| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Regular Article |
a Department of Animal Science and Reproductive Biology Program, University of Wyoming, Laramie, Wyoming 82071
ABSTRACT
Oxidative base (8-oxoguanine) damage, DNA fragmentation, and apoptosis occurred among ovarian surface epithelial cells within the formative site of ovulation in sheep. The incidence of 8-oxoguanine adducts in surviving antiapoptotic Bcl-2/base excision repair polymerase ß-positive cells at the margins of ruptured follicles (which avoid the focal point of the ovulatory assault) was intermediate between apoptotic and outlying healthy epithelium. Cells containing perturbations to DNA expressed the tumor suppressor p53. Localized reactions of DNA injury and programmed cellular death were averted by ovulation blockade with indomethacin. Progesterone enhanced the biosynthesis of polymerase ß in ovarian surface epithelial cells exposed in vitro to a sublethal concentration of H2O2. Ovulation is a putative etiological factor in common epithelial ovarian cancer. A genetically altered progenitor cell, with unrepaired DNA, but not committed to death, could give rise to a transformed phenotype that is hence propagated upon healing of the ovulatory wound; it appears that this incongruity is normally reconciled by up-regulation of the base excision repair pathway during the ensuing luteal phase.
apoptosis, follicle, ovary, ovulation, progesterone
INTRODUCTION
The mammalian ovary is encased by a simple cuboidal to low pseudostratified columnar layer of epithelial cells [1]. 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/tight junctions [1]. Outgrowth of a follicle selected to ovulate brings it into close contact with the ovarian surface epithelium [1, 2]. The ovarian epithelium within the immediate vicinity of the avascular ovulatory stigma becomes apoptotic and is sloughed; bystander proliferative cells mend the surface defect upon involution of the corpus luteum [2, 3]. Most ovarian cancers evidently originate by clonal transformation of a surface epithelial cell that withstands the trauma of ovulation [4–7].
Inflammatory mediators and reactive oxidants are generated during the follicular mechanics of ovulation [8–13]. We hypothesized that ovarian surface epithelial cells located over the apical dome and along the border of ovulatory follicles (i.e., within a limited diffusion radius) contain DNA damaged by oxidative radical attack; that those cells directly apposed to the rupture site (extensive injury) are deleted by apoptosis; and that some surviving circumjacent cells contain sublethal lesions, which could be problematic if not corrected [14]. Circumstances within the periovulatory follicle that plausibly favor the formation or release (or both) of an excess of inflammatory or reactive oxygen molecules include leukocyte infiltration/oxidative burst [15, 16] and ischemia-reperfusion [17, 18]. Cells of the white blood series are attracted into the wall of preovulatory ovine follicles by fragments of degraded collagen; leukocyte chemotaxis, but not apical ovarian ischemia and rupture, was inhibited by intrafollicular injection of collagen peptide antibodies [19]. Follicular collagenolysis, leukocyte accumulation, acute alterations in blood flow, and ovulation were circumvented by the nonsteroidal anti-inflammatory agent indomethacin [20, 21].
The 8-oxoguanine adduct, an effector of spontaneous transversion mutagenesis [22], was used as a marker of oxidative DNA damage in sheep ovarian surface epithelial cells relative to the site of ovulation. Changes in accumulation of 8-oxoguanine were related to occurrences of apoptotic internucleosomal DNA fragmentation [23], expression of the apoptosis-mediating p53 and cell survival Bcl-2 proteins [24–26], and induction of the base excision repair polymerase ß [27]. In vivo response variables also were determined after preovulatory intrafollicular collagen peptide immunoneutralization and systemic indomethacin administration. Finally, direct effects of progesterone on the DNA repair mechanism were tested on cells exposed in vitro to H2O2.
MATERIALS AND METHODS
Experiments were performed with the approval of the University of Wyoming Animal Care and Use Committee. Reagents were purchased from Sigma Chemical Co. (St. Louis, MO) unless indicated otherwise.
In Vivo Studies
Mature Western-range ewes were observed twice daily for estrous behavior in the presence of vasectomized rams. Day 0 was considered the first day of estrus. Only animals that exhibited regular (16–17 days) estrous cycles were assigned to experiments. Luteal regression was synchronized by injection of prostaglandin (PG) F2
(5 mg dinoprost tromethamine i.m.; Upjohn, Kalamazoo, MI) on Day 14. A preovulatory surge of gonadotropins was elicited by administration of an agonistic analogue of GnRH (5 µg i.m. des-Gly10-Ala6-ethylamide) at 36 h after PGF2 injection. The ovarian follicle with the greatest diameter forms an ovulatory stigma 24 h following GnRH; cellular alterations and subsequent luteal function associated with induced ovulations are representative of normal (unmanipulated) cycles [2, 3, 28]. Four ewes were included in each experimental group.
Animals were killed (i.v. Beuthanasia-D; Schering-Plough Animal Health, Kenilworth, NJ) at 0, 12, 18, and 24 h and on Days 2, 4, 8, and 12 after GnRH. Surface epithelial cells were isolated (>95% purity) from the follicular apex, follicular perimeter (
4 mm wide), and a distant area on the same ovary using a modified polytetrafluoroethylene scraper designed to dislodge adherent cells from culture flasks [29]. Samples were divided for 8-oxoguanine and DNA fragmentation analyses.
Reproductive organs were exteriorized using aseptic technique via midventral abdominal laparotomy (i.v. sodium thiopental anesthesia; Abbott Laboratories, North Chicago, IL). Affinity-purified sheep antibodies raised against the glycine-proline-hydroxyproline repeat of collagens or normal immunoglobulin (Ig) G were injected at 12 h after GnRH into the antrum of the dominant follicle as described previously [19]. Ovarian surface epithelial cells for 8-oxoguanine and DNA fragmentation evaluations were obtained at 18 and 24 h, and on Day 2 post-GnRH. Immune inhibition of leukocyte influx into apical follicular tissues was confirmed by histological examination [19].
Ewes were treated 12 h following GnRH with a suspension of indomethacin (800 mg) or injection vehicle (8 ml PBS i.m.). Ovarian surface epithelial cells were recovered for 8-oxoguanine and DNA fragmentation determinations at 18 and 24 h, and on Day 2; and for p53, Bcl-2, and polymerase ß immunostaining at 0, 12, 18, and 24 h, and on Days 2, 4, 8, and 12 after GnRH. The 800-mg dose of indomethacin selected for this study was 100% effective in the suppression of ovulation [30]. Lengths of estrous cycles and circulatory progesterone profiles were unaffected in indomethacin-treated ewes bearing a luteinized, unruptured follicle [31].
In Vitro Model
Pools of surface epithelial cells were obtained from ovaries of lambs killed in the University of Wyoming Meat Science abattoir. Treatments were replicated four to five times.
In a preliminary experiment, the effects of H2O2 on cellular 8-oxoguanine accumulation and DNA fragmentation were established. Cells (1 x 103/0.1 ml Medium-199) were subjected to H2O2 (0, 1 µM, or 1 mM; 10 min) and incubated (in fresh medium) for 6 h (37°C in an atmosphere of 5% CO2 and air; 96-well plates); there was no gross light microscopic (Zeiss 4655422, Oberkochen, Germany) evidence of apoptotic death at 6 h. A trial was therefore conducted to assess cellular morphology after a 12-h incubation.
Incubations following pretreatment with H2O2 (0 or 1 µM) were carried out with progesterone (0, 0.1, 0.5, or 2.5 ng/0.1 ml) for 6, 12, and 24 h. Cells were harvested and analyzed for contents of 8-oxoguanine, DNA fragmentation, p53, Bcl-2, and polymerase ß. Doses of progesterone were chosen to represent the range of circulatory and local ovarian concentrations of the periovulatory transition and luteal phase [32, 33]. Polymerase ß immunostaining was then evaluated in cells (0 or 1 µM H2O2) incubated for 24 h with progesterone (0 or 2.5 ng) and the progesterone receptor antagonist mifepristone/RU486 [34] (National Institute of Mental Health Chemical Synthesis Program; Research Biochemicals International, Natick, MA) or the transcriptional inhibitor actinomycin D [35] (0 or 25 ng). Progesterone was initially dissolved in ethanol, from which stock solutions were made. The final concentration of ethanol in incubations was 0.2%.
Avidin Detection of 8-Oxoguanine
Oxidative lesions in DNA were monitored directly by avidin binding [36]. Cells were fixed in ice-cold 4% paraformaldehyde, transferred onto microscope slides treated with subbing solution (0.025% chromium potassium sulfate, 0.25% gelatin), air-dried, washed in PBS, dehydrated and permeabilized in ice-cold methanol (70% for 3 min, 90% for 3 min, 99% for 30 min), rehydrated to PBS, incubated with avidin-fluorescein isothiocyanate (FITC; 1:200, 37°C, 1 h), washed in two changes of PBS, and covered with Vectashield Medium (Vector Laboratories, Burlingame, CA). Control reactions were performed using avidin-FITC preabsorbed with 100-fold molar excess oligonucleotides containing 8-oxoguanine, a nondamaged counterpart (3850-100-01; Trevigen, Gaithersburg, MD), or ultrapure guanine (G 6779).
8-Oxoguanine, p53, Bcl-2, and Polymerase ß Immunocytochemistry
A mouse monoclonal anti-8-oxoguanine was purchased from Trevigen (clone IF7; 4355-MC-100). Purified antibodies to p53 (mouse monoclonal KAM-CC002) and Bcl-2 (rabbit polyclonal AAP-070) were obtained from StressGen Biotechnologies (Victoria, BC, Canada).
A synthetic peptide (>95% purity, RP-high performance liquid chromatography and mass spectral analysis) corresponding to the amino terminus of polymerase ß (MSKRKAPQETLNGGITDML-) was synthesized by Multiple Peptide Systems (San Diego, CA) and coupled through a C-terminal cysteine thiol to keyhole limpet hemocyanin with the heterobifunctional agent maleimidobenzoyl-N-hydroxysuccinimide in a ratio of 1:1 (w/w). A polyclonal rabbit antipeptide serum was generated (primary and boost immunizations were in complete and incomplete Freunds adjuvant, respectively) and titered by ELISA. Antibodies were purified by affinity chromatography with 5 mg peptide (coupled through the cysteine residue) to 5 ml of Sulfo-Link gel (Multiple Peptide Systems). Molecular consistency of the eluted product was verified by SDS-PAGE.
Cells were fixed, adhered to slides, permeabilized, and analyzed by indirect immunofluorescence microscopy. Slides were incubated for 30 min with 10% normal goat serum and for 1 h with 8-oxoguanine, p53, Bcl-2, or polymerase ß antibodies (1 µg/ml), washed in two changes of PBS, incubated for 30 min with secondary goat anti-rabbit (F 0382) or anti-mouse (F 0257) IgG-FITC (1:40), and washed in two changes of PBS. Negative controls were carried out without primary antibodies and with primary antibodies preabsorbed with 8-oxoguanine nucleotide, human recombinant p53 (Santa Cruz Biotechnology, Santa Cruz, CA), Bcl-2 peptide (amino acids 41–54; StressGen), or recombinant polymerase ß (Trevigen).
DNA Fragmentation Analysis
End-labeling of fragmented DNA was used as an index of progressive (nuclear) apoptosis in fixed/permeabilized cells [3, 37]. Briefly, 3'-OH ends of DNA were linked with digoxigenin-11-d uridine triphosphate by terminal deoxynucleotidyl transferase (TdT) catalysis. Incorporated nucleotide heteropolymers were localized with antidigoxigenin Fab-FITC (ApopTag Kit S7110; Intergen Co., Purchase, NY). Conjugate or TdT were omitted in negative control reactions.
Image Analyses
Ovarian surface epithelial cells were observed under an Olympus BH-2 microscope equipped with a reflected light fluorescence attachment (Tokyo, Japan). Images of cells were captured (400x; subsamples = 18) by computer-interfaced digital photography (1.2 million pixel resolution; Pixera, Los Gatos, CA) and assessed for luminance intensities (gray-scale = 0–5, 0 = black background; Optimas Software, Bothell, WA).
Statistics
Assignments to treatments and selections of fields of microscopic inspection were made at random. Subsample data were averaged. Mean comparisons were made by the Student t-test or ANOVA and protected least significant difference or the Dunnett test. Contrasts were considered significant at P < 0.05.
RESULTS
Ovulatory Dynamics and Ovarian Surface Epithelial DNA Damage
Binding of avidin-FITC or anti-8-oxoguanine in ovarian surface epithelial cells removed from the apical dome of preovulatory follicles (Fig. 1A) did not change from 0 to 12 h, and then increased (P < 0.001) at 18 h after administration of GnRH (Fig. 1, B and C). Surface epithelial cells were not recovered (apoptotic exfoliation) from the developmental ovulation stigma (impending follicular rupture) at 24 h post-GnRH (Fig. 1A). The 8-oxoguanine content of ovarian surface epithelial cells obtained from the perimeter of preovulatory follicles was elevated (P < 0.05) at 24 h; this condition persisted into and subsided during the early and late luteal phases, respectively (Fig. 1, B and C). Fragmentation of DNA indicative of apoptosis-onset was restricted (P < 0.01) to 18-h cells associated with the follicular apex (Fig. 1D). There were no significant temporal alterations in low levels of 8-oxoguanine or DNA strand breaks in surface cells removed from nonfollicular (extrinsic) regions of ovaries (Fig. 1). Binding of avidin or anti-8-oxoguanine in 18-h apical cells was inhibited by preabsorption with an 8-oxoguanine-containing oligonucleotide (fluorescence scores <1.5), but not with an unmodified oligonucleotide or free guanine. Avidin-FITC was used to detect 8-oxoguanine in subsequent experiments.
|
Intrafollicular injection of collagen peptide antibodies at 12 h after GnRH did not affect periovulatory ovarian surface epithelial patterns of 8-oxoguanine (Fig. 2, A and B) or apoptosis (Fig. 2, C and D).
|
Inhibition of Ovulation and Ovarian Surface Epithelial DNA Damage
Stigma formation, follicular rupture, and contiguous oxidative (Fig. 3, A and B) and fragmentation (Fig. 3, C and D) insults to DNA of the ovarian epithelium were negated by administration of indomethacin at 12 h after GnRH. Thus, ovarian surface epithelial cells (which do not become apoptotic) subsisted over the apex of unruptured follicles of indomethacin-treated ewes (Fig. 3, B and D). Moreover, the apical wall of anovulatory follicles displayed an elaborate vascular network (i.e., there were no signs of blood vessel destruction and acute ischemia emblematic of ovulatory ovarian rupture).
|
Cyclical Alterations in p53, Bcl-2, and DNA Polymerase ß in Ovarian Surface Epithelium: Attenuation by Indomethacin
Immunoreactive p53 increased (P < 0.01) in ovarian surface epithelial cells isolated from the apex and perimeter of periovulatory follicles; levels within extant cells of the perimeter decreased after Day 2 of the luteal phase (Fig. 4A). Elevations in immunostaining for Bcl-2 occurred in marginal cells of ovulatory (P < 0.01) and postovulatory (P < 0.05) follicles (Fig. 4C). A linear increase in immunoreactive polymerase ß accumulation was detected within circumjacent ovarian epithelium during ovulation (P < 0.05) and luteinization (P < 0.01; Fig. 4E). Bcl-2 (Fig. 4C) and polymerase ß (Fig. 4E) levels were unaffected in apical cells of preovulatory follicles that presented excessive levels of DNA fragmentation and were destined for apoptotic death. Ovarian surface epithelial cells not associated with periovulatory follicles (Fig. 4, A, C, and E; extrinsic) and of animals given indomethacin (Fig. 4, B, D, and F) contained basal amounts of p53, Bcl-2, and polymerase ß.
|
In Vitro Simulation of the Periovulatory Period: Augmentative Effect of Progesterone on Polymerase ß in Ovarian Surface Epithelial Cells Exposed to H2O2
Aliquots of a pool of surface cells removed at slaughter from ovaries of prepubertal lambs that were challenged with H2O2 and then incubated for 6 h exhibited oxidative base damage (P < 0.05 at 1 µM; P < 0.01 at 1 mM) without (1 µM) or with (1 mM; P < 0.001) DNA fragmentation. Coincident expression of p53 was detected (P < 0.05) in base-compromised cells. Levels of Bcl-2 were elevated (P < 0.05) in cells exposed to the lower concentration of H2O2; polymerase ß tended to be higher (P < 0.1; Fig. 5). Cells treated with the high dose of H2O2 formed apoptotic bodies and degenerated between 6 and 12 h of incubation.
|
Progesterone increased (P < 0.05), in a time- and concentration-dependent manner, the polymerase ß immunostaining in ovarian surface epithelial cells that had been treated with the sublethal dose of H2O2 (Fig. 6: A, 6 h; B, 12 h; C, 24 h); a corresponding decline (P < 0.05) in 8-oxoguanine lesions was noted at 24 h (Fig. 6C). There were no significant effects of progesterone on cellular p53 or Bcl-2 immunofluorescence values, with the exception that p53 was reduced to a near basal level in the high-progesterone group at 24 h (Fig. 6C). Finally, the positive polymerase ß response to progesterone (24 h) in cells subjected to H2O2 was negated (P < 0.05) by RU486 (Fig. 7A) or actinomycin D (Fig. 7B).
|
|
DISCUSSION
Oxidative base damage to DNA is an inevitable byproduct of physiological metabolism (e.g., leakage of radicals associated with the reduction of oxygen to water during mitochondrial respiration). To combat this predicament, animals have evolved elaborate enzymatic antioxidant defense mechanisms (superoxide dismutase, glutathione perioxidase, catalase); however, these are less than perfect, and some oxidants find their way to DNA targets [38].
This is the first report indicating that 8-oxoguanine lesions arise at a higher than normal frequency within ovarian surface epithelial cells juxtaposed to the ovulatory site. Those surface cells within the immediate area of ovarian rupture that manifested extensive base damage were deleted by apoptosis. Ovarian epithelial cells somewhat removed from the ovulatory stigma with a lesser degree of base damage, and not symptomatic of apoptotic DNA fragmentation, were preserved; it appears that the antiapoptotic Bcl-2 was involved, and that consequently, a progesterone-sensitive/base-excision process restored genomic fidelity. Concomitant production of p53 was indiscriminate of cellular outcome—be it death or survival. Expression of p53 in ovaries of gonadotropin-treated mice was maximal around the time of ovulation [39]. Regardless of the obligate mode of action, accurate repair or proficient removal of damaged cells is essential to prevent accumulation of potentially harmful mutations.
Apical follicular ischemia-reperfusion remains a possible determinant of the genotoxic insult to ovarian epithelial DNA. The inhibition of infiltration of leukocytes into sheep follicles by collagen peptide antibodies was unrelated to the processes of DNA damage and ovulatory rupture. Indeed, whether leukocytes are incidental invaders or essential effectors of mammalian ovulation remains equivocal [40]. Superoxide production within preovulatory follicles of rats was largely of leukocytic origin [13]. The inflammatory agents, prostaglandins and tumor necrosis factor , which are produced during the preovulatory period by follicular cells and are down-regulated by indomethacin [30], induced apoptosis in sheep ovarian surface epithelial cells [41, 42].
8-Oxoguanine has become the benchmark for oxidative DNA modification [43]; it is arguably the most important mutagenic lesion in DNA (mispairing with adenine during replication causes GC 
TA transversion often detected in tumor cells) [22, 44]. Most techniques (e.g., HPLC with electrochemical detection and gas chromatography mass spectroscopy) to measure 8-oxoguanine (or its free deoxynucleoside) are cumbersome and lack (single-cell) sensitivity [45]. That avidin binds to 8-oxoguanine (imidazolidone group) with high avidity and selectivity was exploited following the serendipitous observations that nuclei of free radical-damaged and necrotic cells reacted (in the absence of biotin) with avidin [36]. Cytochemical results of this investigation using avidin were corroborated with a specific 8-oxoguanine antibody.
Oxidative stress (and consequent disturbances to DNA) is a typical prelude to p53, Bcl-2, and polymerase ß up-regulation [46–49]. Cells respond to p53 activation by growth arrest (which prevents the replication of damaged DNA until repair can be completed) or apoptosis. Many factors, to include the intensity of stress signals and survival stimuli, affect the complex cellular response to p53, and thus dictate the pivotal choice between viable growth cessation and programmed death [50–54]. In the presence of Bcl-2, low-level DNA damage is normally repaired before the checkpoint p53 suicidal program can be executed; Bcl-2 impedes the subcellular trafficking of p53, inhibits downstream adapters necessary for stimulation of the apoptotic pathway caspases, and can (albeit controversial) act as an antioxidant [24, 55–58]. Increased production of Bcl-2 and polymerase ß by ovarian surface epithelial cells is bypassed in the face of irreparable harm to DNA.
Polymerase ß is a penultimate mediator of mammalian DNA base excision repair. The base excision cascade is generally limited to the repair of small lesions in DNA (e.g., single nucleotide modifications). Short-patch reconstruction is initiated by a proofreading glycosylase that hydrolyzes the N-glycosylic bond linking an improper base to deoxyribose. The abasic sugar-phosphate backbone is then cleaved by an apurinic/apyrimidinic endonuclease or lyase. Polymerase ß fills the nucleotide gap created in DNA with the deoxyribonucleoside triphosphate complementary to the template. Finally, the nick is sealed by a DNA ligase [27, 59]. The base excision pathway is a principal contributor to the amendment of 8-oxoguanine corruptions in DNA [60, 61].
Outcomes of the in vitro experiments indicate that the stimulatory effect of progesterone on polymerase ß in ovarian surface epithelium was receptor-mediated and exerted at the transcriptional level—typical of steroid hormone actions in general [62]. In fact, progesterone receptors have been localized within ovarian surface epithelial cells [63]. Antiapoptotic effects of progesterone on the uterus, breast, and corpus luteum also are receptor-mediated [64]. Doses of progesterone required to augment polymerase ß production exceeded those characteristic of the systemic circulation; this is interpreted to imply that in vivo, sufficient amounts of progesterone are delivered to neighboring surface ovarian cells by simple (paracrine) diffusion from the corpus luteum. That progesterone increased polymerase ß only in the presence of 8-oxoguanine lesions suggests cross-talk among the DNA damage recognition/repair and hormonal control systems. Bioactivity of polymerase ß was androgen-induced in the testis and prostate gland [65, 66].
Progesterone likewise enhanced poly(ADP-ribose) polymerase (PARP) production in sheep ovarian surface epithelial cells [67]. PARP may serve as an adjunct in DNA repair. Binding of PARP (i.e., synthesis of branched polymers of ADP-ribose) in areas adjacent to a single-strand interruption functions as an antirecombinogenic element [59, 68]. Findings that progesterone can augment modes of DNA repair within ovarian surface epithelial cells adjoining the ovulatory stigma are of fundamental pathophysiological significance. Progestogens can apparently reduce the risk of developing ovarian carcinoma in postmenopausal women who have undergone estrogen replacement therapy [69]. Progestin treatment of macaque ovarian surface epithelial cells promoted (in contrast to our findings) apoptosis; it was suggested that this is a mechanism to prevent the development of ovarian cancer [70].
The course of episodes that lead to common epithelial ovarian cancer are multifactorial and not entirely understood—several aberrant stages are undoubtedly required to yield a malignant phenotype with a distinct growth advantage [71, 72]. It seems that the first step in tumorigenesis involves disturbances to the ovarian surface stemming from ovulation [4–7]. Circumstances that avoid ovulation (e.g., oral contraceptive use, pregnancy, lactation) certainly protect against ovarian neoplasia [73–75]. Disease progression has been associated with a host of genetic dysfunctions; for example, more than half of ovarian adenocarcinomas have discernible mutations in p53 [76, 77]. Epithelial ovarian cancer, which carries a 1-in-70 lifetime risk, is the most frequent cause of fatality from a gynecologic malignancy. Less than 15% of patients with advanced disease (peritoneal metastasis) survive beyond 5 years [78].
Results of these studies provide evidence supporting the concept that ovulation is a predisposing (i.e., potentially mutagenic) factor in ovarian cancer. Nevertheless, cause-effect associations between ovulatory disturbances to DNA and ovarian epithelial carcinogenesis have not been established.
FOOTNOTES
1 Supported by U.S. Department of Agriculture-NRI grant 95-37203-2131. 
2 Correspondence: W.J. Murdoch, Department of Animal Science, P.O. Box 3684, University of Wyoming, Laramie, WY 82071. FAX: 307 766 2355; wmurdoch{at}uwyo.edu
Accepted: June 26, 2001.
Received: April 20, 2001.
REFERENCES
-induced sheep ovarian cell apoptosis. Biol Reprod 1999; 61:1655-1659 in the ovulatory mechanism of ewes. J Anim Sci 1997; 75:1601-1605This article has been cited by other articles:
|
|
 |
|
  P. P. Szotek, H. L. Chang, K. Brennand, A. Fujino, R. Pieretti-Vanmarcke, C. Lo Celso, D. Dombkowski, F. Preffer, K. S. Cohen, J. Teixeira, et al. Normal ovarian surface epithelial label-retaining cells exhibit stem/progenitor cell characteristics PNAS, August 26, 2008; 105(34): 12469 - 12473. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. E. Burdette, R. M. Oliver, V. Ulyanov, S. M. Kilen, K. E. Mayo, and T. K. Woodruff Ovarian Epithelial Inclusion Cysts in Chronically Superovulated CD1 and Smad2 Dominant-Negative Mice Endocrinology, August 1, 2007; 148(8): 3595 - 3604. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 N. Ahmed, S. Maines-Bandiera, M. A. Quinn, W. G. Unger, S. Dedhar, and N. Auersperg Molecular pathways regulating EGF-induced epithelio-mesenchymal transition in human ovarian surface epithelium Am J Physiol Cell Physiol, June 1, 2006; 290(6): C1532 - C1542. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. E. Burdette, S. J. Kurley, S. M. Kilen, K. E. Mayo, and T. K. Woodruff Gonadotropin-Induced Superovulation Drives Ovarian Surface Epithelia Proliferation in CD1 Mice Endocrinology, May 1, 2006; 147(5): 2338 - 2345. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. A Slot, M. Voorendt, M. de Boer-Brouwer, H. H van Vugt, and K. J Teerds Estrous cycle dependent changes in expression and distribution of Fas, Fas ligand, Bcl-2, Bax, and pro- and active caspase-3 in the rat ovary J. Endocrinol., February 1, 2006; 188(2): 179 - 192. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 W. J. Murdoch Carcinogenic Potential of Ovulatory Genotoxicity Biol Reprod, October 1, 2005; 73(4): 586 - 590. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 W. J. Murdoch, E. A. Van Kirk, and B. M. Alexander DNA Damages in Ovarian Surface Epithelial Cells of Ovulatory Hens Experimental Biology and Medicine, June 1, 2005; 230(6): 429 - 433. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M Gaytan, M A Sanchez, C Morales, C Bellido, Y Millan, J Martin de las Mulas, J E Sanchez-Criado, and F Gaytan Cyclic changes of the ovarian surface epithelium in the rat Reproduction, March 1, 2005; 129(3): 311 - 321. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. T. Rae, D. Niven, H. O. D. Critchley, C. R. Harlow, and S. G. Hillier Antiinflammatory Steroid Action in Human Ovarian Surface Epithelial Cells J. Clin. Endocrinol. Metab., September 1, 2004; 89(9): 4538 - 4544. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
W. J. Murdoch and J. F. Martinchick Oxidative Damage to DNA of Ovarian Surface Epithelial Cells Affected by Ovulation: Carcinogenic Implication and Chemoprevention Experimental Biology and Medicine, June 1, 2004; 229(6): 546 - 552. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. C. McDonnel, E. A. Van Kirk, D. D. Isaak, and W. J. Murdoch Inhibitory Effects of Progesterone on Plasma Membrane Fluidity and Tumorigenic Potential of Ovarian Epithelial Cancer Cells Experimental Biology and Medicine, March 1, 2003; 228(3): 308 - 314. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. Y.K. Yong, C. Harlow, K.J. Thong, and S. G. Hillier Regulation of 11{beta}-hydroxysteroid dehydrogenase type 1 gene expression in human ovarian surface epithelial cells by interleukin-1 Hum. Reprod., September 1, 2002; 17(9): 2300 - 2306. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
