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Original Studies |
Department of Obstetrics and Gynecology, Institute for the Health of Women and Children, Göteborg University, 413 45 Göteborg, Sweden
Address correspondence and requests for reprints to: Eva Runesson, Department of Obstetrics and Gynecology, Göteborg University, Sahlgrenska University Hospital, 413 45 Göteborg, Sweden. E-mail: eva.runesson{at}medfak.gu.se
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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and
IL-1ß, but not tumor necrosis factor , enhanced IL-8 secretion
from both cell types. IL-8 levels in cultures of granulosa-lutein cells
from hyperstimulated in vitro fertilization
cycles were not affected by either gonadotropins or steroids. These
data provide evidence that ovarian IL-8 is gonadotropin and cytokine
induced and may be involved in the hormonally regulated stages of
follicular development and ovulation. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A common phenomenon in inflammatory reactions is the gathering of leukocytes at the site of inflammation. Studies on the presence of these cells in ovarian tissue have revealed increased concentrations of some specific subsets of leukocytes in the preovulatory follicle at the time of ovulation (4, 5). A functional role of these cells in ovulation is likely, because the addition of leukocytes to in vitro perfused rat ovaries increases the LH-induced ovulation rate (6) and because depletion of circulating neutrophils in the rat decreases the ovulation rate in vivo (7).
A network of locally produced cytokines orchestrates the regulation and
recruitment of the leukocytes to any inflammatory site. Several of
these cytokines have been proposed to act as regulators in different
processes in reproductive physiology. In ovarian physiology,
interleukin 1 (IL-1), IL-2, IL-6, IL-8, tumor necrosis factor ,
colony-stimulating factor-1 and granulocyte/monocyte colony-stimulating
factor may be of particular significance (8, 9, 10, 11, 12). A
distinct subgroup of cytokines is the chemokines, which mainly act as
leukocyte chemoattractants. These chemokines and other specific
chemotactic factors usually work in concert to accumulate and activate
the subsets of leukocytes, appropriate for each specific tissue and
condition.
Neutrophilic granulocytes, which make up 
60% of the total leukocyte
pool in human peripheral blood (13), are only present in
antral follicles (14) and accumulate in the theca layer of
the ovulating follicle (5). Neutrophils contribute to
tissue degradation by release of a panel of proteolytic and vasoactive
factors, and this cell type has been proposed to be of importance in
the ovulatory process. Activation of these cells in the ovary, by the
preovulatory LH surge, was demonstrated in the rabbit ovary, where the
levels of myeloperoxidase and neutrophil elastase increased after human
CG injection (15).
One of the most potent neutrophil chemoattractants in the human is
IL-8. This chemokine attracts and activates mainly neutrophils but has
also some minor activity on other types of leukocytes. By its capacity
to attract and activate neutrophils into sites of tissue damage, IL-8
is of considerable interest in the mechanisms of tissue degradation
occurring in the follicular wall at ovulation. Previously, we and
others have evaluated the presence of this chemokine in follicular
fluid and granulosa-lutein cells from hyperstimulated women undergoing
in vitro fertilization (IVF) (10, 16).
Most of these functional studies concerning proposed intraovarian
mediators in human ovulation and regulation of corpus luteum function
have been carried out on fluids and cells obtained from patients
undergoing gonadotropin hyperstimulation. Because this is a
nonphysiological situation, results obtained in these studies may not
always be in agreement with the events occurring in the normal
menstrual cycle, as exemplified by the differences in prostaglandin
F2 receptor regulation by luteal cells
(17, 18).
The purpose of the present study was to evaluate the presence and regulation of IL-8 in follicles from the normal menstrual cycle and to compare some of the results with those of the hyperstimulated cycle.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Estradiol and progesterone were obtained from
Sigma-Aldrich Corp. GmbH (Steinheim, Germany); recombinant
human FSH (rhFSH) was from Organon (Oss, The Netherlands);
rhLH was from Serono (Rome, Italy); fetal bovine serum was from
Life Technologies, Inc. (Paisley, UK); and recombinant
IL-1, IL-1ß, and tumor necrosis factor were from Peprotec EC.
(London, UK). The polyclonal rabbit antiserum against human IL-8 was a
kind gift from Dr. R. W. Kelly (MRC Reproductive Biology Unit
Center for Reproductive Biology, Edinburgh, UK). The monoclonal
antihuman antibody for leukocyte common antigen (CD45) was purchased
from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA), and
the monoclonal antihuman antibody for macrophage detection (CD 68) was
from DAKO Corp. (Glostrup, Denmark).
Subjects
Tissue samples and follicular fluids for the evaluation of IL-8
in normal menstrual cycles were obtained from 28 women undergoing
surgery for nonovarian diseases. Only women (mean age, 44 yr; range,
29–54) who—based on menstrual data, hormonal profiles, and
intraoperative findings—were operated on during the follicular phase
were included. None of the women were taking any medication known to
affect the normal physiological follicular state. All women had given
their informed consent before surgery, and the study was approved by
the Human Research Ethics Committee at the Faculty of Medicine,
Göteborg University. Serum and plasma samples were collected from
each woman at the time of surgery. According to menstrual data and the
levels of estradiol, LH, and progesterone in serum, the women were
grouped into midfollicular phase [ cycle days -6 to -4 in
relation to predicted LH surge (day 0); estradiol, <0.6 nmol/L; LH,
<8 IU/L; progesterone, <6 nmol/L], late follicular/early ovulatory
phase [ cycle days -3 to -0; estradiol, >0.6 nmol/L; LH, >8
IU/L; progesterone, <10 nmol/L], or late ovulatory phase ( cycle
days 0 to +1; LH, <8 IU/L; progesterone, >10 nmol/L]. All follicles
collected were more than 9 mm in diameter, and collections from
midfollicular phase were performed later than cycle day -6, to
ascertain that material from the dominant follicle (19)
was obtained. Follicular fluids and granulosa lutein cells (GLCs) from
hyperstimulated cycles were obtained at oocyte pick-up of 13 patients
(mean age, 31 yr; range, 24–37 yr) undergoing standardized controlled
ovarian hyperstimulation (10) for subsequent IVF treatment
in our unit.
Follicular fluid
Follicles were removed as the initial procedure during surgery, and follicular fluids were then immediately aspirated. Follicular fluids from IVF cycles were sampled at the time of aspiration at oocyte pick-up, as described previously (10). The fluid volumes were recorded, and this was followed by centrifugation at 200 x g for 10 min to remove cells, and subsequently storage of aliquots at -70 C.
Tissue isolation and cell cultures from normal menstrual cycles
After surgical removal of the follicle, it was placed in ice-chilled PBS and then immediately brought to the laboratory. Follicular fluid was aspirated from the follicle and centrifuged to collect the granulosa cells (GCs). The interior wall of the follicle was gently scraped with an operating instrument (Strabismus Hook; PMS, Tuttlingen, Germany) to further harvest the GCs, which would still be attached to the interior of the follicle in accordance with our previous experience (20). The two cell preparations were pooled, transferred to medium 199 (M199; Life Technologies, Inc.) supplemented with NaHCO3 (0.026 M) and gentamicin (50 µg/mL). Cells were then washed four times, and their viability was examined using Trypan Blue exclusion. Viability of GCs was between 50% and 60% in all samples. The theca interna cell layer was pulled away from the underlying theca externa cell layer using watchmaker’s forceps. Theca cells (TCs) were isolated after enzymatic treatment as described previously (20), but with some minor modifications. Briefly, TCs were cut into 1 x 1-mm pieces, incubated in PBS with collagenase type I (3 mg/mL; Worthington Biochemical Corp., Freehold, NJ), bovine pancreatic DNase grade II (5 µg/mL; Roche Molecular Biochemicals GmbH, Tutzing, Germany), and hyaluronidase (1 mg/mL; Sigma-Aldrich Corp. GmbH, Steinheim, Germany) for 45 min at 37 C. Every 15 min of incubation, undigested cells were mechanically separated using a Pasteur pipette. Theca cells were rinsed three times in M199, counted in a Bürker chamber, and resuspended in medium. Cell viability was determined by trypan blue exclusion and was found to be between 80% and 95% in all experiments.
GLCs from hyperstimulated cycles
GLCs from IVF patients were collected at the time of follicular aspiration and prepared for culture as described previously (10). The aspirates generally contain a considerable number of red blood cells (21), and, to obtain a purer GLC preparation, the cell suspensions were subjected to isotonic percoll centrifugation. After centrifugation, GLCs were collected and were further prepared in the same manner as for GCs of the normal cycle (see above).
Cell culture condition
GCs, TCs, and GLCs (3 x 104
cells/well) were seeded in 0.5-mL medium supplemented with fetal bovine
serum (10%) on 24-well plates (Falcon; Becton Dickinson, Meylan,
France) and cultured for 24 h to allow attachment of the cells.
The media were then changed, and the cells were exposed to either
estradiol (10 ng/mL), progesterone (10 ng/mL), rhLH (10 ng/mL), or
rhFSH (10 ng/mL) for 48 or 96 h. A dose response (1, 10, and 100
ng/mL) experiment with rhFSH was performed in four GC cultures. In a
subset of experiments, the cytokines IL-1, IL-1ß, and TNF- were
added at a concentration of 3 ng/mL. At the end of each experiment, the
supernatants were collected, aliquoted, and stored at -70 C until
analyzed.
Immunohistochemistry
Follicles were frozen in OCT embedding medium (Tissue Tek; Miles Inc., Elkhart, IL) and stored at -70 C until used. Tissue was cut into 8-µm thick sections, placed on microscope slides (SuperFrostPlus, Menzel-Gläser, Germany), air-dried, and fixed in acetone for 5 min. Following peroxidase reduction (0.3% H2O2 in methanol, 30 min), the sections were washed with PBS, incubated with serum for 30 min, followed by IL-8 antiserum (1:500) or CD 45 antibody (1:50) at 4 C overnight. An avidin-biotin-peroxidase developing system (Vectastain ABC-kit; Vector Laboratories, Inc., Burlingame, CA) was used, with final detection by 3,3'-diaminobenzidine tetrahydrochloride (Sigma-Aldrich Corp. Gmbh) as the peroxidase substrate. The slides were counterstained with hematoxylin and mounted in Pertex (Histolab, Göteborg, Sweden). Human tonsil tissue was used as positive control tissue for IL-8 and CD 45. As a negative control, the primary antibody was substituted with an unspecific rabbit IgG (R&D, Abingdon, UK) and an unspecific mouse IgG (R&D). The specificity of the IL-8 antibody has previously been confirmed (22).
To evaluate the proportion of immune cells in the different cell cultures, the harvested GCs, TCs, and GLCs were cultured in Petri dishes for 48 h, fixed in methanol, and then stored at -20 C until analyzed. Cells were incubated with monoclonal antihuman CD 45 (common leukocyte antigen) antibody (1:50) and CD 68 (activated macrophages; 1:50) at room temperature overnight. Primary antibody binding was visualized using a biotinylated horse antimouse antibody and a streptavidin-avidin fluorescein isothiocyanate detection system (Amersham Pharmacia Biotech, Buckinghamshire, UK). Two separate experiments on each type of cell culture were evaluated.
Immunoassays
IL-8 was quantified using an enzyme-linked immunosorbent assay (R&D) that previously has been used and validated for measurements of IL-8 in follicular fluid and GLC cultures (16). According to the manufacturer, no significant cross-reactivity or interference with other cytokines was observed for the IL-8 assay, and the sensitivity was 10 pg/mL. Serum estradiol and LH were measured with a microparticle enzyme immunoassay (Abbott Laboratories, Abbott Park, IL). The sensitivities were 25 pg/mL and 0.5 IU/L, respectively. Progesterone in serum was measured with a solid phase fluoroimmunoassay (Delfia Wallac, Inc., Turku, Finland). The sensitivity was 1 nmol/L. The intra-assay variations of all assays were less than 5%, and the interassay variations were less than 10%.
Statistics
Treatments were performed in duplicates or triplicates. The data are presented as mean ± SEM. All statistical analyses were performed on absolute values. Multiple comparisons were made by one-way ANOVA, followed by Fisher’s post hoc test, and pairwise comparisons were made by paired Student’s t test. Pearson’s linear correlation was used for correlation analyses. Significance was assumed at P less than 0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The levels of IL-8 in follicular fluid from dominant
follicles from three different phases (midfollicular, late
follicular/early ovulatory, late ovulatory) of the menstrual cycle were
investigated and compared with the levels of IL-8 in blood plasma
obtained at the same time. IL-8 levels in plasma were similar (mean
values between 15–30 pg/mL) at the three phases. There was no
difference between plasma and follicular fluid levels at midfollicular
phase, whereas higher levels were found in follicular fluid than in
blood plasma of late follicular/early ovulatory phase and late
ovulatory phase (Fig. 1
). Levels of IL-8
in follicular fluid from dominant follicles of late follicular/early
ovulatory phase (259 ± 132 pg/mL) and late ovulatory phase
(175 ± 61 pg/mL) were significantly higher than in follicular
fluid from dominant follicles of midfollicular phase (34 ± 18
pg/mL)(Fig. 1). At the end of gonadotropin hyperstimulation for IVF,
the levels of IL-8 in blood plasma (<5pg/mL) and follicular fluid
(275 ± 35 pg/mL) were comparable with those of the late
follicular phase of the natural cycle. A positive correlation (r =
0.50, P < 0.05) was found between follicular fluid
volume and follicular fluid IL-8 levels in follicles when all three
stages of the natural cycle were included (Fig. 2A).
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, 
, dotted
line). A subgroup analysis of the stages of the natural cycle that
would be of comparable developmental stage (late follicular/early
ovulatory and late ovulatory stage) to the IVF samples did not show any
correlation (Fig. 2B, •, solid line). IL-8 levels in cultures of GCs, TCs, and GLCs; effects of gonadotropins and steroids
The levels of IL-8 in the media of cultured GCs (Fig. 3, A and B) and TCs (Fig. 4, A and B) were somewhat higher after
the first 48 h of culture compared with the second 48 h of
culture. The levels of IL-8 were significantly increased by the
presence of rFSH (10 ng/mL) in GC culture both during the first and
second 48-h period (Fig. 3B). In all eight individual experiments,
higher IL-8 levels were seen after FSH treatment compared with control.
A similar effect in GCs was seen by rLH (10 ng/mL) (Fig. 3B), which
caused increased IL-8 levels in all five cultures, although the mean
value was not significantly higher than the mean value of control. The
levels of IL-8 under control conditions were about 10-fold higher in
cultures of TCs compared with GCs. No effect by gonadotropins was seen
in the TC cultures (Fig. 4B). Estradiol and progesterone did not affect
the IL-8 levels in the media conditioned by either of the two cell
types (Figs. 3A and 4A).
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). The IL-8 levels were lower after
96 h of culture, and the maximal stimulatory effect was seen at
100 ng/mL (data not shown).
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Effects of proinflammatory cytokines on IL-8 secretion from GCs and TCs
The effects of the proinflammatory cytokines IL-1 and TNF- on
IL-8 secretion were tested at a cytokine concentration of 3 ng/mL, a
concentration that previously has been shown to be in the range where a
clear stimulation of mediator products of human GLCs was seen
(11, 16).
The cytokine TNF- (3 ng/mL) did not affect IL-8 secretion in any
cell type, whereas both IL-1 (3 ng/mL) and IL-1ß (3 ng/mL)
markedly enhanced IL-8 secretion in GC and TC cultures. The levels in
the IL-1-stimulated cultures were 5-fold higher than in control
culture after 48 h. During the second 48-h culture period, the
levels were up to 10-fold higher when compared with the control levels
at that time, although the absolute levels were similar to those of the
first 48-h culture (Fig. 6).
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The distribution of IL-8 and total leukocytes (CD
45+) in the dominant preovulatory follicle was
investigated by immunohistochemistry. To evaluate any differences
between dominant and nondominant follicle, the distribution of IL-8 and
leukocytes in follicles (5 mm) taken from women in the secretory
phase of the menstrual cycle was also studied.
Positive staining for IL-8 was seen in the TC layer of
preovulatory follicles, with a higher intensity localized around blood
vessels. Moderate IL-8 staining could be seen in the GC layer (Fig. 7A). In the nondominant follicle,
moderate staining for IL-8 was seen in the TC compartment, but no
staining was seen in the GC layer (Fig. 7B). Positive staining for CD
45 was seen predominantly in the TC layer of preovulatory
follicles (Fig. 7C), whereas very few CD 45-positive cells could be
localized in small follicles (Fig. 7D).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The IL-8 levels in follicular fluid of the dominant follicle were higher than in blood plasma during late follicular/ovulatory phase, and this difference was not seen during midfollicular phase. The increased levels of IL-8 in the preovulatory follicles, compared with the follicles of midfollicular phase, were further demonstrated by the existence of a positive correlation between follicular fluid volume and follicular fluid IL-8 concentration in dominant follicles. The concentration of IL-8 in follicular fluid from late follicular/ovulatory phase of the natural cycle was comparable with those measured in follicles punctured for IVF in hyperstimulated cycles in the present study and what have been previously reported (16). Interestingly, only a small portion of the different cytokines that have been found in human follicular fluid are present in higher concentrations in this compartment compared with blood (reviewed in Ref. 23). Most of these studies have been carried out on follicular fluids from IVF patients. The results of the present study clearly show that there also exists a concentration gradient of IL-8 from the follicle to the blood in the natural cycle, further pointing out that this is a true physiological phenomenon. The higher concentration of IL-8 in follicular fluid of preovulatory follicles, compared with the follicles of midfollicular phase, is most likely due to increased IL-8 secretion by the follicular cells of this late differentiation stage. We and others (10, 16) have previously shown that the GLCs of the hyperstimulated cycle is a source of IL-8. In the present study, we present data that both GCs and TCs of the preovulatory follicle obtained from physiological cycles can secrete IL-8 in accordance with previous studies on RNA expression (10, 14). It is well known that the follicular fluids of the hyperstimulated cycle contains immune cells (24, 25), probably because the follicles are punctured at a very late stage of the ovulatory process. The immune cells produce large quantities of various cytokines, including IL-8, so that the possibility exists that follicular fluid IL-8, at least from the follicles of hyperstimulated cycles, can be derived from these cells.
The present study shows that both GCs and TCs can secrete IL-8,
with the secretion from the TCs being 10 times greater than the GC
secretion. Thus, it can be assumed that the main source of IL-8 in the
preovulatory follicle is cells of the theca layer. The
immunohistochemical observation of the present study, with positive
IL-8 staining in the theca area, and results of a recent study where
both protein and messenger RNA for IL-8 were detected in the theca
compartment from antral follicles (14), support this
suggestion. The theca layer of the human follicle contains leukocytes
with some time-dependent fluctuations in the distribution pattern of
certain leukocyte subsets (5, 26). Because there is an
extensive vascular network in this layer (27, 28), with
ongoing redistribution of IL-8-responsive leukocytes, a high basal
secretion of IL-8 from TC cultures of our study was expected. The TC
preparation most likely contains theca layer-derived leukocytes and
fibroblasts, with IL-8-secreting properties, and about 3–4% of CD
45-positive cells were found in the TC cultures. Therefore, a
proportion of the IL-8 found in TC cultures may be due to secretion
from these cells.
In the present study, the basal IL-8 secretion from cultured GCs from normal cycles was approximately one tenth of that from cultured TCs. This is in accordance with a previous study, where immunoreactive IL-8 protein and its messenger RNA were found in the granulosa layer from antral follicles, but to a lesser extent compared with the theca layer (14). Although the secretion of IL-8 from the TCs is higher than from GCs, the GCs should still be considered as a possible source of IL-8 with a physiological role, because most functions of chemokines require very low concentrations.
Because the peak in follicular fluid IL-8 concentrations was associated with late follicular/ovulatory phase follicles and because the levels of IL-8 are high in follicles of IVF cycles, it can be hypothesized that gonadotropins and/or steroids may regulate IL-8 expression. The midcycle gonadotropin surge is reflected in the follicular fluid compartment of the human follicle, and both LH and FSH in follicular fluid of large follicles are at their highest levels during this stage (29). The estradiol levels in follicular fluid rise in parallel with the development of the follicle and the acquisition of an increasing pool of GCs expressing aromatase. The peak levels of estradiol are present during late follicular phase (30), whereas progesterone levels rise in concert with and after the LH surge. The IL-8 secretion from GCs of normal cycles was found to be stimulated by FSH and most likely by LH. This stimulation by FSH was found to be dose dependent. LH did not affect the IL-8 secretion from the TCs, and neither did FSH, which was expected due to the lack of FSH-receptor expression on TCs. It seems as if the GC responsiveness to FSH, in terms of IL-8 secretion, is only present when these cells have developed under physiological conditions, because FSH did not influence IL-8 secretion from cultured GLCs of hyperstimulated cycles. The latter finding is in agreement with a previous study, where FSH did not affect IL-8 secretion from GLCs (16). One possible explanation to this difference regarding FSH responsiveness is that the FSH receptor in GLCs is down-regulated because these cells have been exposed to substantially higher FSH levels in vivo during the controlled ovarian hyperstimulation. There seems to exist some uncertainties concerning the effect of LH on regulation of IL-8 secretion in ovarian cells. Thus, human CG/LH has previously been demonstrated to stimulate IL-8 secretion by human ovarian stroma cells and GLCs (16). The results of the present study in cultured GLCs did, however, not show any LH-induced IL-8 production. Even if the effect of LH on GLCs could be expected to be negligible due to LH receptor occupancy and down regulation, any differences in the IVF stimulation protocols used could account for the discrepancies in results.
Progesterone has previously been suggested to be a negative regulator of IL-8 secretion in cultured GLCs, stromal cells, and endometrial cells (16, 31). Progesterone receptors are not present in GCs and TCs, which have not yet initiated their luteinization by LH (32, 33). The results of the present study, demonstrating a lack of progesterone influence on IL-8 secretion, are in line with this concept.
Proinflammatory cytokines are proposed to be involved in ovarian
processes, and TNF- as well as IL-1 are present in human follicular
fluid (34) and stimulate ovulation in the perfused rat
ovary (35, 36). These cytokines induce IL-8 secretion from
immune cells as well as from several other cell types (37, 38). In the present study, we found a pronounced effect on IL-8
secretion by IL-1 and IL-1ß in both GC and TC cultures. The
stimulatory effect by IL-1 on IL-8 secretion from GCs and TCs was found
to be conserved during the second 48-h culture period, whereas the IL-8
levels in control cultures decreased by 50% during the second 48-h
period. In contrast, there was a parallel decreased IL-8 secretion in
both FSH-stimulated and control cultures of GCs. This may indicate a
stimulatory effect of IL-1 on the viability of the GCs and TCs.
The effect of these cytokines on IL-8 secretion in cells from the theca
compartment is most likely an effect on both TCs and other cells, such
as the immune cells, residing in the theca layer. On the contrary, the
effects of IL-1 and IL-1ß in GC cultures are most likely directly
on the GCs because no CD 45-positive cells were seen in these
cultures.
In the present study, there was no effect of the cytokine TNF- on
any cell type. Thus, selectivity in cytokine modulation of IL-8
secretion in both GCs and TCs exist. Even though IL-1 and TNF- in
most cases would similarly induce a chemokine response, there are
cells, such as neural cells (39), that do not respond to
TNF-. Effects by TNF- on ovarian steroid-producing cells may
first appear at luteinization because IL-8 secretion from GLCs are
reported to be induced by TNF- (16).
The main function for IL-8 in the preovulatory follicle may be to act as a chemoattractant and activator of neutrophils. Thus, the LH surge with secondary effects on follicular IL-8 levels could contribute to the massive invasion of leukocytes to the ovulating follicle (4, 5). The ovarian-bound pool of leukocytes, of which some are specialized in tissue degradation, can in turn contribute to the inflammatory-like ovulatory process by release of proteolytic enzymes, such as plasminogen activator, kallikrein, elastase, collagenase, and also vasoactive substances. The action of IL-8 as a neutrophil chemotatic and activating factor in the mammalian ovary is supported by the findings that neutralization of IL-8 activity reduces the levels of both myeloperoxidase, an indicator of neutrophil accumulation, and neutrophil elastase, an indicator of neutrophil activity, in the rabbit ovary (15). Interestingly, these activities were not fully blocked, suggesting that IL-8 may not be the sole component in this process. Other chemokines probably act in combination with IL-8, because recruitment of leukocytes to any inflammatory tissue is in general orchestrated by several chemokines working together to accumulate the different immune cells needed. Furthermore, administration of an IL-8 antiserum led to a 30% reduction of the ovulation rate in the rabbit (15), which is similar to that caused by neutrophil depletion in the rat (7), indicating a role for IL-8 on neutrophils in mammalian ovulation.
The main hypothesis concerning IL-8 in ovarian function involves IL-8 as a potent mediator of ovulation by recruitment and activation of neutrophils. However, this chemokine could also, as shown in other tissues (40, 41), be a component involved in regulation of proliferation and angiogenesis. These two processes are important for growth of the follicle during the final stages of folliculogenesis and in the transition of the preovulatory follicle into a corpus luteum. The findings that gonadotropins induce IL-8 production in GCs but not in TCs suggests specific actions of IL-8 in the different cell layers.
It can be speculated that IL-8 function may be stage specific in the human ovary and that gonadotropin- and cytokine-induced IL-8 secretion from follicle cells could lead to different actions. The demonstration of IL-8 as a proliferative agent on endometrial stromal cells (42), and the evidence that FSH induces follicular growth through an indirect regulation of growth factor expression in GCs (43), supports the hypothesis that the FSH-induced IL-8 production from GCs found in the present study could contribute to the proliferative events occurring in the developing follicle.
At the time of ovulation, IL-8 function may mainly be as an attractant and activator of neutrophils because leukocyte invasion into the ovulating follicle is seen after administration of LH/human CG (4).
In summary, this study has revealed that the concentrations of the chemokine IL-8 are elevated in follicular fluid of late follicular/ovulatory phase follicles of the natural cycle and that GCs and TCs secrete IL-8. The differences in regulation of IL-8 secretion from the GCs and TCs by gonadotropins and cytokines indicate a stage-specific and pleiotropic IL-8 function and further suggest a role for IL-8 in follicular development and/or ovulation.
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Footnotes |
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Received December 15, 1999.
Revised June 28, 2000.
Accepted July 27, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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 K. H. H. Wong, H. Negishi, and E. Y. Adashi Expression, Hormonal Regulation, and Cyclic Variation of Chemokines in the Rat Ovary: Key Determinants of the Intraovarian Residence of Representatives of the White Blood Cell Series Endocrinology, March 1, 2002; 143(3): 784 - 791. [Abstract] [Full Text] [PDF]
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) and follicular fluid (
) during three different stages of the
follicular phase of the human menstrual cycle [MF, midfollicular phase
(n = 13); LF/EO, late follicular/early ovulatory phase (n =
3); LO, late ovulatory phase (n = 6)]. Data are presented as
mean ± SEM. *, P < 0.05; **,
P < 0.01.
