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Endocrinology Vol. 142, No. 10 4515-4521
Copyright © 2001 by The Endocrine Society


ARTICLES

Eotaxin Is Required for Eosinophil Homing into the Stroma of the Pubertal and Cycling Uterus

Valérie Gouon-Evans and Jeffrey W. Pollard

Departments of Developmental and Molecular Biology (V.G.-E., J.W.P.), Obstetrics & Gynecology and Women’s Health (J.W.P.) and Center for the Study of Reproductive Biology and Women’s Health, Albert Einstein College of Medicine, New York, New York 10461

Address all correspondence and requests for reprints to: Jeffrey W. Pollard, Ph.D., Albert Einstein College of Medicine, Department of Developmental and Molecular Biology, 1300 Morris Park Avenue, Bronx, New York 10461. E-mail: pollard{at}aecom.yu.edu


    Abstract
 Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The presence of eosinophils in the endometrium of rodents during the estrous cycle or after E2 administration to ovariectomized animals is well documented. Nevertheless, the chemoattractant for eosinophils and the function of E-dependent eosinophils during the estrous cycle remain unknown. Using mice homozygous for a null mutation in the gene for eotaxin, a specific chemokine for eosinophils, we have identified eotaxin as being necessary for eosinophil homing into the uterine stroma, and regulated by E2 during the estrous cycle. In the absence of eosinophils, the onset of estrous cycle displayed a 2-wk delay along with the first age of parturition, suggesting a possible local role of eosinophils present in the pubertal uterus in preparing the mature uterus for pregnancy. However, despite the absence of eosinophils, once the mice reach maturity, their estrous cycles as well as their reproductive functions were normal. Our results demonstrate that E2 acts through eotaxin to recruit eosinophils to the uterine stroma during the estrous cycle in mice, but that these cells do not have a function in regulating either the duration of the estrous cycle or fertility of mice.


    Introduction
 Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
DURING THE ESTROUS cycle, the heterogeneous cell types of the uterus undergo continuous synchronized waves of proliferation, differentiation, and recruitment in response to the rise and fall of E2 and progesterone (1, 2). Serum E2 levels increase at proestrus to stimulate proliferation of the epithelial compartment. When the serum E2 level drops at estrus, the luminal and glandular epithelial cells undergo apoptosis to return to original cell number (2, 3). Along with the induction of epithelial cell proliferation, E2 is also responsible for recruitment of inflammatory leukocyte cells from the blood stream into the stromal compartment (4, 5). Macrophages, MHC class II{dagger} dendritic cells, eosinophils, and neutrophils are the predominant leukocytes homing to the cycling uterus (6, 7, 8, 9, 10, 11).

Considerable literature exists showing that eosinophils infiltrate cyclically the rodent uterus. Infiltration of the rat uterus by eosinophils, coincident with the estrus cycle (12), was observed during the 1950s, and this observation has been confirmed and extended to different species such as mouse and human (9, 10, 13, 14). Since then, numerous investigations have shown that: 1) injection of E2 into ovariectomized or immature rodents causes a dramatic increase in uterine eosinophils (5) and an increase in uterine peroxidase activity (15) that was shown later to be restricted to eosinophils (16, 17); 2) uterine eosinophil numbers vary more than 100-fold during the normal estrous cycle (18); 3) eosinophils undergo lysis in the uterus around the time of estrus releasing their contents into the extracellular spaces (10, 19); and 4) the E-induced uterine eosinophilia is associated with marked uterine edema (20). Despite the fact that eosinophil homing into the cycling uterus is well established, the role of these cells and their chemoattractant remain to be elucidated.

Of the cytokines implicated in modulating eosinophilic inflammation, only IL-5 and eotaxin have been identified to selectively regulate eosinophil trafficking (21, 22, 23). IL-5 is responsible for the proliferation, differentiation, recruitment, and activation of eosinophils. Studies in mice lacking IL-5 caused by passive immunization against IL-5 or by the generation of a null mutation of the IL-5 gene showed that these mice are virtually devoid of eosinophils in the blood stream and that consequently their uterine eosinophil population was depleted strongly (24, 25, 26). Nevertheless, these studies did not demonstrate the physiological chemoattractant effect of IL-5 for eosinophil homing from the blood stream into the uterus. Eotaxin, a member of the C-C chemokine family, has been originally identified as a novel chemotactic agent for eosinophils into the lung in a guinea pig model of eosinophilic airway hypersensitivity (22). Eotaxin mRNA is induced in multiple animal models of eosinophilic inflammation, in human tissue in response to allergen challenge (23, 27, 28), and also in the mouse mammary gland during postnatal development (29). Recently, the expression of eotaxin and its receptor have been reported in human endometrium through the menstrual cycle (30). However, the functions of eotaxin during the estrus cycle remain unknown.

The purpose of this study was to examine the uterine eosinophil population in eotaxin-deficient mice and determine the physiological importance of eotaxin and eotaxin-dependent eosinophils in reproduction.


    Materials and Methods
 Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Animals and injection schedule for estimation of E2 responsiveness
Eotaxin-deficient mice maintained on an inbred 129/SvEv background were a kind gift from Dr. Marc E. Rothenberg (Division of Pulmonary Medicine, Allergy and Clinical Immunology, Children’s Hospital Medical Center, Cincinnati, OH) (31). 129/SvEv wild-type and mutant mice were mated under pathogen-free conditions in a barrier facility at the Albert Einstein College of Medicine. All studies were performed under NIH guidelines for the care and treatment of experimental laboratory rodents.

The onset of puberty was assessed in two ways: 1) vaginal opening and 2) the onset of complete estrous cycles (two cycles). Mice were examined daily from 4 wk of age to determine whether vaginal opening had occurred. Analysis of estrous cycles was performed from 5 wk of age. Vaginal smears were obtained on a daily basis, stained with hematoxylin/eosin, and examined for cellular content. Four stages of the cycle were defined as followed: proestrus (100% intact and alive epithelial cells), estrus (100% cornified epithelial cells), metestrus (~50% cornified epithelial cells and 50% leukocytes), and diestrus (80–100% leukocytes). For determination of the age of first parturition, seven females of each group were mated with males from the same group from 6 wk of age, and the age of the first parturition was determined. Successful pregnancy interval was also determined in weeks by mating nine wild-type females and eight mutant female with males from the same group. The male was maintained with the female from the mating period to d 2 after parturition, then separated, and finally replaced with the female when the weaning period occurred (3 wk after parturition). Interval times between two successful pregnancies were noted for two to six consecutive pregnancies for each female per group.

Adult female mice (10–12 wk) were bilaterally ovariectomized on d 1, primed at d 7 and d 8 by sc injection of 100 ng of E2 in 0.1 ml of peanut oil, and then treated with 3 consecutive sc injections of 100 ng of E2 in 0.1 ml of peanut oil at d 15, 16, and 17, or with 0.1 ml of peanut oil for the control mice. Mice were killed 24 h or 42 h after the last injection, and uteri removed for analysis. The wet weight of uteri in gram was measured for 6–8 mice of each group, and statistical evaluations using standard deviations were performed with a two-tailed t test.

Histochemistry
Uteri were fixed overnight in formalin (Sigma, St. Louis, MO) and paraffin wax embedded for hematoxylin/eosin staining, or OCT embedded (Tissue Tek; Bayer Corp., Elkart, IN) and frozen for peroxidase activity analysis. Five-micrometer paraffin sections were consecutively stained with hematoxylin and lightly with eosin Y to identify eosinophils by their pink cytoplasmic granules and segmented nucleus. Five-micrometer frozen sections were used to detect the endogenous peroxidase activity restricted to eosinophils in uterine tissue (17). Briefly, frozen sections were fixed in acetone at 4 C for 10 min, rinsed in PBS, and then incubated for 2–5 min at room temperature in diaminobenzidine in presence of hydrogen peroxide in a ratio determined by the Vector Laboratories, Inc. peroxidase kit (Burlingame, CA). Sections were finally rinsed in water and counterstained with hematoxylin.

Northern blot analysis
Total RNA from uteri of one to three mice from each group was isolated by the method of Chomczynski and Sacchi (32). Ten micrograms of total RNA was separated by formaldehyde-agarose gel electrophoresis, transferred to nylon filters, and probed with a [32P]dCTP-labeled cDNA probe for eotaxin [ATCC (Manassas, VA) No. 1463042; GenBank accession no. AA711712] using the method previously described (33).

Western blot analysis
Each individual uterus was homogenized in RIPA buffer (9.1 mM dibasic sodium phosphate, 1.7 mM monobasic sodium phosphate, 150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, pH 7.4), and protein concentration determined by a BCA protein assay (Pierce Chemical Co., Rockford, IL). Equal amount of protein (80 µg) under reducing conditions were separated by electrophoresis on 15% SDS-polyacrylamide gels and transferred onto polyvinylidendifluoride Immobilon-P membranes (0.45 µm; Millipore Corp., Bedford, MA). The membranes were blocked with 5% nonfat dry milk and 0.1% Tween 20 in Tris-buffered saline (TBS: 150 mM NaCl, 20 mM Tris, pH 7.6) for 1 h at RT. They were then incubated with the IgG-purified rabbit antimurine eotaxin antibody (generously provided by Dr. Steven Kunkel, University of Michigan, Ann Arbor, MI) at 0.5 µg/ml in blocking solution for 1 h at RT, washed 3 times for 15 min in TBS-Tween buffer, and subsequently incubated with a 1:3,000 dilution of horseradish peroxidase-linked secondary antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) for 45 min at RT. After washing with TBS-Tween, immunodetection was achieved with an enhanced chemiluminescence system (ECL; Amersham Pharmacia Biotech, Piscataway, NJ). Recombinant murine eotaxin (Chemicon International Inc., Temecula, CA) was used as a reference control and an {alpha}-guanine nucleotide dissociation inhibitor (GDI) (a gift from Dr. Perry Bickel, Washington University, St. Louis, MO) was used as internal control for protein abundance.


    Results
 Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Eotaxin-dependent eosinophils in the pubertal and cycling uterus
Eosinophil invasion of the uterine stroma during the estrous cycle is well documented. However, the chemoattractant agent for these cells has not been identified. Because eotaxin is a specific chemokine for eosinophils and usually acts as a local chemoattractant, we examined its transcript levels in the uterus. Eotaxin transcripts were detected as early as 3 wk of age and throughout the pubertal uterine development at 5 wk of age (Fig. 1AGo). In the adult, they were elevated during proestrus (P) returning to a significantly lower level at diestrus (D) (P = 0.0004, two-tailed t test) (Fig. 1BGo). In ovariectomized mice, transcript levels of eotaxin were almost undetectable, but they were induced dramatically by E2 treatment, 39-fold at 24 h and 31-fold at 42 h after injection (Fig. 1CGo, top and bottom panels). As predicted from the size of the eotaxin cDNA, the major mRNA transcript was about 1 kb. It is of interest that in the adult uterus but not in the peripubertal uterus, smaller transcripts were systematically detected with a major one at 0.6 kb after E2 stimulation either during the estrous cycle (Fig. 1BGo) or following E2 treatment of ovariectomized mice (Fig. 1CGo). In the latter case, the induction of the 0.6-kb transcript paralleled perfectly the induction of the larger transcript, although it was virtually undetectable in the untreated ovariectomized uterus (Fig. 1CGo, bottom panel). Even though this smaller transcript has never been described in mice, it has been reported in certain human tissues such as the small intestine, colon, and heart (28). The nature of the 0.6-kb transcript is not certain, but is sufficiently large to encode the 8.4-kDa eotaxin protein and may represent use of an alternative polyadenylation site (34). The eotaxin protein levels from total uterine homogenates during the estrous cycle were analyzed in parallel to the transcript levels by Western blot analysis using an anti-murine eotaxin antibody. A band at approximately 8 kDa, which corresponded to the reference control protein, indicated the presence of mouse eotaxin in the uterus. Interestingly, eotaxin protein was only detected during the proestrus phase (P/E), which was in this case at late proestrus, a stage of maximal transcript expression. The inability to detect protein in estrus or diestrus may reflect the sensibility of the antibody or rapid degradation of the protein.



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Figure 1. Eotaxin mRNA and protein levels in the uterus. Northern Blot analysis of eotaxin mRNA transcript levels in total uterine RNA from wild-type mice at 3 and 5 wk of age (A), during the estrous cycle (P, proestrus; E, estrus; D, diestrus) (B), and after E2 treatment following ovariectomy (C). Each lane contains the total uterine RNA from 1 mouse. Uniformity of RNA loading is shown by ethidium bromide staining of 28S rRNA (bottom panels). Arrows point to the 1-kb and 0.6-kb transcripts. Means of the global induction of both large and small transcripts during the estrous cycle are significantly different between P and D (P = 0.0004, two-tailed t test). After E2 treatment, means of intensity of both 1 kb and 0.6 kb eotaxin transcript levels are represented in the graph (mean ± SEM), as well as the fold number of the global induction of both large and small transcripts (C) (*/** significant differences between the control no E2 and E2-treated samples, * P = 0.0431 ** P = 0.0035, two-tailed t test). Western blot analysis (D) of eotaxin protein levels in total uterine protein extracts from wild-type mice during the estrous cycle (P/E: advanced proestrus, beginning of E, estrus; D, diestrus). Each lane contains the uterine homogenate from one mouse. The membrane was also probed with the anti-GDI antibody as a protein loading control. Eot, 10 ng recombinant murine eotaxin control.

 
Two independent histologic methods were performed to detect eosinophils in the uterus: hematoxylin/eosin staining, revealing the eosin-positive cytoplasmic granules and the multilobular nucleus of eosinophils, and the detection of endogenous peroxidase activity that has been previously shown to be restricted to eosinophils in uterus (16, 17). In pubertal mice, at 5 wk of age, eosin-positive granule containing polynuclear eosinophils were highly recruited into the uterine stroma of wild-type mice (Fig. 2AGo), whereas they were totally absent in eotaxin-deficient mice (Fig. 2BGo). In adult mice, eosinophil recruitment was analyzed following E2 treatment of ovariectomized mice. In untreated mice, eosinophils were virtually absent (data not shown). However, in confirmation of previous studies (5, 15), upon E2-stimulation, the number of eosin-positive or peroxidase-positive eosinophils increased gradually, reaching the highest number at a time corresponding to an experimental estrus (determined by vaginal smear) in wild-type mice 42 h after E2 treatment (Fig. 2Go, C, E, and F). Eosinophils were recruited into the uterine stroma in the vicinity of the luminal epithelium (Fig. 2Go, C and E) and in the myometrium surrounding the stroma (Fig. 2FGo). In contrast, no eosin-positive or peroxidase-positive eosinophils could be seen in the uterus of eotaxin-deficient mice at the experimental estrus at any location (Fig. 2Go, D and G). Thus, eotaxin transcript level correlates with the recruitment of eosinophils into the uterus of the pubertal and adult mice. It is of interest to note that eotaxin protein is detected early during the estrous cycle at proestrus when eosinophil homing into the uterus begins, whereas the protein is undetectable by Western blot at estrus at the time when eosinophil recruitment is the highest. This suggested that eotaxin is an early signal for eosinophil homing into the uterine stroma. In the absence of eotaxin, eosinophils are completely absent at every stage of development.



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Figure 2. Effect of the lack of eotaxin on eosinophil populations in the uterus. A–D, Hematoxylin/eosin staining of endometrial cross-sections from wild-type +/+ (A, C) and eotaxin-deficient -/- (B, D) mice at 5 wk of age (A, B) and in adult ovariectomized mice 42 h after E2 treatment (C, D). Most of the eosinophils are indicated with arrowheads. E–G, Eosinophil peroxidase activity (dark brown coloration) detected on frozen uterine cross-section of wild-type (E, F) and mutant (G) adult ovariectomized mice 42 h after E2 treatment. (E, G) Endometrium; (F) Endometrium and Myometrium. Note the absence of eosinophils in mutant mice shown by the lack of eosin staining and eosinophil specific-peroxidase activity. *, Luminal epithelium. Original magnification: A–D, 1000x; E–G, 250x.

 
Together these data indicate that eotaxin is the E-regulated chemokine required for eosinophil recruitment in both pubertal and the cycling uterus. Consequently, the eotaxin-deficient mouse is a useful model to study the role of eosinophils in uterine development.

Reproductive phenotypes of eotaxin-deficient mice
Because of the early presence of eosinophils in the uterus of pubertal mice, we examined the consequence of their absence on pubertal uterine development, as well as on the onset of puberty and on the reproductive functions of eotaxin-deficient mice.

The number of uterine glands is a useful parameter to evaluate the maturation of uterine morphogenesis upon puberty (35). The number of glands was enumerated in uterine cross-sections of wild-type and mutant 5-wk-old mice. The gland number was similar in both groups, indicating that the morphologic development of the uterus of mutant mice seemed normal (Fig. 3AGo). Vaginal opening, as one measure of the onset of puberty, was examined, and found to occur at a similar age in both groups between 4.5–5 wk of age. The age of the onset of estrous cycle was next determined by analyzing hematoxylin/eosin staining of daily vaginal smears starting from 5 wk. Once the estrus phase was followed by metestrus, diestrus, and proestrus phases, the estrous cycle was considered established. The first estrous cycle started at 8.6 ± 0.2 wk in eotaxin-deficient mice, significantly delayed by 2 wk compared with 6.6 ± 0.2 wk in wild-type mice (Table 1Go). The age of first pregnancy was assessed by mating seven females from each group at the age of 6 wk and by determining the age of their first parturition. Consistent with the delay in the onset of estrous cycle, the age of the first parturition was significantly delayed by 2 wk in the mutant mice occurring at 11.3 ± 1.5 wk of age compared with 9.7 ± 1.6 wk of age in wild-type mice (Fig. 3BGo). However, once the onset of puberty occurred in mutant mice, the estrous cycle was not disturbed, and it displayed a normal length compared with wild-type mice (Table 1Go). Consequently, once mice reached adulthood, the reproduction function of eotaxin-deficient females was normal with similar litter size and interval time between consecutive successful pregnancies compared with wild-type mice (Table 1Go).



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Figure 3. Effect of the lack of uterine eosinophils on the stromal gland formation, the age of the first parturition and on uterine edema. A, Uterine glands were enumerated in cross sections of the upper third part of uteri from wild-type (+/+) and mutant (-/-) 5 wk of age mice after hematoxylin staining. Bars indicate the mean ± SD from six mice per group. No statistical difference was found (two-tailed t test). (B) The age of the first parturition was assessed by mating seven females of each group at the age of 6 wk with males from the same genotype. The age of parturition in weeks was defined by the day one of the litter birth. Each triangle represents the age of one female (*, significant difference between genotypes; P = 0.0158, two-tailed t test). C, Uterine edema was assayed by measuring the uterine wet weight. Six to eight mice of each genotype ovariectomized at 10–12 wk of age were treated with E2 three times for 3 d consecutively and killed 24 h (corresponding to an experimental proestrus) or 42 h (corresponding to an experimental estrus) after the last injection. Control mice (CTL) were ovariectomized but not E2 treated. Wet weight of the entire uteri was measured in gram. Bars indicate the mean ± SD from three different experiments. No statistical difference was found between two groups (two-tailed t test).

 

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Table 1. Diverse reproductive phenotypes

 
Eosinophil recruitment is concurrent with the E-induced edema during the estrous cycle, leading the suggestion that they may mediate this edema (20). Thus, the effect of the lack of eosinophils on the physiologic fluid accumulation occurring at proestrus and estrus was examined. Uterine edema was experimentally mimicked by reconstituting the estrogenic hormonal environment in ovariectomized mice as indicated in Materials and Methods. Wet uterine weight, which is commonly considered as an accurate parameter for edema (2, 20, 36), was measured at different time points after estrogenic stimulation. The E2 treatment reconstituted perfectly proestrus and estrus as assessed by vaginal smear analysis after 24 h and 42 h, respectively, in both wild-type and mutant mice. After E2 treatment, the wet uterine weights increased similarly and significantly compared with their respective control (P < 0.0001 for both genotypes, two-tailed t test). However, no difference of water imbibition was seen in eotaxin-deficient mice at both experimental-proestrus and -estrus compared with wild-type mice, indicating that the lack of eosinophils did not affect E-induced edema (Fig. 3CGo).

Our data demonstrate that even though eosinophils influence uterine development upon the onset of puberty, the lack of eosinophils does not affect the cycling uterus and hence the reproductive functions of the adult mutant mice. These data suggest that eosinophils are not required for the proper development of the cycling uterus.


    Discussion
 Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Eosinophil invasion of the uterine stroma during the estrous cycle is well documented. It is also well accepted that the infiltration of eosinophils into the cycling uterus requires E2 stimulation (10, 16, 17). However the E-dependent chemoattractant agent for these cells has not been identified. In this study, we have demonstrated that it is eotaxin.

A previous study has shown the existence in the immature rat uterus of an eosinophil chemotactic factor (ECF-U) whose synthesis is E2 receptor mediated (37). After isolation and characterization of uterine proteins displaying eosinophil chemoactivity, an amino acid sequence analysis indicated that one of these proteins could be cyclophilin, a protein that specifically binds cyclosporin A (38). Obviously ECF-U/cyclophilin may contribute to the uterine eosinophil infiltration; nevertheless there is no evidence showing that they are necessary for this purpose. In the present study, we demonstrate clearly that eotaxin is the chemokine necessary for uterine eosinophil recruitment. It may be that eotaxin is in fact ECF-U, because the apparent molecular mass of mouse eotaxin under nonreducing condition is 17 kDa, similar to the 20-kDa rat ECF-U. However, because cyclophilin is a peptidyl propyl-isomerase that can act as a chaperone (39), it may be that eotaxin copurifies with it under nonreducing conditions. Eotaxin also meets the E-dependent requirement of the expected uterine eosinophil chemofactor because its mRNA level is dramatically induced in ovariectomized mice after E2 treatment. Moreover, both eotaxin protein and mRNA are detected at proestrus, when eosinophil homing into uterus begins. Our findings are consistent with the detection of eotaxin messengers by RT-PCR at estrus in mice (40) and detection of eotaxin protein in human endometrium (30).

Among the known eosinophil chemotactic factor, IL-5 was also considered as a candidate acting in the uterus. Although the eosinophil population was strongly depleted in the blood of IL-5-deficient mice, the temporal fluctuations in eosinophil infiltration and localization in the uterine stroma exhibited by the small residual population was unchanged in the IL-5-deficient mice during the estrous cycle (26). Moreover, the coadministration of the monoclonal antibody anti-IL-5 with E2 to ovariectomized mice did not affect the increase in the eosinophil chemotactic activity in the mouse uterus (25). Therefore, it seems that the uterus illustrates a cooperative and not synergistic role of IL-5 and eotaxin in recruiting eosinophils in a specific organ, which has been previously reported in other systems such as lung and skin (41, 42). These observations agree with the notion that IL-5 provides the signal for the release of a pool of eosinophils from the bone marrow, whereas eotaxin remains the critical local chemoattractant for E-dependent recruitment of eosinophils into the uterine stroma.

The eotaxin-deficient mouse, as a uterine eosinophil-free mouse, was therefore a useful model to study the role of eosinophils in uterine development. In these mice, eosinophils are at normal levels in the bone marrow and peripheral blood (31, 43). Moreover, the overexpression of IL-5 transgene is associated with an increased level of gastrointestinal eosinophils in the absence of eotaxin (43). Consequently, impaired eosinophil homing in tissues or release in the circulation are unlikely to account for reduced eosinophil uterine levels or for nonresponsiveness of eosinophils to other chemotactic agents such as IL-5 in eotaxin-deficient mice. The early detection of eotaxin mRNA at 5 wk of age was in line with the infiltration of eosinophils in the pubertal uterus. In the absence of eosinophils, there was a 2-wk delay in the onset of estrous cycles. This finding could be the result of a defect of the hypothalamic/pituitary axis or ovarian functions that are necessary for a proper establishment of the onset of puberty (see Ref. 44 for review). However, the vaginal opening, which is also regulated by the hypothalamic/pituitary axis, occurs at a similar time in the eotaxin-deficient mice compared with wild-type mice. This argues for a functional hypothalamic/pituitary axis in eotaxin-deficient mice. Moreover, eosinophils are completely absent from ovarian tissue of wild-type mice at puberty (at 5 and 7 wk of age, data not shown), excluding a local role of eosinophils on ovarian-dependent steroid hormonal levels, necessary for the onset of puberty (see Ref. 44 for review). Thus, the delay of the onset of estrous cycles in eotaxin-deficient mice, consistent with the delay of the age of first parturition, does not seem to be due to a systemic hormonal defect but rather suggests a local role of eosinophils in the pubertal uterus in preparing the mature uterus for pregnancy.

In contrast to this novel finding of the role of eosinophil in pubertal uterine development, investigations into the role of the E-induced infiltration of eosinophils into the uterine stroma during the estrus cycle have been extensive. Because other events such as increases in uterine growth, edema, protein synthesis, and epithelial C3 synthesis are also E-dependent in cycling mice, it has been thought for long time that uterine recruitment of eosinophils was directly associated with these processes (20, 45, 46, 47, 48). However, our data show very clearly that in the absence of eosinophils, the increase in wet weight as a measure of uterine edema was not affected. Our observations were consistent with others studies using different mouse models that strongly depleted uterine eosinophils by using pertussis toxin-treated mice, IL-5-deficient mice, or anti-IL-5 antibody-treated mice (11, 25, 26). In the two last studies, increase of complement C3 synthesis and changes in uterine morphology during estrus cycle were not modified in relative absence of eosinophils. In addition, a precise study of reproduction capacities of IL-5-deficient mice indicated no major abnormality (26), as judged by a normal estrus cycle length, although the estrus phase was significantly longer in the mutant mice, and even though the time to observe the copulatory plug in mutant mice was significantly reduced by 2 d, the length of gestation and the outcomes of pregnancies were normal. In our uterine eosinophil-free mouse model, displaying a different background (129/SvEv), we confirmed the normal estrus cycle length and litter size as well as the ability to have successive pregnancies occurring at a normal frequency. Consequently, eosinophils do not have a nonredundant role in regulating cyclicity in the adult uterus. This surprising conclusion does still not rule out a potential role for eotaxin in the estrous cycle, because, in humans, the eotaxin receptor (CCR3) is not only expressed on eosinophils but also on endometrial epithelial cells (30). If this is similar in mice, then chemokines other than eotaxin that are known ligands for CCR3 (RANTES, MCP-3) (49) could act directly on the luminal epithelium to control the morphologic modifications occurring upon the estrus cycle, even in absence of eosinophils.

In summary, in this study, we analyzed the uterus of eotaxin-deficient mice, and identified eotaxin as the necessary E-induced chemokine for eosinophil homing to the uterine stroma during puberty and adulthood. Our data suggest that eosinophils have a local role in the pubertal uterus in preparing the mature uterus for pregnancy but demonstrate that these cells are not required for regulating the duration of the estrous cycle and fertility of mice.


    Acknowledgments
 
We thank Dr. Marc E. Rothenberg for providing us the eotaxin-deficient mice. We are grateful to Mr. Jim Lee for his technical assistance in the mouse facility.


    Footnotes
 
This work was supported by NIH Grant R-O1-HD-30280, the Einstein Cancer Center Grant P30-13330, and a DOD postdoctoral fellowship to Dr. Valérie Gouon-Evans. (J.W.P. is the Sheldon and Betty E. Feinberg Senior Faculty Scholar in Cancer Research.)

Abbreviations: ECF-U, Immature rat uterus of an eosinophil chemotactic factor; TBS, Tris-buffered saline.

Received March 14, 2001.

Accepted for publication July 6, 2001.


    References
 Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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