

Figure 2

Optical densities (OD) of CAPZA2 in fresh and cryopreserved sperm after 15 min of incubation in NC or CAP conditions assessed using ELISA. * p < 0.05.

Figure 3

Classification of the identified proteins in fresh and cryopreserved ram sperm incubated in both media (CAP and NC) over time, according to their (A) molecular function, (B) subcellular location, and (C) biological process using Blast2GO and UniProtKB.

Figure 3

Classification of the identified proteins in fresh and cryopreserved ram sperm incubated in both media (CAP and NC) over time, according to their (A) molecular function, (B) subcellular location, and (C) biological process using Blast2GO and UniProtKB.

Figure 4

Biological process categorization of ram sperm proteins identified in different treatments. (A) Distribution of those proteins identified in fresh (outer circle) or in cryopreserved sperm (inner circle). (B) Distribution of those proteins present in sperm incubated in CAP (outer circle) or NC conditions (inner circle).

Figure 5

Different representations of an apoptosis-stress response and other biological processes directly or indirectly involved in reproduction between treatments. (A) Comparison of fresh versus cryopreserved ram sperm regardless of the incubation time and media. (B) Comparison of sperm incubated in CAP versus NC conditions, regardless of the incubation time and treatment (fresh or cryopreserved sperm). Peptide spectral matches (PSMs) of those proteins involved in each process were normalized against the total number of PSMs and compared using the Bonferroni test, * p < 0.05.