Localization of SRC in Fresh and Capacitated Bull Spermatozoa.
Bibliographic record
Abstract
Capacitation is a molecular transformation process which enables spermatozoa to undergo the acrosome reaction, and be capable of fertilization. Protein tyrosine phosphorylation is an indicator of capacitation. SRC, a non-receptor protein tyrosine kinase, has been implicated in both protein tyrosine phosphorylation in human sperm capacitation and signaling cascades associated with Na+K+ATPase in somatic cells. Na+K+ATPase acts as a signaling molecule when its specific ligand ouabain binds and activates cytoplasmic protein kinases. We have shown that ouabain induces dose-dependent protein tyrosine phosphorylation during capacitation in bull spermatozoa. To test the hypothesis that Na+K+ATPase activates SRC through a ouabain-induced signaling pathway to promote capacitation in bovine sperm, this study's objectives were to identify and localize SRC in bull sperm and to investigate the effect of ouabain-induced capacitation on SRC. Immunocytochemistry with anti-SRC (rabbit polyclonal anti-SRC, n=3 bulls) detected no SRC in intact unfixed sperm, but sperm with compromised integrity of the plasma membrane (by fixation in 1% formaldehyde or by methanol permeabilization) localized SRC to the acrosomal region, and some permeabilized sperm also showed punctuate localization of SRC in the equatorial region. Therefore SRC does exist in bull sperm, either on the internal surface of the head plasma membrane, or on the outer acrosomal membrane. Western immunoblotting with the same antibody confirmed SRC presence, detecting a 60 kDa band in fresh bull sperm (n=3 bulls) similar to that of the positive control (HeLa Cell Lysate), with bull sperm also showing four higher kDa SRC-positive bands. Sperm were then incubated under capacitating conditions (SP-TALP ± 10 µg/ml heparin or 50 mM ouabain, 5 hours; 37°C; 5% CO2,; n=4 bulls) and identical gels were assessed by Western immunoblotting for protein tyrosine phosphorylation (to evaluate capacitation) and for SRC content. Both heparin and ouabain stimulated tyrosine phosphorylation, confirming capacitation, although ouabain caused significantly more phosphorylation in all affected proteins, and uniquely phosphorylated a 254 kDa protein. The differences in protein tyrosine phosphorylation between heparin and ouabain support our earlier suggestion that these two agents induce capacitation via different mechanisms. Incubation in capacitating conditions affected SRC. All sperm incubated for 5h contained a 24 kDa SRC not present at 0 hours, which is likely a fragment of SRC. Control sperm at 5 hours (0 heparin, 0 ouabain) had more 60 kDa SRC than 0 hour control (P<0.05); inclusion of heparin or ouabain rendered the increase non-significant. These findings demonstrate that SRC is present in bull sperm, primarily located in the anterior acrosomal region, that SRC expression is altered during capacitation and that heparin and ouabain induce capacitation via different pathways that may involve SRC. (poster)
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How this classification was reachedexpand
Full frame machine prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. The Gemma side is a direct model label for every work in the frame, read from the title-only record. The Codex side is a classifier learned from the 10,348 direct Codex labels and calibrated to design-weighted sample rates; fields without enough sample support carry no Codex call. Candidate is the union of the two sides; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels.
Distilled classifier scores by category (both heads)
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.000 | 0.000 |
| 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 | 0.000 |
| Insufficient payload (model declined to judge) | 0.003 | 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 source (direct Gemma or distilled Codex), 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".