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BOR - Papers in Press, published online ahead of print March 5, 2003.
Biol Reprod 2003, 10.1095/biolreprod.102.010611
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BIOLOGY OF REPRODUCTION 69, 224–233 (2003)
DOI: 10.1095/biolreprod.102.010611
© 2003 by the Society for the Study of Reproduction, Inc.

Female Reproductive Tract

Interleukin-5 Transgene Expression and Eosinophilia Are Associated with Retarded Mammary Gland Development in Mice1

Amanda N. Sferruzzi-Perri3,4, Sarah A. Robertson4, and Lindsay A. Dent2,3

School of Molecular and Biomedical Science3 Department of Obstetrics and Gynaecology,4 University of Adelaide, Adelaide 5005, Australia


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Eosinophils are prevalent in the female reproductive tract, where they may contribute to regulation of development and maintenance of epithelial integrity. The present study examined the effects of constitutive interleukin-5 (IL-5) expression and overabundance of eosinophils on the development and function of the mammary gland, uterus, and ovary in mice. Eosinophils were up to 13-fold and 4-fold more abundant in the uterus and mammary gland, respectively, in female IL-5 transgenic (IL-5Tg) mice than in wild-type (Wt) animals. Eosinophils were present in large numbers in regressing corpora lutea in IL-5Tg mice but not in ovaries from Wt mice. Postpubertal mammary gland development was retarded in IL-5Tg mice, with impaired terminal end bud formation and an altered pattern of epithelial cell proliferation across the mammary fat pad coincident with disrupted ductal branching and extension. By 10 wk of age, the ductal tree was complete in both genotypes. Onset of first estrus was also delayed in IL-5Tg mice, but once IL-5Tg mice reached puberty, serum estrogen content across the cycle and estrous cycle duration were normal. The histology of uterine tissue and epithelial cell turnover were unchanged. Capacity to mate and achieve pregnancy was not affected by maternal IL-5 transgene expression, although at Day 18 of gestation, a modest decrease in the fetal:placental weight ratio was observed. Furthermore, parturition and ability to lactate and nurture postnatal pup development were not compromised. These data demonstrate an effect of IL-5 overexpression on ductal morphogenesis during postpubertal mammary gland development that is consistent with a direct regulatory role for eosinophils in these events, but these data also show that eosinophil excess does not have long-term consequences for adult reproductive function.

cytokines, immunology, mammary glands, pregnancy, uterus


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Eosinophils arise in the bone marrow from hematopoietic stem cells under the control of growth or colony-stimulating factors derived from T lymphocytes and mesenchymal cells [1]. Under normal physiological conditions, eosinophil production and activation status are tightly regulated by cytokines, including granulocyte-macrophage colony-stimulating factor, interleukin (IL)-3, and IL-5 [2, 3]. Mature eosinophils comprise less than 1% to 5% of peripheral blood leukocytes but are common in the submucosa of epithelial tissues, such as the gastrointestinal, respiratory, and lower genitourinary tracts [4, 5]. In allergy and tissue-invasive helminthic infections and some other disease states, bone marrow eosinophilopoiesis is dramatically increased [6], and eosinophil survival in tissues is considerably enhanced [7]. This results in an accumulation of eosinophils in the blood and tissues, which is known as eosinophilia.

Although viewed primarily as effectors of immunity, eosinophils in peripheral tissues express several growth factors, including transforming growth factor (TGF) ß, TGF{alpha}, and epidermal growth factor, and therefore might contribute to regulation and maintenance of epithelial integrity during the development, maturation, repair, and remodeling of tissues [8, 9]. This is particularly relevant to reproductive tissues that, unlike most other peripheral tissues, do not complete development until adult life and even then undergo continued cycles of growth and remodeling to facilitate the reproductive process. Eosinophils have been identified among the plentiful leukocyte populations that home to the rodent uterus. Their numbers vary dramatically across the estrous cycle, peaking in the endometrium at estrus [10] and after estrogen administration to immature animals [11]. Exposure to semen after mating further increases the abundance of eosinophils within the uterine stroma, where they accumulate in areas subjacent to the luminal and glandular epithelium [12–15]. By the time of implantation, uterine eosinophils dissipate and remain relatively few in number for the duration of pregnancy [15–17]. At parturition in rats, eosinophils infiltrate cervical and decidual tissues [18], but their association with cervical ripening is less evident in mice [15].

Recent studies associate eosinophils with postnatal mammary gland morphogenesis [19]. The mammary gland develops predominantly in peri- and postpubertal life, expanding to fill the mammary fat pad with a characteristic pattern of branching epithelial ducts [20]. The ductal network is created as specialized epithelial structures, the terminal end buds (TEBs), advance across the stromal fat pad, bifurcating at regular intervals to produce the ductal tree. The epithelial cells of the TEB have high proliferative potential, and the pattern of ductal elongation and branching that occurs between 4 and 8 wk of age is a consequence of TEB proliferation driven by ovarian steroid hormones, locally acting growth factors, and interaction with stromal cells [21–23]. At approximately 9 to 10 wk of age, the TEBs are replaced by terminal end ducts, which eventually differentiate into milk-secreting, lobuloalveolar structures during final maturation of the tissue. Eosinophils are a prominent feature of the developing mammary gland, accumulating in large numbers within the stromal tissue adjacent to the head of TEBs for the duration of their persistence [19].

Several recent studies indicate that deficiencies in eosinophil generation or recruitment are associated with altered reproductive tissue development and function. Mice that are genetically deficient in the eosinophil chemokine eotaxin are almost entirely devoid of peripheral tissue eosinophils and exhibit delayed onset of puberty and retardation of mammary gland development [16, 19]. The IL-5-null mutant mice, in which eosinophil abundance is diminished by approximately 90% in reproductive tract tissues, have extended estrous cycles and altered placental growth [15].

Since the T lymphocyte-derived cytokine IL-5 selectively regulates eosinophil growth and differentiation [24, 25], availability of eosinophilic IL-5 transgenic (IL-5Tg) mice provides a useful tool for examining the effect of overabundance of eosinophils on reproductive tissues. The IL-5Tg mice have been generated by ligating the genomic IL-5 sequence to the human CD2 regulatory sequence, driving constitutive IL-5 overexpression in T lymphocytes [24]. These mice are characterized by constitutive eosinophilia in the blood, with eosinophils comprising 50%–60% of peripheral blood leukocytes, and accumulation of large numbers of eosinophils in several tissues, including the spleen, bone marrow, peritoneal cavity, and lamina propria of the gut. Despite eosinophilia and splenomegaly, major gross abnormalities are not evident in these mice, and changes in leukocytes other than eosinophils are not detected [24]. Several investigations have been undertaken to explore the effect of IL-5 transgenesis and subsequent eosinophilia in asthma, allergy [26–28], and during infections with helminths [26, 29, 30], but to our knowledge, the precise effect of the genotype on development and homeostasis of epithelial tissues has not been examined. The aim of the present study was to undertake a detailed evaluation of the physiological impact of IL-5 transgenesis on postpubertal maturation and functional competence of epithelial tissues in the female reproductive tract and mammary gland.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Mice, Genotyping, and Breeding Experiments

Heterozygous female CBA/Ca mice carrying approximately 49 copies of the IL-5 transgene (Tg5C2, abbreviated IL-5Tg) and their phenotypically normal littermates (wild type [Wt]) [24] were bred from heterozygous parents housed in the specific pathogen-free facility at the University of Adelaide Medical School Animal House. Mice were provided with food and water ad libitum and treated according to University of Adelaide Animal Ethics Committee guidelines.

The genotype of IL-5Tg mice was determined either by diagnostic polymerase chain reaction (PCR) on tail DNA or on the basis of peripheral blood eosinophil counts. The PCR detected a 457-base pair fragment in the transgene that spans the junction between the 5' end of the noncoding IL-5 genomic sequence and the 3' end of the dominant controlling region of the human CD2 gene. Forward (5'-CAA CAG CAT GAG CAA GGC TCA TT-3') and reverse (5'-GAG CTC TTG GCT AGA CAT AAC TGA-3') primers were used with a Perkin-Elmer GeneAmp PCR System 2400 (Norwalk, CT) thermal cycler with an initial denaturation cycle at 94°C (3 min); 25 cycles at 95°C (40 sec), 62°C (40 sec), and 72°C (1 min); and then a final extension cycle at 72°C (40 sec).

When appropriate, female offspring were analyzed daily from weaning (3 wk) to define the age at which the vagina opened and the onset of puberty. Following vaginal opening, daily vaginal smears were prepared at 0900 h and examined by phase-contrast microscopy. According to the cellular content of these smears, mice were assigned to one of the four stages of the estrous cycle: proestrus (>50% intact, viable epithelial cells), estrus (100% cornified epithelial cells), metestrus (~50% cornified epithelial cells and ~50% leukocytes), or diestrus (>70% leukocytes plus cornified or intact epithelial cells). Estrous cycles were tracked until a total of three or more cycles were completed. In additional 5- to 6-wk-old cycling Wt and IL-5Tg mice, serum estrogen content was evaluated across the cycle. Vaginal smears were taken at 0900–1000 h, blood was drawn retro-orbitally under anesthesia with avertin (1 mg/ml tribromoethyl alcohol in tertiary amyl alcohol [Sigma, St. Louis, MO] diluted to 2.5% [v/v] in saline; 15 µl/g body wt injected i.p.), and serum estrogen content was determined by commercial estradiol RIA kit (Diagnostic Serum Laboratories, Webster, TX) according to the manufacturer's instructions. The limit of detection was 17 nmol/L, and the intra- and interassay variabilities were less than 9% and less than 12%, respectively.

For breeding experiments, virgin adult females (age, 8–12 wk; IL-5Tg or Wt) were housed 2:1 with adult stud males (IL-5Tg or Wt) and allowed to mate naturally. When a plug was observed, this was designated as Day 1 of pregnancy, and the interval between placing females with males and Day 1 of pregnancy was recorded. To assess the impact of maternal and paternal IL-5 transgene expression on mating and pregnancy, females were killed by carbon dioxide inhalation on Day 18 of pregnancy, and viable and resorbing implantation sites were enumerated. Viable fetuses and placentae were dissected free of decidua and fetal membranes and weighed, and the fetal:placental ratio was then calculated. To determine the effect on pregnancy and postpartum development of maternal IL-5 transgene expression, pregnant females were allowed to proceed until term, when the date and time of parturition (to the nearest 0.5 day) and the number of live pups were recorded. Pups were weighed 14–20 h after birth and then at 8, 15, and 22 days of age.

Histological Analysis of Eosinophils

Additional groups of IL-5Tg and Wt female mice (age, 7 or 10 wk) used for histological analyses were killed by carbon dioxide inhalation at 1200–1600 h on each day of the estrous cycle for analysis of eosinophil numbers. The fourth (abdominal) mammary glands, uterus, and ovaries were removed. Uterine and ovarian tissues were embedded in OCT (Tissue-Tek; Sakura, Tokyo, Japan), and snap-frozen in isopentane cooled by liquid nitrogen. Sections (thickness, 5 µm) were cut and fixed in 96% ethanol (v:v in H2O) for detection of endogenous peroxidase activity and counterstained with hematoxylin. Whole mammary glands were fixed in 4% neutral buffered paraformaldehyde, embedded in paraffin, sectioned (5 µm), and stained with hematoxylin and eosin (H&E).

Eosinophils in fresh-frozen tissues were detected on the basis of their endogenous peroxidase activity [15, 31] by incubating slides in diaminobenzidine (DAB; 5 mg/ml in 0.05 M Tris-HCl [pH 7.2]; Sigma) plus 0.02% hydrogen peroxide for 10 min at room temperature or by staining with Congo Red [32] and then counterstaining in hematoxylin.

The density of positive staining in tissue sections (expressed as a function of the area of total staining) was determined using video image analysis (VIA) software (Video Pro 32; Leading Edge Pty Ltd, Adelaide, Australia), and direct area measurements of uterine glands were performed by calibrating the video image to micrometers with the aid of a hemocytometer. Ten fields under high magnification (66x) were analyzed for each section, and the mean area of positive staining was calculated (area of positive staining divided by total stained area multiplied by 100). The numbers of eosinophils associated with TEB areas were counted manually, and the mean number of cells per 0.01 mm2 was calculated from at least three TEBs per mammary gland section.

Analysis of Cell Proliferation

Additional groups of IL-5Tg and Wt female mice were used to analyze epithelial cell proliferation in the mammary gland at 5 and 7 wk of age and in the uterus and ovary at 7 wk of age. 5-Bromo-2'-deoxyuridine (BrdU; Sigma) was injected i.p. 2 h before the mice were killed (100 µg of BrdU in sterile saline per 1 g body wt). Mice were killed by carbon dioxide inhalation at 1200–1600 h at 5 wk of age and on the day of metestrus for 7-wk-old mice, and the fourth (abdominal) mammary glands, uterus, and ovaries were retrieved and fixed overnight in 10% neutral buffered formalin and embedded in paraffin wax. Serial paraffin sections (thickness, 5 µm) were cut and mounted on silane-coated slides for BrdU immunohistochemistry and standard H&E staining. To detect BrdU incorporation, paraffin-embedded tissue sections were dewaxed, rehydrated, and then incubated with 0.1% trypsin at 37°C for 5 min. The DNA was then partially denatured using 2 M HCl at 37°C for 1 h, followed by two 5-min washes in borax buffer (pH 8.5). Following a further wash in PBS and 2-min incubation in 1% BSA in PBS at room temperature, samples were incubated overnight at room temperature with biotinylated mouse anti-BrdU (Clone ZBU30; 1:400 dilution in PBS containing 0.05% Tween 20; Zymed Laboratories, Inc., San Francisco, CA). Staining was visualized by incubation with streptavidin-horseradish peroxidase (DAKO, Carpinteria, CA), then DAB and H2O2 as described above, and counterstained in hematoxylin.

For analysis of epithelial proliferation in uterine sections, the luminal epithelium and the glandular epithelium were examined with surrounding stromal areas excluded. To evaluate epithelial proliferation in the mammary gland at 5 wk of age, entire sections were assessed for BrdU incorporation, whereas in 7-wk-old mammary tissue, two regions, designated as proximal and distal according to distance from the nipple, were analyzed separately. Entire sections of ovary were analyzed for BrdU incorporation. The density of positive staining in tissue sections (expressed as a function of the area of total staining) was determined using VIA as described above.

Preparation of Whole-Mount Mammary Glands

From all mice killed, the left fourth (abdominal) mammary gland was fixed overnight in Carnoy fixative (60% ethanol, 30% chloroform, 10% glacial acetic acid) and prepared as a whole mount. Fixed mammary tissues were then washed in 70% ethanol, rehydrated with distilled water, and stained for 4 h in carmine alum stain [19]. Mammary glands were then dehydrated and cleared in two separate 5-min washes in Safsolvent (APS Chemicals, Seven Hills, Australia) before mounting in DPX (Ajax Chemicals, Sydney, Australia) mounting medium. To determine ductal length (in mm), the three longest ducts in each mammary gland whole-mount preparation were measured from the nipple area to the tip, proximal to the lymph node. An estimate of the extent of ductal branching (i.e., numbers of branches) was calculated from the mean number of branch points on the three longest ducts. The total number of TEBs in the entire mammary gland was determined by manual counting.

Statistical Analysis

Data were compared by nonparametric statistical tests (SPSS Software, Chicago, IL). To evaluate differences between treatment groups, data were analyzed by Kruskal-Wallis one-way ANOVA followed by Mann-Whitney U-test or, when data were normally distributed, by one-way ANOVA followed by Bonferroni t-tests. The effect of maternal genotype on weight gain trajectory in pups was analyzed by univariate ANOVA using litter size as a covariate. Differences between groups were considered to be statistically significant at P < 0.05.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Effect of IL-5 Overexpression on Uterine, Ovarian, and Mammary Gland Eosinophil Populations

The effect of IL-5 overexpression on tissue histology and eosinophil abundance and distribution was determined in sections of uteri, ovaries, and mammary glands taken from IL-5Tg and Wt mice at 7 and 10 wk of age. In preliminary studies using serial sections of fresh uteri, identical staining patterns were found regardless of whether eosinophils were detected on the basis of endogenous peroxidase or using Congo Red or H&E. Endogenous peroxidase and H&E staining were subsequently used for quantitative analyses of eosinophils in reproductive tract tissues and mammary tissues, respectively. Uteri and ovaries recovered from IL-5Tg mice were similar in gross dimensions and morphological appearance to those obtained from Wt mice (data not shown). The number and size of uterine glands were also analyzed, and both parameters were similar regardless of genotype at both 7 and 10 wk of age (not shown).

In the uterus, abundant eosinophils were identified as endogenous peroxidase-positive cells with distinctive "donut"-shaped nuclei, accumulating at estrus in the endometrial-myometrial junction and in the endometrial stroma subjacent to the luminal and glandular epithelium. This pattern of distribution was evident in both Wt and IL-5Tg mice (Fig. 1, A and D, respectively) and is consistent with previous reports [10, 33]. In the uteri of IL-5Tg mice, eosinophils were significantly greater in number than in Wt uteri, with mean values approximately 13-fold and 4-fold higher at 7 and 10 wk, respectively (Fig. 2A). In IL-5Tg mice, eosinophils were particularly prominent in close proximity to the luminal and glandular epithelium, and uncharacteristically, eosinophils were also occasionally seen situated between epithelial cells in the luminal epithelial surface (Fig. 1D). Analysis of uterine eosinophils during other phases of the estrous cycle revealed they were 3- to 5-fold more abundant in IL-5Tg mice across the cycle and that the characteristic decline in abundance at diestrus was retained regardless of genotype (not shown).



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  		FIG. 1. Localization of eosinophils in uteri (A and D), ovaries (B and E), and mammary glands (C and F) of 7-wk-old Wt (A–C) and IL-5Tg (D–F) mice in estrus. Eosinophils (arrowheads) are identified as endogenous peroxidase-positive cells (stained black) in fresh-frozen uterine and ovarian tissue counterstained with hematoxylin. A high-magnification view of eosinophils situated between luminal epithelial cells is shown (D inset). Eosinophils in paraffin-embedded mammary gland tissue stained with H&E are identified as cells with characteristic eosin-positive cytoplasmic granules and distinctive, donut-shaped nuclei (arrowheads). CL, Mature corpus luteum; En, endometrium; Ep, epithelium; F, follicle; Fp, fatty tissue; M, myometrium; rCL, regressing corpus luteum. Bar = 100 µm (A, B, D, and E) and 25 µm (C, D inset, and F)



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  		FIG. 2. Effect of IL-5 transgene expression on abundance of eosinophils in the uterus and mammary gland. Eosinophils were identified in uterine (A) and mammary tissues (B) recovered from Wt and IL-5Tg mice in estrus at 7 and 10 wk of age. The area of staining in uterine sections was determined using VIA software, and data are given as the mean percentage positivity ± SEM (n = 5–8 per group). Data for mammary glands were generated by manual counting of eosinophils associated with TEBs, and data are represented as the mean ± SEM (n = 5–10 per group). All data were compared by Kruskal-Wallis one-way ANOVA and Mann-Whitney U-test. *Significantly different from Wt data (P < 0.05), #significantly different from 7-wk data for same genotype (P = 0.05)

Eosinophils were rarely seen in ovarian tissue obtained from Wt mice, whereas high numbers of eosinophils were evident in the ovaries of IL-5Tg mice (Fig. 1, B and E). In IL-5Tg mice, eosinophils were distributed sparsely throughout the ovarian stroma but were densely localized in regions identified morphologically as regressing corpora lutea (Fig. 1E).

Eosinophils were identified as cells containing eosin-positive cytoplasmic granules and distinctive nuclei surrounding TEBs in H&E-stained mammary glands at 7 wk of age in both IL-5Tg and Wt mice (Fig. 1, C and F). In both genotypes, eosinophils associated with the mammary gland declined in number by approximately 50%–60% over the period from 7 to 10 wk of age (Fig. 2B). At both 7 and 10 wk of age, mammary glands recovered from IL-5Tg mice contained substantially more eosinophils than glands from Wt mice, with a 4-fold difference in the abundance of eosinophils associated with TEBs (Fig. 2B). In 7-wk-old IL-5Tg mice, eosinophils accumulated not only in TEBs but also in the connective tissue adjacent to the fat pad, with fewer cells sparsely distributed between mammary adipocytes (not shown). Unlike in Wt animals, large numbers of eosinophils were also seen distributed along the length of ducts. Furthermore, eosinophils were identified in the inguinal lymph node located in the middle of the mammary gland, with increased numbers evident in IL-5Tg mice (not shown).

Effect of IL-5 Transgene Expression on Morphometric Measures of Mammary Gland Development

To evaluate the effect of IL-5 transgenesis on mammary gland morphogenesis, epithelial duct elongation and branching were assessed in mammary gland whole mounts prepared from 5-, 7-, and 10-wk-old IL-5Tg and Wt mice. Significant delays in several measures of development were observed in IL-5Tg mice. At 5 wk of age, the mean number of branch points was reduced by 38% (P = 0.001) and the mean number of TEBs by 43% (P = 0.001) in IL-5Tg mice. The reduction in the number of ductal branches persisted in mammary tissue at 7 wk of age (P = 0.01), and at this stage, mean ductal length was also reduced by 33% (P = 0.001) (Fig. 3). At 10 wk of age, however, branch numbers and ductal elongation were comparable between genotypes, and glands from both groups of mice were laced with ductal trees, displaying terminal end ducts and lobuloalveolar buds (not shown). No overt differences were found between Wt and IL-5Tg mice in the structure of TEBs or lobuloalveolar buds.



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  		FIG. 3. Effect of IL-5 transgene expression on morphometric measures of mammary gland development. The number of epithelial branch points (A), ductal length (B), and number of TEBs (C) were quantified in 5-, 7-, and 10-wk-old Wt and IL-5Tg mice (n = 9–17 mice per group). Data are presented as the mean ± SEM and were analyzed by Kruskal-Wallis one-way ANOVA and Mann-Whitney U-test. *Significantly different from Wt data (P < 0.05), **significantly different from Wt data (P < 0.005), ***significantly different from Wt data (P < 0.001), #significantly different from 5- and 7-wk data for same genotype (P < 0.005)

Effect of IL-5 Transgene Expression on Epithelial Proliferation in the Uterus, Ovary, and Mammary Gland

To elucidate the impact of IL-5 transgene expression and the abundance of eosinophils on epithelial cell turnover in the uterus, ovaries, and mammary gland, BrdU incorporation was analyzed in 7-wk-old IL-5Tg and Wt mice at metestrus, when proliferation of uterine luminal and glandular epithelial cells is expected to be prominent [34]. The abundance of BrdU-labeled cells was comparable in luminal and glandular epithelium of uterine tissues obtained from Wt and IL-5Tg mice (Figs. 4A and 5, A and D). In both IL-5Tg and Wt uterine tissues, the proportion of proliferating cells was greater in the glandular epithelium than in the luminal epithelium, and this reached statistical significance in Wt uteri (Fig. 4A). Very few BrdU-positive cells were observed in the endometrial stroma or myometrium (Fig. 5, A and D).



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  		FIG. 4. Effect of IL-5 transgene expression on epithelial proliferation. Epithelial proliferation was assessed by BrdU incorporation in sections of uterus (A) and ovary (B) taken from Wt and IL-5Tg mice in metestrus at 7 wk of age and of mammary glands (C) retrieved at 5 and 7 wk of age. Uterine luminal and glandular epithelium were assessed separately. In the 7-wk-old mammary glands, tissue was classified as proximal or distal to the nipple. The area of staining in sections was determined using VIA software. Data are given as the mean percentage positivity ± SEM (n = 5–9 per group) and were analyzed by Kruskal-Wallis one-way ANOVA and Mann-Whitney U-test. *Significantly different from Wt data (P < 0.05), #significantly different from proportion of proliferating cells in uterine luminal epithelium (P = 0.005), ##significantly different from proportion of proliferating epithelial cells in proximal mammary tissue (P < 0.05)



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  		FIG. 5. Proliferating epithelial cells detected by BrdU staining in the uterus (A and D), ovary (B and E), and mammary gland (C and F) of Wt (A–C) and IL-5Tg (D–F) mice at metestrus. The BrdU-labeled cells are marked by arrowheads in paraffin-embedded tissue counterstained with hematoxylin. CL, Mature corpus luteum; En, endometrium; Ep, epithelium; F, follicle; Fp, fatty tissue; G, gland; Ln, lymph node; M, myometrium. Bar = 100 µm

Morphological analysis of ovarian sections from IL-5Tg and Wt mice revealed epithelial and theca cells surrounding follicles were positive for BrdU (Fig. 5, B and E), and quantitative evaluation indicated that IL-5 transgene expression did not influence the proportion of BrdU-positive cells (Fig. 4B).

In mammary tissues of both IL-5Tg and Wt mice, ductal epithelial cells and the cap cells of TEBs were BrdU-positive (not shown), which is consistent with the expected pattern of proliferation in mammary tissue at 7 wk of age [20]. Assessment of BrdU incorporation in entire sections of mammary tissue taken from 5-wk-old mice revealed that BrdU positivity was approximately 50% lower in IL-5Tg mice compared to Wt mice; however, this did not reach significance (P = 0.058) (Fig. 5C). In 7-wk-old mammary glands, BrdU incorporation was quantified in the regions proximal and distal to the nipple, corresponding to areas spanning from the nipple to halfway across the fat pad and from the border of the proximal region to the end of the ductal tissue, respectively. In IL-5Tg mice, epithelial proliferation proximal to the nipple was significantly increased, with average BrdU positivity 3-fold higher than in Wt mice (P = 0.028) (Figs. 4C and 5, C and F). In Wt tissue, 4-fold more epithelial cells were proliferating in the area distal to the nipple than in the areas close to the nipple, a spatial distinction not evident in IL-5Tg mice (Fig. 4C). Indeed, relative to Wt mice, a trend was observed toward fewer proliferating cells in the distal region in IL-5Tg mice.

Effect of IL-5 Transgene Expression and Eosinophilia on the Estrous Cycle

To investigate whether IL-5 transgene expression and eosinophil abundance influence ovarian cyclicity, the time of puberty onset and duration of the estrous cycle were analyzed in IL-5Tg and Wt mice. Vaginal opening, an indication of the onset of puberty, occurred at the same age in Wt and IL-5Tg females (mean ± SEM; Wt mice, 29.9 ± 0.8 days, n = 14; IL-5Tg mice, 29.7 ± 0.8 days, n = 16). Daily vaginal smears were then taken from Wt and IL-5Tg mice as a further measure of sexual maturity. IL-5Tg mice completed the first estrous cycle later than Wt mice (mean ± SEM; Wt mice, 5.0 ± 0.1 wk of age; IL-5Tg mice, 6.0 ± 0.2 wk of age; P < 0.05) (Fig. 6), with approximately 14% of IL-5Tg mice not cycling at 7 wk of age. Despite this delay in the onset of puberty, once IL-5Tg mice reached sexual maturity, the estrous cycle was normal in duration (mean ± SEM; Wt mice, 4.4 ± 0.2 days, n = 15; IL-5Tg mice, 4.7 ± 0.4 days, n = 11; three to four cycles tracked per mouse). Moreover, no effect of IL-5 genotype on ovarian estrogen synthesis was seen when serum estradiol was measured across the estrous cycle in mice bled at 6–8 wk of age, with IL-5Tg mice displaying the expected cycle-related fluctuations in serum estradiol content and the mean level at the estrus peak being comparable to that seen in Wt mice (Table 1).



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  		FIG. 6. Age at completion of first estrous cycle. Daily vaginal smears were assessed following vaginal opening to determine the age of sexual maturation. Symbols represent data from individual mice; bar denotes the median value. *Significantly different from Wt data, Mann-Whitney U-test (P < 0.05)


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  		TABLE 1. Effect of IL-5 transgene expression on serum estradiol content.a

Effect of Parental IL-5 Transgene Expression on Litter Size, Conceptus Viability, and Fetal and Placental Weights at Day 18

To determine whether IL-5 transgene expression influenced survival of the conceptus during pregnancy, virgin Wt and IL-5Tg females mated naturally with males of the same genotype were killed on Day 18 of pregnancy. All six stud males of each genotype were fertile, as judged by generation of viable litters with one or more females. The proportion of plugged mice with implantation sites on Day 18 of gestation and the total number and viability of implantation sites were not affected by genotype (Table 2). To evaluate whether parental IL-5 transgene expression influenced the growth of the placenta or fetus, viable fetuses and placentae from pregnant Wt and IL-5Tg females were weighed on Day 18. Placental weight was 7% greater in IL-5Tg matings (P < 0.001), but fetal weight was unchanged, resulting in an 8% decrease in the fetal:placental weight ratio in IL-5Tg matings (mean ± SEM; Wt matings, 8.2 ± 0.1; IL-5Tg matings, 7.6 ± 0.1; P < 0.001) (Table 2). Fetal tissue was genotyped by PCR, and the proportion of viable implantation sites in IL-5Tg litters carrying IL-5 transgenes was found to be 72%, which is not different from the predicted mendelian frequency.


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  		TABLE 2. Effect of IL-5 transgene expression on fetal and placental parameters at Day 18 of pregnancy.a

Effect of Maternal IL-5 Transgene Expression on Parturition and Postnatal Growth

Virgin Wt and IL-5Tg females were mated naturally with stud Wt males, and gestation was allowed to proceed to term to determine the effect of maternal IL-5 transgene expression on parturition, neonatal viability, and lactation. The mating interval, defined as the number of days between placement with a stud male and detection of a vaginal plug, was unaffected by genotype (Table 3). No effect of maternal IL-5 transgene expression on the duration of gestation was observed (mean ± SEM; Wt mice, 19.6 ± 0.2 days, n = 8; IL-5Tg mice, 19.4 ± 0.2 days, n = 10). Litter sizes and survival of pups during the perinatal period were also comparable between groups (Table 3), as was gender distribution (not shown). Maternal IL-5 transgene expression did not compromise lactation, as indicated by the growth trajectories of pups from litters of Wt and IL-5Tg female matings. Indeed, the mean weight of pups born to IL-5Tg mothers was 10% larger at 2 wk (P = 0.008) and 11% larger at 3 wk (P = 0.001) than the mean weight of Wt progeny (Table 4).


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  		TABLE 3. Effect of maternal IL-5 transgene expression on paturition and term pregnancy outcomes.a


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  		TABLE 4. Effect of maternal IL-5 transgene expression on postpartum growth trajectory.a


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Recent studies link deficiencies in eosinophils or defects in eosinophil recruitment with delayed postnatal mammary gland morphogenesis [19] and perturbations in the estrous cycle [15, 16]. Availability of a transgenic mouse model for systemic eosinophilia, in which constitutive overexpression of IL-5 in T lymphocytes causes eosinophil numbers in the blood and tissues to increase up to 10-fold [24], prompted us to examine the effect of overabundance of eosinophils on these processes. The effects of IL-5 transgenesis on eosinophil abundance and immune function in the gut and lung after challenge with helminthic parasites and aeroallergens can be substantive and are well documented [26–30]. However, to our knowledge, the effects of IL-5 transgenesis on reproductive tract eosinophil populations and reproductive function have not previously been examined.

The present study shows that constitutive overexpression of IL-5 profoundly increases accumulation of eosinophils in the uterus, ovaries, and mammary gland. Overabundance of eosinophils is associated with a delay in the onset of the first estrous cycle and retarded ductal morphogenesis during the pubertal phase of mammary gland development. Reproductive performance in adult mice was largely unaffected by IL-5 transgene expression.

Despite up to 10-fold increases in the number of eosinophils in reproductive tissues, their spatial and temporal patterns of distribution were broadly similar regardless of transgene expression, supporting earlier findings that chemotactic signals other than IL-5 regulate recruitment of bloodborne eosinophils into these tissues [15, 16, 19]. In uterine tissues of both Wt and IL-5Tg mice, eosinophils were most numerous in the endometrial stroma subjacent to the luminal and glandular epithelium and at the endometrial-myometrial junction, and numbers fluctuated across the estrous cycle in the expected manner. In the mammary gland, eosinophils accumulated adjacent to epithelial cells of the TEBs during pubertal development and showed the expected decline between 7 and 10 wk of age, coincident with the completion of postpubertal development. Smaller numbers of eosinophils were evident in ectopic locations in IL-5Tg mice. For example, significant numbers were seen along the length of the ducts and in the connective tissue adjacent to the fat pad, with occasional cells scattered through the adipose tissue.

Most unexpectedly, high numbers of eosinophils accumulated within regressing corpora lutea in ovarian tissue from IL-5Tg mice, whereas eosinophils are rarely detected in ovaries of Wt mice [35]. T lymphocytes in IL-5Tg mice constitutively express IL-5, and because these cells are present in the corpus luteum [36], they may be responsible for infiltration of eosinophils. However, it is notable that eosinophils do not accumulate in large numbers in all tissues containing T cells, so eosinophil-specific recruitment signals may be another feature of this compartment. A candidate signal is eotaxin, a key chemotactic agent for eosinophils expressed in the uterus and mammary gland that is identified as a principal and necessary requirement for recruitment into those tissues [16, 19]. In fact, IL-5 is thought to prime eosinophils to respond to chemoattractants, such as eotaxin [3, 6, 25, 37], so together, they may act to potentiate local chemotactic responses in IL-5Tg mice. Further experiments are required to define the precise combination of molecular signals that account for the compartment-specific accumulation of eosinophils in the ovary.

Mammary gland morphogenesis was studied during the pubertal phase when highly proliferative epithelial cells surrounding the TEB accelerate ductal extension and, with regular bifurcation, form the ductal tree [22]. Tissues examined across the pre- and peripubertal period showed clear evidence of an effect of overexpression of IL-5 on the number of ductal branches and length of ducts. The number of TEBs was reduced by almost half in 5-wk-old IL-5Tg mice but were nearly recovered by 7 wk, suggesting that the effects on ductal extension may stem from a delay in their development. The reduction in proliferating epithelial cells seen at 5 wk in transgenic mice, the immature ductal branching pattern seen at 7 wk, and the evidence of proliferation still evident in areas proximal to the nipple are consistent with this. However, retardation in development was transient, and by 10 wk of age, the ductal tree was complete in both genotypes. No overt differences in lobuloalveolar morphology were noted in mature IL-5Tg mice, indicating that aberrations in growth were only temporary.

These findings are interesting in view of the recent report of altered mammary morphogenesis in the event of disrupted eosinophil recruitment secondary to null mutation in the eotaxin gene. Eotaxin-deficient mice show normal ductal extension but a reduction in the number of branches, suggesting that the role of eosinophils is to define the branching pattern [19]. The molecular mechanisms mediating interaction between eosinophils and epithelial cells are not known, but eosinophil expression of growth factors of the TGFß family likely plays a key role [9]. Several studies involving manipulation of local TGFß activity using exogenous growth factor or transgenic models [38–40], including a recent experiment showing accelerated ductal outgrowth in mice heterozygous for null mutation in the TGFß1 gene [41], indicate an inhibitory role for TGFß1 in mammary gland epithelial cell proliferation. In normal animals, eosinophils might thus provide a means for targeting TGFß secretion in spatially precise positions on the developing ductal tree. Overabundant and inappropriately localized eosinophils in IL-5Tg mice would be expected to result in amplified inhibitory signals at the TEB and, perhaps, also along the length of the elongating duct. It is reasonable to postulate that inhibition of differentiation of epithelial cells into TEB is similarly the result of overabundance of eosinophil-derived growth factors.

Estrogen derived from the ovary is also a principle regulator of mammary gland development. First estrus was delayed by 1 wk in IL-5Tg mice. However, once established, estrous cycles were normal in duration, and the interval between caging with males and mating was not affected by genotype. We were unable to demonstrate a significant correlation between parameters of mammary gland morphogenesis and time of first estrus (not shown). Furthermore, serum estradiol measurements during the first few estrous cycles revealed no evidence of altered ovarian estrogen synthesis resulting from IL-5 overexpression. Thus, on the basis of the existing data, we cannot rule out an ovarian contribution to the observed mammary gland changes, but we interpret the data to favor a direct effect of IL-5 transgenesis and local eosinophilia in the mammary gland rather than altered endocrine parameters.

It is not clear how IL-5 transgenesis or eosinophil abundance delay the onset of puberty. A local effect of eosinophils within the ovary is highly unlikely, because eosinophils are rarely found in ovarian tissues of Wt mice [35], and even in the transgenic mice, in which eosinophil accumulation was evident, eosinophils were restricted to corpora lutea, which do not arise until after first ovulation. An intricate balance of hypothalamic, pituitary, and ovarian hormones regulates the onset of puberty [42], so it is possible that effects of transgenesis are exerted distal to the ovary, such as in tissues of the hypothalamic-pituitary axis. In the endometrium, eosinophils degranulate at estrus [43, 44] and, through releasing a variety of mediators capable of binding and inactivating estrogen [45], may influence tissue growth and onset of puberty from this compartment. These hypotheses are supported by observations of extended estrous cycles in IL-5-deficient mice [15] and delayed onset of estrous cycling in eotaxin-deficient animals [16]. Vaginal opening, a second measure of puberty, was unchanged by IL-5 transgenesis, perhaps reflecting differential roles of eosinophils in the cellular processes that culminate in uterine and vaginal maturity. For example, it might be expected that, as a less-prevalent cell in the vaginal epithelium [15], eosinophils have little impact on maturation of this tissue.

Histological experiments and assessment of uterine epithelial proliferation in IL-5Tg mice revealed that despite the dramatically increased number of eosinophils, no change occurred in the structure or remodeling of the epithelium during the estrous cycle. The development and growth of uterine glands were also unaffected. A further increase in eosinophil infiltration of the uterine endometrium occurs following mating, when these cells are thought to play an immunoregulatory role in preparing the uterus for blastocyst implantation and protection against infectious agents [14]. By the time of implantation, eosinophils are sparse in the vicinity of the developing embryo [14].

Because eosinophils are phagocytic and can also secrete a variety of metabolites capable of causing tissue degeneration, inappropriate uterine tissue remodeling or accelerated regression of the corpus luteum might have been expected to interfere with progesterone synthesis or to impact negatively on implantation success. However, IL-5 transgenesis and an overabundance of eosinophils in the uterus and corpora lutea of ovaries appeared to have little effect on reproductive performance. The proportion of mated IL-5Tg mice achieving successful pregnancy and the resultant litter sizes were comparable to those of Wt controls.

Maternal IL-5 transgene expression was seen to result in increased growth of the placenta and in decreased fetal:placental weight ratios, suggesting impaired placental function. In mice, IL-5 has been reported to be present at high levels in decidual and placental tissues during pregnancy [46], and studies in women have revealed that uterine natural killer cells [47] and type-2 T lymphocytes [48] are responsible for its synthesis. Thus, the usual elevation in uterine IL-5 during pregnancy might be amplified in the IL-5Tg mice, where T cells are responsible for constitutive IL-5 overproduction. Whether an abnormal eosinophil presence in the placenta and decidua of pregnant IL-5Tg mice accounts for altered placental development remains to be examined. It is notable that a similar placental phenotype was evident in eosinophil-deficient, IL-5-null mutant mice [15].

Studies in rats show that at parturition, eosinophils are recruited into the cervical and decidual stroma, where they are thought to play a role in cervical ripening and/or the recovery of this tissue after birth [18], although similar changes have proven hard to detect in mice [15]. In the present study, maternal IL-5 transgene expression did not influence the timing of parturition or successful delivery of viable pups. Finally, lactation in the postpartum period clearly was not compromised by the kinetics of morphogenesis of mammary tissue, because pups born to IL-5Tg mothers thrived and did not show any compromise in postnatal growth.

Collectively, these studies show that IL-5 overexpression and eosinophilia do not severely impair the final development or adult function of reproductive tissues in mice, but they do support an effect of IL-5 in regulating the onset of first estrus and postnatal mammary gland development. We argue that the effects of IL-5 overexpression are almost certainly mediated by the associated excess of eosinophils in the reproductive tract and mammary gland tissues. The principal impact of IL-5 transgenesis in vivo is undisputedly on eosinophilopoiesis, with only limited evidence for effects in other cell lineages [24, 49, 50]. Although expansion of the minor B-1 B lymphocyte population coincident with altered immunoglobulin synthesis has been observed in some IL-5Tg mouse models, changes in these parameters are negligible in the genetic line used in the present study [49, 50]. Moreover, B lymphocytes have not been shown to have significant roles in reproductive tissues other than in host defense against pathogens. This proposition is further supported by the spatial and temporal association between eosinophils and mammary development together with the mounting independent evidence from eotaxin- and IL-5-null mutant mice. The relevance of these findings to pubertal changes in reproductive tract tissues and reproductive competence in human and livestock species, in which high parasite burdens can cause dramatic eosinophilia, remains to be determined.


   ACKNOWLEDGMENTS
 
We thank Ann Hallett, Leanne Srpek, Chris Cursaro, and Kate Pensa for technical assistance and Claire Roberts, Sarah Hudson-Keenihan, and Wendy Ingman for helpful comments and advice.


   FOOTNOTES
 
1 Supported by NHMRC (Australia) project grants (L.A.D. and S.A.R.), ARC (S.A.R.), NHMRC Fellowship Scheme (S.A.R.), and the Cooperative Research Centre for Pest Animal Control (L.A.D. and S.A.R.). Back

2 Correspondence. FAX: 618 8303 7532; lindsay.dent{at}adelaide.edu.au Back

Received: 30 August 2002.

First decision: 19 September 2002.

Accepted: 26 February 2003.


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 RESULTS
 DISCUSSION
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