help button home button Endocrine Society Endocrinology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Purchase Article
Right arrow View Shopping Cart
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 Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Wong, K. H. H.
Right arrow Articles by Adashi, E. Y.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Wong, K. H. H.
Right arrow Articles by Adashi, E. Y.
Endocrinology Vol. 143, No. 3 784-791
Copyright © 2002 by The Endocrine Society

GROWTH FACTORS-CYTOKINES-ONCOGENES

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

Kenneth H. H. Wong, Hiroaki Negishi1 and Eli Y. Adashi

Division of Reproductive Sciences, Department of Obstetrics and Gynecology, University of Utah Health Sciences Center, Huntsman Cancer Institute, Salt Lake City, Utah 84112

Address all correspondence and requests for reprints to: Dr. Eli Y. Adashi, Division of Reproductive Sciences, Department of Obstetrics and Gynecology, University of Utah Health Sciences Center, Huntsman Cancer Institute, 2000 Circle of Hope, Room 5221, Salt Lake City, Utah 84112. E-mail: . eadashi{at}hsc.utah.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
A growing body of evidence suggests that mammalian ovulation bears similarities to local inflammatory reactions. Monocytes/macrophages, eosinophils, and neutrophils are known to infiltrate the area surrounding the dominant follicle before ovulation. Candidate local chemoattractants may include a family of small cytokines, also known as chemokines. In the present study, quantitative RT-PCR was used to initially identify and quantify the chemokines expressed in the preovulatory rat ovary. The chemokines monocyte chemotatic protein 1 (MCP-1), MCP-3, macrophage inflammatory protein 1{alpha} (MIP-1{alpha}), MIP-1ß, MIP-1{gamma}, regulated upon activation normal T cell expressed and secreted, eotaxin, interferon-inducible protein of 10 kDa, growth-regulated oncogene, lymphotactin, and fractalkine were all expressed in the PMSG-primed rat ovary 6 h post human CG. C10, T cell activation gene 3, exodus, exodus-2, cytokine-induced neutrophil chemoattractant-2, MIP-2, and lipopolysaccharide-induced C-X-C were not expressed in the PMSG-primed rat ovary 6 h post human CG. The cyclic variation of the ovary-positive chemokines was also evaluated throughout the course of a superovulated ovarian cycle. Significant preovulatory up-regulation relative to the untreated control state was documented for MCP-1 (18-fold), MCP-3 (12-fold), and growth-regulated oncogene (25-fold). In contrast, the preovulatory ovarian expression of eotaxin, fractalkine and regulated upon activation normal T cell expressed and secreted was not increased. These observations suggest that intraovarian chemokines may be responsible for the cyclic intraovarian residence of representatives of the white blood cell series.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
IT HAS LONG been observed that the process of ovulation bears significant similarities to the inflammatory process. There now exists a growing body of evidence suggesting that ovulation may indeed constitute a local inflammatory reaction (1). In particular, serious consideration is being given to the possibility that leukocytes and cytokines may play key roles in normal ovarian physiology (2, 3).

Many investigators have documented the ovarian presence of different subsets of leukocytes, i.e. neutrophils, macrophages, lymphocytes, and eosinophils at different phases of the ovarian life cycle in various animal species (4, 5, 6, 7). Special mention is made of the evidence for a large influx of leukocytes (both macrophages and neutrophils) into the ovary in apparent response to the LH surge (3). Thus, ovarian leukocytes and their secreted products may constitute integral components of the ovulatory cascade. An equally important influx of monocytes into the corpus luteum characterizes the late postovulatory phase (8).

As is evident from the above, the ovary is not an immunologically privileged site. That is to say, an increase in leukocyte density within the ovary during the periovulatory and postovulatory periods can only occur as a result of influx of such cells. Thus, leukocytes are inevitably being attracted away from the circulation and into the ovary, presumably by way of local, possibly intraovarian, chemoattractants. Candidate local chemoattractants, which may be involved in the periovulatory and postovulatory influx of leukocytes, may include a family of small cytokines, also known as chemokines.

Over the last 5–10 yr, chemotactic cytokines (or chemokines) have been discovered with increasing frequency. Chemokines constitute a family of structurally related, small, inducible, secreted proinflammatory cytokines involved in a variety of immune responses especially as chemoattractants and activators of specific types of leukocytes (9). Approximately 40 chemokines, grouped in distinct families, are known to date. Chemokines are secreted by a variety of cells and thus appear to play important roles in many inflammatory reactions.

There is a growing body of evidence that chemokines may be directly or indirectly involved in follicular development, ovulation, as well as in corpus luteum formation, function, and demise (10). The expression of IL-8, monocyte chemotactic protein-1 (MCP-1), and growth-regulated oncogene {alpha} (GRO-{alpha}) has been reported for the human follicular fluid compartment and for human granulosa/lutein cell cultures (11, 12, 13, 14, 15). In addition, IL-8 appears to be essential to human CG (hCG)-induced ovulation in the rabbit (16, 17, 18). Human granulosa cell IL-8 secretion may also be induced by gonadotropins (19). Finally, several investigators have demonstrated that MCP-1 may play a role in the function of the corpus luteum of the rat (20, 21, 22) and other species (23, 24, 25).

We postulate that ovarian chemokines may influence the periovulatory and postovulatory influx of leukocytes. Our objectives were to determine which chemokines (for which the rat or mouse sequences were known) are present in the rat ovary and to assess whether any of those intraovarian chemokines is the subject of cyclic hormonal regulation.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Immature Sprague Dawley female rats (Zivic-Miller Laboratories, Inc., Zelienople, PA) were used throughout all experiments. The project was approved by the Institutional Animal Care and Use Committee of the University of Utah.

In vivo treatment paradigm
Intact 25-d-old female rats were injected ip with 15 IU of PMSG (2100 IU/mg; Sigma, St. Louis, MO). Ovulation was triggered using 15 IU of highly purified hCG (2500 IU; Sigma) 48 h later. Rats were killed at a time point concordant with PMSG injection (control/untreated), hCG injection (hCG0), as well as 2, 4, 6, 8, 10, 12, 24, and 48 h after hCG administration (hCG2–48 h). At each time point, the ovaries were removed and immediately flash-frozen in liquid nitrogen and stored at -80 C until subsequent processing. The treatment paradigm was repeated twice for a total of three independent experiments.

Construction of control synthetic genes: a template for the quantification of rat ovarian chemokines
Two synthetic genes (ChemoI, ChemoII; Fig. 1Go), comprising 5' and 3' primer sequences, were constructed by the technique of oligonucleotide overlap extension and followed by PCR amplification as previously described (26, 27). Briefly, 0.5 µg each of the oligonucleotides (~100 bp) with overlapping ends were mixed in a standard PCR reaction (100 µl). The reaction mixture consisted of 50 nM KCl, 10 mM Tris-HCl, pH 8.0, 1.5 mM MgCl 2, 200 µM deoxy-nucleotide triphosphates, and 2.5 U of Pfu DNA polymerase (Stratagene, La Jolla, CA). The reaction was denatured at 94 C for 7 min, annealed at 55 C for 2 min, and extended at 72 C for 3 min for one cycle. This, in turn, was followed by seven additional cycles, the denaturation step being reduced to 1.5 min. In the second step, 1 µl of the first PCR product was used as template for the second PCR reaction. Specifically, two primers (0.5 µM each) spanning the 5' and 3' ends of the template formed in the first reaction were added to the standard PCR mixture and amplified for 25 cycles (denatured at 94 C for 1 min, annealed at 55 C for 1 min, and extended at 72 C for 1 min). The resultant PCR products were resolved using ethidium bromide-stained 2% agarose gel electrophoresis, purified, cloned into pBluescript II KS+ (Stratagene), and sequence-verified.



View larger version (20K):
[in this window]
[in a new window]
  		Figure 1. Structure of the Chemo I and Chemo II control plasmids. The two plasmids contain 5' primers of target chemokine genes connected in sequence followed by the complementary sequences of the 3' primers in the order shown. A restriction enzyme linker was placed between the 5' and 3' sequences. The plasmids, Chemo I and II, are 494 and 590 bp in length, respectively.

 
RT-PCR and RT-competitive-PCR
Total cellular RNA from the ovaries of three rats at each time point were pooled together and extracted using the SV Total RNA Isolation System (Promega Corp., Corp., Madison, WI). The first strand of cDNA was synthesized from 1 µg of total RNA using 200 U of the Moloney murine leukemia virus reverse transcriptase (RT; Life Technologies, Inc., Gaithersburg, MD), applied for 1 h at 37 C after oligo(deoxythymidine) priming. Aliquots of the first strand reaction (1 µl) were used as templates for subsequent PCR and competitive PCR reactions.

PCR amplification was performed using the appropriate rat-specific sense and antisense primers for the gene under study (Table 1Go). The sense and antisense primer sequences for the chemokines MCP-3, macrophage inflammatory protein 1{gamma} (MIP-1{gamma}), exodus-2, T cell activation gene 3 (TCA-3), fractalkine, and C10 were mouse specific. For competitive PCR, increasing molar concentrations of synthetic control DNA (Chemo I or II) were added to the cDNA derived from tissues obtained at the appropriate time points.


View this table:
[in this window]
[in a new window]
  		Table 1. Oligonucleotides used to amplify target cDNA and competitor fragment DNA

 
Each PCR reaction mixture (25 µl) contained 0.2 µM of each primer, 0.75 U Taq DNA Polymerase (Perkin-Elmer Corp., Norwalk, CT), 1.5 mM MgCl2, 0.2 mM deoxy-nucleotide triphosphate, and 10x reaction buffer. Amplification was performed under optimal annealing conditions as predetermined for each primer pair. After the final amplification, the reaction was elongated for an additional 7 min at 72 C.

A positive control was derived from spleen tissue samples obtained from rats subjected to ip treatment with lipopolysaccharide (LPS; Sigma). Negative controls consisted of PCR mixtures with the relevant primers wherein water was substituted for cDNA or wherein RT was omitted during the cDNA synthesis. The identities of positive bands were confirmed by direct sequencing of the PCR products.

Quantitative analysis of chemokine amplicons
Quantification was performed as previously described (28). PCR products (10 µl) were electrophoresed in 1.8% agarose gels containing ethidium bromide, visualized with UV, and digitally photographed with a gel documentation system (Bio-Rad Laboratories, Inc., Hercules, CA). The quantities of the competitor and target cDNA were compared using the Molecular Analyst software (Bio-Rad Laboratories, Inc.). The log of the ratio (target/competitor) was plotted against the log of the competitor concentrations using CricketGraph 3 program (Computer Associates International, Inc., Islandia, NY). A linear regression for each curve was calculated. The data generated from these curves were used to extrapolate the number of molecules in each sample. Calculated amounts of PCR products, as determined by competitive PCR, were corrected by the amounts of the calculated ß-actin PCR product. Two separate competitive PCR amplifications were performed for each pooled RNA sample.

Statistical analysis
Quantitative results concerning intraovarian ß-actin concentration and chemokine fold expression are expressed as the mean with SD per time point. Statistical analyses were performed by using ANOVA fitted for the time points, the experimental animal sets (3) and the repetitive competitive PCR amplifications nested within each animal set (two separate PCRs for each animal set) as covariates. For the data sets found to be significant, a comparison between control and the significant time points was performed using Dunnett’s adjustment for multiple comparisons (29). Significance of the data sets was determined by comparison of the control and significant time points by Dunnett’s adjustment for multiple comparisons (29). All statistical analyses were performed with the SAS statistical program (SAS Institute, Inc., Cary, NC). A P value of <0.05 was considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Preovulatory expression of chemokines in the rat ovary
To assess the preovulatory ovarian expression of the chemokine under study, ovaries were obtained from PMSG-primed rats 6 h post hCG and processed for RT-PCR as described. Chemokine transcripts belonging to all four families of chemokines were expressed in the preovulatory ovary (Table 2Go). In contrast, other chemokines (C10, TCA-3, exodus, exodus-2, cytokine-induced neutrophil chemoattractant-2, MIP-2, and LPS-induced C-X-C), belonging to the CC and CXC families, although clearly expressed in the rat spleen (positive control, data not shown), could not be detected in the PMSG-primed rat ovary (6 h post hCG).


View this table:
[in this window]
[in a new window]
  		Table 2. Expression of chemokine transcripts in the ovary

 
Rat ovarian expression of ß-actin throughout a simulated/superovulated cycle
To establish possible cyclic variation in ovarian chemokine expression, use was made of the housekeeping gene ß-actin as a putative normalizing probe for cDNA concentrations. As a normalizing probe, ß-actin must show no variation in ovarian expression during the simulated cycle. As shown (Fig. 2Go), superovulation did not significantly affect the expression of ß-actin in the rat ovary, thereby validating this housekeeping gene as a normalizing probe for the experimental settings under study.



View larger version (29K):
[in this window]
[in a new window]
  		Figure 2. Cyclic expression of ß-actin in the rat ovary. The expression of ß-actin was quantified by using RT-competitive PCR. Ovaries of 25-d-old immature rats were obtained from unstimulated (control), PMSG (15 IU ip)-injected (48 h), or PMSG (15 IU ip)-injected/hCG (15 IU ip)-triggered animals 48 h later (2–48 h after hCG). RNA was extracted, reverse-transcribed into cDNA and subjected to competitive PCR as described. Quantification was performed as described in Materials and Methods. Data depict, in bar graph form, the mean concentration (pg/µl) of ß-actin transcripts ± SD of three independent experiments. *, P < 0.05 vs. control (unstimulated).

 
Up-regulated expression of chemokines in the preovulatory rat ovary
Competitive PCR was performed as described to quantify possible cyclic variation in the ovarian expression of the chemokines under study. As shown (Fig. 3Go), the ovarian expression of MCP-1 significantly (P < 0.05) increased (18-fold) 6 h after hCG administration as compared with control. Ovarian MCP-3 transcripts (Fig. 4Go) were similarly and significantly (P < 0.05) increased (12-fold) at the 2- and 6-h time points post hCG. GRO also displayed significantly (P < 0.05) increased ovarian expression relative to the control at distinct preovulatory time points [at 4 h (25-fold) and 6 h (26-fold) after hCG]. In contrast, two other chemokines (Fig. 4Go), interferon-inducible protein of 10 kDa (IP-10) and fractalkine, displayed ovarian expression at preovulatory time points although not statistically significant (Fig. 4Go). Eotaxin, a chemokine predominantly involved in attracting eosinophils, displayed no statistically significant change from the control (Fig. 5Go). The ovarian expression of RANTES (regulated upon activation normal T cell expressed and secreted) also did not significantly vary relative to the control (Fig. 6Go).



View larger version (29K):
[in this window]
[in a new window]
  		Figure 3. MCP-1 gene expression in the rat ovary. The expression of MCP-1 was quantified by using RT-competitive PCR. Ovaries of 25-d-old immature rats were obtained from unstimulated (control), PMSG (15 IU ip)-injected (48 h), or PMSG (15 IU ip)-injected/hCG (15 IU ip)-triggered animals 48 h later (2–48 h after hCG). RNA was extracted, reverse-transcribed into cDNA and subjected to competitive PCR as described in Materials and Methods. Ten microliters of the PCR reactions were electrophoresed through 1.8% agarose gels and visualized with ethidium bromide. Quantification was performed as described in Materials and Methods. The top panel depicts in bar graph form the fold expression relative to control ± SD of three independent experiments. *, P < 0.05 vs. control (unstimulated). A representative gel is shown below the bar graph. Each group of five gel lanes corresponds to the competitive PCR for a particular time point. Five serial logarithmic dilutions of the synthetic competitive DNA and a fixed amount of first strand cDNA were coamplified. The amplified MCP-1 product (upper band) is 312 bp and the competitor (lower band) is 205 bp.

 


View larger version (32K):
[in this window]
[in a new window]
  		Figure 4. MCP-3, IP-10, GRO, and fractalkine gene expression in the rat ovary. The expression of MCP-3, IP-10, GRO, and fractalkine was quantified by using RT-competitive PCR as described in Fig. 3Go. The four panels depict in bar graph form the fold expression relative to control ± SD of three experiments. *, P < 0.05 vs. control (unstimulated).

 


View larger version (32K):
[in this window]
[in a new window]
  		Figure 5. Eotaxin gene expression in the rat ovary. The expression of eotaxin was quantified by using RT-competitive PCR as described in Fig. 3Go. The top panel depicts in bar graph form the fold expression relative to control ± SD of three experiments. A representative gel is shown below the bar graph. *, P < 0.05 vs. control (unstimulated). Each group of five gel lanes corresponds to the competitive PCR for a particular time point. Five serial logarithmic dilutions of the synthetic competitive DNA and a fixed amount of first strand cDNA were coamplified. The amplified MCP-1 product (upper band) is 369 bp, and the competitor (lower band) is 246 bp.

 


View larger version (28K):
[in this window]
[in a new window]
  		Figure 6. RANTES gene expression in the rat ovary. The expression of RANTES was quantified by using RT-competitive PCR as described in Fig. 3Go. The top panel depicts in bar graph form the fold expression relative to control ± SEM. *, P < 0.05 vs. control (unstimulated). A representative gel is shown below the bar graph. Each group of five gel lanes corresponds to the competitive PCR for a particular time point. Five serial logarithmic dilutions of synthetic competitive DNA and a fixed amount of first strand cDNA were coamplified. The amplified RANTES product (lower band) is 105 bp, and the competitor (upper band) is 179 bp.

 
Expression of chemokines in the postovulatory rat ovary
The ovarian expression of MCP-3, fractalkine, and eotaxin was detected at 48 h after hCG administration, a point in time corresponding to the diestrous stage of the unstimulated estrous cycle. Although not reaching statistical significance, MCP-1, IP-10, GRO, and RANTES displayed ovarian expression at the 48-h-post-hCG time point.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The experiments presented herein were designed to ascertain the expression of members of the chemokine family in the rat ovary and to assess their hormonal dependence and cyclic variation.

We initially concentrated our efforts on identifying which chemokines were expressed in the PMSG-primed rat ovary 6 h post hCG. This time point was chosen to focus on identifying chemokines expressed in the preovulatory ovary. In line with previous reports (20, 21, 22), MCP-1, a member of the CC chemokine group, was detected in the rat ovary. Additionally, we report, to the best of our knowledge, for the first time, the expression of other members of the CC chemokine group including MCP-3, MIP-1{alpha}, MIP-1ß, MIP-1{gamma}, RANTES, and eotaxin in the rat ovary. Furthermore, representative chemokines, not previously reported, from other chemokine families (C, CXC, CX3C) were also detected in the ovary. Several of the chemokines studied (C10, TCA-3, exodus, exodus-2, cytokine-induced neutrophil chemoattractant-2, MIP-2, and LPS-induced C-X-C) belonging to the CC and CXC families, however, could not be detected in the preovulatory rat ovary by RT-PCR analysis. Our results thus suggest that chemokines from all four chemokine families are expressed in the preovulatory rat ovary and may thus play a role in ovarian physiology.

It is recognized that additional chemokines belonging to all four chemokine families may be expressed in the rat ovary. The initial screening for ovarian chemokines using the PMSG-primed rat ovary 6 h post hCG may have limited the identification of chemokines expressed primarily in the postovulatory phase of the ovarian cycle and as such may constitute a relative shortcoming of this study.

To further assess the role chemokines may play in normal ovarian physiology, we evaluated the cyclic ovarian expression of the ovary-positive chemokines during a simulated estrous cycle. The CC chemokine, MCP-1, displayed an 18-fold increase in its ovarian expression 6 h after hCG administration when compared with the control unstimulated state (Fig. 3Go). MCP-3, a CC chemokine related to MCP-1, also displayed increased (12-fold) preovulatory ovarian expression (Fig. 4Go). The CC chemokines, similar in their DNA sequences, are distinguished by the first two conserved cysteine residues, which are adjacent to each other (9). The CC chemokines predominantly attract leukocytes of the monocyte/macrophage lineage. Given the preovulatory increase in the ovarian expression of MCP-1 and MCP-3, our data suggest that the leukocyte influx into the ovary during ovulation may be due, in part, to the preovulatory increase in the ovarian expression of the above CC chemokines. Indeed, the timing of the increased expression of the CC chemokines appears related to the LH surge (simulated by hCG in the current experiments). Taken together, CC chemokines may constitute intermediary modulators responsible for attracting leukocytes into the ovary following the initiation of the mid-cycle LH surge.

In addition to the CC chemokines, a CXC chemokine was also ascertained to display increased preovulatory ovarian expression. In contrast to the CC chemokines, the CXC chemokines are distinguished by the separation of the first two-cysteine residues of the mature protein by a single amino acid. A neutrophilic attractant, GRO, displayed markedly increased preovulatory ovarian expression (25-fold over control, Fig. 4Go). Our results are similar to the findings of Ushigoe et al. (30) demonstrating increased ovarian mRNA expression of GRO at 6 h post hCG. GRO was also previously detected in human follicular fluid and implicated in the recruitment of leukocytes to the preovulatory human follicle (15). These data suggest that the leukocytic influx occurring during ovulation may be attributable, in part, to both CC and CXC chemokines and that the leukocytic cells attracted may be derived from several different lineages including monocytes, lymphocytes, and neutrophils.

In contrast to the elevated preovulatory expression levels of GRO, eotaxin, a CXC chemokine and an eosinophilic attractant, displayed a constant ovarian expression pattern throughout the estrous cycle (Fig. 5Go). Although the exact role of eotaxin in ovarian physiology is unknown, the steady-state levels of this CXC chemokine may be required to maintain the resident eosinophils of the ovary. This possible ovarian role of eotaxin is further evidenced by the constitutive presence of eosinophils, which appear to depend on eotaxin as demonstrated by its targeted disruption (31).

Chemokines belonging to two other families, the C and CX3C families, were also investigated for possible temporal fluctuations in their ovarian expression patterns during a simulated estrous cycle. Fractalkine, a CX3C chemokine, displayed a constant level of ovarian expression (Fig. 4Go). The ovarian expression of lymphotactin, a C chemokine, however, could not be measured accurately by competitive PCR due to low expression levels (data not shown). Both of these chemokines may, in concert with the other previously mentioned chemokines, account for the influx of leukocytes into the ovary as observed after the LH surge.

The data suggesting ovarian expression of MCP-1 (Fig. 3Go) and RANTES (Fig. 6Go) during the postovulatory phase of the ovarian cycle are consistent with the previous detection of MCP-1 in rat corpora lutea (20, 21, 22) and of RANTES in bovine corpora lutea (32). Our data, i.e. an absent preovulatory increase in the ovarian expression of RANTES, are consistent with the findings of Karstrom-Encrantz and colleagues (15) in which low levels of RANTES were observed in human follicular fluid. Therefore, the ovarian expression of MCP-1 and RANTES in the postovulatory phase suggests a role for these chemokines in mediating the recruitment of monocytes/macrophages into the regressing corpus luteum, an established physiologic phenomenon (3, 5, 8, 22).

Our findings of widely different ovarian expression levels of various chemokines (Figs. 3Go and 4Go) may be responsible for the differential recruitment of subpopulations of leukocytes at different phases of the ovarian life cycle. Chemokine expression levels have been demonstrated to be differentially regulated in human endothelial and colon epithelial cells and may be responsible for the various types of leukocytes recruited during an inflammatory response (33, 34). Functional groups of chemokines may coordinate the recruitment of leukocyte subsets and generate complete inflammatory response (35).

In conclusion, we have provided evidence that chemokines from all four families are expressed in the rat ovary. Furthermore, the ovarian expression of MCP-1, MCP-3, and GRO is increased during time points corresponding to the periovulatory window of the estrous cycle. In contrast, the ovarian expression of eotaxin and fractalkine throughout the estrous cycle proved relatively constant. The expression of MCP-1 and RANTES was increased during the postovulatory phase. These observations suggest that chemokines may be responsible for the intraovarian accumulation of leukocytes as depicted in Fig. 7Go, both in and around ovulation as well as during corpus luteum regression, and as such may be intimately involved in the normal physiology of the ovarian life cycle.



View larger version (25K):
[in this window]
[in a new window]
  		Figure 7. Summary of introvarian chemokine expression patterns. Resident white blood cells are present in the interstitial ovarian compartment (A). Following the LH surge, there is an increased infiltration of white blood cells culminating in the release of the oocyte (B, C). Postovulatory, the number of white blood cells increases and eventually the white blood cells invade the corpus luteum (D). The intraovarian expression of the chemokines MCP-1, MCP-3, and GRO corresponds with the increased numbers of white blood cells following the LH surge. The postovulatory intraovarian expression of MCP-1 and RANTES may lead to the accumulation of white blood cells in the corpus luteum. The constant expression of intraovarian eotaxin may be necessary to maintain ovarian eosinophilia.

 


   Acknowledgments
 
The authors would like to acknowledge Dr. Aniko Szabo, Biostatistics Resource at the Huntsman Cancer Institute, for her invaluable help with the statistical analysis. In addition, the authors would like to thank Ms. Michelle Lewandowski for her administrative assistance in the preparation of this manuscript.


   Footnotes
 
This work was supported, in part, by NIH Research Grant HD-30288 (to E.Y.A.).

1 Current address: Department of Obstetrics and Gynecology, Hokkaido University School of Medicine, North 15 West 6 Kitaku, Sapporo City, 606 Japan. Back

Abbreviations: GRO, Growth-regulated oncogene; hCG, human CG; IP-10, interferon-inducible protein of 10 kDa; LPS, lipopolysaccharide; MCP-1, monocyte chemotatic protein 1; MIP1, macrophage inflammatory protein 1; RANTES, regulated upon activation normal T cell expressed and secreted; RT, reverse transcriptase; TCA, T cell activation gene.

Received July 31, 2001.

Accepted for publication November 20, 2001.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Espey LL 1980 Ovulation as an inflammatory reaction—a hypothesis. Biol Reprod 22:73–106[CrossRef][Medline]
  2. Adashi EY 1990 The potential relevance of cytokines to ovarian physiology: the emerging role of resident ovarian cells of the white blood cell series. Endocr Rev 11:454–444[Medline]
  3. Brannstrom M, Norman RJ 1993 Involvement of leukocytes and cytokines in the ovulatory process and corpus luteum function. Hum Reprod 8:1762–1775[Abstract/Free Full Text]
  4. Brannstrom M, Mayrhofer G, Robertson SA 1993 Localization of leukocyte subsets in the rat ovary during the periovulatory period. Biol Reprod 48:277–286[Abstract]
  5. Brannstrom M, Pascoe V, Norman RJ, McClure N 1994 Localization of leukocyte subsets in the follicle wall and in the corpus luteum throughout the human menstrual cycle. Fertil Steril 61:488–495[Medline]
  6. Standaert FE, Zamora CS, Chew BP 1991 Quantitative and qualitative changes in blood leukocytes in the porcine ovary. Am J Reprod Immunol 25:163–168
  7. Suzuki T, Sasano H, Takaya R, Fukaya T, Yajima A, Date F, Nagura H 1998 Leukocytes in normal-cycling human ovaries: immunohistochemical distribution and characterization. Hum Reprod 13:2186–2191[Abstract/Free Full Text]
  8. Brannstrom M, Giesecke L, Moore IC, van den Heuvel CJ, Robertson SA 1994 Leukocyte subpopulations in the rat corpus luteum during pregnancy and pseudopregnancy. Biol Reprod 50:1161–1167[Abstract]
  9. Luster AD 1998 Chemokines—chemotactic cytokines that mediate inflammation. N Engl J Med 338:436–445[Free Full Text]
  10. Garcia-Velasco JA, Arici A 1998 Chemokines and human reproduction. Fertil Steril 71:983–993
  11. Runesson E, Bostrom EK, Janson PO, Brannstrom M 1996 The human preovulatory follicle is a source of the chemotactic cytokine interleukin-8. Mol Hum Reprod 2:245–250[Abstract/Free Full Text]
  12. Arici A, Oral E, Bukulmez O, Buradagunta S, Engin O, Olive DL 1996 Interleukin-8 expression and modulation in human preovulatory follicles and ovarian cells. Endocrinology 137:3762–3769[Abstract]
  13. Arici A, Oral E, Bukulmez O, Buradagunta S, Bahtiyar O, Jones EE 1997 Monocyte chemotactic protein-1 expression in human preovulatory follicles and ovarian cells. J Reprod Immunol 32:201–219[CrossRef][Medline]
  14. Chang RJ, Gougeon A, Erickson GF 1998 Evidence for a neutrophil-interleukin-8 system in human folliculogenesis. Am J Obstet Gynecol 178:650–657[CrossRef][Medline]
  15. Karstrom-Encrantz L, Runesson E, Bostrom EK, Brannstrom M 1998 Selective presence of the chemokine growth-regulated oncogene {alpha} (GRO{alpha}) in the human follicle and secretion from cultured granulosa-lutein cells at ovulation. Mol Hum Reprod 4:1077–1083[Abstract/Free Full Text]
  16. Ujioka T, Matsukawa A, Tanaka N, Matsuura K, Yoshinaga M, Okamura H 1998 Interleukin-8 as an essential factor in the human chorionic gonadotropin-induced rabbit ovulatory process: interleukin-8 induces neutrophil accumulation and activation in ovulation. Biol Reprod 58:526–530[Abstract/Free Full Text]
  17. Ujioka T, Matsukawa A, Tanaka N, Matsuura K, Yoshinaga M, Okamura H 1998 Analysis of the cytokine interaction among interleukin-1ß, interleukin-8, and interleukin-1 receptor antagonist in the rabbit ovulatory process. Fertil Steril 70:759–765[CrossRef][Medline]
  18. Belayet HM, Kanayama N, Khatun S, Asahina T, Okada Y, Kitamura K, Kobayashi T, Terao T 2000 Pharmacologic doses of interleukin 8 suppositories induce follicular maturation in rabbits. Cytokine 12:361–367[CrossRef][Medline]
  19. Runesson E, Ivarsson K, Janson PO, Brannstrom M 2000 Gonadotropin- and cytokine-regulated expression of the chemokine interleukin 8 in the human preovulatory follicle of the menstrual cycle. J Clin Endocrinol Metab 85:4387–4395[Abstract/Free Full Text]
  20. Bowen JM, Keyes PL, Warren JS, Townson DH 1996 Prolactin-induced regression of the rat corpus luteum: expression of monocyte chemoattractant protein-1 and invasion of macrophages [published erratum appears in Biol Reprod 1996 Jul;55(1):224]. Biol Reprod 54:1120–1127[Abstract]
  21. Townson DH, Warren JS, Flory CM, Naftalin DM, Keyes PL 1996 Expression of monocyte chemoattractant protein-1 in the corpus luteum of the rat. Biol Reprod 54:513–520[Abstract]
  22. Bowen JM, Towns R, Warren JS, Landis Keyes P 1999 Luteal regression in the normally cycling rat: apoptosis, monocyte chemoattractant protein-1, and inflammatory cell involvement. Biol Reprod 60:740–746[Abstract/Free Full Text]
  23. Haworth JD, Rollyson MK, Silva P, McIntush EW, Niswender GD 1998 Messenger ribonucleic acid encoding monocyte chemoattractant protein-1 is expressed by the ovine corpus luteum in response to prostaglandin F2{alpha}. Biol Reprod 58:169–174[Abstract/Free Full Text]
  24. Penny LA, Armstrong DG, Baxter G, Hogg C, Kindahl H, Bramley T, Watson ED, Webb R 1998 Expression of monocyte chemoattractant protein-1 in the bovine corpus luteum around the time of natural luteolysis. Biol Reprod 59:1464–1469[Abstract/Free Full Text]
  25. Senturk LM, Seli E, Gutierrez LS, Mor G, Zeyneloglu HB, Arici A 1999 Monocyte chemotactic protein-1 expression in human corpus luteum. Mol Hum Reprod 5:697–702[Abstract/Free Full Text]
  26. Ho SN, Hunt HD, Horton RM, Pullen JK, Pease LR 1989 Site-directed mutagenesis by overlap extension using the polymerase chain reaction. Gene 77:51–59[CrossRef][Medline]
  27. Horton RM, Hunt HD, Ho SN, Pullen JK, Pease LR 1989 Engineering hybrid genes without the use of restriction enzymes: gene splicing by overlap extension. Gene 77:61–68[CrossRef][Medline]
  28. Souaze F, Ntodou-Thome A, Tran CY, Rostene W, Forgez P 1996 Quantitative RT-PCR: limits and accuracy. Biotechniques 21:280–285[Medline]
  29. Dunnett CW 1955 A multiple comparisons procedure for comparing several treatments with a control. J Am Stat Assoc 50:1096–1121[CrossRef]
  30. Ushigoe K, Irahara M, Fukumochi M, Kamada M, Aono T 2000 Production and regulation of cytokine-induced neutrophil chemoattractant in rat ovulation. Biol Reprod 63:121–126[Abstract/Free Full Text]
  31. Rothenberg ME, MacLean JA, Pearlman E, Luster AD, Leder P 1997 Targeted disruption of the chemokine eotaxin partially reduces antigen-induced tissue eosinophilia. J Exp Med 185:785–790[Abstract/Free Full Text]
  32. Aust G, Brylla E, Lehmann I, Kiessling S, Spanel-Borowski K 1999 Cloning of bovine RANTES mRNA and its expression and regulation in ovaries in the periovulatory period. FEBS Lett 463:160–164[CrossRef][Medline]
  33. Parks E, Lukacs NW, Strieter RM, Kunkel SL 1998 Chemokine expression in endothelial cells and monocytes is differentially regulated. Pathobiology 66:64–70[CrossRef][Medline]
  34. Yang SK, Eckmann L, Panja A, Kagnoff MF 1997 Differential and regulated expression of C-X-C, C-C, and C-chemokines by human colon epithelial cells. Gastroenterology 113:1214–1223[CrossRef][Medline]
  35. Gutierrez-Ramos JC, Lloyd C, Kapsenberg ML, Gonzalo JA, Coyle AJ 2000 Non-redundant functional groups of chemokines operate in a coordinate manner during the inflammatory response in the lung. Immunol Rev 177:31–42[CrossRef][Medline]



This article has been cited by other articles:


Home page
 EndocrinologyHome page
P. Zhao, A. De, Z. Hu, J. Li, S. M. Mulders, M. D. Sollewijn Gelpke, E.-K. Duan, and A. J. W. Hsueh
Gonadotropin Stimulation of Ovarian Fractalkine Expression and Fractalkine Augmentation of Progesterone Biosynthesis by Luteinizing Granulosa Cells
Endocrinology, June 1, 2008; 149(6): 2782 - 2789.
[Abstract] [Full Text] [PDF]

Home page
 J. Leukoc. Biol.Home page
A. J. Lengi, R. A. Phillips, E. Karpuzoglu, and S. A. Ahmed
Estrogen selectively regulates chemokines in murine splenocytes
J. Leukoc. Biol., April 1, 2007; 81(4): 1065 - 1074.
[Abstract] [Full Text] [PDF]

Home page
 Hum ReprodHome page
P. Fedorcsak, M. Raki, and R. Storeng
Characterization and depletion of leukocytes from cells isolated from the pre-ovulatory ovarian follicle
Hum. Reprod., April 1, 2007; 22(4): 989 - 994.
[Abstract] [Full Text] [PDF]

Home page
 Mol Hum ReprodHome page
P. Dahm-Kahler, E. Runesson, A. K. Lind, and M. Brannstrom
Monocyte chemotactic protein-1 in the follicle of the menstrual and IVF cycle
Mol. Hum. Reprod., January 1, 2006; 12(1): 1 - 6.
[Abstract] [Full Text] [PDF]

Home page
 J EndocrinolHome page
M. C Gieske, G. Y. Na, Y. Koo, M. Jo, T. E Curry Jr, and C. Ko
Decay-accelerating factor in the periovulatory rat ovary
J. Endocrinol., August 1, 2005; 186(2): 303 - 313.
[Abstract] [Full Text] [PDF]

Home page
 Biol. Reprod.Home page
K. Sayasith, N. Bouchard, D. Boerboom, K. A. Brown, M. Dore, and J. Sirois
Molecular Characterization of Equine P-Selectin (CD62P) and Its Regulation in Ovarian Follicles During the Ovulatory Process
Biol Reprod, March 1, 2005; 72(3): 736 - 744.
[Abstract] [Full Text] [PDF]

Home page
 J. Clin. Endocrinol. Metab.Home page
N. J. Hannan, R. L. Jones, H. O. D. Critchley, G. J. Kovacs, P. A. W. Rogers, B. Affandi, and L. A. Salamonsen
Coexpression of Fractalkine and Its Receptor in Normal Human Endometrium and in Endometrium from Users of Progestin-Only Contraception Supports a Role for Fractalkine in Leukocyte Recruitment and Endometrial Remodeling
J. Clin. Endocrinol. Metab., December 1, 2004; 89(12): 6119 - 6129.
[Abstract] [Full Text] [PDF]

Home page
 Biol. Reprod.Home page
N. Bonello, M. J. Jasper, and R. J. Norman
Periovulatory Expression of Intercellular Adhesion Molecule-1 in the Rat Ovary
Biol Reprod, October 1, 2004; 71(4): 1384 - 1390.
[Abstract] [Full Text] [PDF]

Home page
 Hum Reprod UpdateHome page
R. Wu, K. H. Van der Hoek, N. K. Ryan, R. J. Norman, and R. L. Robker
Macrophage contributions to ovarian function
Hum. Reprod. Update, March 1, 2004; 10(2): 119 - 133.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Purchase Article
Right arrow View Shopping Cart
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 Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Wong, K. H. H.
Right arrow Articles by Adashi, E. Y.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Wong, K. H. H.
Right arrow Articles by Adashi, E. Y.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology Endocrine Reviews J. Clin. End. & Metab.
Molecular Endocrinology Recent Prog. Horm. Res. All Endocrine Journals