Nitric Oxide Synthase Activity Is Critical for the LH-Induced Ovulatory Cascade in Bovine Granulosa Cells.
Bibliographic record
Abstract
Fertility in dairy and beef cows is negatively affected by stressors such as lamness and high milk yield, and one site of action of these stressors is the process of ovulation. Rupture of the follicle is initiated by the preovulatory surge of LH, which stimulates granulosa cell release of the EGF-like ligands epiregulin (EREG) and amphiregulin (AREG), which activate EGF receptors in granulosa and cumulus cells to stimulate prostaglandin synthase 2 (PTGS2, also known as COX2) expression, prostaglandin secretion and downstream expression of cumulus expansion genes. Various factors have been implicated in the control of this process, including endogenous nitric oxide (NO). In rodents, NO has been associated either positively or negatively with ovulation. The objectives of the present study were to determine (1) if endogenous NO is critical for expression of genes critical for the ovulatory cascade in bovine granulosa cells, (2) and to determine if nitric oxide synthase (NOS) enzymes are regulated by LH. We employed a short-term bovine granulosa cell (GC) culture system in which AREG, EREG and PTGS2 mRNA levels are acutely upregulated by LH. The first series of experiments was performed to determine the importance of NO in the preovulatory cascade. Pretreatment of cells with a NOS inhibitor, L-NAME, effectively blocked the effect of LH on EREG, AREG and PTGS2 mRNA abundance at 6 hours post-LH, and on PTGS2 protein levels at 12 hours post LH challenge (P<0.05). To determine if NO is necessary for downstream, EGFR-dependent signaling, cells were pretreated with L-NAME and challenged with EGF; EGF alone increased mRNA encoding EREG within 1 h, and this was inhibited by pretreatment with L-NAME. EGF alone increased abundance of mRNA encoding AREG and the nuclear orphan receptors NR5A1 and NR5A2 after 8 h, and L-NAME inhibited the effect of EGF. A second series of experiments was performed to determine the regulation of NOS mRNA abundance in bovine GC. Time- and dose-response experiments demonstrated that LH had a significant stimulatory effect on NOS3 (eNOS) mRNA abundance (P<0.05), although transcriptional regulation of NOS3 was not observed until 12 h of LH treatment, much later than that of EREG mRNA (1-3 h). Messenger RNA encoding NOS2 (iNOS) was detected at very low levels and was not investigated further. Further experiments demonstrated that EGF stimulated NOS3 mRNA levels in a dose-dependent manner (P<0.05). As some studies report a positive feedback loop between prostaglandin E2 (PGE2) and EGF-like factors in cumulus cells, we tested whether PGE2 stimulates NOS3 mRNA levels in mural granulosa cells. The results confirmed that PGE2 stimulates EREG and also NOS3 mRNA levels (P<0.05). Moreover, when cells were pretreated with indomethacin, a PTGS2 inhibitor, EGF was not capable of inducing NOS3 and EREG mRNA levels (P<0.05), suggesting that the transcriptional regulation of NOS3 is prostaglandin dependent. To our knowledge, this is the first study indicating an interaction between nitric oxide and EGF-like factors during the preovulatory period. Taken together, these results suggest that endogenous NOS activity is critical for LH-induced ovulatory cascade in granulosa cells and NO may be essential for ovulation in cattle. Supported by FRQNT and NSERC Canada.
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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 | 0.000 |
| 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".