Biol Reprod Track the topics, authors and articles important to you
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

BOR - Papers in Press, published online ahead of print June 23, 2004.
Biol Reprod 2004, 10.1095/biolreprod.104.030650
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
71/4/1384    most recent
biolreprod.104.030650v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Bonello, N.
Right arrow Articles by Norman, R. J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Bonello, N.
Right arrow Articles by Norman, R. J.
Agricola
Right arrow Articles by Bonello, N.
Right arrow Articles by Norman, R. J.
BIOLOGY OF REPRODUCTION 71, 1384–1390 (2004)
DOI: 10.1095/biolreprod.104.030650
© 2004 by the Society for the Study of Reproduction, Inc.

Ovary

Periovulatory Expression of Intercellular Adhesion Molecule-1 in the Rat Ovary1

Nigel Bonello2, Melinda J. Jasper, and Robert J. Norman

Research Centre for Reproductive Health, Department of Obstetrics and Gynaecology, University of Adelaide, Queen Elizabeth Hospital, Woodville, South Australia 5011, Australia


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Leukocytes, especially neutrophils and macrophages, traditional cellular regulators of the immune system, reside within the tissue architecture of the rodent and human ovary and dramatically increase in number, in response to gonadotropin, in the theca of preovulatory follicles. Evidence strongly suggests a modulatory role for leukocytes in ovarian tissue remodeling events, such as ovulation, luteinization, and luteolysis. The present study investigates the ovarian localization and potential gonadotropin regulation of intercellular adhesion molecule-1 (ICAM-1), an important factor in neutrophil and monocyte attachment to endothelium. Reverse transcription-polymerase chain reaction and immunohistochemical detection and quantification of ICAM-1 mRNA and protein were carried out in ovaries of immature and eCG/hCG-primed rats during the periovulatory period (0, 6, 12, and 24 h post-hCG). While whole ovarian ICAM-1 mRNA levels did not vary significantly during the preovulatory period, ovarian follicles exhibited ICAM-1 mRNA and protein specifically within the thecal region, where mRNA expression increased 5-fold and protein expression increased 6-fold when comparing pre-hCG levels with those at the estimated time of ovulation (12 h post-hCG). Thecal ICAM-1 was most prevalent in highly vascularized regions as evidenced by serial staining with an endothelium-specific antibody. Granulosa layer ICAM-1 immunoactivity was acquired only during/after follicle rupture. These results show ICAM-1 is localized within the ovarian theca and its expression is associated with follicular development in periovulatory follicles, peaking in expression at the time of rupture. Additionally, ICAM-1 is expressed among granulosa-lutein cells of the ovulating follicle and developing corpus luteum. Taken together, these findings suggest rat ovarian ICAM-1 may be instrumental in the active recruitment of leukocytes into the preovulatory ovary and may have a role in corpus luteum formation.

follicle, ICAM-1, immunology, ovary, ovulation, theca cells


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The ovary is recognized as an important site of immune-endocrine-reproductive interactions, where leukocytes and their secreted products play a substantial role in hormonally regulated, inflammation-like ovarian transitions leading to and following follicle rupture [1, 2]. With some exceptions, evidence shows neutrophils and monocyte/macrophages in particular, infiltrate the vascularized thecal region of mammalian Graafian follicles, including the rat, rabbit, and human, where maximal leukocyte density occurs just prior to ovulation [2–5]. The relevance of such leukocytes to ovarian function at ovulation is demonstrated through rodent studies, where leukocyte supplementation or depletion results in enhanced or reduced ovulation rates, respectively [6–9]. Activated leukocytes are a rich source of ovulation-facilitating collagenases, plasminogen activators, and reactive oxygen species involved in follicle wall connective tissue breakdown prior to rupture and subsequent luteal cellular reorganization. Evidence suggests other proinflammatory leukocyte products, including eicosanoids, platelet-activating factor (PAF), nitric oxide (NO), and cytokines (e.g. interleukin 1ß [IL-1ß] and tumor necrosis factor {alpha}), may also exert pro-ovulation and proluteinization effects through vascular (flow, permeability, and angiogenic) or steroidogenic pathways [10–12]. Additionally, macrophage phagocytosis of luteal cellular debris is a key component of the subsequent tissue remodeling process in luteolysis [13].

The preovulatory ovarian sequestration of leukocytes is assumed to parallel events observed elsewhere in the body, involving leukocyte activation, adhesion on endothelium-expressing adhesion molecules, transendothelial migration, and chemoattraction into "inflamed" areas of ovarian tissue [14]. However, while the potential chemotaxins involved in the preovulatory recruitment of leukocytes have received attention [15–20], ovarian studies have not yet adequately investigated the periovulatory expression of adhesion molecules primarily responsible for leukocyte-endothelial interactions, such as intercellular adhesion molecule type-1 (ICAM-1). ICAM-1 is a cytokine-inducible, or constitutively expressed but cytokine-upregulatable, 76–114-KDa, single-chain transmembrane glycoprotein of five extracellular immunoglobulin-like domains [21–23]. Through binding of its ß2-integrin ligands, lymphocyte function associated antigen-1 (LFA-1) and Mac-1 [24, 25], ICAM-1 plays a significant role in the inflammatory attachment of granulocytes and mononuclear blood cells to endothelial cells, although the systemic distribution of ICAM-1 is varied and also includes hematopoietic, epithelial, and fibroblast cell types, in addition to endothelium [26]. In rat mesenteric venules, enhanced leukocyte-endothelial interactions resulting from ovulation induction are attenuated by ICAM-1 antibody administration [27]. Ovarian cells thus far reported to express ICAM-1 protein include the oocyte [28], luteinized granulosa cells [29], and vascular structures or parenchyma of regressing corpora lutea [30–32]. These studies have shown leukocyte binding function is retained with ovarian expressed ICAM-1 [29], and areas of ovarian ICAM-1 staining appear to colocalize with leukocyte distribution [32]. Furthermore, a soluble form of ICAM (sICAM-1) exists in follicular fluid and serum [33, 34], and we have shown serum sICAM-1 levels are maximal in the menstrual cycle's follicular phase [35].

Hence, the aims of this study were 2-fold: first, to track any changes in whole ovarian ICAM-1 mRNA expression in equine chorionic gonadotropin (eCG)-/human CG (hCG)-primed rats across the periovulatory period, and, second, to locate and quantify expression of ICAM-1 mRNA and protein within specific regions of the ovary. It is envisaged such information will contribute to our understanding of the mechanisms leading to leukocyte recruitment into the ovary prior to ovulation and early luteinization.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animal Treatments and Tissue Collection

Experimental numbers and interventions outlined in this project were approved by the University of Adelaide Animal Ethics Committee. Immature female Sprague-Dawley rats, weighing 50–75 g, were maintained under controlled temperature and photoperiod (14L:10D) with access to water and pelleted food ad libitum. At 27 days of age, rats received 16 IU eCG (Intervet, Boxmeer, Netherlands) s.c. to promote the growth and maturation of a first generation of antral follicles, followed 48 h later by administration of 10 IU hCG (Organon, Oss, Netherlands) i.p. to induce ovulation after approximately 12 h [36]. Whole ovaries were rapidly excised and snap frozen for reverse transcription-polymerase chain reaction (RT-PCR) analysis (n = 2–4 rats/stage) and immunohistochemistry (n = 4 rats/stage) when animals were killed by cervical dislocation at the following times/ovarian stages relative to hCG administration: –48 h (immature, Imm), 0 h (eCG-matured, eCG-Mat), 6 h (preovulatory, PreOv), 12 h (ovulatory, Ov), and 24 h post-hCG (luteal, Lut). Crude preparations of immature (largest on ovary; ~0.3–0.5 mm diameter) and ovulatory stage whole follicles (ImmWF, OvWF), granulosa cells (ImmGC, OvGC), and theca shells (ImmTS, OvTS) were also collected and snap frozen for RT-PCR analysis (n = 2 rats/stage). Ovaries were quickly isolated and pooled in a Petri dish containing 0.9% NaCl on ice. Using a dissecting microscope and watchmaker's forceps to pull aside the bursa, follicles were trimmed of stroma, ruptured, and scraped with a 30-G needle, and granulosa cells were aspirated with a silicon-coated Pasteur pipette. For collection of theca shells, dissected follicles were ruptured, thoroughly scraped, and rinsed of the granulosa layer in 0.9% NaCl.

RNA Preparation, Reverse Transcription into cDNA, and Primer Design

Total cellular RNA from whole ovaries and specific ovarian-derived tissue compartments was isolated and purified using an established guanidinium thiocyanate-phenol-chloroform extraction method [37], with modifications described previously [38]. Final RNA content (OD260) and relative purity (OD260:OD280) was determined with a Beckman DU-600 spectrophotometer (Beckman Instruments, Somerset, NJ). First-strand cDNA was synthesized from 1 µg total RNA using a Superscript RNase H-reverse transcriptase (RT) kit (Gibco, Grand Island, NY), with 500 mg/ml random hexamer (Geneworks, Adelaide, Australia) and 10 mM dNTPs (Pharmacia Biotech, Uppsala, Sweden). Rat ICAM-1 (5'-3', 160-GAGTGGACACAACTGGAAGC-180, 400-ACGGAGCAGCACTACTGAGA-380) and ß-actin (1343-CGTGGGCCGCCCTAGGCACCA-1363, 1672-TTGGCCTTAGGGTTCAGAGGGG-1651) oligonucleotide primer pairs (Gibco) were designed using Primer Design (Scientific and Educational Software, State Line, PA) and published nucleotide sequences [39, 40]. The ß-actin gene provided a constitutively expressed internal control for cDNA quantity and integrity, while both primer sets amplified intron-spanning regions of their respective genes to control for genomic DNA contamination.

Whole Ovarian mRNA: PCR Amplification and Quantification with Phospho Image Analysis

Quantification of relative mRNA amounts by RT-PCR was performed according to a previously described method [41], with subsequent adaptations [38]. The PCR amplification employed reagents supplied in a Taq DNA polymerase kit (Biotech International, Perth, Australia). Each 25-µl final reaction mixture consisted of buffer (67 mM Tris-HCl [pH 8.8], 16.6 mM [NH4]2SO4, 0.2 mg/ml gelatin, 0.45% Triton X-100), 2.5 mM MgCl2, 0.2 mM dNTPs (Pharmacia Biotech), 0.1 µCi 32P-labeled dCTP (Geneworks), 2 µM 5' and 3' primers, and 2 µl cDNA overlaid with two drops of paraffin oil. For each time point, 15-µl reaction mixture/well (including cDNA) were aliquoted down eight well strips of a 96-well reaction plate. PCR was performed in a plate thermal cycler (Hybaid, Teddington, UK) for 27–41 cycles (ICAM-1) or 17–31 cycles (ß-actin) and was initiated by the addition, at two-cycle intervals beginning at the highest cycle number, of 10 µl Taq DNA polymerase (0.55 U) at the 94°C denaturation step. Optimized PCR conditions for both primer pairs were 5 min (94°C), repeated 3 x 1 min (94°C/60°C/72°C) cycles, with a 7 min (72°C) final extension step. The negative control included in each reaction consisted of H2O substituted for cDNA. PCR reaction products were separated by electrophoresis in 2% agarose gels containing 0.5 µg/ml ethidium bromide, and their size was determined by comparison to Hpa II-digested pUC19 molecular weight marker (Biotech International), prior to rapid alkaline transfer (0.6 M NaCl, 0.4 M NaOH) onto a nylon membrane (Schliecher and Schuell, Dassel, Germany) and overnight exposure to a phosphor imaging plate. Reaction product 32P-dCTP incorporation was quantified using Image Quant v3.3 (Molecular Dynamics, Sunnyvale, CA) and the product yield (Y) was plotted against cycle number (n) as a semilogarithmic curve. SigmaPlot v3 (Jandel Corporation, San Rafael, CA) was used to estimate the initial amount of mRNA (I) by using the linear portion of the curve to fit the linear regression equation Y = I x En, where E, the efficiency across the linear portion, approaches 2 (i.e., doubling of the product at each cycle step). Data were normalized for ß-actin mRNA expression, then expressed relative to the Imm stage ovary. The PCR assay was replicated three times, with interassay coefficient of variation being 17.7%.

Ovarian Compartment mRNA: Amplification and Quantification Using Real-Time PCR

Real-time PCR amplification employed reagents supplied in a 2x SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA). Each 20-µl reaction consisted of 0.5–1 µM 5' and 3' primer and 4 µl of cDNA (20 ng/µl), where each sample was amplified in duplicate. The negative control included in each run consisted of H2O substituted for cDNA. Optimized PCR conditions utilized in the ABI Prism 5700 Sequence Detection System (Applied Biosystems), which allowed amplicon quantification for both primer pairs, were 2 min (50°C), 10 min (95°C), and 40 subsequent cycles of 20 sec (95°C), 20 sec (59°C), and 1 min (72°C). A validation experiment was performed to examine the efficiencies of each primer pair in the PCR. Cycle threshold (Ct) values, defined as the cycle number in which the detected fluorescence exceeds the threshold value, were determined for serial 10-fold dilutions of cDNA in a range of 0.08–80 ng total RNA. For each primer pair, the linearity of detection was confirmed to have a correlation coefficient of >0.99 over the detection area, when plotted as Ct versus log of RNA concentration. Delta Ct ({Delta}Ct = CtICAM-1 – Ctß-actin) was then calculated for each given RNA concentration and plotted versus log RNA concentration, yielding a slope of <0.1 (y = –0.0239x + 9.302), confirming similar efficiencies. Specificity of the PCR was confirmed by detection of a single distinct peak on examination of the dissociation curve profile of the reaction product. In addition, reaction products were analyzed by electrophoresis in 2% agarose gel containing 0.5 µg/ml ethidium bromide and visualized over an ultraviolet light box. Relative data were normalized for ß-actin mRNA expression and expressed relative to the immature theca shell (ImmTS) sample using the arithmetic equation 2{Delta}Ct x 100/K (Applied Biosystems User Bulletin #2, updated 2001), where K is the constant used to normalize data to an ImmTS mRNA value of 100.

Immunohistochemistry

Dissected ovaries were immediately embedded in Tissue Tek OCT compound (Miles Inc., Elkhart, IN) and frozen with isopentane (BDH, Poole, UK) in liquid nitrogen (N2), prior to storage at –80°C. Air-dried 5-µm-thick cryostat sections underwent indirect immunohistochemical procedures using mouse anti-rat CD54 (ICAM-1; 1:2000 dilution; Genzyme, Boston, MA) and anti-rat endothelial cell antigen-1 (RECA-1; 1:15 dilution; Serotec, Oxford, UK) monoclonal antibodies (mAb), with a polyclonal sheep anti-mouse IgG1 horseradish peroxidase conjugated secondary antibody (1:100 dilution; Amersham International, Amersham, UK) as described previously [12], with normal sheep serum used to block nonspecific binding. The specificity of both mAbs has been confirmed by previous studies [42, 43]. Bound antibody was visualized with 0.5 mg/ml 3,3'diaminobenzidine tetrahydrochloride (Sigma, St. Louis, MO) in 0.05 M Tris-HCl (pH 7.6) containing 1% H2O2. Positive control tissue included rat heart, liver, and Peyer patch. Negative antibody controls included primary antibody omission, normal mouse serum, and equal titer mouse IgG1 or unrelated isotype-matched antibody (mouse anti-human leukocyte common antigen; Serotec).

Quantification of ICAM-1 Protein Expression

Ovarian ICAM-1 protein staining was quantified using a video image analysis (VIA) system, Video Pro 32 software (Leading Edge, Adelaide, Australia), and a calibrator slide. Intra- and interassay coefficients of variation were 2.6% and 9.4%, respectively. Potential batch variations in fixation and antibody penetration were controlled by processing slides in a single session. Percentage positive ICAM-1 stain was determined at each periovulatory time point in the theca of two follicle-size subsets (0.3 mm– 0.6 mm and ≥0.6 mm), as well as in corpora lutea. Rat ovarian follicles of diameter ≥0.6 mm can be considered to have escaped atresia and thus are destined to ovulate [44]. Percentage positive ICAM-1 stain was defined as the extent of brown stain (area 1) in adjacent, nonoverlapping fields of view (2–5/structure) of hematoxylin-stain (area 2) and was calculated for each follicular thecal region or corpus luteum structure using the equation % positive ICAM-1 stain = (sum of area 1/sum of area 2) x 100. The number of thecal regions or corpora lutea analyzed per size subset per rat ovary ranged from 5 to 12 at each time point.

Data Analysis and Statistics

Data are presented as mean + SEM after analysis using Instat version 2.04a (Graphpad Software, San Diego, CA). Where appropriate, Kruskal-Wallis nonparametric ANOVA statistical tests were utilized, as data covered a broad range of values and were not always normally distributed. Dunn multiple comparisons post hoc tests were used to identify significant differences between the specific periovulatory stages. Statistical significance was accepted when P < 0.05.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Whole Ovarian ICAM-1 mRNA Quantification

Levels of ICAM-1 mRNA synthesis were measured in Imm, eCG-Mat, PreOv, Ov, and Lut stage whole ovaries to determine whether ovarian ICAM-1 expression is regulated at the mRNA transcription level during follicular development and rupture (Fig. 1, a and b). Messenger RNA data were normalized to ß-actin mRNA content at each stage and graphically expressed relative to ICAM-1 mRNA levels observed at the Imm stage of ovarian development (Fig. 1c). Levels of ICAM-1 mRNA in whole ovaries were moderately increased following eCG administration, peaking in the hours prior to ovulation, but these fluctuations did not reach a level of statistical significance. Each cDNA generated a single product of the expected size (244 bp), with no evidence of contaminating genomic DNA.



View larger version (31K):
[in this window]
[in a new window]
  		FIG. 1. Quantitative RT-PCR of periovulatory ICAM-1 mRNA in whole rat ovaries. A representative example of an individual run is shown where ICAM-1 products (244 bp) of RT-PCR amplifications initiated with Taq DNA polymerase addition at two-cycle intervals were simultaneously electrophoresed on a 2% agarose gel (a) and the amount of product at each stage was quantified by phosphor image densitometry following transfer to a nylon membrane (b). Analysis of the linear portion of each periovulatory stage regression curve (Imm stage depicted) in replicated experiments allowed quantification of the relative amounts of starting material, where whole ovarian periovulatory ICAM-1 mRNA levels (mean + SEM % expression at Imm stage), normalized to ß-actin, were not significantly different between the five stages of ovarian development (c). Assay repeated three times. Abbreviations defined in Materials and Methods

Ovarian Compartment ICAM-1 mRNA Localization and Quantification

As changes in ICAM-1 mRNA expression within follicular granulosa or theca may be masked in whole ovary samples because of large numbers of ICAM-1 mRNA negative cell types, quantitative real-time RT-PCR analysis was used to examine potential changes at the follicular compartment level. Data revealed the presence of ICAM-1 mRNA in whole follicles (ImmWF and especially in OvWF), almost completely confined to the follicular thecal region (Fig. 2), where time course studies showed a dramatic 5-fold increase in relative expression in OvTS compared with ImmTS. No ICAM-1 mRNA was detected in crude preparations of isolated granulosa cells until the ovulatory stage (OvGC), by which time the luteinization process is presumed to have commenced.



View larger version (13K):
[in this window]
[in a new window]
  		FIG. 2. Quantitative RT-PCR detection of ICAM-1 mRNA in specific follicular tissue compartments. Whole follicles (ImmWF, OvWF) and their individual constituent granulosa cells (ImmGC, OvGC) and theca shells (ImmTS, OvTS) were dissected from immature pooled primed rat ovaries (~10 follicles/ovary) and subjected to RT-PCR. All data were normalized to ß-actin and expressed relative to ImmTS ICAM-1 mRNA expression. Whole follicle ICAM-1 mRNA expression was present in immature ovaries (ImmWF) but was greatest at ovulation (OvWF). There was some granulosa cell expressed ICAM-1 mRNA in ovulatory ovaries (OvGC), but generally theca shells were responsible for most or all follicular ICAM-1 mRNA expression, with OvTS being 5-fold more than ImmTS expression

ICAM-1 Protein Localization and Quantification

Minimal levels of immunodetectable ICAM-1 were observed in some arterioles or venules outside follicles and in the stroma of Imm stage ovaries, as shown by RECA localization of endothelium, but ICAM-1 staining was absent in the granulosa layer of both atretic and nonatretic follicles (Fig. 3). In matured ovaries of eCG-Mat stage rats, a weak but apparent stain was localized to the thecal region of antral follicles (Fig. 4, a and b). At the PreOv and Ov stages, ICAM-1 protein remained absent in the granulosa yet was dispersed prominently in the theca (Fig. 4, c–e), where it was associated at least partly with the concentric inner and outer vascular networks of this layer, as revealed by endothelium localization by RECA (Fig. 4f). By the Lut stage (24 h post-hCG), ICAM-1 was detectable in the periphery of corpora lutea and for the first time scattered among luteinizing granulosa cells, both within the parenchyma and adjacent to the antral space of developing corpora lutea (Fig. 4g). As expected, RECA stain identified strong angiogenic activity within developing corpora lutea (Fig. 4h), which broadly colocalized with ICAM-1. Nonspecific staining was not present in sections where primary antibody was replaced by equal titer mouse IgG1, antibody diluent, normal mouse serum or human leukocyte common antigen as an isotype-matched unrelated antibody (data not shown).



View larger version (157K):
[in this window]
[in a new window]
  		FIG. 3. Immunolocalization of ICAM-1 and endothelial cells in ovaries of immature rats (unprimed, 27 days old). Endothelial cells of larger vessels in the ovarian stroma (a; arrows) and of a small portion of the external thecal vasculature of primary and small secondary follicles (c; arrows) express ICAM-1, as confirmed by respective serial sections stained with the specific endothelial cell marker, RECA (b and d). Negligible ICAM-1 staining was observed in endothelia of atretic (AT) follicles (a and b). Bar (d) = 40 µm in all plates



View larger version (149K):
[in this window]
[in a new window]
  		FIG. 4. Ovarian immunolocalization of ICAM-1 in eCG-primed rats at various times relative to an ovulation-inducing dose of hCG. Faint ICAM-1 staining was dispersed throughout the thecal layer of eCG-matured follicles at the time of hCG administration (a and b) but became progressively more pronounced in preovulatory and ovulatory follicles at 6 h (c and d) and 12 h (e) following hCG, respectively. While ICAM-1 expression appeared scattered throughout the entire theca at these stages (d), it was often most concentrated in stromal vessels (c and e; arrows) and in the inner and outer capillary wreaths of the thecal region (c and e; arrowheads), adjacent to the known location of the basal laminae. Plate f, a serial section of e, clearly shows RECA positive endothelial cells in the ovarian stroma and theca of ovulatory follicles, including a longitudinal array of capillaries of the outer capillary wreath (f) that correspond with dense ICAM-1 expression in e (arrowheads). Noticeably, ICAM-1 expression is totally absent in the follicular granulosa layer (a–e), confirmed as avascular in f, but ICAM-1 is present among luteinizing cells adjacent to the antral space (g; arrowheads) during corpus luteum (CL) formation at 24 h post-hCG administration. RECA stain showed neovascularization within developing corpora lutea at 24 h post-hCG (h). Bars = 40 µm in all plates

Video image analysis of ICAM-1 stain for quantification of relative thecal region protein expression levels revealed low-level, constitutive ICAM-1 expression in Imm and eCG-Mat ovaries that increased significantly following hCG administration in the subset of follicles programmed for ovulation (i.e., diameter ≥0.6 mm). Peak ICAM-1 expression occurred at the Ov stage, where there had been a dramatic 6-fold rise compared with the eCG-Mat stage (Fig. 5). Relative expression levels in this follicle size subset then decreased significantly again in the theca of luteinizing follicles and in the parenchyma of newly forming corpora lutea, which together made up the Lut stage. Expression of ICAM-1 in eCG-exposed follicles, not necessarily destined for ovulation (i.e., 0.3–0.6 mm diameter) appeared to also increase following hCG, but not to a significant degree (Fig. 5). Hence, the expression of ICAM-1 protein would appear to correlate with ICAM-1 mRNA expression detectable in corresponding tissue compartments.



View larger version (15K):
[in this window]
[in a new window]
  		FIG. 5. Quantification by video image analysis of periovulatory ICAM-1 immunodetectable protein expression (mean ± SEM % positive ICAM-1 stain) in the thecal region of follicles and in developing corpora lutea of two diameter subsets. There were no follicles of dimensions ≥0.6 mm at the Imm stage. PreOv and/or Ov stage % positive ICAM-1 stain were significantly (P < 0.001) elevated compared with levels observed at eCG-Mat (*) and Lut (#) stages in follicles/CLs ≥0.6 mm in diameter


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In this study we have sought to more fully understand the mechanisms controlling the preovulatory ovarian leukocyte invasion by localizing and tracking relative changes in rat ovarian ICAM-1 mRNA and protein expression across the periovulatory period. ICAM-1 is a critical adhesion molecule in the recruitment of neutrophils and monocyte/macrophages, which constitute the bulk of invading immune cells in the rat and human ovary. Following on from our demonstration of sICAM-1 changes across the menstrual cycle in the peripheral blood of women, this study's results clearly demonstrate a marked upregulation in rat ICAM-1 protein expression following ovulation induction with hCG that peaks at the expected time of ovulation, is restricted to the highly vascularized theca, and coincides with a raised level of ICAM-1 mRNA observed when transcription levels increased in the theca following hCG priming. Importantly, the post-hCG increase in ICAM-1 protein expression was significant only in the subset of follicles with a diameter greater than 0.6 mm, a size for leading follicles in the rat that is indicative of programming for ovulation [44]. To our knowledge, this is the first evidence of leukocyte adhesion receptor expression in the preovular ovary being associated with follicular development, and it supports the concept that the preovulatory recruitment of leukocytes in this organ is an active mechanism, most likely initiated by gonadotropin actions.

Some degree of ICAM-1 mRNA expression and immunoactivity was detectable in the ovary at each periovulatory stage examined in this study, consistent with previous reports of constitutive ICAM-1 expression in a number of tissues [22, 23], including the ovary [32]. At each periovulatory stage, ICAM-1 protein colocalized with the specific rat endothelial cell marker, RECA, in a proportion of thecal and stromal blood vessels, indicating a definite endothelial origin, but at no point was ICAM-1 staining seen in all vessels. Accordingly, immunoactive ICAM-1 in developing follicles exposed to gonadotropin was strongest and strictly contained within the abundantly vascularized thecal region of large-diameter follicles and was completely absent from the avascular granulosa layer. The stain was not confined to thecal endothelia but appeared broadly dispersed throughout the entire thecal region, suggesting that ovarian thecal cells themselves express ICAM-1, as may the heterogeneous cells that constitute the theca, including leukocytes, fibroblasts, and smooth muscle cells. The diffuse ICAM-1 staining pattern we observed, which makes identifying specific cells expressing ICAM-1 antigen difficult, is well recognized in other cell systems and may be attributable to a patchy/punctate cell surface stain, binding of ICAM-1 to cytoskeleton, or evidence of sICAM-1 in the interstitium [23, 42, 45, 46].

Theca-restricted localization of ICAM-1 protein and mRNA until follicle rupture and the initiation of luteinization, as shown in our study, is of interest when considering the known colocalization of other regulatory factors in this compartment and the opportunities for paracrine interactions. In addition to the preovulatory thecal confinement of resident and invading leukocytes, the expression of the rat neutrophil specific chemotaxin CINC/gro has been shown to be theca specific, to follow a similar time course of expression as ICAM-1, and to be secreted dose dependently by cytokines, including IL-1ß [18]. IL-1ß itself promotes follicle rupture [47], is expressed at protein and mRNA levels within the rodent theca [48, 49], and is a potent inducer or upregulator of ICAM-1 [22, 23, 26]. Hence, the thecal region of ovarian follicles may be the primary site of cytokine-induced adhesion and chemotactic events that result in the increased leukocyte density observed in this tissue layer by the time of rupture.

It is also interesting that ovulatory stage crude granulosa cell preparations were only marginally positive for ICAM-1 mRNA and that all stages were negative for protein expression until the luteal phase (24 h post-hCG), when antral layers of granulosa-lutein cells in early-developing corpora lutea began to express ICAM-1 antigen, corroborating reports of ICAM-1 immunoactivity on human granulosa cells luteinized in culture [29]. This abrupt acquisition of stain in transformed granulosa cells during luteinization of ovulated follicles has been observed previously with IL-1ß [49], suggesting that the IL-1ß/ICAM-1 component of the previously proposed network may also have a role in corpus luteum formation and function, perhaps applied through angiogenic or steroidogenic means.

Unlike the adhesion molecule P-selectin, no storage granules of ICAM-1 protein exist, so induced or up-regulated protein expression depends on the induction of mRNA synthesis. A typical time course between endothelial ICAM-1 message transcription and peak translated protein expression is approximately 12 h [23], which is consistent with the time span we observed between the rise above constitutive expression levels of ICAM-1 mRNA (at 0 h post-hCG) first becoming apparent and peak ICAM-1 immunoactivity at 12 h post-hCG.

In conclusion, through this study we have presented the first evidence that ICAM-1, present in the rat ovary throughout the periovulatory period as both mRNA and protein, undergoes changes in expression that support ICAM-1 having an integral role in the preovulatory influx of neutrophils and monocyte/macrophages. Furthermore, follicular ICAM-1 antigen expression was limited to endothelial cells and other cells that constitute the heterogeneous population of the thecal region, but subsequent ICAM-1 immunoactivity among luteinizing granulosa cells suggests an additional role in corpus luteum formation and/ or function.


   ACKNOWLEDGMENTS
 
We thank Amanda Magaletta and Rosemary Bonello for assistance with manuscript preparation.


   FOOTNOTES
 
1 N.B. was supported by the University of Adelaide Medical Faculty and the Queen Elizabeth Hospital Research Foundation. Back

2 Correspondence: Research Centre for Reproductive Health, Department of Obstetrics and Gynaecology, University of Adelaide, Queen Elizabeth Hospital, Woodville SA 5011, Australia. FAX: 61 8 82227521; nigel.bonello{at}adelaide.edu.au Back

Received: 6 April 2004.

First decision: 26 April 2004.

Accepted: 27 May 2004.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Adashi EY. The potential relevance of cytokines to ovarian physiology: the emerging role of resident ovarian cells of the white blood cell series. Endocr Rev 1990 11:454-464[Medline]
  2. Brännström M, Norman RJ. Involvement of leukocytes and cytokines in the ovulatory process and corpus luteum function. Hum Reprod 1993 8:1762-1775[Abstract/Free Full Text]
  3. Bjersing L, Cajander S. Ovulation and the mechanism of follicle rupture. I. Light microscopic changes in rabbit ovarian follicles prior to induced ovulation. Cell Tissue Res 1974 149:287-300[Medline]
  4. Brännström M, Mayrhofer G, Robertson SA. Localization of leukocyte subsets in the rat ovary during the periovulatory period. Biol Reprod 1993 48:277-286[Abstract]
  5. Brännström M, Pascoe V, Norman RJ, McClure N. Localization of leukocyte subsets in the follicle wall and in the corpus luteum throughout the human menstrual cycle. Fertil Steril 1994 61:488-495[Medline]
  6. Hellberg P, Thomsen P, Janson PO, Brännström M. Leukocyte supplementation increases the luteinizing hormone-induced ovulation rate in the in vitro-perfused rat ovary. Biol Reprod 1991 44:791-797[Abstract]
  7. Brännström M, Bonello N, Norman RJ, Robertson SA. Reduction of ovulation rate in the rat by administration of a neutrophil-depleting monoclonal antibody. J Reprod Immunol 1995 29:265-270[CrossRef][Medline]
  8. Van der Hoek KH, Maddocks S, Woodhouse CM, van Rooijen N, Robertson SA, Norman RJ. Intrabursal injection of clodronate liposomes causes macrophage depletion and inhibits ovulation in the mouse ovary. Biol Reprod 2000 62:1059-1066[Abstract/Free Full Text]
  9. Araki M, Fukumatsu Y, Katabuchi H, Shultz LD, Takahashi K, Okamura H. Follicular development and ovulation in macrophage colony-stimulating factor-deficient mice homozygous for the osteopetrosis (op) mutation. Biol Reprod 1996 54:478-484[Abstract]
  10. Norman RJ, Bonello N, Jasper MJ, Van der Hoek KH. Leukocytes: essential cells in ovarian function and ovulation. Reprod Med Rev 1997 6:97-111
  11. Bonello N, McKie K, Jasper M, Andrew L, Ross N, Braybon E, Brännström M, Norman RJ. Inhibition of nitric oxide: effects on interleukin-1 beta-enhanced ovulation rate, steroid hormones, and ovarian leukocyte distribution at ovulation in the rat. Biol Reprod 1996 54:436-445[Abstract]
  12. Bonello NP, Norman RJ, Brännström M. Interleukin-1ß inhibits luteinizing hormone-induced plasminogen activator activity in rat preovulatory follicles in vitro. Endocrine 1995 3:49-54
  13. Paavola LG. The corpus luteum of the guinea pig. IV. Fine structure of macrophages during pregnancy and postpartum luteolysis, and the phagocytosis of luteal cells. Am J Anat 1979 154:337-364[CrossRef][Medline]
  14. Butcher EC. Leukocyte-endothelial cell recognition: three (or more) steps to specificity and diversity. Cell 1991 67:1033-1036[CrossRef][Medline]
  15. Runesson E, Boström EK, Janson PO, Brännström M. The human preovulatory follicle is a source of the chemotactic cytokine interleukin-8. Mol Hum Reprod 1996 2:245-250[Abstract/Free Full Text]
  16. Karström-Encrantz L, Runesson E, Boström EK, Brännström M. Selective presence of the chemokine growth-regulated oncogene alpha (GROalpha) in the human follicle and secretion from cultured granulosa-lutein cells at ovulation. Mol Hum Reprod 1998 4:1077-1083[Abstract/Free Full Text]
  17. Harkin DG, Bignold LP, Herriot-Warnes DM, Kirby CA. Chemotaxis of polymorphonuclear leukocytes towards human pre-ovulatory follicular fluid and serum using a "sparse-pore" polycarbonate filtration membrane. J Reprod Immunol 1994 27:151-155[CrossRef][Medline]
  18. Ushigoe K, Irahara M, Fukumochi M, Kamada M, Aono T. Production and regulation of cytokine-induced neutrophil chemoattractant in rat ovulation. Biol Reprod 2000 63:121-126[Abstract/Free Full Text]
  19. Murdoch WJ, McCormick RJ. Mechanisms and physiological implications of leucocyte chemoattraction into periovulatory ovine follicles. J Reprod Fertil 1993 97:375-380
  20. Wong KH, Negishi H, Adashi EY. 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 2002 143:784-791[Abstract/Free Full Text]
  21. Rothlein R, Dustin ML, Marlin SD, Springer TA. A human intercellular adhesion molecule (ICAM-1) distinct from LFA-1. J Immunol 1986 137:1270-1274[Abstract]
  22. Malik AB, Lo SK. Vascular endothelial adhesion molecules and tissue inflammation. Pharmacol Rev 1996 48:213-229[Medline]
  23. Carlos TM, Harlan JM. Leukocyte-endothelial adhesion molecules. Blood 1994 84:2068-2101[Abstract/Free Full Text]
  24. Marlin SD, Springer TA. Purified intercellular adhesion molecule-1 (ICAM-1) is a ligand for lymphocyte function-associated antigen 1 (LFA-1). Cell 1987 51:813-819[CrossRef][Medline]
  25. Diamond MS, Staunton DE, Marlin SD, Springer TA. Binding of the integrin Mac-1 (CD11b/CD18) to the third immunoglobulin-like domain of ICAM-1 (CD54) and its regulation by glycosylation. Cell 1991 65:961-971[CrossRef][Medline]
  26. Dustin ML, Rothlein R, Bhan AK, Dinarello CA, Springer TA. Induction by IL-1 and interferon-gamma: tissue distribution, biochemistry, and function of a natural adherence molecule (ICAM-1). J Immunol 1986 137:245-254[Abstract]
  27. Katayama T, Kusanagi Y, Kiyomura M, Ochi H, Ito M. Leukocyte behaviour and permeability in the rat mesenteric microcirculation following induction of ovulation. Hum Reprod 2003 18:1179-1184[Abstract/Free Full Text]
  28. Campbell S, Swann HR, Seif MW, Kimber SJ, Aplin JD. Cell adhesion molecules on the oocyte and preimplantation human embryo. Hum Reprod 1995 10:1571-1578[Abstract/Free Full Text]
  29. Viganò P, Gaffuri B, Ragni G, Di Blasio AM, Vignali M. Intercellular adhesion molecule-1 is expressed on human granulosa cells and mediates their binding to lymphoid cells. J Clin Endocrinol Metab 1997 82:101-105[Abstract/Free Full Text]
  30. Suzuki T, Sasano H, Takaya R, Fukaya T, Yajima A, Nagura H. Cyclic changes of vasculature and vascular phenotypes in normal human ovaries. Hum Reprod 1998 13:953-959[Abstract/Free Full Text]
  31. Olson KK, Anderson LE, Wiltbank MC, Townson DH. Actions of prostaglandin F2alpha and prolactin on intercellular adhesion molecule-1 expression and monocyte/macrophage accumulation in the rat corpus luteum. Biol Reprod 2001 64:890-897[Abstract/Free Full Text]
  32. Olson KK, Townson DH. Prolactin-induced expression of intercellular adhesion molecule-1 and the accumulation of monocytes/macrophages during regression of the rat corpus luteum. Biol Reprod 2000 62:1571-1578[Abstract/Free Full Text]
  33. Viganò P, Fusi F, Gaffuri B, Bonzi V, Ferrari A, Vignali M. Soluble intercellular adhesion molecule-1 in ovarian follicles: production by granulosa luteal cells and levels in follicular fluid. Fertil Steril 1998 69:774-779[CrossRef][Medline]
  34. Rothlein R, Mainolfi EA, Czajkowski M, Marlin SD. A form of circulating ICAM-1 in human serum. J Immunol 1991 147:3788-3793[Abstract]
  35. Bonello N, Norman RJ. Soluble adhesion molecules in serum throughout the menstrual cycle. Hum Reprod 2002 17:2272-2278[Abstract/Free Full Text]
  36. Larson L, Olofsson J, Hellberg P, Brännström M, Selstam G, Hedin L. Regulation of prostaglandin biosynthesis by luteinizing hormone and bradykinin in rat preovulatory follicles in vitro. Prostaglandins 1991 41:111-121[CrossRef][Medline]
  37. Chomczynski P, Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 1987 162:156-159[Medline]
  38. Robertson SA, Mayrhofer G, Seamark RF. Ovarian steroid hormones regulate granulocyte-macrophage colony-stimulating factor synthesis by uterine epithelial cells in the mouse. Biol Reprod 1996 54:183-196[Abstract]
  39. Kita Y, Takashi T, Iigo Y, Tamatani T, Miyasaka M, Horiuchi T. Sequence and expression of rat ICAM-1. Biochim Biophys Acta 1992 1131:108-110[Medline]
  40. Nudel U, Zakut R, Shani M, Neuman S, Levy Z, Yaffe D. The nucleotide sequence of the rat cytoplasmic beta-actin gene. Nucleic Acids Res 1983 11:1759-1771[Abstract/Free Full Text]
  41. Chelly J, Kaplan JC, Maire P, Gautron S, Kahn A. Transcription of the dystrophin gene in human muscle and non-muscle tissue. Nature 1988 333:858-860[CrossRef][Medline]
  42. Tamatani T, Miyasaka M. Identification of monoclonal antibodies reactive with the rat homolog of ICAM-1, and evidence for a differential involvement of ICAM-1 in the adherence of resting versus activated lymphocytes to high endothelial cells. Int Immunol 1990 2:165-171[Abstract/Free Full Text]
  43. Duijvestijn AM, van Goor H, Klatter F, Majoor GD, van Bussel E, van Breda Vriesman PJ. Antibodies defining rat endothelial cells: RECA-1, a pan-endothelial cell-specific monoclonal antibody. Lab Invest 1992 66:459-466[Medline]
  44. Osman P. Rate and course of atresia during follicular development in the adult cyclic rat. J Reprod Fertil 1985 73:261-270
  45. Stoll G, Jander S, Jung S, Archelos J, Tamatani T, Miyasaka M, Toyka KV, Hartung HP. Macrophages and endothelial cells express intercellular adhesion molecule-1 in immune-mediated demyelination but not in Wallerian degeneration of the rat peripheral nervous system. Lab Invest 1993 68:637-644[Medline]
  46. Kanagawa K, Ishikura H, Takahashi C, Tamatani T, Miyasaka M, Togashi M, Koyanagi T, Yoshiki T. Identification of ICAM-1-positive cells in the nongrafted and transplanted rat kidney: an immunohistochemical and ultrastructural study. Transplantation 1991 52:1057-1062[Medline]
  47. Brännström M, Wang L, Norman RJ. Ovulatory effect of interleukin-1 beta on the perfused rat ovary. Endocrinology 1993 132:399-404[Abstract]
  48. Simón C, Frances A, Piquette G, Polan ML. Immunohistochemical localization of the interleukin-1 system in the mouse ovary during follicular growth, ovulation, and luteinization. Biol Reprod 1994 50:449-457[Abstract]
  49. Hurwitz A, Ricciarelli E, Botero L, Rohan RM, Hernandez ER, Adashi EY. Endocrine- and autocrine-mediated regulation of rat ovarian (theca-interstitial) interleukin-1 beta gene expression: gonadotropin-dependent preovulatory acquisition. Endocrinology 1991 129:3427-3429[Abstract]



This article has been cited by other articles:


Home page
 Hum Reprod UpdateHome page
D. L. Russell and R. L. Robker
Molecular mechanisms of ovulation: co-ordination through the cumulus complex
Hum. Reprod. Update, May 1, 2007; 13(3): 289 - 312.
[Abstract] [Full Text] [PDF]

Home page
 Mol Hum ReprodHome page
M. J. Jasper, K. P. Tremellen, and S. A. Robertson
Primary unexplained infertility is associated with reduced expression of the T-regulatory cell transcription factor Foxp3 in endometrial tissue
Mol. Hum. Reprod., May 1, 2006; 12(5): 301 - 308.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
71/4/1384    most recent
biolreprod.104.030650v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Bonello, N.
Right arrow Articles by Norman, R. J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Bonello, N.
Right arrow Articles by Norman, R. J.
Agricola
Right arrow Articles by Bonello, N.
Right arrow Articles by Norman, R. J.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS