1 Division of Reproductive Sciences, Oregon National Primate Research Center, 505 NW 185th Avenue, Beaverton, OR 97006, USA and 2 Department of Physiology and Pharmacology, Oregon Health & Science University, 3181 Sam Jackson Road, Portland, OR 97201, USA
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Abstract |
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Key words: follicle rupture/ovary/ovulation/prostaglandin
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Introduction |
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Systemic administration of COX inhibitors blocks ovulatory events in various species, including primates. Administration of general COX inhibitors to monkeys (Wallach et al., 1975) and women (Killick and Elstein, 1987
) around the time of administration of the ovulatory dose of gonadotrophin can cause the development of luteinized, unruptured follicles (LUF). Recently, oral administration of a COX-2 selective inhibitor was shown to delay follicle rupture in women experiencing natural menstrual cycles (Pall et al., 2001
). Minor or no disruption of subsequent luteal function was noted following administration of COX inhibitors around the time of ovulation in these studies (Killick and Elstein, 1987
; Athansiou et al., 1996
; Pall et al., 2001
). These data indicate that, while PG may play an essential role in follicle rupture, they may not be required for luteinization of the follicle and normal luteal function.
The local action of PG within the follicle to mediate the ovulatory effects of the mid cycle gonadotrophin surge has been demonstrated for some mammalian species. Using perfused ovaries from rats (Mikuni et al., 1998) and rabbits (Zanagnolo et al., 1996
), the ovulatory effects of gonadotrophin were inhibited by administration of a COX inhibitor but could be restored by co-administration of the COX inhibitor and PGE2 or PGF2
. However, an essential role for locally produced PG in mediating ovulatory processes has not been studied in primates. In addition, studies examining the role of PG in ovulatory events in monkeys and women have relied on visual assessment of a breach of the ovarian surface or ultrasound assessment of follicle size as indicators of follicle rupture (Killick and Elstein, 1987
; Athansiou et al., 1996
; Pall et al., 2001
); oocyte release was not confirmed. The current studies were designed to determine if locally produced PG are necessary for ovulatory events in a primate species, the rhesus macaque. Vehicle or the COX inhibitor indomethacin was injected directly into large, spontaneously developed follicles near the time of the ovulatory LH surge. Injected ovaries were subsequently removed to determine if follicle rupture and oocyte release occurred. Co-injection of indomethacin and PGE2 was performed on additional animals to determine if replacement of PGE2 could restore indomethacin-inhibited ovulatory function.
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Materials and methods |
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The follicular injection model used for administration of test compounds directly into the follicular fluid of the pre-ovulatory follicle in rhesus macaques has been previously described (Hazzard et al., 2002). Briefly, daily serum estradiol levels were used initially to predict the day of the ovulatory LH surge. The peak in serum LH concentrations is known to occur most often on the day of or the day after peak serum estradiol levels (Weick et al., 1973
). On the day of or the day after the serum estradiol peak, an aseptic laparotomy was performed on the anaesthetized monkey to identify the ovary bearing the dominant follicle. A syringe with a 28 G needle and a very small hub volume, was loaded with 50 µl vehicle or test compound, and the needle of this syringe was inserted at the end of the ovary opposite to the follicle and into the antrum of the dominant follicle. An equal amount (50 µl) of follicular fluid was aspirated into the syringe, and preliminary experiments confirmed excellent mixing of the pre-loaded liquid and follicular fluid within the syringe. Without withdrawing the needle of the syringe from the follicle, 50 µl of follicular fluid mixed with vehicle or test compound was injected back into the follicle, restoring the original volume of the follicle. The needle of the syringe was then withdrawn from the ovary. Careful placement of the syringe needle resulted in minimal leakage from the injected follicle, and a sterile cotton swab was placed over the injection site for several minutes to ensure closure. Previous studies employing follicle aspirations determined that the volume of follicular fluid contained within the ovulatory follicle in macaques is ~200 µl; therefore, the final concentration of vehicle or test compound within the follicle following injection was calculated based on this estimate. Final concentrations of test compounds in macaque follicles were 10-6 and 10-5 mol/l indomethacin (Sigma Chemical Co., St Louis, MO, USA) in a vehicle of dimethylsulphoxide (DMSO, <0.02% final concentration; Sigma) and 1 µg/ml PGE2 (Cayman Chemical, Ann Arbor, MI, USA) in a vehicle of ethanol (0.1% final concentration). All vehicle and test compounds were diluted in phosphate-buffered saline, pH 7.4.
For all animals, an aseptic laparoscopic surgery was performed 3 days after follicle injection. In some experiments, the appearance of the ovulatory stigmata was recorded by digital video, and animals continued to be bled daily for determination of serum estradiol and progesterone. In these experiments, the date of first menstruation was also recorded. In additional experiments, injected ovaries were removed 3 days after follicle injection at laparoscopic surgery. These ovaries were fixed in 4% paraformaldehyde, embedded in paraffin, and serial sectioned at 5 µm; every fifth section was stained with haemotoxylin and eosin. The presence of a rupture site as well as an oocyte within the injected follicle was determined by examination of each stained section.
Data analysis
Analyses of serum LH and steroid concentrations were performed using a two-way analysis of variance (ANOVA) with one repeated measure, followed by NewmanKeuls test when indicated. All data were assessed for heterogeneity of variance using Bartletts test and log-transformed when necessary before ANOVA. In all studies where serum LH, estradiol and progesterone levels were determined during the ovulatory period and luteal phase of the menstrual cycle, animals were administered follicular injection of vehicle during one menstrual cycle. In a subsequent menstrual cycle, these animals received follicular injection of a test compound. In this way, each animal served as its own control, and all serum hormone data were analysed using repeated measures to compare control and treatment cycles within animal. The interval from the LH surge to the onset of menstruation (i.e. length of the luteal phase) was analysed using paired t-test. Detection of oocytes in follicles was compared using 2-test with correction for small sample size (Goldstein, 1964
). Data are presented as mean ± SEM, and significance was assumed at P < 0.05.
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Results |
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Ovarian morphology
When laparoscopic evaluations of the ovarian surface were performed 3 days after follicular injection, all vehicle-treated monkeys showed ovulatory stigmata on the injected ovaries (Figure 2A; n = 6). These ovulatory stigmata featured red, luteinizing tissue protruding through an opening in the ovarian surface; they were often observed leaking bloody fluid. Histological examination of sections of vehicle-injected ovaries (Figure 2F
; n = 3) showed luteinizing granulosa cells with notable stromal infoldings, typical of luteinizing follicles 23 days after exposure to peak LH levels. An ovulatory canal connected the collapsing follicular antrum with the exterior of the ovary, and luteinizing tissue protruded through a breach in the ovarian surface epithelium (Figure 2D
).
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To determine if PG replacement could reverse the effects of indomethacin treatment on follicle structure, additional animals received follicular injection to achieve final concentrations of 10-5 mol/l indomethacin + 1 µg/ml PGE2 within the follicular fluid, followed by removal of the ovary 3 days after injection as described above (n = 3). Ovaries injected with indomethacin + PGE2 formed large protruding stigmata similar to those observed on vehicle-injected ovaries, except that these structures appeared paler in colour and less vascular (Figure 2C). Examination of ovarian tissue sections showed well-luteinized follicles (Figure 2H
). These follicles contained stromal infoldings and luteinizing granulosa cells similar to those seen in vehicle- and indomethacin-injected ovaries.
Oocyte detection
Serial sections of each ovary collected were examined for the presence of an oocyte trapped within the luteinizing follicle. Oocytes were never observed within the luteinizing ovulatory follicle of vehicle-injected ovaries (Table I). However, oocytes were identified within the luteinizing follicles of 50% of ovaries injected with either 10-6 or 10-5 mol/l indomethacin (Table I
; P < 0.05 when comparing all indomethacin-injected ovaries versus vehicle). The retained oocytes were not detached from the follicle wall but were surrounded by luteinizing granulosa cells and encased within the developing corpus luteum. (Figure 2I
). Oocytes were never observed within or near the antrum of the luteinizing follicles. One of these four trapped oocytes was found within the 10-5 mol/l indomethacin-injected ovary lacking a breach in the ovarian surface epithelium. However, three oocytes were found within indomethacin-injected follicles with rupture sites. No oocytes were located within follicles injected with indomethacin + PGE2 (Table I
; n = 3).
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Discussion |
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Systemic administration of COX inhibitors, including indomethacin and others, to monkeys and women demonstrated that these drugs can cause delayed ovulation (Hedin et al., 1987; Pall et al., 2001
), failure of the follicle to rupture (Hedin et al., 1987
; Killick and Elstein, 1987
), and infertility (Akil et al., 1996
; Mendonca et al., 2000
). In these studies, methods including ultrasound or visual inspection of the ovaries were used to determine if COX inhibitor treatment resulted in LUF or delayed ovulation; the location of the oocyte was not determined. In the present study, ovulation failure was assessed by direct examination of ovarian tissue sections to determine if an oocyte was present within the luteinizing follicle. The absence of an identifiable oocyte occurred in 50% of indomethacin-injected ovaries and none of the vehicle-injected ovaries. Failure to detect oocytes in indomethacin-injected ovaries probably reflects oocyte release but could also result from oocyte atresia. In most cases, indomethacin injection did not eliminate the formation of the ovulatory opening; only one luteinized, unruptured follicle was observed. Data from previous studies, along with those presented here, indicate that a variety of ovulatory defects can result from COX inhibitor administration. Factors such as the specific inhibitor used, dosage, method of administration, or timing of administration relative to the time of the ovulatory gonadotrophin surge may be responsible for these differences in ovulatory defects. Data from the present study demonstrate that follicle rupture does not ensure oocyte release and support the use of direct measures of ovulatory success, such as localization of the oocyte within the reproductive tract, in future studies.
Indomethacin administered by follicular injection probably prevents oocyte release by inhibition of ovarian PG synthesis. The best-characterized action of indomethacin is the inhibition of COX activity, but indomethacin can also affect other cellular functions (Tegeder et al., 2001). For this reason, additional animals received follicle injection of indomethacin plus PGE2 at levels similar to the peak concentration of PGE2 measured in monkey follicular fluid 4 h before the expected time of ovulation (Duffy and Stouffer, 2001
). In the present study, follicle injection with indomethacin and PGE2 restored oocyte release when compared with injection of indomethacin alone. Studies using knockout mice demonstrated that mice lacking COX-2 expression have defects in ovulation (Lim et al., 1997
), and treatment of these mice with PGE2 restored ovulatory function (Davis et al., 1999
). An essential role for the PGE receptor subtype EP2 in cumulus expansion has been demonstrated using mice lacking expression of this receptor (Hizaki et al., 1999
). In the present study, oocytes were observed surrounded by luteinizing granulosa cells within indomethacin-treated ovaries, suggesting that failure of cumulus expansion may have prevented the oocytecumulus complex from detaching from the follicle wall in these ovaries. These data are consistent with a role for PGE2 in cumulus expansion in primates as well as rodent species, though additional studies will be required to fully address this question.
Follicular injection of indomethacin does not appear to alter some essential ovarian processes including luteal formation, luteal function, or timely luteal regression. Luteinizing granulosa cells streaming into the antrum of the follicle and stromal infoldings were observed in serial sections of ovaries injected with vehicle, indomethacin, and indomethacin + PGE2, suggesting that formation of the corpus luteum proceeded normally following all treatments. In addition, little disruption of luteal function was noted following indomethacin injection. Few differences in circulating estrogen and progesterone between indomethacin- and vehicle-injected animals during the luteal phase suggests that inhibition of PG synthesis during the ovulatory interval does not prevent processes necessary for luteal formation and subsequent steroidogenic function, consistent with previous reports of studies performed in women (Killick and Elstein, 1987; Athansiou et al., 1996
; Pall et al., 2001
). However, other studies show that manipulation of luteal PG concentrations do alter luteal function and life span in primates (Auletta and Flint, 1988
). Additional studies will be required to determine if intraovarian administration of COX inhibitors throughout the entire menstrual cycle alters ovarian function and/or menstrual cyclicity.
Because the cells of the primate ovulatory follicle express COX-2, but not COX-1 (Duffy and Stouffer, 2001), indomethacin most likely blocks oocyte release by inhibition of ovarian COX-2 activity. However, the specific mechanism(s) by which indomethacin prevents normal ovulatory function in primates is unknown. Studies using the isolated perfused ovary model demonstrated that infusion of the COX-2 selective inhibitor NS-398 prevented follicle rupture in rats (Mikuni et al., 1998
), and oral administration of the COX-2 selective inhibitor rofecoxib delayed follicle rupture in women (Pall et al., 2001
), supporting the hypothesis that ovarian COX-2 activity is essential for normal ovulation to occur. The poor solubility and high IC50 of NS-398 when compared with indomethacin has hindered our efforts to compare the ability of these COX inhibitors when injected into the monkey follicle in advance of the expected time of follicle rupture (D.M.Duffy and R.L.Stouffer, unpublished data). Based on our current studies, further studies are warranted using isoform selective COX inhibitors to evaluate the roles of COX-1 and COX-2 in ovulatory events in primates.
The data presented here are consistent with the hypothesis that inhibition of ovarian COX-2 activity and subsequent prevention of PGE2-regulated cumulus expansion results in the trapping of the oocyte within the granulosa cells of the luteinizing follicle, resulting in the failure to ovulate. PG may be involved in the formation of the ovulatory stigmata, but follicular PG such as PGE2 do not appear to be essential for luteinization of the follicle or subsequent luteal function. Enhanced understanding of the mechanism by which PGE2 and possibly other PG mediate follicle rupture and oocyte release in monkeys and women may lead to improved treatment for infertility due to ovulation failure. In addition, inhibition of PG production may form the basis for the development of novel contraceptive strategies.
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Acknowledgements |
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Notes |
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4 To whom correspondence should be addressed. E-mail: duffydm{at}evms.edu
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References |
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Submitted on April 23, 2002; accepted on July 25, 2002.