Failure of Elastic Fiber Homeostasis Leads to Pelvic Floor Disorders
2006; Elsevier BV; Volume: 168; Issue: 2 Linguagem: Inglês
10.2353/ajpath.2006.050399
ISSN1525-2191
AutoresXiaoqing Liu, Yun Zhao, Basil S. Pawlyk, Margot S. Damaser, Tiansen Li,
Tópico(s)Kidney Stones and Urolithiasis Treatments
ResumoPelvic floor disorders, a group of conditions affecting adult women, include pelvic organ prolapse and urinary incontinence. Vaginal childbirth and aging are risk factors, and weakening of the pelvic support structures is a major aspect of the pathology. However, the underlying molecular mechanism remains unknown. Female reproductive organs are rich in elastic fibers that turn over slowly in most adult tissues but undergo massive remodeling in the reproductive organs through pregnancy and birth. Here we show that a failure to maintain elastic fiber homeostasis in mice causes pelvic floor disorders. Lysyl oxidase-like-1 (LOXL1), a protein essential for the postnatal deposition of elastic fibers, was highly expressed and regulated in the reproductive tract of the mouse, and its expression was diminished during aging. LOXL1 deficiency caused an inability of reproductive tissues to replenish elastic fibers after parturition, leading to pelvic organ prolapse, weakening of the vaginal wall, paraurethral pathology, and lower urinary tract dysfunction. These data demonstrate the importance of elastic fibers for maintaining structural and functional integrity of the female pelvic floor. Our findings raise the possibility that a failure of elastic fiber homeostasis, either due to genetic predisposition or advancing age, could underlie the etiology of pelvic floor dysfunction in women. Pelvic floor disorders, a group of conditions affecting adult women, include pelvic organ prolapse and urinary incontinence. Vaginal childbirth and aging are risk factors, and weakening of the pelvic support structures is a major aspect of the pathology. However, the underlying molecular mechanism remains unknown. Female reproductive organs are rich in elastic fibers that turn over slowly in most adult tissues but undergo massive remodeling in the reproductive organs through pregnancy and birth. Here we show that a failure to maintain elastic fiber homeostasis in mice causes pelvic floor disorders. Lysyl oxidase-like-1 (LOXL1), a protein essential for the postnatal deposition of elastic fibers, was highly expressed and regulated in the reproductive tract of the mouse, and its expression was diminished during aging. LOXL1 deficiency caused an inability of reproductive tissues to replenish elastic fibers after parturition, leading to pelvic organ prolapse, weakening of the vaginal wall, paraurethral pathology, and lower urinary tract dysfunction. These data demonstrate the importance of elastic fibers for maintaining structural and functional integrity of the female pelvic floor. Our findings raise the possibility that a failure of elastic fiber homeostasis, either due to genetic predisposition or advancing age, could underlie the etiology of pelvic floor dysfunction in women. Pelvic floor disorders refer to a group of conditions that include pelvic organ prolapse, urinary incontinence, and other sensory and emptying abnormalities of the lower urinary tract. 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It begins as a rule with slight leakage, which gradually grows worse, leading to complete incontinence with all its unfortunate and repellent sequelae."12Kelly HA Dumm WM Urinary incontinence in women without manifest injury to the bladder.Surg Gynecol Obstet. 1914; 18: 444-450Google Scholar Effective closure of the urethra requires the concerted action of various pelvic floor structures in addition to proper function of urethral musculature. It is widely accepted that the suburethral vaginal wall and the paraurethral connective tissues are key factors in maintaining continence.4Keane DP O'Sullivan S Urinary incontinence: anatomy, physiology and pathophysiology.Baillieres Best Pract Res Clin Obstet Gynaecol. 2000; 14: 207-226Abstract Full Text PDF PubMed Scopus (49) Google Scholar, 5Cheater FM Castleden CM Epidemiology and classification of urinary incontinence.Baillieres Best Pract Res Clin Obstet Gynaecol. 2000; 14: 183-205Abstract Full Text PDF PubMed Scopus (85) Google Scholar, 8Ulmsten U Falconer C Connective tissue in female urinary incontinence.Curr Opin Obstet Gynecol. 1999; 11: 509-515Crossref PubMed Scopus (51) Google Scholar Two epidemiological factors most strongly associated with stress urinary incontinence are vaginal childbirth10Thomas TM Plymat KR Blannin J Meade TW Prevalence of urinary incontinence.Br Med J. 1980; 281: 1243-1245Crossref PubMed Scopus (782) Google Scholar, 13Turan C Zorlu CG Ekin M Hancerliogullari N Saracoglu F Urinary incontinence in women of reproductive age.Gynecol Obstet Invest. 1996; 41: 132-134Crossref PubMed Scopus (50) Google Scholar and advancing age.10Thomas TM Plymat KR Blannin J Meade TW Prevalence of urinary incontinence.Br Med J. 1980; 281: 1243-1245Crossref PubMed Scopus (782) Google Scholar Vaginal delivery can injure the nerve, muscle, and connective tissues responsible for maintaining continence.14Retzky SS Rogers Jr, RM Urinary incontinence in women.Clin Symp. 1995; 47: 2-32PubMed Google Scholar, 15Lin AS Carrier S Morgan DM Lue TF Effect of simulated birth trauma on the urinary continence mechanism in the rat.Urology. 1998; 52: 143-151Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar Physiological changes with age also contribute to the development of urinary incontinence,14Retzky SS Rogers Jr, RM Urinary incontinence in women.Clin Symp. 1995; 47: 2-32PubMed Google Scholar, 16Makinen JI Pitkanen YA Salmi TA Gronroos M Rinne R Paakkari I Transdermal estrogen for female stress urinary incontinence in postmenopause.Maturitas. 1995; 22: 233-238Abstract Full Text PDF PubMed Scopus (17) Google Scholar although the detailed mechanism is unclear. Other risk factors may include genetic predisposition, lifestyle, and certain medical conditions.17Weber AM Buchsbaum G Chen B Clark A Damaser M Daneshgari F Davis G DeLancey J Kenton K Weidner AC Word RA Basic science and translational research in female pelvic floor disorders: proceedings of an NIH-Sponsored Meeting.Neurourol Urodyn. 2004; 23: 288-301Crossref PubMed Scopus (51) Google Scholar, 18Hannestad YS Lie RT Rortveit G Hunskaar S Familial risk of urinary incontinence in women: population based cross sectional study.BMJ. 2004; 329: 889-891Crossref PubMed Scopus (92) Google Scholar, 19Koduri S Sand PK Recent developments in pelvic organ prolapse.Curr Opin Obstet Gynecol. 2000; 12: 399-404Crossref PubMed Scopus (19) Google Scholar, 20Casey BM Schaffer JI Bloom SL Heartwell SF McIntire DD Leveno KJ Obstetric antecedents for postpartum pelvic floor dysfunction.Am J Obstet Gynecol. 2005; 192: 1655-1662Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar Pelvic organ prolapse, another major clinical manifestation of pelvic floor disorders, has a prevalence and associated risk factors similar to those of urinary incontinence.3Olsen AL Smith VJ Bergstrom JO Colling JC Clark AL Epidemiology of surgically managed pelvic organ prolapse and urinary incontinence.Obstet Gynecol. 1997; 89: 501-506Crossref PubMed Scopus (2700) Google Scholar, 21Hendrix SL Clark A Nygaard I Aragaki A Barnabei V McTiernan A Pelvic organ prolapse in the Women's Health Initiative: gravity and gravidity.Am J Obstet Gynecol. 2002; 186: 1160-1166Abstract Full Text Full Text PDF PubMed Scopus (900) Google Scholar, 22Snooks SJ Swash M Mathers SE Henry MM Effect of vaginal delivery on the pelvic floor: a 5-year follow-up.Br J Surg. 1990; 77: 1358-1360Crossref PubMed Scopus (517) Google Scholar, 23Gilpin SA Gosling JA Smith AR Warrell DW The pathogenesis of genitourinary prolapse and stress incontinence of urine. A histological and histochemical study.Br J Obstet Gynaecol. 1989; 96: 15-23Crossref PubMed Scopus (233) Google Scholar Pelvic organ prolapse and urinary incontinence frequently occur together or at different times in the same patients. Urinary incontinence may be masked in a patient with pelvic organ prolapse due to urinary retention from obstruction of the urethra by the prolapsed organ. Such complications have been well described in the literature.24Grody MH Urinary incontinence and concomitant prolapse.Clin Obstet Gynecol. 1998; 41: 777-785Crossref PubMed Scopus (48) Google Scholar, 25Romanzi LJ Chaikin DC Blaivas JG The effect of genital prolapse on voiding.J Urol. 1999; 161: 581-586Abstract Full Text Full Text PDF PubMed Scopus (225) Google Scholar It has been suggested that the etiology of urinary incontinence and pelvic organ prolapse is multifactorial, with different factors acting or interacting to produce clinical conditions in different women.17Weber AM Buchsbaum G Chen B Clark A Damaser M Daneshgari F Davis G DeLancey J Kenton K Weidner AC Word RA Basic science and translational research in female pelvic floor disorders: proceedings of an NIH-Sponsored Meeting.Neurourol Urodyn. 2004; 23: 288-301Crossref PubMed Scopus (51) Google Scholar Yet, a clear understanding of the pathophysiology of pelvic floor disorders is lacking. Animal models could be particularly useful in studying female pelvic floor disorders because these conditions are described by a wide variety of symptoms and have multiple causative factors whose interrelationships are not fully understood. A number of injury-induced rat models of pelvic floor disorders have been studied and were recently reviewed by Weber and colleagues.17Weber AM Buchsbaum G Chen B Clark A Damaser M Daneshgari F Davis G DeLancey J Kenton K Weidner AC Word RA Basic science and translational research in female pelvic floor disorders: proceedings of an NIH-Sponsored Meeting.Neurourol Urodyn. 2004; 23: 288-301Crossref PubMed Scopus (51) Google Scholar No genetic animal models, however, have thus far been described.17Weber AM Buchsbaum G Chen B Clark A Damaser M Daneshgari F Davis G DeLancey J Kenton K Weidner AC Word RA Basic science and translational research in female pelvic floor disorders: proceedings of an NIH-Sponsored Meeting.Neurourol Urodyn. 2004; 23: 288-301Crossref PubMed Scopus (51) Google Scholar Connective tissues in the pelvic floor are critical for its tensile strength and provide support to the pelvic organs that are subjected to intra-abdominal pressures. Research into the role of connective tissues in pelvic floor disorder has traditionally focused on changes in fibrillar collagens.8Ulmsten U Falconer C Connective tissue in female urinary incontinence.Curr Opin Obstet Gynecol. 1999; 11: 509-515Crossref PubMed Scopus (51) Google Scholar Published reports indicate decreased collagen content in vesicovaginal fascia,26Rechberger T Donica H Baranowski W Jakowicki J Female urinary stress incontinence in terms of connective tissue biochemistry.Eur J Obstet Gynecol Reprod Biol. 1993; 49: 187-191Abstract Full Text PDF PubMed Scopus (59) Google Scholar abdominal skin, and round ligament8Ulmsten U Falconer C Connective tissue in female urinary incontinence.Curr Opin Obstet Gynecol. 1999; 11: 509-515Crossref PubMed Scopus (51) Google Scholar in women with urinary incontinence compared with controls. There have also been reports suggesting increased prevalence of pelvic floor disorders in genetic conditions characterized by collagen and connective tissue defects, such as Ehlers-Danlos and Marfan syndromes.27McIntosh LJ Mallett VT Frahm JD Richardson DA Evans MI Gynecologic disorders in women with Ehlers-Danlos syndrome.J Soc Gynecol Investig. 1995; 2: 559-564Crossref PubMed Scopus (40) Google Scholar, 28Carley ME Schaffer J Urinary incontinence and pelvic organ prolapse in women with Marfan or Ehlers Danlos syndrome.Am J Obstet Gynecol. 2000; 182: 1021-1023Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar It remains unclear if a primary defect in collagen metabolism constitutes a contributing factor to the development of clinical pelvic floor disorders. In addition to collagens, female pelvic tissues are extremely rich in elastic fibers. Elastic fibers are components of the extracellular matrix and confer resilience.29Mecham RP Davis E Yurchenco PD Birk DE Mecham RP Elastic Fiber Structure and Assembly. Academic Press, New York1994: 281-314Google Scholar The latter property is presumably important for reproductive tissues to accommodate the enormous expansion in pregnancy and involution after parturition. Elastic fibers are turned over slowly in most adult tissues30Davis EC Stability of elastin in the developing mouse aorta: a quantitative radioautographic study.Histochemistry. 1993; 100: 17-26Crossref PubMed Scopus (89) Google Scholar except for the female reproductive organs, where they undergo massive remodeling.31Woessner JF Brewer TH Formation and breakdown of collagen and elastin in the human uterus during pregnancy and post-partum involution.Biochem J. 1963; 89: 75-82Crossref PubMed Scopus (94) Google Scholar, 32Starcher B Percival S Elastin turnover in the rat uterus.Connect Tissue Res. 1985; 13: 207-215Crossref PubMed Scopus (37) Google Scholar The major component of elastic fibers is an amorphous polymer composed of the protein elastin, known as tropoelastin in its monomeric form. Polymerization requires an initial step of oxidative deamination of lysine residues catalyzed lysyl oxidases (LOXs).33Kagan HM Li W Lysyl oxidase: properties, specificity, and biological roles inside and outside of the cell.J Cell Biochem. 2003; 88: 660-672Crossref PubMed Scopus (711) Google Scholar Mammalian genomes have five related genes coding for the prototypic LOX and four LOX-like proteins (LOXL1, LOXL2, LOXL3, and LOXL4).33Kagan HM Li W Lysyl oxidase: properties, specificity, and biological roles inside and outside of the cell.J Cell Biochem. 2003; 88: 660-672Crossref PubMed Scopus (711) Google Scholar Recently, we have shown that LOXL1 is essential for elastic fiber homeostasis in multiple tissues including the female pelvic organs.34Liu X Zhao Y Gao J Pawlyk B Starcher B Spencer JA Yanagisawa H Zuo J Li T Elastic fiber homeostasis requires lysyl oxidase-like 1 protein.Nat Genet. 2004; 36: 178-182Crossref PubMed Scopus (521) Google Scholar In all tissues examined, LOXL1 always co-localizes with elastic fibers. Mice lacking LOXL1 are unable to synthesize elastin polymers in adult tissues, whereas collagen synthesis appears to proceed normally. These observations suggest that the function of LOXL1 is dedicated to elastic fiber homeostasis. Because elastic fibers undergo active remodeling throughpregnancy and parturition in the female genito-urinary organs, these tissues are expected to be sensitive to gene defects affecting elastogenesis. In this study, we examined the impact of failed elastic fiber homeostasis on female pelvic organs and development of voiding abnormalities. Generation of LOXL1-deficient mice was described previously.34Liu X Zhao Y Gao J Pawlyk B Starcher B Spencer JA Yanagisawa H Zuo J Li T Elastic fiber homeostasis requires lysyl oxidase-like 1 protein.Nat Genet. 2004; 36: 178-182Crossref PubMed Scopus (521) Google Scholar Both the mutant and wild-type (WT) control mice were of a mixed C57BL/6 and 129Sv backgrounds. Loxl1−/− mice manifest a host of pathologies that can be attributed to elastic fiber defects.34Liu X Zhao Y Gao J Pawlyk B Starcher B Spencer JA Yanagisawa H Zuo J Li T Elastic fiber homeostasis requires lysyl oxidase-like 1 protein.Nat Genet. 2004; 36: 178-182Crossref PubMed Scopus (521) Google Scholar Approximately one-third of female Loxl1−/− mice develop severe pelvic organ prolapse after the first litter, and all of the remaining two-thirds develop prolapse after the second litter. No female Loxl1−/− mice were ever found to give birth to a third litter. The acute stage of prolapse, in which a long stretch of vaginal/uterine tissues is exposed outside of the body cavity, typically lasts 1 to 2 weeks. This is followed by a permanent and moderate prolapse, as indicated by the descent of pelvic organs forming a bulge at the urogenital region, which remains little changed for the remainder of the animal's lifespan (stable stage). Female Loxl1−/− mice that had given birth to one or two litters, and were between 4 and 7 months of age, were selected at random for the examination of pelvic organ pathology (n = 32), and for urinary behavior measurement (n = 8; from within the group of 32). This group did not include any mouse with apparent signs of urinary retention (see below). At the time of study, mice were in the stable stage of pelvic organ prolapse and were between 3 and 10 weeks after their most recent parturition. Age-matched WT females that had given birth to two or three litters were randomly chosen and included as controls in the study of pelvic organ pathology (n = 30) and urinary behavior measurement (n = 7). The WT control females matched or exceeded the parity of the mutant mice. On rare occasions, female Loxl1−/− mice showed signs of urinary retention as indicated by an enormous pelvic bulge in the pelvic region that far exceeded those typically seen in mice with prolapse. They also had difficulty walking. These mice were visually identified and tested for voiding behavior followed by gross pathology examinations (n = 3; with results shown in Figure 4). The mutant and WT mice were processed in parallel and under identical conditions, and the results from the two groups of animals were compared. For studies examining expression of LOXL1 at different gestational time points, nulliparous WT females were used. Mice were mated and gestational days were calculated after the detection of a vaginal plug. For studies examining expression of LOXL1 at different ages, WT mice, at 2 and 18 months of age, were used. Blood urea nitrogen was analyzed at Anilytics, Inc. (Gaithersburg, MD). All experiments involving animals were performed following protocols approved by the institutional Animal Care and Use Committee. Mice were euthanized by CO2 inhalation, and pelvic organs were examined and photographed under a dissecting microscope. Afterward, tissues were rinsed in phosphate-buffered saline (PBS) and fixed in 4% formaldehyde/PBS overnight. Tissues were embedded in paraffin. Transverse sections through the middle portion of the vagina/urethra were cut at 4-μm thickness. Sections were stained with hematoxylin and eosin (H&E). A total of 32 Loxl1−/− mice and 30 WT mice were examined. A mouse micturition chamber, designed to measure urinary output in real time, was custom built by Columbus Instruments (Columbus, OH). This chamber was adapted from the standard mouse metabolic cage offered by Columbus Instruments. Key features of the micturition chamber included a wire mesh bottom (mesh 4), which was connected to a funnel. The bottom of the chamber was designed for unobstructed collection of urine droplets, but it would not sequester solid droppings. The inside surface of the funnel was coated with molten paraffin and was recoated after several uses to minimize trapping of liquid droplets inside the funnel. Directly below the bottom opening of the funnel was a collection tube, which was placed on a balance (Mettler Toledo electronic balance, model PL83 with 0.001 g weight resolution). The data port of the balance was connected to a computer. The bottom of the funnel and the collection tube were encased in a Plexiglas outer casing, which served to cut down evaporation and reduce the effect of air draft. Changes in the weight of the collection tube were recorded at a sampling speed of six times/minute. Initial tests of this system had confirmed that urine droplets as small as 50 μl in volume could be reliably collected and recorded. Before placement inside the chamber, mice were given a residue-free diet (Lactaid brand whole milk, lactose-free) for 24 hours. The liquid diet was given to prevent feces droppings from interfering with measurement of urine output. During the entire test period, mice continued to have free access to this liquid diet. Pilot tests had confirmed that this diet was well accepted by mice and that it produced no apparent adverse effects (in contrast, regular milk that contained lactose produced diarrhea). Loxl1−/− mutant and WT control mice were tested in this chamber one at a time, each lasting a duration of 24 hours. Mutant and WT mice were tested in an alternating sequence. The climate control included 12 hours of light and 12 hours of darkness that were synchronized with the light/dark cycle in the animal facility. Data were analyzed and plotted using the Multi-Device Interface software provided by Columbus Instruments. LOXL1N and LOXL1C antibodies, which recognize the N- and C-termini of LOXL1, respectively, were described previously.34Liu X Zhao Y Gao J Pawlyk B Starcher B Spencer JA Yanagisawa H Zuo J Li T Elastic fiber homeostasis requires lysyl oxidase-like 1 protein.Nat Genet. 2004; 36: 178-182Crossref PubMed Scopus (521) Google Scholar Elastin antibodies were obtained from Elastin Products Company (Owensville, MO): PR385 (rabbit anti-mouse elastin, exons 6 to 17) and RT675 (goat anti-rat elastin). Immunoblotting and immunofluorescence staining of unfixed cryosections were performed as described.34Liu X Zhao Y Gao J Pawlyk B Starcher B Spencer JA Yanagisawa H Zuo J Li T Elastic fiber homeostasis requires lysyl oxidase-like 1 protein.Nat Genet. 2004; 36: 178-182Crossref PubMed Scopus (521) Google Scholar For immunoblotting analysis, 5 μg of total proteins were loaded per lane, separated on sodium do-decyl sulfate-polyacrylamide gels, and transferred to polyvinylidene difluoride membranes. For immunostaining on frozen sections, transverse sections were cut at 10 μm through the middle portion of the vaginas/urethras. Unfixed frozen sections were used in immunofluorescence procedures. Cell nuclei were stained blue with Hoechst dye 33342. Total RNA was isolated using the TRIzol reagent (Life Technologies, Inc., Grand Island, NY). First-strand cDNA synthesis was primed with oligo(dT)20 using the ThermoScript RT-PCR system (Invitrogen, Carlsbad, CA). PCR primers for amplifying LOXL1 were P1 (5′-CGCGTTACGAGGACTACGGAG-3′) and P2 (5′-GACCATTCTGGTTGGGTCGGT-3′). PCR primers for LOX were P7 (5′-GCAGGAACCGACCTGGATACGGCAC-3′) and P8 (5′-CAGCCTGAGGCATAGGCATGATGTC-3′). PCR primers for elastin were P3 (5′-CTGGATCGCTGGCTGCATCCA-3′) and P4 (5′-GTCCAAAGCCAGGTCTTGCTG-3′). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was amplified together with LOXL1 and elastin targets in the same tube as an internal standard for quantification. PCR primers for GAPDH were P5 (5′TGAAGGTCGGTGTGAACGGATTTGGC-3′) and P6 (5′-CATGTAGGCCATGAGGTCCACCAC-3′). Pilot experiments were done to determine the optimal primer concentrations in these mixed PCR reactions. Finally, P1, P2, P3, P4, P7, and P8 primers were used at 0.15 μmol/L. P5 and P6 primers were used at 0.1 μmol/L. PCR products were separated on 1.5% agarose gels and the images were captured by Fluor-S MultiImager. PCR reactions were terminated at different cycle numbers (20, 25, 30, and 35) to ensure that amplifications did not reach a plateau. Quantification was performed using the Multi-Analyst software (Bio-Rad Laboratories, Hercules, CA). Mean volume per urinary event was calculated for each animal. Data analyses included univariate statistics to calculate group means, standard deviations, and plots of frequency distributions. Mean group differences were evaluated by t-test. P < 0.05 indicated a significant difference between groups. The pelvic organ defects in the LOXL1 mutant animals were striking. Loxl1 mutant mice developed pelvic organ prolapse after giving birth to either their first or second litter of pups. Severe prolapse was seen 1 to 3 days postpartum. Exposed vaginal/uterine tissues varied from a quarter to one inch in length (Figure 1A, left). The prolapsed tissues typically retracted throughout a period of 1 to 2 weeks, but a large bulge remained apparent in the urogenital region indicating internal pelvic organ descent (Figure 1A, middle). Mice would remain in this stable state of moderate prolapse indefinitely. Loxl1 mutant females were also prone to develop mild rectal prolapse. Rectal prolapse generally appeared later than vaginal prolapse and was not seen in all of the mutant mice that had developed vaginal prolapse (∼50%). Female Loxl1−/− mice appeared to lose fecundity afterward, as none had been found to give birth for a third time. Parturition appeared to be the single most important trigger for pelvic organ prolapse in female Loxl1−/− mice because virgin females did not develop prolapse in this age range (3 to 7 months). Spontaneous pelvic floor problems did develop slowly in nulliparous Loxl1−/− females, so that by 1 year of age ∼50% of them showed sign of pelvic organ descent that appeared similar to the one shown in Figure 1A (middle). Pelvic floor defects in nulliparous Loxl1−/− females were not examined further. WT females, up to 18 months of age and regardless of parity, showed no sign of pelvic organ prolapse or descent. A cohort of female Loxl1 mutant mice, which had recovered from acute prolapse after parturition, was examined for gross pelvic organ pathology and histopathology in comparison to the WT control group. On dissection of the pelvic cavity, WT controls were found to maintain well-defined uterine, cervical, and vaginal structures. The urethra was tightly adhered to the suburethral vaginal wall along its entire length, and the urinary bladder was firmly attached at a position near uterine cervix (Figure 1B, left). In contrast to the WT mice, all mutant mice showed descent of the uterine, bladder, and upper vaginal tissues into the lower vaginal cavity, creating a ring-like fold in the upper middle position of the vaginal wall (Figure 1B, middle and right; Figure 1C). The severity of the pelvic organ descent was such that the urinary bladder was seen trapped into this vaginal fold (Figure 1B, right). The ring-shaped fold in the vaginal wall appeared to mark the position where uterine and vaginal tissues folded inside out during the acute phase of prolapse. Without exception, mutant mice had enormously distended lower vaginal walls (Figure 1B). The upper portion of the urethra in the mutant was typically detached from the vaginal wall, potentially allowing for a much greater degree of movement of the urethra and bladder. In addition, the lower portion of the vaginal walls in the mutant had a totally different texture and appearance compared to the WT (Figure 1D). Whereas the WT tissues appeared thick and exhibited considerable tensile strength during dissection, the mutant tissues were membrane-thin and tore at the slightest application of force. After parturition, t
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