268 ROLE OF ATPase MOTOR CYTOPLASMIC DYNEIN AND PRIMARY CILIA IN TESTICULAR MORPHOGENESIS
Bibliographic record
Abstract
In vertebrates, the primary cilium is a nearly ubiquitous organelle present in somatic cells, but little is known about its function in the male gonad. We investigated the role of primary cilia in testis cells using in vitro formation of seminiferous tubules and in vitro culture of testicular somatic cells by inhibiting the primary cilium with CiliobrevinD, a cell-permeable, reversible chemical modulator that inhibits the major component of the organelle: ATPase motor cytoplasmic dynein. We analysed in vitro cultures for the presence of primary cilia and the activation of hedgehog signalling through translocation of Gli2 to the nuclei; in vitro tubule formation was evaluated by length and width of tubules formed. Methods: testicular cells were harvested from neonatal pigs by 2-step enzymatic digestion. Cells (50 × 106 mL–1) were plated on 100 mm Petri dishes in 15 mL of DMEM + 5% FBS + 50 U of penicillin and incubated at 37°C in 5% CO2 in air overnight, cells remaining in suspension and those slightly attached were removed and the somatic cells attached were trypsinized to obtain a single cell suspension, and then submitted to two different protocols: in vitro culture (A) or in vitro tubule formation (B), n = 5 replicates each. For A, somatic cells were replated on coverslips in 24-well plates and cultured in serum free media for 48 h, then for the treated group, 10 mM of CiliobrevinD was added for 24 h, attached cells from control and treated groups were fixed in 4% PFA and characterised by immunocytochemistry for ARL13B, Vimentin, and Gli2. For B: 1 × 106 cells were added to 24-well plates coated with 1 : 1 diluted Matrigel, the control group was kept in serum free media and to the treated group was added 20 mM CiliobrevinD at Day 0. Results: A) primary cilia were present in 89.3 ± 2.3% of cells cultured in serum-free media for the control group and Gli2 was located in the nuclei of 90.2 ± 1.2% of cells; in the CiliobrevinD-treated group the percentage of primary cilia decreased (P < 0.05) to 3.1 ± 2.5% and nuclear Gli2 to 3.9 ± 0.7; B) tubules formed in the control group were significantly longer and wider than the ones formed when CiliobrevinD was added (9.91 ± 0.35 v. 5.540 ± 1.08 mm and 339.8 ± 55.78 v. 127.2 ± 11.9 µm, respectively, P < 0.05 by Student's t-test). In conclusion, the inhibition of ATPase motor cytoplasmic dynein perturbs formation of primary cilia in testicular somatic cells, blocks Hedgehog signalling, and impairs in vitro tubule formation. Therefore, primary cilia on testicular somatic cells appear to be essential for testicular morphogenesis. Research was supported by 5 R01 OD016575-13.
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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 | 0.000 | 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) | 0.000 | 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".