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Dec 23, 2011 - modification of the fibrocartilage into an interpubic ligament (IpL), followed by its relaxation prior to parturition. It is recognized that nitric oxide ...
RESEARCH ARTICLE Molecular Reproduction & Development 79:272–282 (2012)

High iNOS mRNA and Protein Localization During Late Pregnancy Suggest a Role for Nitric Oxide in Mouse Pubic Symphysis Relaxation CAMILA FERNANDES MORO, SI´LVIO ROBERTO CONSONNI, RENATA GIARDINI ROSA, ´ LIA CAVINATO NASCIMENTO, AND PAULO PINTO JOAZEIRO* MARIA AMA Department of Histology and Embryology, State University of Campinas (Unicamp), Campinas, SP, Brazil

SUMMARY Remodeling and relaxation of the mouse pubic symphysis (PS) are central events in parturition. The mouse PS remodels in a hormone-controlled process that involves the modification of the fibrocartilage into an interpubic ligament (IpL), followed by its relaxation prior to parturition. It is recognized that nitric oxide synthase (NOS) and consequently nitric oxide (NO) generation play important roles in extracellular matrix modification, and may promote cytoskeleton changes that contribute to the remodeling of connective tissue, which precedes the onset of labor. To our knowledge, no studies thus far have investigated inducible nitric oxide synthase (iNOS) expression, protein localization, and NO generation in the mouse PS during pregnancy. In this work, we used a combination of the immunolocalization of iNOS, its relative mRNA expression, and NO production to examine the possible involvement of iNOS in remodeling and relaxation of the mouse IpL during late pregnancy. The presence of iNOS was observed in chondrocytes and fibroblast-like cells in the interpubic tissues. In addition, iNOS mRNA and NO production were higher during preterm labor on day 19 of pregnancy (D19) than NO production on D18 or in virgin groups. The significant increase in iNOS mRNA expression and NO generation from the partially relaxed IpL at D18 to the completely relaxed IpL at D19 may indicate that NO plays an important role in late pregnancy during relaxation of the mouse IpL. Mol. Reprod. Dev. 79: 272–282, 2012. ß 2011 Wiley Periodicals, Inc.

Received 22 July 2011; Accepted 13 December 2011

INTRODUCTION Remodeling of connective tissue preceding the onset of labor is a hallmark of the major histoarchitectural adaptations in the reproductive tract and musculoskeletal elements that synergistically facilitate successful parturition. Such musculoskeletal elements are responsible for genitourinary and digestive tract support, and include the pelvic skeleton, which forms the margins of the birth canal (Yiou et al., 2001; Delancey et al., 2008; Weaver and Hublin, 2009; Becker et al., 2010). Morphological changes in the ß 2011 WILEY PERIODICALS, INC.

* Corresponding author: Department of Histology and Embryology State University of Campinas CP 6109 13083-970 Campinas, SP, Brazil. E-mail: [email protected] Grant sponsor: Conselho Nacional de Desenvolvimento Cientıfico e gico—CNPq; Grant number: Tecnolo 308169/2009-3; Grant sponsor: ~o de Amparo a  Pesquisa do Fundac¸a ~o Paulo—FAPESP; Estado de Sa Grant number: 2008/56492-0

Published online 23 December 2011 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/mrd.22020

female pelvic girdle during pregnancy have been examined in the guinea pig (Talmage, 1947) and mouse (Hall, 1947). Indeed, both animals have been explored as models for understanding the hormonal induction of symphyseal widening, although this phenomenon is less conspicuous in humans (Becker et al., 2010) and rats (Ortega et al., Abbreviations: D#, day number of pregnancy; iNOS, inducible nitric oxide synthase; IpL, interpubic ligament; NO, nitric oxide; NOS, nitric oxide synthase; PS, pubic symphysis; a-SMA, a-smooth muscle actin.

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2003). The widening and increased mobility of the pubic symphysis (PS) in some mammals allow for a disproportionately large fetus to negotiate a birth passage that would otherwise be too small to accommodate the offspring. One striking example of this can be found in bats, where a single, large full-term fetus stretches the ligament to more than 15 times its original length (Crelin and Newton, 1969). Previous histological and ultrastructural studies have demonstrated that the mouse PS passes through discrete preparatory modifications during the early period of pregnancy (Hall, 1947; Crelin, 1969; Ortega et al., 2003). By day 12 of pregnancy (D12), the typical histoarchitecture is quite similar to that observed in the virgin mouse, comprising an interpubic fibrocartilaginous disk placed between thin layers of hyaline cartilage caps at both sides of the pubic bones. The joint increases from 0.15 to 0.2 mm between D12 and D15, and an interpubic ligament (IpL) grows in a process classically described as ‘‘separation’’ of the PS. The IpL further enlarges to 2.4 mm on D18, then to about 3 mm on D19 (the day of delivery). As the period of the helical collagen fiber structure is decreased, there is a final lengthening of the fiber. This process is known as ‘‘relaxation,’’ which refers to a breakdown and reorganization of the connective tissue (Pinheiro et al., 2004). The target tissue of these hormonal actions, that is, mouse PS fibrocartilaginous cells and differentiated fibroblasts, expresses higher concentrations of a and b estrogen and the relaxin receptors LGR7 and LGR8 (Wang et al., 2009). LGR7 and LGR8 are also able to induce cellular expression of the nitric oxide synthase (NOS) isoenzymes, such as inducible NOS, which sustains nitric oxide (NO) synthesis (Nistri and Bani, 2003). NO is a biologically active gas that functions as a potent signaling molecule in a number of physiological and pathophysiological processes. It is synthesized by the three isoforms of NOS—endothelial NOS (eNOS), inducible NOS (iNOS), and neural NOS (nNOS)—that co-localize directly or indirectly with elements of the cytoskeleton, including actin microfilaments, microtubules, and intermediate filaments. The co-localization of the NOS isoforms with the cytoskeleton permit optimal NO production and help the NOS isoforms to perform their functions (Su et al., 2005). The observed changes in NOS expression in organs of the reproductive system and gestational tissues are spatially and temporally regulated in each species. Because NO is known to represent the final metabolic mediator of cervical ripening, studies focusing on the regulatory role of NOS or the NO system have shown that an improvement in labor induction can be achieved by the application of NO donors. Likewise, NO inhibitors can block cervical ripening and may be used for the prevention and treatment of preterm birth or cervical incompetence (Chwalisz and Garfield, 1998a). The mRNA levels of NOS are controlled by a paracrine regulator, which decreases the expression levels of NOS in the rat and human term uterus while simultaneously increasing it in the cervix, or vice versa (Buhimschi

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et al., 1996; Purcell et al., 1997; Chwalisz and Garfield, 1998b; Tschugguel et al., 1999; Tornblom et al., 2005). Although it is recognized that relaxin increases the expression of NOS and can act as a regulator of the production of NO in mouse uterine tissues (Bani et al., 1999), there is little data regarding the involvement of the NOS isoforms in cervical and PS remodeling during pregnancy and parturition in the mouse. Thus, NO plays important roles in altering the extracellular matrix, and may cause cytoskeletal changes that contribute to the remodeling of connective tissue preceding the onset of labor, as described for uterine cervix ripening. To our knowledge, however, there are no studies that have investigated iNOS expression, protein localization, or NO generation in the mouse PS. Thus, the purpose of this study was to investigate whether NO could be involved in the relaxation of the mouse PS during late pregnancy.

RESULTS Tissue Remodeling in the Pregnant Mouse Pubic Symphysis A morphological analysis revealed that the virgin female PS consisted of two pubic bones connected by a fibrocartilaginous disk (Fig. 1A). During pregnancy, the growth of an IpL between the pubic bones was observed, along with tissue relaxation on D19 (Fig. 1B). This relaxed IpL consisted of collagen fibers parallel to the long axis of the ligament and fibroblast-like cells with proliferative capacity (Fig. 1C,D). Localization of iNOS in the Mouse Pubic Symphysis In all three groups (virgin, D18, and D19 groups), a faint but specific immunoreaction for iNOS was detected in cell populations from both the PS and IpL by light microscopy. In virgin cycling mice, a diffuse reaction for iNOS could be observed in some hypertrophic chondrocytes in the PS hyaline cartilage (Fig. 2A). Cells from the IpL of pregnant mice at D18 (Fig. 2B,D) and D19 (Fig. 2E) labeled with anti-iNOS revealed the morphology of chondrocytes and fibroblast-like cells. Some dividing fibroblast-like cells also remained positive for anti-iNOS (Fig. 2D). No staining was detected in negative control samples (Fig. 2C,F). Subcellular localization by immunoelectron microscopy revealed that iNOS staining in the fibroblast-like cells of the IpL was preferentially distributed into well-defined cell compartments: perinuclear areas associated with the nuclear envelope and heterochromatin-associated regions (Fig. 3A); regions associated with the cytoplasmic face of the endoplasmic reticulum membrane (Fig. 3A,B); and minute vesicle membranes-associated with the cortical cytoplasm, where two gold particles were observed next to each other (Fig. 3A,B). Negligible background labeling, with no apparent staining of the extracellular matrix or fibroblast-like cells, was observed in the IpL negative control on D18 (Fig. 3C). 273

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Figure 1. Representative light microscopy images of a virgin mouse pubic symphysis (A) and the interpubic ligament of pregnant mice at D18 (C) and D19 (B and D). A: The virgin mouse pubic symphysis is formed by pubic bones (PB) connected by hyaline cartilage (HC) and fibrocartilage (FC) with typical fibrochondrocytes. B: On D19, the interpubic ligament (IpL) developed between the pubic bones (PB). C: Before parturition (on D18), the IpL is formed by wavy collagen fibers organized in a crimp (arrow) and spindle-shaped fibroblast-like cells (arrowhead). D: On the day of parturition, fibroblast-like cells could be found in mitosis in the IpL (arrows). Masson’s Trichrome stain. Bars: A, 100 mm; B, 600 mm; C and D, 30 mm.

Nitrite Production During Pregnancy in Mouse Pubic Symphysis Interpubic tissue nitrite production (Fig. 4) was similar in the virgin (median 2.35 mmol/g) and D18 groups (median 1.90 mmol/g). In contrast, a substantial increase in nitrite production was observed on the morning of D19 (median 3.60 mmol/g, P < 0.05 vs. virgin). Quantitative iNOS Gene Expression Quantitative, real-time PCR revealed iNOS gene expression in the virgin PS and IpL during late pregnancy (D18 and D19). On D19, the expression of iNOS was significantly higher compared to either the virgin (P < 0.01) or D18 groups (P < 0.05). There were no significant differences between the virgin and D18 groups (Fig. 5), however.

DISCUSSION Our evaluation of the morphological, biochemical, and molecular characteristics of iNOS in mouse PS and IpL revealed general aspects of the temporally regulated changes in the extracellular matrix microstructure, the

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localization of iNOS, nitrite production (NO generation), and quantitative iNOS gene expression. In this model, NO may represent one of the metabolic mediators of the relaxation phase in the IpL on the day of delivery. As such, fibrocartilaginous cells and differentiated fibroblasts isolated from the mouse PS showed enhanced expression of estrogen and the relaxin receptors (Wang et al., 2009), which enabled the cells to promote the expression of iNOS and NO generation (Nistri and Bani, 2003). In the virgin PS, the fibrocartilage histoarchitecture showed characteristics of a tissue suited for contact force and responsivity to high-rate loading or impact. The wavy design (crimp) of collagen fibers in the IpL reflects a very efficient arrangement that is able to maintain the integrity and mechanical functions of the symphysis (Pinheiro et al., 2004). This morphology is related to a variable degree of compressive or even tensional forces, thereby providing a mechanism for the smooth transfer of forces while efficiently protecting the birth canal (Pinheiro et al., 2004). Moreover, modification of the fibrocartilaginous cell phenotype from an oval in the PS to a spindle-shaped, fibroblastlike cell in the IpL that may be proliferating suggests that cells undergo adaptations to support the changing regional forces produced by alterations in the IpL extracellular matrix

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Figure 2. Light microscopy of representative immunohistochemistry for iNOS on the pubic symphysis (virgin—A and C) and interpubic ligament (D18–B and D; D19–E and F). Hypertrophic chondrocytes (A and B, arrow), fibroblast-like cells in mitosis (D, arrowhead), and spindle-shaped fibroblast-like cells (E, arrowhead) could be seen as positive for iNOS. Negative controls showed no reaction for iNOS in hypertrophic chondrocytes (C; arrow) or in fibroblast-like cells (F; arrowhead). Bars: A, 5 mm; B and C, 10 mm; D, 20 mm; E, 10 mm; and F, 5 mm.

composition during pregnancy. These changes allow for the safe passage of the fetus during labor (Moraes et al., 2004). This remodeling is controlled by hormones, such as estrogen and relaxin, which promote substantial growth in the lower reproductive tract of rodents (Parry and Vodstrcil, 2007; Yao et al., 2010). Slight changes in the density of collagen fibers of interpubic connective tissue were observed in LGR7-knockout mice, resulting in a loss of PS elongation at late pregnancy. These mice also exhibited abnormal cervical, vaginal, and mammary gland morphology (Zhao et al., 1999; Parry et al., 2005). In relaxin receptor RXFP1- or LGR7-knockout mice, histopathological abnormality in cervix, vagina, and mammary gland was proposed to be a factor that results in parturition defects (Kamat et al., 2004; Krajnc-Franken et al., 2004). No pregnancyrelated, lower reproductive tract connective tissue remodeling was shown in RXFP1-knockout mice, but dead pups entrapped within the birth canal in some of these animals may have been a result of this histopathological abnormality (Kamat et al., 2004; Krajnc-Franken et al., 2004).

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Figure 3. iNOS subcellular localization by immunoelectron microscopy of representative fibroblast-like cells in the interpubic ligament on D18 (A–C). The presence of iNOS was detected as isolated gold particles (arrows) at the perinuclear region, on heterochromatin in the nucleus (A), and in association with the cytosolic face of ER membranes (A and B). Detailed electron micrographs (A and B, upper right) reveal two gold particles next to each other (arrowhead), which were predominantly on minute vesicles localized at the cortical cytoplasm. Negative control (C and inset) showed no iNOS staining in interpubic ligament fibroblast-like cells on D18. Bars: 500 mm.

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Figure 4. Nitrite production (NO generation) in the virgin mouse pubic symphysis and the interpubic ligament on D18 and D19 (D19 ¼ the morning of the delivery). Each value is described in the box plot, with bars indicating the median level. *P < 0.05 versus virgin.

Relaxin induces high expression levels of matrix metalloproteinases (MMPs) as well as estrogen, and relaxin receptors have been observed in mouse pubic fibrocartilaginous tissue (Kapila et al., 2009; Wang et al., 2009). Combined elevation of MMP expression and activity in the absence of granulocytic cell migration can degrade components of the PS extracellular matrix at D12, mainly via MMP-8, and from D15 to delivery, via MMP-8, -2, and -9

Figure 5. Inducible NOS (iNOS) mRNA levels were measured by realtime PCR using mRNA extracted from connective tissues of the interpubic articulation in the virgin, D18, and D19 animals, and normalized to the 36B4 levels. **P < 0.01 versus virgin. *P < 0.05 versus D18.

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(Rosa et al., 2008, 2011a). At this stage, the IpL undergoes connective tissue remodeling, resulting in rapid and pronounced expansion and flexibility, which will transform it into a ‘‘relaxed’’ structure at its maximum length. Our immunolocalization study demonstrates that iNOS is present in spindle-shape, fibroblast-like cells of the IpL, which agrees with observations made during bovine prelabor cervical ripening that found iNOS in areas containing predominantly fibroblasts (Aalberts et al., 2007). According to Moraes et al. (2004), spindle-shaped fibroblast-like cells in the IpL during late pregnancy often possess large bundles of intermediate filaments, and thin filaments in close proximity to the cytoplasmic cortex, termed the fibronexus. The fibronexus propagates the transmission of contractile forces within the tissue to numerous minute and pinocytotic vesicles. Thus, our subcellular localization study of spindle-shaped fibroblast-like cells showed that the immunolabeled minute vesicles may be associated with a strategic distribution of iNOS, consistent with a previous study (Heijnen et al., 2006). Heijnen et al. observed specific labeling of iNOS-associated proteins in chondrocytes that produce NO constitutively, and in fibroblasts that play a role in mechanotransduction in their respective tissues. In their study, however, both NO and superoxide anion were produced in response to iNOS induction. Interestingly, we identified two gold particles next to each other in our study. An additional structural analysis could be used to determine if those two gold particles were a dimer, which would correspond to the active form of iNOS (Ratovitski et al., 1999; Saini et al., 2006). Although our results identified cell proliferation in the IpL of late pregnancy, studies in other systems have shown that oxidative stress induces NO production at levels that lead to cell death (Bustamante et al., 2002). Oxidative stress occurs when there is an imbalance between oxidants and antioxidants (Peng et al., 2010). Thus, it is possible that the mechanisms resulting in oxidative stress in the IpL at D19 induce cell death after a peak of cell proliferation, as demonstrated by Veridiano et al. (2007). Taken together, our data suggest that iNOS may be associated with the hormonally regulated process responsible for the adaptive changes in the interpubic tissue during late pregnancy. Specifically, we show that iNOS was localized in some cells of the PS and IpL, but there were no significant alterations in the relative iNOS gene expression or the generation of NO between virgin interpubic tissue and D18 IpL, despite the drastic changes in the histoarchitecture of the interpubic tissue observed between those groups. In addition, we observed that the concentration of NO and NOS expression in the D18 IpL was closer to the values measured for these elements in the placenta of mice C57 in late pregnancy (Ma et al., 2011). These findings show a similar temporal regulation to the uterine cervix, where iNOS expression and NO generation were low until cervical ripening at the onset of labor. However, iNOS increases NO generation at term in the rat (Buhimschi et al., 1996; Ali et al., 1997; Purcell et al., 1997; Chwalisz and Garfield, 1998a; Tschugguel et al., 1999; Ledingham et al., 2000; Shi et al., 2000) but decreases NO levels during

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late pregnancy and parturition in the bovine cervix (Aalberts et al., 2007). The mouse cervical remodeling process passes through four progressive and overlapping phases, termed softening, ripening, dilation/labor, and postpartum repair (Read et al., 2007). The physiological process of cervical ripening is controlled by mechanisms that can act independently, although these mechanisms converge on the same final pathway. Namely, there is a breakdown and decrease in cross-linked collagen and a reduction in the organization of long collagen fibers. This occurs without a decline in collagen content, but with an increased high molecular hyaluronic acid synthesis (Straach et al., 2005; Timmons and Mahendroo, 2006; Ruscheinsky et al., 2008; Timmons et al., 2009, 2010; Akins et al., 2011). Studies have highlighted that NO is an important element in controlling cervical function during the transition from pregnancy to the conditioning phase of labor (for review, see Chwalisz and Garfield, 1998b). Inducible NO may be required to increase the expression of MMP-8 and MMP-9 in the later stages of gestation, as these enzymes break down collagen during cervical ripening, leading to a decrease in cross-linked collagen and reduced organization of long collagen fibers, as described above. Consequently, a steady decrease in the birefringence due to changes in collagen organization can be observed as pregnancy progresses to term (Marx et al., 2006). According to Marx et al., the mechanisms regulating cervical ripening and parturition under normal conditions might independently regulate the expression of the iNOS and COX-2 genes. On one hand, it was recognized that mRNA levels of NOS are controlled by a paracrine regulator, which decreases the expression levels of NOS in the rat and human term uterus while simultaneously increasing it in the cervix during labor (Buhimschi et al., 1996; Purcell et al., 1997; Chwalisz and Garfield, 1998b; Tschugguel et al., 1999; Tornblom et al., 2005; Kuon et al., 2011). On the other hand, it was observed that treatment with the systemic NOS inhibitor (L-NAME) at middle and late pregnancy causes preterm delivery in mice (Tiboni and Giampietro, 2000; Tiboni et al., 2008) and guinea pig, but not in rat (see Chwalisz and Garfield, 1998b, for original contributions). Interestingly, both guinea pig and mouse relax the PS during pregnancy, but rats do not (Ortega et al., 2003). Thus, the NO contribution for mouse PS relaxation, as proposed in this study, became an outstanding question, at least in part, in view of studies showing that NO inhibitors (L-NAME) cause mouse preterm delivery (Tiboni and Giampietro, 2000; Tiboni et al., 2008). This controversy was also reported by Baccari and Bani (2008) when discussing the pathophysiological actions of relaxin in cervical softening, strictly related to its ability to activate endogenous pathways of NO. Keeping in mind the general similarities between cervical and PS connective tissue remodeling and the histoarchitectural adaptations of the reproductive tract as mentioned above, we propose some possible explanations for this outstanding question brought up by studies using NO inhibitors in the mouse. First, given that L-NAME was administered on D16–17 and

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the preterm delivery occurred until D18 in mouse (Tiboni and Giampietro, 2000; Tiboni et al., 2008), this treatment overlapped the softening to ripening phase in the cervix (Read et al., 2007) and the elongation to relaxed phase in PS (Pinheiro et al., 2004). Nevertheless, without direct iNOS involvement in the softening or elongation phases, preterm delivery occurred. Because NOS enzymatic activity rises significantly on the day of delivery, NO inhibitors may require several days to prevent ripening (Shi et al., 2000). Second, it would be difficult to stop fetal growth in L-NAME-treated, late pregnant C57 mice, which deliver pups of similar weight to those delivered by control females (Ma et al., 2011). Yet, all pups delivered preterm at 18 days of pregnancy were about 15% lighter when compared to those of the same lineage born at term (Murray et al., 2010). So, probably the maximal expansion and flexibility of the IpL at the time of labor is not needed during premature birth in mice when the pups are smaller. Remodeling and relaxation of the mouse PS requires a fine balance between deposition and degradation of the extracellular matrix. On one hand, thick collagen and newly forming elastic fibers (Moraes et al., 2003; Pinheiro et al., 2004), high molecular weight hyaluronic acid, and proteoglycans (Pinheiro et al., 2004, 2005; Garcia et al., 2008; Rosa et al., 2011b) are produced and contribute to IpL maximal expansion and flexibility. On the other hand, the balance between the expression of MMP-8 from D12 to D15, MMP-2 and -9 from D15 to D19, and cathepsin B and tissue inhibitor of metalloproteases (TIMP)-1 and -2 throughout pregnancy indicate that tight regulation of protein expression plays an important role in remodeling of the interpubic tissue to support its dynamic adaptation to the forces present during labor (Rosa et al., 2008, 2011ab). According to Marx et al. (2006), a decrease in iNOS expression and NO production may be required to increase MMP-8 and MMP-9 in the rat cervix at later stages of gestation. Thus, the expression of iNOS and NO production in the IpL of a pregnant mouse may be temporally regulated and may correlate with MMP-8 and MMP-9 expression, similar to the rat cervix. Both histoarchitectural changes and an elevation in MMP expression or activity are involved in disrupting the collagen fiber architecture in the mouse PS. These changes are particularly notable in the crimp structure, which is at least partially responsible for the late ligament enlargement observed during pregnancy (Pinheiro et al., 2004; Rosa et al., 2008, 2011a). There is an increased synthesis of high molecular weight hyaluronic acid and versican along with a decline in expression of the protease ADAMTS1 in the IpL on gestation D18. These patterns of expression are consistent with a stabilized hyaluronic acid-rich matrix and the increased tissue viscoelasticity found in ‘‘relaxed’’ IpL at its maximum length (Garcia et al., 2008; Rosa et al., 2011b). Furthermore, the interpubic fibroblast-like cells showed a permanent and homogeneous expression of asmooth muscle actin (a-SMA) and a transient pattern of vimentin and desmin expression with ultrastructural features of a typical myofibroblast during pregnancy (Moraes et al., 2004). Indeed, they present ultrastructural features

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consistent with the characteristics of both cell types, and also the ability to produce elastic fibers (Moraes et al., 2003). Changes in the extracellular matrix may cause a disruption in the actin cytoskeleton that would increase the G-actin level. This, in turn, would upregulate IL-1b-induced iNOS expression. In addition, NO may shift the steady-state balance of fibrillar (F-actin) toward globular actin (G-actin), which further upregulates IL-1b-induced iNOS and NO production in a positive feedback loop that can affect mesangial cell contraction (Zeng and Morrison, 2001) and can prevent the epithelial–mesenchymal transition and the generation of myofibroblasts (Vyas-Read et al., 2007). Interestingly, iNOS upregulation and a significant enhancement in the generation of NO in the IpL were detected on D19, the day of delivery. This is coincident with a time when the extracellular matrix is susceptible to profound changes, such as widespread collagen remodeling, and follows D18, when IpL cells acquire vimentin, desmin, and a-SMA (Moraes et al., 2004). In addition to vimentin and a-SMA, the differential expression of desmin in myofibroblast-like cells at D18 (Moraes et al., 2004) is closely associated with the plasma concentration of relaxin, which reaches high levels during pregnancy, specifically at D18 (Sherwood, 1994). Likewise, a similar expression pattern of a-SMA has been observed in human and bovine uterine cervical tissue (Montes et al., 2002; Aalberts et al., 2007; Malmstrom et al., 2007), and both a-SMA and desmin have been observed in rat cervical tissues during pregnancy (Varayoud et al., 2001). Recently it has been demonstrated that relaxin-induced NO production in lung (myo)fibroblasts may inhibit cell contractility (Huang et al., 2011). These findings suggest the regulation of a-SMA and desmin dynamics may be regulated by NO in the D19 IpL fibroblast-like cells, which might contribute to the complex adaptations of connective tissue cells observed in the relaxation phase. Although pregnancy-related PS modifications in humans are or less conspicuous when compared to rodent models, the mechanisms underlying pregnancy-related pubic symphyseal pain are a neglected, yet relatively common cause of pubic pain (Becker et al., 2010). Recently, accurate studies in primiparous women showed that not only pelvic floor muscle injury but also pubic fractures, capsular ruptures, and bone marrow edema occurred along the posterior and inferior para-symphyseal regions, where distractive forces may be most pronounced. These findings highlight the vulnerability to PS injury (Brandon et al., 2011; Miller et al., 2011). Thus, it is important to understand and recognize differences in pregnancy between mice and humans (Ratajczak and Muglia, 2008; Timmons et al., 2010). In summary, our study shows that NO generation and iNOS mRNA levels increase significantly between the partially relaxed IpL at D18 and the completely relaxed IpL at D19, which may indicate that NO plays a role during IpL relaxation in the mouse, as shown for rat uterine cervix ripening (Buhimschi et al., 1996; Ali et al., 1997; Shi et al., 2000). Cervix ripening during late pregnancy marks the transition from pregnancy to the conditioning phase of

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labor (see Chwalisz and Garfield, 1998a, for original contributions). Overall, we postulate that the significant increase in NO generation by iNOS in the mouse IpL at D19 may facilitate efficient relaxation of the mouse pelvic girdle, thereby contributing to an optimal accommodation by the birth canal and a safe delivery. A better understanding of NOS isoforms and their influences on the myofibroblast cytoskeleton and extracellular matrix could allow us to better monitor the behavior of NO targets, which may promote changes in the biomechanical characteristics of the extracellular matrix similar to the weakness of the birth canal caused by improperly formed elastic fibers (Liu et al., 2004; Drewes et al., 2007; Abramowitch et al., 2009; Rahn et al., 2009). These studies highlight the impaired connective tissue remodeling and the significant musculoskeletal histoarchitectural disruption elements of birth canal and pelvic organ support. The understanding of these elements is important for successful parturition, and for maintaining homeostasis of the genitourinary and digestive tract support and the pelvic skeleton after parturition.

MATERIALS AND METHODS Animals Inbred female C57Bl/6 mice (90 days old) obtained from the Multidisciplinary Center for Biological Investigation at UNICAMP were housed in cages in a room maintained at 25 C with controlled light (lights on from 06:00 to 18:00 hr), and pellet laboratory chow and water were available ad libitum. Females were mated with fertile males, and the presence of a vaginal plug was considered as day 1 of pregnancy (D1). Pubic symphyses or IpLs were obtained from the following groups between 11:00 a.m. and 12:00 p.m.: virgins (n ¼ 15) in estrus (Shorr, 1941); D18 (n ¼ 15); and D19 (n ¼ 15). As detailed below, three animals per group were used in each experiment, resulting in a total of 45 animals for this study. All of the surgical procedures were performed using aseptic technique, and the experimental protocols were approved by the Institutional Committee for Ethics in Animal Research (State University of Campinas, Protocol no. 1831-1). Light Microscopy Three mice from each group (virgin, D18, and D19) were used for light microscopy. The interpubic tissue (symphyses and ligaments) was fixed in situ in 4% paraformaldehyde (Merck, Darmstadt, Germany) in 0.1 M phosphate buffer at pH 7.4. The tissue was then removed and immediately immersed in the same fixative solution for 24 hr at 4 C. The tissue was decalcified in 5% (w/v) EDTA with 2% (v/v) paraformaldehyde in 0.1 M phosphate buffer, pH 7.4, for 5 days at 4 C. For immunohistochemistry, the samples were fixed for 7 hr and did not undergo the decalcification step. The samples were then dehydrated in a graded ethanol series and embedded in paraffin at 58 C. Five-micron sections were mounted on glass slides, deparaffinized, and stained with Masson’s Trichrome. Mol Reprod Dev 79:272–282 (2012)

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Immunohistochemistry Immunohistochemical procedures were performed according to the manufacturer’s recommendations. Briefly, non-decalcified sections were deparaffinized, and endogenous peroxidase activity was blocked using 0.3% hydrogen peroxide. Incubation in 5% bovine serum albumin (BSA) at pH 7.8 was used to block non-specific IgG binding. The sections were then incubated with anti-iNOS primary antibody at 1:50 (N9657—Sigma Company, St. Louis, MO) diluted in 0.1 M Tris-buffered saline (TBS) and 2% BSA in a humidified chamber overnight at 4 C. Next, the sections were incubated with the anti-mouse IgG-HRP secondary antibody at 1:500 (A9309 Sigma Company) for 1 hr. The reactions were developed using a mixture of 3.3diaminobenzidine (0.5 mg/ml; Sigma Company) and 0.3% hydrogen peroxide as a substrate. The sections were counter-stained with Harris’ hematoxylin and mounted in permanent mounting buffer. For negative controls, the primary antibody was replaced by non-immune mouse/ rabbit serum. The sections were examined and photographed with a Nikon Eclipse E800 light microscope. Immunoelectron Microscopy Interpubic tissues were collected and fixed in a solution containing 4% paraformaldehyde in 0.1 M PBS, pH 7.4, for 1 hr. Samples were dehydrated in an increasing gradient of dimethyl formamide and embedded in LR White resin (Electron Microscopy Sciences, Fort Washington, PA). Ultrathin sections (70 nm) were captured on nickel grids. The grids were immersed for 5 min in ultra-pure filtered water for hydration and incubated for an additional 5 min with 0.1 M Tris–HCl, pH 7.8. Non-specific reactions were blocked with 1% BSA in Tris–HCl 0.1 M for 40 min. Next, the grids were washed in 0.1 M Tris–HCl, pH 7.8, and incubated overnight with monoclonal anti-iNOS primary antibody (1:50 in 0.1% BSA and 0.1 M Tris–HCl, pH 7.8) in a humidified chamber. The next morning, grids were washed with 0.1 M Tris–HCl, pH 7.8, and incubated with anti-mouse IgG secondary antibody (1:50 in 0.1% BSA and 0.1 M Tris–HCl, pH 7.8) coupled to 10-nm gold particles (Electron Microscopy Sciences) for 1 hr. After washing with 0.1 M Tris–HCl, pH 7.8, the grids were placed on filter paper for drying. Sections were counterstained with 0.5% uranyl acetate for 2 min and with 2% lead citrate for 20 sec. Grids were analyzed using a FEI Tecnai 12 transmission electron . Brazil). microscope (State University of Londrina, Parana The negative control consisted of the omission of iNOS antiserum in the staining procedure, and incubation in buffer with non-immune serum diluted to the same concentration as the iNOS antibody. Nitrite Analysis Production of NO by cells in the interpubic tissue was determined by measuring the amount of nitrite, a stable metabolic product of NO. Interpubic tissue homogenates (0.1 g/ml of tissue wet weight) were assessed as described for the Griess reaction (Nitrate/Nitrite Colorimetric Assay Mol Reprod Dev 79:272–282 (2012)

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Kit, Cayman Chemical Company, Ann Arbor, MI), according to Collins et al. (2001). Ultra-filtrate tissue homogenates, standard curves, and reagent aliquots were employed according to the manufacturer’s recommendations. For each determination, 40 ml of tissue homogenate was mixed with 160 ml of Griess reagent in a round-bottom 96-well tissue culture plate, and the absorbance at 540 nm was measured on a microplate reader (MR 5000; Dynatech Labs, Inc., Chantilly, VA). The amount of nitrite was then determined by comparison of each sample with a NaNO2 standard curve. The NO detection limit was approximately 2.5 mM. We expressed the results of the NO detection assay as the amount of NO/g wet weight of tissue, as previously reported for the uterus and cervix in rats by Buhimschi et al. (1996).

Real-Time PCR Gene expression was assessed by quantitative, realtime PCR on the virgin, D18, and D19 groups. Total RNA was extracted from frozen tissue (n ¼ 3 mice per time point) using the Trizol Reagent (Invitrogen Life Technologies, Carlsbad, CA), and cDNA was synthesized using a RevertAid H Minus First Strand cDNA Synthesis Kit (Fermentas, Glen Burnie, MD). Both procedures were carried out according to the manufacturer’s recommendations. Real-time quantitative RT-PCR was performed using SYBR Green (Applied Biosystems, Foster City, CA) in the Applied Biosystems 7300 cycler. Each gene was normalized to the expression of the housekeeping gene, ribosomal protein 36B4 (Laborda, 1991). The primers for iNOS (nitric oxide synthase 2; NOS2, forward-50 GCAAACCCAAGGTCTACGTTCA-30 and reverse-50 GAGCACGCTGAGTACCTCATTG-30 , NM_010927.3) and 36B4 (forward-50 CACTGGTCTAGGACCCGAGAAG-30 and reverse-50 GGTGCCTCTGGAGATTTTCG-30 NM_007475.5) were purchased from Applied Biosystems. All primers were optimized and dissociation curves were analyzed to ensure that only one product was amplified. In each reaction, 20 ng of cDNA was used, according to the universal cycling conditions for the SYBR Green system. The results were normalized using the CT (threshold cycle) values of the housekeeping gene 36B4 on the same plate. To quantify and acquire the fold-increase of iNOS, the 2DDCt mathematical model was utilized, and the virgin group was considered as the calibrator. Efficiency of the iNOS assays was calculated using the equation E ¼ 10(1/slope), with resulting values of 0.95. All reactions were performed in triplicate on the same plate for each animal.

Statistical Analysis The results for the Griess reaction and quantitative real-time PCR were expressed as the mean  standard error mean. Statistical comparisons were done using ANOVA followed by the Tukey test, with P < 0.05 indicating significance. 279

Molecular Reproduction & Development ACKNOWLEDGMENTS This work is part of a Master’s dissertation submitted by C.F.M. to the Institute of Biology, State University of Campinas, in partial fulfillment of the requirements for a master’s degree. The authors would like to thank Mrs. Liliam Panagio for excellent administrative assistance, Dr. Hernandes F. €hne for their support during the Carvalho and Fabiana Ku lia Guadalupe Tardeli de Jesus real-time PCR and Dr. Ce Andrade for her support during immunoelectron microscopy analysis.

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