Effects of cortisol on LPS- and hydrogen peroxide-challenged bovine cumulus-oocyte complexes during in vitro maturation

Ovulation is a complex inflammatory response that creates an oxidative environment within the follicle. Additionally, bacterial infections that increase during the perinatal period trigger inflammation via lipopolysaccharide (LPS), an endotoxin. Follicles are routinely exposed to both endogenous and exogenous inflammation and the accompanying reactive oxygen species (ROS). LPS and ROS are known to disrupt various reproductive processes, such as follicular and oocyte maturation, and alleviating these stresses may improve reproductive efficiency. In bovine cumulus-oocyte complexes (COCs), local production of cortisol from cortisone via 11β-hydroxysteroid dehydrogenase type 1 (HSD11B1) significantly increases during maturation. Meanwhile, cortisol derived from the adrenal cortex is consistently present in follicular fluid. Bovine COCs express glucocorticoid receptors (GR) regardless of maturation stage, suggesting that both locally produced and endocrine delivered cortisol regulates COC function via GR. Cortisol is known for its anti-inflammatory and antioxidant properties in various tissues and may protect COCs from stress caused by LPS and ROS. This study employed a bovine in vitro maturation (IVM) system to examine the inhibitory effects and underlying mechanisms of both locally produced and externally supplemented cortisol on oocyte maturation impaired by exposure to LPS and ROS, across three experimental chapters. In the Chapter 3, the effects of cortisol on oocyte maturation under LPS exposure was examined. COCs were aspirated from 2–5 mm follicles of bovine ovaries obtained from a local slaughterhouse and subjected to IVM for 21 hours in the presence or absence of LPS (0.001–1 μg/ml), cortisol (0.1–10 μM), cortisone (0.1 μM; inactive glucocorticoid and cortisol precursor), and RU486 (10 μM; antagonist of GR and progesterone receptor (PR). LPS significantly reduced oocyte maturation (MII rate) without affecting MI rate. This inhibitory effect of LPS (1 μg/ml) was suppressed by the addition of cortisol (0.1 μM). During IVM, approximately 10% of added cortisone was converted to cortisol (about 0.01 μM) by the COCs. Locally produced cortisol suppressed the inhibitory effects of LPS. The addition of RU486 blocked the effects of cortisol, indicating that cortisol acts via GR. On the other hand, LPS did not affect cortisol production in COCs. Taken together, these results suggest that cortisol functions as a potent anti-inflammatory agent, mitigating the harmful effects of LPS on oocyte maturation, and that COCs protect oocytes by locally producing cortisol. As oocyte maturation progresses, the synthesis of progesterone (P4) and cortisol in COCs increases. Previous experiments conducted in our laboratory have reported that P4 induces the expression of HSD11B1, thereby enhancing cortisol production. Furthermore, P4 is suggested to be essential for bovine oocyte maturation and to possess anti-inflammatory properties, potentially working in conjunction with cortisol to mitigate the inhibitory effects of LPS. Chapter 4 investigates this hypothesis. COCs were subjected to IVM for 21 hours in the presence or absence of LPS (0.001–1 μg/ml), RU486 (10 μM), trilostane (10 μM; a P4 synthesis inhibitor), nomegestrol acetate (0.001–1 μM, NA; a synthetic progesterone), and cortisone (0.1 μM). Trilostane suppressed P4 production in COC to below the detection limit. RU486 and trilostane inhibited oocyte maturation regardless of LPS presence. LPS inhibited oocyte maturation irrespective of the presence of P4 or NA. Trilostane suppressed HSD11B1 expression and local cortisol production. In the absence of P4, cortisol could not suppress the inhibitory effect of LPS on oocyte maturation. On the other hand, NA reversed the trilostane-induced suppression of HSD11B1 expression, regardless of dosage. On the other hand, LPS did not affect P4 production in COCs, nor did it influence cumulus expansion. These results indicate that P4 is essential for bovine oocyte maturation. Although P4 alone cannot suppress the effects of LPS, it protects oocytes by encouraging local cortisol synthesis Chapter 5 examined the role of cortisol in oocyte maturation under oxidative stress induced by hydrogen peroxide (H2O2). H2O2 is a relatively stable reactive oxygen species (ROS) produced in various pathological and physiological processes. In this study, bovine COCs were subjected to IVM in the presence or absence of H2O2 (12.5, 50, 200 μM), cortisol (0.1 μM), and cortisone (0.1 μM). H2O2 significantly inhibited oocyte maturation at all concentrations. Co-treatment with cortisol (0.1 μM) suppressed the inhibitory effect induced by H2O2 (200 μM). During IVM, approximately 10% of the added cortisone was converted to cortisol (approximately 0.01 μM) by the COCs. However, the locally produced cortisol couldn’t counteract the negative impact of H2O2 on oocyte maturation. Since local cortisol production in COCs mainly occurs in the latter half of IVM, a time-dependent experiment was conducted in which cortisol (0.1 μM) was added at 0, 7, or 14 h following H2O2 (200 μM) treatment. Cortisol added at 0 h suppressed the inhibitory effect of H2O2, whereas delayed addition at 7 or 14 h failed to do so. When added at 0 h, cortisol suppressed the H2O2-induced increase in intracellular ROS levels, but this effect was not observed when cortisol was added at 7 or 14 h. H2O2 increased the expression of two opposing genes: catalase (CAT), an H2O2-degrading enzyme, and BAX, a pro-apoptotic protein. Cortisol added at 0 h suppressed the upregulation of both CAT and BAX. When cortisol was added at 7 or 14 h, only BAX suppression was observed at 7 hours, with no other significant effects. These results suggest that cortisol can prevent oxidative stress caused by H2O2 during IVM. However, unlike the case with LPS, cortisol must be added immediately after the start of IVM to suppress the effects of H2O2. Similar to LPS, H2O2 did not affect P4 and cortisol production or cumulus expansion in COCs. Overall, this study suggests that both locally produced and exogenous cortisol protect bovine oocytes from two maturation-inhibiting factors: LPS and H2O2. Since both factors suppressed MII rates without affecting MI rates, the inhibitory response likely occurs during the transition from MI to MII. However, the differing effects of cortisol on LPS and H2O2 suggest that distinct mechanisms are involved. In vivo, COCs are constantly exposed to adrenal-derived cortisol, and local cortisol production surges in ovulatory follicles. This implies that COCs are protected by cortisol against LPS and ROS regardless of the developmental stage. Furthermore, this study revealed that P4 and cortisol production, as well as cumulus functions such as expansion, are not inhibited by LPS or H2O2. It also demonstrated that P4 is essential for cortisol production and the expression of cumulus expansion-related factors. These findings indicate that bovine COCs possess a robust defense system against LPS and ROS, driven by P4 and cortisol.