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Biology of Reproduction 67, 1439-1449 (2002)
© 2002 Society for the Study of Reproduction, Inc.

Regular Article

Differential Expression and Estrogen Response of Lactoferrin Gene in the Female Reproductive Tract of Mouse, Rat, and Hamster

Christina T. Teng1,a, Clara Bearda, and Wesley Gladwella

a Gene Regulation Section, Laboratory of Reproductive and Developmental Toxicology, National Institute> of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina 27709


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Lactoferrin, an iron-binding glycoprotein, kills bacteria and modulates inflammatory and immune responses. Presence of lactoferrin in the female reproductive tract suggests that the protein may be part of the mucosal immune system and act as the first line of defense against pathogenic organisms. We have discovered that lactoferrin is a major estrogen-inducible protein in the uterus of immature mice and is up-regulated by physiological levels of estrogen during proestrous in mature mice. In the present study, we examined lactoferrin gene expression and its response to estrogen stimulation in the female reproductive tract of several strains of immature mouse, rat, and hamster. The lactoferrin expression in the cycling adult female rat was also evaluated. Lactoferrin gene polymorphism exists among the different mouse strains. In the three inbred mouse strains studied, lactoferrin gene expression is stimulated by estrogen in the immature uterus, although it is less robust than in the outbred CD-1 mouse. We found that the lactoferrin gene is constitutively expressed in the epithelium of the vagina and the isthmus oviduct; however, it is estrogen inducible in the uterus of immature mice and rats. Furthermore, lactoferrin is elevated in the uterine epithelium of the mature rat during the proestrous and estrous stages of the estrous cycle. Estrogen stimulation of lactoferrin gene expression in the reproductive tract of an immature hamster is limited to the vaginal epithelium. The present study demonstrates differential expression and estrogen responsiveness of the lactoferrin gene in different regions of the female rodent reproductive tract and variation among the rodent species studied.

estradiol, female reproductive tract, menstrual cycle, uterus, vagina


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Lactoferrin, a member of the transferrin gene family, is expressed in the mucosal epithelium and is present in the milk, tear, saliva, seminal plasma, and secretions of the uterus and vagina. It is a major protein in the secondary granule of the neutrophil (see [1] and references therein). The protein was found to kill bacteria, viruses, and fungi. In addition, it modulates the inflammatory and immune responses (see [2–7] and references therein). Lactoferrin protein binds iron molecules with high affinity [8–10]. This property enables lactoferrin to sequester free iron in the environment, which thereby inhibits bacterial growth. Furthermore, the highly basic region (amino acids 2–5) of the human lactoferrin N-terminus is responsible for binding to the receptor and heparin [2, 11–14], and the loop region within the antibacterial "lactoferricin" H (amino acids 28–34) is responsible for the high-affinity binding with lipopolysaccharide of the gram-negative bacteria [15, 16]. In addition to the bacteriostatic and bactericidal properties, lactoferrin is involved in modulating the immune and inflammatory response by down-regulating several cytokines, such as tumor necrosis factor {alpha}, interleukin 6, and granulocyte-macrophage colony-stimulating factor [17–19]. Based on the expression pattern and the biological properties, lactoferrin serves as a first line of defense and is part of the innate immune system [3, 20].

We have discovered that lactoferrin is an estrogen-inducible protein in the mouse uterus [21–23] and that its expression varies during the estrous cycle, with the highest level at the proestrous stage when the blood estrogen level is known to be elevated [24]. Recently, the lactoferrin gene in the human and monkey endometrium was shown as estrogen responsive [25]. We have characterized the lactoferrin gene promoters of the mouse and human [26–28] and identified the potential estrogen-response element in these genes [21, 28–31]. Thus, the molecular studies support the in vivo observations. The biological role of lactoferrin in the reproductive tract is yet to be defined. Evidence exists that sex hormones and cytokines are involved in the control of mucosal immunity in the female reproductive tract [32, 33]. The secretions of mucosal-immune systems, such as the immunoglobulin (Ig) A, IgG, and secretory components, increase sharply at proestrous in the uterus and decline relative to other stages of the cycle [32–35], which correlates with the expression pattern of lactoferrin. Therefore, lactoferrin could play a role in the innate host defense of the reproductive tract.

It is well established that the lactoferrin gene in the uterus of the outbred CD-1 mouse is estrogen responsive. To our knowledge, however, few studies regarding lactoferrin gene expression in other rodent species have been reported [36–39], and no such study has been reported for the inbred mouse strains. Genetic variability and strain differences in response to estrogen stimulation as well as susceptibility to infection among the inbred and outbred mice have been documented [40, 41]. Because lactoferrin plays an important role in mucosal immunity of the uterus, its response to estrogen stimulation in the uterus of inbred strains of mice might provide insight regarding the involvement of estrogen in host defense of the reproductive tract.

The present study examined the expression of lactoferrin in the reproductive tract of immature inbred strains of mouse, rat, and hamster treated with diethylstilbestrol (DES), the synthetic estrogen. Lactoferrin expression in the uterus of adult rats at different stages of the estrous cycle was also investigated. We found that lactoferrin is differentially expressed in selective regions of the reproductive tract and is variable in estrogen response among the different species.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals and Tissue Preparation

Immature female SJL, SWR, and SM mice (21 days old) were purchased from the Jackson Laboratory (Bar Harbor, ME). Immature female CD-1 mice (21 days old), immature female CD rats (35 days old), adult CD rats (3 mo old), and immature Fischer (F344) rats (21 days old) were obtained from Charles River Laboratories (Wilmington, MA). Immature female Han:Aura hamsters (21 days old) were obtained from Harlan Spague Dawley (Indianapolis, IN). All animals were kept in the National Institute of Environmental Health Sciences (NIEHS) animal facilities laboratory at 72 ± 2°F, 40–60% humidity, and a 12L:12D photoperiod and were handled using methods approved by the Animal Care and Use Committee. Drinking water was processed through reverse osmosis and deionization. Animals received NIH 31 chow as food. Animals were kept 3–4 days before injection with DES in a corn oil vehicle given at 300 µg/kg bodyweight every 24 h. For the immature female outbred CD-1 and inbred strains of mice, four animals were injected with DES and two with vehicle twice before they were killed. Six immature CD rats per group received either DES or vehicle three times before they were killed. The immature CD rat studies were repeated once. To investigate the effect of endogenous estrogen on the lactoferrin gene expression in the rat uterus, 28-day-old immature F344 rats (three animals) were treated with one dose (5 IU) of eCG to induce estrogen production of the ovary for 24 h before tissue collection. To study the lactoferrin expression during the estrous cycle of the rat, reproductive tracts of the adult female rats were collected and prepared for histological study. Based on the morphology of the vaginal epithelium from these rats, various stages of the estrous cycle were determined. Among the 12 female rats, 4 were at estrous, 2 at proestrous, and 2 at diestrous. The other four rats were in between stages. Immature hamsters (five per group) were treated similarly as the rats, and the experiments were repeated twice. All animals were killed with CO2 before tissues were removed. Uterine fluid was collected by aspiration with a needle and syringe as previously described [22].

Tissues collected for immunohistochemistry studies were placed immediately into 10–13 ml of Bouin solution. Tissue was rocked on an Adams Nutator (Clay Adams, Parsippany, NJ) in the Bouin solution for 24–30 h before the Bouin solution was disposed of and 70% ethanol was added to the tissue. Tissues were embedded in paraffin once the yellow color did not appear in the 70% (w/v) ethanol for several hours. Embedded tissue was sectioned (thickness, 7 µm) and placed onto microscope slides in preparation for staining. For Western blot analysis and immunodetection, tissue samples were minced with scissors on ice and then homogenized with a Teflon-to-glass homogenizer in 500 µl of RIPA lysis buffer (10 mM Tris-HCl [pH 7.4], 150 mM NaCl, 0.1% SDS, 1% CHAPS, and 1 mM EDTA) containing 1% (w/v) proteinase-inhibitor cocktail (104 mM AEBSF, 80 µM aprotinin, 2.1 mM leupeptin, 3.6 mM bestatin, 1.5 mM pepstatin A, and 1.4 mM E-64) from Sigma (St. Louis, MO). The tissue homogenates were sonicated with a Sonic Dismembrator (Fisher, Pittsburgh, PA) at a setting of 15 for a 5-sec burst at least three times before centrifugation at 4°C in a Beckman Coulter Microfuge R (Schaumburg, IL) for 40 min at 14 000 rpm. The supernates were removed and stored at -70°C until use. Bone marrow of the rat was collected from six femurs by flashing the bone marrow tissue with Hanks balanced salt solution without phenol red indicator. The marrow cells were collected by centrifugation, and the cells were lysed in RIPA buffer before Western blot analysis.

Genomic DNA Preparation and Southern Blot Analysis

Genomic DNA from SJL, SWR, SM and CD-1 strains were prepared from mouse liver according to a previously reported method [42]. Ten micrograms of genomic DNA were digested with restriction enzyme EcoRI or BamHI. The DNA fragments were resolved on 0.8% agarose gel and transferred onto nitrocellulose membrane. The membrane was probed with 32P-labeled mouse lactoferrin cDNA (T267) [23] according to the standard method. After hybridization, the membrane was exposed to Kodak x-ray film (Eastman Kodak, Rochester, NY) with the presence of intensify screen for 3 days at -70°C. After developing the x-ray film, the lactoferrin cDNA probe was stripped from the membrane and reprobed with 32P-labeled p45015{alpha} hydroxylase cDNA [43].

Deparaffination and Immunohistochemistry

The slides were deparaffinized in xylene, rinsed through a series of decreasing concentrations of ethanol (95%, 70%, and 50%), and finally washed three times in phosphate buffer solution. Before immunostaining, the tissue samples on the slides were first circled with a hydrophobic PAP pen. The circled area was filled with ImmunoPure Peroxidase Suppressor (Pierce, Rockford, IL) to block the endogenous peroxidase activity. The blocking was done in a humid box for 15 min. The immunostaining was carried out with a Histostain SP Kit (Zymed, South San Francisco, CA) according to the manufacturer's specifications. The IgG fraction of mouse lactoferrin (mLF) 8344 [22] at a concentration of 6 µg/ml was used as the primary antibody, and the incubation was carried out overnight at 4°C. Nonimmune serum or its IgG fraction was used as negative control for the immunostaining study. After the slides were immunostained, the tissues were counterstained with sterile modified hematoxylin (Sigma). The slides were rinsed in distilled water to remove excess hematoxylin, air-dried, and mounted by liquid mount with a coverslip.

The staining intensity for lactoferrin in the uterine epithelium of the mice was quantitatively estimated (0 = negative, 1 = weak, 2 = moderate, 3 = strong, and 4 = intense) according to previous studies [44]. Three different areas of the uterus (top; close to the oviduct, middle portion, and bottom; and close to the vagina) were randomly scored from four animals in two independent immunostainings, and the data were subjected to ANOVA and the Dunnett test [45].

Western Blot Analysis

The mouse lactoferrin was isolated from the uterine fluid of an estrogen-treated mouse as described previously [22]. The bovine milk lactoferrin, human milk lactoferrin, and mouse transferrin were obtained commercially (Sigma). Equal amounts of uterine fluid from the inbred mouse strains or different concentrations of purified proteins were loaded onto precast 4–12% Bis-Tris NuPAGE gels. The electrophoresis was carried out in the Novex XCell II Mini-Cell system in a 1x MOPS buffer (Novex, San Diego, CA) at 120 mA and 200 V for 1.25 h. Proteins were transferred from a gel onto an Immobilon-P polyvinylidene difluoride membrane (Millipore, Marlboro, MA) at 200 mA and 25 V for 2 h with a 1x NuPAGE transferring buffer containing 20% methanol. Immunodetections were carried out with an enhanced chemiluminescence (ECL) kit (Amersham, Piscataway, NJ) according to the manufacturer's specifications. Primary antibodies were diluted (1:10 000 ;obv/v;cb) and incubated with the protein blots overnight at 4°C. After washing, the blots were incubated with second antibody (diluted 1:10 000 [v/v]) for 1 h before ECL detection. X-ray film was exposed 1 min and developed on a Konica (Wayne, NJ) SRX-101.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Lactoferrin Gene Polymorphisms and Estrogen Response among Different Mouse Strains

Lactoferrin gene in the CD-1 mouse has been studied extensively, and its estrogen response has been well established in the uterus (see [21] and references therein). However, to our knowledge, whether the lactoferrin gene in the uterus of inbred mouse strains also responds to estrogen stimulation has not been examined. To characterize the lactoferrin gene in inbred strains, we first analyzed the restriction enzyme digestion patterns of the lactoferrin gene from several inbred mouse strains and the outbred CD-1 mouse by Southern blot analysis (Fig. 1). We found that lactoferrin gene polymorphisms existed between the inbred (SJL, SWR, and SM) and outbred (CD-1) mouse strains (Fig. 1, left). Three major lactoferrin gene fragments resulted from the EcoRI digestion of the genomic DNA from SJL (lane 1), SWR (lane 2), and SM (lane 4), whereas only two fragments were from the CD-1 DNA (lane 3). Similarly, BamHI digestion resulted in four major fragments from the inbred strains, SJL (lane 5), SWR (lane 6), and SM (lane 8), whereas the CD-1 DNA gave five fragments (lane 7). Origin of the minor fragments from the inbred mouse DNA (minor band in lanes 1, 2, 4, 6, and 8) was unknown. The restriction enzyme polymorphisms were not found in the P45015{alpha} hydroxylase gene of these mice (Fig. 1, right). The EcoRI restriction enzyme pattern of lactoferrin gene in B6, C3H, NZB, DBA, BALB, and A/J inbred strains was similar to that in the CD-1 strain (data not shown).



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  		FIG. 1. Detection of lactoferrin gene polymorphism in various strains of mice. Ten micrograms of liver DNA from SJL (1 and 5), SWR (2 and 6), CD-1 (3 and 7), and SM (4 and 8) mice were digested with restriction enzyme EcoRI (1–4) or BamHI (5–8), separated on an 0.8% agarose gel, and Southern transferred onto nitrocellular membrane. The lactoferrin gene was detected by hybridization to lactoferrin cDNA (LF) [23] and the 15{alpha}-hydroxylase gene by P45015{alpha} cDNA probe [43]. Sizes of the various DNA markers are labeled. Restriction enzymes and the probes used for hybridization are indicated on top

The three inbred strains of mice (SM, SJL, and SWR) were selected to examine the estrogen responsiveness of the lactoferrin gene in the uterus. Lactoferrin in the uterine fluid after DES treatment was analyzed by Western blot analysis (Fig. 2), and the protein in the uterine epithelium was detected by immunohistochemistry (Fig. 3) with anti-mouse lactoferrin rabbit serum. Purified mouse uterine lactoferrin served as the standard in Western blot analysis (Fig. 2, lane 1) and the serum protein used as negative control (lane 7). In addition, nonimmune rabbit serum was used as the negative control in both Western blot analysis and the immunostaining study (data not shown). Differential estrogen response was found among the different strains of mice. High levels of lactoferrin were found in the uterine fluid and uterine epithelium of CD-1 mice after DES treatment (Fig. 2, lane 2, and Fig. 3A, panel d). Lower levels of lactoferrin were measured in the uterine fluid of DES-treated inbred strains (Fig. 2, lanes 3–6 and 8), and less intense immunostaining of the uterine epithelium was found as well (Fig. 3, A, panels f, h, and j, and B). To semiquantify the lactoferrin expression in the uterus of different strains of mice, we scored three regions of the uterus (top; near the oviduct, middle, and bottom; and near the vagina) with arbitrary numbers (0 = negative, 1 = weak, 2 = moderate, 3 = strong, and 4 = intense), and the data were analyzed by both ANOVA and the Dunnett test. The mean ± SEM and the significant differences between DES-treated and untreated mice are presented in Figure 3B. We found that DES induced an intense immunostaining of the lactoferrin in CD-1 mice along the entire epithelial of the uterus and glands; however, we also found variability of lactoferrin staining along the uterine epithelium in the inbred strains. The bottom part of the uterus exhibited stronger lactoferrin immunostaining than the top part of the uterus. Lactoferrin was not detected in the uterus of vehicle-treated CD-1 mice (Fig. 3A, panel b) and the inbred strains (data not shown). It was interesting to find that lactoferrin is present in the oviduct of the various strains of mice regardless of hormone treatment (Fig. 3A, panels a, c, e, g, and i, pink stain). The results showed strain differences in estrogen response of the lactoferrin gene in the uterus.



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  		FIG. 2. Western blot detection of lactoferrin in the uterine fluid from various strains of mice after DES treatment. Five micrograms of purified mouse lactoferrin standard (1); 5 µl of uterine fluid from CD-1 (2), SJL (3), SM (4–6, Brown or Black), and SWR (8) mice; and control serum (7) were separated with the Novex gel system and examined by Western blot analysis with rabbit anti-mouse lactoferrin serum (mLF45)



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  		FIG. 3. Immunohistochemical detection of lactoferrin in the oviduct and uterine epithelium from various strains of immature mice after DES treatment. A) Immunostaining. Positive lactoferrin immunostaining (red or pink) was detected in the oviducts (arrows in panels a, c, e, g, and i) in both control and DES-treated mice. Lactoferrin immunostaining was detected in both luminal and glandular epithelium of the uterus from mice treated with DES (arrows in panels d, f, h, and j; red or pink). Control oviduct and uterus were from CD-1 mice treated with vehicle. The inbred mice treated with vehicle were identical to the CD-1 control (data not shown). CD-1, Outbred strain; SM, SJL, and SWR, inbred strains. Bar = 200 µm, x200. B) Stain intensity of the lactoferrin in the uterus from A was scored with an arbitrary number (0 = negative, 1 = weak, 2 = moderate, 3 = strong, and 4 = intense). The number scored for each bar was 15, and the data were subjected to ANOVA and the Dunnett test. Results are presented as the mean + SEM. *P < 0.05 vs. control (Dunnett test)

Characterization of Antilactoferrin AntibodyThat Cross-Reacts to Lactoferrin but Not Transferrin of the Rat and Hamster

Our laboratory has generated a number of rabbit polyclonal antibodies against human or mouse lactoferrin [22, 25]. The polyclonal anti-mouse lactoferrin antibody from three different rabbits immunized at the same time with the same protein isolates showed a wide range of specificity toward lactoferrin or transferrin in different species (Fig. 4). Three lots of anti-mouse lactoferrin serum were characterized, and we found that the mLF44 cross-reacts with the human and bovine lactoferrin and, to a lesser degree, with the mouse transferrin (Fig. 4A). The mLF45 and mLF46 showed specificity to mouse lactoferrin (Fig. 4, B and C). The mLF44 antiserum was tested for cross-reactivity to rat transferrin. As a control, the highly specific anti-mouse lactoferrin serum mLF46, the anti-mouse transferrin, and the commercial rat transferrin antibodies were included in the Western blot analysis (Fig. 4D). The anti-mouse transferrin and mLF46 were specific to the mouse transferrin (lanes 1–3) and mouse lactoferrin (lanes 7–9), respectively. As shown earlier, the mLF44 cross-reacted weakly with the mouse transferrin (lane 5) but not with the rat transferrin (lane 4). The rat transferrin antibody detected rat transferrin as doublets with equal intensity (lane 10). The rat transferrin antibody also detected the mouse transferrin (lane 11) but not the mouse lactoferrin (lanes 12). By Western blot analysis, the mLF44 did not recognize the rat transferrin; however, at immunohistochemistry, mLF44 produced background in the reproductive tract of the rat (data not shown). To ensure the lactoferrin specificity in immunostaining studies, we included a neutralizing level of rat transferrin protein in the primary antibody reaction step. This step has enhanced the staining of the lactoferrin and reduced the background staining caused by the transferrin (data not shown).



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  		FIG. 4. Characterization of the rabbit anti-mouse lactoferrin sera by Western blot analysis. Purified lactoferrin protein from mouse (mLF), human (hLF), bovine (bLF), and mouse transferrin (mTF) were separated on a the Novex gel system and examined by Western blot analysis with anti-mouse lactoferrin serum from rabbit 44 (A), 45 (B), and 46 (C). The concentration of lactoferrin or transferrin protein (ng), the molecular marker (kDa), and the locations of lactoferrin (LF) and transferrin (TF) are indicated. Relative mobility of the mTF, mLF, and rat transferrin (rTF) in the Novex gel system and the respective antiserum specificity were also examined by Western blot analysis (D). Three hundred nanograms of protein were loaded onto each lane in D. Antisera used to determine the specificity are indicated on top

Detection of Lactoferrin in Rat and Hamster Tissues

To identify the lactoferrin protein in the tissues of rat and hamster, we analyzed the tissue homogenates by Western blot analysis (Fig. 5). Tissue choice was based on the abundance of lactoferrin protein detected in the human and mouse tissues [46–48]. Various sizes of lactoferrin were found in DES-treated rat uterus (Fig. 5A, lane 2) and uterine fluid (lane 5) and in adult male rat prostate (lane 3) and bone marrow (lane 4). A 65-kDa lactoferrin was detected in the uterus of DES-treated immature rat and in bone marrow of the adult rat, whereas a 60-kDa lactoferrin was found in the rat prostate. Lactoferrin secreted into the uterine fluid of DES-treated animals is less then 60 kDa in size. All the rat lactoferrins in these tissues are smaller than the mouse lactoferrin in the uterine fluid, which is 70 kDa (compare lanes 2–5 to lane 6). The origin of the minor bands, which run faster or slower than the lactoferrin, is not known. It could be the aggregation or degradation products of the lactoferrin.



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  		FIG. 5. Detection of lactoferrin in the rat and hamster tissues by Western blot analysis. Tissue homogenate (50–100 µg) from rat (A) or hamster (B) were separated on the Novex gel system and examined by Western blot analysis with mLF44 IgG. For the rat samples, the primary antibody was diluted 1:1000 and preblocked with 10 µg/ml of rat transferrin overnight at 4°C. Uterus was collected from rats treated with vehicle (Cont Ut) or DES (DES Ut). Uterine fluid (Ut Flu) was collected from DES-treated animals. Other tissues were collected from adult rats. Hamster tissues were collected from the untreated immature females. Prostate (prost), bone marrow (BM), spleen (Spl), and seminal vesicle (SV) are labeled. Locations of lactoferrin (LF), transferrin (TF), and molecular markers are indicated

Lactoferrin of the hamster is similar to that of the mouse in size (Fig. 5B, lanes 5 and 6). Hamster prostate (lane 8) contains a low level of lactoferrin, but the size of the protein is similar to that in the uterus (lane 5) and spleen (lane 6). The seminal vesicle has a smaller-size lactoferrin (lane 7), and all tissues examined contain transferrin (lanes 1–4). Apparently, mLF44 also recognized the hamster transferrin (lower band in lanes 5–8). No measurable difference was found between lactoferrin in the uterus of control and DES-treated animals (data not shown).

Differential Estrogen Response of the Lactoferrin Gene in the Reproductive Tract of Immature Rat, Adult Rat, and Immature Hamster

The reproductive tract of the immature rat was examined for lactoferrin gene expression and its response to estrogen stimulation (Fig. 6). Lactoferrin was expressed in the oviduct at the isthmus region (Fig. 6a) and the vaginal epithelium (Fig. 6, c and d, pink and red stain) of the immature 35-day-old CD rat. No lactoferrin was present in the luminal and glandular epithelium of the uterus (Fig. 6, b and c, arrows). After estrogen treatment, lactoferrin expression was induced in the uterine epithelium (Fig. 6, f and g, arrows). Thus, under the influence of estrogen, lactoferrin was detected in the epithelium of all sections of the reproductive tract of an immature rat (Fig. 6, e–h). Lactoferrin in the uterine epithelium of the F344 rat was induced by eCG treatment (Fig. 6, j and k, arrows). Because eCG induces estrogen secretion by the ovary, the expression of lactoferrin in the uterine epithelium was assumed to likely be caused by an estrogenic effect. The intensity of the lactoferrin immunostaining in the uterine epithelium of eCG-treated F344 rat (Fig. 6, j and k, light pink) was less than that of DES-treated CD rat (Fig. 6, f and g, red).



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  		FIG. 6. Immunohistochemical detection of lactoferrin in the reproductive tract of immature rat with or without hormonal treatment. Pink or red color indicates positive immunostaining of lactoferrin protein. Uterine luminal and glandular epithelium are indicated by arrows. Oviduct (Ov), uterus (Ut), uterus and vaginal junction (Ut/Vg), and vaginal epithelium (Vg) are labeled. CD rats and F344 rats are indicated on top. CD rats treated with vehicle (a–d) or DES (e–h) and F344 rats treated with eCG (i–l) are indicated. Pictures were taken at different portions of the reproductive tract of the same animal. Bar = 100 µm, x100.

The uterus and vaginal epithelium of the adult cycling CD rats were examined (Fig. 7). Lactoferrin was detected in the uterine luminal and glandular epithelium during the proestrous (Fig. 7, d and e, arrows) or estrous (Fig. 7, g and h, arrows) stage of the cycle. No lactoferrin staining was found in the uterus while the rats were in diestrous (Fig. 7, a and b, arrows). Lactoferrin staining was detected in the vaginal epithelium and the infiltrating neutrophils at all stages of the cycle (Fig. 7, c, f, and i). Among the 12 adult rats, several of them fell into each stage of the cycle that we have examined, and the above observations were verified. Results from the present study demonstrated that lactoferrin in the rat uterus was responsive to both endogenous natural estrogen and the exogenous synthetic estrogen.



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  		FIG. 7. Immunohistochemical detection of lactoferrin in the reproductive tract of adult rats at various stages of the estrous cycle. Stages of the cycle and location of the reproductive tract are indicated. Diestrous (a–c), proestrous (d–f), estrous (g–i), uterine and epithelium (a, d, and g), uterine and vaginal junction (b, e, and h), vaginal epithelium (c, f, and i). Pink or red color indicates positive immunostaining of lactoferrin. Arrows indicate the uterine luminal and glandular epithelium. Bar = 100 µm, x100

Lactoferrin expression in the reproductive tract of the immature hamster was also examined (Fig. 8). The neutralizing rat transferrin was eliminated from the immunohistochemistry procedures of this study, because presence of rat transferrin did not improve the specificity of staining. To our knowledge, hamster transferrin is not commercially available; therefore, mLF44 IgG was used alone as the primary antibody in the hamster studies. It was surprising to find that lactoferrin was not detectable in the reproductive tract of the immature hamster except in the neutrophils (Fig. 8, a–c). Lactoferrin expression was induced in the vaginal epithelium (compare Fig. 8, c and f) after three injections of DES, whereas no increase of lactoferrin staining in other parts of the reproductive tract (Fig. 8, d and e) was seen. Several other antilactoferrin sera were tested, and no positive staining in the uterus was obtained (data not shown).



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  		FIG. 8. Immunohistochemical detection of lactoferrin in the reproductive tract of immature hamsters with or without DES treatment. Treatment and location of the reproductive tract are indicated. Control (a–c), DES-treated (d–f), oviduct (a and d), uterine epithelium (b and e), vaginal epithelium (c and f). Pink or red color indicates positive immunostaining of lactoferrin. Arrows in the uterine epithelium indicate the luminal and glandular epithelium. Arrows in the DES-treated vaginal epithelium indicate the positive staining of lactoferrin. Bar = 100 µm, x 100 (a, b, d, and e) and 200 µm, x200 (c and f)


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
It has been well established that the lactoferrin gene in the uterus of an immature or cycling female adult CD-1 mouse is a sensitive target for estrogen action (see [1] and references therein). However, it is not known whether the lactoferrin gene in the uterus of inbred strains of mice is sensitive to estrogen stimulation. Although lactoferrin has been cloned from various farm animals, to our knowledge only mouse lactoferrin cDNA and its gene have been isolated and characterized from laboratory animals [23, 49, 50]. The lack of suitable reagents for lactoferrin from laboratory animals other than mouse may explain the limited investigation that has been conducted on rat [36, 37], hamster [38, 39], or guinea pig [51]. Because of the high sequence homology at certain regions of the lactoferrin gene and its protein product among different species and the close family member transferrin [1, 23], the polyclonal rabbit antiserum generated against the human or mouse lactoferrin may cross-react with both lactoferrin and transferrin when applied to other rodent species. Therefore, to further investigate lactoferrin expression in the rat and hamster reproductive tract, we first characterized a polyclonal anti-mouse lactoferrin serum that cross-reacts with lactoferrin but only minimally with the transferrin of rats and hamsters. The present study provides some insight regarding the variability of lactoferrin gene expression and its differential response to estrogen stimulation in the reproductive tract of the mouse, rat, and hamster.

A wide range of genetic variability exists among the different mouse strains and their response to the synthetic estrogen DES [40, 41, 52]. The lactoferrin gene in the three inbred strains examined (SJL, SM, and SWR) showed similar patterns of EcoRI and BamHI restriction enzyme polymorphisms that differ from the outbred CD-1 mouse. These immature inbred mice responded to estrogen stimulation and increased lactoferrin gene expression in the uterus; however, their responses were less robust than those of CD-1 mice. Among the three inbred strains, SWR was more resistant to estrogen stimulation. It has less fluid accumulated in the uterine lumen and a lower level of lactoferrin expression in the epithelium after DES treatment. Interestingly, the lactoferrin detected in the oviduct was also low in the SWR strain (Fig. 3A, panel i). Both SWR and SLJ strains were highly susceptible to infection as compared to other strains [53]. It will be interesting to know whether lactoferrin expression in the neutrophils and other surface mucosa epithelium of these two strains of inbred mice is also low. Nonetheless, the present findings demonstrated that the lactoferrin gene in the uterus of mice is estrogen inducible and likely occurs through the well-characterized estrogen response element (ERE) of the gene [21] regardless of gene polymorphisms. Methylation pattern surrounding the ERE [48, 54] and the presence of estrogen receptor-related receptor [31, 55] could play an important role in determining the estrogen response of the lactoferrin gene in these inbred strains of mice.

Lactoferrin in the rat has been a subject of controversy [23, 36–39, 51, 56]. Although lactoferrin was originally found in human milk [57] and was then shown to be present in the milk of various other species [51], it has not been detected in the milk of rats [51, 56, 58]. In addition, the rat has a consistently low amount of lactoferrin in neutrophils as measured by a variety of techniques [39]. Using a mouse lactoferrin cDNA probe, however, we have detected cross-hybridization with mRNA from the lactating mammary gland of the rat but not from the estrogen-stimulated uteri [23]. By using the established antibody and the presence of a neutralizing level of rat transferrin in the present study, we found various sizes of lactoferrin in different tissues of the rat, including the DES-treated uterus (Fig. 5A). Previous studies also found various sizes of lactoferrin among rhesus monkey tissues [25]. Posttranslational modification of the protein and differential protein processing in the tissues could explain such differences. The immunoreactive lactoferrin of the hamster from uterus, spleen, and prostate did not show size differences (Fig. 5B). It ran at the 76-kDa region on an SDS-PAGE, which is slightly smaller than the human protein and agrees with what has been previously published [39]. However, a smaller size of lactoferrin was detected from the seminal vesicle.

The estrogen responsiveness of the lactoferrin gene in the reproductive tract of the immature and adult cycling rat was also evaluated. The 35-day-old CD rats, whose developmental stage is comparable to that of the 21-day-old mouse, were used for the present study. We found that the lactoferrin gene in the rat uterus is estrogen inducible (Fig. 6), although lactoferrin gene in younger rats (21 days old) was unresponsive to the estrogen stimulation [23]. In addition to the injection of immature rats with exogenous DES, enhancing the endogenous estrogen production by eCG-treatment of the immature F344 rats also induced lactoferrin expression (Fig. 6). However, the intensity of the lactoferrin immunostaining in the uterine epithelium of eCG-treated F344 rats was less than that of DES-treated CD rats. This may reflect the lower level of estrogen induced by the eCG treatment in 24 h. Taken together, these results demonstrated that the lactoferrin gene was sensitive to estrogen stimulation in the immature rat at the uterine portion of the reproductive tract, whereas in the oviduct and vaginal regions, lactoferrin was present regardless of the hormonal status. Lactoferrin was detectable in the uterine epithelium (both luminal and glandular) of adult CD rats at the proestrous and estrous stages and was undetectable during the diestrous stage of the estrous cycle. Estrogen levels rise at the proestrous stages and start to fall during the estrous stage, which is followed by a wave of rising and falling progesterone levels during the estrous cycle. Progesterone antagonism of estrogen actions in the uterus has long been recognized, and the balance between these two hormones is critical for normal uterine physiology. Detection of lactoferrin expression during the proestrous and estrous phases, but not during the metestrous and diestrous phases, of the cycle suggests that lactoferrin gene in the adult rat uterus was sensitive to the endogenous level of estrogen, whereas the endogenous progesterone suppressed lactoferrin expression. Recent studies with stromal/epithelial tissue recombination between wild-type, estrogen receptor-null (ERKO) and progesterone receptor-null (PRKO) mice demonstrated that estrogen and progesterone receptors in both the epithelium and stroma are required for maximum lactoferrin expression and suppression, respectively [59, 60]. In contrast to the uterus, lactoferrin is constantly present in the isthmus oviduct and the vaginal epithelium of the rat regardless of the hormonal status and age of the rat. The present study has demonstrated that the lactoferrin gene in the reproductive tract of the rat is subject to spatiotemporal regulation by estrogen.

The lactoferrin gene in the reproductive tract of the immature 21-day-old hamster has a very different expression pattern from that of the rat and mouse. Lactoferrin was not detected in either oviductal or uterine epithelium despite the hormonal treatment. However, abundant infiltrating neutrophils with high lactoferrin content were found in the stromal region of the oviduct and uterus. Therefore, the lactoferrin that was found in the uterus by Western blot analysis (Fig. 5B, lane 5) could originate from the many neutrophils in this tissue. The high background staining in the stromal region of the uterine epithelium could be contributed by the transferrin in this region. The vaginal stroma of the untreated immature hamster is also infiltrated with neutrophils. After estrogen treatment, lactoferrin gene expression is induced in the vaginal epithelium (Fig. 8). Whether age plays a role in the estrogen responsiveness of the lactoferrin gene in the hamster uterus is yet to be investigated. Limited information regarding hamster lactoferrin was found in the literature [38], and to our knowledge, no information regarding the characterization of the protein has been reported. Therefore, the detection of positive lactoferrin immunostaining in the neutrophils and vaginal epithelium after estrogen treatment does not exclude the possibility that mLF44 IgG may not recognize the uterine form of hamster lactoferrin. It is interesting that neonatal treatment with DES, but not with estradiol, resulted in an up-regulated expression of the lactoferrin protein throughout the endometrial epithelial cell compartment of estradiol-stimulated adult hamsters [38]. Lactoferrin is present throughout the hamster reproductive tract, although not in the epithelium. Whether estrogen enhances neutrophil infiltration into the reproductive tract is unclear. Ample evidence suggests that the first estrogen response in the uterus is infiltration of serum protein and immune cells [32, 33]. As long as lactoferrin is present in the reproductive tract, it could function in host defense and play a role in mucosal immunity in the female reproductive tract [35, 61–63].


   ACKNOWLEDGMENTS
 
We gratefully acknowledge the excellent preparation of the histology samples by the Pathology Support Group of the Laboratory of Experimental Pathology, NIEHS. Dr. B. Davis kindly provided the eCG-treated Fischer (F344) rats for the study, and Drs. M. Negishi and B. Burkhart supplied the P45015{alpha} cDNA probe. We thank Drs. Darlene Dixon and Barbara Davis for critically reading the manuscript and providing useful comments, Dr. Joseph Haseman for the statistical analysis, and Ms. Loretta Moore for editing the paper.


   FOOTNOTES
 
1 Correspondence: Christina Teng, NIEHS/NIH, PO Box 12233, Research Triangle Park, NC 27709. FAX: 919 541 0696; teng{at}niehs.nih.gov Back

Received: 30 November 2001.

First decision: 2 January 2002.

Accepted: 7 June 2002.


   REFERENCES
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 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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K. Stokes, B. Alston-Mills, and C. Teng
Estrogen response element and the promoter context of the human and mouse lactoferrin genes influence estrogen receptor {alpha}-mediated transactivation activity in mammary gland cells
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