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Department of Molecular and Integrative Physiology (S.Z., O.D.S.) and College of Medicine (O.D.S.), University of Illinois at Urbana-Champaign, Urbana, Illinois 61801; and Department of Structural/Cellular Biology (P.A.F.), University of South Alabama College of Medicine, Mobile, Alabama 36688
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 Avenue, Urbana, Illinois 61801. E-mail: od-sherw{at}uiuc.edu
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Abstract |
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Introduction |
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The cellular mechanism(s) that regulates the accumulation of cervical and vaginal cells during the second half of pregnancy is not known. Homeostatic control of cell number is thought to result from the dynamic balance between programmed cell death (apoptosis) and cell proliferation (12). Apoptosis affects scattered single cells and involves cell shrinkage, chromatin condensation, and the formation of apoptotic bodies that contain nuclear fragments (13, 14). A striking feature that occurs in nearly all cases of apoptosis is the activation of calcium/magnesium-dependent endonuclease activity, which specifically cleaves cellular DNA between regularly spaced nucleosomal units. The end result is the generation of DNA fragments that can be stained immunohistochemically and localized in situ (15). There are reports that apoptosis occurs in the female reproductive tract in both normal cycling (16, 17) and pregnant rats (18, 19).
The first hypothesis tested in this study is that the rate of apoptosis in cervical and vaginal cells declines during the second half of rat pregnancy. Finding that it does, we tested the hypothesis that relaxin inhibits apoptosis by determining whether the rate of apoptosis in the cervix and the vagina increases after the passive neutralization of circulating endogenous relaxin.
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Materials and Methods |
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Exp 1: Rate of apoptosis during pregnancy
To test the hypothesis that the rate of apoptosis in the lower
reproductive tract declines during the second half of pregnancy,
cervices and vaginas were collected throughout most of gestation and
early postpartum. Four rats were anesthetized with ether and killed by
cervical dislocation between 0900–1100 h on each of days 5, 10, 15,
18, and 21 of pregnancy and days 1 and 2 postpartum. The cervices and
vaginas were quickly removed, cleaned, and weighed. Tissues were fixed
for 24 h in 10% neutral buffered formalin. After fixation,
tissues were dehydrated in an ascending series of ethanol, cleared with
xylene, embedded in paraffin, and sectioned to obtain 5-µm
sections.
Exp 2: Influence of endogenous relaxin on apoptosis during late
pregnancy
To test the hypothesis that relaxin inhibits the rate of
apoptosis in cervical and vaginal cells, endogenous relaxin was
passively neutralized. A monoclonal antibody for rat relaxin designated
MCA1 (21) was administered on days 19–21 of pregnancy,
when relaxin levels are maximal in the peripheral circulation
(8). The rationale for this experiment is that
immunoneutralization of the putative survival factor relaxin, during
the period when its effects are likely maximal, will cause pronounced
cell death in the cervix and the vagina. Additionally, the
neutralization of relaxin, at least 3 days before delivery, enabled
analysis of the effect of treatment over a 3-day period.
On day 9 of pregnancy, rats were laparotomized under ether anesthesia, and the number of implantation sites was determined. Only rats with eight or more implantation sites were used because serum relaxin levels are directly related to the number of conceptuses in rats with small litters (22). Animals were randomly divided into three treatments, with 20 rats per treatment. Unanesthetized rats were placed in a restraining device and injected via tail vein. One group received 10 mg MCA1 at 0900 h daily from day 19 to day 21 of pregnancy. The control groups received either 10 mg monoclonal antibody for fluorescein (MCAF, monoclonal antibody control; 11), or 1 ml PBS (vehicle control).
To determine the time course over which apoptosis occurs after the withdrawal of relaxin, cervices and vaginas from four rats per treatment were collected 6, 12, 24, 48, and 72 h after the initial injection of MCA1, MCAF, and PBS. Skeletal muscle, which is not a target for relaxin, was also collected from two rats per group, 24 h after treatment, as a negative tissue control. Tissues were fixed, embedded, and sectioned as described for Exp 1.
In situ localization of apoptosis in cervical and vaginal cells
Terminal deoxynucleotidyl transferase-mediated deoxyuridine
5'-triphosphate nick end-labeling (TUNEL), in conjunction with
morphometric analysis, was employed to detect and quantify cells
undergoing apoptosis. Sections were stained immunocytochemically by
TUNEL using the method described by Gavrieli et al.
(15). A commercial kit (ApopTag in situ
Apoptosis Detection; Intergen, Purchase, NY), which links
digoxigenin-nucleotide to DNA by terminal deoxynucleotidyl transferase
(TdT), was used. Sections were deparaffined with xylene, rehydrated
with a descending series of ethanol, incubated with proteinase K,
immersed in 3% aqueous hydrogen peroxide, and then pretreated with
equilibration buffer. DNA was labeled at the 3'-end by incubating
sections with a mixture of digoxigenin deoxynucleotide triphosphate,
unlabeled deoxynucleotide triphosphate, and TdT enzyme at 37 C for
1 h. Slides were washed with PBS and incubated with
antidigoxigenin antibody conjugated to peroxidase at room temperature
for 30 min. Slides were washed again in PBS, incubated with 3,3'
diaminobenzidine, counterstained with methyl green, mounted, and
sealed. Positive control slides were treated with deoxyribonuclease I
before the labeling reaction. Negative control slides were incubated
with labeling reaction solution devoid of TdT enzyme.
Sections were examined morphometrically with a BH-2 light microscope (Olympus Corp., Mellville, NY) equipped with a video camera and connected to a personal computer running a Stereo Investigator program (MicroBrightField, Inc., Colchester, VT). The Stereo Investigator program automatically controls the movement of the microscope stage to provide unbiased determination of the fields of analysis. For both the cervix and the vagina, epithelial cells and stromal cells were analyzed independently. The percentage of TUNEL-labeled cells (labeling index, LI) was determined by counting TUNEL-labeled cells divided by total cells in the same field and then multiplying by 100. Data were obtained from 4 rats/group, 3 sections (approximately 20µm apart)/rat, and at least 500 cells/section. Thus, at least 6000 cells were analyzed per group for the epithelium, and 6,000 cells were analyzed per group for the stroma.
Ultrastructural analysis of apoptosis in cervical and vaginal
cells
To confirm that cervices and vaginas demonstrating
elevated TUNEL-labeling contain apoptotic cells, transmission electron
microscopy was used to examine the morphological characteristics of
cells. Cervices and vaginas were collected 24 h after the initial
injection of MCA1 or MCAF, on day 19 of pregnancy, as described for Exp
2. Evaluation of cervical tissue was conducted by collecting 3 areas of
tissue from 2 MCA1-treated and 2 MCAF-treated pregnant rats. The small
pieces of tissue were placed in Karnovsky’s fixative (2%
glutaraldehyde and 2% paraformaldehyde in 0.1 M PBS) for
24 h, washed 3 times in 0.1 M cacodylate buffer, and
then shipped to the University of South Alabama College of Medicine.
There, the pieces of tissue were cut in half and processed for electron
microscopy evaluation as previously described for the rat uterus
(23). Thin sections were cut from 3 depths of each tissue
half, and 50 epithelial cells and 50 stromal cells were evaluated from
each section, for a total of 900 epithelial cells and 900 stromal cells
per animal. Two areas of vaginal tissue were collected from one
MCA1-treated and one MCAF-treated pregnant rat and handled as described
above. Therefore, 600 epithelial cells and 600 stromal cells were
evaluated per animal. A nucleus had to be observed in the cell in order
for the cell to be counted. The percent of apoptotic cells was
calculated.
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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. The cervical and vaginal wet weights
did not differ on days 5 and 10. However, the tissue weights increased
progressively between days 10 and 23 of pregnancy (P <
0.05). On the day of delivery (day 23 of pregnancy or day 1
postpartum), the cervical and vaginal wet weights were maximal, and
they decreased dramatically by the following day (day 2
postpartum).
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) was
approximately 2% on days 5 and 10, declined to approximately 0.5% on
days 15–21, and then rose dramatically to 18% by the second day after
delivery. In the cervical stroma, the LI was significantly lower on day
21 than earlier in pregnancy or days 1 and 2 postpartum. As with the
epithelium, the LI increased dramatically during the first 2 days
postpartum (Fig. 2B).
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). The LI in the vaginal epithelium and
stroma were lower on days 18 and 21 than either days 5 and 10 of
pregnancy or days 1 and 2 postpartum.
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). The maximal LI occurred 12 and
24 h after treatment. Whereas the LI in the cervical stroma did
not increase as markedly as in the epithelium, it was greater than in
controls 6 and 12 h after the neutralization of endogenous relaxin
(Fig. 4B). The LI in vaginal epithelial cells was greater 6, 12, and
24 h after initiation of neutralization of endogenous relaxin than
it was in controls. The maximal LI occurred 24 h after treatment
(Fig. 5A). Treatment did not influence
the LI in the vaginal stroma (Fig. 5B). The LI in skeletal muscle,
24 h after treatment, was only about 0.05%, and there was no
difference among treatment groups (data not shown).
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).
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). Cytoplasmic organelles, such as
mitochondria, were intact. The apoptotic stromal cells seemed to be
fibroblasts, as indicated by the large amount of rough endoplasmic
reticulum and by a lack of dense filaments found in smooth muscle
cells. The cervical epithelium of all samples from MCA1-treated animals
seemed disrupted, as indicated by spacing of adjacent epithelial cells
(Fig. 8A). This was not observed in the
tissues from MCAF-treated animals (Fig. 8B). In the epithelium, an
occasional apoptotic cell seemed to have been phagocytized by an
adjacent cell (Fig. 8A).
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).
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Discussion |
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One function of apoptosis is to maintain steady cell numbers in tissues (12). However, when tissues must grow, as is the case during pregnancy, a mechanism that can contribute to increased cell numbers is the reduction in the rate at which cells undergo apoptosis. This report indicates that during the first half of rat pregnancy, when the lower reproductive tract is not growing extensively, the rate of apoptosis is moderate and steady. However, during the second half of pregnancy, when cervical and vaginal growth increase progressively, the rate of apoptosis is reduced severalfold. Consistent with the view that apoptosis plays a role in regulating cell numbers is the observation that the rate of apoptosis is extremely high after parturition, when the size of the cervix and vagina decrease markedly.
Whereas the roles that hormones play to reduce the rate of apoptosis in the cervix and vagina during the second half of rat pregnancy are poorly understood, this study provides evidence that relaxin may act as a cell survival factor during this period. The observation that the rates of apoptosis are relatively low during the second half of pregnancy, when relaxin is secreted into the blood, provides indirect evidence that relaxin may inhibit the rate of apoptosis. The strongest evidence that relaxin is a cell survival factor is the finding that the rate of apoptosis increases markedly after the neutralization of endogenous relaxin during late pregnancy. Additionally, the finding that both cervical and vaginal wet weights were lower than controls after neutralization of relaxin is consistent with the view that relaxin promotes growth of the lower reproductive tract, at least in part, by inhibiting apoptosis.
Findings from the analysis of the effects of relaxin on apoptosis in cervical and vaginal tissue by electron microscopy were in general agreement with those obtained by the TUNEL method. There was an increase in the rate of apoptosis in epithelial cells and stromal cells in both the cervix and the vagina after immunoneutralization of relaxin. The rates of apoptosis after immunoneutralization of relaxin tended to be somewhat higher when determined by electron microscopy than when determined by the TUNEL method. A likely explanation for this is that fragmentation of the chromosomes has not occurred in nuclei with condensed chromatin. An increase in the rate of apoptosis after neutralization of relaxin was found in vaginal stromal cells by means of electron microscopy but not by the TUNEL method.
Relaxin is not the only hormone that regulates growth of the cervix and vagina in rats. Studies with ovariectomized nonpregnant rats demonstrated that relaxin’s growth-promoting effects on the cervix are estrogen-dependent (24, 25). Moreover, estrogen alone promotes growth of the cervix (24, 25) and vagina (26) in nonpregnant rats. Estrogen has also been demonstrated to prevent apoptosis in the rat vagina (16, 17, 27, 28). During the second half of rat pregnancy, developing ovarian follicles secrete increasing quantities of estrogen (29). Therefore, it seems likely that estrogen not only contributes to relaxin’s role as a cell survival factor but also, in and of itself, is a cell survival factor in the lower reproductive tract during rat pregnancy.
This report may provide insight concerning a mechanism associated with relaxin’s effects on other rat tissues and/or target tissues in other species during pregnancy. Specifically, relaxin-induced growth of the rat nipple (30, 31) and pig vagina, cervix, uterus, and mammary glands (32, 33, 34) during pregnancy may be attributable, at least in part, to a reduction in the rate of apoptosis.
Presently, the possibility that relaxin also increases the rate of cell proliferation in the cervix and vagina during rat pregnancy cannot be ruled out. Indeed, studies of porcine ovarian cells by Bagnell and co-workers (35, 36) provide evidence that relaxin promotes cell proliferation in an in vitro biological system. These workers reported that relaxin increased the rate of [3H]thymidine incorporation into cultures of porcine follicular granulosa cells and thecal cells. The hypothesis that relaxin both inhibits the rate of apoptosis and increases the rate of cell proliferation in the rat cervix and vagina is attractive because it provides complimentary mechanisms to account for the dramatic growth of the lower reproductive tract that occurs during the second half of pregnancy.
Studies concerning the changes that occur in the rate of cervical cell apoptosis during rat pregnancy are not in agreement. Previously, another laboratory reported that cervical DNA content declines steadily and by about 80% between day 12 and term (18). This laboratory reported that apoptosis increases progressively in cervical smooth muscle and fibroblast cells during the second half of rat pregnancy (18, 19). About 5% of both smooth muscle and fibroblast cells were reported to be apoptotic on day 5 of pregnancy, and the rate of apoptosis in both cell types was in the range of 30–50% by days 18 and 21 of pregnancy. In contrast, we previously reported that the cervical DNA content increases by more than 50% during the second half of pregnancy (5). Additionally, in the present study, we report that the rate of apoptosis declines during the second half of rat pregnancy in both the cervical epithelium and the cervical stroma. We report that the LI is not greater that 4% during pregnancy, and we found no evidence that smooth muscle cells undergo notable apoptosis. We cannot presently account for the differences in the findings between the two laboratories.
In summary, this study provides evidence that the rate of apoptosis in the cervix and vagina declines during the second half of rat pregnancy. Moreover, this study provides evidence that the reduction in the rate of apoptosis is attributable, at least in part, to the influence of relaxin. Therefore, it is concluded that relaxin-induced growth of the cervix and the vagina is likely attributable, at least in part, to inhibition of apoptosis.
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Acknowledgments |
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Footnotes |
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Received June 19, 2000.
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