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. 2023 Nov 6;165(1):bqad163. doi: 10.1210/endocr/bqad163

Classical Estrogen Signaling in Ciliated Epithelial Cells of the Oviduct Is Nonessential for Fertility in Female Mice

Emily A McGlade 1,✉, Kalli K Stephens 2, Sarayut Winuthayanon 3, Prashanth Anamthathmakula 4, Michael J Holtzman 5, Wipawee Winuthayanon 6,✉
PMCID: PMC10658216  PMID: 37942801

Abstract

Ciliary action performs a critical role in the oviduct (Fallopian tube) during pregnancy establishment through sperm and egg transport. The disruption of normal ciliary function in the oviduct affects oocyte pick-up and is a contributing factor to female infertility. Estrogen is an important regulator of ciliary action in the oviduct and promotes ciliogenesis in several species. Global loss of estrogen receptor α (ESR1) leads to infertility. We have previously shown that ESR1 in the oviductal epithelial cell layer is required for female fertility. Here, we assessed the role of estrogen on transcriptional regulation of ciliated epithelial cells of the oviduct using single-cell RNA-sequencing analysis. We observed minor variations in ciliated cell genes in the proximal region (isthmus and uterotubal junction) of the oviduct. However, 17β-estradiol treatment had little impact on the gene expression profile of ciliated epithelial cells. We also conditionally ablated Esr1 from ciliated epithelial cells of the oviduct (called ciliated Esr1d/d mice). Our studies showed that ciliated Esr1d/d females had fertility rates comparable to control females, did not display any disruptions in preimplantation embryo development or embryo transport to the uterus, and had comparable cilia formation to control females. However, we observed some incomplete deletion of Esr1 in the ciliated epithelial cells, especially in the ampulla region. Nevertheless, our data suggest that ESR1 expression in ciliated cells of the oviduct is dispensable for ciliogenesis and nonessential for female fertility in mice.

Keywords: cilia, epithelium, estrogen, estrogen receptor, fertility, oviduct, scRNA-seq

The oviduct, or Fallopian tube in humans, is part of the female reproductive tract structure that connects the ovary to the uterus. The oviduct is the site of sperm migration, fertilization, preimplantation embryo development, and egg/embryo transport to the uterus. Multiciliated (ciliated) epithelial cells are present in the ventricles, the respiratory tract, and the oviduct (1). These ciliated epithelial cells are motile and possess a 9 + 2 axoneme ultrastructure at the ciliary stalk, unlike a 9 + 0 structure in the primary cilium (2). The ciliary beating activity of the ciliated epithelial cells has been postulated for their role in reproduction (3). In women, disruption of ciliary function by infection or cigarette smoking is associated with an increase in the risk of ectopic pregnancy (4, 5). Ectopic pregnancy is a condition in which the embryo implants outside of the uterus, most commonly in the Fallopian tube, and is the leading cause of maternal death during the first trimester (5). Primary ciliary dyskinesia is a disorder that affects the efficiency and synchronicity of ciliary beating and the overall function of the oviduct during pregnancy establishment (6-9). However, other studies indicate that some women with primary ciliary dyskinesia have normal fertility (10). A recent report in mice showed that oocyte pick-up was impaired, whereas sperm and embryo transport were unaffected in the absence of functional motile cilia (11). These studies suggest that ciliary activity may play an important role in oviductal function during early pregnancy, but compensatory mechanisms may exist in the oviduct and could aid in gamete/embryo transport when ciliary function is disrupted.

17β-estradiol (E2), one of the female steroid hormones, exhibits its classical action through estrogen receptors (ESR) α and β (encoded by Esr1 and Esr2 genes, respectively). It has been established that E2 regulates the histoarchitecture and function of the female reproductive tract in mammalian species. In the oviduct, E2 stimulates ciliogenesis and the expression of ciliated cell marker, Forkhead Box J1 (FOXJ1) in rhesus macaques (12), rats (13), and chicks (14). Previous studies reported that Foxj1 expression was detected at postnatal day (PND) 5 and was highly expressed from PND 10 to 20 (15-17) in the mouse oviduct. During PND 5, the treatment of E2 increased the Foxj1 expression and levels of β-tubulin IV (one of the ciliated cell markers) in the oviduct. Cotreatment with an ESR antagonist, ICI182780, abolished E2-mediated Foxj1 and β-tubulin IV levels in ciliated cells (16), suggesting that E2 promotes ciliated cell differentiation in an ESR-dependent pathway. Interestingly, a global deletion of Esr1 (Esr1−/− mouse model) had no effect on cilia formation (16). However, the impact of the loss of Esr1 on ciliary function remains unclear. Global deletion of Esr2 (Esr2−/−; specifically expressed in granulosa cells of the ovary), resulted in a subfertility phenotype due to a folliculogenesis defect (18, 19), but not because of an embryo transport defect in the oviduct (20).

We previously showed that ESR1 in the entire oviductal epithelial cell layer (both secretory and ciliated epithelial cells) was absolutely required for female fertility (21) as Wnt7aCre/+;Esr1f/f female mice had 100% embryo retention in the oviduct (20). Specifically, cilia were significantly longer and beating at lower frequencies compared with control females (20). However, we were not able to distinguish whether the embryo transport defect in Wnt7aCre/+;Esr1f/f female mice was due to a loss of ESR1 in ciliated cells and/or secretory cells. Therefore, in this study, we aimed to assess the classical E2 signaling and its transcriptional profile in the ciliated epithelial cells, as well as functionally test the role of ESR1 directly in ciliated epithelial cells during preimplantation embryo development and embryo transport function in the oviduct.

Materials and Methods

Animals

Animals were maintained at Washington State University (WSU) and University of Missouri-Columbia and were handled according to Animal Care and Use Committee guidelines using approved protocols #6147 and #6151 (WSU) and #38961 and #38927 (University of Missouri-Columbia). Foxj1Cre/+ mice (22) were crossed with Esr1f/f mice (23) to create a conditional deletion of ESR1 from ciliated epithelial cells (Foxj1Cre/+;Esr1f/f). Esr1f/f mice were used as littermate controls for all experiments. Genotyping protocols for Esr1f/f and universal Cre were performed as described (23). C57B6/J mice were purchased from Jackson Laboratory (Stock #:000664, Bar Harbor, ME). Adult mice (approximately 8 to 16 weeks old) were used in this study and had access to water and food ad libitum. The number of animals in each experiment is indicated in the figure legends.

Collection of Oviduct Samples During Estrous Cycle and Early Pregnancy

Vaginal cytology was performed using vaginal wash at ∼08:00 hours using sterile 0.89% normal saline in adult female mice. Vaginal cells collected were blotted and dried on the slide. Samples were incubated in methanol for 5 minutes, then stained in hematoxylin for 30 seconds and rinsed in diH2O to remove excess hematoxylin. Then, samples were dehydrated in 70% and 95% EtOH for 2 minutes each. Samples were then stained in eosin for 30 seconds and dehydrated in 95% and 100% EtOH for 2 minutes each. Vaginal cells were then evaluated using light microscopy (DMi8, Leica Microsystems Inc, Deerfield, IL) to stage the estrous cycle. The mice were then sacrificed, and oviducts were collected approximately at ∼09:00 hours. To collect the oviduct samples during pregnancy, adult C57B6/J females were mated with proven breeder males overnight. The next morning, the presence of copulatory plug was considered 0.5 days post coitus (dpc). Females were euthanized using CO2 asphyxiation and cervical dislocation at 0.5, 1.5, 2.5, and 3.5 dpc. The oviducts were fixed in 10% normal buffered formalin and processed for histological analyses.

Ovariectomy and E2 Supplement

Ovariectomy (OVX) was performed in adult C57B6/J female mice as previously described (24). Approximately 2 weeks after OVX, E2 at a concentration of 0.25 μg in sesame oil was injected subcutaneously. Sesame oil (100 μL) was used as a vehicle (Veh) control. Oviducts were either collected at 2 hours after the injection for single-cell RNA-sequencing (scRNA-seq) analysis or at 24 hours for histological analysis as described in the following section.

Cell Isolation and scRNA-seq Analysis

A total of 4 females per treatment were used and pooled for cell isolation. The oviduct was dissected into distal (infundibulum and ampulla [Inf/Amp]) or proximal (isthmus and uterotubal junction [UTJ] [Isth/UTJ]) regions. Trypsin-EDTA (0.25%, Sigma, T4049) was used for oviductal cell dissociation as previously described (24). A total of 8000 cells/run was used and processed in 10× Chromium Controller using Single Cell 3′ v3 kit (10× Genomics Inc, Pleasanton, CA). Libraries were generated using manufacturer's protocol at the Center for Reproductive Biology, WSU, and then were sequenced at the Genomics & Cell Characterization Core Facility, University of Oregon, using an Illumina NovaSeq 6000, targeting 1 billion reads for the pool in 1 lane, paired-end, and 100-bp read length. Raw data were processed using CellRanger-6.0.1 with mouse reference genome mm10-r-102 for sequence alignment. Web summaries for each sample are shown in Table 1. scRNA-seq data were then processed using similar pipeline, analysis packages, and cutoffs as our previous work (24), except “highly variable genes” was set to 6000 genes.

Table 1.

Summary of data from scRNA-seq analysis for each sample

Sample name Estimate number of cells Total reads Means reads/cell Mean gene/cell Median UMI counts/cell Total detected gene # Sequencing saturation
V2Amp 6857 149M 21 804 2366 5678 20 829 25.6%
V2Isth 7603 122M 16 085 1861 4610 20 635 27.3%
E2Amp 5901 138M 23 395 2465 6011 20 720 27.9%
E2Isth 5569 114M 20 573 1671 3957 20 566 34.7%
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Abbreviations: scRNA-seq, single-cell RNA-sequencing; UMI, unique molecular identifier.

Immunohistochemistry

Samples were deparaffinized with xylene, hydrated in 100%, 95%, and 70% EtOH, and rinsed in automation buffer (AB; containing 945.5 mL diH2O, 0.5 mL Tween-20, and 50 mL Tris-HCl). Heat-induced epitope retrieval was performed using the Decloaking chamber (BioCare Medical, Pacheco, CA) with 1× citrate buffer (10 mM sodium citrate, 0.05% Tween 20, pH 6.0) at 110°C for 15 minutes. Samples were treated with 3% H2O2 against endogenous peroxidase for 15 minutes at room temperature (RT). Samples were then blocked with 10% normal horse serum (NHS) in AB for 20 minutes at RT. Samples were incubated with anti-ESR1 (BioCare Medical, #ACA054C, RRID: AB_2651037) at 1:400, or with anti-acetylated α-tubulin (Ace α-Tub; Sigma-Aldrich, #T6793, RRID: AB_477585) at 1:16,000 in 10% NHS in AB overnight at 4°C, as indicated in Table 2. After rinsing with AB, samples were incubated with horse-anti-mouse secondary antibody (BA-2000, Vector Laboratories, Burlingame, CA) at 1:1000 in 10% NHS in AB for 30 minutes at RT, then Vectastain R.T.U. Elite (Vector Laboratories) for 30 minutes at RT. Next, Chromogen (ImmPACT, Vector Laboratories) was applied for 2 to 5 minutes. Samples were counterstained with hematoxylin for 1 minute and agitated in AB until samples turned blue. Samples were then dehydrated in EtOH, incubated in xylene, and then cover-slipped using Permount. For Ace α-Tub immunohistochemistry (IHC), tissues underwent the same steps for deparaffinization, heat-induced epitope retrieval, 3% H2O2 blocking, dehydration, and mounting. However, samples were blocked with 10% NHS + 1% BSA + 1% milk, as well as an additional blocking step using Avidin D and Biotin for 15 minutes each at RT, before incubating with the primary antibody.

Table 2.

Antibody table

Protein target Name of antibody Manufacturer, catalog no. Species raised in; monoclonal or polyclonal Dilution used RRID
Estrogen receptor α Estrogen Receptor (ER) [1D5] BioCare Medical, #ACA054C Mouse Monoclonal 1:400 AB_2651037
Acetylated α-tubulin Anti-Tubulin, Acetylated Antibody produced in mouse, clone 6-11B-1, ascites fluid Sigma-Aldrich, #T6793 Mouse Monoclonal 1:16,000 AB_477585
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Abbreviation: RRID, research resource identifier.

Breeding Trial

Ciliated Esr1d/d (Foxj1Cre/+;Esr1f/f) females were paired with control (Esr1f/f) males for 6 months. Number of litters and number of pups per litter were recorded. After 6 months, the males were removed and once it was determined the females were not pregnant, the reproductive tract was collected in 10% normal buffered formalin for paraffin embedding for future analyses.

Embryo Collection

Control (Esr1f/f) and ciliated Esr1d/d (Foxj1Cre/+;Esr1f/f) females were mated with proven C57BL/6 wild-type male breeders overnight. Females were euthanized at 3.5 dpc. Oviduct, uterus, and ovary were dissected as 1 piece of tissue and collected in Leibovitz 15 (41 300 070, ThermoFisher Scientific, Carlsbad, CA) media supplement with 1% fetal bovine serum and kept at 37°C. To collect embryos from different regions of the reproductive tract, the uterus was flushed with Leibovitz 15 media using a 24-gauge needle in a culture dish on a temperature-controlled dissecting scope (MZ10f, Leica Microsystems). The oviduct was then separated and flushed in a new dish with a 30.5-gauge blunted needle. The number of embryos from each region was counted and imaged using light microscopy (DMi8, Leica Microsystems).

Cilia Measurements

Images from Ace α-Tub IHC staining was used for cilia analysis. The measurement was performed blind as the phenotypes were masked from the images. Ciliary length was determined using ImageJ software, as previously described (20). A total of 5 areas and 5 ciliated epithelial cells from each area were measured. Hematoxylin and eosin images from infundibulum and ampulla regions from a total of 3 to 6 mice/group were measured.

Statistical Analysis

All graphs represent mean ± standard error of the mean. Individual values from each mouse were included in a scatter plot when applicable. Statistical analysis was performed using GraphPad Prism v8.4.0 for Mac OS X (GraphPad Software, Inc, La Jolla, CA). Statistical significance is considered when P < .05 using 2-tailed unpaired Student t-test with Welch correction for simple comparison or 2-way ANOVA with Sidak multiple comparisons test, unless otherwise indicated.

Results

Hormonal Changes do not Impact the Presence of Acetylated α-tubulin in Ciliated Cells

To understand the impact of hormonal changes on ciliated cells, we determined whether the presence of cilia was impacted by different levels of E2 and progesterone (P4) during the estrous cycle and early pregnancy. IHC analysis of Ace α-Tub, a motile ciliary stalk marker, was performed on oviducts during each stage of the estrous cycle and during early pregnancy. Oviducts were collected at diestrus, proestrus, estrus, and metestrus as well as at 0.5, 1.5, 2.5, and 3.5 dpc. Consistent with a previous report (25), we also found that the cells with Ace α-Tub-positive staining were mostly present in the distal portion (infundibulum and ampulla) of the oviduct, whereas cells at the isthmus region showed minimally positive signal for Ace α-Tub (Fig. 1). We also demonstrated that Ace α-Tub staining was present throughout the infundibulum and ampulla regardless of the estrous cycle (Fig. 1A) or stage of early pregnancy (Fig. 1B). However, we observed that Ace α-Tub distribution and intensity were different between diestrus and metestrus in both ampulla and isthmus regions—visually more intense and denser in diestrus. This suggests that different levels of E2 and P4 during the estrous cycle and early pregnancy may impact the presence of motile cilia in the mouse oviduct.

Figure 1.

Figure 1.

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Expression of Ace α-Tub protein using IHC analysis of the infundibulum, ampulla, and isthmus regions of the oviducts from adult female mice during different stages of the estrous cycle and early pregnancy. Inset: negative control. Representative images from n = 3-4 mice per time point, all scale bars = 50 μm and 10 μm for higher magnification images at the infundibulum region.

Hormone Depletion and Estrogen Replacement has Minimal Impact on the Oviductal Cilia

Previous studies have shown that E2 increases Foxj1 (a ciliogenesis marker) gene expression during postnatal development in the mouse oviduct (16). We also found that loss of classical ESR1 signaling in the entire oviductal epithelium (both ciliated and secretory cells) increased ciliary length (20). To further test whether E2 could modulate changes in ciliated cells of the oviduct during adulthood, we performed an OVX and E2 replacement experiment. Here, oviducts were collected 24 hours following Veh or E2 treatment and stained for Ace α-Tub. We found that levels of Ace α-Tub staining were less intense in E2-treated compared with Veh-treated oviducts (Fig. 2A and 2B). Ciliary length at the infundibulum and ampulla regions was comparable between Veh- and E2-treated oviducts (Fig 2C), suggesting that E2 does not regulate overall ciliary morphology or length in the oviduct during adulthood.

Figure 2.

Figure 2.

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Expression of Ace α-Tub protein in the oviduct of ovariectomized (OVX) mice after the treatment of vehicle (sesame oil) or E2 (0.25 μg/mouse) for 24 hours. (A) Ace α-Tub expression via IHC in the infundibulum, ampulla, and isthmus regions. Representative images from n = 3 mice per treatment, scale bars = 50 μm. (B) Higher magnification of Ace α-Tub IHC images. Scale bars = 10 μm. (C) Ciliary length measured using ImageJ analysis at the infundibulum and ampulla regions in female mice treated with vehicle (Veh) or E2, n = 3 mice per treatment.

E2 Mediates Transcript Levels in Ciliated Epithelial Cells of the Oviduct

Previously, Cerny et al demonstrated the ESR1-dependent transcriptome of mouse oviduct using bulk RNA-seq analysis (26). Studies have shown that steroid hormones, such as E2 or P4, recruit ESR1 (27) and progesterone receptor (28) to the promotor/enhancer regions of target genes in mouse uteri within 1 hour. Therefore, we specifically focused on a 2-hour timepoint for the assessment of changes in the gene expression profile after E2 treatment in the oviduct and chose to evaluate the morphological changes at 24 hours. Here, we assessed whether classical E2 signaling modulates the gene expression profile in oviductal ciliated cells of adult mice using scRNA-seq analysis. In this experiment, OVX mice were treated with vehicle control or E2 for 2 hours. Because we were interested in the ciliated epithelial cell population, other cell types were not included in the analysis. Nevertheless, a separate dataset with all cell types included is readily available, as indicated in the Methods.

Three populations of ciliated cells were detected: ciliated cells of the Inf/Amp region and 2 populations of ciliated cells in the Isth/UTJ (Fig. 3A-3C). Here, we show that these ciliated cell populations express ciliated cell-specific marker, Foxj1, and do not express secretory cell-specific marker, Pax8. Known region-specific markers including Pdxk (Inf/Amp) and Serpina1e (Isth/UTJ) (24) were used to validate these cell populations in each region (Fig. 3D). The expression profiles of ciliated cell-related genes were similar between each cluster (Fig. 3E). The only noticeable differences were in levels of Tubulin α1b (Tuba1b), Foxj1, and Coiled-Coil Domain Containing 153 (Ccdc153) that appeared to be expressed at lower levels in the Isth/UTJ compared with Inf/Amp regions (Fig. 3F). Additionally, ciliated cell-associated genes such as Tuba1b, intraflagellar Transport 46 (Ift46), and MYB Proto-Oncogene Transcription Factor (Myb) were expressed at comparable levels in E2-treated ciliated cells compared with Veh-treated ciliated cells. Tubulin α1a (Tuba1a) was expressed in a small subset of cells at very low levels in both Veh- and E2-treated oviducts (Fig. 3G). Gene ontology terms including negative regulation of microglial cell activation, prostaglandin biosynthetic process, amino acid import across plasma membrane, morphogenesis of an epithelium, and epithelial cell differentiation were enriched in Veh-treated oviducts (Fig. 3H), whereas ureter urothelium development, vitamin D3 metabolic process, regulation of fibroblast growth factor production, glandular epithelial cell differentiation, and regulation of epithelial cell differentiation were upregulated in E2-treated oviducts (Fig. 3I). These findings indicate that transcriptional signatures of ciliated cells in the Inf/Amp are similar to that in Isth/UTJ regions and that E2 has minimal impacts on gene expression of ciliated epithelial cells of the mouse oviduct.

Figure 3.

Figure 3.

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scRNA seq analysis of oviducts collected from ovariectomized females that were treated with Veh or E2 for 2 hours. Only ciliated cells identified using Foxj1+ and Ccdc153+ cell makers were included in the analysis. (A) Three clusters of ciliated cells in the oviduct were separated into (B) region and (C) treatment. (D) Cell-type (Foxj1+, Ccdc153+ = ciliated cells, Pax8 = secretory cells) and region-specific markers (Pdxk = InfAmp, Serpina1e = IsthUTJ) are shown. Dot plots of genes related to ciliated cells and ciliogenesis in each (E) ciliated cell cluster, (F) region, and (G) treatment. Gene ontology analyses of the top 1000 genes enriched in (H) Veh-treated and (I) E2-treated oviducts. (J-H) UMAPs of ciliated cells (Foxj1+) from mice treated with Veh, E2 for 2 hours combined with E2 for 24 hours. Dataset from E2 for 24 hours was reanalyzed from our previously published dataset (24). n = 4 mice/treatment.

Because we observed minimal changes in the transcriptional signature of ciliated epithelial cells after 2 hours of E2 treatment, we combined our previously published E2 24-hour treatment dataset (24) and evaluated whether the impact of E2 on ciliated cells was more pronounced at 24 hours than at 2 hours. As expected, the majority of the transcriptional changes in ciliated cells were observed at 24 hours at a higher level than that of 2 hours of E2 treatment as observed by a shift of cell populations from Veh-treated cells (Fig. 3J-3L). These analyses suggest a time-dependent modulation of transcription in ciliated cells by E2. Note that we did not perform gene ontology analysis in this 24-hour dataset because it was previously reported in our prior work (24).

Role of ESR1 in Ciliated Cells During Pregnancy Establishment

To directly investigate whether classical ESR1 signaling in oviductal ciliated epithelial cells is functionally required during pregnancy establishment, we conditionally ablated Esr1 from ciliated (Foxj1+) cells. We found that ESR1 was specifically ablated ciliated epithelial cells in ciliated Esr1d/d oviducts using ESR1 IHC analysis (Fig. 4), whereas expression of ESR1 in other cell types remained intact. However, we noticed that ESR1 protein remains expressed in the nuclei of some ciliated epithelial cells in the ampulla region, in which some ciliated epithelial cells still showed ESR1 staining. Nevertheless, motile cilia were present in the ESR1-depleted cells. This suggests that ESR1 is dispensable for ciliogenesis.

Figure 4.

Figure 4.

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Validation of ESR1 deletion in ciliated epithelial cells of the oviduct. ESR1 immunohistochemistry in control and Ciliated Esr1d/d oviduct. Oviducts were collected from females at estrus (determined by vaginal smears). Inset: negative control (secondary antibody only). Representative images from n = 3 mice/genotype, scale bars = 10 μm; arrowheads are ESR1-negative ciliated cells.

To evaluate the functional requirement of ESR1 in ciliated epithelial cells for female fertility, we performed a 6-month breeding trial in adult female mice. Control and ciliated Esr1d/d females were mated with control males. There was no significant difference in the number of litters or number of pups per litter per dam produced by ciliated Esr1d/d females compared with controls over the 6-month trial period (Fig. 5A and 5B). Furthermore, a similar number of blastocysts and morula stage embryos were collected from control and ciliated Esr1d/d females at 3.5 dpc (Fig. 5C and 5D). Although some embryos collected from ciliated Esr1d/d females were detected in the oviduct region, we did not identify a significant number of retained embryos, suggesting that there was no embryo transport defect compared with controls (Fig. 5E). As anticipated, serum E2 and P4 levels at 3.5 dpc in control and ciliated Esr1d/d females were comparable (Fig. 5F and 5G). In addition, no significant difference in cilia morphology nor length was observed between ciliated Esr1d/d females and controls (Fig. 5H and 5I). Together, these data suggest that classical E2 signaling through ESR1 in ciliated epithelial cells of the oviduct is not essential for cilia formation, embryo development, embryo transport, and female fertility. However, because of a limitation due to an incomplete deletion of Esr1 in some of the ciliated cells in the ampulla region, we conclude that a loss of ESR1 in the majority of ciliated cells of the oviduct had no impact on female fertility in mice.

Figure 5.

Figure 5.

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The effect of ESR1 deletion in ciliated cells during pregnancy establishment. (A-B) 6-month fertility trial showed no fertility defect in ciliated Esr1d/d mice. Control and ciliated Esr1d/d females were paired with control males for 6 months. Number of litters (A) and pups per litter (B) were quantified. (C) Control and ciliated Esr1d/d females were mated with control males and embryos were collected from the oviduct and uterus at 3.5 dpc. (D) Percentage of blastocysts, morulae, and underdeveloped/fragmented embryos (shown as “< morula”) were quantified. (E) Percentage of embryos retrieved from the oviduct and uterus were quantified. Serum levels of E2 (F) and P4 (G) were measured in control and ciliated Esr1d/d females at 3.5 dpc. (H) Cilia morphology (hematoxylin and eosin staining) and Ace α-Tub levels were observed in control and ciliated Esr1d/d oviducts. (I) Cilia was measured in control and ciliated Esr1d/d oviducts. n = 3-6 mice per genotype.

Discussion

In our previous study, we showed that classical E2 signaling through the exogenous treatment of E2 for 24 hours increased progesterone receptor expression in ciliated epithelial cells of the ampulla region of the oviduct in mice (24). Furthermore, E2 also decreased nuclear ESR1 expression, but increased cytoplasmic ESR1 expression in oviductal epithelial cells (24). However, we found E2 did not affect proliferation of oviductal epithelial cells (ie, no significant changes in Ki67+ cells) (24), unlike the effect of E2 in the uterus (23). In this study, we showed that ciliary length was not impacted by an acute treatment of E2. However, there were slight changes in the overall transcriptional profile of the ciliated cells, especially in the distal region of the oviduct. Ciliated cells at the infundibulum and ampulla regions in mice that were treated with E2 for 2 hours were enriched for epithelial cell differentiation, especially glandular development. This is likely due to an increase in the expression of Foxa2 in ciliated epithelial cells of the oviduct (29), which is a marker for glandular epithelial cell differentiation (30, 31).

In female rhesus macaques, ciliary length increases during the follicular phase (when E2 levels are at the highest) and decreases during the luteal phase (when P4 levels peak) (31). In rabbits, extensive loss of ciliated cells and shortening of ciliary length are observed after OVX (32), suggesting that loss of ovarian hormones dramatically affects ciliated cell morphology. Here, we found that the ciliated cells in mouse oviducts did not drastically change their overall morphology nor length throughout the different stages of estrous cycle. However, we observed a high density (staining intensity of Ace α-Tub) during the diestrus stage. Nevertheless, electron microscopy is necessary to accurately quantify ciliary density in the oviduct. These findings indicate that the impact of E2 on ciliated cell morphology and ciliary length may be species-specific. Wang et al demonstrated that ciliary activity is responsible for the circular movement of cumulus oocyte complexes in the ampulla region of the oviduct at 0.5 dpc, which was postulated to be crucial for the breaking up of cumulus cells surrounding the zygotes (33). In addition, the ciliary beat frequency of ciliated epithelial cells in vivo changes from ∼8 Hz at 0.5 dpc to ∼4 Hz at 2.5 dpc in mice (34). Yet, we found that there was no apparent change in the histoarchitecture of ciliated cells during different stages of early pregnancy at 0.5, 1.5, 2.5, and 3.5 dpc. These data indicate that changes in ciliary activity of the oviduct during early pregnancy was not due to the changes in overall histoarchitecture of the cilia itself, at least in mice.

In our prior study, deletion of Esr1 in all epithelial cells of the oviduct led to an increase in ciliary length in the infundibulum region compared with controls (20). However, in the present study, there was no change in cilia length when Esr1 was exclusively ablated in the ciliated epithelial cells. Our findings are consistent with the previous finding using global Esr1−/− mice (16), indicating that ESR1 was not required for ciliogenesis. Ghosh et al show that, during the development, secretory cells give rise to ciliated epithelial cells of the oviduct (15). As such, if the deletion of Esr1 in the epithelial cells (using Wnt7aCre/+ mouse model) takes place in secretory epithelial cells before the differentiation of ciliated cells, ciliogenesis may be impacted. Unfortunately, the previous study in Esr1−/− did not report the measurement of ciliary length in the oviduct (16). Therefore, we could not test this hypothesis at this time. Furthermore, because the deletion of ESR1 in all epithelial cells impacted cilia length and beat frequency, but not in the ciliated cell specific deletion of ESR1, an E2-regulated paracrine signaling mechanism may exist between ciliated and secretory cells that may affect cilia function in the mouse oviduct. We therefore plan to assess ESR1 function in secretory epithelial cells alone to test this hypothesis.

Species-specific differences in oviductal function, such as ciliated cell morphology and regulation, may contribute to differences in physiological and pathological conditions, such as ectopic pregnancy (embryo implantation outside the uterus, most commonly in the oviduct). We currently lack a viable animal model for studying ectopic pregnancy because ectopic pregnancies are very rare, or nonexistent, in rodent models (35). Clinically, however, ectopic pregnancies occur in ∼2% of pregnancies in women (36). Although mice do not exhibit ectopic pregnancy, proper timing of embryo transport to the uterus is still necessary for a successful pregnancy (37). In mice, embryo transport is dependent on ovarian hormones because OVX prevents oviduct transport of the embryos whereas replacement of E2 and P4 after OVX rescues embryo transport (38). It was previously shown that E2 could increase or decrease the embryo transport rate depending on the dose administered. In rats, E2 accelerates transport rates of the eggs in vivo and ESR antagonist, ICI182,780 inhibited E2-induced acceleration of egg transport (39). Although rare, guinea pigs do sometimes exhibit ectopic pregnancy (40). Ciliary beat frequency in guinea pigs is accelerated by the treatment of E2 and suppressed by ICI182,780 in vitro (41). These data using pharmacological inhibition of ESR1 demonstrated that ESR1 is required for cilia beating activity and that modulation of ESR1 function disrupts embryo transport function of the oviduct. However, the reproductive function of classical E2 signaling through ESR1 specifically in the ciliated cells of the oviduct has not been tested in vivo. Therefore, we generated a conditional knockout mouse model to functionally test the role of ESR1 in ciliated cells of the oviduct. We found that a loss of ESR1 in ciliated epithelial cells did not lead to defective female fertility, fertilization process, nor embryo development or transport. Orihuela et al and others showed that E2 modulated ciliated cell activity of the oviduct through nonclassical estrogen signaling mainly through the regulation of cAMP-PKA and inositol triphosphate signals in rats (39, 42-44). Together, these findings suggest that E2's action in vitro is different than that of in vivo and that the ESR1 signaling pathway in ciliated cells of the oviduct could be dispensable for female reproduction in mice.

In Esr1−/− mice, females are infertile (45). However, it is unclear how distinct cell populations are involved in the function of the oviduct during pregnancy establishment. Our previous studies found that ESR1 in the oviduct is required for pregnancy, specifically that of the epithelial layer. In the absence of ESR1 in all epithelial cells (both ciliated and secretory cells), female mice are infertile partly because of an embryo transport defect and an imbalance of the protease activities (21). However, this epithelium consists of both ciliated and nonciliated secretory cells. Therefore, it is still unclear whether ESR1 in secretory and/or ciliated cells are crucial for reproductive function of the oviduct. Additionally, our recent study showed that a loss of ESR1 in all cell types specifically in the proximal region of the oviduct, including the isthmus and the UTJ, caused 100% embryo retention in the oviduct (37), suggesting the importance of ESR1 in the epithelial, stromal, and muscle cells in the proximal region for embryo transport function. In this study, we showed that a lack of ESR1 in the ciliated epithelial cells of the oviduct had no impact on fertility (Fig. 6). We found that ESR1 was not ablated in all ciliated epithelial cells using this mouse model. It is possible that the histological section contains secretory (peg) cells that were normally wedged between ciliated cells. It is also highly likely that Foxj1 was not present in all ciliated epithelial cell in mouse oviduct as shown at the transcriptional level in Fig. 3D. Therefore, to definitively determine whether ESR1 in ciliated epithelial cells is required for the oviduct function, more studies need to be performed.

Figure 6.

Figure 6.

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Summary of genetic mouse models to evaluate the function of ESR1 in different cell types of the oviduct from others and this present study. Fertility phenotypes showed that a lack of ESR1 in all epithelial cells (Wnt7aCre/+;Esr1f/f) of the oviduct leads to embryo death before the 2-cell stage. Deletion of Esr1 in the proximal region of the oviduct (PgrCre/+;Esr1f/f) leads to embryo retention in the oviduct but does not impact preimplantation embryo development. Deletion of Esr1 only in the ciliated epithelial cells (Foxj1Cre/+;Esr1f/f) does not impact embryo development or transport function of the oviduct. Cells with dark brown nuclei indicate normal expression of ESR1, whereas cells with light nuclei indicate a deletion of ESR1 in specific cell types. Light pink cells, ciliated epithelial cells; purple cells, secretory epithelial cells; yellow cells, fibroblasts; and blue cells, smooth muscle cells. UTJ, uterotubal junction. Made with BioRender.

In our whole oviduct dataset after 2 hours of E2 treatment shown in https://www.winuthayanon.com/genes/ve2h_allclusters/, we found that E2 shifted the transcriptional signature of genes in secretory, stromal, and muscle cells. Therefore, our data point to the importance of the role of ESR1 in the secretory epithelial cells, stroma, and muscle cells of the oviduct that might be crucial for fertilization processes, preimplantation embryo development, and embryo transport function. In the future, we plan to further address the role of ESR1 specifically in these other cell populations in the Wnt7aCre/+;Esr1f/f model to gain a better understanding of how E2/ESR1 tightly regulates these distinct oviductal epithelial cell populations during preimplantation embryo development and embryo transport.

Acknowledgments

We thank Dr. Kenneth Korach at the National Institutes of Health, the National Institute of Environmental health Sciences (NIH/NIEHS) for the Esr1f/f mice.

Abbreviations

AB

automation buffer

dpc

days post coitus

E2

17β-estradiol

ESR

estrogen receptor

IHC

immunohistochemistry

Inf/Amp

infundibulum and ampulla

Isth/UTJ

isthmus and uterotubal junction

NHS

normal horse serum

OVX

ovariectomy

P4

progesterone

PND

postnatal day

RT

room temperature

scRNA-seq

single-cell RNA-sequencing

Veh

vehicle

WSU

Washington State University

Contributor Information

Emily A McGlade, Obstetrics, Gynecology and Women's Health, School of Medicine, University of Missouri, Columbia, MO 65211, USA.

Kalli K Stephens, Obstetrics, Gynecology and Women's Health, School of Medicine, University of Missouri, Columbia, MO 65211, USA.

Sarayut Winuthayanon, Division of Animal Sciences, University of Missouri, Columbia, MO 65211, USA.

Prashanth Anamthathmakula, School of Medicine, University of Missouri-Kansas City, Kansas City, MO 64108, USA.

Michael J Holtzman, Pulmonary and Critical Care Medicine, Department of Medicine, Washington University School of Medicine, St.Louis, MO 63110, USA.

Wipawee Winuthayanon, Obstetrics, Gynecology and Women's Health, School of Medicine, University of Missouri, Columbia, MO 65211, USA.

Funding

This study is supported by the Eunice Kennedy Shriver National Institute of Child Health & Human Development (NIH/NICHD) award numbers R01HD097087 to W.W., R01HD097087-02S1 and F31HD107807 to E.A.M., the NIH/National Heart, Lung, and Blood Institute, award numbers R35HL145242 and Department of Defense TTDA W81XWH2010603 and W81XWH2210281 to M.J.H., and NIH-funded WSU Maximizing Access to Research Careers (MARC) program and Barry Goldwater Scholarship to K.K.S.

Disclosures

M.J.H. is the founder of NuPeak Therapeutics. However, there are no financial or other interests related to the submitted work that could affect or have the perception of affecting the author's objectivity or influence or have the perception of influencing the content of the article. All other authors declare no competing interests.

Data Availability

Original data generated and analyzed during this study are included in this published article and deposited in the public repositories as the following. All analyses in this study were saved in JupyterLab Notebook and deposited on GitHub at https://github.com/winuthayanon/ovx_ve2h. A gene search function for all cell types is available at https://www.winuthayanon.com/genes/ve2h_allclusters/and ciliated-cell-specific genes at https://www.winuthayanon.com/genes/ve2h_cilia/. In addition, the combined 2-hour and 24-hour E2 treatment dataset is available at https://www.winuthayanon.com/genes/ve2_24h_allclusters/. Raw data as fastq files are deposited at Gene Expression Omnibus (GSE244422).

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Data Availability Statement

Original data generated and analyzed during this study are included in this published article and deposited in the public repositories as the following. All analyses in this study were saved in JupyterLab Notebook and deposited on GitHub at https://github.com/winuthayanon/ovx_ve2h. A gene search function for all cell types is available at https://www.winuthayanon.com/genes/ve2h_allclusters/and ciliated-cell-specific genes at https://www.winuthayanon.com/genes/ve2h_cilia/. In addition, the combined 2-hour and 24-hour E2 treatment dataset is available at https://www.winuthayanon.com/genes/ve2_24h_allclusters/. Raw data as fastq files are deposited at Gene Expression Omnibus (GSE244422).

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