Utilization of Different Aquaporin Water Channels in the Mouse Cervix during Pregnancy and Parturition and in Models of Preterm an
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《内分泌学杂志》
Department of Pediatrics (J.A., N.B., J.R.), Vanderbilt University Medical Center, Nashville, Tennessee 37232
Department of Obstetrics and Gynecology (M.S.M.), University of Texas Southwestern Medical Center, Dallas, Texas 75390
Abstract
Biochemical changes of cervical connective tissue, including progressive disorganization of the collagen network and increased water content, occur during gestation to allow for cervical dilatation during labor, but the mechanisms that regulate cervical fluid balance are not fully understood. We examined whether aquaporins (AQPs), a family of membrane channel proteins that facilitate water transport, help mediate fluid balance in the mouse cervix during parturition. Of the 13 known murine AQPs, AQP0–2, 6, 7, 9, 11, and 12 were absent or at the limits of detection. By Northern blot and real-time PCR, AQP3 expression was low in nongravid and mid-pregnancy cervices with peak expression on d 19 and postpartum d 1 (PP1). AQP4 expression was generally low throughout pregnancy but showed a small upward trend at the time of parturition. AQP5 and AQP8 expression were significantly increased on d 12–15 but fell to nongravid/baseline by d 19 and PP1. By in situ hybridization and immunohistochemistry, AQP3 was preferentially expressed in basal cell layers of the cervical epithelium, whereas AQP4, 5, and 8 were primarily expressed in apical cell layers. Females with LPS-induced preterm labor had similar trends in AQP4, 5, and 8 expression to mice with natural labor at term gestation. Mice with delayed cervical remodeling due to deletion of the steroid 5-reductase type 1 gene showed significant reduction in the levels of AQP3, 4, and 8 on d 19 or PP1. Together, these studies suggest that AQPs 3, 4, 5, and 8 regulate distinct aspects of cervical water balance during pregnancy and parturition.
Introduction
THE CERVIX IS composed of approximately 10–15% smooth muscle with an underlying connective tissue stroma composed primarily of collagen fiber bundles interposed with glycosaminoglycan and proteoglycan molecules, the most abundant of which are chondroitin and dermatan sulfate (1, 2, 3). The interaction between collagen fiber bundles and glycosaminoglycans is important for providing cervical structure and mechanical strength to retain the developing fetus during pregnancy. Changes in cervical connective tissue, such as reorganization of the collagen network, increased cervical water content, and a shift in glycosaminoglycan composition with a relative increase in hyaluronic acid and decrease in chondroitin and dermatan sulfate, take place in gestation to allow for cervical dilatation during labor (1, 3, 4). Despite a wealth of information on structural and biochemical changes in the ripening cervix, the mechanisms for cervical water balance have not been fully elucidated. With its hydrophilic properties, the increase in hyaluronic acid and other proteoglycans could partly explain increased cervical water content observed during pregnancy (2, 4, 5, 6, 7). Prostaglandins, which are commonly used to induce cervical ripening and are potent vasoactive mediators, have also been shown to increase hyaluronic acid and water content in the cervix (8, 9, 10, 11). Estrogen and progesterone have significant effects on vascular permeability and tissue edema throughout pregnancy via direct effects on the vessel wall and indirect effects on multiple pathways in the female reproductive tract (12). Relaxin stimulates cervical softening and increased cervical water content in rats, mice (13), and most other species studied (14) and acts via prostaglandin-independent pathways (15). However, relaxin-null mice have decreased water content in the pubic symphysis but not in the cervix (16), suggesting more complex mechanisms for regulation of cervical water content.
Aquaporins (AQPs) are a family of membrane channel proteins that allow selective, rapid transport of water across biological membranes. The AQPs consist of a relatively conserved group of six transmembrane helical domains predicted to form barrel-like channels that function as pores for water transport (17, 18). AQPs exist in plants, bacteria, insects, and among diverse members of the animal kingdom. To date, 13 mammalian AQPs (numbered 0–12) have been identified (17, 18, 19, 20, 21). All 13 are highly permeable to water, whereas AQPs 3, 7, 9, and 10 are also permeable to glycerol and some small solutes and, therefore, have been referred to as aquaglyceroporins (17, 19, 20).
Recent studies show that AQPs 1, 4, and 5 are present in distinct cell types of the peri-implantation uterus at times when the uterus displays edema and hyperemia (22, 23, 24, 25). We also showed that baseline AQP1 expression in the myometrium of ovariectomized mice was enhanced by estrogen treatment of progesterone-primed uteri, along with hormone-induced appearance of AQP1 in the uterine stromal vasculature (23). AQP5 expression in the glandular epithelium was low in response to either hormone alone, but was markedly increased in progesterone-primed, estrogen-stimulated uteri (23). Other studies on the role of AQPs in the female reproductive tract have focused on the uterus, vagina, ovary, and oviduct, but there is no information available on AQPs in the cervix during pregnancy or in the cervical ripening process. We hypothesized that AQPs help regulate fluid balance in the cervix during pregnancy and parturition and that increasing AQP levels would parallel the increase in cervical water content with advancing gestation. Thus, we examined the expression of AQP0–12 in wild-type mice from mid-gestation through the postpartum period. AQP10, the most recent AQP reported in humans, was omitted because it is considered a pseudogene in the mouse (26). The relationship of temporal changes in AQP expression to changes in cervical water content during pregnancy and parturition was evaluated. Cell-specific localization patterns of mRNA and protein were determined for AQPs that were significantly expressed during pregnancy. AQPs with pregnancy-specific expression were also examined in an LPS-induced model of preterm labor. Finally, the role of AQPs was also evaluated in mice with targeted deletion of the steroid 5-reductase type 1 gene, where impaired cervical progesterone catabolism results in failure of cervical ripening despite normal uterine contractility (27). These mice have significant reductions in hyaluronic acid synthesis in the cervix, reduced cervical distensibility, and fail to give birth (7, 27).
Materials and Methods
Animals
Mice were housed in Assessment and Accreditation of Laboratory Animal Care-approved facilities. Adult female CD-1 mice (7–8 wk old; Charles River, Raleigh, NC) were bred with fertile males for timed pregnancies. The morning of copulation plug detection was considered d 1 of pregnancy, with delivery at term typically occurring on the evening of d 19 (d 19.5) or in the early morning hours of gestation d 20. Mice were killed between 0800 and 1000 h on gestation d 12, 15–19, and postpartum d 1 (PP1) using isoflurane inhalational anesthesia and subsequent cervical dislocation. Cervix and lower uterine tissue and various positive control tissues were collected by sharp dissection, snap frozen, and stored at –80 C for later analysis. Cervical and uterine tissues were also collected from 6- to 8-wk-old virgin CD-1 mice for comparison to gravid females.
Mice with targeted deletion of the steroid 5-reductase type 1 (Srd5a1) gene were generated and genotyped as described previously (28). These animals and their wild-type controls were maintained on a mixed genetic background (C57BL6/129SvEv). For these mice, timed matings were carried out from 0800 h until 1300 h. Females were checked at midday for the presence of a vaginal plug (d 1 of pregnancy), with birth occurring in the early hours on d 20. Wild-type C57BL6/129SvEv mothers have completed parturition by the morning of d20 (designated PP1). However, Srd5a1-deficient mice do not give birth on d 20 and are thus still pregnant at this time point (designated d 20). All studies were conducted in accordance with the standards of humane animal care described in the National Institutes of Health "Guide for the Care and Use of Laboratory Animals" using protocols approved by an institutional animal care and research advisory committee.
In one series of experiments, pregnant CD-1 mice were treated on gestation d 15 (n = 8) with a single ip injection of LPS (100 μg/100 μl in PBS; Escherichia coli serotype 0111:B4; Sigma, St. Louis, MO) to induce preterm labor (29, 30). Time to delivery was assessed by Kaplan-Meier survival analysis to determine the time point at which 50% of mice deliver. Pregnant mice were then treated with LPS as above and killed at either 1 h (n = 8) or 8 h (n = 9). Cervical tissue was collected from these mice as well as untreated d 15 (n = 9) and d 19 (n = 8) mice, snap frozen, and stored at –80 C until further analysis.
Determination of tissue water content
The wet weights of adult CD-1 mouse cervical and uterine tissues were separately measured in nongravid (NG) and pregnant females on gestation d 12, 15, 19, and PP1 (n = 6–10 per group). Samples were desiccated at 65–67 C for 3–5 d, and dry weights were obtained. Percent water content was calculated as (wet – dry weight)/wet weight. NG cervical samples deemed to be in estrous phase at the time of dissection were excluded.
RT-PCR
Total RNA was extracted from cervical tissue on gestation d 15, d 19, and PP1 as well as from adult eye, kidney, brain, lung, testis, pancreas, and liver to serve as positive control tissues (TRIzol; Invitrogen, Carlsbad, CA). RNA samples were DNase treated (DNase I; Invitrogen) at room temperature for 15 min, the reaction was terminated at 65 C for 15 min, then the samples were chilled on ice and stored at –80 C for later analysis. Oligo-dT-primed reverse transcription (RT) was performed according to the manufacturer’s recommendations (Superscript II; Invitrogen). For initial expression screening, 2 μl of RT product was amplified by semiquantitative PCR with primers specific for each AQP transcript. General thermocycling conditions included denaturation at 95 C for 5 min; 40 cycles of amplification at 94 C for 30 sec, 50–66 C for 60 sec (depending on AQP), 72 C for 60 sec; then elongation at 72 C for 10 min. AQP-specific primers for AQP0–9 and cycling conditions were adapted from our prior work and published literature (23, 31, 32). Primers for AQP11 and AQP12 were derived from sequence information in public databases: AQP11: 5'-TCTAGCTACCTTCCAGCTCTGC–3' (sense), 5'-AGACACCTTCCACAGAGAAAGC-3' (antisense) (Tm = 60 C), 5'-GGAGCCTGAGTCTGACCAAG-3' (internal) (GenBank accession no. NM_175105); AQP12: 5'-GTCCTTGCTCCTTGTAGAACC-3' (sense), 5'-CTTGGCGTCCACAGAACC-3' (antisense) (Tm = 64 C), 5'-GAATGTGTCCCTCTGTTTCTTTTT-3' (internal) (GenBank accession no. NM_177587). RT-PCR products were visualized in 2% agarose gels then transferred to nylon membranes. Southern hybridization was performed with 32P-labeled internal oligonucleotides to verify the accuracy of target gene amplification.
Real-time PCR (Roche Diagnostics, Indianapolis, IN) was subsequently performed to quantify gene expression levels of select AQPs that were detected by screening. Fresh cervical tissues were obtained from NG and pregnant females on gestation d 12, d 15–19, and PP1 (n = 3–5 pooled cervices per group x three groups) and total RNA was extracted. Oligo-dT-primed RT products (2 μl) were amplified under conditions adapted from above. The concentration of gel-purified RT-PCR products from positive control tissues for AQP3, AQP4, AQP5, AQP8, and the housekeeping gene ribosomal protein L7 (rpL7) was determined (2100 BioAnalyzer; Agilent Technologies, Palo Alto, CA) and serial dilutions (100 pg to 0.1 fg) were made for use as standards in real-time PCR analysis. FastStart PCRs (Roche Diagnostics) were optimized to determine primer and template concentrations, magnesium concentration and resolve melting curve artifacts. Quantification of cervical AQP expression was determined in relation to serial dilutions of target sequence standards and normalized to rpL7 expression (as a loading control) according to the manufacturer’s recommendations (LightCycler software version 3.3). Real-time PCR was also performed on cervices from LPS-treated mice as well as on 5-reductase-type 1 null mice and wild-type controls of the same genetic strain. For these experiments, total RNA obtained from each cervix was analyzed individually rather than pooled (n = 4–6 cervices per condition).
Northern hybridization
For Northern hybridization, poly(A)+ RNA was extracted (Oligotex mRNA Mini Kit; Qiagen Inc., Valencia, CA) from cervical tissue on gestation d 12, d 15–19, PP1, as well as NG cervix (n = 10–16 per time point) and positive control tissues (kidney, AQP3 and AQP4; lung, AQP5; liver, AQP8). Poly(A)+ RNA (0.5 μg) was denatured, separated by formaldehyde-agarose gel electrophoresis, transferred, cross-linked to the membranes by UV irradiation, and stored at 4 C until hybridization. Each AQP cDNA was subcloned into plasmid vectors containing a promoter for SP6 RNA polymerase and used as template for synthesis of antisense 32P-labeled cRNA probes using SP6 polymerase. Northern blots were prehybridized, hybridized, and washed. Hybridization was carried out for 20 h at 68 C in SET (50 mM NaCl, 5 mM EDTA, 10 mM Tris-HCl, pH 8.0), 0.1% SDS, 20 mM phosphate buffer (pH 7.2), 250 μg/ml transfer RNA, 10% dextran sulfate, and 1 x 106 cpm of 32P-labeled cRNA probe per milliliter of hybridization buffer. Hybridization was performed with probes for mouse AQP3, AQP4, AQP5, AQP8, and rpL7. Membranes were stripped by boiling for 5 min in 0.5x SET and 0.1% SDS before subsequent rehybridization. Transcripts were detected by autoradiography, and hybridized bands were quantified by densitometry. AQP results were standardized to rpL7 expression.
In situ hybridization
Based on RT-PCR screening results, AQP3, AQP4, AQP5, AQP7, AQP8, and AQP9 amplification products were cloned into a suitable vector (TOPOII; Invitrogen), and their identity and orientation were determined by T7 or M13-primed sequencing. These cDNAs were used to create 35S-labeled sense and antisense cRNA probes for in situ hybridization. Utero-cervical segments from NG and gravid mice on gestation d 15, d 19, and PP1 were positioned with the appropriate controls on the same glass slides. Positive control tissues included adult kidney (AQP3), brain (AQP4), lung (AQP5), testis (AQP7), and liver (AQP8, AQP9). In situ hybridization was performed as previously described (23, 33). Briefly, 10 μm sections of frozen tissues were thaw mounted onto poly-L-lysine-coated slides and fixed in 4% paraformaldehyde/PBS for 10 min at 4 C. Tissue sections were then acetylated, prehybridized, and hybridized at 45 C for 4 h in buffer containing a 35S-labeled antisense cRNA probe. After hybridization and washing, the slides were incubated with RNase A (20 μg/ml) at 37 C for approximately 15 min. RNase A-resistant hybrids were detected by autoradiography using Kodak NTB2 emulsion (Eastman Kodak, Rochester, NY). Slides were developed after 5- to 30-d exposure periods. Parallel tissue sections hybridized with 35S-labeled sense probes served as negative controls. Sections were briefly poststained with hematoxylin and eosin. Experiments were performed in triplicate with tissues from three to four mice at each time point.
Immunohistochemistry
Goat antihuman polyclonal antibodies against AQPs 3–5 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) and a rabbit antirat AQP8 polyclonal antibody (Calbiochem, La Jolla, CA) were used to examine the cellular localization of AQP proteins. Immunohistochemistry was performed as previously described (23). Briefly, frozen sections of NG and gravid d 15, d 19, and PP1 mouse utero-cervical segments and appropriate controls were thaw mounted onto poly-L-lysine-coated slides, fixed in acetone or 4% paraformaldehyde at 4 C for 10 min, nonspecific staining blocked with 10% nonimmune serum for 10 min, and incubated overnight with primary antibody for either AQP3 (2 μg/ml), AQP4 (1 μg/ml), AQP5 (2 μg/ml), or AQP8 (23 μg/ml) at 4 C according to the manufacturer’s recommendations (Zymed Laboratories, Inc., San Francisco, CA). Slides were then washed, incubated with biotinylated secondary antibody for 10 min, briefly exposed to 0.23% periodic acid to block endogenous peroxidase activity, washed, and exposed to peroxidase substrate under direct visualization to determine maturity of the reaction. Immunoreactive protein was detected as red-brown deposits. Sections were lightly counterstained with hematoxylin. Negative controls were similarly treated, except they were incubated overnight with 10% nonimmune serum without addition of the primary antibody. Experiments were performed in triplicate with tissues from two to three mice at each time point.
Statistical analysis
Water content results and gene expression studies were analyzed by Student’s t test with P < 0.05 considered significant. Data are presented as mean ± SEM.
Results
Tissue water content
Increased cervical water content is present in various species during late gestation in preparation for labor and delivery. Water content in the mouse cervix was significantly increased by d 15 when compared with NG and d 12 and remained constant between d 15 and 19 (Fig. 1). By PP1, cervical water content decreased nearly to the NG baseline. In contrast, uterine water content remained stable throughout gestation.
Selective expression of AQPs during late gestation
Because cervical water content was increased during late gestation, RT-PCR was used to screen for the expression of AQP water channel genes in the cervix during mid- to late-gestation on pregnancy d 15, d 19, and PP1. Results showed the presence of AQP3, AQP5, and AQP8 at all three time points. A faint band was present for AQP4 on d 15 and PP1, but was below the limits of detection on d 19. AQP7 and AQP9 were both present on d 15, but AQP7 bands were very faint at the remaining time points, whereas AQP9 was not detectable on d 19 and PP1. There was no evidence of AQP0, AQP1, AQP2, AQP6, AQP11, or AQP12 expression by Southern hybridization of RT-PCR blots (Fig. 2).
Differential expression of AQP3, AQP4, AQP5, and AQP8 in the cervix from mid-gestation through parturition
To better evaluate the time course of expression of AQP3, AQP4, AQP5, and AQP8 during pregnancy and parturition, Northern hybridization of poly(A)+ RNA and real-time PCR was carried out using cervices from NG and pregnant females on gestation d 12, d 15–19, and PP1 (Fig. 3). A distinct expression pattern for each AQP was evident during pregnancy and the peripartum period. AQP3 was lowest in NG and d-12 cervices, with an upward trend during late gestation and peak expression after parturition on PP1. AQP4 levels remained relatively low throughout gestation with a small incremental trend on d 19 and PP1. AQP5 levels were significantly increased from NG to d 15 and continued an elevated trend throughout mid-gestation. Levels abruptly decreased after d 18, reaching baseline NG levels by PP1. AQP8 levels also showed an upward trend on d 12 and were significantly elevated on d 15. Levels remained elevated over baseline throughout mid-gestation, then displayed a downward trend in mid- to late-gestation, decreasing to NG levels by d 19 and PP1.
Localization of AQP3, AQP4, AQP5, and AQP8 mRNA in the cervical epithelium from mid-gestation through parturition
AQPs that were detected by initial RT-PCR screening were examined by RNA in situ hybridization to determine the cell-specific patterns of gene expression on pregnancy d 15, d 19, and PP1 and in NG mice. Antisense probes to AQP7 and AQP9 failed to detect autoradiographic signals despite extended exposure times and accumulation of hybridization signals in positive control tissues (data not shown). In contrast, specific localization patterns were noted for AQP3, AQP4, AQP5, and AQP8. AQPs were generally localized to the cervical epithelium with low level signals noted for some AQPs in the cervical stroma. AQP3 signal accumulation was similar at all time points except for d 15, which showed slightly less signal intensity than NG, d 19, or PP1 (Fig. 4). AQP3 mRNA expression was highly localized to the basal layer of cells in the stratified squamous cervical epithelium. AQP4 signal accumulation was most prominent in the apical cell layers of cervical epithelium in the NG cervix. Expression was near the limits of detection on d 15, but was again noted in the apical epithelium on d 19 and PP1, although hybridization was less intense than in the NG cervix (Fig. 4). Extended hybridization periods were required to visualize AQP4 expression compared with the other AQPs. AQP5 gene expression was primarily localized in the apical layers of the cervical epithelium but was also present in basal cells. AQP5 signals showed less intensity in NG cervices, with a notable increase in signal accumulation in d-15 and d-19 cervices, particularly in the endocervical canal, and a relative decrease in postpartum tissues (Fig. 5). However, accumulation of AQP5 signals in the stroma was present and most notable in the NG cervix, decreasing with gestation. AQP8 expression was at the limits of detection in NG mice, increased on d 15 and 19 in the apical regions of the cervical epithelium, and decreased postpartum, similar to the pattern of AQP5 expression (Fig. 5). Diffuse, nonspecific localization of AQP8 was also noted in the cervical stroma, but was not as pronounced as AQP5. For each AQP probe, sense slides were negative at sites of specific hybridization.
Localization of AQP3, AQP4, AQP5, and AQP8 protein in the cervix during late gestation and parturition
AQPs detected by in situ hybridization were examined by immunohistochemistry in the cervix of NG and pregnant females on gestation d 15 and 19 and on PP1. Distinct spatial and temporal expression patterns were noted for AQP3, AQP4, AQP5, and AQP8 in the cervical epithelium. AQP3 was present at all time points mainly in the basal cell layers of the epithelium (Figs. 4 and 6). Immunostaining patterns were consistent with cell membrane localization of AQP3 in basal epithelial cells. Immunoreactive AQP4 was present mainly in the apical cell layers in NG, d 19, and PP1 samples, although NG was significantly more intense; staining was minimal on d 15 (Figs. 4 and 6). AQP4 staining was most intense at the apical surface and along mucus-secreting cell surfaces. AQP5 was present at all time points (Figs. 5 and 6). In the NG cervix, light staining was observed predominantly in the basal layers of the epithelium in a pattern suggestive of cell membrane localization, along with scant staining in stromal cells. For d 15, d 19, and PP1, AQP5 staining was prominent in both apical and basal cell layers. For the most apical layer of cells, staining was most intense along the luminal surface. In the remaining cell layers, the staining pattern was more consistent with a cell membrane distribution. AQP8 immunostaining in the NG cervix was similar to AQP5, with light staining in the basal epithelial layers and scant staining noted in the cell membrane of some stromal cells (Figs. 5 and 6). Day-15 cervical tissues had minimal AQP8 expression whereas d 19 and PP1 showed well-demarcated staining in the basal cell layers, in contrast to the apical localization of AQP8 mRNA that was observed by in situ hybridization. AQP8 staining appeared more intense in the cell membranes and cytoplasm of basal layers of the cervical epithelium. Localization of AQP8 in the cytoplasm or in unspecified intracellular organelles has been observed in intestine, liver, testis, airway, kidney, and numerous other tissues (34, 35). The mechanism by which AQP8 facilitates water transport in these systems is unclear.
Altered cervical AQP expression during LPS-induced preterm labor
LPS-induced preterm delivery occurred in 50% of females approximately 8–10 h after a single dose of E. coli LPS (Fig. 7), thus, cervical tissues were collected at 1 and 8 h after LPS injection. Northern analysis and real-time RT-PCR were performed to determine whether this type of artificially induced preterm labor affects the expression of AQP3, AQP4, AQP5, and AQP8 in the cervix (Fig. 8). AQP3 expression was unchanged after 1 or 8 h of LPS treatment when compared with nontreated mice on d 15; an upward trend in expression was noted between d 15 and 19 control cervices, similar to the previous Northern/real-time PCR results in untreated females (Fig. 3). AQP4 expression was also unaffected by LPS exposure. Levels of cervical AQP4 expression in untreated females were similar on d 15 and 19, in agreement with prior results (extended exposure times were required to detect AQP4 by poly(A)+ Northern blot). AQP5 expression was similar in the cervix of d-15 control mice and d-15 mice after 1 h of LPS treatment. Eight hours after LPS exposure, a decreased trend was noted in AQP5 expression, although this did not reach statistical significance. AQP8 levels also showed a decreasing trend at 1 and 8 h after LPS-induced preterm labor on d 15, with the 8-h post-LPS treatment group expressing AQP8 at approximately the same levels as untreated d-19 mothers. Greater changes in AQP levels might have been observed if LPS-treated dams had been evaluated at a later time point. However, significantly fewer mice remained pregnant after longer periods of LPS exposure, in agreement with previous reports (30).
Altered AQP expression in the cervix of mice with delayed cervical ripening
Mice with targeted deletion of the steroid 5-reductase type 1 gene fail to give birth due to impaired progesterone catabolism in the cervix and subsequent failure of cervical ripening (27). Thus, the expression of AQP3, 4, 5, and 8 were examined in these mice to determine whether high local progesterone levels or inhibition of cervical ripening is associated with altered AQP expression in the cervix (Fig. 9). A similar overall pattern of increasing AQP3 and AQP4 expression with advancing gestation was noted in both wild-type and 5-reductase type 1 null mice. However, AQP3 and AQP4 levels were both significantly reduced in the cervix of 5-reductase type 1 null mice on d 20 of gestation, at a time when these AQPs should reach their peak level of expression. There were no differences in AQP5 expression levels between wild-type and 5-reductase-1 null cervices at three representative time points (Fig. 9). In contrast, similar to wild-type, AQP8 expression was appropriately elevated on d 16 in the 5-reductase type 1-deficient mouse; however, AQP8 levels declined prematurely as AQP8 was significantly reduced in the cervix of 5-reductase type 1 null mice on the evening of d 19, at the time of expected delivery. No difference was noted in AQP8 mRNA expression between wild-type and 5-reductase type 1 null cervices on PP1/d 20.
Discussion
Cervical ripening encompasses a variety of biochemical changes in cervical connective tissue, including disorganization of the stromal connective tissue network, shifts in glycosaminoglycan composition, increased cervical water content, and infiltration of inflammatory cells and their mediators (1, 36, 37, 38, 39, 40, 41). To better understand the mechanisms that underlie cervical edema during pregnancy and parturition, we examined the role of AQP water channels in the mouse cervix from mid-gestation to the postpartum period. Our results show that AQP3, AQP4, AQP5, and AQP8 are expressed in the cervix and are primarily localized to the cervical epithelium. AQP3 and AQP5 increase with advancing gestation, with steady levels of AQP3 until PP1, whereas AQP5 is high in mid- to late gestation with peak expression on d 18–19, and returns to NG levels by PP1. AQP4 expression is low throughout gestation with a small upward trend on PP1, similar to the overall pattern of AQP3 expression. AQP8, in contrast, peaks on d 12–15, with steady decline to NG levels by d 19. LPS-stimulated preterm labor on d 15 of pregnancy induced a premature decline in AQP8 expression, similar to declining expression patterns with advancing gestation. In addition, the expression of AQP3, AQP4, and AQP8 was significantly decreased on d 19 or d 20 in mice with delayed cervical ripening due to deletion of the steroid 5-reductase type 1 gene. Together, these results suggest that AQPs 3, 4, 5, and 8 are involved in fluid homeostasis in the pregnant and peri-partum cervix and facilitate distinct aspects of water transport across the cervical epithelium.
In contrast to the rapid changes that occur in the uterine myometrium in preparation for parturition at term, the cervix undergoes a more gradual process of remodeling during the quiescent period of pregnancy. Cervical softening and increased extensibility can be detected as early as 7–10 d before delivery in the mouse (42, 43, 44) and is recognized as a prolonged process that begins on d 12–13 in rats and during the latter half of pregnancy in women (1, 45, 46). We observed an increase in cervical water content between d 12 and 15 of pregnancy without a concomitant change in uterine water content. An increase in cervical water content was also noted by other investigators between d 12 and 13 of pregnancy in the mouse (47) and between d 13 and 16 of pregnancy in the rat without associated changes in uterine water content (13). Edematous changes of the cervix may be localized, because superficial and deep layers of the bovine cervical stroma have different water content (48) and cervical softening occurs progressively along the longitudinal axis of the cervix in sheep and pigs (49, 50). Our results show an increase in AQP5 and AQP8 expression during this period corresponding to the increase in water content that occurs after d 12 of pregnancy, and are in agreement with numerous studies showing increased cervical softening and extensibility that begins around the mid-point of gestation. Although a causal relationship cannot be established, these findings suggest that early stages of cervical softening in the mouse may be mediated by water flux associated with these two AQP channels. The underlying hydrophilic molecules or oncotic forces that result in increased water accumulation in the mid-gestation cervix are unclear.
Increased cervical water content in late-gestation is due in part to the shift in glycosaminoglycan content resulting in increased concentrations of hyaluronic acid (reviewed in Ref.7) and the large proteoglycan versican (51) that form strong hydrophilic forces in the cervix. Hyaluronic acid is present diffusely throughout cervical connective tissue at baseline; however, immunostaining is stronger in pregnant rather than nonpregnant cervices, particularly around blood vessels, cervical glands, and beneath the cervical epithelium (52). Similar to other species, hyaluronic acid concentration increases in the mouse cervix with the approach of labor and decreases rapidly postpartum (7, 53). Although all three enzymes for hyaluronic acid synthase (HAS) are present, increased hyaluronic acid content appears to be the result of increased HAS2 activity in mice and women and cannot be explained by alterations in metabolism by the hyaluronidase enzymes Hyal1 or Hyal2 (7). AQP3 and AQP4 showed increasing trends (by Northern, quantitative RT-PCR) at the end of pregnancy and in the postpartum cervix, along with continued, albeit declining, expression of AQP5 and AQP8 during this time. It is possible that AQP water channels increase with advancing gestation as the concentration of hyaluronic acid increases, because hyaluronic acid levels are gradually elevated on d 15–17, with a marked increase on d 18 and 19 (7), thereby allowing transport of water into the cervical stroma toward this hydrophilic substance. Alterations in steroid hormones represent one potential mechanism for coregulation of these events because HAS and AQP expression patterns coincide with falling serum progesterone (or rising estrogen) levels in the late-gestation mouse. Progesterone regulates the expression of HAS1 and HAS2 in the mouse cervix (7), and AQP expression in the mouse uterus is influenced by estrogen and progesterone (23, 24). In addition, the expression of HAS2 (7) and AQP3, AQP4, and AQP8 (Fig. 6) is significantly diminished in 5-reductase type 1 knockout mice, where progesterone levels remain high in the cervix and cervical ripening does not occur (7, 27). Thus, AQP expression and hyaluronic acid accumulation are influenced by local progesterone levels within the cervix, resulting in coordinated mechanisms for water movement during the later stages of pregnancy and parturition. Together, these results suggest that AQP3 and AQP4 contribute to the final stages of cervical ripening at the time of delivery and may also play a role in the formation of cervical mucus or facilitate water efflux from the cervical stroma into the cervical lumen as hyaluronic acid decreases and the edematous cervix returns to its NG state postpartum. In contrast, AQP5 and AQP8 might be more responsible for early water influx as cervical softening progresses and hyaluronic acid concentration increases.
The process of labor depends on regulated interactions between anatomic, biochemical, and molecular pathways in the uterus and cervix. In addition to the effects of estrogen and progesterone, components of the inflammatory response are also involved in cervical ripening during term and preterm labor (40, 41, 54). Decreasing trends in AQP5 and AQP8 levels were observed in mice with LPS-induced preterm labor, similar to the patterns of AQP5 and AQP8 expression in mice with natural labor at term. In contrast, AQP3 and AQP4 expression levels were unaffected by LPS treatment. It is possible that the latter AQPs serve a different role, or that levels were unchanged because they are not already elevated on d 15 like AQP5 and AQP8. Alternatively, although induction of preterm labor by LPS has characteristics similar to naturally occurring labor at term, differences in the mechanisms of parturition between physiological and pathological induction of labor may cause cervical changes for one set of AQPs but not the other. LPS treatment on d 15 of pregnancy results in cervical edema, dissociation of collagen fibers, inflammatory cell infiltration, and significant increases in cervical softness (43). Thus, fluid shifts via AQP5 and AQP8 are likely contributors to cervical softening in response to LPS, corresponding to their postulated role in early cervical softening during mid-gestation in normal pregnancy.
The mechanism of action for LPS-induced changes in cervical AQPs is unclear. LPS-induced preterm labor can occur as the result of declining progesterone levels (55), with potential alterations in AQP expression due to shifts in steroid hormone signaling. However, intrauterine infection can also stimulate excessive production of proinflammatory cytokines and prostaglandins resulting in uterine contractions and cervical ripening independent of effects on serum progesterone levels (56). Proinflammatory cytokines impair the expression of AQPs in other tissues (57, 58) but are important mediators of cervical ripening (36, 37, 38, 39, 54). Thus, decreasing trends in AQP5 and AQP8 expression at term gestation or after LPS treatment on d 15 of pregnancy may correspond to cytokine-mediated effects on cervical ripening. Prostaglandins also play important roles in cervical ripening and are an important adjunct for induction of labor (59). Prostaglandins alter cervical extracellular matrix by increasing collagenase activity, increasing hyaluronic acid and versican synthesis, stimulating inflammatory cell recruitment and cytokine synthesis, and promoting local vasodilation (8, 9, 10, 11, 59, 60). The prostaglandin receptor signaling pathways that mediate these effects are not fully defined. The prostaglandin E2 (PGE2) receptors EP2 and EP4 are present in cultured cervical fibroblasts from cycling women, although only EP4 stimulation causes changes in glycosaminoglycan synthesis (61). In pregnant baboons, EP2 is down-regulated in the cervix as gestation progresses, allowing EP3 to mediate PGE2 effects on cervical dilatation, as EP2 and EP3 have opposite effects on adenylate cyclase activity (62). This study also showed increasing cervical EP1 expression with advancing gestation. A study in rats revealed that cervical EP4 mRNA and protein expression both peak on the day of parturition (63). Treating the surgically isolated rat cervix with PGE2 also leads to increased hyaluronic acid concentrations and increased hydration similar to events occurring normally in late gestation (11). Our data are consistent with a potential link between prostaglandin receptor signaling and AQP activity in cervical ripening, leading to increased cervical edema in mid- to late-gestation. At the onset of pregnancy in the mouse, AQP1 is colocalized with EP3, suggesting that these signaling pathways may interact to produce uterine edema at the time of implantation (23). Our current findings also suggest that prostaglandin and AQP functions may be related in the cervix; however, the ontogeny of prostaglandin receptors in the mouse cervix is unknown. Further studies are required to determine the relationship of prostaglandins and AQPs during the cervical remodeling process.
In summary, the present studies support a role for specific AQP water channels during cervical softening as well as the later stages of cervical ripening at the time of delivery. Although reproductive abnormalities have not been reported in mice with targeted deletion of AQP genes, the presence of AQPs in various regions of the female reproductive tract suggest that subtle abnormalities may exist that require further inspection. The development of pharmacological agents to perturb the function of specific AQPs may provide additional therapeutic strategies to help induce cervical ripening or to inhibit the progression of cervical softening in women with threatened preterm delivery.
Footnotes
This work was supported by National Institutes of Health Grants HD43154 (to M.S.M.) and HD40221 (to J.R).
First Published Online September 22, 2005
Abbreviations: AQP, Aquaporin; HAS, hyaluronic acid synthase; NG, nongravid; PGE2, prostaglandin E2; PP1, postpartum d 1; rpL7, ribosomal protein L7; RT, reverse transcriptase.
Accepted for publication September 13, 2005.
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Department of Obstetrics and Gynecology (M.S.M.), University of Texas Southwestern Medical Center, Dallas, Texas 75390
Abstract
Biochemical changes of cervical connective tissue, including progressive disorganization of the collagen network and increased water content, occur during gestation to allow for cervical dilatation during labor, but the mechanisms that regulate cervical fluid balance are not fully understood. We examined whether aquaporins (AQPs), a family of membrane channel proteins that facilitate water transport, help mediate fluid balance in the mouse cervix during parturition. Of the 13 known murine AQPs, AQP0–2, 6, 7, 9, 11, and 12 were absent or at the limits of detection. By Northern blot and real-time PCR, AQP3 expression was low in nongravid and mid-pregnancy cervices with peak expression on d 19 and postpartum d 1 (PP1). AQP4 expression was generally low throughout pregnancy but showed a small upward trend at the time of parturition. AQP5 and AQP8 expression were significantly increased on d 12–15 but fell to nongravid/baseline by d 19 and PP1. By in situ hybridization and immunohistochemistry, AQP3 was preferentially expressed in basal cell layers of the cervical epithelium, whereas AQP4, 5, and 8 were primarily expressed in apical cell layers. Females with LPS-induced preterm labor had similar trends in AQP4, 5, and 8 expression to mice with natural labor at term gestation. Mice with delayed cervical remodeling due to deletion of the steroid 5-reductase type 1 gene showed significant reduction in the levels of AQP3, 4, and 8 on d 19 or PP1. Together, these studies suggest that AQPs 3, 4, 5, and 8 regulate distinct aspects of cervical water balance during pregnancy and parturition.
Introduction
THE CERVIX IS composed of approximately 10–15% smooth muscle with an underlying connective tissue stroma composed primarily of collagen fiber bundles interposed with glycosaminoglycan and proteoglycan molecules, the most abundant of which are chondroitin and dermatan sulfate (1, 2, 3). The interaction between collagen fiber bundles and glycosaminoglycans is important for providing cervical structure and mechanical strength to retain the developing fetus during pregnancy. Changes in cervical connective tissue, such as reorganization of the collagen network, increased cervical water content, and a shift in glycosaminoglycan composition with a relative increase in hyaluronic acid and decrease in chondroitin and dermatan sulfate, take place in gestation to allow for cervical dilatation during labor (1, 3, 4). Despite a wealth of information on structural and biochemical changes in the ripening cervix, the mechanisms for cervical water balance have not been fully elucidated. With its hydrophilic properties, the increase in hyaluronic acid and other proteoglycans could partly explain increased cervical water content observed during pregnancy (2, 4, 5, 6, 7). Prostaglandins, which are commonly used to induce cervical ripening and are potent vasoactive mediators, have also been shown to increase hyaluronic acid and water content in the cervix (8, 9, 10, 11). Estrogen and progesterone have significant effects on vascular permeability and tissue edema throughout pregnancy via direct effects on the vessel wall and indirect effects on multiple pathways in the female reproductive tract (12). Relaxin stimulates cervical softening and increased cervical water content in rats, mice (13), and most other species studied (14) and acts via prostaglandin-independent pathways (15). However, relaxin-null mice have decreased water content in the pubic symphysis but not in the cervix (16), suggesting more complex mechanisms for regulation of cervical water content.
Aquaporins (AQPs) are a family of membrane channel proteins that allow selective, rapid transport of water across biological membranes. The AQPs consist of a relatively conserved group of six transmembrane helical domains predicted to form barrel-like channels that function as pores for water transport (17, 18). AQPs exist in plants, bacteria, insects, and among diverse members of the animal kingdom. To date, 13 mammalian AQPs (numbered 0–12) have been identified (17, 18, 19, 20, 21). All 13 are highly permeable to water, whereas AQPs 3, 7, 9, and 10 are also permeable to glycerol and some small solutes and, therefore, have been referred to as aquaglyceroporins (17, 19, 20).
Recent studies show that AQPs 1, 4, and 5 are present in distinct cell types of the peri-implantation uterus at times when the uterus displays edema and hyperemia (22, 23, 24, 25). We also showed that baseline AQP1 expression in the myometrium of ovariectomized mice was enhanced by estrogen treatment of progesterone-primed uteri, along with hormone-induced appearance of AQP1 in the uterine stromal vasculature (23). AQP5 expression in the glandular epithelium was low in response to either hormone alone, but was markedly increased in progesterone-primed, estrogen-stimulated uteri (23). Other studies on the role of AQPs in the female reproductive tract have focused on the uterus, vagina, ovary, and oviduct, but there is no information available on AQPs in the cervix during pregnancy or in the cervical ripening process. We hypothesized that AQPs help regulate fluid balance in the cervix during pregnancy and parturition and that increasing AQP levels would parallel the increase in cervical water content with advancing gestation. Thus, we examined the expression of AQP0–12 in wild-type mice from mid-gestation through the postpartum period. AQP10, the most recent AQP reported in humans, was omitted because it is considered a pseudogene in the mouse (26). The relationship of temporal changes in AQP expression to changes in cervical water content during pregnancy and parturition was evaluated. Cell-specific localization patterns of mRNA and protein were determined for AQPs that were significantly expressed during pregnancy. AQPs with pregnancy-specific expression were also examined in an LPS-induced model of preterm labor. Finally, the role of AQPs was also evaluated in mice with targeted deletion of the steroid 5-reductase type 1 gene, where impaired cervical progesterone catabolism results in failure of cervical ripening despite normal uterine contractility (27). These mice have significant reductions in hyaluronic acid synthesis in the cervix, reduced cervical distensibility, and fail to give birth (7, 27).
Materials and Methods
Animals
Mice were housed in Assessment and Accreditation of Laboratory Animal Care-approved facilities. Adult female CD-1 mice (7–8 wk old; Charles River, Raleigh, NC) were bred with fertile males for timed pregnancies. The morning of copulation plug detection was considered d 1 of pregnancy, with delivery at term typically occurring on the evening of d 19 (d 19.5) or in the early morning hours of gestation d 20. Mice were killed between 0800 and 1000 h on gestation d 12, 15–19, and postpartum d 1 (PP1) using isoflurane inhalational anesthesia and subsequent cervical dislocation. Cervix and lower uterine tissue and various positive control tissues were collected by sharp dissection, snap frozen, and stored at –80 C for later analysis. Cervical and uterine tissues were also collected from 6- to 8-wk-old virgin CD-1 mice for comparison to gravid females.
Mice with targeted deletion of the steroid 5-reductase type 1 (Srd5a1) gene were generated and genotyped as described previously (28). These animals and their wild-type controls were maintained on a mixed genetic background (C57BL6/129SvEv). For these mice, timed matings were carried out from 0800 h until 1300 h. Females were checked at midday for the presence of a vaginal plug (d 1 of pregnancy), with birth occurring in the early hours on d 20. Wild-type C57BL6/129SvEv mothers have completed parturition by the morning of d20 (designated PP1). However, Srd5a1-deficient mice do not give birth on d 20 and are thus still pregnant at this time point (designated d 20). All studies were conducted in accordance with the standards of humane animal care described in the National Institutes of Health "Guide for the Care and Use of Laboratory Animals" using protocols approved by an institutional animal care and research advisory committee.
In one series of experiments, pregnant CD-1 mice were treated on gestation d 15 (n = 8) with a single ip injection of LPS (100 μg/100 μl in PBS; Escherichia coli serotype 0111:B4; Sigma, St. Louis, MO) to induce preterm labor (29, 30). Time to delivery was assessed by Kaplan-Meier survival analysis to determine the time point at which 50% of mice deliver. Pregnant mice were then treated with LPS as above and killed at either 1 h (n = 8) or 8 h (n = 9). Cervical tissue was collected from these mice as well as untreated d 15 (n = 9) and d 19 (n = 8) mice, snap frozen, and stored at –80 C until further analysis.
Determination of tissue water content
The wet weights of adult CD-1 mouse cervical and uterine tissues were separately measured in nongravid (NG) and pregnant females on gestation d 12, 15, 19, and PP1 (n = 6–10 per group). Samples were desiccated at 65–67 C for 3–5 d, and dry weights were obtained. Percent water content was calculated as (wet – dry weight)/wet weight. NG cervical samples deemed to be in estrous phase at the time of dissection were excluded.
RT-PCR
Total RNA was extracted from cervical tissue on gestation d 15, d 19, and PP1 as well as from adult eye, kidney, brain, lung, testis, pancreas, and liver to serve as positive control tissues (TRIzol; Invitrogen, Carlsbad, CA). RNA samples were DNase treated (DNase I; Invitrogen) at room temperature for 15 min, the reaction was terminated at 65 C for 15 min, then the samples were chilled on ice and stored at –80 C for later analysis. Oligo-dT-primed reverse transcription (RT) was performed according to the manufacturer’s recommendations (Superscript II; Invitrogen). For initial expression screening, 2 μl of RT product was amplified by semiquantitative PCR with primers specific for each AQP transcript. General thermocycling conditions included denaturation at 95 C for 5 min; 40 cycles of amplification at 94 C for 30 sec, 50–66 C for 60 sec (depending on AQP), 72 C for 60 sec; then elongation at 72 C for 10 min. AQP-specific primers for AQP0–9 and cycling conditions were adapted from our prior work and published literature (23, 31, 32). Primers for AQP11 and AQP12 were derived from sequence information in public databases: AQP11: 5'-TCTAGCTACCTTCCAGCTCTGC–3' (sense), 5'-AGACACCTTCCACAGAGAAAGC-3' (antisense) (Tm = 60 C), 5'-GGAGCCTGAGTCTGACCAAG-3' (internal) (GenBank accession no. NM_175105); AQP12: 5'-GTCCTTGCTCCTTGTAGAACC-3' (sense), 5'-CTTGGCGTCCACAGAACC-3' (antisense) (Tm = 64 C), 5'-GAATGTGTCCCTCTGTTTCTTTTT-3' (internal) (GenBank accession no. NM_177587). RT-PCR products were visualized in 2% agarose gels then transferred to nylon membranes. Southern hybridization was performed with 32P-labeled internal oligonucleotides to verify the accuracy of target gene amplification.
Real-time PCR (Roche Diagnostics, Indianapolis, IN) was subsequently performed to quantify gene expression levels of select AQPs that were detected by screening. Fresh cervical tissues were obtained from NG and pregnant females on gestation d 12, d 15–19, and PP1 (n = 3–5 pooled cervices per group x three groups) and total RNA was extracted. Oligo-dT-primed RT products (2 μl) were amplified under conditions adapted from above. The concentration of gel-purified RT-PCR products from positive control tissues for AQP3, AQP4, AQP5, AQP8, and the housekeeping gene ribosomal protein L7 (rpL7) was determined (2100 BioAnalyzer; Agilent Technologies, Palo Alto, CA) and serial dilutions (100 pg to 0.1 fg) were made for use as standards in real-time PCR analysis. FastStart PCRs (Roche Diagnostics) were optimized to determine primer and template concentrations, magnesium concentration and resolve melting curve artifacts. Quantification of cervical AQP expression was determined in relation to serial dilutions of target sequence standards and normalized to rpL7 expression (as a loading control) according to the manufacturer’s recommendations (LightCycler software version 3.3). Real-time PCR was also performed on cervices from LPS-treated mice as well as on 5-reductase-type 1 null mice and wild-type controls of the same genetic strain. For these experiments, total RNA obtained from each cervix was analyzed individually rather than pooled (n = 4–6 cervices per condition).
Northern hybridization
For Northern hybridization, poly(A)+ RNA was extracted (Oligotex mRNA Mini Kit; Qiagen Inc., Valencia, CA) from cervical tissue on gestation d 12, d 15–19, PP1, as well as NG cervix (n = 10–16 per time point) and positive control tissues (kidney, AQP3 and AQP4; lung, AQP5; liver, AQP8). Poly(A)+ RNA (0.5 μg) was denatured, separated by formaldehyde-agarose gel electrophoresis, transferred, cross-linked to the membranes by UV irradiation, and stored at 4 C until hybridization. Each AQP cDNA was subcloned into plasmid vectors containing a promoter for SP6 RNA polymerase and used as template for synthesis of antisense 32P-labeled cRNA probes using SP6 polymerase. Northern blots were prehybridized, hybridized, and washed. Hybridization was carried out for 20 h at 68 C in SET (50 mM NaCl, 5 mM EDTA, 10 mM Tris-HCl, pH 8.0), 0.1% SDS, 20 mM phosphate buffer (pH 7.2), 250 μg/ml transfer RNA, 10% dextran sulfate, and 1 x 106 cpm of 32P-labeled cRNA probe per milliliter of hybridization buffer. Hybridization was performed with probes for mouse AQP3, AQP4, AQP5, AQP8, and rpL7. Membranes were stripped by boiling for 5 min in 0.5x SET and 0.1% SDS before subsequent rehybridization. Transcripts were detected by autoradiography, and hybridized bands were quantified by densitometry. AQP results were standardized to rpL7 expression.
In situ hybridization
Based on RT-PCR screening results, AQP3, AQP4, AQP5, AQP7, AQP8, and AQP9 amplification products were cloned into a suitable vector (TOPOII; Invitrogen), and their identity and orientation were determined by T7 or M13-primed sequencing. These cDNAs were used to create 35S-labeled sense and antisense cRNA probes for in situ hybridization. Utero-cervical segments from NG and gravid mice on gestation d 15, d 19, and PP1 were positioned with the appropriate controls on the same glass slides. Positive control tissues included adult kidney (AQP3), brain (AQP4), lung (AQP5), testis (AQP7), and liver (AQP8, AQP9). In situ hybridization was performed as previously described (23, 33). Briefly, 10 μm sections of frozen tissues were thaw mounted onto poly-L-lysine-coated slides and fixed in 4% paraformaldehyde/PBS for 10 min at 4 C. Tissue sections were then acetylated, prehybridized, and hybridized at 45 C for 4 h in buffer containing a 35S-labeled antisense cRNA probe. After hybridization and washing, the slides were incubated with RNase A (20 μg/ml) at 37 C for approximately 15 min. RNase A-resistant hybrids were detected by autoradiography using Kodak NTB2 emulsion (Eastman Kodak, Rochester, NY). Slides were developed after 5- to 30-d exposure periods. Parallel tissue sections hybridized with 35S-labeled sense probes served as negative controls. Sections were briefly poststained with hematoxylin and eosin. Experiments were performed in triplicate with tissues from three to four mice at each time point.
Immunohistochemistry
Goat antihuman polyclonal antibodies against AQPs 3–5 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) and a rabbit antirat AQP8 polyclonal antibody (Calbiochem, La Jolla, CA) were used to examine the cellular localization of AQP proteins. Immunohistochemistry was performed as previously described (23). Briefly, frozen sections of NG and gravid d 15, d 19, and PP1 mouse utero-cervical segments and appropriate controls were thaw mounted onto poly-L-lysine-coated slides, fixed in acetone or 4% paraformaldehyde at 4 C for 10 min, nonspecific staining blocked with 10% nonimmune serum for 10 min, and incubated overnight with primary antibody for either AQP3 (2 μg/ml), AQP4 (1 μg/ml), AQP5 (2 μg/ml), or AQP8 (23 μg/ml) at 4 C according to the manufacturer’s recommendations (Zymed Laboratories, Inc., San Francisco, CA). Slides were then washed, incubated with biotinylated secondary antibody for 10 min, briefly exposed to 0.23% periodic acid to block endogenous peroxidase activity, washed, and exposed to peroxidase substrate under direct visualization to determine maturity of the reaction. Immunoreactive protein was detected as red-brown deposits. Sections were lightly counterstained with hematoxylin. Negative controls were similarly treated, except they were incubated overnight with 10% nonimmune serum without addition of the primary antibody. Experiments were performed in triplicate with tissues from two to three mice at each time point.
Statistical analysis
Water content results and gene expression studies were analyzed by Student’s t test with P < 0.05 considered significant. Data are presented as mean ± SEM.
Results
Tissue water content
Increased cervical water content is present in various species during late gestation in preparation for labor and delivery. Water content in the mouse cervix was significantly increased by d 15 when compared with NG and d 12 and remained constant between d 15 and 19 (Fig. 1). By PP1, cervical water content decreased nearly to the NG baseline. In contrast, uterine water content remained stable throughout gestation.
Selective expression of AQPs during late gestation
Because cervical water content was increased during late gestation, RT-PCR was used to screen for the expression of AQP water channel genes in the cervix during mid- to late-gestation on pregnancy d 15, d 19, and PP1. Results showed the presence of AQP3, AQP5, and AQP8 at all three time points. A faint band was present for AQP4 on d 15 and PP1, but was below the limits of detection on d 19. AQP7 and AQP9 were both present on d 15, but AQP7 bands were very faint at the remaining time points, whereas AQP9 was not detectable on d 19 and PP1. There was no evidence of AQP0, AQP1, AQP2, AQP6, AQP11, or AQP12 expression by Southern hybridization of RT-PCR blots (Fig. 2).
Differential expression of AQP3, AQP4, AQP5, and AQP8 in the cervix from mid-gestation through parturition
To better evaluate the time course of expression of AQP3, AQP4, AQP5, and AQP8 during pregnancy and parturition, Northern hybridization of poly(A)+ RNA and real-time PCR was carried out using cervices from NG and pregnant females on gestation d 12, d 15–19, and PP1 (Fig. 3). A distinct expression pattern for each AQP was evident during pregnancy and the peripartum period. AQP3 was lowest in NG and d-12 cervices, with an upward trend during late gestation and peak expression after parturition on PP1. AQP4 levels remained relatively low throughout gestation with a small incremental trend on d 19 and PP1. AQP5 levels were significantly increased from NG to d 15 and continued an elevated trend throughout mid-gestation. Levels abruptly decreased after d 18, reaching baseline NG levels by PP1. AQP8 levels also showed an upward trend on d 12 and were significantly elevated on d 15. Levels remained elevated over baseline throughout mid-gestation, then displayed a downward trend in mid- to late-gestation, decreasing to NG levels by d 19 and PP1.
Localization of AQP3, AQP4, AQP5, and AQP8 mRNA in the cervical epithelium from mid-gestation through parturition
AQPs that were detected by initial RT-PCR screening were examined by RNA in situ hybridization to determine the cell-specific patterns of gene expression on pregnancy d 15, d 19, and PP1 and in NG mice. Antisense probes to AQP7 and AQP9 failed to detect autoradiographic signals despite extended exposure times and accumulation of hybridization signals in positive control tissues (data not shown). In contrast, specific localization patterns were noted for AQP3, AQP4, AQP5, and AQP8. AQPs were generally localized to the cervical epithelium with low level signals noted for some AQPs in the cervical stroma. AQP3 signal accumulation was similar at all time points except for d 15, which showed slightly less signal intensity than NG, d 19, or PP1 (Fig. 4). AQP3 mRNA expression was highly localized to the basal layer of cells in the stratified squamous cervical epithelium. AQP4 signal accumulation was most prominent in the apical cell layers of cervical epithelium in the NG cervix. Expression was near the limits of detection on d 15, but was again noted in the apical epithelium on d 19 and PP1, although hybridization was less intense than in the NG cervix (Fig. 4). Extended hybridization periods were required to visualize AQP4 expression compared with the other AQPs. AQP5 gene expression was primarily localized in the apical layers of the cervical epithelium but was also present in basal cells. AQP5 signals showed less intensity in NG cervices, with a notable increase in signal accumulation in d-15 and d-19 cervices, particularly in the endocervical canal, and a relative decrease in postpartum tissues (Fig. 5). However, accumulation of AQP5 signals in the stroma was present and most notable in the NG cervix, decreasing with gestation. AQP8 expression was at the limits of detection in NG mice, increased on d 15 and 19 in the apical regions of the cervical epithelium, and decreased postpartum, similar to the pattern of AQP5 expression (Fig. 5). Diffuse, nonspecific localization of AQP8 was also noted in the cervical stroma, but was not as pronounced as AQP5. For each AQP probe, sense slides were negative at sites of specific hybridization.
Localization of AQP3, AQP4, AQP5, and AQP8 protein in the cervix during late gestation and parturition
AQPs detected by in situ hybridization were examined by immunohistochemistry in the cervix of NG and pregnant females on gestation d 15 and 19 and on PP1. Distinct spatial and temporal expression patterns were noted for AQP3, AQP4, AQP5, and AQP8 in the cervical epithelium. AQP3 was present at all time points mainly in the basal cell layers of the epithelium (Figs. 4 and 6). Immunostaining patterns were consistent with cell membrane localization of AQP3 in basal epithelial cells. Immunoreactive AQP4 was present mainly in the apical cell layers in NG, d 19, and PP1 samples, although NG was significantly more intense; staining was minimal on d 15 (Figs. 4 and 6). AQP4 staining was most intense at the apical surface and along mucus-secreting cell surfaces. AQP5 was present at all time points (Figs. 5 and 6). In the NG cervix, light staining was observed predominantly in the basal layers of the epithelium in a pattern suggestive of cell membrane localization, along with scant staining in stromal cells. For d 15, d 19, and PP1, AQP5 staining was prominent in both apical and basal cell layers. For the most apical layer of cells, staining was most intense along the luminal surface. In the remaining cell layers, the staining pattern was more consistent with a cell membrane distribution. AQP8 immunostaining in the NG cervix was similar to AQP5, with light staining in the basal epithelial layers and scant staining noted in the cell membrane of some stromal cells (Figs. 5 and 6). Day-15 cervical tissues had minimal AQP8 expression whereas d 19 and PP1 showed well-demarcated staining in the basal cell layers, in contrast to the apical localization of AQP8 mRNA that was observed by in situ hybridization. AQP8 staining appeared more intense in the cell membranes and cytoplasm of basal layers of the cervical epithelium. Localization of AQP8 in the cytoplasm or in unspecified intracellular organelles has been observed in intestine, liver, testis, airway, kidney, and numerous other tissues (34, 35). The mechanism by which AQP8 facilitates water transport in these systems is unclear.
Altered cervical AQP expression during LPS-induced preterm labor
LPS-induced preterm delivery occurred in 50% of females approximately 8–10 h after a single dose of E. coli LPS (Fig. 7), thus, cervical tissues were collected at 1 and 8 h after LPS injection. Northern analysis and real-time RT-PCR were performed to determine whether this type of artificially induced preterm labor affects the expression of AQP3, AQP4, AQP5, and AQP8 in the cervix (Fig. 8). AQP3 expression was unchanged after 1 or 8 h of LPS treatment when compared with nontreated mice on d 15; an upward trend in expression was noted between d 15 and 19 control cervices, similar to the previous Northern/real-time PCR results in untreated females (Fig. 3). AQP4 expression was also unaffected by LPS exposure. Levels of cervical AQP4 expression in untreated females were similar on d 15 and 19, in agreement with prior results (extended exposure times were required to detect AQP4 by poly(A)+ Northern blot). AQP5 expression was similar in the cervix of d-15 control mice and d-15 mice after 1 h of LPS treatment. Eight hours after LPS exposure, a decreased trend was noted in AQP5 expression, although this did not reach statistical significance. AQP8 levels also showed a decreasing trend at 1 and 8 h after LPS-induced preterm labor on d 15, with the 8-h post-LPS treatment group expressing AQP8 at approximately the same levels as untreated d-19 mothers. Greater changes in AQP levels might have been observed if LPS-treated dams had been evaluated at a later time point. However, significantly fewer mice remained pregnant after longer periods of LPS exposure, in agreement with previous reports (30).
Altered AQP expression in the cervix of mice with delayed cervical ripening
Mice with targeted deletion of the steroid 5-reductase type 1 gene fail to give birth due to impaired progesterone catabolism in the cervix and subsequent failure of cervical ripening (27). Thus, the expression of AQP3, 4, 5, and 8 were examined in these mice to determine whether high local progesterone levels or inhibition of cervical ripening is associated with altered AQP expression in the cervix (Fig. 9). A similar overall pattern of increasing AQP3 and AQP4 expression with advancing gestation was noted in both wild-type and 5-reductase type 1 null mice. However, AQP3 and AQP4 levels were both significantly reduced in the cervix of 5-reductase type 1 null mice on d 20 of gestation, at a time when these AQPs should reach their peak level of expression. There were no differences in AQP5 expression levels between wild-type and 5-reductase-1 null cervices at three representative time points (Fig. 9). In contrast, similar to wild-type, AQP8 expression was appropriately elevated on d 16 in the 5-reductase type 1-deficient mouse; however, AQP8 levels declined prematurely as AQP8 was significantly reduced in the cervix of 5-reductase type 1 null mice on the evening of d 19, at the time of expected delivery. No difference was noted in AQP8 mRNA expression between wild-type and 5-reductase type 1 null cervices on PP1/d 20.
Discussion
Cervical ripening encompasses a variety of biochemical changes in cervical connective tissue, including disorganization of the stromal connective tissue network, shifts in glycosaminoglycan composition, increased cervical water content, and infiltration of inflammatory cells and their mediators (1, 36, 37, 38, 39, 40, 41). To better understand the mechanisms that underlie cervical edema during pregnancy and parturition, we examined the role of AQP water channels in the mouse cervix from mid-gestation to the postpartum period. Our results show that AQP3, AQP4, AQP5, and AQP8 are expressed in the cervix and are primarily localized to the cervical epithelium. AQP3 and AQP5 increase with advancing gestation, with steady levels of AQP3 until PP1, whereas AQP5 is high in mid- to late gestation with peak expression on d 18–19, and returns to NG levels by PP1. AQP4 expression is low throughout gestation with a small upward trend on PP1, similar to the overall pattern of AQP3 expression. AQP8, in contrast, peaks on d 12–15, with steady decline to NG levels by d 19. LPS-stimulated preterm labor on d 15 of pregnancy induced a premature decline in AQP8 expression, similar to declining expression patterns with advancing gestation. In addition, the expression of AQP3, AQP4, and AQP8 was significantly decreased on d 19 or d 20 in mice with delayed cervical ripening due to deletion of the steroid 5-reductase type 1 gene. Together, these results suggest that AQPs 3, 4, 5, and 8 are involved in fluid homeostasis in the pregnant and peri-partum cervix and facilitate distinct aspects of water transport across the cervical epithelium.
In contrast to the rapid changes that occur in the uterine myometrium in preparation for parturition at term, the cervix undergoes a more gradual process of remodeling during the quiescent period of pregnancy. Cervical softening and increased extensibility can be detected as early as 7–10 d before delivery in the mouse (42, 43, 44) and is recognized as a prolonged process that begins on d 12–13 in rats and during the latter half of pregnancy in women (1, 45, 46). We observed an increase in cervical water content between d 12 and 15 of pregnancy without a concomitant change in uterine water content. An increase in cervical water content was also noted by other investigators between d 12 and 13 of pregnancy in the mouse (47) and between d 13 and 16 of pregnancy in the rat without associated changes in uterine water content (13). Edematous changes of the cervix may be localized, because superficial and deep layers of the bovine cervical stroma have different water content (48) and cervical softening occurs progressively along the longitudinal axis of the cervix in sheep and pigs (49, 50). Our results show an increase in AQP5 and AQP8 expression during this period corresponding to the increase in water content that occurs after d 12 of pregnancy, and are in agreement with numerous studies showing increased cervical softening and extensibility that begins around the mid-point of gestation. Although a causal relationship cannot be established, these findings suggest that early stages of cervical softening in the mouse may be mediated by water flux associated with these two AQP channels. The underlying hydrophilic molecules or oncotic forces that result in increased water accumulation in the mid-gestation cervix are unclear.
Increased cervical water content in late-gestation is due in part to the shift in glycosaminoglycan content resulting in increased concentrations of hyaluronic acid (reviewed in Ref.7) and the large proteoglycan versican (51) that form strong hydrophilic forces in the cervix. Hyaluronic acid is present diffusely throughout cervical connective tissue at baseline; however, immunostaining is stronger in pregnant rather than nonpregnant cervices, particularly around blood vessels, cervical glands, and beneath the cervical epithelium (52). Similar to other species, hyaluronic acid concentration increases in the mouse cervix with the approach of labor and decreases rapidly postpartum (7, 53). Although all three enzymes for hyaluronic acid synthase (HAS) are present, increased hyaluronic acid content appears to be the result of increased HAS2 activity in mice and women and cannot be explained by alterations in metabolism by the hyaluronidase enzymes Hyal1 or Hyal2 (7). AQP3 and AQP4 showed increasing trends (by Northern, quantitative RT-PCR) at the end of pregnancy and in the postpartum cervix, along with continued, albeit declining, expression of AQP5 and AQP8 during this time. It is possible that AQP water channels increase with advancing gestation as the concentration of hyaluronic acid increases, because hyaluronic acid levels are gradually elevated on d 15–17, with a marked increase on d 18 and 19 (7), thereby allowing transport of water into the cervical stroma toward this hydrophilic substance. Alterations in steroid hormones represent one potential mechanism for coregulation of these events because HAS and AQP expression patterns coincide with falling serum progesterone (or rising estrogen) levels in the late-gestation mouse. Progesterone regulates the expression of HAS1 and HAS2 in the mouse cervix (7), and AQP expression in the mouse uterus is influenced by estrogen and progesterone (23, 24). In addition, the expression of HAS2 (7) and AQP3, AQP4, and AQP8 (Fig. 6) is significantly diminished in 5-reductase type 1 knockout mice, where progesterone levels remain high in the cervix and cervical ripening does not occur (7, 27). Thus, AQP expression and hyaluronic acid accumulation are influenced by local progesterone levels within the cervix, resulting in coordinated mechanisms for water movement during the later stages of pregnancy and parturition. Together, these results suggest that AQP3 and AQP4 contribute to the final stages of cervical ripening at the time of delivery and may also play a role in the formation of cervical mucus or facilitate water efflux from the cervical stroma into the cervical lumen as hyaluronic acid decreases and the edematous cervix returns to its NG state postpartum. In contrast, AQP5 and AQP8 might be more responsible for early water influx as cervical softening progresses and hyaluronic acid concentration increases.
The process of labor depends on regulated interactions between anatomic, biochemical, and molecular pathways in the uterus and cervix. In addition to the effects of estrogen and progesterone, components of the inflammatory response are also involved in cervical ripening during term and preterm labor (40, 41, 54). Decreasing trends in AQP5 and AQP8 levels were observed in mice with LPS-induced preterm labor, similar to the patterns of AQP5 and AQP8 expression in mice with natural labor at term. In contrast, AQP3 and AQP4 expression levels were unaffected by LPS treatment. It is possible that the latter AQPs serve a different role, or that levels were unchanged because they are not already elevated on d 15 like AQP5 and AQP8. Alternatively, although induction of preterm labor by LPS has characteristics similar to naturally occurring labor at term, differences in the mechanisms of parturition between physiological and pathological induction of labor may cause cervical changes for one set of AQPs but not the other. LPS treatment on d 15 of pregnancy results in cervical edema, dissociation of collagen fibers, inflammatory cell infiltration, and significant increases in cervical softness (43). Thus, fluid shifts via AQP5 and AQP8 are likely contributors to cervical softening in response to LPS, corresponding to their postulated role in early cervical softening during mid-gestation in normal pregnancy.
The mechanism of action for LPS-induced changes in cervical AQPs is unclear. LPS-induced preterm labor can occur as the result of declining progesterone levels (55), with potential alterations in AQP expression due to shifts in steroid hormone signaling. However, intrauterine infection can also stimulate excessive production of proinflammatory cytokines and prostaglandins resulting in uterine contractions and cervical ripening independent of effects on serum progesterone levels (56). Proinflammatory cytokines impair the expression of AQPs in other tissues (57, 58) but are important mediators of cervical ripening (36, 37, 38, 39, 54). Thus, decreasing trends in AQP5 and AQP8 expression at term gestation or after LPS treatment on d 15 of pregnancy may correspond to cytokine-mediated effects on cervical ripening. Prostaglandins also play important roles in cervical ripening and are an important adjunct for induction of labor (59). Prostaglandins alter cervical extracellular matrix by increasing collagenase activity, increasing hyaluronic acid and versican synthesis, stimulating inflammatory cell recruitment and cytokine synthesis, and promoting local vasodilation (8, 9, 10, 11, 59, 60). The prostaglandin receptor signaling pathways that mediate these effects are not fully defined. The prostaglandin E2 (PGE2) receptors EP2 and EP4 are present in cultured cervical fibroblasts from cycling women, although only EP4 stimulation causes changes in glycosaminoglycan synthesis (61). In pregnant baboons, EP2 is down-regulated in the cervix as gestation progresses, allowing EP3 to mediate PGE2 effects on cervical dilatation, as EP2 and EP3 have opposite effects on adenylate cyclase activity (62). This study also showed increasing cervical EP1 expression with advancing gestation. A study in rats revealed that cervical EP4 mRNA and protein expression both peak on the day of parturition (63). Treating the surgically isolated rat cervix with PGE2 also leads to increased hyaluronic acid concentrations and increased hydration similar to events occurring normally in late gestation (11). Our data are consistent with a potential link between prostaglandin receptor signaling and AQP activity in cervical ripening, leading to increased cervical edema in mid- to late-gestation. At the onset of pregnancy in the mouse, AQP1 is colocalized with EP3, suggesting that these signaling pathways may interact to produce uterine edema at the time of implantation (23). Our current findings also suggest that prostaglandin and AQP functions may be related in the cervix; however, the ontogeny of prostaglandin receptors in the mouse cervix is unknown. Further studies are required to determine the relationship of prostaglandins and AQPs during the cervical remodeling process.
In summary, the present studies support a role for specific AQP water channels during cervical softening as well as the later stages of cervical ripening at the time of delivery. Although reproductive abnormalities have not been reported in mice with targeted deletion of AQP genes, the presence of AQPs in various regions of the female reproductive tract suggest that subtle abnormalities may exist that require further inspection. The development of pharmacological agents to perturb the function of specific AQPs may provide additional therapeutic strategies to help induce cervical ripening or to inhibit the progression of cervical softening in women with threatened preterm delivery.
Footnotes
This work was supported by National Institutes of Health Grants HD43154 (to M.S.M.) and HD40221 (to J.R).
First Published Online September 22, 2005
Abbreviations: AQP, Aquaporin; HAS, hyaluronic acid synthase; NG, nongravid; PGE2, prostaglandin E2; PP1, postpartum d 1; rpL7, ribosomal protein L7; RT, reverse transcriptase.
Accepted for publication September 13, 2005.
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