Ovarian surface epithelium during ovulatory and anovulatory ovine estrous cycles.код для вставкиСкачать
THE ANATOMICAL RECORD 240:322-326 (1994) Ovarian Surface Epithelium During Ovulatory and Anovulatory Ovine Estrous Cycles WILLIAM J. MURDOCH Department of Animal Science, University of Wyoming, Laramie, Wyoming 82071 ABSTRACT Background: Ovarian surface epithelial cells have been implicated in the mechanisms of ovulation and development of common ovarian cancers. An early indication of predisposition to neoplasia is the formation of ovarian epithelial inclusion cysts. It was unclear whether morphological alterations along the ovarian surface are related directly to ovulation per se or associated endocrine parameters of reproductive cyclicity. Methods: Light microscopic disturbances in ovarian surface epithelium were monitored during synchronous ovulatory and anovulatory estrous cycles of sheep. Ovulation blockade accompanied by normal luteal phases was induced by administration of indomethacin, a prostaglandin synthase inhibitor. Results: Degenerative cells were sloughed from the apical dome of periovulatory follicles. The resultant stigma of luteinizing follicles was void of surface epithelium. Repair of the ovulatory wound by epithelium did not occur until complete involution of the corpus luteum during the subsequent estrous cycle. In a few cases inclusions containing entrapped ovarian surface epithelium were noted within adjacent stroma. Epithelia covering luteinized unruptured follicles remained intact and was not incorporated into the ovary during luteal resorption. Conclusion: Localized damage to and subsequent remodelling of the ovarian surface occurs in a cyclic fashion conjoined with the physical process of follicular rupture. 0 1994 Wiley-Liss, Inc. Key words: Ovarian surface epithelium, Ovulation, Indomethacin, Sheep The mammalian ovary is encased by a layer of epithelial cells (Anderson et al., 1976) derived from the same peritoneal mesothelium that forms the embryonic Mullerian ducts (Byskov, 1986). These cells are supported by a basal lamina located along the surface of the tunica albuginea, the organized cortical connective tissue matrix of the ovary. Ovarian surface epithelial cells have been implicated in the mechanics of fqllicular rupture; they evidently release lysosomal enzymes that facilitate the breakdown of the tunica albuginea (Cajander, 1976). It has been hypothesized that aberrations in the proliferative wound repair of the ovarian surface following ovulation can lead to expression of a malignant phenotype and neoplasia. The majority of ovarian cancers arise from surface epithelium that had presumably been traumatized at ovulation (Rao and Slotman, 1991; Bast et al., 1992; Godwin e t al., 1993). Despite the pathophysiological significance of the ovarian surface epithelium, fundamental information concerning cyclic structural alterations in this cell layer is lacking. The focus of the following experiment was to examine surface epithelium associated with dominant follicles and luteal glands during ovulatory and anovulatory estrous cycles of sheep. Ovulation was 0 1994 WILEY-LISS. INC. blocked using indomethacin, a n inhibitor of the cyclooxygenase pathway of arachidonate metabolism (Flower, 1974). Intrafollicular injection of indomethacin during the preovulatory period caused formation of luteinized unruptured follicles (Murdoch and Dunn, 1983). MATERIALS AND METHODS Mature western-range ewes were penned with vasectomized rams daily and observed for estrous behavior (average cycle lengths = 17 days). Luteal regression and the onset of the preovulatory surge of luteinizing hormone (LH) were synchronized by intramuscular (IM) treatment with 10 mg of prostaglandin (PG) F,a tromethamine (Lutalyse; The Upjohn Co., Kalamazoo, MI) on Day 14 of the estrous cycle followed 36 h later by 5 p,g of a n agonistic analogue of LH-releasing hormone (des-Ala6-Glyl0 ethylamide; LHRH; Sigma Chemical Co., St. Louis, MO). Follicles 6-8 mm in diameter will consistently ovulate about 24 h after administration of LHRH (Roberts et al., 1985). Received March 1, 1994; accepted May 16, 1994 OVARIAN SURFACE EPITHELIUM AND OVULATION 323 Eight hours following injection of LHRH, ewes were treated with 500 mg IM of indomethacin (Sigma Chemical Co., St. Louis, MO) suspended in 8 ml of phosphatebuffered saline solution (PBS) or that vehicle alone (controls). Drug was given after the anticipated completion of the LH surge and before the preovulatory follicular rise in prostaglandin production (Murdoch et al., 1986). Animals (sidgroup) were killed by barbiturate overdose a t 24 h after injection of LHRH (Day l), Day 4, Day 12, or Day 4 of the ensuing (synchronized) estrous cycle (N = 48). Ovaries were removed immediately, rinsed in chilled PBS, and immersed in 10% buffered formalin. Blocks of fixed tissues containing surface-oriented periovulatory follicles or luteal structures were isolated from ovaries using a single-edged razor blade. Special care was taken not to handle the surface of the ovaries (Gillett, 1991) during removal or preparation for histology. Specimens were dehydrated in a graded series of ethanol, cleared, and infiltrated with and embedded in paraffin. Tissues were serially cross-sectioned at 3-6 pm thickness, rehydrated to water, and stained in hematoxylin and eosin. Sections were examined under a n Olympus Vanox light microscope. Sera samples for radioimmunoassay of progesterone (Murdoch and Dunn, 1983) were collected by jugular venipuncture each day from ewes in which tissue collections were made on Day 4 of the second estrous cycle after treatment (control vs. indomethacin). These data were evaluated by split-plot analysis of variance. RESULTS Follicles of control ewes obtained at 24 h after LHRH had either recently ruptured or exhibited a n ovulatory papilla. Luteal tissues of controls characteristically protruded from the surface of the ovary. There was no evidence of impending ovulation in indomethacintreated ewes. Enlarged, hyperemic follicles of these animals were smooth surfaced, which would have formed unruptured, fluid-containing luteal glands (Fig. 1). One-to-three dominant follicles or corpora lutea and corpora albicantia (Day 1 and Days 4) were obtained from the pair of ovaries of each animal for histological inspection. Epithelia covering sheep ovaries was simple or pseudostratified cuboidal. Epithelial cells were generally absent from the ovarian surface juxtaposed to periovulatory follicles. Strands of degenerative cells were sometimes observed sloughing from the ovarian surface. This reaction was localized; it did not extend to the ovary in general. Loosely affixed cells were often noted a t the margins of the follicular protrusion. Exposed surfaces of luteinizing ruptured follicles and developed corpora lutea were not covered by epithelium. The ovulatory defect was mended only after structural regression of the corpus luteum was achieved by Day 4 of the subsequent estrous cycle. Small nests of intraovarian surface epithelial cells were observed in proximity to a corpus albicans in a few cases (3/34 [Day 41 specimens of different animals examined over two cycles). Epithelial coverings of anovulatory luteinized follicles of indomethacin-treated ewes remained intact and appeared healthy at each time of tissue collection; there was no detection of epithelial invaginations or Fig. 1 . Ovaries of control (At and indomethacin-treated(B) ewes on Day 4 after injection of LHRH. CL, corpus luFum. LUF, luteinized unruptured follicle. intraovarian inclusions associated with retrogressive luteal tissues in these animals (Fig. 2). Circulatory levels of progesterone did not differ significantly (P > 0.05) between control and indomethacin-treated ewes (Fig. 3). DISCUSSION This study indicates that during the process of ovulation in sheep epithelial cells are shed from the surface of the ovary. A similar phenomenon has been observed in hamsters and mice (Talbot et al., 1987). It appears th a t ovarian surface epithelial cells undergo localized death following the surge of LH. Biochemical interaction, within a limited diffusion radius of the preovulatory follicle, is evidently a prerequisite of this response. Prostaglandins (mainly PGE,) induced chromatin condensation and DNA fragmentation in incubated surface epithelial cells isolated from sheep ovaries (Ackerman and Murdoch, 1993). Indeed, indomethacin, at a dose known to effectively inhibit preovulatory follicular prostaglandin biosynthesis (Murdoch and McCormick, 1991), prevented the loss of surface epithelial cells typical of ovulatory follicles. A human adenocarcinoma ovarian cancer cell line (OVCAR-3)resistant in vitro to clinically relevant concentrations of adriamycin, cisplatin, and cyclophosphamide was not sensitive to the lethal effect of prostaglandins (Ackerman and Murdoch, 1993). Cancerous transformations of ovarian surface epithelial cells may Fig. 2. 325 OVARIAN SURFACE EPITHELIUM AND OVULATION PROGESTERONE (ng/ml) 1 2 3 4 5 6 7 8 9 101112131415 1 2 3 4 D A Y POST-LHRH Fig. 3. Mean patterns of sera concentrations of progesterone during ovulatory (control)and anovulatory (indomethacin) ovine estrous cycles. Abrupt declines in progesterone are indicative of PGF,a-induced functional luteolysis. be related to escape from programmed death ordinarily man, 1992). Apparently the undisturbed ovarian suraffiliated with follicular prostaglandin production and face of luteinized unruptured follicles is not likely to ovulatory tissue dissolution. become involved in inclusion cyst formation. There has been debate over whether the causative LITERATURE CITED factor in common ovarian epithelial cancers is ovulation or some endocrine factor (e.g., gonadotropic or ste- Ackerman, R.C. and W.J. Murdoch 1993 Prostaglandin-induced apoproid) associated with ovarian cyclicity (Balasch and tosis of ovarian surface epithelial cells. Prostaglandins, 45r475485. Barri, 1993). Pregnancy and oral contraceptive use E., G. Lee, R. Letourneau, D.F. Albertini, and S.M. Meller confer significant protection against epithelial neopla- Anderson, 1976 Cytological observations of the ovarian epithelium in mamsia (Piver e t al., 1991). Cyclic alterations in ovarian mals during the reproductive cycle. J . Morphol., 150:135-166. surface epithelium in sheep were specifically inte- Balasch, J . and P.N. Barri 1993 Follicular stimulation and ovarian cancer? Human Reprod., 8r990-996. grated with the actual process of follicular rupture, and I. Jacobs, and A. Berchick 1992 Malignant transformation not to the hormonal environments of estrous cycles. Bast,ofR.C., ovarian epithelium. J . Natl. Cancer Inst., 84t556-558. Timings of luteal regression and levels of preovulatory Byskov, A.G. 1986 Differentiation of mammalian embryonic gonad. gonadotropin secretion were managed by exogenous Physiol. Rev., 66r71-117. PGF,a and LHRH. And luteal phases of anovulatory Cajander, S. 1976 Structural alterations of rabbit ovarian follicles after mating with special reference to the overlying surface epi(indomethacin) cycles were essentially identical to conthelium. Cell Tiss. Res., 173r437-449. trol ovulatory cycles. Infertile menstrual cycles during Flower, R.J. 1974 Drugs that inhibit prostaglandin biosynthesis. which ovulation in response to the gonadotropin surge Pharmacol. Rev., 26t33-67. fails but luteal progesterone output progresses nor- Gillett, W.R. 1991 Artefactual loss of human ovarian surface epithelium: Potential clinical significance. Reprod. Fertil. Dev., 3t93mally (luteinized unruptured follicle syndrome) closely 98. resemble unaffected estrous cycles observed in experi- Godwin, A.K., J.R. Testa, and T.C. Hamilton 1993 The biology of mental mammals after inhibition of preovulatory folovarian cancer development. Cancer, 71r530-536. licular prostaglandin production (Killick and Priddy, Killick, S.R. and A.R. Priddy 1988 Eicosanoids and the mechanism of ovulation. Med. Sci. Res., 161257-1260. 1988; Murdoch et al., 1993). Murdoch, W.J. and T.G. Dunn 1983 Luteal function after ovulation Ovarian inclusions of epithelial cells were noted in a blockade by intrafollicular injection of indomethacin in the ewe. minority of histological sections of involuted corpora J. Reprod. Fertil., 69r671-675. lutea. Transient invaginations probably form during Murdoch, W.J. and R.J. McCormick 1991 Dose-dependent effects of indomethacin on ovulation in the sheep: Relationship to follicular ovarian resorption of nonfunctional luteal tissues. prostaglandin production, steroidogenesis, collagenolysis, and These structures can harbor malignant cells (Talerleukocyte chemotaxis. Biol. Reprod., 45r907-911. Fig. 2. Representative light photomicrographs of ovarian sections. A: Normal appearance of ovarian surface epithelium. B Degenerative epithelial cells becoming detached from the ovary, control Day 1. C: Exposed ovarian surface basement membrane, control Day 1. D: Perimeter of ovulatory follicle, control Day 1. E: Surface of CL, control Day 12. F Surface invagination, control Day 4. G Inclusion of epithelial cells, control Day 4. H: Intact epithelium, indomethacin Day 1. x 400, except F, x 180. Murdoch, W.J., T.A. Peterson, E.A. Van Kirk, D.L. Vincent, and E.K. Inskeep 1986 Interactive roles of progesterone, prostaglandins, and collagenase in the ovulatory mechanism of the ewe. Biol. Reprod., 35r1187-1194. Murdoch, W.J., T.R. Hansen, and L.A. McPherson 1993 Role of eicosanoids in vertebrate ovulation. Prostaglandins, 46t85-115. Piver, M.S., T.R. Baker, M. Piedmonte, and A.M. Sandecki 1991 Epidemiology and etiology of ovarian cancer. Semin. Oncol., 18t177185. Rao, B.R. and B.J. Slotman 1991 Endocrine factors in common epithelial ovarian cancer. Endo. Rev., 12r14-26. Roberts, A.J., T.G. Dunn, and W.J. Murdoch 1985 Induction of ovu- 326 W.J. MURDOCH lation in proestrous ewes: Identification of the ovulatory follicle and functional status of the corpus luteum. Domest. Anim. Endocrinol., 2:207-210. Talbot, P., G.G. Martin, and H. Ashby 1987 Formation of the rupture site in preovulatory hamster and mouse follicles: Loss of the surface epithelium. Gamete Res., 17r287-302. Talerman, A. 1992 Ovarian pathology. Curr. Opin. Obstet. Gynecol., 4t608-615.