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a Department of Cell Biology, Physiology and Immunology
b Department of Pathology, School of Medicine, University of Cordoba, 14004 Cordoba, Spain
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, or vehicle (saline) at 1900 h in proestrus. The ovulatory response was analyzed on the morning of estrus by evaluating follicle rupture and the location of the oocytes in serially sectioned ovaries. The number of oocytes in the oviducts was also counted in rats treated with the highest prostaglandin doses. In indomethacin-treated rats, most newly formed corpora lutea showed abnormal follicle rupture at the basolateral sides. In addition, invasion of the ovarian stroma and blood and lymphatic vessels by granulosa cells and follicular fluid was observed. Prostaglandins of the E series, and especially PGE1, inhibited abnormal follicle rupture and restored ovulation, although the number of oocytes in the oviducts were significantly decreased. PGF2 was only partially effective in inhibiting abnormal follicle rupture and restoring ovulation. These data suggest that prostaglandins of the E series, and particularly PGE1, play a crucial role in ovulation by determining the targeting of follicle rupture at the apex, thus allowing release of oocytes to the periovarian space.
follicle, ovulation
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Most previous studies have assumed that indomethacin prevents follicle rupture [7–9], most likely by inhibiting collagenolytic activity [2, 10]. In a recent study [11] we reported that abnormal follicle rupture, but not its inhibition, was responsible for the ovulatory failure in rats treated with indomethacin. More than 90% of follicles showed one or more points of rupture, and most oocytes were released to the ovarian interstitium or were retained inside the corpus luteum, although about 20% of oocytes were released to the periovarian space, and were therefore effectively ovulated. In addition, invasion of the ovarian stroma and blood vessels was observed, suggesting the existence of uncontrolled, but not inhibited, collagenolytic activity.
Indomethacin is a powerful inhibitor of prostaglandin synthesis [12, 13] and induces a rapid decrease in ovarian prostaglandin concentrations [14]. It is generally accepted that prostaglandin plays some still-undefined role in ovulation (reviewed in [4, 15]). The involvement of prostaglandin in ovulation is based on several lines of evidence. First, prostaglandins (PGs) are formed in preovulatory follicles in response to the preovulatory LH surge, and reach their highest concentrations around the time of ovulation [14, 16, 17]. Second, nonsteroidal anti-inflammatory (indomethacin-like) drugs inhibit both prostaglandin synthesis and ovulation [1–4], and third, mice genetically deficient in cyclooxygenase-2 (COX-2) or PGE2 receptors also show ovulatory failure [18, 19]. However, whether indomethacin blocks ovulation through COX inhibition or by alternative mechanisms, as well as the precise role of prostaglandin in ovulation remain to be elucidated. Indomethacin also affects other processes such as Ca2+ flux [20, 21] and this, together with inconclusive data from prostaglandin replacement studies (reviewed in [4]), has raised some doubts about the role of COX inhibition in the antiovulatory action of indomethacin-like drugs.
Several studies have been devoted to analyze whether the inhibitory action of indomethacin can be reversed by prostaglandin replacement. However, these studies have provided contradictory results; whereas some studies [6, 22] have found that PGE2, PGF2, or both can overcome the inhibitory effect of indomethacin, other studies [23, 24] have reported that these prostaglandins cannot restore ovulation in indomethacin-treated animals (reviewed in [4]).
In this context, the objective of this study was to analyze the effect of prostaglandin replacement on follicle rupture and ovulation in indomethacin-treated rats. This would provide information on the mechanism underlying the antiovulatory action of indomethacin, and on the role of prostaglandins in ovulation.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Adult Wistar cycling rats were maintained under controlled light (14L:10D, lights-on at 0500 h) and temperature conditions, and had free access to rat chow and tap water. Vaginal smears were taken daily, and only animals displaying at least two consecutive 4-day estrous cycles were used. Experiments were performed according to the Guide for Care and Use of Laboratory Animals, and were approved by the Ethical Committee of the University of Córdoba.
Rats were injected s.c. at 1200 h in proestrus with 1 mg of indomethacin (Sigma Chemical Co., St. Louis, MO) in olive oil or vehicle (200 µl). Indomethacin-treated rats were injected with vehicle (200 µl of saline) or PGE1 (50, 250, or 500 µg/rat), PGE2 (50, 250, or 500 µg/rat), or PGF2 (50, 250, or 500 µg/rat; Sigma) at 1900 h in proestrus. This time schedule was selected because the expected time of the preovulatory surge, in our colony, was 1830 h in proestrus [25]. Prostaglandin doses in this range have been previously reported to be effective in inducing luteolysis or reversing indomethacin effects on ovulation [18, 26–28]. Animals (five per group) were killed at 0900 h in estrus.
Tissue Processing
The ovaries were dissected and fixed in Bouin-Hollande fluid for at least 24 h and processed for paraffin embedding. In animals injected with vehicle, indomethacin, and with the highest prostaglandin doses (500 µg/rat), the number of eggs in the oviducts were counted and expressed according to the number of newly formed corpora lutea in the corresponding ovaries. Whole ovaries were serially sectioned (6 µm thick), stained with hematoxylin and eosin, and all sections were scored under a microscope.
Classification of Newly Formed Corpora Lutea
Newly formed corpora lutea were classified into three types depending on the characteristics of the rupture sites in the theca externa, and the degree of invasion of the ovarian interstitium by granulosa cells and follicular fluid. The first type was normally ruptured corpora lutea (NRCL), showing only one rupture site at the apex. The oocyte was released to the periovarian space (Fig. 1, A and B). The second type was abnormally ruptured corpora lutea grade I (ARCL-I), showing several small rupture sites at the basolateral sides, including or not including a rupture site at the apex (Fig. 1, C–E). Granulosa cells and small amounts of follicular fluid invaded adjacent areas of ovarian stroma. The oocyte was released to the periovarian space or, in most cases, it remained entrapped inside the corpus luteum. The third type was abnormally ruptured corpora lutea grade II (ARCL-II), showing at least a large rupture site at the basolateral sides. Oocytes were released to the ovarian interstitium (Figs. 2 and 4) or, less frequently, were entrapped inside the corpus luteum. Granulosa cells, cumulus cells, and abundant follicular fluid invaded the ovarian interstitium, eroded the blood and lymphatic vessel wall (Figs. 2–4), producing emboli of granulosa cells and follicular fluid (Figs. 2–4). Oocytes were frequently observed inside blood or lymphatic vessels (Fig. 4, B and C).
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The percentages of the different types of corpora lutea were determined by two independent observers who were blind to the treatment. The percentages were established for the total number of newly formed corpora lutea.
Statistical Analysis
Statistical analysis was performed by ANOVA to test the existence of significant differences among groups. When significant differences existed, it was followed by the Dunnett method for multiple comparison among means Significance was considered at the 0.05 level, for n = 5.
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RESULTS |
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The effects of PGE2 administration were roughly similar, although it was less effective and does not completely reverse ovulation or abnormal follicle rupture. The highest doses of PGE2 were needed to inhibit large follicle rupture (ARCL-II) and to almost completely restore the number of oocytes released to the periovarian space. However, small follicle ruptures (ARCL-I) were still found, and about 10% of oocytes remained entrapped inside the corpus luteum. The number of oocytes recovered from the oviducts was significantly (P < 0.05) lower than in vehicle-treated rats.
PGF2 was partially effective in inhibiting abnormal follicle rupture. The number of oocytes released to the periovarian space was significantly (P < 0.05) larger, although about 20% of oocytes remained entrapped inside the corpus luteum or were released to the interstitium, even with the highest dose. The number of oocytes in the oviducts was significantly (P < 0.05) smaller.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In a recent study [11], we proposed an alternative hypothesis to the mechanism underlying targeting follicle rupture at the apex, based on the effects of indomethacin in cycling rats. Under this hypothesis, the balance between proteolytic enzymes and protease inhibitors is tilted toward proteolytic inhibition throughout the follicle wall. Local disruption of proteolytic inhibition at the apex could be the result of the release of local factors by the surface epithelium, by the fibroblasts of the tunica albuginea, or both. In the presence of indomethacin, proteolytic activity seems to be maintained throughout the follicle wall, thus allowing abnormal follicle rupture at the basolateral sides.
In the present study, prostaglandin replacement restored ovulation in indomethacin-treated rats, suggesting that the antiovulatory action of indomethacin is mediated by COX inhibition. Prostaglandins of the E series, especially PGE1, were the most effective in restoring ovulation. Several lines of evidence suggest that prostaglandins of the E type are those involved in ovulation. Prostaglandin E2 concentrations show a 30-fold to 70-fold increase during the ovulatory process and are higher than those of PGF2 at any time during ovulation [14, 31]. Mice with targeted disruption of the PGE2 receptor (EP2) show ovulatory defects [19, 32], and defective ovulation in COX-2 deficient mice can be restored by treatment with PGE2 [18].
Otherwise, PGF2 was only partially effective in restoring ovulation. This agrees with previous studies suggesting that this prostaglandin does not play a key role in ovulation. Consistent with the data of the present study, PGF2 does not completely restore ovulation in COX-2 deficient mice [18]. In addition, mice lacking the PGF2 receptor do not show ovulatory defects [33]. The partial increase in the number of oocytes released to the periovarian space found in the present study as well as in previous studies [18] after PGF2 replacement, could be due to its ability to bind to prostaglandin E receptors [34].
Previous studies using prostaglandin replacement in indomethacin-treated animals have provided contradictory results. Whereas in some studies [6, 22] treatment with PGE2, PGF2, or both overcame the inhibitory effect of indomethacin; in others [23, 24], prostaglandin treatment did not bring about any recovery (reviewed in [4]). The reasons for these discrepancies are not known, but they could be due to the use of different species and experimental models, as well as different ways to evaluate the ovulatory response. In most studies, ovulation was detected by counting oocytes in the oviduct, and few [11, 35] have exhaustively examined the location of the oocytes in the ovary. In the present study, each corpus luteum was systematically scored in serial sections to look for the existence of follicle ruptures and the location of the oocyte. However, whether oocytes released to the periovarian space (in indomethacin-treated rats) were located in the oviduct or retained inside the periovarian cavity, was not directly assessed because intact ovarian bursa was not processed. However, counting oocytes in the oviduct indicated that even in those cases in which all (PGE1-treated rats) or nearly all (PGE2-treated rats) oocytes were released to the periovarian space, the percentage of oocytes found in the oviducts was significantly decreased. This agrees with the observation that oocytes adhered to the ovarian surface in indomethacin-treated rats in the present study (data not shown), and with similar findings reported previously in indomethacin-treated rats supplemented with PGE2 [36]. This suggests the existence of a failure in the transport of eggs to the oviduct in indomethacin-treated rats that was not completely reversed by prostaglandin supplementation. In this sense, the evaluation of the ovulation rate by counting eggs in the oviduct could be inadequate, and could be responsible for some inconsistencies in the literature. Additional studies analyzing oocyte transport to the oviduct in indomethacin-treated rats are needed.
Abnormal follicle rupture is responsible for the antiovulatory action of indomethacin [11]. Prostaglandins acted in a dose-dependent manner by inhibiting abnormal follicle rupture. At lower doses, PGE1, and to a lesser extent PGE2, inhibited large follicle ruptures at the basolateral sides, thus preventing release of the oocytes to the interstitium, as well as invasion of the ovarian stroma by granulosa cells. At higher doses, follicle rupture at the basolateral sides was almost completely inhibited. The different degree of follicle rupture and invasion of the stroma (grades I and II) is suggestive of the existence of a different proteolytic activity. Espey [3, 37] introduced the concept of ovulation as an acute inflammatory reaction, based on the histological and biochemical similarities between both processes. The data of the present study suggest that prostaglandins of the E series play a regulatory role in ovulation by inhibiting follicle rupture at the basolateral sides, probably by modulating proteolytic activity. Prostaglandins play both proinflammatory and anti-inflammatory roles during inflammatory processes. Particularly, PGE1 has been found to be effective as an anti-inflammatory agent and may act by inhibiting collagenase gene expression [38, 39]. The anti-inflammatory effects of prostaglandins could explain the apparently paradoxical effect of PGE2, which has been reported to inhibit ovulation in about 50% of gonadotropin-primed immature rats [24].
The invasive capacity of granulosa cells and follicular fluid in indomethacin-treated rats was impressive. Invasion of the ovarian stroma and of blood and lymphatic vessels provides evidence of their destructive capacity. Some data of this study suggest that blood vessel walls were eroded by follicular fluid (see Fig. 2), which agrees with the presence of metalloproteinases in follicular fluid [40]. Such a proteolytic activity of follicular fluid and granulosa cells should be tightly regulated to prevent proteolytic damage to the ovary while allowing the tissue degradation needed for oocyte release. PGE1 seems to play a key role in controlling proteolytic activity during ovulation, as well as in the mechanism underlying spatial targeting of follicle rupture at the ovarian surface.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: FAX: 57 34 218288; bc1galuf{at}uco.es
Received: 25 February 2002.
First decision: 11 March 2002.
Accepted: 2 May 2002.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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This article has been cited by other articles:
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  M Gaytan, C Bellido, C Morales, J E Sanchez-Criado, and F Gaytan Effects of selective inhibition of cyclooxygenase and lipooxygenase pathways in follicle rupture and ovulation in the rat. Reproduction, October 1, 2006; 132(4): 571 - 577. [Abstract] [Full Text] [PDF]
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Q. Li, F. Jimenez-Krassel, Y. Kobayashi, J. J Ireland, and G. W Smith Effect of intrafollicular indomethacin injection on gonadotropin surge-induced expression of select extracellular matrix degrading enzymes and their inhibitors in bovine preovulatory follicles. Reproduction, March 1, 2006; 131(3): 533 - 543. [Abstract] [Full Text] [PDF]
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 F. Gaytan, C. Bellido, M. Gaytan, C. Morales, and J. E. Sanchez-Criado Differential Effects of RU486 and Indomethacin on Follicle Rupture During the Ovulatory Process in the Rat Biol Reprod, July 1, 2003; 69(1): 99 - 105. [Abstract] [Full Text] [PDF]
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