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Ovary |
Department of Anatomy and Structural Biology, University of Otago School of Medical Sciences, Dunedin 9001, New Zealand
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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aging, ovary, ovulation
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Studies regarding proliferation in the OSE in vivo have been performed only in young animals [2–11], with the results of earlier studies suggesting that new oocytes are formed during each cycle from surface epithelial cells. To our knowledge, age-comparison studies have not been performed. Bullough [7] counted mitotic cells in the OSE in serial sections of mouse ovaries arrested in metaphase. The dividing OSE cells were classified according to their proximity to small follicles and stroma/interstitial cells, larger growing follicles, and corpora lutea in different stages of development. Mitosis was most commonly observed in cuboidal and columnar OSE cells. Dividing cells in the OSE of proestrous ovaries were more common close to rapidly growing follicles, whereas in preovulatory estrous ovaries, mitosis was restricted to a narrow, circular area surrounding the base of the now large, presumably preovulatory follicle. The maximum number of mitotically active OSE cells occurred shortly after ovulation in cells at the base of the now-ruptured follicle and later in the OSE over newly forming corpora lutea. The number of mitotic cells decreased during metestrus, and the lowest number of dividing OSE cells was observed at diestrus [7].
Wound healing takes longer in elderly people [12–15], and aged mice have been reported to take longer each cycle to ovulate all their oocytes [16]. This suggests that the re-epithelialization response to ovulation may be altered with age. Recent papers by Johnson et al. [17] and Bukovsky et al. [18] described the presence of mitotically active germline stem cells in the surface epithelium of the adult mouse ovary. Although the role of these cells in the adult ovary remains unclear, it is conceivable that such cells are involved in OSE replenishment or proliferation as well as primordial follicle renewal.
More than 90% of ovarian cancers are epithelial in origin and thought to arise from the OSE. Factors that decrease the total lifetime ovulation number, such as pregnancy and oral contraceptive use, also decrease the risk of epithelial ovarian cancer development, whereas risk increases with age [19–21]. The age of the individual and the number of ovulations previously experienced therefore may affect the response of the OSE to ovulation re-epithelialization.
The aims of the present study were to determine the following: 1) if a specific type of OSE cell (e.g., flat or cuboidal) is responsible for re-epithelialization, 2) the position of proliferating cell nuclear antigen (PCNA) immunoreactive cells on the ovarian surface relative to the ovulation site, 3) the time around ovulation at which the majority of PCNA immunoreactivity occurred, and 4) any changes in the pattern of PCNA staining with age and total lifetime ovulation number. The PCNA is a highly conserved, auxiliary protein of DNA polymerase that is involved in DNA synthesis and repair [8, 22]. An increase in the expression of the PCNA protein occurs if appropriate stimuli, such as growth factors, are received during the G1 phase of the cell cycle. Expression of PCNA protein increases during the G1 to S phases and reaches a plateau during the G2 phase, after which levels decrease considerably during the M phase and in quiescence [23]. Transcription of PCNA is not a direct result of DNA synthesis, but it is highly correlated with proliferation in normal cells [17, 23–26]. Studies comparing cell division using immunohistochemistry for PCNA and Ki67, another proliferation marker, with tritiated thymidine incorporation show comparable results [27, 28]. Furthermore, experiments blocking PCNA expression using antisense oligonucleotides suggest it is essential for DNA replication [23]. Therefore, PCNA immunohistochemistry was utilized to observe cells in the G1 and S phases of the cell cycle.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Experiments were approved by the Otago University Animal Ethics Committee. Ovaries were collected from three groups of Swiss Webster mice at various time points (approximately every 5 h) postovulation. The groups were 3-mo virgins (estimated median total lifetime ovulation number, 92; range, 13–234; n = 40 mice), 12-mo breeders (estimated median total lifetime ovulation number, 208; range, 97–324; n = 35 mice), and 8-mo virgin animals (estimated median total lifetime ovulation number, 652; range, 455–907; n = 15 mice) subjected to incessant ovulation by housing in a divided cage, alongside but separated from a male mouse [29]. Vaginal cytology was used to determine the stage of the estrous cycle for each mouse, and animals were mated and checked for a sperm plug to confirm estrus. A reference point of 0100 h was chosen to calculate time postovulation, because ovulation has been shown to occur around the midpoint of darkness [30]. Ovaries from six prepubertal (1-mo) animals were also collected. One ovary from each animal was viewed under the scanning electron microscope, and the remaining ovary was sectioned for histology and immunohistochemistry. Housing of animals, vaginal cytology, collection and processing of tissue, and estimation of total lifetime ovulation number have been described previously [29].
Scanning Electron Microscopy
Following dissection and bursal removal, the ovaries were fixed in 2.5% (v/v) gluteraldehyde and 1% (v/v) osmium and then processed for scanning electron microscopy. Ovaries were viewed using the Cambridge Stereoscan 360 SEM, and measurements of the diameter of all visible ovulation sites were taken. More detailed methods have been described previously [29]. The diameters of ovulation sites were plotted against time for each of the different animal groups, and the rates of ovulation site closure were compared using regression analysis.
Immunohistochemistry
Serial sections (thickness, 4 µm) of each ovary were cut from one to three animals per time point. Every 10th section was mounted and stained with hematoxylin and eosin. The number of ovulation sites seen was counted, and between two and eight ovulation sites per time point were randomly chosen for immunohistochemical analysis. Two sections through the middle of the point of rupture for each chosen ovulation site were used for immunohistochemistry against PCNA (monoclonal; PC10; Novocastra Laboratories Ltd., Newcastle-upon-Tyne, U.K.). Deparaffinized sections were rehydrated, and antigen retrieval was performed using an 800-W microwave for 15 min in 350 ml of 0.01 M sodium citrate buffer (pH 6.0). Slides were left to stand for 15 min in the hot buffer before washing in PBS. Endogenous peroxidase was blocked with 3% hydrogen peroxide in methanol for 5 min, and after further washes, nonspecific binding was minimized by incubation with normal goat serum (1:20 in PBS, 30 min; Sigma-Aldrich, Inc., St. Louis, MO). Sections were incubated for 1 h at room temperature in a moist chamber with the anti-PCNA antibody (1:50 in 0.01 M phosphate buffer, 0.2% Tween 20, 1% sodium chloride, and 1% bovine serum albumin). Sections of small intestine were used as a positive control [24]. A negative control without PCNA antibody was performed on a serial section on each slide. Sections were washed in PBS before incubation in biotinylated goat anti-mouse secondary antibody for 30 min (1:200 in PBS; Amersham Pharmacia Biotech, U.K.), followed by incubation in streptavidin biotinylated horseradish peroxidase for 30 min (1: 100 in PBS; Amersham Pharmacia Biotech). Antibody complex was detected with diaminobenzidine (Vector Laboratories, Inc., Peterborough, U.K.) for 2 min. Sections were counterstained with Gill hematoxylin. Staining with PCNA was quantified by scoring OSE cells for staining (positive or negative), cell shape (cuboidal or flattened), and position on the ovary (ovulation site, adjacent area or random area of OSE). Cells were defined as flattened if the length of the cell was longer than the height. On each section, all OSE cells overlying the chosen ovulation site, approximately 100 OSE cells adjacent to the site, and approximately 100 OSE cells from a random area not associated with an ovulation site (chosen using a grid and a random number table) were assessed for PCNA staining.
Data Analysis
The percentage of cells stained positive for PCNA was compared with age, cell shape, area, and time. The data were log transformed before ANOVA (SPSS 10 for the Macintosh). Post-hoc analysis using the Bonferroni test was performed to determine which of the specific groups were different. Each ovulation site was chosen randomly for study and was considered to be an independent data point, because within-animal and between-animal variations in data from ovulation sites were comparable.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Ovulation site closure can be represented by the equation y = –ax + b, where y is the ovulation site diameter (µm) at x, which is the time (h) after ovulation. The decrease in ovulation site diameter is shown in Figure 1, and the rates of closure, correlation coefficients, and times required for re-epithelialization in the three animal groups are shown in Table 1. The rates of ovulation site closure were not significantly different from each other (P > 0.10). The maximum size of the ovulation site (170–180 µm) also did not vary significantly between the groups (P > 0.10). A few small, denuded areas were observed around ovulation sites at later time points in ovaries from the 8- and 12-mo animals, suggesting that the process of re-epithelialization may become less efficient in older animals (data not shown).
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PCNA Immunohistochemistry
Nuclear staining for PCNA was detected in ovaries from all ages. Figure 2 shows representative images of PCNA staining in 3-mo and 8-mo ovaries. Just after ovulation, the number of cells positive for PCNA in the OSE surrounding a freshly ruptured follicle (Fig. 2A) was high, and most stained cells were cuboidal (Fig. 2B). Fewer cells were stained adjacent to the ovulation site (Fig. 2B), and stained cells were rare in areas not associated with ovulation sites (Fig. 2C). Approximately 2 days following ovulation, cuboidal OSE cells on the side of the newly developing corpora lutea showed positive PCNA staining (Fig. 2E), whereas other areas of OSE contained few PCNA-immunoreactive cells (Fig. 2F). When overall percentages of PCNA-immunoreactive cells were compared with age and total lifetime ovulation number, regardless of cell shape, area, and time, the prepubertal 1-mo ovaries showed significantly higher percentages than the other three groups (P < 0.05). The 3-mo ovaries also showed significantly higher numbers of PCNA-immunoreactive cells than both the 8-mo and 12-mo animals (F = 56.49, P < 0.01) (Fig. 3).
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In the 3-mo animals only, significantly more cuboidal than flattened OSE cells stained positive for PCNA (P < 0.05) (Fig. 4). The PCNA immunoreactivity was significantly higher at the site of ovulation in the 3-mo animals and when data from all groups were combined (P < 0.05) (Fig. 5). The PCNA immunoreactive cells were observed in the extraovarian mesothelium, when present in some sections, and at the hilus (the neck of the ovary), where the mesothelium is continuous with the OSE (Fig. 2H), in all groups. In addition, PCNA staining was seen in the granulosa and thecal cells of follicles, with fewer stained cells in atretic follicles (Fig. 2G) than in healthy follicles (Fig. 2E) and in corpora lutea, in all groups (Fig. 2, C, F, and H).
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The numbers of PCNA-immunoreactive cells varied with the relative time after ovulation in the 3-mo and 12-mo groups and in the combined data. Significantly more staining (P < 0.05) was seen on Days 4 and 5 (90 and 100 h), when data from all groups were combined (Fig. 6). The position on the ovary (ovulation site, adjacent to the ovulation site, or away from ovulation sites) was analyzed separately for differences in PCNA staining density with time using data from all groups combined (Fig. 7). Higher numbers of cells overlying the ovulation site stained positive for PCNA on the day of estrus (5–20 h). A second, smaller peak occurred at the end of the second day (50 h). A peak was also found in PCNA staining in the OSE adjacent to the ovulation site at this time. The OSE overlying all three areas contributed to the peak in the combined data seen on Day 4 (90 h), whereas the peak on Day 5 (100 h) was related to higher densities of PCNA-immunoreactive cells in the OSE adjacent to and away from the ovulation site (Fig. 7).
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Significant differences in PCNA staining were also found between cell shape and position when the data were combined, regardless of age or ovulation number. Cuboidal cells overlying ovulation sites and adjacent areas were more likely to be positive than were flattened cells at the same sites. Similar staining levels were seen in cuboidal and flattened cells from randomly chosen areas of OSE away from ovulation sites (data not shown).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In the present study, low densities of PCNA-immunoreactive cells were observed in OSE not associated with an ovulation site, whereas the highest PCNA immunoreactivity was seen in cuboidal cells at the ovulation site the day following ovulation, during re-epithelialization. These results are similar to those of other studies showing proliferation in rodent or rabbit OSE to be highest at estrus and metestrus [2, 4, 10] or immediately postovulation [6, 9]. Other studies have shown maximum proliferation at 6–12 h [7] or at 24 h postovulation [11]. An increase in PCNA immunoreactivity was also observed in the present study on Days 4 and 5 following ovulation, in OSE adjacent to the ovulation site and in random areas of OSE. This equates to the time when complete re-epithelialization of the ovulation site has occurred. As the mice in the present study were mated and, therefore, assumed to be pregnant, the increase in PCNA staining may reflect proliferation of the OSE to accommodate corpora lutea or even the next wave of developing follicles, because large follicles have been found in the initial stages of pregnancy in mice [31].
The rise in cell division in the OSE just after estrus or before ovulation may be related to the relative concentrations of estrogen and progesterone to which the OSE is subjected. Estrogen stimulates OSE cell proliferation in vitro [32], whereas progesterone inhibits estradiol stimulation of OSE proliferation in vitro [28, 33]. This suggested to Murdoch and Van Kirk [33] that progesterone secretion from the corpora lutea, during the luteal phase of the menstrual cycle, inhibits OSE proliferation, allowing the repair of genetically damaged OSE. The OSE then proliferates on regression of the corpora lutea in response to the estradiol from the next preovulatory follicle [33]. Our observations with scanning electron microscopy and histology do not support this idea, because the mouse ovary appears to be completely re-epithelialized 3 days following ovulation in the presence of corpora lutea.
The small number of OSE cells positive for PCNA seen in the 8-mo and 12-mo ovaries appeared to provide too few new cells to cover the ovulation sites. These data lead us to question from where the cells come from to cover the site. Mechanisms to re-epithelialize the ovulation site could involve proliferation before ovulation and migration of new cells to the ovulation site. Given the speed of closure in the absence of apparent cell division, this mechanism is likely. Proliferation before ovulation has been shown by others [34] and is thought to accommodate the increasing size of the preovulatory follicle. Wrinkles and layers of OSE were seen in the 8-mo and 12-mo animals under the scanning electron microscope [29], but it is not possible to show whether these are new or migrating cells ready to cover the ovulation site. As the animal ages, the rates of OSE cell division and/or apoptosis may be decreased [12, 35, 36], because wrinkles and layers are rarely observed in 1-mo and 3-mo animals [29].
Proliferation may also occur in the extraovarian mesothelium, followed by the migration of new cells onto the ovarian surface. Proliferation of the extraovarian mesothelium has been reported [5, 7], and Beller et al. [5] suggest that this is how the ovulation site is covered. More than twice the amount of proliferation seen in the OSE has been found in the extraovarian mesothelium surrounding the uterine horns and in nearby peritoneum in superovulated prepubertal mice as measured by incorporation of tritiated thymidine [5]. This proliferation occurred following injection of eCG but before hCG injection, and it continued until after ovulation. This also suggests that cells are dividing in advance of ovulation. Furthermore, cells in the extraovarian mesothelium and around the hilum of the ovary have been shown to divide in response to both diethylstilbestrol [5] and estrone [11]. Estrogen levels peak just before ovulation, possibly stimulating proliferation in the OSE and the extraovarian mesothelium.
In summary, the process of ovulation site closure in 3-mo animals has been shown to be similar to the findings reported by others, with most PCNA immunoreactivity being noted in cuboidal cells at the site of ovulation on the day of estrus. Lower numbers of PCNA-stained cells were observed in the 8-mo and 12-mo animals, suggesting that the process of ovulation site re-epithelialization may be altered in these older animals. However, both scanning electron microscopy and histology showed that ovulation site closure was complete within 3 days in all age groups. We speculate that either most cell division occurs in the OSE before ovulation or that the cell division occurs in the extraovarian mesothelium, and cells migrate to cover the ovulation site.
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FOOTNOTES |
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2 Correspondence: Jean. S. Fleming, Department of Anatomy and Structural Biology, University of Otago School of Medical Sciences, P.O. Box 913, Dunedin 9001, New Zealand. FAX: 64 3 479 7254; jean.fleming{at}stonebow.otago.ac.nz
Received: 31 March 2004.
First decision: 16 April 2004.
Accepted: 21 June 2004.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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100 cells) were scored from six animals. In 3-mo animals, 226 areas were scored from 21 animals. In 8-mo animals, 178 areas were scored from 15 animals, and in 12-mo animals, 257 areas were scored from 25 animals. Data are expressed as the mean ± SD. Different letters indicate a significant difference between age groups. Data were log transformed, followed by a one-way ANOVA and then a Bonferroni test (P < 0.05)
