Src Is a Signaling Molecule in Ouabain-Na+K+-ATPase-Stimulated Signaling Pathway During Bull Sperm Capacitation.
Bibliographic record
Abstract
Na+K+-ATPase acts as a signaling molecule when ouabain binds, inducing dose-dependent protein tyrosine phosphorylation (p-tyr) during sperm capacitation. Src is present in head membranes of bull sperm and its expression alters during ouabain-induced p-tyr, so this study investigated Src's role in capacitation. Fresh bull sperm (n=3-6 ejaculates per trial) were pre-incubated with or without Src inhibitors herbimycin A (inhibits kinase) or SU6656 (prevents ATP binding to Src), followed by incubation in the presence or absence of ouabain or heparin. Extracted proteins were assessed by western blotting, band intensity measured, and quantities of each protein band compared by ANOVA. Both protein p-tyr and LPC-induced acrosome reaction increased (p<0.05) upon exposure of sperm to ouabain (242, 176, 81, 49 kDa) or heparin (176kDa). Inhibition of Src activity by herbimycin or SU6656 antagonized (P<0.05) various ouabain- and heparin-induced p-tyr, and herbimycin inhibited the ouabain-induced acrosome reaction, confirming Src's integral role in capacitation. Herbimycin actually reduced some Src itself (p<0.05) during ouabain- or heparin-induced capacitation incubation. However, since Src was activated by auto-phosphorylation in the presence of ouabain or heparin, it was of greater interest that herbimycin significantly reduced ouabain-enhanced Src autophosphorylation of the 59kDa phospho-Src and the other ouabain- (166-31kDa) and heparin-induced phospho-Src (130, 40kDa) (P<0.05) implying that those are coupling proteins or substrates of Src. Src can also be activated by serine phosphorylation (p-ser) and both ouabain and heparin significantly stimulated p-ser of various sperm proteins (254-18kDa). SU6656 inhibited ouabain-induced p-ser at 40 and 32kDa, but heparin overcame this inhibition, implying that the Na+K+-ATPase pathway requires ATP binding to Src. In contrast, heparin is less ATP sensitive, perhaps primarily stimulating the cAMP/PKA pathway. Ouabain appears to induce capacitation through the ERK pathway, since phospho-ERK1/2 (Thr202/Tyr204, Thr185/Tyr187) during capacitation were ouabain-sensitive. SU6656 antagonized ouabain-enhanced phosphorylation of ERK2, and other ERKs (39-17kDa) at 2h and herbimycin inhibited ERK1 in control sperm incubated for 5h. Heparin overcame the Src inhibitors' effect on ERKs possibly through the heparin-induced cAMP/PKA pathway. In summary, this study demonstrates Src's activation and involvement in tyrosine phosphorylation-dependent signaling through involving and activating ERK1/2 (potentially ERK2) in ouabain-induced bull sperm capacitation or acrosome reaction. The ouabain-sensitive Na+K+-ATPase signaling cascade is unique and distinct from that of heparin, but this novel research does identify for first time that Src cross talks with the heparin signaling pathway to induce capacitation. (poster)
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How this classification was reachedexpand
Full frame distilled prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. Learned from the 10,348 direct Codex labels and 10,348 direct Gemma labels. Candidate is the union of thresholded teacher heads; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels or direct frontier model labels.
Codex and Gemma teacher scores by category
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.001 | 0.001 |
| Meta-epidemiology (narrow) | 0.000 | 0.000 |
| Meta-epidemiology (broad) | 0.000 | 0.000 |
| Bibliometrics | 0.000 | 0.000 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.000 | 0.000 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.000 | 0.001 |
| Insufficient payload (model declined to judge) | 0.000 | 0.000 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedMachine predicted; a candidate call from one teacher head, not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".