Temporal and regional intestinal changes in permeability, tight junction, and cytokine gene expression following ovariectomy‐induced estrogen deficiency

Abstract Estrogen deficiency that occurs during menopause is associated with wide‐ranging consequences, including effects on the gastrointestinal system. Although previous studies have implicated a role for estrogen in modulating colonic permeability and inflammatory gene expression, the kinetics of these changes following loss of estrogen and whether they are intestinal region specific are unknown. To test this, we performed sham or ovariectomy (OVX) surgery in BALB/c mice and examined permeability (in vivo and ex vivo) and gene expression changes in the duodenum, jejunum, ileum, and colon at 1, 4, and 8 weeks postsurgery. In vivo permeability, assessed by FITC‐dextran gavage and subsequent measures of serum levels, indicated that OVX significantly increased whole intestinal permeability 1 week postsurgery before returning to sham levels at 4 and 8 weeks. Permeability of individual intestinal sections, measured ex vivo by Ussing chambers, revealed specific regional and temporal responses to OVX, with the most dynamic changes exhibited by the ileum. Analysis of gene expression, by qPCR and by mathematical modeling, revealed an OVX‐specific effect with tight junction and inflammatory gene expression elevated and suppressed with both temporal and regional specificity. Furthermore, ileal and colonic expression of the tight junction protein occludin was found to be significantly correlated with expression of TNF α and IL‐1β. Together, our studies reveal previously unappreciated effects of estrogen deficiency in specific intestinal segments and further demonstrate temporal links between estrogen deficiency, inflammatory genes, and intestinal permeability.


Introduction
Natural menopause, the permanent cessation of the menstrual cycle following the loss of ovarian follicular activity, is a process that typically occurs in women between 47 and 52 years of age (Davis et al. 2015). Menopause causes a rapid decline in circulating estrogen, which can have a detrimental effect on reproductive and nonreproductive tissues resulting in osteoporosis, urogenital atrophy, metabolic changes leading to muscle atrophy, lower basal metabolic rate and redistribution of body fat, increased risk of stroke, cognitive decline, and increased risk of depression (Burger et al. 1999;Khosla et al. 2011;Davis et al. 2012;Lisabeth and Bushnell 2012;Portman and Gass 2014;Weber et al. 2014). Menopause also increases serum levels of proinflammatory cytokines, including tumor necrosis factor a (TNFa), interleukin-1b (IL-1b), IL-8, and IL-6, as well as the osteoclastogenic cytokine receptor activator of nuclear factor-jB ligand (RANKL) (Jilka et al. 1992;Jabbar et al. 2011;Malutan et al. 2014). In addition, loss of estrogen is associated with intestinal changes that include decreased calcium absorption and changes in intestinal permeability which suggest that estrogen deficiency has direct effects on gastrointestinal homeostasis (Heaney et al. 1989;Li et al. 2016).
Ovariectomy (OVX), removal of the ovaries, is the gold standard murine model of estrogen deficiency (Jee and Yao 2001). While OVX causes a rapid loss of estrogen, similar to hysterectomy, the results produced using this model mirror the endpoints observed during menopause. Like menopause, ovariectomized mice exhibit a period of rapid bone loss (Jee and Yao 2001), urogenital atrophy (Ohlsson et al. 2014), and redistribution of body fat (Babaei et al. 2010;Davis et al. 2015). Thus, OVX provides a good model for understanding the effects of estrogen loss on organ function. The OVX model is routinely performed in adult animals, rather than an aged individual, as a plateau in the rate of bone growth has been reached. While aged animals would be more representative of human menopause, complicating factors including age have been shown to have a significant role in the regulation of the intestine and immune system, independent of estrogen loss (Pinchuk and Filipov 2008;Khosla et al. 2011). In rodent models of estrogen deficiency (ovariectomy, OVX), previous studies identified increased colonic permeability in both in vivo and ex vivo assays (Braniste et al. 2009;Li et al. 2016). These changes were associated with decreased expression of tight junction proteins occludin, members of the claudin family, and junction-associated adhesion molecule (JAM) 3 in the colon (Braniste et al. 2009;Li et al. 2016). Furthermore, estrogen receptor-b (ER-b) knockout mice exhibit decreased cell adhesion molecules and a disrupted tight junction in the colon as well as abnormal colon architecture (Wada-Hiraike et al. 2006). Additionally, colonic inflammation is associated with decreased ER-b expression and increased colonic permeability (Looijer-van Langen et al. 2011). These studies suggest that estrogen signaling is important in colonic homeostasis; however, the role of estrogen signaling or the consequence of estrogen deficiency on other intestinal segments is unknown.
Estrogen is also a critical regulator of intestinal immune cell functions, loss of which has been shown to increase expression of proinflammatory cytokines in immune cells isolated from the small intestine (Li et al. 2016). Estrogen receptors ER-a and ER-b are expressed to varying degrees in the gut-associated immune cells (Phiel et al. 2005;Kovats 2015). Signaling through ER-a in CD4 + T cells has been shown to have anti-inflammatory properties inhibiting Th1/Th17 priming (L elu et al. 2011). Correspondingly, ER-a-deficient macrophages and dendritic cells express higher levels of TNFa in response to lipopolysaccharide (Lambert et al. 2004). Together, these studies indicate that estrogen deficiency could lead to local intestinal changes in inflammatory cytokine expression akin to those seen in the blood and bone marrow.
Although previous studies suggest that estrogen deficiency can modulate intestinal permeability and inflammatory gene expression, the development of these changes subsequent to estrogen depletion, and whether the changes occur in the various regions of the intestine or are localized to specific intestinal segments, is not known. Here, we define the kinetics of expression of tight junction and inflammatory genes in the different segments of the small and large intestine following estrogen deficiency. We find that changes in in vivo and ex vivo permeability are time and region dependent, and that expression of tight junction and inflammatory genes are modulated in both temporal and regional specificity. Using modeling approaches, we also provide models of changes in these genes following estrogen deficiency. These findings reveal that care should be taken when extrapolating data from single time-point measurements. Furthermore, they demonstrate complex and multidimensional effects on the intestine, consequent to estrogen deficiency while providing targets and regions for future investigation.

Ethical approval
All animal procedures were approved by the Michigan State University Institutional Animal Care and Use Committee and conformed to NIH guidelines.

Animals and experimental design
Female BALB/c mice 11 weeks of age were obtained from The Jackson Laboratory (Bar Harbour, Maine). Mice were allowed to acclimate to animal facility for 1 week prior to start of experiment. Animals were randomly split into two groups: sham control or OVX. For sham and OVX surgeries, mice were anesthetized with isofluorane and a 2 cm lower mid-dorsal incision was made extending through the skin and muscle layers. Ovaries were isolated in both sham and OVX groups; ovaries were removed from the OVX cohort and incision sites closed using surgical staples in both sham and OVX mice. Mice were given Teklad 2019 chow (Madison, WI) and water ad libitum and were maintained on a 12-h light/dark cycle. Mice were sacrificed at 1, 4, and 8 weeks postsurgery.

In vivo permeability
In vivo intestinal permeability was evaluated by measuring paracellular permeability to 4 kDa fluorescein isothiocyanate (FITC)-dextran as described previously (Laukoetter et al. 2007;Lee et al. 2015;Li et al. 2016). Briefly, mice were administered 300 mg/kg FITC-dextran in a total volume of 150 lL by oral gavage. Serum was obtained 4 h after administration by cardiac puncture under isoflurane anesthesia and fluorescence intensity measured with a microplate reader (excitation, 485 nm; emission, 530 nm; Tecan, Morrisville, NC). FITC-dextran concentrations were determined using a standard curve and normalized against time.

Ex vivo Ussing chamber intestinal permeability
Mice were sacrificed at designated time points and proximal segments of the duodenum, jejunum, and ileum and distal colon were removed. Sections were mounted in Lucite chambers and placed in Ussing chambers (Physiologic Instruments, San Diego, CA) exposing mucosal and serosal surfaces to oxygenated (95% O 2 , 5% CO 2 ) Krebs bicarbonate ringer buffer (Sigma, St. Louis, MO). Intestinal sections were not stripped of underlying muscle. Buffer was maintained at 37°C by a heated water jacket and samples were allowed to equilibrate for 30 min. Transepithelial conductance (G t ) was measured by clamping the voltage and recording the change in the short-circuit current (I sc ) following a pulsed command voltage every 20 sec.
For measurements of tissue flux, 4 kDa FITC-dextran (2.2 mg/mL final concentration) was added to the mucosal chamber; 10 kDa rhodamine B isothiocyanate (RITC)dextran (0.55 mg/mL final concentration) was also added to the mucosal chamber and used as a control for tissue integrity. Serosal chamber samples were taken at 0 and 60 min, and fluorescence intensity determined (FITC excitation,485 nm;emission,530 nm;RITC excitation,595 nm;emission,615 nm;Tecan). FITC-dextran/RITCdextran concentrations were determined using a standard curve and FITC-dextran flux in OVX mice normalized against sham mice and reported as fold change.

Intestine RNA analysis
Sections from the proximal duodenum, jejunum, and ileum and the distal colon were frozen and crushed under liquid nitrogen conditions with a Bessman Tissue Pulverizer (Spectrum Laboratories, Rancho Dominguez, CA). RNA was isolated from frozen samples using TriReagent (Molecular Research Center, Cincinnati, OH) and integrity assessed by formaldehyde-agarose gel electrophoresis. cDNA was synthesized by reverse transcription using Superscript II Reverse Transcriptase Kit and oligo dT (12-18) primers (Invitrogen, Carlsbad, CA). cDNA was amplified by quantitative qPCR with iQ SYBR Green Supermix (BioRad, Hercules, CA), and gene-specific primers (synthesized by Integrated DNA Technologies, Coralville, IA; Table 1). Hypoxanthine guanine phosphoribosyl transferase (HPRT) mRNA levels were used as an internal control. Real-time PCR was carried out for 40 Table 1. qPCR primers.

Gene
Forward primer (5 0 -3 0 ) Reverse primer (5 0 -3 0 ) cycles using the iCycler (Bio-Rad) and data evaluated using the iCycler software. Each cycle consisted of 95°C for 15 sec, 60°C for 30 sec, and 72°C for 30 sec. Negative controls included primers without cDNA. Expression of all genes was normalized to 1 week sham duodenum.

Statistical analysis
Significant outliers were removed using the Grubbs' test for outliers. The data were transformed to fold change relative to week 1 expression levels in the duodenum to allow determination of relative gene expression across the length of the intestine. Hierarchical clustering was performed using Euclidean pairwise distance metrics for temporal expression and correlation pairwise distance metrics for gene expression. Distance metrics for generating clusters were computed using the furthest distance between each cluster. Dendrograms were then computed from the clustered data for both gene expression and time points. Heat maps were designed to cluster together tight junctions/inflammatory cytokines that have similar changes in gene expression. Furthermore, the heat maps were designed to cluster time points based on similarity; therefore, depending on the outcomes, the different heat maps have different timings. To compute the OVX effect, the P values from the paired t-tests comparing control versus ovariectomy data were transformed to log10 values, and nonparametric, smoothing splines were computed from the data.

Effect of sham and OVX on general body parameters
In humans, loss of ovarian function during menopause is associated with a rapid increase in adipose tissue mass, redistribution of fat to the abdomen, and decreased uterine size (Merz et al. 1996;Davis et al. 2012). We examined these parameters in the murine OVX model of estrogen deficiency at 1, 4, and 8 weeks following surgery (Table 2). Although at 1 week postsurgery, there was no significant difference in body weight between OVX and sham groups, at 4 and 8 weeks postsurgery, body weights were significantly higher in the OVX (22.4 AE 0.6 g and 21.6 AE 0.4 g, respectively) compared to the sham cohort (20.7 AE 0.4 g and 20.2 AE 0.3 g, respectively). Inguinal and retroperitoneal fat pad weights in OVX mice decreased at 1 week, but increased at 8 weeks postsurgery. Specifically, at 1 week postsurgery in OVX versus sham mice the inguinal fat mass displayed a 42% decrease (104 AE 15 mg vs. 177 AE 13 mg, respectively) and the retroperitoneal fat mass displayed a 58% decreased (14 AE 3 mg vs. 33 AE 3 mg, respectively). By 4 weeks postsurgery, there were no differences in fat pad weights. In contrast, by 8 weeks postsurgery, the OVX inguinal fat mass was increased by 27% (150 AE 12 mg vs. 118 AE 8 mg, respectively) and the retroperitoneal fat mass was increased by 32% (65 AE 4 mg vs. 47 AE 4 mg, respectively) over sham. These changes in fat mass were present even when corrected for body weight, with the exception of the inguinal fat at 8 weeks (data not shown).
As expected, uterine weights were significantly decreased in OVX animals (compared to sham) as early as 1 week postsurgery, which persisted throughout all the time points tested.

Temporal changes in intestinal permeability following OVX
To systematically establish the onset of changes and longterm effects of OVX on intestinal barrier function, we examined in vivo intestinal permeability (as a measure of overall gut permeability) as well as region-specific ex vivo permeability of duodenum, jejunum, ileum, and colon at 1, 4, and 8 weeks following surgery. At 1 week postsurgery, a significant increase in in vivo intestinal permeability was observed in the OVX cohort compared to the sham controls (Fig. 1A). Interestingly, at 4 and 8 weeks postsurgery, no significant differences in in vivo intestinal  Significantly increased permeability was detected in the OVX ileum at 1 week (P < 0.05) and the OVX duodenum at 4 weeks (P < 0.05). A significant decrease in permeability was identified in the OVX ileum 8 weeks postsurgery (P < 0.05), while permeability in the OVX colon trended lower across the time course. Representative results for the (C) fold change in intestinal section transepithelial conductance (G t ) for the duration of FITC-dextran measurement in the Ussing chambers. Mean baseline sham G t recorded as: duodenum, 4.02; jejunum, 4.8; ileum, 5.9; and colon, 4.8 mS/cm 2 . Statistical analysis performed by multiple t-test corrected for multiple comparisons by the Holm-S ıd ak method.
permeability were observed between sham and OVX mice, suggesting that estrogen deficiency affects intestinal permeability only at early onset, and that by 4 weeks permeability is restored to sham levels. Analysis of regionspecific permeability, by assaying individual gut sections ex vivo, revealed both temporal and regional differences (Fig. 1b). One week postsurgery, only the ileum exhibited a significant increase in permeability in OVX compared to sham. Other segments, at this point, did not show any marked differences except for a modest decrease in the OVX colon (P = 0.08). Interestingly, 4 weeks after surgery, permeability was significantly increased only in the OVX duodenum (P < 0.01) with no significant differences observed in the jejunum, ileum, or colon. By 8 weeks postsurgery, there was a significant decrease in permeability in the OVX compared to sham ileum, while other sections did not have any differences in permeability. ANOVA comparing the OVX and sham intestinal sections revealed that OVX had a significant effect on duodenal (P < 0.05) and colonic permeability (P < 0.01) across the time course. Transepithelial conductance (G t ) was recorded as a measure of tissue integrity, no significant change in G t was observed in any of the segments during the time period of permeability measurements (Fig. 1C). Together, these data suggest that changes in ileal permeability early after OVX surgery is likely responsible for the observed increase in in vivo overall gut permeability in the estrogen-deficient mice. These results also demonstrate that the overall changes in intestinal permeability are not only time-dependent following estrogen deficiency, they are dependent on specific intestinal segments.

Estrogen deficiency alters tight junction protein gene expression
Intestinal paracellular permeability and barrier function are controlled through the expression of numerous tight junction proteins including members of the claudin family, occludin, the junctional adhesion molecules (JAM) family, and zonula occludens (ZO) ( Fig. 2; Suzuki 2013). Therefore, we measured gene expression of tight junction proteins in the various segments of the intestine to determine whether estrogen deficiency alters intestinal permeability through modulation of the tight junction. Tight junction gene profiles were first organized using hierarchical clustering. Figure 3 shows the clustered expression levels, relative to week 1 expression in the duodenum of the sham control, for the nine tight junction protein genes and the four time points, intact (0) to 8 weeks postsurgery (8), in each of the gut sections for sham and OVX mice. In response to surgery, changes in tight junction gene expression were observed across all regions of the intestine. Large increases in relative gene expression of occludin, claudin-3, and JAM3 were observed in both the sham and OVX in all regions relative to the sham 1 week duodenum. Modest changes in gene expression were detected for the clusters: claudin-1, claudin-4, claudin-5, and ZO-1; and claudin-2 and claudin-8 in the duodenum, jejunum, and ileum. However, in the colon, differing gene clustering patterns and expression were observed in relation to the rest of the intestine. Analysis of temporal clustering identified differences between the intestinal regions and between sham and OVX. However, across all intestinal regions, in both sham and OVX mice, the intact time point was furthest removed except for the sham jejunum. The heat maps reveal that both surgery and OVX have a profound effect on tight junction gene expression throughout the intestine and that these effects are long-lasting. Furthermore, they identify that certain genes are clustered together in the small intestine, whereas the large intestine exhibits a differing response.
To identify the specific effect of OVX over sham surgery on tight junction gene expression, statistical testing was employed (Fig. 4). The results were then related back to the hierarchical analysis and qPCR analysis to  surgery. Intestinal regions were isolated and tight junction gene expression analyzed by qPCR. Statistical testing was used to identify the specific effect of OVX on tight junction gene expression. Graphs represent regional and temporal changes (upregulated and downregulated) in OVX gene expression compared to the sham control. Changes greater than 95% confidence level was determined to be significant. N = 5-11. determine whether OVX resulted in an upregulation or suppression of gene expression over the sham control. In the duodenum, OVX affected expression of occludin, JAM3, and claudin-5, which rose significantly early on in the time course (1 week; CI > 99%), while levels of claudin-3 (CI > 99%) increased at 4 weeks. In contrast, claudin-8 (CI > 95%) was significantly downregulated 4 weeks postsurgery. Interestingly, 8 weeks postsurgery no significant differences in gene expression was observed between the sham and OVX duodenum with the exception of claudin-1, which was significantly lower in the OVX cohort (CI > 95%). A similar pattern in gene expression was observed in the OVX jejunum. Occludin and JAM3 were again significantly (CI > 99%) upregulated at 1 week. In addition, claudin-1, claudin-3, claudin-8 (CI > 99%), and claudin-4 (CI > 95%) were also observed to be elevated, while ZO-1 (CI > 99%) was suppressed. Four weeks postsurgery, occludin was still significantly elevated in the OVX cohort in contrast to claudin-8 expression which was significantly reduced compared to the sham. As with the duodenum, the majority of jejunal tight junction gene expression was comparable between sham and OVX at 8 weeks; however, expression of JAM3 was significantly reduced (CI > 95%) in the OVX mice.
The expression analysis of these various tight junction genes reveals that OVX has an effect over and above that of surgery alone demonstrating that estrogen deficiency profoundly affects tight junction genes in both a temporal and intestinal segment-specific manner. Furthermore, the data also suggest that changes in the expression of some of these tight junction genes track with changes in permeability (especially ileum), while in some segments (especially colon), the relationship between the expression of these tight junction genes and permeability is not as clear.

Changes in intestinal pro-and antiinflammatory cytokine expression following estrogen deficiency
Cytokines are known to play an important role in the regulation of tight junction protein expression under pathological conditions (Lee 2015). While estrogen deficiency increases expression of proinflammatory cytokines in the blood and bone marrow, the local effect on the regions of gut remains less clear. Therefore, we analyzed regional intestinal inflammatory gene expression to determine whether estrogen deficiency-induced changes in pro-and anti-inflammatory genes can be correlated with observed changes in temporal and regional tight junction gene expression and intestinal permeability (Fig. 5).
As with the tight junction proteins, inflammatory cytokine gene expression and time points were organized via hierarchical clustering for the different intestinal segments (duodenum, jejunum, ileum, and colon) in the sham and OVX mice (Fig. 6). In some cases, cytokine gene expression levels were up-and downregulated throughout the intestine in both sham and OVX, suggesting that surgery itself can influence intestinal cytokine expression levels. These changes, however, were region specific as no consistent clustering of genes between sections was observed, except for IFNc which was separate from the other cytokines. Members of the IL-10 family (IL-10 and IL-22) tended to be increased in the ileum and the colon throughout the experimental time course, with some exceptions observed in the OVX mice. Interestingly, in relation to other proinflammatory cytokines such as IL-17A and IL-1b, TNFa, expression only exhibited modest changes throughout the intestine. Temporal clustering was identified to be region and condition specific with no discernible pattern observed across the whole intestine. However, in the sham duodenum, ileum, and colon and in the OVX jejunum, ileum, and colon, the cytokine gene expression profiles in intact (0) and 8 weeks were observed to cluster together, suggesting that inflammation returns to presurgery levels by 8 weeks. Comparable to the tight junction analysis, both surgery and OVX resulted in both regional and temporal changes in intestinal cytokine gene expression. In contrast to the tight junctions, however, no discernible pattern in gene clustering was observed between the different intestinal sections indicating that the inflammatory response to surgery and OVX is region specific.
We next sought to distinguish the effect of OVX on intestinal cytokine gene expression from that of sham surgery by statistical analysis (Fig. 7), with the results related back to the hierarchical analysis and qPCR analysis and are noted below in detail for both the small and large intestine. Specific time-and site-dependent cytokine changes were observed in the small intestine. In the duodenum, at 1 week post-OVX, expression of IL-17A was significantly elevated (CI > 95%) compared to the sham. Levels of IL-10 and IFNc (CI > 95%) were observed to be elevated at the 4-week time point. Eight weeks postsurgery, no differences in gene expression were observed between the sham and the OVX. Analysis of the jejunum identified even more significant changes in cytokine gene expression. IL-1b, TGFb (CI > 99%), and IL-10 (CI > 95%) were upregulated, while TNFa and IL-23p19 were downregulated 1 week post-OVX surgery. At 4 weeks postsurgery, IL-1b (CI > 99%) and TGFb (CI > 95%) expression were still elevated in addition to increased TNFa and IFNc (CI > 99%). Expression of IL-23p19 was suppressed (CI > 95%). Similar to the duodenum, gene expression between the sham and the OVX was comparable at the 8 week time point. In contrast to the proximal small intestine, the ileum displayed only minor OVX-induced changes in gene expression after 1 week, except for a significant increase in IL-23p19 expression (CI >99%). However, by the 4 week time point, expression of TNFa, IFNc (CI >99%), and IL-1b (CI >95%) were significantly increased, whereas IL-10 (CI >99%) and IL-23p19 (CI >95%) were decreased. Again, no difference was observed at 8 weeks except for decreased IL-23p19 expression (CI > 99%), comparable with the other sections of the small intestine, supporting the concept that with extended time the estrogen deficiency-induced inflammatory processes likely return to control levels in the small intestine.
In the large intestine, significant OVX-induced changes in gene expression were revealed at 1 week for TNFa (CI > 99%), IL-17A, and IFNc (CI > 95%). Four weeks postsurgery, IFNc was still upregulated (CI > 95%) in addition to IL-1b (CI > 99%). Expression of IL-10 (CI > 99%), however, was suppressed. By 8 weeks, while many of the gene changes reverted to normal, the OVX colon still displayed several significant changes: IL-1b expression was reduced, whereas IL-22 expression was elevated by 13-fold (CI >95%). Taken together, gene expression analysis of the temporal-and region-specific cytokines suggest that while the small intestine displays only acute (1 and 4 week) cytokine changes to OVX, the colon exhibits long-term (8 weeks) changes involving IL- 22. Analysis of cytokine gene expression identified that OVX has profound effects on the intestine over surgery, significantly increasing proinflammatory gene expression while suppressing anti-inflammatory gene expression. The data further revealed that 4 weeks postsurgery is a critical time as this is when the largest OVX effects were observed in all sections of the intestine. Interestingly, the data suggest that 8 weeks postsurgery, the initial effects of estrogen depletion on cytokine expression have returned to levels comparable to the sham mice.

Discussion
Loss of estrogen leads to many adverse consequences on both reproductive and nonreproductive tissues, including urogenital atrophy and osteoporosis (Burger et al. 1999;Khosla et al. 2011;Davis et al. 2012;Lisabeth and Bushnell 2012;Portman and Gass 2014;Weber et al. 2014). Historically, the main effect associated with estrogen deficiency on the intestine has been calcium malabsorption (Heaney et al. 1989). Recent studies, however, have suggested that loss of estrogen has a more complex effect on intestinal permeability and proinflammatory cytokine expression (Braniste et al. 2009;Li et al. 2016). However, studies to date have not examined the intestinal changes that occur at the onset of estrogen deficiency. In addition, there are no studies that have determined whether these changes are localized to specific intestinal segments and when these changes begin and how long they persist. In the present study, we demonstrate time-dependent changes in in vivo intestinal permeability following loss of estrogen. We further reveal, ex vivo, that estrogen deficiency results in temporal-and regional-specific changes in permeability, tight junction gene expression, and proand anti-inflammatory cytokine gene expression.
The intestinal epithelium provides a critical physiological barrier between the luminal gut microbiome and the host (Turner 2009). Dysregulation of this barrier allows the permeation of pathogens, toxins, and antigens across the mucosal tissue into the systemic sites which can lead to adverse consequences, as seen in inflammatory bowel disease (IBD), celiac disease, and type I diabetes (Arrieta et al. 2006;Turner 2009;Lee 2015). Estrogen receptor beta (ERb) is the predominant ER type in the intestinal tract, though expression of ERa and the G proteincoupled estrogen receptor (GPER) have also been reported (Choijookhuu et al. 2016). While the direct effect of OVX on intestinal ER expression is not clear, changes in estrogen levels as observed during the menstrual cycle and pregnancy have been observed to modulate ER levels, with elevated estrogen levels corresponding to increased ERb and ERa expression (Choijookhuu et al. 2016). Previous studies on the effects of estrogen deficiency on intestinal permeability have not been consistent. Using mice, Li et al. (2016) showed that in vivo intestinal permeability is increased 4 weeks after OVX surgery. However, in a rat model of OVX, no difference in in vivo permeability was observed after 5 weeks (Cox-York et al. 2015). Our results demonstrate that the onset of enhanced intestinal permeability occurs early after OVX (1 week), but normalizes over time (by 4 and 8 weeks), suggesting that inconsistencies in previous studies may be attributed to studying a single time-point measurement following surgery. The precise difference in time points of enhanced intestinal permeability between ours and Li et al. could be due to different mouse strains (ours was BALB/c vs. C57BL/6J used by Li et al.). Although such strain-specific difference in OVX-induced intestinal permeability has not been reported, differences in BALB/c and C57BL/6 bone density in responses to OVX (Beamer et al. 1996;Bouxsein et al. 2005), susceptibility to organ fibrosis (Walkin et al. 2013), and immune response (Watanabe et al. 2004) have been demonstrated. The current study thus builds upon previous findings by showing that ablation of estrogen modulates in vivo intestinal permeability in a time-dependent manner, and occurs early after induction of estrogen deficiency.
We demonstrate here for the first time that estrogen deficiency induces region-specific effects on intestinal permeability. Furthermore, analyses of regional permeability ex vivo correlated with the overall in vivo permeability. Interestingly, while the permeability status of the duodenum (increased) and colon (decreased) was maintained across the time course, the effects on the jejunum and ileum were more dynamic. The time frame of changes in OVX ileal ex vivo permeability, from increased to decreased (compared to sham), matches that of the in vivo changes. This suggests that, in this model, ileum barrier function may have a critical contribution to overall intestinal permeability. The decreased colonic permeability observed in the current study is contrary to those previously reported (Braniste et al. 2009). However, care should be taken before making direct comparisons. Braniste et al. (2009) used Wistar rats that were ovariectomized for an 11-day period before distal colonic permeability measurements were taken. While the difference could be related to species-specific response to estrogen deficiency, other possibilities exist as well. In the present study, a distal section of the colon was analyzed for permeability. Differences in proximal and distal colon permeability have been observed both by our laboratory (data not shown) and by other groups in rodents (Fihn and Jodal 2001;Busche et al. 2002). This raises the possibility that dynamic changes in permeability occur within as well as between the sections of the intestine.
To decipher the mechanistic basis of changes in intestinal permeability following estrogen deficiency, we investigated expression of the tight junctions. These are multiple protein complexes located at the apical end of the epithelial cells and determine the paracellular permeability to solutes (Suzuki 2013). Four key transmembrane proteins have been identified: occludin, claudins, JAMs, and tricellulin (Ulluwishewa et al. 2011;Suzuki 2013). Intracellular proteins like the zonula occludins (ZO) are crucial to the assembly of the tight junction as they have multiple sites that interact with occludin and members of the claudin family (Fanning et al. 1998). These transmembrane In summary, the results from this study demonstrate for the first time that loss of estrogen has regional and temporal effects on the intestine. We show, in vivo, that intestinal permeability increases early after OVX surgery before returning to sham levels, and ex vivo, the permeability of the different intestinal regions differs. Furthermore, we reveal temporal and regional changes in tight junction gene expression correlating with inflammatory genes and subsequent changes in permeability. These results demonstrate that estrogen deficiency induces a new and altered state in the gastrointestinal system that eventually normalizes for some physiological observations, likely due to complex compensatory mechanisms. Given that estrogen affects almost all physiological processes, our studies indicate that the effects of estrogen deficiency are time dependent in the intestine and single time-point measurements of intestinal function could be misleading.