In Stallion Spermatozoa, Superoxide Dismutase (Cu–Zn) (SOD1) and the Aldo-Keto-Reductase Family 1 Member b (AKR1B1) Are the Proteins Most Significantly Reduced by Cryopreservation

Abstract

Although cryopreservation is widely used in animal breeding, the technique is still suboptimal. The population of spermatozoa surviving the procedure experiences changes attributed to alteration in their redox regulation. In order to expand our knowledge regarding this particular aspect, the proteome in fresh and frozen thawed aliquots of equine spermatozoa was studied to identify the proteins most severely affected by the procedure. If alteration of redox regulation is a major factor explaining cryodamage, proteins participating in redox regulation should be principally affected. Using a split sample design, 30 ejaculates from 10 different stallions were analyzed as fresh spermatozoa, and another aliquot from the same ejaculate was analyzed as a frozen thawed sample. The proteome was studied under both conditions using UHPLC-MS/MS and bioinformatic analysis conducted to identify discriminant variables between both conditions. Data are available through the ProteomeXchange Consortium with identifier PXD022236. The proteins most significantly reduced were Aldo-keto reductase family 1 member B ( p = 2.2 × 10 –17 ) and Superoxide dismutase (Cu–Zn) ( p = 4.7 × 10 –14 ). This is the first time that SOD1 has been identified as a discriminating variable using bioinformatic analysis, where it was one of the most highly significantly different proteins seen between fresh and frozen thawed semen. This finding strongly supports the theory that alteration in redox regulation and oxidative stress is a major factor involved in cryodamage and suggests that control of redox regulation should be a major target to improve current cryopreservation procedures.

Aldose Reductase B1 in Pig Sperm Is Related to Their Function and Fertilizing Ability

“…The presence or activity of AKR1B1 in ejaculated sperm has been demonstrated in equine (26), bovine (17,25) and porcine species (24). Similarly, the results of the peptide competition assay of the Western Blot showed a specific double-band pattern at 36 kDa and ~80 kDa in all ejaculated and epididymal sperm samples, thus suggesting that different forms of this protein could be physiologically present in pig sperm.…”

“…Although aldose reductases have been identified in bovine (17,25), equine (26) and porcine sperm (24), the origin of this protein in mature sperm cells is still unknown. In this regard, while translation during spermatogenesis could be a possibility, no previous study has confirmed if the relative content of AKR1B1 is higher in ejaculated than in epididymal sperm.…”

Sperm cryopreservation impacts the early development of equine embryos by downregulating specific transcription factors

“…Some of these proteins may be critical for gene expression regulation after embryo genome activation; the integrative analysis of the sperm, oocyte and blastocyst proteomes and transcriptomes revealed a set of embryo proteins with an exclusive paternal origin, some of which are crucial for correct embryogenesis and, possibly, for modulation of the offspring phenotype (Castillo et al 2018). In relation to this, cryopreservation is known to impact the sperm proteome (Bogle et al 2017; Martin-Cano et al 2020b; Gaitskell-Phillips et al 2021b; Gaitskell-Phillips et al 2021a). Taking all these findings in account, is likely that embryos produced with cryopreserved spermatozoa may experience alterations resulting of changes induced by cryopreservation in the proteome of the spermatozoa.…”

“…This mitochondrial damage causes redox imbalance and accelerates different forms of sperm death, including necrotic, apoptotic (Aitken and Koppers 2011; Aitken and Baker 2013) and probably ferroptotic sperm mortality (Ortiz-Rodriguez et al 2020). In addition to protein degradation during cryopreservation, redox deregulation may induce oxidative damage to proteins causing notable modifications of the sperm proteome (Bogle et al 2017; Martin-Cano et al 2020a; Gaitskell-Phillips et al 2021b). All these modifications may affect proteins participating in the regulation and or survival of the early embryo (Castillo et al 2018).…”

Dataset of the sperm proteome of stallions with different motility

“…The same protocol as described in [3] , [4] , [5] , [6] , [7] , [8] , [9] was used. Stallion ejaculates were washed in PBS (600gx 10’), then the pellet formed by the spermatozoa was frozen immediately in liquid nitrogen and kept frozen at -80 °C.…”