Reactive oxygen species are indispensable in ovulation

Abstract

Ovulation is stimulated by the preovulatory surge of the pituitary luteinizing hormone (LH). Because the ovulatory response is commonly identified with inflammation, we explored the involvement of reactive oxygen species (ROS) in this process. Our experiments show that administration of broad-range scavengers of oxidative species into the ovarian bursa of mice, hormonally induced to ovulate, significantly reduced the rate of ovulation. LH-induced cumulus mucification/expansion, a necessary requirement for ovulation, was prevented by antioxidants both in vivo and in an ex vivo system of isolated intact ovarian follicles. Along this line, H(2)O(2) fully mimicked the effect of LH, bringing about an extensive mucification/expansion of the follicle-enclosed cumulus-oocyte complexes. Impaired progesterone production was observed in isolated follicles incubated with LH in the presence of the antioxidant agents. Furthermore, LH-stimulated up-regulation of genes, the expression of which is crucial for ovulation, was substantially attenuated upon ROS ablation. This system was also used for demonstrating the role of ROS in phosphorylation and activation of the EGF receptor as well as its downstream effector, p42/44 MAPK. Together, our results provide evidence that ovarian production of ROS is an essential preovulatory signaling event, most probably transiently triggered by LH.

Fig. 1.

Intrabursal administration of antioxidants significantly reduces ovulation rate. (A) Ovaries of eCG-primed, sexually immature mice were injected with two different antioxidant agents, BHA (250 μM) and NAC (100 mM), 3 h before hCG administration. Ovaries of control mice were injected with the vehicle. Ovulated oocytes were flushed from the oviductal ampulae and counted 22 h later. Means having asterisks are significantly different from the control (P < 0.05). (B and C) The ovaries were then collected, weighted (B), examined for abnormalities (C Left), and further processed for histological analysis. Arrow points to an oocyte (C Right). n, Number of animals in each experimental sample; F, follicle; AF, antral follicle. (Scale bars: 100 μm.)

Fig. 2.

Cumulus expansion depends on ROS formation. Large antral ovarian follicles were cultured with either LH (1 μg/mL) or H₂O₂ (20 μM) in the presence or absence of BHA (125 μM). (A) After 8 h, the COCs were released, and expansion was monitored by using differential interference contrast microscopy. (B) The fraction of expanded cumuli under each treatment was calculated. A total of 12–18 follicles/COCs from each group were analyzed (n = 3).

Fig. 3.

ROS play a role in preovulatory progesterone production. Follicles were incubated in the presence of LH (1 μg/mL) with or without a preincubation with BHA (125 μM). Follicles incubated in LH-free medium for 6 h served as control. Progesterone concentrations were determined in the medium at the indicated time points. Data are representative of three independent experiments. Columns with no common superscripts are significantly different (P < 0.05).

Fig. 4.

ROS mediate LH-induced expression of preovulatory genes. (A) Groups of 35 follicles were incubated with LH (1 μg/mL) in the presence or the absence of one of the following antioxidant agents—NAC (10 mM), BHA (125 μM), and GSH (40 mM)—for 1 h before the addition of LH to the culture medium. After 4 h, the follicles were collected and homogenized, and mRNA was extracted for determination of gene expression by using quantitative real-time PCR. (B) Follicles were incubated in the presence of H₂O₂ or LH with or without either H89 (25 μM) or UO126 (10 μM) for 1 h. Gene expression profiles were determined by real-time PCR (n ≥ 3).

Fig. 5.

LH-induced phosphorylation of both EGFR and p44/42 MAPK is reduced by ROS scavengers. (A and B) Follicles were preincubated for 1 h with either BHA (125 μM) or NAC (10 mM) or for 30 min with the EGFR tyrosine kinase inhibitor AG1478 (10 μm) before the addition of LH (1 μg/mL) for an additional 30 min of incubation. Total protein was extracted and subjected to Western blot analysis. Representative experiment is shown (n = 4).

Fig. 6.

H₂O₂ induces phosphorylation of p42/44 MAPK. The EGFR kinase inhibitor AG1478 (0.5 μM) was added to follicles incubated with either LH (1 μg/mL, control) or H₂O₂ (2 mM). The samples were subjected to Western blot analyses with anti–phospho-p42/44 MAPK antibodies followed by total MAPK antibodies. Representative experiment is shown (n = 4).

Fig. 7.

The proposed mechanism for ROS involvement in ovulation. Interaction of LH with its receptor provokes an inflammatory-like response involving generation of ROS. ROS might be involved in activation of the EGFR by inhibition of either EGFR-specific phosphatases (PTPs) or MAPK phosphatases (PPs). The activated EGFR initiates a subsequent signaling cascade, which brings about p42/44 MAPK phosphorylation. Other signaling pathways take part in the activation of p42/44 MAPK as well. ROS may also be involved in activation of the PKA signaling and the MEK signaling cascade.