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Biol Reprod. 2012 July; 87(1): 10.
Published online 2012 May 2. doi:  10.1095/biolreprod.112.099846
PMCID: PMC3406554

Neonatal Phytoestrogen Exposure Alters Oviduct Mucosal Immune Response to Pregnancy and Affects Preimplantation Embryo Development in the Mouse1

ABSTRACT

Treatment of neonatal mice with the phytoestrogen genistein (50 mg/kg/day) results in complete female infertility caused in part by preimplantation embryo loss in the oviduct between Days 2 and 3 of pregnancy. We previously demonstrated that oviducts of genistein-treated mice are “posteriorized” as compared to control mouse oviducts because they express numerous genes normally restricted to posterior regions of the female reproductive tract (FRT), the cervix and vagina. We report here that neonatal genistein treatment resulted in substantial changes in oviduct expression of genes important for the FRT mucosal immune response, including immunoglobulins, antimicrobials, and chemokines. Some of the altered immune response genes were chronically altered beginning at the time of neonatal genistein treatment, indicating that these alterations were a result of the posteriorization phenotype. Other alterations in oviduct gene expression were observed only in early pregnancy, immediately after the FRT was exposed to inflammatory or antigenic stimuli from ovulation and mating. The oviduct changes affected development of the surviving embryos by increasing the rate of cleavage and decreasing the trophectoderm-to-inner cell mass cell ratio at the blastocyst stage. We conclude that both altered immune responses to pregnancy and deficits in oviduct support for preimplantation embryo development in the neonatal genistein model are likely to contribute to infertility phenotype.

Keywords: blastocyst, developmental patterning, female reproductive tract, immunoglobulin

INTRODUCTION

The mammalian female reproductive tract (FRT) is highly sensitive to developmental disruption by estrogenic chemicals. We previously demonstrated in a mouse model that neonatal exposure to the phytoestrogen genistein results in complete infertility in female adults [1]. Causes of the infertility include anovulation resulting from hypothalamic-pituitary-ovarian axis dysfunction, a reduction in the ability of the oviduct to support preimplantation embryo development, and failure of the uterus to support effective implantation of blastocyst stage embryos [2]. However, the mechanistic basis for failure of the oviduct and uterus to support embryo development and implantation in this model system is not clear.

The FRT contributes to immunological homeostasis by defending against pathogens that routinely enter the vagina and endocervix and periodically enter the upper (or anterior) FRT regions, the uterus and oviduct. Like other epithelial tissues that provide mucosal immunity, the FRT uses multiple immune mechanisms, including serving as a barrier to infection, secreting antimicrobial molecules, presenting antigens to lymphocytes, and transporting immunoglobulins [3]. At the same time, the FRT must temper its immune responses to allow for the survival of commensal organisms, sperm, and the semi-allogeneic fetus. These tasks are accomplished in part by differential expression of immune response mediators along the FRT. For example, specific toll-like receptors and lymphocyte antigen 96 (also known as MD2) are differentially expressed in vaginal and cervical tissues, which are normally colonized by commensal organisms, compared to the normally sterile uterus and oviduct [4, 5].

Mammals have also evolved a complex system for precise steroid hormone regulation of FRT immune functions that is characterized by different sites within the FRT having differential responses to the same steroid hormones [6, 7]. For example, in mice, the antimicrobial molecule lactoferrin is most highly expressed in the uterus during estrus in response to high estradiol levels, whereas lactoferrin expression in the oviduct and vagina are much lower and relatively constant throughout the estrous cycle [8, 9]. In women, two immune regulators, secretory leukocyte protease inhibitor (SLPI) and human intestinal defensin-5 (HD-5), are secreted in cervical mucus in a hormonally regulated fashion; HD-5 expression does not vary in the vagina or ectocervix regardless of menstrual cycle timing [1013].

Modulation of the upper FRT mucosal immune response in the periovulatory period facilitates fertilization and subsequent survival of the preimplantation embryo while continuing to inhibit survival of pathogens [3, 14]. At the same time, some degree of inflammatory response appears to promote preimplantation embryo development and implantation [1518]. This requirement for a carefully balanced immune response suggests that altered immune responses in the oviduct could interfere with pregnancy establishment, including preimplantation embryo development.

In an effort to determine the mechanistic basis of the oviduct's contribution to infertility in this model, we analyzed oviducts from neonatal genistein-treated and control mice. Oviduct tissues were collected from superovulated mice on Pregnancy Day 2, the day of gestation immediately preceding the observed loss of preimplantation embryos in the oviduct. We found that neonatal genistein treatment caused morphological abnormalities characterized by oviduct wall thickening, pseudogland formation, excessive epithelial proliferation, and epithelial disorganization [19]. Microarray analysis and subsequent validation by real-time PCR and protein-based analyses indicated that neonatal genistein treatment caused permanent “posteriorization” of the FRT. This effect is characterized by abnormal expression in the oviduct of numerous genes normally expressed only in the cervix and vagina, including several homeobox transcription factors with critical roles in prenatal development. In this study, we delved further into our previous microarray findings and tested the hypothesis that altered inflammatory responses in the oviduct environment during early pregnancy explained the poor survival of preimplantation embryos in this region of the FRT.

MATERIALS AND METHODS

Animals and Treatments

Female CD-1 pups and adult CD-1 males were obtained from the in-house colony at National Institute of Environmental Health Sciences (NIEHS, Research Triangle Park, North Carolina). CD-1 males were used for mating in all experiments except those requiring vasectomized males. For some experiments, fertile or vasectomized adult male B6SJLF1/J mice (8–16 weeks of age; Jackson Laboratories, Bar Harbor, ME) were used for mating. Vasectomized males were verified to be infertile prior to use. All animal procedures complied with NIH/NIEHS animal care guidelines. Animal treatments were described in detail previously [19]. Briefly, female pups were injected subcutaneously on Postnatal Days 1–5 (PND1–5) with either corn oil (control) or genistein, 50 mg/kg/day (Sigma, St. Louis, MO); the estrogen receptor antagonist ICI-182780 (ICI; Sigma), 1 mg/kg/day; or ICI, 1 mg/kg/day, 30 min prior to genistein, 50 mg/kg/day. The dosing strategy for genistein was chosen because it results in a serum genistein concentration in the same range as that measured in infants fed soy-based infant formulas [20]; a complete discussion of the rationale for this model system is reviewed in the report by Jefferson et al. [21]. Females, age 6–8 weeks, underwent superovulation alone or superovulation and mating as described previously [19] or were mated to vasectomized males. Only vaginal plug-positive females were used in experimental endpoints. Where possible, females were confirmed to be pregnant either by flushing embryos from the oviduct on Pregnancy Day 2 or from the uterus on Pregnancy Day 4.

Real-Time RT-PCR

Real-time PCR was carried out as previously described [19]. Briefly, oviducts were collected from neonatal females on PND5 and PND22, from adult superovulated females at 8 and 15 h after human chorionic gonadotropin (hCG) administration, and from adult superovulated and mated females on Pregnancy Days 2 and 4 (48 and 96 h after hCG, respectively). Oviducts were also collected from superovulated adult CD-1 females (6–8 wks of age) that were mated to vasectomized males. Reproductive tract tissues were collected from untreated adult CD-1 females (6–8 wks of age). cDNA was generated from 1 μg of total RNA, amplified using primers crossing exon-intron junctions, and detected using SYBR Green (Invitrogen, Carlsbad, CA). Graphs show expression relative to that of peptidylprolyl isomerase A (Ppia).

Microarray Analysis

Microarray analysis of oviductal mRNA from control and genistein-treated mice on Pregnancy Day 2 was described previously [19]. Briefly, four independent biological replicates for each group were analyzed using Agilent Whole Mouse Genome arrays (Agilent Technologies, Santa Clara, CA). Data were processed using Rosetta Resolver software (Rosetta Biosoftware, Kirkland, WA), analyzed using Ingenuity Pathway Analysis software (Ingenuity Systems, Redwood City, CA), and deposited in the NCBI Gene Expression Omnibus under accession no. GSE27639.

Histology and Immunohistochemistry

Formalin-fixed female reproductive tract tissues were paraffin embedded and sectioned at 5 μm. Sections were stained with periodic acid-Schiff stain (PAS) and Alcian blue according to standard protocols. Sections were immunostained for factor VIII (diluted 1:500; Biocare Medical, Concord, CA) using the Vectastain Elite kit (Vector Laboratories, Burlingame, CA) following the manufacturer's instructions. Diaminobenzamine (DAB) was used for visualization, and hematoxylin was used as a counterstain. Additional sections were immunostained with biotinylated CD45 (diluted 1:250; GeneTex, San Antonio, TX) followed by peroxidase-conjugated streptavidin (Biogenex, San Ramon, CA); DAB was used for visualization, and hematoxylin was used as a counterstain. For Oil Red O stain, oviducts were frozen in OCT compound, sectioned at 4 μm and stained with Oil Red O and counterstained with hematoxylin. Slides were scanned using a Scanscope scanner and Imagescope software (Aperio Technologies, Vista, CA). Unless otherwise indicated, all reagents were from Sigma (St. Louis, MO).

Immunoglobulin A ELISA

Oviducts were collected on Pregnancy Days 2 and 4; one oviduct was flushed to confirm pregnancy and the other oviduct was frozen at −80C until used for the ELISA. The non-flushed oviduct was pulverized on dry ice and then homogenized in 200 μl of 1× diluent supplied in the mouse immunoglobulin A (IgA) ELISA kit (Innovative Research, Novi, Michigan). Samples were centrifuged for 5 min at 10 000 rpm to pellet cellular debris, and the supernatant was removed for use in the assay. Each oviduct sample was diluted 1:10 in the diluent buffer, and then IgA was measured in individual oviducts (4–7 mice per group) following the manufacturer's instructions. All test plates included a standard series of IgA dilutions for use in calculating sample IgA concentrations. The presence of green dye in the diluent buffer precluded direct measurement of protein concentrations, so to confirm that the sample protein concentrations were similar, we immunoblotted 10 μl of each sample for actin as described previously [19]. For serum IgA analysis, blood was collected from the vena cava on Pregnancy Day 4. The blood was allowed to clot at room temperature for 30 min and then centrifuged for 10 min at 3000 rpm to remove blood cells. Serum was diluted 1:40 with diluent buffer, and IgA ELISA was performed using a procedure similar to that for the oviduct samples (n = 6–8 mice per group).

Blastocyst Transfers and Cell Counts

Control recipient females were bred with vasectomized males to stimulate pseudopregnancy. Blastocysts were flushed from the uterus of neonatal genistein-treated or control mice on Pregnancy Day 4 (96 h post-hCG) and then transferred to control recipients on Day 3 of pseudopregnancy according to standard procedures. Twelve blastocysts were transferred into one uterine horn, and recipients were allowed to deliver at term. For differential cell counts, blastocysts were flushed from oviducts 96 h post-hCG. Between three and five fully cavitated, non-expanded blastocysts from each mouse were differentially stained as described [22] and imaged in three dimensions using a Leica confocal microscope. Blastocyst cell counts were performed by an observer blinded to the treatment group.

Statistical Analysis

Data were analyzed using GraphPad Prism version 5.0c software (GraphPad Software, Inc.). Two-tailed Mann-Whitney tests were used to compare control and genistein-treated samples at each time point, except for experiments with vasectomized mice, which were analyzed with a one-tailed Mann-Whitney test (P < 0.05). Multiple comparisons were carried out using ANOVA followed by Tukey's test (P < 0.05).

RESULTS

Neonatal Genistein Alters Expression of Inflammatory Response Genes During Early Pregnancy

Data from a microarray analysis of oviducts from control and genistein-treated mice on Pregnancy Day 2 were previously reported [19]. These data, combined with that from subsequent real-time PCR and protein analyses, demonstrated that neonatal genistein treatment caused oviduct “posteriorization,” or abnormal expression of genes usually restricted to the lower (posterior) female reproductive tract, the cervix and vagina. After the posteriorization findings, the most notable finding in the microarray analysis of oviducts on Pregnancy Day 2 was that neonatal genistein treatment resulted in significant alterations in genes within “immune response” biological function categories (Table 1). In addition to genes categorized by the Ingenuity analysis software into immune response functions, 35 distinct immunoglobulin genes were upregulated, including the IgA and IgM-specific joining chain, IgJ, whereas no immunoglobulin genes were downregulated (Supplemental Table S1; all supplemental data are available online at www.biolreprod.org). Of these, expression levels of 34 genes were increased more than twofold, representing 17% (34 of 200) of the significantly altered genes identified by microarray analysis to be upregulated by at least this amount. Many genes important for FRT mucosal immunity had altered expression, including secretory leukocyte peptidase inhibitor (Slpi), CD44 antigen (Cd44), surfactant associated protein D (Sftpd), and toll-like receptor 1 (Tlr1). The most highly up- or downregulated genes identified included six different clade A serpins, which encode extracellular α1-protease inhibitors that function to limit the activity of serine proteases involved in the inflammatory response [23]. The chemokine (C-X-C motif) ligand 15 (Cxcl15) was also highly upregulated; expression of this gene is increased in lung tissues during inflammatory responses and is expressed in uterine epithelium [24, 25].

TABLE 1.
Significantly altered genes in inflammatory response categories.*

To determine if the altered immune response genes could be detected at the protein level, we focused on immunoglobulins because of the large number of upregulated immunoglobulin mRNAs. In addition, immunoglobulins are normally secreted from reproductive tract mucosal epithelium as IgA polymers, and therefore, we anticipated that protein levels could be measured despite the limited material available. Single oviducts were collected on Pregnancy Days 2 and 4, and IgA was quantified by ELISA. There was a significant increase in IgA in the oviducts of genistein-treated mice compared to that in controls on Pregnancy Day 2; this difference was even greater on Pregnancy Day 4 (Fig. 1A). Although the IgA was measured on the same quantity of protein from each oviduct based on the extraction method used, immunoblots for actin were also obtained to document equivalent testing conditions for the ELISA (Fig. 1B). These data indicated that oviducts of genistein-treated mice had significantly increased amounts of IgA protein on Pregnancy Days 2 and 4. The increase in oviduct IgA was not a result of systemic alterations in IgA production because serum IgA levels were similar in control and genistein-treated mice on Pregnancy Day 4 (means ± SEM in control 5.50 ± 1.04 vs. genistein 8.46 ± 1.53 ng/ml; P = 0.35, Mann-Whitney test).

FIG. 1.
Increased oviductal IgA in genistein-treated mice. A) IgA levels in oviducts of control and genistein-treated mice on Pregnancy Days 2 and 4. Means ± SEM were plotted. White bars, controls; black bars, genistein-treated. *P < 0.05. B) ...

Abnormal Oviduct Histology Following Neonatal Genistein Treatment

Although a description of histological findings in the oviduct of genistein-treated mice was published previously [19], we performed additional morphological characterization of the oviduct histology to determine if there was evidence of inflammatory changes. An accumulation of amorphous material was observed in the oviduct lumen and within many epithelial cells in genistein-treated mice (Fig. 2, A–D). Some of this material stained positively with Alcian blue, indicating that it contained glycosaminoglycans and/or glycoproteins. There was little to no staining of this material with Oil Red O (data not shown), documenting the lack of a significant lipid component. The vascular supply to the oviduct was also dramatically increased in genistein-treated mice. Enlarged blood vessels were observed during oviduct dissections; this finding was confirmed using factor VIII to label endothelial cells (Fig. 2, E–H). Although the number of blood vessels in cross-sections of similar oviduct regions did not appear to differ, the blood vessels were greatly dilated in genistein-treated mice when compared to controls. The presence of leukocytes was examined in oviduct sections by immunostaining for the leukocyte marker, CD45. There were no positively stained cells observed in any oviduct sections from either control or genistein-treated mice (three oviduct sections were observed in each of three mice per group); vaginal tissue on the same slides served as internal positive controls (data not shown).

FIG. 2.
AD) PAS/Alcian blue stain. Two-cell embryos (e) present in oviductal lumen in A and B. Note increased Alcian blue staining in epithelium (arrow) and lumen (asterisk) in B and D. EH) Factor VIII immunohistochemistry; blood vessels indicated ...

Time Course of Alterations in Immune Response Genes

We next determined whether the immune response genes were permanently altered in genistein-treated mice beginning during the time of neonatal genistein treatment or whether they were altered later in response to hormonal or other changes associated with pregnancy. The Cxcl15, Slpi, Cd44, and Ltf genes were chosen for this analysis because there is good documentation of their involvement in epithelial immune responses [24, 2628]. Expression of these genes was examined using real-time PCR at PND5, PND22, before and after ovulation, and on Pregnancy Days 2 and 4. Cxcl15 expression was highly upregulated in oviducts of genistein-treated mice at all time points examined (Fig. 3A). This pattern of expression was similar to that of several homeobox transcription factors (Pitx1, Six1, and Nkx3-1) in the oviducts of genistein-treated mice [19]. In control adults, Cxcl15 was much more highly expressed in the cervix and vagina than in the uterus or oviduct (25-fold and 10-fold upregulated, respectively) (Fig. 3B), so its upregulation in oviducts of neonatal genistein-treated mice was consistent with oviduct posteriorization.

FIG. 3.
Expression of Cxcl15, Slpi, Cd44, and Ltf in oviduct following neonatal genistein-treatment and in control FRT. A) Relative expression of the indicated genes in the oviducts of control and genistein treated mice at indicated time points. Mean relative ...

In contrast to Cxcl15 expression, the remaining immune response genes examined had altered expression only after ovulation and mating. Slpi expression was significantly higher than that in controls beginning immediately after ovulation, whereas Cd44 and Ltf expression levels were significantly increased only on Pregnancy Days 2 and 4 (Fig. 3A). In adult control mice, Slpi, Cd44, and Ltf had significant expression levels throughout the FRT and were not restricted to the cervix and vagina, although the expression levels varied among the different regions (Fig. 3B). These findings suggest that unlike Cxcl15, these genes were upregulated abnormally in the oviduct in response to factors associated with ovulation and/or establishment of pregnancy rather than permanently upregulated as a result of oviduct posteriorization.

Mechanisms Underlying Altered Oviduct Immune Responses

To determine whether the altered oviduct immune response could be an abnormal response to the presence of sperm or embryos, which carry paternal antigens, control and genistein-treated mice were bred to vasectomized males, and expression of the altered genes was examined in oviducts collected on Day 4 after mating. Following breeding to vasectomized males, expression of Cxcl15, Slpi, Cd44, and Ltf was significantly increased in genistein-treated mice compared to that in controls (Fig. 4). These data suggest that the altered gene expression is a response to factors associated with early pregnancy other than sperm or embryos (e.g., the hormonal milieu of early pregnancy or components in the semen besides sperm).

FIG. 4.
Expression of Cxcl15, Cd44, Slpi, and Ltf in control or genistein-treated mice following breeding to vasectomized males. Oviducts were collected 96 hours post hCG and breeding. Mean relative expression ± SEM plotted. White bars, controls; black ...

Although phytoestrogen actions are mediated mainly by the classical estrogen receptors (ER), ERα and ERβ, phytoestrogens can also serve as ligands for alternative signaling pathways such as those downstream of the estrogen-related, aryl hydrocarbon or peroxisome proliferator-activated receptors [29, 30]. To determine whether the altered immune response phenotype was due to genistein's ER-mediated activity, we treated mice with the ER antagonist ICI just prior to genistein treatment on Neonatal Days 1–5. Expression of the altered immune response genes was then evaluated by quantitative PCR in the adult females on Pregnancy Day 4. Pretreatment with ICI prevented genistein-induced alterations in Slpi, Cd44, and Cxcl15 expression; Pitx1 was included as a control for ICI efficacy (Fig. 5). These findings indicate that alterations in these three immune response genes are mediated by ER action.

FIG. 5.
Inhibition of alterations in gene expression by estrogen receptor antagonist. Expression of Pitx1, Cxcl15, Slpi, and Cd44 in oviduct of control (Co), ICI, genistein (Gen), or genistein plus ICI (G+I)-treated mice on Pregnancy Day 4. Mean relative expression ...

Altered Embryo Development after Neonatal Genistein Treatment

In neonatal genistein-treated mice, there is a delay of several hours in fertilization of ovulated eggs, and only approximately half of the embryos survive in the oviduct beyond Pregnancy Day 2 [2]. We hypothesized that abnormalities in oviduct morphogenesis combined with upregulation of the inflammatory response to ovulation and mating would cause alterations in development of the surviving embryos. To test this idea, we examined the rate of preimplantation embryo development in vivo and the morphology and developmental competence of blastocyst stage embryos. Embryos were flushed from the FRT on Days 2, 3, and 4 of pregnancy (48, 72, and 96 h after hCG, respectively). There were no differences in the average number of embryos per mouse on Pregnancy Day 2, and most embryos had reached the 2-cell stage (Fig. 6A). However, on Pregnancy Day 3, in addition to having approximately half as many total embryos [2], genistein-treated mice had a lower percentage of 6- to 8-cell embryos and a higher percentage of embryos with >8 cells than controls, demonstrating that despite the delay in fertilization, the embryos were cleaving more rapidly. On Pregnancy Day 4, most of the embryos in both groups were blastocysts, and there were no significant differences in the total number of cells per embryo (Fig. 6, A and B). However, the cell distribution was altered in that the trophectoderm-to-inner cell mass (TE:ICM) ratio was significantly lower in blastocysts from genistein-treated mice (Fig. 6B).

FIG. 6.
Alterations in embryo development in vivo following neonatal genistein-treatment. A) Mean percentage of surviving embryos in each category flushed from the FRT at indicated time points are plotted. n = 46–133 embryos from 4 mice/group/time point. ...

Blastocyst developmental competence was determined by transferring morphologically indistinguishable, fully cavitated blastocysts flushed from control and genistein-treated mice to the uteri of pseudopregnant control females. There were no differences between the number of live pups delivered in either group (Table 2). These findings indicate that the differences in preimplantation development rate and blastocyst cell distribution did not impact the surviving embryos sufficiently to affect development to term.

TABLE 2.
Live pups produced following transfer of blastocysts derived from control and genistein-treated mice.

DISCUSSION

Results presented here combined with our previously published data [19] demonstrate that in mice, a brief neonatal exposure to the phytoestrogen genistein causes substantial permanent changes in oviduct morphology, gene expression, and function in adulthood. A unifying explanation for the changes observed is oviduct posteriorization with consequent misregulation of mucosal immune response genes and abnormal expression of proteins normally associated with the squamous epithelium of the lower FRT. Some of these altered immune response genes are chronically altered beginning at the time of neonatal genistein treatment; however, others are altered only in early pregnancy, immediately after the FRT is exposed to inflammatory or antigenic stimuli including follicular fluid, semen, and the early embryo. Together, these changes lead to altered immune responses to pregnancy and to deficits in oviduct support for preimplantation embryo development, both of which are likely to contribute to the infertility phenotype.

FRT epithelium is exposed to a large bolus of paternal antigens during mating, particularly in rodents in which the ejaculated semen, and not just sperm, enters the uterine cavity. In addition to clearing the semen and supernumerary sperm from the uterus and oviduct, a critical aspect of the FRT mucosal immune response after mating is to mediate upregulation of T regulatory (Treg) cells that recognize paternal antigens that entered the FRT [3133]. These Treg cells are responsible for subsequent creation of an immune-privileged environment at the fetal-maternal interface that prevents fetal rejection at implantation [34]. Neonatal genistein treatment clearly alters the balance of immune response mediators in the oviduct during early pregnancy, and could also affect local or systemic Treg cell generation. Indeed, neonatal exposure to estrogenic chemicals is firmly established as a cause of alterations in thymus development and function [3538], and thymus-derived Treg cells are essential regulators of autoimmune responses, allograft rejection, and antimicrobial responses [39, 40]. However, it is unlikely that alterations in Treg cell function explain the loss of preimplantation embryos in the oviduct of genistein-treated mice because the timing of the loss between Days 2 and 3 of pregnancy is not consistent with previously observed timing of Treg-mediated postimplantation embryo loss [32, 34]. In addition, leukocyte infiltration into the oviduct was not observed, so T-cell mediated embryo loss would be difficult to envision.

Many of the upregulated immune response genes in the oviduct of genistein-treated mice encoded immunoglobulins and antimicrobials that are secreted into the FRT lumen as part of the epithelial cell innate immune response. These findings are consistent with our observations of increased amounts of material in the oviduct lumen and the fact that the major changes in oviduct morphology were observed in the epithelial compartment (Fig. 2). Indeed, many oviductal epithelial cells were filled with large amounts of intracellular material that could represent secretory products. Although antimicrobials are generated in oviduct epithelial cells [8, 13, 41], the source of the increased immunoglobulin protein in oviducts of genistein-treated mice is not clear. Based on extensive work in the intestine, IgA is generated by lymphoid tissue-associated plasma cells that migrate into the lamina propria below the epithelium [42]. IgA is then taken up from the basolateral surface of the epithelial cells via secretory component receptor-mediated endocytosis, transported across the cell, and secreted from the apical region into the lumen. In the oviduct, IgA and IgG are detected in the stromal tissue underlying the epithelium, along with sparse plasma cells, but only in the infundibulum and not in other regions [43], and we found no evidence of any type of leukocyte, including plasma cells, in the oviducts of genistein-treated or control mice. However, given the presence in whole oviduct tissue of mRNAs encoding many immunoglobulins, a local source of immunoglobulins is likely even though the cell type responsible is not clear. Another potential source of oviductal immunoglobulin protein is transudation from circulating serum. In the rat, both IgG and IgA appear to enter uterine tissue via serum transudation during the water imbibition response to estradiol administration [44]. Absence of plasma cells combined with the presence of dilated blood vessels in the oviducts of genistein-treated mice (Fig. 2) suggests that serum transudation could be a source of the increased oviductal IgA. Given that circulating serum IgA levels are similar in genistein-treated and control mice, this would only be true if IgA was more efficiently transferred into or maintained in the oviduct of genistein-treated mice.

Based on the well-documented microbicidal and fungicidal activities of lactoferrin and SLPI [26, 28, 45, 46], one explanation for the loss of preimplantation embryos in the oviduct is upregulation of antimicrobials that cause direct embryocidal action. There are certainly similarities in structure between bacterial and fungal peptidoglycan cell walls and the embryo's zona pellucida (ZP), which is composed of cross-linked filaments of glycoproteins rich in O- and N-linked glycans [47]. Furthermore, ZP thinning normally occurs during embryo development as a result of factors derived from both the FRT and the embryo [48, 49], so ZP degradation could be a normal physiological function of secreted oviduct antimicrobials. We did not observe any gross differences in the ZPs of embryos from genistein-treated or control mice, and found no differences in the blastocyst ZP dissolution rate in response to pronase treatment (unpublished observations). These experiments are difficult to interpret because one explanation is that only some embryos, not including the surviving blastocysts, are susceptible to these factors. Alternatively, the antimicrobials may affect the ZP but only in a subtle fashion not detectable by the dissolution assay. Of note, disruption of the ZP in human embryos by assisted hatching is associated with very low implantation rates after embryo transfer unless immunosuppressive and antibiotic treatments are administered [50]. This finding suggests that increased oviduct-derived lytic activity by antimicrobials could be detrimental to fertility by increasing access of immune response mediators to the embryo proper.

The oviductal environment of genistein-treated mice alters preimplantation embryo development by increasing the cleavage rate (after an initial delay in fertilization) and altering the TE:ICM ratio. Of note, embryos from genistein-treated mice do not cleave more quickly than embryos from controls if flushed from the oviduct at the 1-cell stage and cultured in vitro [2]. Genistein-induced increases in activity of any of the numerous embryotropic factors present in the oviductal environment could account for these developmental differences. For example, granulin precursor (previously acrogranin) is secreted by both the embryo and the reproductive tract epithelium and promotes preimplantation embryo cleavage, trophectoderm cell proliferation, and cavitation [5153]. SLPI prevents elastase-mediated granulin precursor proteolysis, which would theoretically increase granulin precursor's downstream effects. The increased expression of SLPI in oviducts of genistein-treated mice could therefore contribute to the faster embryo cleavage rate and altered TE:ICM ratio. Although these alterations were subtle and did not affect full term development after embryo transfer, it is possible that they could have long-term effects on offspring health as has been observed in other models of altered preimplantation embryo development [5456].

The findings reported here raise the possibility that exposure to low levels of estrogenic environmental chemicals or to phytoestrogens during sensitive developmental windows can alter the balance between FRT mucosal immunity and FRT support of fertilization and preimplantation embryo development. In humans, the late first trimester and full second trimester of pregnancy is the time window of FRT development when regional and cellular differentiation occurs in a fashion similar to that in the neonatal period in the mouse [57, 58]. However, in humans FRT cellular differentiation is much more protracted because it continues through the onset of puberty [58, 59]. As a result, estrogenic chemical exposures to the female fetus, infant, child, and adolescent all have the potential to impact FRT mucosal immunity and fertility, and limiting such exposures could improve female reproductive health.

ACKNOWLEDGMENT

We thank Donald Cook and Joy Winuthayanon (NIEHS) for critical reading of the manuscript, Elizabeth Ney (NIEHS) for graphics support, and Grace Kissling (NIEHS) for assistance with statistical analysis.

Footnotes

1Supported by the Intramural Research Program of the National Institutes of Health, National Institutes of Environmental Health Sciences grant Z01-ES102405.

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