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Department of Molecular and Integrative Physiology (S.Z., O.D.S.) and the College of Medicine (O.D.S.), University of Illinois at Urbana-Champaign, Urbana, Illinois 61801
Address all correspondence and requests for reprints to: Dr. O. D. Sherwood, Department of Molecular and Integrative Physiology, University of Illinois at Urbana-Champaign, 524 Burrill Hall, 407 South Goodwin Ave, Urbana, Illinois 61801. E-mail: od-sherw{at}uiuc.edu
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Abstract |
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There were three treatment groups (N = 4/group). Five milligrams of a monoclonal antibody for rat relaxin, designated MCA1, were injected iv daily on days 12–21 of gestation to treatment group MCA1. Control groups received either 5 mg of monoclonal antibody for fluorescein (MCAF; monoclonal antibody control) or 0.5 ml PBS (vehicle control). Vaginas were removed on day 22 of pregnancy, fixed in 10% neutral-buffered formalin, and embedded in paraffin. Tissue sections (5 µm) were stained with Gomori’s trichrome to visualize collagen, or orcein to visualize elastin. Measurements were performed with a light microscope equipped with a video camera connected to a computer. Within the vaginal stroma, the density of collagen fiber bundles was lower, the length of elastin fibers was shorter, and the cross-sectional area and wall thickness of arteries were greater in relaxin-replete control rats than in relaxin-deficient MCA1-treated rats. These relaxin-induced changes in the stroma appear to account, at least in part, for the hormone’s softening effect on the vagina. Within the epithelium, there were approximately 2-fold more basal and mucus-secreting cells in relaxin-replete control rats than in MCA1-treated rats. The relaxin-induced accumulation of epithelial cells appears to contribute to vaginal growth.
We conclude that relaxin plays a role in preparing the vagina as well as the cervix for rapid and safe delivery in pregnant rats.
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Introduction |
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A fundamental step toward a better understanding of the mechanism(s) whereby relaxin brings about its actions on the vagina is to examine its effects on the histological characteristics of the tissue. The histological changes associated with relaxin-induced growth and softening of the rat cervix during pregnancy were reported (8). Qualitative and quantitative analysis of cervical histological characteristics showed that in relaxin-deficient MCA1-treated rats, the epithelial cell density was lower, the collagen fiber bundles were more compact, the elastin fibers were longer, and the arteries had smaller cross-sectional area than in controls (8). Relaxin had no apparent effect on the height of the epithelial cell layer (8). The vagina and cervix are contiguous and share similar structural features, such as a lumen lined with epithelial cells and a stroma rich in collagen and elastin fibers.
We hypothesized that relaxin promotes vaginal softening and does so by inducing histological changes that are similar to those that occur in the cervix. Accordingly, there were two objectives to this study. The first objective was to determine if relaxin promotes vaginal softening during the second half of pregnancy. The second objective was to determine the effects of relaxin on the histological characteristics of the vagina during this period. To accomplish both objectives, endogenous circulating rat relaxin was neutralized with monoclonal antibody MCA1 throughout the second half of pregnancy.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Treatments
Pregnant rats were randomly divided into three groups: MCA1,
MCAF (monoclonal antibody for fluorescein; monoclonal antibody
control), and PBS (vehicle control). Endogenous relaxin was neutralized
with a monoclonal antibody for rat relaxin designated MCA1 as
previously described (4, 5, 7, 8, 11, 12). In brief, five
milligrams of MCA1 were injected into unanesthetized restrained rats
once daily via tail vein between 0900–1100 h from days 12–21 of
pregnancy. Control rats received either 5 mg MCAF or 0.5 ml PBS.
Effects of endogenous relaxin on vaginal softening
There were six rats per treatment group. Rats were anesthetized
with ether and killed by cervical dislocation at 0900 h on day 22.
The fetuses were counted, removed from the uterus, cleaned, and
weighed. Both the vagina and the cervix were quickly removed, trimmed
of fat and connective tissue, and weighed. Vaginas were placed in
ice-cold Krebs-Ringer bicarbonate buffer (pH 7.5) until their
extensibilities were determined approximately 1 h later.
Vaginal extensibility was determined as described previously for cervices (3) with modifications. In brief, a 1.5 x 1 cm strip was cut from each vagina. The strip was suspended between two 1 cm wide stainless steel tissue clamps (constructed by the University of Illinois School of Life Sciences Machine Shop), so that 1 cm x 1 cm of tissue was suspended between the two clamps. The vaginal strip was placed in a 60 ml organ bath containing Krebs-Ringer bicarbonate buffer oxygenated with 95% O2 –5% CO2. The lower clamp was fixed to the bottom of the organ bath, and the upper clamp was mobile and connected to a Grass FT03 force displacement transducer (Grass Instruments, Quincey, MA). The temperature was maintained at 37 C by circulating water through the outer chamber of the organ bath. Transducers were calibrated in grams before the measurements were started, and tension generated within 1 cm2 strips of vaginal tissue was expressed in grams. Outputs from the transducers were recorded on a Macintosh computer. For each vaginal strip, the distance between the two clamps was gradually increased until a slight increase in tension was recorded. This was the resting tension of the tissue. Each vaginal strip was allowed to equilibrate for 15 min. The distance between the two hooks was then increased by 2-mm increments at 15-min intervals until a total of 14 mm extension was applied to each vaginal strip. Tension developed within the vaginal tissue immediately after extension was recorded as initial tension, and that tension remaining within the tissue 15 min after extension was recorded as final tension. Tension that developed at extension was plotted vs. millimeters of extension. The slope of the linear regression of grams tension/millimeter extension for each treatment group was determined.
Effects of endogenous relaxin on vaginal histology
Four rats from each of groups MCA1, MCAF, and PBS were
anesthetized with ether and killed by cervical dislocation at 0900
h on day 22 of pregnancy. Cervices and vaginas were quickly removed,
cleaned, and weighed. Fetal number and fetal weights were determined.
Each vagina was fixed whole in 10% neutral-buffered formalin (NBF) for
2 h. After this short prefixation, the vagina was cut in
cross-section at two places to obtain a cephalic piece, middle piece,
and caudal piece. Vaginal pieces were fixed in NBF for an additional
24 h, then dehydrated in an ascending series of ethanol, cleared
with xylene, embedded in paraffin, and sectioned to obtain 5 µm thick
sections (13). Sections were then stained with Gomori’s trichrome for
collagen or orcein for elastin (13).
Morphometric analysis of the vagina was performed with an Olympus Corp. (Mellville, NY) BH-2 light microscope equipped with a video camera connected to a Power Macintosh 7100/66 computer. Images were analyzed digitally using the public domain NIH image program (developed at NIH and available from the Internet by anonymous FTP from zippy.nimh.nih.gov or on floppy disk from the National Technical Information Service (Springfield, VA), part number PB95–500195GEI). Data were obtained from four rats/group, three vaginal blocks (cephalic, middle, and caudal pieces)/rat, and four sections (approximately 20 µm apart)/block. Since five randomly selected fields/section were examined for each histological feature, a total of 60 fields per animal and 240 fields per group were analyzed.
The influence of relaxin on vaginal stroma or stroma plus adventitia was determined by analyzing the following: total cross-sectional area and thickness of the stroma, density of collagen fiber bundles in the stroma, length of elastin fibers in the stroma plus adventitia, cross-sectional area of arterial lumina in the stroma plus adventitia, and thickness of arterial walls in the stroma plus adventitia. To measure collagen density all the collagen fibers in a field were highlighted, and the number of highlighted pixels was divided by the total number of pixels in the field and expressed as a percentage of the field. To measure elastin fiber length, more than 10 elastin fibers were measured in each field. Thus, the lengths of more than 2,400 elastin fibers were measured per group. The cross-sectional area of 240 arteries (one per field) were determined per group. The arterial walls appeared to be of uniform thickness. Therefore, one determination was made of wall thickness per artery.
Analysis of vaginal stroma cells is complicated by the presence of
several cellular components. These include isolated stromal cells
(fibroblasts, mast cells, polymorphonuclear leukocytes, etc.), cells
associated with blood vessels and extensive smooth muscle cell bundles
that are located near the periphery of the vagina (Fig. 1
). A recent study in our laboratory
demonstrated that smooth muscle cells in cervical and vaginal stroma do
not proliferate extensively during the second half of rat pregnancy
(14). Accordingly, only isolated stromal cells located between the
epithelium and the smooth muscle cell bundles were analyzed without
regard to cell type. Analysis of this predominant stromal area included
density of stromal cells (cells/mm2) and total stromal
cells per cross-sectional area of the vagina.
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Statistical analysis
All data were analyzed by one-way ANOVA and Tukey’s test.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). Consistent with
previous findings, the mean vaginal wet weights and cervical wet
weights were much greater (P < 0.01), and mean fetal
weights were slightly lower (P < 0.05) in controls
than in MCA1-treated rats (7, 11, 15).
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shows the tensile
properties of vaginas obtained from MCAF-, PBS-, and MCA1-treated rats.
With all three groups, the initial tension did not increase until the
tissue was extended 6 mm. At subsequent extensions, the initial rise in
tension at extension was followed by a marked decay in tension over the
15-min period between extensions. Over the range from 8 mm to 14 mm
extension, the mean tensions generated within vaginas obtained from
MCAF- and PBS-treated control rats were significantly lower at both
initial tension (Fig. 2A), and final tension (Fig. 2B) than those
generated within vaginas obtained from MCA1-treated rats.
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Morphometric analysis of relaxin’s effects on the vaginal stroma are
shown in Fig. 3. The total
cross-sectional area of vaginal stroma did not differ among treatment
groups (Fig. 3A). However, in vaginas obtained from control rats the
thickness of the stroma (Fig. 3B), the density of collagen fiber
bundles (Fig. 3C), and the length of elastin fibers (Fig. 3D) were
lesser; and the cross-sectional area of arterial lumina (Fig. 3E) and
thickness of arterial walls (Fig. 3F) were greater than in vaginas
obtained from relaxin-deficient MCA1-treated rats. Relaxin also
influenced the cellular composition of the vaginal stroma. Both the
density (Fig. 4A) and total number of
stromal cells per cross-sectional area of vagina (Fig. 4B) were greater
in relaxin-replete controls than in relaxin-deficient MCA1-treated
rats.
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contains photomicrographs
of sections of vaginas obtained from PBS- and MCA1-treated
animals. Differences in the histological characteristics of both the
stroma and the epithelium of vaginas obtained from control and
relaxin-deficient rats were so marked they were readily apparent by
visual examination. Note the lower density of collagen fiber bundles in
relaxin-replete control rats (Fig. 5, A and B) than in
relaxin-deficient MCA1-treated rats (Fig. 5, C and D). Also it is
obvious that the height of the entire vaginal epithelium in
relaxin-replete rats (Fig. 5A) was greater than that in
relaxin-deficient rats (Fig. 5C). The difference in height of the
epithelium was attributable to differences in the thickness of both the
mucus-secreting cells (compare Fig. 5A with Fig. 5C) and basal cells
(compare Fig. 5B with Fig. 5D).
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and 7. Both the height of entire epithelium
and the circumference of the vaginal lumen were greater in controls
than in relaxin-deficient MCA1-treated rats (Fig. 6). Analysis of the
two epithelial cell strata indicated that the height of both the basal
cells and mucus-secreting cells were greater in relaxin-replete rats
than in relaxin-deficient rats (Fig. 7, A and B). This finding is
attributable, at least in part, to more layers of both basal and
mucus-secreting cells in the relaxin-replete groups (Fig. 7, C and D).
Because both the circumference of the lumen and the number of cell
layers per stratum are greater in relaxin-replete animals, the total
epithelial cells in each stratum per circumference of the vagina in
relaxin-replete animals is more than twice that in the
relaxin-deficient MCA1-treated animals (Fig. 7, E and F). There was no
difference among the three treatment groups in the ratio of
mucus-secreting epithelial cells and basal epithelial cells (data not
shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The observation that the tension generated with extension of vaginas obtained from relaxin-deficient MCA1-treated rats on day 22 of pregnancy was significantly greater than that generated in vaginas obtained from controls can be interpreted to indicate that relaxin facilitates the passage of fetuses though the vagina as well as the cervix (5, 15) at delivery in rats.
Several of the relaxin-induced changes in the histological characteristics of the vaginal stroma may account for relaxin’s effects on vaginal extensibility (softening). These histological changes include reduction in the thickness of the stroma, density of collagen fiber bundles, and length of elastin fibers. The mechanisms whereby relaxin reduces the density of collagen fiber bundles and length of elastin fibers in its target tissues are poorly understood. The finding that vaginal blood vessels in MCA1-treated rats are smaller than in controls is consistent with previous evidence that relaxin causes blood vessel dilation in mouse uterine endometrium (16), rat myometrium (17), rat and pig cervix (8, 18), mouse mammary gland (19), and rat mammary nipple (12). Relaxin-induced enlargement of blood vessels could increase blood supply to support vaginal growth during the second half of pregnancy. The mechanism whereby relaxin causes blood vessel dilation is not understood. It has been postulated that relaxin may mediate its effects on vascular smooth muscle by increasing the production nitric oxide. Bani (20) recently reported that bovine vascular smooth muscle cells respond to relaxin in vitro by increasing the production of nitrites and intracellular levels of cGMP, the mediator of the cell response to nitric oxide (21). The finding that relaxin increases the thickness of arterial walls can be interpreted to indicate that relaxin induces proliferation of blood vessel cells. Burger and Sherwood (14) recently reported that the percentage of new cells surrounding cervical blood vessels at term was greater in relaxin-replete rats than in relaxin-deficient rats.
The finding that relaxin increases the number of stromal cells in the vagina is consistent with a study (16) that demonstrated that relaxin may synergize with steroids to promote proliferation of endometrial stromal cells. The influence of relaxin on the function of stromal cells is not clear. It was reported that relaxin promotes collagenase activity in cultures of human cervical stromal cells (22) and induces decidual transformation of endometrial stromal cells in mice (16).
Whereas relaxin-induced changes in the histological characteristics of the stroma appear to be largely responsible for softening of the vagina, the relaxin-induced accumulation of epithelial cells contributes to vaginal growth (6, 7). The observation that relaxin promotes epithelial cell growth by increasing both the number of epithelial cell layers and the total epithelial cells by about 2-fold is consistent with our previous finding that relaxin-replete rats have approximately a 2-fold greater vaginal DNA content than do relaxin-deficient rats (6, 7). The mechanism whereby relaxin brings about an increased in the number of vaginal epithelial cells is not known. The present findings can be interpreted to indicate that relaxin promotes cell proliferation and/or prevents apoptosis in the vaginal epithelium during the second half of pregnancy. Regardless of the mechanism whereby relaxin increases the accumulation of vaginal epithelial cells, the finding that the ratio of basal to mucus-secreting cells appears to remain constant seems noteworthy. We cannot presently explain this finding. The physiological significance of the dramatic increase in vaginal epithelial cells is not known with certainty. It seems likely that the increase in total epithelial cells contributes to the increase in the circumference of the vagina, which in turn contributes to rapid and safe delivery. Also, the increase in layers of mucus-secreting cells may facilitate birth by increasing lubrication of the birth canal. The relaxin-induced increase in vaginal epithelial cells may be beneficial to the mother rat. The increase in layers of both basal and mucus-secreting cells may protect the lower reproductive tract during the delivery.
Though the mechanisms whereby relaxin alters histological characteristics are presently unclear, two lines of evidence indicate that relaxin promotes tissue remodeling by similar mechanisms in the vagina, cervix, mammary gland, and nipples during the second half of rat pregnancy. First, relaxin-induced histological changes of the vagina are similar to those in the cervix (8), mammary gland (11), and mammary nipples (12). In all four tissues, the collagen density was lower; elastin fibers were shorter; and blood vessels were larger in control rats than in relaxin-deficient animals. Second, in all four tissues, specific relaxin-binding sites were found associated with the same cell types; namely, the epithelial cells, smooth muscle cells, and blood vessels (7, 23, 24, 25).
In summary, this study demonstrates that relaxin promotes softening of the vagina during the second half of rat pregnancy. Morphometrical analysis demonstrated that relaxin induces changes in the histological characteristics of the vaginal stroma that are similar to those the hormone induces in the cervical stroma. These changes likely contribute to vaginal softening. Relaxin also promotes a marked increase in vaginal epithelial cells. The increased epithelial cells may facilitate birth by contributing to increased vaginal lumen circumference and increased secretion of mucus into the birth canal. We conclude that relaxin plays a role in preparing the vagina as well as the cervix for rapid and safe delivery of the pups in pregnant rats.
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Acknowledgments |
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Received June 2, 1998.
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