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BOR - Papers in Press, published online ahead of print December 11, 2002.
Biol Reprod 2002, 10.1095/biolreprod.102.012138
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BIOLOGY OF REPRODUCTION 68, 1748–1754 (2003)
DOI: 10.1095/biolreprod.102.012138
© 2003 by the Society for the Study of Reproduction, Inc.

Ovary

In Vivo Effect of Epidermal Growth Factor, Interleukin-1ß, and Interleukin-1RA on Equine Preovulatory Follicles1

Alain Martoriati, Guy Duchamp, and Nadine Gérard2

I.N.R.A.-Haras Nationaux, Equipe de Reproduction Equine, P.R.C., F-37380 Nouzilly, France


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Paracrine factors have significant effects during folliculogenesis. Because of various morphological features, the mare is a convenient model to study in vivo the effects of factors involved in periovulatory events. In the present work, epidermal growth factor (EGF; experiment 1, n = 49 mares) and interleukin-1ß and interleukin-1RA (IL-1ß and IL-1RA, respectively; experiment 2, n = 80 mares) were injected intrafollicularly to evaluate the influence of these factors on in vivo maturation of equine preovulatory follicles. A transvaginal ultrasound-guided injection was performed when the diameter of the dominant follicle reached 30–34 mm. In experiment 1, the four experimental groups were 1) EGF group, intrafollicular (i.f.) injection of EGF (2 ml; 0.5 µg/ml) plus i.v. injection of physiological serum; 2) control group, no injection; 3) PBS group, i.f. injection of 2 ml of PBS plus i.v. injection of physiological serum; 4) crude equine gonadotropins (CEG) group, i.f. injection of PBS plus i.v. injection of CEG (20 mg). In experiment 2, groups 3 and 4 were the same as in experiment 1, but groups 1 and 2 were changed as follows: 1) IL-1ß group, i.f. injection of IL-1ß (2 ml; 0.5 µg/ml) plus i.v. injection of physiological serum; 2) IL-1RA group, i.f. injection of IL-1RA (2 ml; 0.5 µg/ml) plus i.v. injection of physiological serum. In each experiment, cumulus-oocyte complexes from dominant/injected follicles were collected by transvaginal ultrasound-guided aspiration 38 h after intrafollicular injection. Cumulus morphology and oocyte nuclear stage were assessed. Additionally, in experiment 2, 40 mares were used to determine the time of ovulation after treatments. Our results indicate that intrafollicular injection of EGF or PBS induced lower cumulus expansion and oocyte maturation rates compared with the CEG group (P < 0.05). In experiment 2, the IL-1ß and CEG groups showed the same expansion rate, the same oocyte maturation rate, and the same ovulation distribution. On the other hand, the intrafollicular injection of IL-1RA, as PBS, did not induce follicle and cumulus-oocyte complex (COC) maturation. In conclusion, we confirmed that the technique of intrafollicular injection can be used in the mare to study the role of specific molecules. We demonstrated for the first time in mares that the injection of EGF did not influence in vivo COC maturation. In contrast, IL-1ß injection into the dominant follicle induced in vivo oocyte maturation and the ovulation process whereas IL-1RA seemed to block these mechanisms.

epidermal growth factor, equine, interleukin 1, intrafollicular injection, ovulation


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
It is well known that ovarian activities are regulated not only by endocrine hormones but also by autocrine and paracrine factors [1]. Among them, growth factors such as epidermal growth factor (EGF) and cytokines, such as interleukin-1, seem to be important regulators of the ovarian physiology.

EGF is a peptide originally described in the submaxillary glands of male mice [2]. It has been shown to be involved in a number of developmental events and in the differentiation of various organ systems [3]. It also stimulates the in vitro proliferation of numerous cell types. Recent studies have documented roles for EGF in the ovarian physiology. Indeed, EGF, its receptor, and mRNA were detected in the hamster, rat, and human granulosa and theca cells [4–6]. EGF was also detected in human and porcine follicular fluid [7, 8]. It was localized in porcine and human oocytes [9, 10]. The effect of EGF at the ovarian level was mainly studied in vitro. EGF stimulates the proliferation of rat and porcine granulosa cells [11, 12]. This effect is likely regulated by LH and FSH [13]. There is increasing evidence that EGF regulates the in vitro cellular activity of granulosa cells either by inhibiting the FSH-induced aromatase activity [14], LH receptor production [15], or inhibin secretion [16], or by stimulating FSH-receptor expression and progesterone production [17]. Several studies have clearly demonstrated the positive effect of EGF on in vitro oocyte maturation (bovine [18], porcine [19], equine [20]).

Interleukins are polypeptide cytokine components of the immune system originally defined by their action between leukocytes. Interleukin-1 (IL-1) was the first interleukin discovered and has been the most widely studied. The IL-1 system is composed of two bioactive ligands, IL-1{alpha} and IL-1ß [21], two types of receptors, IL-1R1 and IL-1R2 [22, 23], and a receptor antagonist, IL-1RA, which regulates the IL-1 biological activity by a competitive fixation on receptors [24]. All of them are expressed or have effects on a large range of tissues, including ovarian cells [25–28]. In vitro studies have shown that IL-1ß regulates some cellular activities of granulosa and theca cells, such as steroidogenesis [29, 30] and the synthesis of proteases [31], plasminogen activator [32, 33], and prostaglandins [34, 35]. Moreover, it has been demonstrated that IL-1ß promotes the ovulation process in the rat [36] and the rabbit [37] models. IL-1ß increases the germinal vesicle breakdown of oocytes in the rabbit model [37]. In contrast, we demonstrated recently that IL-1ß inhibits the eLH-induced in vitro meiosis resumption in equine oocytes [38], suggesting a potential role of IL-1ß in oocyte nuclear maturation. These observations led us to hypothesize that, in mammalian species, 1) EGF is an important factor involved in oocyte maturation and 2) IL-1 is a paracrine factor that is involved in the cascade of events leading to ovulation [39] and in the maturation of oocytes. The large size of the equine follicle and the presence of a thick surrounding tunica albugina allowed studying the in vivo effect of molecules of interest by transvaginal ultrasound-guided intrafollicular injection [40, 41].

In this context, the purpose of the present work was to study the in vivo effect of EGF, IL-1ß and IL-1RA on cumulus-oocyte complex (COC) maturation and on ovulation.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals and Treatments

Adult cyclic pony mares in good body condition, kept indoors and fed with concentrates, were used. Mares received a prostaglandin F2{alpha} analogue (Cloprostenol [Estrumate], 125 µg/mare i.m.; Scherring-Plough, Levallois-Peret, France) during the midluteal phase to induce luteolysis. Ovarian activity was then assessed by routine rectal ultrasound scanning. Intrafollicular injections were performed in the dominant follicle, between 30–34 mm in diameter, at the end of the follicular phase (see below). Before intrafollicular injection, the mares were sedated with detomidine (0.6 mg/100 kg body weight [BW] i.v., Domosedan; Pfizer, Amboise, France) and propantheline bromide (20 mg/100 kg BW i.v.; Sigma, St. Louis, MO) was administered to achieve rectal relaxation. After intrafollicular injection, mares were treated with an antibiotic (Intramicine, 1 600 000 IU penicillin/100 kg BW and 1.3 g dihydrostreptomycin/100 kg BW i.m.; Sanofi, Libourne, France). The dominant/injected follicle was aspirated 38 h after intrafollicular injection. Before and after puncture sessions, the mares were sedated, relaxed, and treated with antibiotics as described above.

Intrafollicular Injection Procedure

The injection was performed using a transvaginal ultrasound-guided system. A 60-cm-long single lumen needle of 1.1-mm outer diameter was inserted into the dominant follicle the day it reached 30–34 mm in diameter. Follicular fluid (2.5 ml) was aspirated in a syringe directly connected to the needle. Two milliliters of the studied molecule diluted in sterile PBS (Dulbecco ‘A’; Unipath, Dardilly, France) and maintained at 37°C were injected in the follicle by using a syringe directly connected to the needle. Follicular fluid (0.5 ml) from the first syringe was then injected back into the follicle to rinse the needle and reestablish the initial follicular volume.

Detection of Ovulation

Injected follicles were examined by ultrasonography 8 h after intrafollicular injection, then twice a day for 2 days, and then once a day until ovulation, (i.e., absence of a large dominant follicle and presence of a corpus luteum).

COC Recovery

COCs were recovered from injected follicles 38 h after the intrafollicular injection (i.e., 34 h post-i.v. injection) by transvaginal ultrasound-guided follicle puncture, as previously described [42]. Briefly, a single lumen needle (18-mm outer diameter) was used to aspirate follicular fluid. The follicle was then flushed several times with PBS containing heparin (50 IU/ml; LEO S.A., St. Quentin Yvelines, France) at 37°C and scraped with the needle in order to pick up COCs. All aspirated fluids were examined with a stereomicroscope for COC recovery.

COCs were classified morphologically as compact or expanded.

Nuclear Examination of Oocytes

Recovered COCs were rinsed twice and stripped of their cumulus cells with a small glass pipette in PBS at 37°C, as previously described [38]. Totally denuded oocytes were rinsed in PBS, stained with 1 µg/ml bis-benzimide solution (Hoechst 33342; Sigma), and observed in a drop on a slide under a fluorescence microscope in order to determine their nuclear stage. As illustrated in several works from our lab [43, 44], oocytes were considered in metaphase II (MII) when they showed a polar body, an intact nuclear membrane (light microscopy), and two distinct spots of chromosomes stained by Hoechst (fluorescence microscopy). The other nuclear stages (metaphase I, dense chromatin, germinal vesicle) were classified as described by Goudet et al. in 1997 [43].

Experiment 1: Effect of EGF Intrafollicular Injection on COC In Vivo Maturation

In experiment 1, 49 cyclic mares were used. They were divided into four groups. In the first group (EGF group; n = 12), the dominant follicle received intrafollicular (i.f.) injection of 2 ml of mouse EGF (0.5 µg/ml in PBS; Sigma) and jugular injection (i.v.) of 5 ml of physiological serum. In the second group (control group; n = 13), mares did not receive any injection. In the third group (PSB group; n = 12), mares were i.f. injected with 2 ml of PBS and received an i.v. injection of 5 ml of physiological serum. In the last group (CEG group; n = 12), mares received an i.f. injection of 2 ml of PBS and i.v. injection of 20 mg of crude equine gonadotropin (CEG) in 5 ml of physiological serum. This fourth group was the positive control group since CEG i.v. injection induces ovulation and oocyte maturation [45].

COCs were collected by transvaginal ultrasound-guided aspiration 38 h after intrafollicular injection. Cumulus expansion and oocyte nuclear stage were assessed stereomicroscopically and under fluorescence, respectively.

Experiment 2: Effect of IL-1ß and IL-1RA Intrafollicular Injection on Ovulation and COC In Vivo Maturation

In experiment 2, 80 cyclic mares were used. They were divided into four groups. Mares from the first group (IL-1ß group; n = 20) received an i.f. injection of 2 ml of recombinant human IL-1ß (0.5 µg/ml in PBS, rhIL-1ß; R&D System, Abingdon, UK) and an i.v. injection of 5 ml of physiological serum. Mares from the second group (IL-1RA group; n = 20) received an i.f. injection of 2 ml of recombinant human interleukin-1ra (0.5 µg/ml in PBS, rhIL-1RA; R&D System) and an i.v. injection of 5 ml of physiological serum. Mares from the third group (PBS group; n = 20) received an i.f. injection of 2 ml of PBS and an i.v. injection of physiological serum. Mares from the last group (CEG group; n = 20) received an i.f. injection of 2 ml of PBS and an i.v. injection of 20 mg of CEG in 5 ml of physiological serum.

Within each group, injected follicles were either punctured 38-h postinjection or monitored by ultrasonography until ovulation. The collected COCs from punctured follicles were assessed for cumulus expansion and oocyte nuclear stage, as described above.

Statistical Analysis

The nonparametric tests of Kruskal-Wallis and Wilcoxon-Mann-Whitney were performed using StatXact 5 software (CYTEL, Cambridge, MA [www.cytel.com]) in order to compare oocyte in vivo maturation rates, expanded cumulus rates, and the time of ovulation.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
To evaluate the effect of EGF on equine COC in vivo maturation (experiment 1) and the effects of IL-1ß and IL-1RA on the time of ovulation and on COC in vivo maturation (experiment 2), intrafollicular injections of these molecules were performed in the dominant follicle when it reached 30–34 mm in diameter. Injected follicles were then either aspirated by transvaginal ultrasound-guided puncture to recover and analyze the enclosed COCs (experiment 1 + experiment 2) or checked by ultrasonography to determine the time of ovulation (experiment 2).

Experiment 1: Effect of Intrafollicular Injection of EGF on COC In Vivo Maturation

During this experiment, 49 follicles were injected and punctured successfully whereas 12 mares were not used either because ovulation occurred before ovarian puncture (n = 3) or because of large leaks of fluid from the injected follicle (n = 9). Large leaks of fluid were illustrated by a large decrease in follicular diameter (15 mm at least, corresponding to more than 50% of the initial size). The 49 successfully injected follicles showed a slight decrease in diameter (1.6 ± 0.7 mm, 7% of the initial size).

Recovery of COCs

Out of the 49 injected/punctured follicles, 32 oocytes were collected and analyzed (66% collection rate). As shown in Table 1, the collection rates were similar in the EGF group (91%, 11/12), CEG group (75%, 9/12), and PBS group (67%, 8/12). A significantly lower collection rate was obtained in the control group (31%, 4/13; P < 0.05).


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  		TABLE 1. Number of punctured follicles, percentage of recovered and expanded cumulus-oocyte complexes, and percentage of degenerate, immature, and metaphase II oocytes in each group of the two experiments.*

Cumulus Morphology at Recovery

The expansion rate of the cumulus was the lowest in the control group, where no expanded cumulus (0/4) was observed (Table 1). It was the highest in the CEG group, where all recovered cumulus were expanded (100%, 9/9). The difference observed between these two groups was significant (P < 0.05). Intermediate cumulus expansion rates were observed in the PBS group (62.5%, 5/8) and the EGF group (82%, 9/11). The cumulus expansion rate was significantly lower in the PBS group than in the CEG group (P < 0.05).

Oocyte Nuclear Stage at Recovery

As shown in Table 1, the rate of MII oocytes was significantly higher in the CEG group (PBS i.f., CEG i.v.) than in the EGF group (EGF i.f., physiological serum i.v.; P < 0.035), PBS group (PBS i.f., physiological serum i.v.; P < 0.05), and control group (no i.f., no i.v.; P < 0.02), which were not different between them. The other oocytes were either degenerated (2/9 in the CEG group, 3/11 in the EGF group, and 2/8 in the PBS group) or immature (4/11 in the EGF group, 3/8 in the PBS group, and 4/4 in the control group).

Experiment 2: Effect of Intrafollicular Injection of IL-1ß and IL-1RA on Ovulation and COC In Vivo Maturation

During this experiment, 7 out of 87 injected mares were not used because of large leaks of fluid from the injected follicles (n = 4; decrease in follicular diameter >15 mm) or ovulation before ovarian puncture (n = 3). The successfully injected follicles showed a decrease in diameter of 1.5 ± 0.8 mm maximum, corresponding to 7% of the initial size.

COC Recovery

In this experiment, 8 to 10 mares were used in each group, 36 follicles were punctured, and 29 oocytes were collected and analyzed (80% collection rate). The collection rate was similar in each group (Table 1).

Cumulus Morphology at Recovery

The expansion rate of cumulus was significantly lower in the PBS group (28.6%, 2/7) compared with the CEG group (100%, 8/8), IL-1ß group, and IL-1RA group (85.7%, 6/7 each) (P < 0.05).

Oocyte Nuclear Stage at Recovery

As shown in Table 1, the rate of MII oocytes was significantly higher in the CEG group (PBS i.f., CEG i.v.) and IL-1ß group (IL-1ß i.f.; physiological serum i.v.) than in the PBS group (PBS i.f., physiological serum i.v.; P < 0.05). Moreover, the IL-1RA group (IL-1RA i.f., physiological serum i.v.) showed significantly fewer mature oocytes than the CEG group (P < 0.035), whereas it only tended to show fewer mature oocytes than the IL-1ß group (P < 0.055). As shown in Figure 1, the other oocytes were either degenerated (1/8 in the CEG group, 1/7 in the IL-1ß group, 4/7 in the IL-1RA group, and 3/7 in PBS group) or immature (2/8 in the CEG group, 2/7 in the IL-1ß group, 2/7 in the IL-1RA group, and 4/7 in the PBS group). The distributions of oocyte nuclear stages were similar between the CEG group and the IL-1ß group but significantly differed from the distributions observed in the IL-1RA group (P < 0.012 and P < 0.02, respectively) and the PBS group (P < 0.009 and P < 0.028, respectively). The distributions of oocyte stages were not different between the IL-1RA group and the PBS group.



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  		FIG. 1. In vivo oocyte maturation after intrafollicular administration of IL-1ß or IL-1RA in equine dominant follicles. IL-1ß (0.5 µg/ml, group IL-1ß; n = 8 mares), IL-1RA (0.5 µg/ml, group IL-1RA; n = 10 mares), or PBS (groups CEG and PBS; n = 9 mares in each) were injected directly into the dominant follicle (i.f. injection; 30–34 mm in diameter) at the end of the follicular phase. The same day, 4 h after i.f. injection, a jugular injection of physiological serum (groups IL-1ß, IL-1RA, and PBS) or 20 mg of CEG (group CEG) was performed. Oocytes were collected by transvaginal ultrasound-guided follicle puncture and their nuclear stages were analyzed and classified according to the following classes: DEG, degenerate; immature, which regroups germinal vesicle, dense chromatin, and metaphase I stages; MII, metaphase II. For each group, histograms represent the percentage of oocytes in each nuclear class. The proportion of oocytes in each class is indicated inside the histograms. The rate of mature oocytes is compared between groups. a, b, c, Indicate groups that are significantly different (P< 0.05)

Time of Ovulation

Ten mares were used in each group. Figure 2 illustrates the ovulation profiles after intrafollicular injection of PBS (2 ml), IL-1ß (1 µg/2 ml), or IL-1RA (1 µg/2 ml). In the CEG group (PBS i.f., CEG i.v.), most of the mares (8/9) ovulated between 31 and 47 h after the intrafollicular injection (i.e., between 28 and 44 h after the CEG injection). Mares from the IL-1ß group (IL-1ß i.f., physiological serum i.v.) ovulated mainly (5/9) in the same interval. The distribution of the time of ovulation was not significantly different between these two groups. In the PBS group (PBS i.f., physiological serum i.v.), the length of time from injection to ovulation was very heterogeneous. Half of the mares (5/10) ovulated more than 55 h after the i.f. injection, 2/10 ovulated between 47 and 55 h, 2/10 ovulated between 31 and 47 h, and 1 ovulated during the first interval (23–31 h). This distribution was significantly different from that of the CEG group (P < 0.0064). Finally, the IL-1RA group (IL-1RA i.f., physiological serum i.v.) showed no ovulation before 47 h. Indeed, most of the ovulations (6/8) were observed in the last period (>55 h) or in the 47–55-h interval (2/8). In spite of these observations, this distribution was not significantly different from that of the PBS group. On the other hand, it differed significantly from the IL-1ß group (P < 0.0265) and the CEG group (P < 0.0005) distributions of ovulation.



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  		FIG. 2. Length of time from injection to ovulation after intrafollicular administration of IL-1ß or IL-1RA in equine dominant follicles. IL-1ß (0.5 µg/ml, group IL-1ß; n = 10 mares), IL-1RA (0.5 µg/ml, group IL-1RA; n = 10 mares), or PBS (groups CEG and PBS; n = 10 mares each) were injected directly into the dominant follicle (i.f. injection; 30–34 mm in diameter) at the end of the follicular phase. The same day, 4 h after i.f. injection, a jugular injection of physiological serum (groups IL-1ß, IL-1RA, and PBS) or 20 mg of CEG (group CEG) was performed. Follicle maturation was monitored by ultrasonography twice a day during 2 days and once a day until ovulation. For each group, histograms represent the percentage of mares that ovulated during each time interval. The proportion of mares is indicated on the top of the histograms


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The aim of the present work was to analyze in vivo the effect of EGF (experiment 1) and IL-1ß and IL-1RA (experiment 2) after intrafollicular injection into the preovulatory follicle. In experiment 1, the COC maturation (cumulus expansion and oocyte nuclear stage) was studied. In experiment 2, the effects on both COC maturation and ovulation were investigated.

In order to study the effect of a factor on oocyte and follicle maturation, the intrafollicular injection technique is an interesting alternative. In the present work, 129 dominant follicles were successfully injected, and only 13 suffered serious damage. The first work describing this approach in the rabbit was published in 1972 [46]. It has been used in the ewe [47], the cow [48], and more recently in the rhesus monkey [49, 50]. This technique was developed in mares in 1991 [40] and was improved in 1995 [41]. In the mare, this approach needs no surgery because the large size of the preovulatory follicle and the presence of a tunica albuginea allow stinging follicles with limited leakage by using the transvaginal ultrasound echo-guided method.

In the first experiment, 1 µg of EGF diluted in 2 ml of PBS or the vehicle only were intrafollicularly injected. To our knowledge, it was the first time that EGF was injected in vivo into an ovarian follicle of an animal species. Concerning the theoretical volume of follicular fluid (about 20 ml), the quantity injected was chosen to represent the concentration of 50 ng/ml of follicular fluid; that concentration improves the oocyte nuclear maturation rate in vitro [20, 43]. We observed the lowest COC recovery and expansion rates in the control group. The closer and broader attachment of immature equine COCs to the follicular wall may prevent them from being aspirated from the follicle [51]. As suggesting by Goudet et al. in 1997 [43], during the maturation of COCs, the follicular wall disorganizes, reducing connections with COCs. Therefore, the collection rate of COCs by follicular aspiration could increase. The intrafollicular injection could promote some local disruption involved in cumulus maturation. The rate of expanded cumulus in intrafollicularly injected groups is in accordance with this hypothesis. We observed that EGF intrafollicular injection tended to increase the cumulus expansion rate compared with PBS injection. Nevertheless, the result on in vivo nuclear maturation of oocytes did not confirm previous in vitro results [20, 43]. Actually, no increase in maturation rate was observed after intrafollicular injection of EGF in comparison with intrafollicular injection of PBS alone. These observations suggest the existence of some local intrafollicular regulation of the EGF effect on oocyte maturation. Although not significant, we observed a higher rate of COC maturation in the PBS group than in the control group (no injection). This slight increase could disclose some local inflammatory reaction after intrafollicular injection. One could hypothesize that this local inflammation could promote COC maturation, thus masking the EGF effect. Interestingly, we observed in the first experiment that CEG i.v. injection coupled with a PBS i.f. injection induced a similar COC maturation rate compared with a single CEG i.v. injection [52]. This confirms that intrafollicular injection has no deleterious effect on COC maturation.

In the second part of this work, the hypothesis of a role played by some inflammatory molecules at the follicular level in the periovulatory events was tested. The influence of intrafollicular administration of IL-1ß was investigated on COC maturation and ovulation. Previous studies performed on perfused ovary in the rat [36] and the rabbit [37] implicated IL-1ß in these two processes. Intrafollicular administration of IL-1RA to competitively inhibit the action of IL-1 in the preovulatory follicle was also tested. To our knowledge, this is the first study of the effect of IL-1 system components on follicle development and oocyte maturation that has been performed in vivo in any species. Concerning the effect of IL-1ß on ovulation, we demonstrated that the intrafollicular injection of IL-1ß coupled with an i.v. injection of vehicle induced synchronized ovulations, in contrast with the intrafollicular injection of PBS. The mechanism of action of IL-1ß remains to be elucidated, but we can hypothesize that the increase in the IL-1ß intrafollicular level after injection mimics the local preovulatory events that precede ovulation. It is worth noting that IL-1ß intrafollicular injection and CEG i.v. injection coupled with PBS intrafollicular injection gave the same result, in terms of ovulation distribution, as a single i.v. injection of CEG in our herd [45]. This observation not only confirmed that the intrafollicular injection did not alter follicle maturation, as previously concluded, but also pointed out the role of IL-1ß in the ovulation process in the mare. In other species, the involvement of IL-1ß in ovulation-associated events such as the modulation of steroidogenesis [53], regulation of prostaglandin [54], and protease synthesis [31, 33] have been demonstrated. Such implications of IL-1ß could also exist in the mare.

Regarding oocyte nuclear maturation, the present work showed the positive effect of IL-1ß when injected intrafollicularly. This effect could be mediated by IL-1 receptors as a direct effect because IL-1R2 receptor mRNAs are expressed by equine oocytes [38]. More probably, it results from an indirect effect mediated by cumulus cells that have been shown to express IL-1R1 receptor mRNA [38].

The positive effect of IL-1ß on oocyte nuclear maturation demonstrated in the present work is consistent with the results obtained in the rabbit model [37] indicating that IL-1ß perfusion in ovary-induced oocyte vesicle germinal breakdown. This result is not in agreement with our previous in vitro study in which a negative effect of IL-1ß was demonstrated on LH-induced equine oocyte nuclear maturation [38]. The discrepancy between in vivo and in vitro results could be explained by the existence of complex local regulatory mechanisms that are not present in vitro.

The distribution of ovulations obtained after IL-1RA intrafollicular injection reinforces the results obtained with IL-1ß. Actually, we observed that intrafollicular injection of IL-1RA did not induce the ovulation process as observed with IL-1ß. IL-1RA, rather, would delay it, as 75% of the ovulations were observed more than 55 h after treatment and no ovulation was observed before 47 h. A retarding effect of IL-1RA on the ovulation process was already suggested by the results of Peterson et al. [55], who used the rat ovarian perfusion model and demonstrated that IL-1RA induced a lower ovulation rate than LH during a period of 20 h of perfusion. Moreover, it has been shown that intrafollicular injection of indomethacin, another anti-inflammatory molecule, disturbs the normal follicle development [40, 56]. In the present study, we presumed that, after injection, the IL-1RA intrafollicular level was maintained during a few hours. The mechanisms for the IL-1RA effect in the preovulatory follicle have to be demonstrated, but one could hypothesize that this effect is as a competitive factor for endogenous IL-1, which could be present during follicle maturation and ovulation. In our work, IL-1RA had no effect on the nuclear maturation rate of oocytes. This absence of effect has to be confirmed because, to our knowledge, it is the first work studying IL-1RA effects on oocyte maturation in vivo or in vitro. It seems important to better understand the potential implication of IL-1RA in oocyte maturation since we demonstrated recently the expression of IL-1RA mRNA in COCs [38].

In conclusion, although the mechanism by which IL-1ß, IL-1RA, and EGF regulate oocyte maturation are unclear, the IL-1 family may play an essential role in the physiology of COCs in the equine species. Moreover, our results showed that the IL-1 family also plays a central role in the ovulation process.


   ACKNOWLEDGMENTS
 
The authors wish to thank Laurence Debray, Ysoline Protin, Maud Caillaud, Bernard Bruneau, and the staff of the experimental study for technical assistance. We are grateful to Annick Lacombe for proofreading and correction of the English.


   FOOTNOTES
 
1 This work was supported by grants from the Institut National de la Recherche Agronomique, France, and the Haras Nationaux, France. A.M. was supported by a fellowship from the Institut National de la Recherche Agronomique, France, and the Région Centre, France. Back

2 Correspondence. FAX: 33 2 47 42 77 43; gerard{at}tours.inra.fr Back

Received: 9 October 2002.

First decision: 3 November 2002.

Accepted: 2 December 2002.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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