Gonadotropin Binding Sites in Human Postmenopausal Ovaries*

FERTILITY AND STERILITY Copyright © 1976 The American Fertility Society

Vol. 27, No.7, July 1976 Printed in U.S.A.

GONADOTROPIN BINDING SITES IN HUMAN POSTMENOPAUSAL OVARIES* JOHN J. PELUSO, PH.D.,t RICHARD W. STEGER, PH.D.,t S. JASZCZAK, M.D.,t AND E. S. E. HAFEZ, PH.D. Reproductive Physiology Laboratories, C. S. Matt Center for Human Growth and Development, Wayne State University School of Medicine, Detroit, Michigan 48201

Glucose-6-phosphate dehydrogenase (G6PD) activity and gonadotropin binding sites were localized within seven postmenopausal ovaries. G6PD was localized in the cells of cortical stroma and hilus using a histochemical technique for the reduction of the tetrazolium salt, Nitro-Blue tetrazolium. Gonadotropin binding sites were localized by autoradiography following incubation of ovarian sections with 125/-labeled gonadotropins. The binding sites for both 1251-labeled luteinizing hormone (1 251-hLH) and 125/-labeled folliclestimulating hormone (1 251-hFSH) were identified in the cortical stroma and hilus cells. Since these cells contain G6PD and other enzymes necessary for steroidogenesis and also have the capacity to bind both hLH and hFSH, steroidogenesis in postmenopausal ovaries appears to be controlled by circulatting gonadotropins. Blood vessels within postmenopausal ovaries also bound both gonadotropins, but 1251-hFSH binding was often more intense than 1251-hLH binding.

The endocrine profile of the postmenopausal women is characterized by elevated gonadotropin levels and reduced serum estrogen levels. 1 • 2 Although there is considerable variation among women, ovariectomy often results in a further reduction of serum estrogen levels, indicating that the postmenopausal ovary still has limited steroidogenic activity. 2 Histochemical studies have shown that the cortical stroma of the postmenopausal ovary possesses several of the enzymes necessary for steroid synthesis. 3 • 4 Judd and co-workers 5 have shown that the postmenopausal ovary actively synthesizes predominately androgens but also produces limited amounts of estrogens.

In addition, there is a high correlation between serum estrogen levels and ovarian glucose-6-phosphate dehydrogenase activity. 4 Although luteinizing hormone and follicle-stimulating hormone levels are elevated in the postmenopausal woman, the ability of these gonadotropins to regulate ovarian function is still in question. Therefore, it is of interest (1) to determine whether the postmenopausal ovary can bind 125 I-labeled gonadotropins, (2) to identify the ovarian structures to which the gonadotropins bind, and (3) to correlate the gonadotropin binding sites with steroid-synthesizing cells as indicated by high glucose-6-phosphate dehydrogenase activity. MATERIALS AND METHODS

Accepted March 5, 1976. *Presented at the Thirty-Second Annual Meeting of The American Fertility Society, April 7 to 9, 1976, Las Vegas, Nev. tFord Foundation Postdoctoral Fellow.

Samples of ovarian tissue were obtained from patients undergoing hysterectomy and bilateral salpingo-oophorectomy. Immediately after surgery, each

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Both hLH and hFSH were iodinated ovarian sample was divided into two pieces. One piece was fixed in Bouin's with 125 I, using the chloramine T method. 6 solution for histologic examination. The After iodination, the reaction mixture other piece was quick-frozen in a Dry was passed through a Bio-Gel P-60 column Ice-ethanol bath and stored at - 20o C. (Bio-Rad, Richmond, Calif.) in order to The Bouin's-fixed tissue was embedded separate the 125 I-labeled gonadotropin in paraffin, sectioned at 10 JLm, and from the free 125!. Approximate values stained with hematoxylin and eosin or for specific activity were 26 and 50 JLCil Masson's trichrome. The quick-frozen JLg of protein for hLH and hFSH, retissue was sectioned at 10 JLm in a cryo- spectively. stat, placed on glass slides, and stored In order to identify the cellular sites at 4° C in a moist chamber until a suf- to which 125I-labeled gonadotropins bind, ficient number of sections were cut. Al- sections were incubated for 1 hour at ternate sections were then used for deter- 37° C with either 125 I-hLH (2.5 x 10 5 mining the localization of gonadotropin cpm/50 JLD or 125 I-hFSH (2.3 x 105 cpm/ binding sites, glucose-6-phosphate dehy- 50 JLD. After incubation the sections drogenase (G6PD) activity, or for hema- were washed for 10 minutes in cold phostoxylin-eosin staining. phate-buffered saline (pH 7 .6), rinsed in Localization of Gonadotropin Binding distilled water for 5 minutes, and airSites. Purified human FSH (hFSH) and dried. The sections were then dipped in LH (hLH) (Calbiochem, La Jolla, Calif.) Kodak NTB-3 nuclear track emulsion, were used in this experiment. The hLH exposed for 15 days at 4° C, and developed. 7 had a biologic potency of 2000 IU/mg, Selected sections were incubated with as judged by the ovarian ascorbic acid 125 I-hLH or 125 I-hFSH in the presence of a depletion bioassay, and 4 IU of FSH activity in the human chorionic gonado- 100-fold excess of the respective unlabeled tropin augmentation bioassay. 125 I-hLH gonadotropin in order to determine binddid not bind to an FSH antiserum which ing specificity. had been preabsorbed with hLH. The Localization of G6PD Activity. G6PD human hFSH preparation had a biologic activity was localized in ovarian sections potency of 3500 IU/mg when tested in by using a histochemical technique inthe human chorionic gonadotropin aug- volving the reduction of the tetrazolium mentation bioassay. The LH contamina- salt, Nitro-Blue tetrazolium. The sections tion of this FSH preparation was less than were incubated for 30 to 40 minutes at 5% when tested in both the ovarian 37" C with the incubation media to idenascorbic acid depletion bioassay and tify the cells that possessed G6PD activity. 8 against preabsorbed LH antisera. TABLE 1. Clinical Data on Seven Postmenopausal Patients Age of patient

Time after menopause

yr

mo

50 50

53 53 54

12 2.5" 60

12 2"

63

132

68

156

Ovarian morphology

Corrugated surface Small, atrophic Small, atrophic Small, atrophic Small, some adhesions Small, atrophic Small, atrOphic

Pathology

Uterine fibroid Uterine fibroid Adenoma to us hyperplasia of endometrium Adenomatous hyperplasia of endometrium Fibroid, cervical carcinoma in situ Uterine fibroid, vaginal carcinoma Adenoma to us hyperplasia of endometrium

"Time of menopause was difficult to establish because of prolonged, irregular, uterine bleeding associated with uterine fibromyomata.

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FIGs. 1 TO 4. Alternate sections from a postmenopausal ovary (x 90). FIG. 1. A section showing cortical stromal cells (S), corpora albicantia (A), and blood vessels (arrowhead) (hematoxylin and eosin). FIG. 2. A section demonstrating G6PD activity within the cortical stromal cells. FIGs. 3 AND 4. Autoradiographs showing '"I-hLH (Fig. 3) and 125!-hFSH (Fig. 4) binding to the cortical stroma. The corpora albicantia do not bind either ' 2 '!-labeled gonadotropin.

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FIGS. 5 TO 7. Hilus cells found within the postmenopausal ovary (Fig. 5) have G6PD activity (Fig. 6) and 1251-hLH binding sites (Fig. 7) (x 360).

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FIGS. 8 AND 9. The blood vessels within the ovary bind both 1251-hLH (Fig. 8) and 1251-hFSH (Fig. 9). The gonadotropins occasionally bind only to the inner and outer surfaces of the ovarian arteries (arrow) (x 270).

ment, but corpora albicantia in various stages of degeneration were often obEvaluation of Clinical Material. The served (Fig. 1). Stromal cells were noted ovaries selected for this investigation in the ovarian cortex. In some ovaries, were obtained from women 50 to 68 years stromal cells were aggregrated in dense of age undergoing hysterectomy and areas and arranged in a pattern resembilateral salpingo-oophorectomy. These bling cortical stromal hyperplasia. Other patients were 2 months to 13 years post- dense clusters of stromal cells were idenmenopausal. All ovaries were free of tified as cortical stromal fibrosis by their any gross pathology. Postoperative ex- dominant collagen staining. Hilus cells amination of the uteri ofthe seven women and blood vessels were observed throughrevealed four cases of uterine fibroids out the medulla of the postmenopausal and three cases of endometrial hyper- ovary. plasia. One patient had cervical carG6PD Activity and Gonadotropin Bindcinoma in situ and another had vaginal ing. Preliminary autoradiographic carcinoma (Table 1). In two patients, studies have shown that 2 to 3 x 10 5 the time of menopause was difficult cpm/50 ILl of either hLH or hFSH were to establish because of prolonged, irregu- required to visualize specific binding lar, uterine bleeding associated with sites within the ovary. When sections fibromyomata, but these women had not were incubated with both labeled and unexperienced uterine bleeding for 2 to 3 labeled gonadotropin, binding to the cortimonths prior to surgery. cal stroma, hilus, and blood vessels was Ovarian Histology. The ovaries did not substantially reduced, indicating binding contain follicles in any stage of develop- specificity. RESULTS

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In all ovaries studied, G6PD activity estrogenic stimulation of the endomewas found in both the cortical stromal trium.3 The present study demonstrates cells and hilus cells (Figs. 2 and 6). that the cortical stromal and hilus cells These cell types also bound both 125 I- of the postmenopausal ovary have both labeled gonadotropins (Figs. 3, 4, and 7). G6PD activity and the ability to bind In contrast, the cells of the corpora al- labeled LH and FSH. In contrast, the bicantia did not demonstrate either G6PD cells of the corpora albicantia do not activity or the ability to bind 125 I-hLH possess either G6PD activity or gonadoor 125I-hFSH (Figs. 2 to 4). tropin binding sites. These observations The arterioles and venules found in suggest that cells of the stroma and postmenopausal ovaries bound both hilus in the postmenopausal ovary can labeled gonadotropins, but 125 I-hFSH bind circulating gonadotropins. Since binding was often more intense than these cells do possess gonadotropin 125 I-hLH binding (Figs. 8 and 9). The binding sites, their ability to synthesize pattern of gonadotropin binding to the steroids may be regulated by circulating arterioles varied within the same ovary. LH and FSH. In some cases, the labeled gonadotropin In postmenopausal women the conbound uniformly to the entire wall of the centrations of testosterone, androstenearteriole, whereas binding to other ar- dione, 17,8-estradiol, and estrone are terioles was confined to the inner and higher in ovarian venous blood than in outer surfaces of the arterial wall. Both peripheral blood. 5 The differences begonadotropins bound uniformly to ovarian tween ovarian and peripheral levels are 15-fold for testosterone, 4-fold for androvenules (Figs. 8 and 9). stenedione, and 2-fold for both 17,8estradiol and estrone. Since LH promotes DISCUSSION testosterone synthesis in ovarian tissue, 13 The postmenopausal ovary does not LH binding to the stromal and hilus cells have follicles capable of ovulating but could stimulate androgen biosynthesis. retains a limited endocrine function. In the rat ovary, FSH stimulates the Numerous reports have shown that post- ovarian aromatizing enzyme system that menopausal women have higher serum converts androgens into estrogens. 14 and urine steroid levels than do women Since a small amount of estrogen is who have undergone bilateral oophorec- produced by the postmenopausal ovary, 5 tomy. However, there is extensive in- the presence of FSH binding sites and dividual variation in the percentage of G6PD activity suggest that the stromal postmenopausal women whose ovaries cells may also be a site of estrogen synsecrete estrogens. The lipid-rich lu- thesis. teinized cells of postmenopausal ovarian The hilus cells of the postmenopausal stromal hyperplasia resemble the theca ovary also have binding sites for hLH interna cells of the follicle and contain and hFSH as well as G6PD activity, sugseveral enzymes, including 3,8-hydroxy- gesting that these cells have steroidosteroid dehydrogenase, which are neces- genic activity. Histochemically, the sary for steroid synthesis:1 • 9-lt It is as- hilus cells are similar to the interstitial sumed that these cells are the sites of cells of the testes and are generally steroid synthesis in the postmenopausal assumed to produce and~gens. 3 The preovary. 12 Other studies have shown a posi- sence of gonadotropin binding sites tive correlation between the number of suggests that gonadotropins may regulate G6PD-active cells in the stroma of the androgen biosynthesis in the hilus cells. postmenopausal ovary and the degree of Ovarian arteries and veins. also bind

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both labeled gonadotropins. The functional significance of this phenomenon is not fully understood. Preliminary studies have shown that the blood vessels within the rat ovary also bind 125 I-labeled gonadotropin. 15 In rat and rabbit ovaries, both LH and FSH can regulate ovarian blood flow. 16 • 17 It is not known whether gonadotropins affect ovarian blood flow by a direct action on the blood vessels or indirectly through increased steroid synthesis. However, the ability of the ovarian blood vessels to bind gonadotropins suggests that gonadotropins could regulate blood flow by a direct effect on the wall of ovarian blood vessels.

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10. Acknowledgments. The authors are indebted to Drs. M. E. Azzam and T. N. Evans for providing the ovarian tissue and to Mr. Bruce Borin for providing the iodinated hormones.

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REFERENCES

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1. Riley GM: Endocrinology of the climacteric. Clin Obstet Gynecol 7:432, 1964 2. Paulsen CA, Leach RB, Sandberg H, Sheinfield S, Maddock WO: Function of the postmenopausal ovary. Comparison of urinary estrogen and gonadotropin excretion and response to administration of FSH in postmenopausal and ovariectomized women. JAm Geriatr Soc 6:803, 1958 3. Novak ER, Goldberg B, Jones GS, O'Toole RV: Enzyme histochemistry of the menopausal ovary associated with normal and abnormal endometrium. Am J Obstet Gynecol 93:669, 1965 4. Brandau H, Brandau L, Mestwerdt W: Endocrine activity in postmenopausal ovaries. Eur J Obstet Gynecol Reprod Bioi [Suppl 1] 4:S187, 1974 5. Judd HL, Judd GE, Lucas WE, Yen SSC:

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