Development and maintenance of a polycystic condition in ovaries autotransplanted under the kidney capsule.код для вставкиСкачать
THE ANATOMICAL RECORD 225118-123 (1989) Development and Maintenance of a Polycystic Condition in Ovaries Autotransplanted Under the Kidney Capsule GINA C. DESJARDINS AND JAMES R. BRAWER Department of Anatomy, McGill Uniuersity, Montreal, P.Q. H3A 2B2, Canada ABSTRACT A single injection of estradiol valerate (EV) produces a polycystic ovarian (PCO) condition in the rat. The development of the PCO condition coincides with alterations in the endogenous plasma gonadotropin patterns, suggesting that PCO may be a response to abnormal gonadotropin stimulation. Other factors, however, such a s direct autonomic innervation, also contribute significantly to the regulation of the ovary and could be important in generating and/or maintaining PCO. We have, therefore, removed and autotransplanted one ovary in each of eight rats under the capsule of the ipsilateral kidney, thus totally disrupting its innervation. The animals were injected with EV and both ovaries of each animal were examined 8 weeks later. In a second group of animals, we induced the PCO condition, autotransplanted one polycystic ovary in each animal under the kidney capsule, and examined the ovaries 2 weeks later. In both groups the autotransplanted ovaries exhibited the full range of polycystic morphology, a s did the intact ovary in each animal. We conclude that since a major perturbation in innervation affects neither the development nor the maintenance of PCO, autonomic innervation does not play a crucial role in this disorder. Anovulatory acyclicity characterized by ovaries containing multiple cystic follicles is a reproductive anomaly that occurs in a wide range of mammalian species (including the human) under a variety of circumstances. It occurs in the laboratory rat as a result of age (Aschheim, 1976), neonatal androgen treatment (Gorski, 1971), and treatment in adulthood with dihydroepiandrosterone (DHEA), (Parker and Mahesh, 19761, estradiol valerate (EV) (Brawer et al., 19781, antibody to LHRH (Fraser and Baker, 1978; Popkin et al., 19831, or thiouracil and hCG (Copmann and Adams, 1981). The condition can also be produced by anterior hypothalamic deafferentation (Halasz, 19691, constant light exposure (Daane and Parlow, 1971), and implantation of estradiol benzoate into the anterior hypothalamus (Kawakami and Vissuvan, 1979). The histologically definitive polycystic condition (PCO) does not appear to reflect intrinsic ovarian pathology. The polycystic ovaries in both the neonatally androgenized and EV-induced models ovulate in response to a n LHRH challenge (Hemmings et al., 1983). In the DHEA-treated and constant-light-induced models, the ovaries revert to normal function and histologic appearance following the cessation of treatment. Moreover, ovulatory cyclicity and normal ovarian histology can be restored in EV-induced PCO by the removal of one polycystic ovary (i.e., hemiovariectomy) (Farookhi et al., 1985). Since we have identified hypothalamic and pituitary impairments in animals with PCO (Brawer e t al., 1986; Simard et al., 1987; Carrier et al., 19891, it seemed most likely that the polycystic condition reflects the response of a normal ovary to abnormal gonadotropic 0 1989 ALAN R. LISS, INC. stimulation. This hypothesis was strengthened by the observation that the development of polycystic ovaries in the EV-treated model is preceded by the emergence of a specific abnormal episodic plasma pattern of LH (McCarthy et al., 1986). This pattern is invariably present in animals with established PCO (Grosser et al., 1987). Although it seems likely t h a t the plasma LH pattern plays a role in PCO, there are other factors, such as direct autonomic innervation, which contribute to the regulation of ovarian function and that may also be significant in generating and maintaining PCO. The rat ovary is richly supplied with both sympathetic (Lawrence and Burden, 1980) and parasympathetic (Burden et al., 1978) innervation. In addition, a beta adrenergic mechanism is involved in steroid-induced increases in ovarian blood flow (Varga et al., 1985) a s well as in the facilitation of LH-induced steroidogenesis in hypertrophied thecal and secondary interstitial cells (Erickson et al., 1985). Moreover, these cells have been shown to receive direct adrenergic innervation (Erickson et al., 1985). In view of the prominence of hypertrophied thecal and secondary interstitial cells in the polycystic ovary (Brawer et al., 1989) we have asked whether direct innervation andlor any other local factor is essential to the production or maintenance of polycystic ovaries. Our approach has been to disrupt the innervation to the ovary and to alter the local environment by remov- Received December 6, 1988; accepted February 21, 1989. 119 PCO IN TRANSPLANTED OVARIES ing the ovaries and implanting them beneath the kidney capsule. We can then determine whether EV treatment will produce the PCO morphology in transplanted normal ovaries and whether the PCO morphology is maintained in transplanted polycystic ovaries. TABLE 1. Variety of follicular structures occurring in initially normal ovaries autotransplanted under the kidney capsule and examined 8 weeks after an injection of estradiol valeratel MATERIALS AND METHODS Animals Corpus Rat No. Precystic Cystic Atretic 2" Healthy 2" luteum 1 +I+ +I+ +I+ +I+ -17 2 +I+I+ +I+ +/+ -I3 +I+I+I+ +/-I4 +I+ +I+ +I+ +/+ -I5 -I+ +I+ +I+ +/+ -I+I+ +I+ 6 +/+ -I+I+ 7 -I+ +I+ +I+ +I+ -I- Female Wistar rats (Charles River, Quebec) were maintained under conditions of controlled light (lights on between 0500 and 1900 hours) and temperature (22°C). They were fed Purina rat chow and permitted to drink water freely. Estrous cycles prior to and after treatment were monitored by daily examination of vaginal smears. Experimental Groups Two groups of animals exhibiting at least two consecutive 4-day estrous cycles were used in this study. One group of eight animals underwent unilateral excision and reimplantation of the ovary under the kidney capsule on the left side only. The contralateral ovary was left in place. Immediately following surgery, each animal was injected intramuscularly with 2 mg of EV (Delestrogen, E.R. Squibb and Sons, Princeton, NJ). The ovaries were fixed by perfusion 8 weeks later to allow for the complete development of the PCO condition (Brawer et al., 1978, 1986). Each of seven animals in the second group was injected with EV and left for 8 weeks time, during which the ovaries became polycystic. One cystic ovary of each animal was then implanted under the capsule of the ipsilateral kidney, while the remaining ovary was left in place. Two weeks after transplantation, the ovaries were fixed by perfusion and examined. Transplantation Rats were anesthetized with ether, and a 1 cm-wide incision was made approximately 2 cm below the last rib in the midclavicular region. The ovary was removed, stripped of adherent connective tissue, and washed in saline. The kidney was then localized through the same incision, and a small aperture was made in the kidney capsule into which the ovary was then inserted. Perfusion and Preparation of Tissues Animals were anesthetized with 0.9 ml of urethane per 100 g body weight, and the descending aorta was exposed through a ventral midline incision. The descending aorta was clamped immediately above the bifurcation. An 18-gauge needle was introduced into the aorta, above the clamp, and perfusion with lactated Ringer's solution was begun. A second clamp was placed on the aorta just below the level of the liver and the renal vein of the kidney containing the ovarian transplant was cut. When the kidney appeared uniformly blanched, the perfusate was switched to fixative. This fixative consisted of 1% formaldehyde and 1% glutaraldehyde solution in 0.12 M phosphate buffer. Both ovaries were removed and cleaned of adherent connective tissue. Each ovary was then cut into halves or thirds and these blocks of tissue were left in fixative Follicle tvDe 'The numbers in the column on the left each designate an individual animal. The categories across the top each indicate a type of follicular structure. The occurrence of a specific type of follicular structure in the ovary of an individual animal is designated by + ; the absence of that structure is indicated by -. The first symbol refers to the control (intact) ovary and the second to the autotransplanted ovary. + / would, therefore, indicate that the intact ovary, but not the autotransplanted ovary in a particular rat, has the designated structure. overnight. The following day, the tissues were postfixed in a solution of 1% Os04, and 1.5% ferrous cyanide for 4 hours. The tissues were then dehydrated in graduated concentrations of methanol, left in a 1:l mixture of propylene oxide overnight, and embedded in Epon; 2-3 pm-thick sections were cut on a Reichert microtome and stained with toluidine blue. RESULTS The transplanted ovaries in all animals of both experimental groups exhibited the typical polycystic morphology that characterized the nontransplanted (control) ovary. Both the transplanted and control ovary of each animal expressed the full complement of morphologic features that exemplify the polycystic condition in the EV-treated model (Hemmings et al., 1983; Brawer et al., 1986,1989).These included the presence of large precystic and cystic follicles, numerous atretic secondary follicles (as well a s a few normal secondary follicles), absence of corpora lutea, and a n extensive stroma composed of hypertrophied secondary interstitial cells. In addition, occasional very large healthy follicles described previously as type I11 large follicular structures (Brawer et al., 1989) were also seen in both control and transplanted ovaries. These results are summarized in Tables 1 and 2. The histological appearance of the cysts and precystic follicles was archetypic of the EV-induced PCO condition. The cystic follicle consisted of a large antrum surrounded by a highly attenuated membrana granulosa (Figs. 1-3). This layer was often no more than one cell thick. Occasional large macrophagelike cells appeared on the antral surface of the membrana granulosa (Fig. 3). Precystic follicles resembled cysts except for irregular, occasionally extensive patches of degenerate granulosa cells occurring at variable intervals along the perimeter of the antrum (Figs. 4, 5). The thecal cells in cystic, precystic, and atretic secondary follicles were generally large, polygonal in shape, and filled with lipid ghosts (Figs. 2, 3). Occasional small fusiform thecal cells typical of normal fol- 120 G.C. DESJARDINS AND J.R. BRAWER TABLE 2. Polycystic ovaries autotransplanted under the kidney capsule and examined 2 weeks later' Follicle type Corpus Rat No. Precvstic Cvstic Atretic 2" Healthy 2" luteum 1 2 3 4 5 6 7 +I+ -I+ -I+ +/+ +I-I+ +I- +I+ -I+ +I+ +I+I+ +/+ +I+ +I+ +I+ +/+ +I+ +I+ +I+ +/+ +/+ +/+ +/+ +/+ +/+ +/+ +I+ -I+I-I-I-/-1-I- 'Results are expressed as the presence ( + 1 or absence (-) of the indicated follicular structure in the intact/autotransplanted ovaries of each animal. licles were also occasionally seen in cystic and precystic theca. Large clusters of hypertrophied, lipid-containing interstitial cells occurred throughout the ovaries (Fig. 2). In addition to the cystic, precystic, and atretic follicles, a unique follicular structure which we have called the type I11 large follicular structure (Brawer et al., 1989) was occasionally observed. Cystic and precystic follicles have been designated as type I and I1 large follicular structures (Brawer et al., 1989). The type I11 large follicular structure was characterized by a very large antrum surrounded by an extremely thick layer of healthy granulosa cells (Figs. 3, 6). The theca is composed of a diffuse layer of small fusiform cells, characteristic of healthy follicles. Occasional large polygonal Fig. 2. Cyst wall and secondary interstitial cells in 2-week ovarian transplant. The membrana granulosa (arrowheads) is one to two cells thick. Immediately beneath the membrana granulosa is a basement membrane beneath which is the hypertrophied thecal cell layer. The thecal cells are large, polygonal, and often filled with lipid ghosts. The left quarter of the field is occupied by secondary interstitial cells (arrows) similar in appearance to the cystic thecal cells. x 800. cells appeared scattered throughout the thecal cell layer (Fig. 3). The type I11 large follicular structure differs from a preovulatory follicle in that it is larger and in that occasional mitotic figures occur in the perimural granulosa region. Furthermore, the absence of corpora lutea in these ovaries indicates that the type I11 large follicular structure is not ovulatory. DISCUSSION Fig. 1. Cystic follicle in 8-week ovarian transplant. The large antrum is surrounded by a single layer of granulosa cells over most of the perimeter. In the upper right quadrant of the cyst the membrana granulosa is slightly thicker (arrowheads). x 130. The polycystic ovary in the estradiol-treated rat exhibits a unique constellation of morphologic features. This includes absence of corpora lutea, a dearth of healthy secondary follicles, although atretic secondary follicles are commonly encountered, and an abundance of secondary interstitial tissue (Brawer et al., 1986, 1989). In addition, there are three types of follicular structures unique to the polycystic ovary. These are the cystic and precystic follicles and the type I11 large follicular structure. This latter structure is of Darticular interest because of its size and the healthy nbrmal appearance Of the granulosa and Moreover, it is the only follicular structure in the PolYcYstic ovary to exhibit LH-binding sites on the perimural granulosa PCO IN TRANSPLANTED OVARIES 121 planted under the kidney capsule sustain normal estrous cyclicity and exhibit the typical range of follicular structures as well as normally appearing corpora lutea (Del Castillo, 1928; Vivien, 1948; Harris and Eakin, 1949; Krohn, 1959, 1962). Although some cyst formation as well as other expressions of pathology have been reported following transplant to spleen or subcutaneous connective tissue at very long postimplantation intervals (months) (Krohn, 1962; Parkes, 19561, no abnormal structures occur in ovaries implanted beneath the kidney capsule (see Krohn, 1962, for review). The appearance of pathologic changes in old ovarian grafts in spleen or connective tissue is attributed to inadequate vascularization. Although the transplant procedure obviously causes a total disruption of ovarian innervation, there is little information on the extent to which the transplanted ovaries may be reinnervated. Hill (1949), using the protargol stain, failed to observe any reinnervation of adult ovaries autotransplanted t o the spleen in mice. On the other hand, Jacobowitz and Laties (1970),using induced catecholamine fluorescence, observed reinnervation of ovarian tissue transplanted to the anterior chamber of the eye in the cat. Although fluorescent fibers were observed 35 days after transplant, no fibers were observed at 15 days. Moreover, the extent of adrenergic innervation in the transplanted ovaries (at 35 or 75 days) was considerably less than that seen in the normal ovary. In the present study, it is possible that reinnervation fig. 3. Segments of a cystic follicle and a type 111 large follicular structure in 8-week ovarian transplant. The antrum of the cystic follicle (C) is surrounded by a single layer of granulosa cells. Peripheral to the basement membrane is the thecal layer composed mostly of large, lipid-filled polygonal cells. The antrum of the type III large follicular structure (T) is surrounded by multiple layers of healthy granulosa cells. The theca consists of scattered cells many of which are small and fusiform. x 400. cells as well as on thecal cells (Brawer et al., 1989).The occurrence of the type I11 large follicular structure indicates that the polycystic ovary engages in a unique form of folliculogenesis in addition to the degenerative processes that produce atretic secondary follicles, the abundance of secondary interstitial tissue, and cysts. This study demonstrates that the complete spectrum of histological characteristics that define the polycystic ovary can be produced and maintained in ovaries transplanted under the kidney capsule. In each animal, the transplanted ovary exhibited the same morphological features as the intact (control) ovary. This suggests that neither direct innervation t o the ovary nor any other local factor need play a significant role in the generation of PCO. This interpretation is based on two assumptions. The first is that the transplant procedure does not itself induce the PolYcYstic condition, and the second is that the transplantation results in a significant disruption in ovarian innervation. The first of these assumpt~onsis well supported by the literature, representing Over 40 Years of experience with ovarian transplants. Healthy ovaries trans- Fig. 4. Segments ofa cystic and precystic follicle in 2-week ovarian transplant. The antrum of the cyst ( C )is surrounded by a n attenuated irregular membrana granulosa. The thecal cells are typically large and contain numerous lipid ghosts. Although the theca of the precystic follicle (PI resembles that of cysts, the membrana granulosa contains numerous cells many of which show clear signs of degeneration. x 4,000. 122 G.C. DESTARDINS AND J.R. BRAWER Fig. 5. Precystic follicle in 8-week nontransplanted (control side) ovary. In the top of the field is a typical follicle (PI. This particular section includes the degenerating ovum. To the immediate left is a segment of a cystic follicle (C).The bottom of the field is occupied by atretic follicles (a) and clusters of secondary interstitial cells (arrowheads). x 52. occurs to some extent in the long-term (8-weekI-transplanted ovaries. It is unlikely, however, that significant reinnervation occurs in the l k d a y transplants. In any event we have shown that even discrete temporary perturbations in the neuroendocrine axis of rats with PCO rapidly produce major alterations in ovarian histology (Hemmings et al., 1983; Carriere et al., 1989). Clearly, a significant disruption in ovarian innervation has no such effect, suggesting that innervation plays a minor, if any, role in PCO. ACKNOWLEDGMENTS The authors gratefully acknowledge the technical assistance of Ms. Dalia Chen. This work was supported by a n operating grant to J. Brawer from the Medical Research Council of Canada. LITERATURE CITED Aschheim, P. 1976 Aging in the hypothalamic hypophyseal ovarian axis in the rat. In: Hypothalamus, Pituitary and Aging. A Everill and J.A. Burgess, eds. Charles C. Thomas, Springfield, 11, pp. 376-418. Brawer, J.R., F. Naftolin, J. Martin, and C. Sonnenshein 1978 Effects of a single injection of estradiol valerate on the hypothalamic arcuate nucleus and on the reproductive function in the female rat. Endocrinology, 103:501-512. Fig. 6. Type I11 large follicular structure in &week transplant. The large antrum is surrounded by multiple layers of healthy granulosa cells and a thecal cell layer of ordinary thickness and appearance. Compare with Figure 1. Brawer, J.R., M. Munoz, and R. Farookhi 1986 Development of the polycystic ovarian condition in the estradiol valerate-treated rat. Biol. Reprod., 35t647-655. Brawer, J.R., M. Richard, and R. Farookhi 1989 Pattern of human chorionic gonadotropin binding in the polycystic ovary. Am. J. Obstet. Gynecol., in press. Burden, H.W., and I.E. Lawrence 1978 Experimental studies on the acetylcholinesterase-positivenerves in the ovary of the rat. Anat. Rec., 190r233-242. Carriere, P.D., Farookhi, R., and Brawer, J.R. 1989 The role of aberrant hypothalamic opiatergic function in generating polycystic ovaries in the rat. Can. J. Phys. Pharm. (in press). Copmann, T.L., and W.C. Adams 1981 Relationship of polycystic ovary induction to prolactin secretion: Prevention of cyst formation by bromocriptine in the rat. Endocrinology, 108t1095-1097. Daane, T.A., and A.F. Parlow 1971 Serum FSH and LH in constant light-induced estrus: Short-term and long-term studies. Endocrinology, 88t964-968. Del Castillo, E.B. 1928 Reapparition et caracteristiques de cycle oestral apres greffe ovarienne chez le rat blanc castre. C. R. SOC.Biol. (Paris), 99t1501-1502. Erickson GF, Magofin DA, Dyer CA, Hofeditz C, 1985. The ovarian androgen producing cells: a review of structure/function relationships. Endocrine Reviews 6371-49. Farookhi, R., R. Hemmings, and J.R. Brawer 1985 Unilateral ovariectomy restores ovulatory cyclicity in rats with a polycystic ovarian condition. Biol. Reprod., 32r530-540. Fraser, H.M., and T.G. Baker 1978 Changes in the ovaries of rats after immunization against luteinizing hormone releasing hormone. J . Endocrinol., 77t85-93. Gorsky, R.A. 1971 Gonadal hormones and the perinatal development of neuroendocrine function. In: Frontiers in Neuroendocrinology . PCO IN TRANSPLANTED OVARIES L. Martini and W.F. Ganong, eds. Oxford University Press, New York, pp. 237-290. Halasz, B. 1969 The endocrine effects of isolation of the hypothalamus from the rest of the brain. In: Frontiers in Neuroendocrinology. L. Martini and W.F. Ganong, eds. Oxford University Press, New York, pp. 307-342. Harris, M., and R.M. Eakin 1949 Survival of transplanted ovaries in rats. J. Exp. Zool., 112:131-153. Hemmings, R., R. Farookhi, and J . Brawer 1983 Pituitary and ovarian responses to luteinizing hormone releasing hormone in a rat with polycystic ovaries. Biol. Reprod., 29:239-248. Hill, R.T. 1949 Adrenal cortical physiology of spleen grafted and denervated ovaries in the mouse. Exp. Med. Surg., 7:86-98. Jacobowitz, D. and A.M. Laites 1970 Adrenergic reinnervation of the cat ovary transplanted to the anterior chamber of the eye. Endocrinology, 86:92 1-924. Kawakami M., and S. Visessuvan 1979 The differing responsiveness of the anterior- and middle-anterior hypothalmic area to estradiol benzoate implant: inhibition of compensatory ovarian hypertrophy. Endokrinologie 74:271-86. Krohn, P.L. 1959 Transplantation of endocrine glands. In: Transplantation of Tissues. Lyndon A. Peer, ed. William and Wilkins, Baltimore, Vol. 11, Chap. 12, pp. 401-461. Krohn, P.L. 1962 Transplantation of the ovary. In: The Ovary 11. Sally 123 Zuckerman, ed. Academic Press, New York, Chap. 21, pp. 435-446. Lawrence, I.E., and H.W. Burden 1980 The origin of the extrinsic adrenergic innervation to the rat ovary. Anat. Rec., 196:51-59. McCarthy, F.G., J.R. Brawer, and R. Farookhi 1986 Plasma LH patterns characterizing the development of the polycystic ovarian condition (PCO) in the estradiol valerate (EV)-treatedmice. Biol. Reprod., 34(Suppl. 11:153. Parker, C.R., and V.B. Mahesh 1976 Interrelationship between excessive circulating androgens in blood and ovulatory failure. J. Reprod. Med., 17r75-90. Parkes, A S . 1956 Survival time of ovarian homografts in two strains of rats. J. Endocrinol., 13r201-210. Popkin, R.M., H.M. Fraser, and R.G. Gosden 1983 Effects of LHRH agonist on LHRH immunoneutralization on the pituitary and ovarian LHRH receptors in female rats. J. Reprod. Fertil., 69r249-252. Varga, B., E. Horvath, G. Folly, and E. Stark 1985 Study of luteinizing hormone induced increase of ovarian blood flow during the estrous cycle in the rat. Biol. Reprod., 32:480-488. Vivien, J.H. 1948 Comportemnet de greffes intra-viscerales de gonades chez la souris albinos: I’Ovaire. C. R. SOC.Biol. (Paris) 142t1006-1007.