Differential effects of RU486 and indomethacin on follicle. rupture during the ovulatory process in the rat

Transcription

1 BOR Papers in Press. Published on February 19, 2003 as DOI: /biolreprod Differential effects of RU486 and indomethacin on follicle rupture during the ovulatory process in the rat Francisco Gaytán 1, Carmen Bellido 1, María Gaytán 1, Concepción Morales 2 and José Eugenio Sánchez-Criado 1 Department of 1 Cell Biology, Physiology and Immunology and Department of 2 Pathology, School of Medicine, University of Cordoba, Spain. Running title: RU486 and indomethacin in ovulation Key words: ovary, ovulation, follicle rupture, RU486, indomethacin Address correspondence to:f. Gaytán Department of Cell Biology, Physiology and Immunology, School of Medicine, University of Cordoba, Cordoba, Spain bc1galuf@uco.es fax: This work has been subsidized by Grant BFI from the DGI, Spain.

2 ABSTRACT Ovulation (i.e. the release of mature oocytes from the ovary) requires spatially-targeted follicle rupture at the apex. Both progesterone and prostaglandins play key roles in the ovulatory process. We have studied follicle rupture and ovulation in adult cycling rats treated with a progesterone receptor antagonist (RU486), an inhibitor of prostaglandin synthesis (indomethacin, IM), or both. All rats were treated with LHRH antagonist on the morning (0900 h) of proestrus to inhibit endogenous gonadotropins, and with 10 µg of ovine LH (olh) at 1700 h in proestrus to induce ovulation. Animals were treated from metestrus to proestrus with 2 mg /day of RU486 or vehicle (olive oil), and on the morning of proestrus (1200 h) with 1 mg of IM or vehicle (olive oil). Some rats treated with vehicle or RU486 were killed on the morning of proestrus to assess preovulatory follicle development. The remaining rats were killed on the morning of estrus, to study follicle rupture and ovulation. In vehicle-treated rats, olh induced ovulation in 98% of follicles. In IM-treated rats, spatial targeting of follicle rupture was disrupted. Most oocytes were released to the ovarian interstitum (50%) or to the periovarian space (39%), and a smaller percentage (11%) of oocytes remained trapped inside the luteinized follicle. RU486-treated rats showed, on the morning of estrus, unruptured luteinized follicles. Only occasionally (2.8%), the oocytes were released to the periovarian space. IM treatment induced follicle rupture in RU486-treated rats, and 25% of oocytes were released to the ovarian interstitium. However, the number of oocytes released to the periovarian space (i.e. ovulated) was not increased by IM treatment in rats lacking progesterone actions. Overall, these data indicate that RU486 and IM have opposite effects on follicle rupture, and suggest that both progesterone and prostaglandins are necessary for the spatial targeting of follicle rupture at the apex.

3 INTRODUCTION Ovulation is a complex, multi-step, process that is triggered, in cycling rats, by the preovulatory LH surge on the evening of proestrus. LH surge induces the expression of multiple genes whose products are involved in the ovulatory process. Two essential LHinduced genes are those encoding progesterone receptor (PR) and prostaglandin synthase-2 (PGS-2) or cyclooxygenase-2 (COX-2) [1-3], and several lines of evidence indicate that both progesterone and prostaglandins are required for successful ovulation. The LH surge stimulates progesterone secretion [4] and induces the transient expression of PR [1,2] in granulosa cells of preovulatory follicles. Treatment with progesterone antiserum [5] or inhibitors of progesterone synthesis such as epostane [6] or trilostane [7] cause ovulatory inhibition, that is overcome by concomitant progesterone administration. Treatment with progesterone receptor antagonists such as RU486 [8,9], ZK299 [10] or ORG [11] also inhibits ovulation. Furthermore, PR knockout female mice lacking PR fail to ovulate and are completely infertile [12]. On the other hand, inhibitors of prostaglandin synthesis, such as indomethacin (IM) (reviewed in [13, 14]), NS-398 [15], or selective COX-2 inhibitors [16], have been repeatedly reported to inhibit ovulation. Mice carrying a null mutation for COX-2 [17] or prostaglandin E receptor (EP2) [18] genes also show defective ovulation. Furthermore, exogenous prostaglandin supplementation restores ovulation in both IM-treated rats [19,20] and COX-2 deficient mice [17]. However, the mechanisms and the precise site of action of progesterone and prostaglandins in preovulatory follicles are not fully understood. Ovulation is often referred to as follicle rupture. However, these terms are not equivalent and deserve precise definition. Follicle rupture consists of the breakdown of the theca layers at one or several sites, allowing the release of granulosa cells, the oocyte 3

4 and/or follicular fluid. Ovulation is the release of mature oocytes to the periovarian space and requires, therefore, spatially-targeted follicle rupture. In physiological conditions, proteolytic degradation of the theca layers and extracellular matrix is limited to the zone of the follicle wall facing the ovarian surface [13,14]. Nevertheless, the mechanisms determining the site of follicle rupture at the apex are not known. Recent studies [20,21], using a morphological approach, have shown that ovulation, but not follicle rupture, is inhibited in IM-treated rats. In these animals, follicle rupture seems to occur at random, at any site of the follicle surface, and oocytes are consequently released to either the ovarian interstitum or the periovarian space. This indicates that the mechanisms underlying spatial targeting of follicle rupture are disrupted in IM-treated rats. Detailed histological analysis allows evaluation of both follicle rupture and ovulation. We have studied, by a morphological approach, follicle rupture and ovulation in rats lacking progesterone and/or prostaglandins actions. For this, cycling rats were treated with a progesterone receptor antagonist (RU486), and/or an inhibitor of prostaglandin synthesis (indomethacin). 4

5 MATERIALS AND METHODS Animals and treatments Female cycling Wistar rats (250 g bw in average) were purchased from Panlab (Barcelona, Spain) used. Animals were maintained under standart light (14 h L: 12 h D) and temperature (22 ºC) conditions and had free access to pelleted food and tap water. Vaginal smears were taken daily, and only animals displaying at least two consecutive fourday estrous cycles were used. Experimental designs were established according to the Guide for the Care and Use of Laboratory Animals and were approved by the Ethical Commetee of the University of Córdoba. The progesterone antagonist RU486 was obatined from Exlegin (Paris, France). Indomethacin (IM) was purchased from Sigma Cem. Co. (St Louis, MD), LHRH antagonist (ORG 30276, Organon, Oss, The Netherlands) and ovine LH (olh) from the NIH (Bethesda, MD). Experimental design All animals were treated, on the morning of proestrus (0900 h), with a sc injection of 1 mg of LHRH-a, to blunt endogenous LH secretion, and on the evening (1700 h of proestrus) with an intrajugular injection of 40 µg of olh to induce ovulation. This was performed to synchronize preovulatory gonadotropin surge, that has been reported to be altered in rats treated with PR antagonists [9,22]. Rats were injected from metestrus to proestrus with a sc injection of 2 mg of RU486 or vehicle (olive oil) at 0900h. This treatment schedule induced, in a pilot experiment, nearly complete ovulation inhibition, without altering the morphological features of preovulatory follicles (data not shown). At 1200h in proestrus, rats were injected with either a sc injection of 1 mg of IM to inhibit prostaglandin synthesis, according to previous studies [20] or vehicle (olive oil). Some rats 5

6 treated with RU486 or vehicle (5 rats per group), were sacrifized on the morning (0900 h) of proestrus to analyse the morphological features of preovulatory follicles. The remaining animals (n = 5 for vehicle- and IM-treated rats, n = 6 for RU486-treated rats, and n = 8 for RU486 plus IM-treated rats) were sacrificed on the morning (0900 h) of estrus for the evaluation of follicle rupture and ovulation. Tissue processing and histological analysis The ovaries were fixed for 24 h in Bouin-Hollande s fluid and routinely processed for paraffin embedding. Serial 6 µm-thick sections were cut and stained with hematoxylin and eosin. This stain allows the identification of the granulosa and theca layers. The location of the oocyte was recorded in each luteinized follicle or corpus luteum. Newlyformed corpora lutea were clearly distinguished from those of previous cycles by the presence of remnants of the follicular antrum, and basophilic, non fully luteinized, granulosa cells, in addition to the presence of regressive changes in CL of previous cycles. Oocytes were found in the oviducts or, occasionally, in the bursal cavity, when released to the periovarian space (i.e., ovulated); in the ovarian stroma or inside blood or lymphatic vessels when released to the ovarian interstitium, or trapped inside the luteinized follicles. The proportion of oocytes in each location was expressed with respect to the total number of corpora lutea (CL) or luteinized follicles (LF) per rat. Statistical Analysis Satistical analysis was performed by ANOVA followed by the Student-Newman-Keuls method for multiple comparison among means. Significance was considered at the 0.05 level. 6

7 RESULTS On the morning of estrus, vehicle-treated rats showed newly-formed corpora lutea (CL) and the oocytes surrounded by cumulus cells were found in the oviducts. In these animals (Fig. 1), all (or nearly all) preovulatory preovulatory follicles ovulated (98.3 ± 1.65 %, mean ± SEM for n = 5). Rats treated with IM, showed, on the morning of estrus, different types of CL, depending on the fate of the oocyte (Fig. 1). In 39% of the CL, rupture had occurred at the ovarian surface, and the oocytes were found in the oviducts or, occasionally, in the bursal cavity. However, in most cases (50%), follicle rupture had happened at the basolateral sides and oocytes were found in the interstitium, in lacunae containing blood and follicular fluid or inside blood or lymphatic vessels (Fig.2A). The remaining oocytes (11%) were trapped inside the CL. The cumulus was dispersed and the oocyte was in the metaphase II stage (Fig.2C). RU486-treated rats showed, on the morning of proestrus, before treatment with an ovulatory dose of olh, preovulatory follicles displaying normal morphological features. The cumulus was compact and the oocyte was in the germinal vesicle stage. On the morning of estrus, these animals showed unruptured luteinized follicles, in which the oocyte was trapped (Fig. 1, Fig. 2B). The cumulus was dispersed (Fig. 2D) and the oocyte was in the metaphase II stage. Only 2.8% of oocytes had been released to the periovarian space (Fig. 1). The total number of LF or CL per rat was equivalent in RU486 and vehicletreated rats (13.8 ± 0.79 vs 14.0 ± 0.58, mean ± SEM for n = 5 and 6, respectively), indicating that the development of preovulatory follicles was not affected in RU486-treated rats. IM treatment induced abnormal follicle rupture in RU486-treated rats (Fig. 1). About 25% of the oocytes were released to the ovarian interstitium (Figs. 3A,B). In some 7

8 cases, the released oocyte was found under the ovarian surface in lacunae containing blood and follicular fluid, but the ovarian surface epithelium was apparently intact (Fig. 3B). Erosion of the blood vessel walls and formation of emboli of granulosa cells and follicular fluid, similar to that found in IM-treated rats, were also observed. About 73% of the oocytes remained trapped in the antrum, although some of these follicles showed rupture of the follicle wall with release of granulosa cells and follicular fluid to the ovarian interstitium (Figs. 3C,D). However, IM treatment did not increase the number of oocytes released to the periovarian space (2.2% in average) in RU486-treated rats (Fig. 1). 8

9 DISCUSSION The nature of the ovulatory process, involving the breakdown of the collagenous tissues of the theca layers, the tunica albuginea, and the ovarian surface epithelium, implicates proteolytic degradation of the extracellular matrix [23,24]. The PA [25-27] and MMP [27-31] protease systems, as well as the PR-dependent proteases ADAMTS-1 and cathepsin-l [32,33] have been proposed to play key roles in ovulation. The histological data of this study, based on the presence of follicle rupture, constitute an objective evidence of the existence in vivo of proteolytic activity high enough to cause the breakdown of the theca layers. The absence of follicle rupture in RU486-treated rats is consistent with previous studies suggesting a role for progesterone in the regulation of proteolytic activity [34,35]. Although morphological alterations were not observed in the preovulatory follicles in RU486-treated rats on the morning of proestrus, its full competence to ovulate cannot be ascertained. However, the ovulatory inhibition reported after acute treatment with progesterone antiserum [5] or progesterone synthesis inhibitors [6,7], suggest that ovulatory inhibition was due to the lack of progesterone actions, rather than to defective preovulatory follicles. Furthermore, IM treatment induced follicle rupture in RU486-treated rats. This indicates that effective proteolytic activity (due to either basal levels of PR-dependent [32,33] or non PR-dependent [25-31] proteases) was present in the absence of PR activation. The mechanisms underlying spatial targeting of follicle rupture at the apex were disrupted in IM-treated rats. In these animals, follicle rupture seems to occur at random at any site of the follicle surface [20,21]. The probability of a follicle to undergo rupture at the ovarian surface should be dependent, at least in part, on the proportion of the follicle wall facing the ovarian surface, that shows large inter-follicle variation. This model predicts that 9

10 a variable number of follicles would be ruptured at the ovarian surface and, therefore, some oocytes would be released to the periovarian space. This contention was supported by the data of the present, and previous [20,21] studies, in which up to 39% of oocytes were effectively ovulated. Previous studies have also reported that complete inhibition of ovulation cannot be achieved, even with the higher possible IM doses [13], and that a limited number of ovulations also occur in COX-2 deficient mice [36]. Surprisingly, IM treatment did not increase the number of oocytes released to the periovarian space in rats lacking progesterone actions, in spite of the induction of follicle rupture in more than 25% of follicles, and the degradation of interstitial extracellular matrix and blood vessel walls by granulosa cells and follicular fluid, that indicates effective proteolytic activity. This suggests that PR-mediated mechanisms play a key role in the breakdown of the ovarian surface tissues. Although mechanical factors likely play a role in stigma formation and final follicle rupture [37], previous proteolytic breakdown of the extracellular matrix seems to be neccessary for the softening of the apical tissues. A role for the ovarian surface epithelium (OSE) in ovulation, involving PA secretion by OSE cells, has recently been demonstrated in the sheep [38]. Plasminogen activators increase preferentially within the apices of rat preovulatory follicles [39], and intrabursal administration of inhibitors of the PA-plasmin system decreases ovulation in rats [25]. This could be related to the results reported by Tsafriri et al [40], indicating the presence of eggs released into the theca layer after intrabursal administration of antibodies against PA and α 2 antiplasmin. Inhibition of the PA-plasmin system at the ovarian surface would prevent stigma formation, and could cause herniation of the follicle at the basolateral sides. Furthermore, treatment with progesterone synthesis inhibitors [35] or PR antagonists [11] decreases PA activity in the 10

11 rat ovary. However, whether progesterone mediates the secretion of proteolytic enzymes by OSE cells is, to our knowledge, unknown. In summary, the differential effects of RU486 and indomethacin on follicle rupture support the hypothetical model that is presented in Fig. 4. During follicle growth, continuous remodeling of the follicle basement membrane, as well as of the surrounding ovarian stroma, have to occur in order to allow follicle growth. This is likely due to the presence of proteolytic enzymes and its associated inhibitors, that maintain basal proteolytic homeostasis just allowing controlled tissue remodeling [26-31]. This tissue remodeling process seems to be independent of both progesterone and prostaglandins, as indicated by the existence of follicle growth in rats treated with PR antagonists or in PR knockout female mice [12,32,33], as well as in rats treated chronically with IM (unpublished data). At the time of ovulation, PA and MMP protease systems are upregulated by the preovulatory LH surge [25-31]. In addition, LH-induced secretion of follicular progesterone [4], together with the transient expression of PR in granulosa cells [1,2], determine the expression of additional (PR-dependent) proteases [32,33]. This is counteracted by COX-2 derived prostaglandins that modulate proteolytic activity by activation of protease inhibitors or by increasing the concentration of plasma derived protease inhibitors [41] through changes in blood flow and vascular permeability. This would maintain proteolytic homeostasis, allowing degradation of the follicular basement membrane, but not the breakdown of the theca layers throughout the follicle wall. At the apex, interactions between the preovulatory follicle and the ovarian surface tissues leads to local disruption of the proteolytic homeostasis, that is tilted toward proteolytic activity. Progesterone seems to play a permissive role in ovarian surface events. Edematization [21] and breakdown of the ovarian surface tissues would create a vulnerable region for stigma 11

12 formation. Thereafter, mechanical factors [37] would facilitate follicle rupture and oocyte release at the apex. 12

13 Acknowledgements. The authors are very grateful to J Molina, P Cano and E Tarradas for their technical assistance. REFERENCES 1. Park OK, Mayo KE. Transient expression of progesterone receptor messenger RNA in ovarian granulosa cells after the preovulatory luteinizing hormone surge. Mol Endocrinol 1991; 5: Natraj U, Richards J. Hormonal regulation, localization, and functional activity of the progesterone receptor in granulosa cells of rat preovulatory follicles. Endocrinology 1993; 133: Sirois J, Simmons DL, Richards JS. Hormonal regulation of messenger ribonucleic acid of prostaglandin endoperoxide synthase in rat preovulatory follicles. J Biol Chem 1992; 267: Uchida K, Kadowaki M, Miyake T. Ovarian secretion of progesterone and 20α-hydroxypregn-4-en-3-one during estrous cycle in chronological relation to pituitary release of luteinizing hormone. Endocrinol Jpn 1969; 16: Mori T, Suzuku A, Nishimura T, Kambegawa A. Inhibition of ovulation in immature rats by antiprogesterone antiserum. J Endocrinol 1977; 73: Snyder B, Beecham G, Schane H. Inhibition of ovulation in rats with epostane, an inhibitor of 3β-hydroxysteroid dehydrogenase. Proc Soc Exp Biol Med 1984; 176:

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19 FIGURE LEGENDS Figure 1. Percentage of oocytes released to the periovarian space (i.e. ovulated), trapped inside the corpus luteum, or released to the ovarian interstitium, in rats treated with RU486 (RU), indomethacin (IM), or both. Different superscripts mean significant (p< 0.05) differences; n = 5 for vehicle- and IM-treated rats, and n = 6 and 8 for RU- and RU plus IM-treated rats respectively. Figure 2. Representative micrographs from the ovary of rats on the morning of estrus, treated with indomethacin (A, C) or RU486 (B,D). In A, interstitial oocytes surrounded by cumulus cells can be observed in the ovarian stroma near to a corpus luteum (CL) or inside blood vessels. B, unruptured leteinized frollicles (ULF) containing the oocyte (arrows). C and D, details of the dispersed cumuli (arrows). Scale bars = 100 µm in A,C, D; 500 µm in B. Figure 3. Micrographs from the ovary of rats on the morning of estrus, treated with RU486 plus indomethacin. A, B, ruptured luteinized follicles (RLF). Oocytes surrounded by cumulus cells (arrows in A and B) in the ovarian interstitium, and absence of breakdown of the ovarian surface tissues (open arrows in B) can be observed. C, D, non-consecutive serial sections showing a luteinized follicle in which the oocyte was trapped (arrow in C), but showing a rupture site (open arrows in D) with release of cumulus cells. Scale bar = 125 µm. Figure 4. Hypothetic model on the role of progesterone (P) and prostaglandins (PGs) on the modulation of proteolytic activity during the ovulatory process. The LH preovulatory surge induces the transient expression of progesterone receptors (PR) and COX-2 in preovulatory follicles. P, through activation of PR, induces the expression of PR-dependent proteolytic enzymes. COX-2 derived PGs modulate proteolytic activity by the secretion/activation of proteolytic inhibitors or by increasing the concentrations of plasma derived protease inhibitors, through changes in blood flow and vascular permeability. This would maintain proteolytic homeostasis just preventing breakdown of the theca layers throuhout the follicle wall. At the apex, interactions between the preovulatory follicle and 19

20 the ovarian surface tissues causes a local disruption of the proteolytic homeostasis, leading to tissue breakdown, favouring stigma formation and follicle rupture, driven by follicle pressure. P seems to play some undefined role in apical changes. The effects of blocking prostaglandin sinthesis with indomethacin (IM), progesterone receptor activation with RU486 (RU), or both, and the most frequent locations of the oocytes (indicated as percentages), are depicted below. 20

21 a Ovulated b c c Percentage of oocytes a b c Trapped d Interstitial 80 a 40 b NF NF Vehicle IM RU RU+IM Fig. 1

22 $ Interstitial oocytes &/ Blood vessel % & ' 8/) 8/) Fig. 2

23 A C RLF B D RLF Fig. 3

24 + PR +? + Proteolytic enzymes LH surge + COX-2 PGs Proteolytic inhibitors + + [ 39 %] PR + P PGs COX-2,0 - [ 50 %] PR + PR + P PGs COX P [ 97 %] [ 73 %] PGs COX-2 -,0 [ 25 %] )LJXUH