Phosphoproteomic profiling of Siberian sturgeon (<i>Acipenser baerii</i>, Brandt, 1869) sperm: insights into cryopreservation-induced changes with DMSO and methanol

Semen cryopreservation is a crucial technique in aquaculture, enabling the long-term preservation of genetic material from endangered species such as the Siberian sturgeon (Acipenser baerii, Brandt, 1869). While methanol (MeOH) and dimethyl sulfoxide (DMSO) similarly affect sturgeon semen quality post-thaw, DMSO significantly reduces hatching rates. Given our prior findings on cryoprotectant-specific differences in the sturgeon sperm proteome, we investigated the molecular basis of DMSO-induced dysfunction using label-free quantitative phosphoproteomics. In fresh sperm, 1649 phosphorylation sites were identified on 736 proteins, forming the most extensive database of phosphorylated sperm proteins in fish. Functional enrichment analysis showed these phosphoproteins were primarily involved in cilium organization, energy metabolism, spermatogenesis, transmembrane transport, vesicle-mediated transport and sperm binding to the zona pellucida, predominantly localizing to cytoplasm, cilia and mitochondria. Cryopreservation profoundly altered the phosphoproteome, with 269 phosphosites showing differential phosphorylation between fresh and cryopreserved samples. DMSO induced phosphorylation changes at 231 sites across 114 proteins, whereas MeOH affected 260 sites across 127 proteins. Distinct phosphorylation patterns were identified for each cryoprotectant, with shared alterations involving cilium assembly, glycolysis, phosphatidylinositol metabolism, chromatin structure and ion channel regulation associated with impaired motility, membrane destabilization and reduced acrosomal integrity. DMSO-specific changes disrupted nucleoporin structure and centriole organization while inhibiting Rho signaling, crucial for acrosomal reaction and actin filament dynamics, which likely contribute to low hatching outcomes. In contrast, MeOH uniquely enhanced AMPK signaling, promoting mitochondrial ATP buffering and energy homeostasis. This study provides the first comprehensive phosphoproteomic analysis of sturgeon sperm, linking DMSO-induced disruptions to impaired fertilization outcomes.