Abstract
A cause-effect relationship between ovulation and common (surface) epithelial ovarian cancer has been suspected for many years. The ovarian surface epithelium apparently becomes exposed to genotoxins that are generated during the ovulatory process. Intensive egg-laying hens readily develop ovarian carcinomatosis. Indeed, elevated levels of potentially mutagenic 8-oxo-guanine adducts were detected in avian ovarian epithelial cells isolated from the apical surfaces and perimeters of pre-and postovulatory follicles, respectively. Internucleosomal DNA fragmentation indicative of apoptosis was evident in ovarian surface epithelial cells associated with the formative site of ovulation (stigma line) and regressive ruptured follicles. It is conceivable that a genetically altered progenitor cell with unrepaired DNA but not committed to death (i.e., a unifocal “escape”) could give rise to a transformed phenotype. Hence, the high rate of ovarian cancer in egg-laying hens could be the consequence of genomic damages to the ovarian surface epithelium associated with incessant ovulations, thereby increasing the likelihood of mutation and clonal expansion.
Ovaries of most vertebrates are covered by a simple layer of epithelial cells. Surface epithelial cells are supported along the ovarian cortical interstitium (tunica albuginea) by a basal lamina and are held together laterally by desmosomes and gap or tight junctions. Outgrowth of a follicle selected to ovulate brings it into apposition with the ovarian surface (1–5). The ovarian surface epithelium is therefore subjected, within a limited diffusion radius, to inflammatory mediators and oxyradicals liberated during the mechanics of ovulatory follicular rupture (6–8).
Base damages to DNA caused by reactive oxygen species are an inevitable by-product of physiological metabolism. To combat this predicament, animals have evolved elaborate enzymatic antioxidant defense mechanisms; however, these are less than perfect, and some oxidants find their way to DNA targets. Oxidative damage products in DNA are a significant contributor to the risk of cancer development (9–11).
The majority (>90%) of cancers of the ovary apparently originate from a surface epithelial cell perturbed by genotoxins produced at ovulation (8, 12–14). Ovarian surface epithelial cells that overlie the formative ovulation site (stigma) in mammals suffer extensive or irreparable damages and become apoptotic. Those cells at the margins of ruptured follicles (sheep, human) that survive the insult of ovulation contain elevated concentrations of 8-oxo-guanine (15, 16). Oxo-guanine is arguably the most important mutagenic adduct in DNA; mispairing with adenine during replication causes GC-to-TA transversions often detected in tumor cells (17–19). Fortunately, sublethal oxidative distresses to DNA caused by ovulation are normally reconciled by tumor suppressor or cell-cycle arrest and base-excision repair mechanisms (15). Notwithstanding, the ovarian surface epithelium may be vulnerable to genetic damages that are not repaired because it has not been under a strong evolutionary pressure to respond to repeated ovulations (13).
Ovarian cancer rates among mammals, which generally have relatively few lifetime ovulations, are low. In contrast, ovarian surface epithelial cancer occurs in egg-laying domestic poultry at a relatively high incidence (4%–40%, depending on ovulation history and age; Refs. 20–22). Because the course of disease resembles that in women, the “avian model” is of possible value for etiological studies and for the preclinical evaluation of chemopreventive and therapeutic agents (22). The objective of this investigation was to characterize oxidative and apoptotic DNA damages in ovarian surface epithelial cells of ovulatory hens.
Materials and Methods
Reagents were purchased from Sigma Chemical Company (St. Louis, MO) unless indicated otherwise.
Ovarian surface epithelial cells were isolated from the apical surface (prospective, formative, or site of ovulation) and a basal area (toward the body of the ovary) of pre- and postovulatory follicles (Fig. 1) of five 18-month-old egg-laying (ovulatory cycles = approximately 24 hrs; more than five clutches) White Leghorn (Welp strain) hens (photo-period = 14:10-hr light:dark cycle) at euthanasia (intravenous Beuthanasia-D, Schering-Plow Animal Health, Kenilworth, NJ) 2–4 hrs before predicted oviposition by using a modified polytetrafluoroethylene scraper designed to dislodge adherent cells from culture flasks (24). All ovaries appeared normal (i.e., no evidence of disease or cancer) and exhibited a full hierarchy of follicles (F1–4, POF1–2).
Cells were fixed in Histochoice (Amresco, Solon, OH), divided into aliquots for 8-oxo-guanine and DNA fragmentation analyses, transferred onto microscope slides treated with subbing solution (0.025% chromium potassium sulfate, 0.25% gelatin), air-dried, washed in phosphate-buffered saline (PBS), permeabilized in ice-cold methanol (70% for 3 mins, 90% for 3 mins, 99% for 30 mins), and rehydrated to PBS.
A mouse monoclonal antibody against 8-oxo-guanine was purchased from Trevigen (Gaithersburg, MD; clone IF7, 4355-MC-100). Specimens were incubated for 30 mins with 10% normal goat serum and for 1 hr with anti–8-oxo-guanine (1 μg/ml), washed in two changes of PBS, incubated for 30 mins with secondary goat anti-mouse immunoglobulin G-fluorescein isothiocyanate (FITC) (F 0257; 1:40), and washed in two changes of PBS. Serum and antibodies were diluted in freshly prepared PBS containing 0.5% bovine serum albumin. Controls were conducted without primary antibody and with primary antibody preincubated (4 hrs) with 100-fold molar excess oligonu-cleotides containing 8-oxo-guanine, a nondamaged counterpart (3850-100-01; Trevigen), or ultrapure guanine (G 6779).
Fragmented DNA was end labeled as a marker of progressive (nuclear) apoptosis (25, 26) by using a commercial kit according to the instructions of the manufacturer (ApopTag S7110; Intergen Co., Purchase, NY). Briefly, 3′-OH ends of DNA were linked with digoxigenin-11-d uridine triphosphate by terminal deoxy-nucleotidyl transferase (TdT) catalysis. Incorporated nucleotide heteropolymers were localized with antidigoxigenin Fab-FITC. Conjugate or TdT were excluded in (negative) control reactions.
Cells were observed under an Olympus BH-2 microscope equipped with a reflected light fluorescence attachment. Images of individual cells were captured (×400 magnification; subsamples = 20 for each biochemical determination [8-oxo-guanine, apoptosis] per specimen [apex and base of F1-4 and POF1-2] per animal) by computer-interfaced digital photography (1.2 million pixel resolution; Pixera, Los Gatos, CA) and analyzed for luminance intensities (continuous inverted gray-scale = 0 [black]–255 [white]; Optimas Software, Bothell, WA). Selections of microscopic fields or cells for analyses were made at random. Within-animal (subsample) data values were averaged. Mean comparisons (apex vs. base and across-follicular classifications) were made by Student’s t test and analysis of variance and protected least significant difference. Contrasts were considered different at P < 0.05.
Ovaries were fixed by overnight immersion in Histo-choice (Amresco). Samples of abraded and unmanipulated surfaces were excised, dehydrated, cleared, infiltrated with paraffin wax, cross-sectioned at 6-μm thickness, floated onto microscope slides, air-dried, deparaffinized in xylene, rehydrated, stained in hematoxylineosin, and examined by light microscopy.
Results
Immunostaining data for 8-oxo-guanine accumulation in ovarian surface epithelial cells of pre- and postovulatory follicles are shown in Figure 2. Fluorescence scores for cells from which 8-oxo-guanine antibody was omitted did not vary because of site of collection (apex vs. base) or follicular status (F1–4, POF1–2; Fig. 1). Elevations in 8-oxo-guanine immunostaining (compared with an averaged background fluorescence value = 51 ± 7) were most evident among cells recovered from the fringes of ovulatory rupture sites (POF1–2). An increase in 8-oxo-guanine of lesser intensity was observed in apical cells of the largest preovulatory (F1) follicles. Anti–8-oxo-guanine binding was blocked by preabsorption with an 8-oxo-guanine–containing oligonucleotide but not with an unmodified oligonucleotide or free guanine. No significant variations were across follicular classifications in 8-oxo-guanine levels in ovarian surface cells recovered from basal walls, and no differences were between apical and basal regions of F2–4.
Fragmentation of DNA (Fig. 3) was detected in apical ovarian surface epithelial cells of immediate pre- (F1) and postovulatory (POF1) follicles (background fluorescence data for negative control reactions = 48 ± 6). Apoptosis was most apparent in cells collected from overtly degenerative POF2 tissues; in this case, no distinction was between apical and basal areas. Fluorescence scores for ovarian surface epithelial cells isolated from apical and basal hemispheres of F2–4 were at baseline.
Examination of sections of ovarian/follicular wall validated that the surface epithelial cells were removed selectively. The basement membrane, which supports the ovarian epithelium and underlying interstitium, theca, and granulosa, was not compromised by the scraping procedure (Fig. 4). The epithelial nature of cellular isolates was confirmed by cytokeratin staining (data not presented).
Discussion
To our knowledge, this is the first report indicating that DNA damage accrues in ovarian surface epithelial cells associated with pre- and postovulatory follicles of egg-laying hens. Reactive oxygen molecules are presumably generated by leukocytes (respiratory burst) that infiltrate the ovulatory stigma and due to apical follicular ischemia reperfusion (7, 27–30). It appears that oxidative base corruptions perpetrated by ovulations in poultry are generally deposed by incidental apoptosis. Proficient removal or accurate repair (in the case of postovulatory mammalian ovaries) of genetically compromised cells is essential to prevent accumulations of potentially harmful mutations.
The “incessant ovulation hypothesis” of ovarian cancer was proposed by Fathalla (31) in 1971. Repeated ovulations (which wound or traumatize the ovarian surface) without long dormant periods to allow for repair and healing were reasoned to cause malignant transformation of the ovarian epithelium. The belief that circumstances that avert ovulation (oral contraceptive use, pregnancy, lactation) afford protection against ovarian carcinoma lends circumstantial support to this supposition (32–34). Induced chromosomal anomalies and metaplasia have been detected in rat and mouse ovarian surface epithelial cells subjected to repetitive subculturing, thereby adding further indirect corroboration to the idea that mitogenic reactions predicated by superfluous ovulations contribute to the pathogenesis of common ovarian cancer (35, 36). Moreover, several epidemiologic surveys have demonstrated positive correlations between increasing lifetime ovulations and ovarian cancer in women (34, 37–42). In a recent study, an overall 6% increase in risk was reported with each ovulatory year; ovulations in the 20- to 29-year-old age group were associated with the greatest liability (34).
Common epithelial ovarian cancer is a deadly insidious disease because it typically remains asymptomatic until it has advanced beyond the ovaries (43, 44). Because the prognosis for patients with ovarian cancer with metastatic disease is so poor, and because early detection has proven elusive, it is imperative that prophylactic measures be explored. Accordingly, the DNA of ovarian surface epithelial cells circumjacent to ovulation stigmata of ewes was protected from oxidative damages by vitamin E; fertility was not affected (16). A supplemental antioxidant could be of particular value in women designated at a preeminent risk for the development of ovarian cancer (e.g., those with a genetic predisposition and not using a contraceptive technique that inhibits ovulation). Results of the present study establish a basis for the use of ovulatory hens as an experimental paradigm to assess putative relationships between genomic disturbances to the ovarian epithelium and carcinogenesis.
Functional (left) ovary of a domestic hen, illustrating the follicular hierarchy. Rapidly growing follicles (selected to ovulate) develop a vascular pedicle and protrude from the ovary. F1 is the dominant follicle, which would have ovulated next. F2–4 represent successively smaller and less mature follicles in the preovulatory cohort. The follicular wall is covered by ovarian surface epithelial cells. Rupture takes place along an avascular band (stigma line) that extends over the apical surface of the follicle. The most recent postovulatory follicle is designated POF1. Avian postovulatory follicles, unlike those in mammals, do not form a corpus luteum but rather regress and are generally resorbed within a few days (23). Immunoreactive 8-oxo-guanine in ovarian surface epithelial cells contiguous with hen follicles. Means ± standard errors are plotted. Statistical contrasts denoted by asterisks indicate increases (*P < 0.01; **P < 0.001). Representative photomicrographs of immunostained (upper panel) and negative control (lower panel) cells obtained from the apex (fringe area) of a POF1 are shown. Bar, 10 μm. Immunofluorescence labeling of DNA fragments (internu-cleosomal degradation) indicative of apoptosis in follicle-associated ovarian surface epithelial cells. Increases (above baseline) are indicated by asterisks (*P < 0.05; **P < 0.001). Representative photomicrographs of immunostained (upper panel) and negative control (lower panel) cells obtained from the apex (fringe area) of a POF2 are shown. Bar, 10 μm. Photomicrographs of representative histologic sections of hen ovary depicting surface epithelial cells (upper panel; magnification: ×400) and a scraped area adjacent to an F1 follicle (lower panel; magnification: ×100).
Footnotes
This work was supported by a grant from the National Institutes of Health Grant RR-016474.