Pelvic organ prolapse in fibulin-5 knockout mice: pregnancy-induced changes in elastic fiber homeostasis in mouse vagina

Abstract

Pelvic organ prolapse is strongly associated with a history of vaginal delivery. The mechanisms by which pregnancy and parturition lead to failure of pelvic organ support, however, are not known. Recently, it was reported that mice with null mutations in lysyl oxidase-like 1 (LOXL1) develop pelvic organ prolapse. Elastin is a substrate for lysyl oxidase (LOX) and LOXL1, and LOXL1 interacts with fibulin-5 (FBLN5). Therefore, to clarify the potential role of elastic fiber assembly in the pathogenesis of pelvic organ prolapse, pelvic organ support was characterized in Fbln5-/- mice, and changes in elastic fiber homeostasis in the mouse vagina during pregnancy and parturition were determined. Pelvic organ prolapse in Fbln5-/- mice was remarkably similar to that in primates. The temporal relationship between LOX mRNA and protein, processing of LOXL1 protein, FBLN5 and tropoelastin protein, and desmosine content in the vagina suggest that a burst of elastic fiber assembly and cross linking occurs in the vaginal wall postpartum. Together with the phenotype of Fbln5-/- mice, the results suggest that synthesis and assembly of elastic fibers are crucial for recovery of pelvic organ support after vaginal delivery and that disordered elastic fiber homeostasis is a primary event in the pathogenesis of pelvic organ prolapse in mice.

Figure 1

Pelvic organ prolapse in Fbln5^−/− mice. Anogenital region of 6-month-old nonpregnant virginal Fbln5^−/− mice (B) is compared with age-matched wild-type females (A). Both mice were of mixed strain (C57BL/6;129SvEv). Note urogenital bulge (arrow) and visible cervix at the vaginal opening in the knockout.

Figure 2

Surgical anatomy of urogenital bulge in Fbln5^−/− mice. In wild-type mice (A), the bifurcation of the uterine horns (Ut), urethra (U), and bladder (B) are superior to the pubic ligament (PL). (The pubic ligament is disarticulated.) In Fbln5^−/− mice with pelvic organ prolapse (B), the bladder and underlying vagina are caudal to the pubic ligament. Bifurcation of uterine horns and vagina are also caudal to the pubic ligament (not visible in photograph). V, vagina; PL, pubic ligament; Ut, uterine horn; U, urethra.

Figure 3

Absence of pelvic support structures in Fbln5^−/− mice. Uterine horns (Ut) from wild-type (left) and Fbln5^−/− (right) mice are dissected from the ovarian suspensory ligaments and brought forward through the disarticulated pubic ligament (PL) to visualize the rectum (R) and uterosacral ligaments that suspend the upper vagina (V) and cervix (CX) to the pelvis posteriorly (arrows). Note absence of uterosacral ligaments and the flattened patulous vagina (V) in the Fbln5 knockout animal. Both mice were of mixed strain (C57BL/6;129SvEv).

Figure 4

Vaginal mass is increased in Fbln5^−/− mice. Vaginal size is increased in vaginal specimens from Fbln5^−/− mice (B) compared with wild-type controls (A). ut, uterus; vag, vagina. C: Bladder and vaginal wet weight was determined in wild-type (WT) and Fbln5^−/− (Fbln5 KO) mice. Each bar represents mean ± SEM of 34 virginal wild-type (C3BL/6) and 26 (bladder) or 33 (vaginal) virginal Fbln5^−/− (C57BL/6;129SvEv) females. **P < 0.001 compared with WT.

Figure 5

Immunoblot analysis of FBLN5 and tropoelastin protein in mouse vagina during pregnancy, parturition, and the puerperium. Total protein (10 μg/lane) from soluble and urea extracts of vaginal tissues were applied to 5 to 20% gradient polyacrylamide gels, separated by electrophoresis, transferred to nitrocellulose, and probed for FBLN5 (top blot) and tropoelastin (middle blot) with specific antibodies. Results were compared with total protein in side-by-side Coomassie-stained gels (bottom). Tropoelastin and FBLN5 were enriched in urea extracts (Urea Ext) but absent in the soluble fraction (Sup). Protein extract from a Fbln5 KO uterus was used as a negative control. The immunoreactive band detected in the Fbln5^−/− lane using the elastin antibody is nonspecific. NP, nonpregnant; D18, pregnant D18; 2h, 24h, 48h, and 72h indicate hours postpartum.

Figure 5

Quantification of FBLN5 and tropoelastin protein and desmosine in mouse vagina during pregnancy, parturition, and postpartum time period. Staining intensity of immunoreactive FBLN5 (A) and tropoelastin (B) was quantified with chemiluminescence and compared with a nonpregnant standard on each blot. Each bar represents mean ± SEM of 3 to 11 samples at each time point. **P ≤ 0.001 compared with nonpregnant; *P ≤ 0.05 compared with pregnant D18. C: Desmosine content was determined in nonextractable pellets from vaginal homogenates and expressed as pmol per mg protein. NP, nonpregnant; Preg D18, pregnant D18; In Labor, tissues collected after delivery of first pup; 2, 4, 12, 24, 48, and 72 hours indicate hours postpartum; 7 days PP, 7 days postpartum.

Figure 7

Elastic fibers in upper and mid vaginal wall from pregnant (D16) and postpartum mice (2 hours after delivery). Hart’s stain was used to visualize elastic fibers (black) in sagittal sections of vaginal tissues from pregnant (D16, A and C) and postpartum (2 hours, B and D) C3/BL6 wild-type mice. Note elastic fiber morphology in vaginal muscularis suspending the vaginal wall to the levator ani muscle (l ani). Sections were obtained from the upper and middle thirds of the vagina. epi, epithelium; m, muscularis. E and F: Masson’s trichrome stain of vaginal wall from pregnant (D16, E) and postpartum (2 hours, F) mice. Arrows in F indicate many of the infiltrating inflammatory cells that are associated with dissolution of the collagenous extracellular matrix (stained blue). Original magnifications: ×250 (A–D); ×200 (E, F).

Figure 8

Regulation of LOX isoform mRNA expression in urogenital tissues during pregnancy and the postpartum time period. The relative abundance of LOX and LOXL1-4 mRNA was determined by real-time PCR in vagina, cervix, uterus, and bladder from nonpregnant (open bar), pregnant D18 (hatched bar), and 24 hours postpartum (closed bar) wild-type (C3BL/6) mice. Transcript levels are expressed relative to that of LOX in nonpregnant vagina. Each bar represents mean ± SEM of 3 to 16 tissues. *P ≤ 0.05 compared with corresponding nonpregnant tissue; **P ≤ 0.05 compared with corresponding postpartum tissue.

Figure 9

Regulation of LOX and LOXL1 in vaginal tissues during pregnancy, parturition, and the puerperium. A and D: Real-time PCR was used to determine the relative abundance of LOX (A) and LOXL1 (D) mRNA in vaginal tissues from nonpregnant (np) and pregnant wild-type (C3/BL6) mice at various time points in gestation or postpartum. Data are expressed relative to β2-microglobulin and normalized to a nonpregnant vagina standard. Each data point represents mean ± SEM of 5 to 13 tissues at each time point except 72 hours (n = 3). Labor, in labor after delivery of the first pup; 24h, 24 hours postpartum; 48h, 48 hours postpartum; 72h, 72 hours postpartum; 1 wk, 1 week postpartum; 2 wk, 2 weeks postpartum. *P ≤ 0.05 compared with early pregnancy time points. B and E: Immunoblot analysis of LOX (B) and LOXL1 (E) with antibodies that recognize pro- and mature enzymes in vaginal tissues from nonpregnant, pregnant D18, and postpartum (12 to 24 hours) mice. The blot in E is overexposed to detect mature LOXL1 protein because epitopes for antibody recognition are fewer in the mature form compared with the proenzymes. C and F: Relative intensity of chemiluminescence of mature LOX (C) and LOXL1 (F) isoforms in vaginal tissues from nonpregnant (NP, open bars), pregnant D18 (hatched bars), and postpartum (PP, solid bars). LOX and LOXL1 indicate proforms of the enzyme, whereas mature LOX and LOXL1 indicate the final processed forms. Ten μg of urea-extracted protein was applied in each lane. Data represent mean ± SEM of six to eight tissues in each group. *P ≤ 0.05 compared with nonpregnant.

Figure 10

Effect of elastase injection on pelvic organ prolapse in postpartum mice. A: Parturient (C3BL/6) mice were injected with saline (left) or 5 U of purified pancreatic elastase (right) on the day of delivery and examined for pelvic organ prolapse. Arrow denotes urogenital bulge in elastase-treated animals. Results are representative of six of eight elastase-treated mice and eight animals in the control group. B: Vaginal wall tissues of postpartum mice injected with saline (control) or elastase (elastase) were obtained 72 hours after injection, mounted in the transverse section, and stained for elastic fibers. Marked dissolution of the extracellular matrix and shortened, frayed elastic fibers were noted in the posterior vaginal wall of elastase-injected animals. epi, epithelium; m, muscularis.