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Regular Article |
a Department of Obstetrics and Gynecology, University of Tokushima School of Medicine, Tokushima 770-8503, Japan
ABSTRACT
A cytokine-induced neutrophil chemoattractant (CINC/gro), which belongs to the interleukin (IL)-8 family, acts as a functional chemoattractant for neutrophils in rats. In the present study, we examined whether CINC/gro contributes to the ovulation process in the rat ovulation system. In rat ovaries, CINC/gro was immunohistochemically recognized in the theca layer of the antral follicle but not in the granulosa cells. To clarify the role of CINC/gro in the ovulation process, CINC/gro protein and mRNA were examined during pregnant mare serum gonadotropin (PMSG)-hCG treatment. CINC/gro protein did not increase as a result of PMSG injection. However, it increased rapidly after hCG injection and peaked at 6 h after hCG. CINC/gro mRNA was also strongly expressed after hCG injection. The increase of CINC/gro protein followed increases in IL-1ß and tumor necrosis factor
(TNF). In the whole ovarian dispersate culture, FSH, hCG, IL-1ß, and TNF stimulated the production of CINC/gro protein in a dose-dependent manner. In particular, the stimulatory effects of IL-1ß and TNF were stronger than those of gonadotropins. These results suggest that CINC/gro plays an important role in the rat ovulation process by attracting neutrophils. CINC/gro increased just prior to ovulation, and it may be regulated directly by cytokines such as IL-1ß and TNF and indirectly by gonadotropins.
cytokines, follicle, ovulation
INTRODUCTION
A cytokine-induced neutrophil chemoattractant (CINC/gro), an interleukin (IL)-8-like neutrophil chemoattractant, has been purified and cloned from the culture fluid of a rat kidney epitheloid cell line NRK-52E [1]. The primary structure of CINC/gro indicates that it is the rat counterpart of human GRO [2] and therefore belongs to the IL-8 family, which acts as a functional chemoattractant for neutrophils in vivo [3]. It is well known that the IL-8 family plays an important role in several types of inflammatory diseases, such as synovitis [4], nephritis, and dermatitis [5]. CINC/gro contributes to neutrophil infiltration into inflammatory sites in lipopolysaccharide-induced inflammation models in rats [6].
The ovulatory process, which is not an inflammatory disease but a physiological process, resembles an inflammatory reaction in that it is characterized by leukocyte infiltration [7] and the participation of cytokines such as IL-1ß and tumor necrosis factor (TNF) [8]. Infiltrating leukocytes, especially neutrophils, have been observed in thecal layer of ovaries in humans [9] and rats [10] just prior to ovulation. Peripheral blood leukocytes, when added to an in vitro-perfused rat ovary, increased the number of LH-induced ovulations [11], and neutrophil-depleted rats have a decreased rate of ovulations [12]. These findings suggest that leukocytes might participate in the ovulatory process.
Earlier reports have clearly shown the presence of chemotactic activity for neutrophils in human follicular fluid [13, 14]. Recently, IL-8 and GRO, which is a member of the IL-8 family, were detected in human follicular fluid, and their production was enhanced by hCG [15, 16]. In rabbits, the ovarian concentration of IL-8 increases rapidly after an injection of hCG. In IL-8-depleted rabbits, the rate of ovulation decreased and neutrophil accumulation and activation in the ovary were suppressed [17].
However, the issue of whether chemokine is an important modulator of rat ovulation has not been clarified. Interleukin-8 itself has not been identified in rats, which are commonly used as a model animal of the ovulatory process. In rats, the predominant chemokine of the IL-8 family appears to be CINC/gro. We therefore postulate that CINC/gro plays a pivotal role in the rat ovulatory process by accumulating and activating neutropils. In the present study, we investigated the profiles of CINC/gro, including the localization in the ovary, changes during ovulation, and regulation.
MATERIALS AND METHODS
Reagents
McCoy 5A medium was obtained from Gibco-BRL (Life Technologies, Tokyo, Japan), human recombinant IL-1ß was from R&D System (Minneapolis, MN), human recombinant TNF was purchased from Genzyme Corp. (Cambridge, MA), pregnant mare serum gonadotropin (PMSG), hCG, human pituitary FSH, 17ß-estradiol, and progesterone were from Sigma Chemical Co. (St. Louis, MO), and, [-32P]dCTP (3000 Ci/mmol) was purchased from Amersham (Bucks, UK). Anti-rat CINC/gro antibody was purchased from Immuno-Biological Laboratories Co. (Fujioka, Gunma, Japan). The rat CINC/gro cDNA probe was generously provided by Dr. Ohtsuka (Institute of Cytosignal Research, Tokyo, Japan).
Animals and Superovulation Procedure
All experiments were conducted in accordance with the ethical standards established by the institutional animal care and use committee of the University of Tokushima. Immature (21 days old) Sprague-Dawley female rats were obtained from Charles River Japan (Yokohama, Japan). They were induced to superovulate by subcutaneous injection of 10 IU of PMSG followed by 10 IU of hCG 48 h later. In this model, ovulation occurs between 12 and 15 h after hCG injection [18]. The above procedure represents a well-established and commonly reported model, which is used in the study of rat ovulation mechanism. This method enabled us to obtain samples in a more timely fashion than using adult female rats with spontaneous estrous cycles. Animals were killed by exposure to 100% CO2, and their ovaries were removed at 24 and 48 h after the injection of PMSG and at 4, 6, 8, 12, 24, and 48 h after hCG injection. The collected ovaries were stored at -80°C until a complete series of experimental samples could be assayed simultaneously. Half of each ovary was used for the determination of cytokine contents, and the other half was used for the determination of CINC/gro mRNA expression by reverse transcription polymerase chain reaction (RT-PCR).
Immunohistochemical Staining
The removed ovaries were immediately transferred to 4% paraformaldehyde in phosphate buffer for fixation. Paraffin-embedded sections (5 µm), which were stored at room temperature, were deparaffinized in xylene and rapidly rehydrated through an alcohol gradient. Endogenous peroxidase activity was quenched with 3% H2O2, and nonspecific antibody binding was blocked by 1% BSA in PBS. Tissue sections were incubated with anti-CINC/gro antibody at a concentration of 2 µg/ml as the primary antibody. Staining was performed by the labeled streptavidin biotin method using a DAKO LSAB kit (DAKO, Kyoto, Japan) and 3'3'diaminobenzidine containing 0.01% H2O2. All sections were counterstained with hematoxylin. For each experiment, normal rabbit serum was substituted for the primary antibody as a negative control.
Whole Ovarian Dispersate Culture Procedures
Whole ovarian dispersates from immature (3 wk old) rats were generally prepared as previously described [19]. Ovaries from intact rats were dissected free from their bursae, divided into four to six pieces, washed with 10 ml McCoy 5A medium, and subjected to enzymatic dispersion for 45 min at 37°C using 0.4% (wt/vol) collagenase, 0.001% (wt/vol) DNase, and 0.1% (wt/vol) BSA (0.1 ml/ovary). In the course of this incubation period, the ovaries were dissociated into a cell suspension by repeated pipetting at 30-min intervals with a graded series of micropipettes (inside diameter 0.5–1.0 mm). At the end of the incubation period, the cells were collected by centrifugation at 250 x g for 5 min, washed (three times) with McCoy 5A medium, and then resuspended in a known volume of the same medium. The dispersion procedure yielded approximately 3 x 106 cells/ovary. Cell viability, as assessed by the trypan blue dye exclusion test, was consistently >85%.
Whole ovarian dispersates were seeded in McCoy 5A medium (modified, without serum) supplemented with L-glutamine (2 mM), penicillin (100 U/ml), and streptomycin sulfate (100 µg/ml). Cells were plated in 24-well plates and culture dishs (35 x 10 mm; Falcon Plastics, Tokyo) at cell densities of 1 x 105 viable cells/well and 6 x 107 viable cells/dish, respectively, which were considered appropriate for the experimental design. Cells were treated with serum-free medium for 24 h before treatment with test agents was initiated. Cell cultures were maintained at 37°C under a mixture of 90% air and 5% CO2 at 100% humidity for 48 h to examine production of CINC/gro protein and for 6 h to study the expression of the corresponding mRNA.
At the end of each experiment, the culture medium was collected and frozen at -40°C for the quantification of CINC/gro by ELISA. Cells were used for isolation of total RNA.
Measurement of Cytokines
Ovaries were transferred to 500 µl of ice-cold PBS, and the samples were then sonicated (20 kHz, 160 W) at 30-sec intervals for 5 min. They were centrifuged at 10 000 x g for 10 min at 4°C to obtain the supernatant. CINC/gro, IL-1ß, and TNF in the ovarian extracts were measured by ELISA using a rat CINC/gro kit (IBL, Fujioka, Gunma, Japan), an IL-1ß kit (BioSource International, Camarillo, CA), and TNF kit (BioSource International), respectively. The lowest levels of detection for CINC/gro, IL-1ß, and TNF were 4.7 pg/ml, 3.0 pg/ml, and 0.7 pg/ml, respectively. The intraassay coefficent of variation (CV) for the three cytokines was 3.4%, 6.7%, and 3.1%, and the interassay CV was 3.5%, 8.7%, and 5.2%, respectively. The content of CINC/gro in culture medium was also determined in the same manner.
RT-PCR Analysis
Total RNA was extracted from the ovaries by the acid guanidinium thiocyanate-phenol-chloroform (AGPC) method. cDNAs were synthesized from 1.0 µg of RNA as previously described [20]. PCR primers for rat CINC/gro and glyceraldehyde-3-phosphate dehydrogenase (G3PDH) were obtained from GenBank and were synthesized by Boehringer Mannheim Biochemica (Tokyo, Japan). The sequences for these primers were as follows: CINC/gro: 5'-ATGGTCTCAGCCACCCGCTCG-3' (sense) and 5'-ACTTGGGGACACCCTTTAGC-3' (antisense); G3PDH: 5'-TGAAGGTCGGTGTCAACGGATTTGGC-3' (sense) and 5'-CATGTAGGCCATGAGGTCCACCAC-3' (antisense). These primers specify PCR products of 289 and 983 base pairs (bp), respectively. Amplification was initiated by 10 min of denaturation at 94°C for one cycle, followed by 33 cycles at 94°C for 30 sec, 60°C for 2 min, and 72°C for 3 min using a GeneAmp PCR System 2400 Thermal Cycler (Perkin Elmer/Cetus, Takara Biochemicals, Tokyo, Japan). After the last cycle of amplification, the samples were incubated at 72°C for 10 min. The specificity of the PCR bands was confirmed by restriction site analysis of the amplified cDNA, which generated restriction fragments of the expected size (data not shown). The final RT-PCR products were separated electrophoretically along with size standards on 1.2% agarose gels and visualized by ultraviolet (UV) illumination after staining with ethidium bromide. Loading equivalence was verified by equal intensities of rat G3PDH mRNA.
Northern Blot Analysis
Cells were harvested at appropriate times after various treatments, and total RNA was extracted by the AGPC method. Total RNA (20 µg) was size-fractionated by electrophoresis on 1% formaldehyde-agarose gels, transferred to nylon membranes (Hybond-N; Amersham, Tokyo, Japan) by capillary blotting, and cross-linked to the membrane using UV light. Hybridizations were conducted for 16 h at 45°C in a buffer that contained the entire 0.9-kilobase (kb) CINC/gro cDNA radiolabeled with [-32P]dCTP using a Megaprime DNA labeling kit (Amersham). After hybridization, the membranes were washed twice with 2x SSPE (1 x SSPE contains 0.18 M NaCl, 10 mM sodium phosphate, 1 mM EDTA, pH 7.4) and 0.1% SDS at room temperature for 15 min, once with 1x SSPE and 0.1% SDS at 50°C for 15 min, and twice with 0.5x SSPE and 0.1% SDS at 50°C for 10 min. The membranes were exposed to Fuji imaging plates, and the radioactivity levels were determined using a bioimage analyzer (Fuji BAS 1500; Fuji Co., Tokyo, Japan). The 1.0-kb fragment of human G3PDH cDNA (Clontech Laboratories, Palo Alto, CA) was used for RNA normalization.
Statistical Analysis
All data are expressed as the mean ± SEM. Statistical analyses were performed using a single-variable ANOVA followed by a Fisher protected least significant difference test. A P value of <0.05 was considered significant.
RESULTS
CINC/gro Immunohistochemical Staining in Rat Ovary
To investigate the production sites of CINC/gro in the rat ovary, we employed immunohistochemical techniques. Immunoreactive CINC/gro was localized in the theca layer, particularly in the antral follicles (Fig. 1). Signals of CINC/gro were mainly present in theca interna rather than theca externa. However, this signal was absent in the granulosa cells and oocytes.
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Change of Ovarian CINC/gro Production during Ovulation
The administration of PMSG had no effect on the ovarian concentration of CINC/gro protein. The first significant increase of CINC/gro was detected 4 h after the administration of hCG (P < 0.001 versus control). The CINC/gro level reached a peak that was 3.7 times the control value at 6 h after hCG was injected (P < 0.0005) and stayed at a significantly high level (P < 0.05 vs. control) until 24 h after hCG injection (Fig. 2A). To examine changes in ovarian CINC/gro mRNA expression, RT-PCR was performed. The expression of CINC/gro mRNA was very weak in the ovaries obtained from the rat that had been treated with PMSG alone and in the ovaries from the untreated rats. However, in the ovaries from the hCG-treated rat, CINC/gro mRNA was expressed strongly (Fig. 2B). Loading equivalence was verified by equal intensities of rat G3PDH mRNA.
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Effects of Hormones on CINC/gro Production in Cultured Whole Ovarian Dispersates
We examined the basal secretion of CINC/gro in cultured whole ovarian dispersates. CINC/gro secretion from unstimulated cells increased in a time-dependent manner from 6 to 48 h of incubation (data not shown). Therefore, whole ovarian dispersates were incubated for 48 h to examine the effects of various reagents on CINC/gro production.
The direct effect of various hormones known to be involved in the ovulatory process on ovarian CINC/gro production is shown in Figure 3. In the whole ovarian dispersate cultures, FSH stimulated the secretion of CINC/gro in a dose-dependent manner (Fig. 3A). Significant increases in comparison with controls were obtained with >0.1 ng/ml FSH. The increase in CINC/gro at 10 ng/ml of FSH was 2.1 times higher than that in the controls (P < 0.0001). Human CG also stimulated the secretion of CINC/gro in a dose-dependent manner (Fig. 3B), and at 1000 mIU/ml of hCG the increase was about 1.8 times the control value (P < 0.0001). However, neither estradiol nor progesterone caused any increase in CINC/gro production in the investigated range (Fig. 3, C and D).
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Effects of IL-ß and TNF on CINC/gro in Cultured Whole Ovarian Dispersates
We further examined the effects of IL-1ß and TNF, which are known to be involved in the ovulatory process, on ovarian CINC/gro production. IL-1ß markedly stimulated the release of CINC/gro (Fig. 4A). The effect of IL-1ß was concentration dependent. The increase of CINC/gro in the presence of 10 ng/ml IL-1ß was approximately 60 times the control value (P < 0.0001). TNF also stimulated the secretion of CINC/gro in a dose-dependent manner (Fig. 4B). In the presence of 10 ng/ml TNF, the CINC/gro value was about 4.4 times the control value (P < 0.0001).
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The expression of CINC/gro mRNA was examined by Northern blot analysis. In a time-course study, the CINC/gro mRNA level induced by IL-1ß and TNF was already elevated by 2 h posttreatment and peaked at 6 h (data not shown). Whole ovarian dispersates were incubated for 6 h to examine the dose-dependent effects. The IL-1ß treatment of cultured whole ovarian cells increased the level of CINC/gro mRNA (Fig. 4C); TNF also increased the level of CINC/gro mRNA in cultured whole ovarian dispersates (Fig. 4D).
Because IL-1ß and TNF increased the level of CINC/gro protein secretion and mRNA expression in cultured rat whole ovarian cells, we examined the IL-1ß and TNF levels during PMSG/hCG induced ovulation in relation to CINC/gro production. The levels of IL-1ß and TNF changed in parallel during the ovulatory process. They rapidly increased after hCG injection, peaked at 4 h after hCG, and returned to baseline levels by 6 h after hCG injection (Fig. 5). The peak levels were significantly higher than the levels prior to hCG injection (P < 0.05 versus baseline) and preceded the peak level of CINC/gro.
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DISCUSSION
The ovulatory process is similar to an inflammatory reaction in that it is characterized by leukocyte, especially neutrophil, infiltration. Neutrophils probably facilitate ovulation by producing several key mediators such as eicosanoids, collagenase, platelet-activating factor (PAF), and plasminogen activators [10, 21]. It has been suggested that prostaglandins cause rupture of the follicle by elevating ovarian contractility and thinning the follicular wall via enhancement of collagenolytic activity [7]. In an earlier study, PAF partially reversed the supression of ovulation caused by inhibitors of eicosanoid synthesis in rats [22]. Plasminogen activators, which are also synthesized by the granulosa cells, convert plasminogen into plasmin, and plasmin activates collagenase from the proenzyme form [7]. Collagenase contributes to the decomposition of conective tissue in the follicle wall. Thus, infiltrating neutrophils are important components of the ovulatory process. We investigated the production and regulation of CINC/gro, a chemoattractant for neutrophils, in rat ovary.
We demonstrated, by means of immunohistochemical techniques, that CINC/gro was present in the theca layer in the rat ovary just prior to ovulation. This is the first demonstration of the existence of CINC/gro, a member of the IL-8 family, in the rat ovary. These data also confirm that the main source of CINC/gro appears to be the theca layer, although we cannot completely exclude the possibility that macrophages, which are known to be a source of CINC/gro [23], also secrete it in the theca layer. Brännström et al. [10] observed infiltrating neutrophils in the thecal layer of rat ovaries just prior to ovulation. A similar localization of CINC/gro and neutrophils provides support for the possibility that CINC/gro attracts neutrophils into the area surrounding the preovulatory follicle.
If CINC/gro were truly involved in follicle rupture by means of attracting neutrophils during the ovulatory process in rats, CINC/gro should increase prior to ovulation. Our results indicate that the ovarian concentration of CINC/gro protein increased immediately after hCG injection. Arici et al. [15] demonstrated the presence of IL-8 in the follicular fluid of women undergoing ovulation induction and found that its concentration was enhanced by hCG. Ujioka et al. [17] reported that the ovarian concentration of IL-8 increased rapidly after hCG injection during the hCG-induced ovulatory process in rabbits. These results suggest that the CINC/gro detected during ovulation in rats is the equivalent of human and rabbit IL-8. The increase in CINC/gro mRNA after hCG injection suggests that the elevated concentration of CINC/gro protein found in the ovary was not due to transfer from peripheral blood into follicular fluid but to increased production at the transcriptional level.
We next investigated the factor that regulates CINC/gro production. We speculated that gonadotropin and sex steroid hormones might stimulate CINC/gro production. Among these hormones, FSH and hCG stimulated the secretion of CINC/gro in whole ovarian dispersate cultures. FSH stimulated CINC/gro secretion in vitro but not in vivo. This discrepancy may be explained by the presence of some inhibitors of CINC/gro production that are present in vivo. Glucocorticoids may inhibit CINC/gro production in rat ovaries. Ohtsuka et al. [24] reported that glucocorticoids inhibited the production of CINC/gro in cultured rat kidney cells. However, further studies will be required to clarify this point.
The effects of FSH and hCG on CINC/gro mRNA expression were also examined by Northern blot analysis. However, the band that hybridized to CINC/gro cDNA was very weak only when high concentrations (10 ng/ml FSH, 1000 mIU/ml hCG, up to 48 h of treatment) were used and films were exposed for long periods of time (data not shown). At high doses of FSH and hCG, CINC/gro mRNA expression was increased in cultured whole ovarian dispersates.
Interleukin-1ß and TNF, which are important cytokines in the ovulatory process, also stimulated CINC/gro protein production and mRNA expression. Their effects were much stronger than those of FSH and hCG. When ovaries from immature rats were treated with PMSG/hCG, IL-1ß and TNF were detected in the perfusate [25]. In particular, IL-1ß mRNA in ovary obtained from PMSG/hCG-treated rats increased after hCG injection and was localized in the thecal layer, particularly in the large preovulatory follicle [26]. These results suggest that these inflammatory cytokines may be a main regulator for CINC/gro production in the rat ovary. The changes of CINC/gro were compared with those of IL-1ß and TNF. CINC/gro, IL-1ß, and TNF were increased after hCG injection, and the peaks of IL-1ß and TNF preceded that of CINC/gro. We hypothesized that hCG stimulated IL-1ß and TNF secretion and that these cytokines then stimulated CINC/gro production. Ujioka et al. [27] reported that blocking the activity of IL-1ß with anti-IL-1ß antiserum inhibited the accumulation of IL-8 in the hCG-induced rabbit ovulatory process. Those findings are consistent with the results reported herein.
Thus, it is likely that CINC/gro production in theca layers is stimulated just prior to ovulation by IL-1ß and TNF induced by hCG injection. Furthermore, CINC/gro probably attracts neutrophils into the area surrounding the preovulatory follicles. Infiltrating neutrophils may contribute to timely follicle wall rupture by secreting specific proteolytic enzymes, prostaglandins, and other factors.
The data herein demonstrate that CINC/gro is produced in the rat ovary and that gonadotropin, IL-1ß, and TNF increased CINC/gro production just prior to ovulation. These findings suggest that CINC/gro plays an important role in the rat ovulatory process by attracting neutrophils to the areas surrounding preovulatory follicles.
ACKNOWLEDGMENTS
We thank Otsuka Cellular Technology Institute for help with the cytokines assays.
FOOTNOTES
First decision: 3 December 1999.
1 Correspondence: Kenjiro Ushigoe, Department of Obstetrics and Gynecology, University of Tokushima School of Medicine, 3-18-15, Kuramoto-cho, Tokushima 770-8503, Japan. FAX: 81 88 631 2630; ushi{at}clin.med.tokushima-u.ac.jp 
Accepted: February 15, 2000.
Received: November 1, 1999.
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X 174 phage DNA HaeIII digest); lane N: negative control (without cDNA); lane P: positive control (cDNA from cells known to express CINC/gro mRNA). Loading equivalence was verified by equal intensities of rat G3PDH mRNA. The expected sizes of the PCR products were 289 bp for CINC/gro and 983 bp for G3PDH
, TNF
, CINC/gro.