Maladaptation of trout spermatozoa to fresh water is related to oxidative stress and proteome changes

in Reproduction
Correspondence should be addressed to J Nynca; Email: j.nynca@pan.olsztyn.pl

Rainbow trout sperm are ‘maladapted’ to freshwater spawning, resulting in shorter duration of sperm motility in fresh water compared to buffered saline solution. We hypothesized that different sperm motility-activating media have various effects on sperm motility characteristics and oxidative stress, as well as on the protein profiles of rainbow trout sperm. We designed an experimental model for activation of rainbow trout sperm motility in different osmotic conditions: (i) isosmotic and (ii) hypoosmotic. Spermatozoa activation with hypoosmotic solution was associated with lower values for sperm motility parameters (52%) and an induced increase in ROS level (19.4%) in comparison to isosmotic activation with isosmotic solution (67 and 9.5% for sperm motility and ROS, respectively). Hypoosmotic activation resulted in a higher number of differentially abundant sperm proteins (out of which 50 were identified) compared to isosmotic conditions, where only two spots of protein disulfide-isomerase 6 were changed in abundance. The proteins are mainly involved in the TCA cycle, tight and gap junction signaling, Sertoli cell–Sertoli cell junction signaling and asparagine degradation. Our results, for the first time, indicate that during hypoosmotic activation of sperm motility, osmotic stress triggers oxidative stress and disturbances mostly to structural proteins and metabolic enzymes. Our results strongly suggest that comparative physiological and biochemical analysis of rainbow trout sperm characteristics in isosmotic and hypoosmotic conditions could be a useful model for studying the mechanism of sperm activation in salmonid fish.

Downloadable materials

  • Supplementary Table 1. 2D DIGE analysis of immobilized and activated rainbow trout spermatozoa. Representative protein spots correspond(4, 11, 16, 29, 30, 46, 48, 52) to proteins identified from 2D DIGE which changed in abundance after activation of immobilized sperm in hypoosmotic conditions. Numbered protein spots (44, 47) correspond to proteins identified from 2D DIGE which changed in abundance after activation of immobilized sperm in isosmotic conditions.
  • SupplementaryTable 2. Proteins that showed a significant change in abundance in activated sperm compared to immobilized sperm of rainbow trout.
  • Suplementary Table 3. Gene ontology of proteins with larger changes in protein expression after hypoosmotic activation of sperm

 

    Society for Reproduction and Fertility

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    Effect of isosmotic and hypoosmotic activation on sperm motility parameters of rainbow trout (n = 7). (A) Percentage of sperm motility, (B) VCL (curvilinear line velocity), (C) VSL (straight line velocity), (D) VAP (average path velocity), (E) ALH (amplitude of lateral head displacement), (F) LIN (linearity). Results are expressed as means ± s.d. Asterisk indicates significant differences between parameters for activated sperm in isosmotic and hypoosmotic conditions (*P < 0.05, **P < 0.01; ***P < 0.001).

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    Effect of sperm activation on viability (A) and ROS-positive cell content of rainbow trout semen (B) (n = 7). Results are expressed as means ± s.d. Asterisk indicates significant differences between ROS-positive cell content for immobilized and activated sperm in isosmotic and hypoosmotic conditions (P < 0.05).

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    2D DIGE analysis of immobilized and activated rainbow trout spermatozoa. Numbered protein spots (1–61) correspond to proteins identified from 2D DIGE which changed in abundance after activation of immobilized sperm (A) in hypoosmotic conditions (B). Numbered protein spots (44, 47) correspond to proteins identified from 2D DIGE which changed in abundance after activation of immobilized sperm (C) in isosmotic conditions (D). The nonlinear pH range of the first-dimension IPG strip is indicated along the top of the gel, acidic pH to the left. The MW (relative molecular mass) scale can be used to estimate the molecular weights of the separated proteins.

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    Gene ontology annotations for rainbow trout sperm proteins according to the GO term ‘molecular function’.

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    Cellular movement as signaling pathways associated with rainbow trout sperm motility activation. Red denotes proteins that increased after sperm motility activation, while green denotes proteins that decreased after sperm motility activation. Color intensity indicates expression level. The proteins filled in ‘red’ and ‘green’ are the ones identified in our study while the remaining ones are the proteins in the Ingenuity Knowledge Base that interact with ours (unfilled were added by IPA software to complete the pathway). Lines indicate interactions, with the arrowheads indicating directionality. Absence of arrowheads refers to a binding interaction. Dotted line indicates an inferred or indirect interaction. Nodes are represented with various shapes to distinguish the functional class of the molecule: ◊ enzyme, □ G-protein coupled receptor, □ growth factor, ▼ kinase, ◊ peptidase, transcription regulator, ○ group or complex, ○ other.

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    Substructures comprising sperm flagellar axoneme with marked identified proteins changed after activation in hypoosmotic conditions. Proteins that show marked changes (−2> and >2) in expression are shown in red. Proteins that show minor changes (−2< and <2) in expression are shown in green.

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