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Reproductive sequelae in female rats after in utero and neonatal exposure to the phytoestrogen genistein
FERTILITY AND STERILITYt VOL. 70, NO. 3, SEPTEMBER 1998 Copyright ©1998 American Society for Reproductive Medicine Published by Elsevier Science Inc. Printed on acid-free paper in U.S.A.
Reproductive sequelae in female rats after in utero and neonatal exposure to the phytoestrogen genistein Caleb A. Awoniyi, Ph.D.,* Dawn Roberts, M.S.,* D. N. Rao Veeramachaneni, D.V.M., Ph.D.,† Bradley S. Hurst, M.D.,* Kathleen E. Tucker, Ph.D.,* and William D. Schlaff, M.D.* University of Colorado Health Sciences Center, Denver, and Colorado State University, Fort Collins, Colorado
Objective: To determine reproductive sequelae in female rats after in utero and lactational dietary exposure to genistein. Design: Experimental animal study. Setting: University laboratory. Animal(s): Sprague Dawley rats. Intervention(s): Pregnant rats were fed control rat chow or rat chow incorporated with genistein (approximately 50 mg/d) beginning on day 17 of gestation and continuing until the end of lactation (postpartum day 21). Genistein-exposed female pups were divided into two groups on day 21. One group continued to receive a genistein-added diet (G70); the other group was changed to a control diet (Ex-G). At necropsy (days 21 and 70), blood and reproductive tissues were collected. Main Outcome Measure(s): Serum levels of gonadotropins and gonadal steroids and histopathologic examination of the ovaries. Received December 18, 1997; revised and accepted April 29, 1998. Supported in part by grant DK48520 from the National Institutes of Health, Bethesda, Maryland, and the Academic Enrichment Fund from the Department of Obstetrics and Gynecology, University of Colorado Health Sciences Center, Denver, Colorado. Reprint requests: Caleb A. Awoniyi, Ph.D., Division of Reproductive Endocrinology, Department of Obstetrics and Gynecology, University of Colorado Health Sciences Center, Denver, Colorado 80262 (FAX: 303-315-8889; E-mail: CALEB.AWONIYI @UCHSC.EDU). * Division of Reproductive Endocrinology, Department of Obstetrics and Gynecology, University of Colorado Health Sciences Center. † Animal Reproduction and Biotechnology Laboratory, Colorado State University. 0015-0282/98/$19.00 PII S0015-0282(98)00185-X
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Result(s): The weight of the ovaries and uterus and serum levels of E2 and progesterone in genistein-exposed rats on day 21 (G21) were significantly reduced compared with control rats. On day 70, serum levels of E2, progesterone, LH, and FSH were similar in all groups. Atretic follicles and secondary interstitial glands were more common in G70 and Ex-G rats compared with control rats. Cystic rete ovarii was observed in some G70 and Ex-G rats. Conclusion(s): Our data indicate that in utero and lactational exposure to dietary genistein adversely affects reproductive processes in the adult female rat. (Fertil Sterilt 1998;70:440 –7. ©1998 by American Society for Reproductive Medicine.) Key Words: Phytoestrogen, genistein, estrous cyclicity, ovary, uterus, gonadotropins, estrogen, progesterone
Isoflavonoids are present in plants and resemble steroidal estrogens and mimic many of their actions (1–3). However, the biologic roles played by isoflavonoids when they are ingested by animals are not fully understood. Genistein and diadzein, the major isoflavones in soybeans (4, 5), are found in high concentrations in the urine of Asian women, a population known to be resistant to breast cancer. Conversely, they are found in low concentrations in patients with breast cancer, suggesting that they may have a role in the prevention of estrogen-dependent carcinoma (6, 7). Although the possible role of isoflavonoids as anticancer agents is being considered, there also is increasing concern that naturally occurring and xenobiotic environmental agents may exert some adverse effects as well (8). In fact, a recent review suggested that the ingestion of
a fungal estrogen may have contributed significantly to the decline of the rural 19th and 20th century European population (9). Given the widespread consumption of these phytoestrogens by humans and the perceived potential for developing therapeutic, anticancer treatments with them, it is imperative to assess the potential impact of these isoflavonoids on reproductive function. Phytoestrogens have been shown to cause both direct and indirect toxicologic effects on the reproductive tract of animals (3, 10 –13). These effects include persistent vaginal cornification, hemorrhagic ovarian follicles, and premature vaginal opening (11, 14). Although it has been known for years that grazing in certain clover fields causes significant infertility in sheep and cattle (15, 16), only recently has it been discussed that the phytoestrogens
contained in clover produce alterations in the hypothalamicpituitary-ovarian axis by binding to hypothalamic estrogen receptors (17–20). To date, the effects of phytoestrogens on sexual development and on the development of the neuroendocrine system are not well understood.
FIGURE 1 Schematic diagram of the experimental design. GD 5 gestation day; PND 5 postnatal day.
Recently, Faber and Hughes (21) observed an increase in the size of the sexually dimorphic nucleus of the preoptic area of the hypothalamus in adult rats after neonatal exposure to various doses of genistein. They also found that low doses of genistein resulted in increased pituitary responsiveness to GnRH, whereas high doses resulted in decreased responsiveness. Available animal studies on phytoestrogens provide little insight into the consequences of the long-term low-dose exposure that is likely to be characteristic of the natural situation in humans. The objective of this study was to examine reproductive sequelae at critical periods of reproductive development in rats after dietary exposure to a phytoestrogen, genistein, at doses relevant to possible human exposure.
MATERIALS AND METHODS Animals Pregnant Sprague Dawley rats (Sasco, Omaha, NE) were purchased at day 10 of gestation. Animals were housed individually in a room maintained at 22°C with a constant light-dark cycle (14 hours of light and 10 hours of darkness), and were provided isoflavonoid-free rat chow and water ad libitum. These studies were performed in accordance with the Guidelines for the Care and Use of Experimental Animals and were approved by the Animal Care Committee of the University of Colorado Health Sciences Center.
Administration of Genistein Because commercial rodent chow contains some isoflavonoid (22), all rats were fed the AIN semipurified rat-mouse diet (Bioserve, Frenchtown, NJ), which is devoid of isoflavonoid and contains casein-high nitrogen (20%), DL-methionine (0.3%), cornstarch (15%), sucrose (50%), fiber-celfil (5%), mineral mixture (3.5%), vitamin mixture (1%), and chlorine bitartrate (0.2%), until day 17 of gestation. We chose to administer genistein in the diet at a dose of 5 mg/kg of feed based on the data of Barnes (23) and of Steele et al. (24). Barnes estimated that human intake of genistein is approximately 50 mg/d. Steele et al. reported that rats given a diet containing 150 mg of genistein per kilogram of feed had a daily intake of 10.45 mg/kg of body weight. On the basis of these observations, we estimated that the required concentration of genistein in the diet is about 2.5 mg/kg of feed to provide 50 mg/d to a 300-g rat. However, because of variations in weight and dietary intake that are certain to occur, we elected to supply a dose of 5 mg/kg of feed to ensure that all animals consumed at least 50 mg of genistein per day. FERTILITY & STERILITYt
Experimental Regimen Because the critical period of steroid-mediated development of the reproductive system in the rat begins around day 17 of gestation, we began dietary genistein exposure (5 mg/kg of feed) in 12 rats on day 17 of gestation. Eight pregnant rats were fed the semipurified control diet. Animals were weighed weekly throughout the experiment. Daily feed consumption was determined by weighing feed daily at 11 AM. Pups were weaned on day 21 and only female rats were used in this study. Pups from four litters in the control (C21) and genisteinexposed (G21) groups were euthanized on day 21 (Fig. 1); their hormones and reproductive organs were evaluated. On postnatal day 21, four litters of genistein-treated rats were given a control diet (Ex-G) and the remaining four continued to receive the genistein-added diet until necropsy was performed on approximately postnatal day 70 (G70). Four litters that had been receiving the control diet continued to receive the same diet and served as controls (C70) for the day 70 groups. The time of vaginal opening was determined, and vaginal smears were taken daily to evaluate estrous cyclicity. All rats were euthanized in proestrus. Trunk blood and reproductive organs were collected. Serum was separated and stored at 220°C for subsequent determination of concentrations of E2, progesterone, LH, and FSH by RIA. Weights of the ovaries and reproductive tracts were recorded, and tissues were processed for histopathologic examination. 441
FIGURE 2 Daily genistein intake in rats fed a genistein-added diet. Values are means 6 SEM.
Briefly, the status of follicular development; the normalcy of ovarian structures such as the surface epithelium, rete ovarii, interstitial glands, and corpora lutea; and the status of the general architecture and the lining epithelium of the oviducts, uterus, cervix, and vagina were evaluated qualitatively.
Statistical Analysis One-way analysis of variance was used to detect significant treatment effects between groups. Scheffe´’s test was used to identify differences between groups (27). The level of statistical significance was P,0.05. The data are reported as means 6 SEM.
RESULTS Genistein Intake The daily mean (6 SEM) consumption of genistein per rat was 52.8 6 1.0 mg during the first week after weaning and 53.0 6 3.0 mg during the second week. This amount of
Evaluation of the Estrous Cycle The vaginal vault was washed with 1–2 drops of sterile phosphate-buffered saline with the use of an eyedropper (Pasteur pipette), and a smear of vaginal cells from the wash was made on a microscopic slide. The smears were air-dried and stained by the Papanicolaou method. Proestrus was identified when the vaginal smear showed nucleated epithelial cells, leukocytes, and occasional cornified cells; estrus when large squamous cornified cells predominated; metestrus when there was a large number of leukocytes and cornified cells, caseous detritus, and large, flat nucleated cells; and diestrus when there were mostly leukocytes (25).
FIGURE 3 The effect of early exposure to genistein on the weight of the ovaries (A) and the uterus (B) at 21 and 70 days of life. Open bars represent the control group, stippled bars represent the genistein-exposed group, and lined bars represent the exgenistein group. For each graph, bars with asterisks are significantly different (P,0.05) from controls.
Radioimmunoassays Serum concentrations of E2 and progesterone were determined in duplicate samples (100 mL/tube) with the use of RIA kits obtained from Diagnostic Products Corporation (Los Angeles, CA), according to the protocol provided with each kit. The sensitivities of hormone detected per assay tube were 8.0 pg/mL and 0.02 ng/mL for E2 and progesterone, respectively. Serum concentrations of gonadotropins (LH and FSH) were measured with the use of a double-antibody RIA as previously described by L’Hernite et al. (26). The samples were measured at one time. The intra-assay coefficients of variation for E2, progesterone, LH, and FSH were 6.5%, 4.4%, 4.7%, and 5.3%, respectively.
Processing and Evaluation of Tissues Tissues from the ovary, oviduct, uterus, cervix, and vagina were immerse-fixed in Bouin’s fluid for 24 –36 hours, dehydrated in a graded series of alcohol, and embedded in paraffin. Five-micron–thick sections were cut, stained with hematoxylin and eosin or periodic acid-Schiff reaction and hematoxylin, and examined with the use of a Nikon Microphot-FXA light microscope (Nikon Inc, Melville, NY). 442
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FIGURE 4 Serum concentrations of LH (A), FSH (B), E2 (C), and progesterone (D) of rats exposed neonatally to genistein on days 21 and 70 of life. Open bars represent the control group, stippled bars represent the genistein-exposed group, and lined bars represent the ex-genistein group. For each graph, bars with asterisks are significantly different (P,0.05) from controls.
genistein approximates our intended dosage. However, on average, these rats consumed more food during the remaining duration of the experiment and hence their genistein intake was higher (Fig. 2). At these dose levels, no adverse effects on the length of gestation, the size of the litter, or the survival of the offspring were observed.
Body and Organ Weights There was a statistically significant (P,0.04) reduction in the body weight of G21 rats (54 6 1 g) compared with that of control rats (58 6 1 g). Similarly, the body weight of G70 rats (215 6 3 g) was significantly reduced compared with that of control rats (240 6 5 g), whereas that of Ex-G rats (281 6 6 g) was significantly higher than that of control rats. The weights of the ovaries and uterus on postnatal days 21 and 70 are presented in Figure 3. Whereas there were significant reductions in the weights of both the ovaries (Fig. 3A) and the uterus (Fig. 3B) of G21 rats, no such reductions were observed in G70 and Ex-G rats.
Serum Hormone Levels Figure 4 shows the effect of in utero exposure to genistein FERTILITY & STERILITYt
on hormone levels on postnatal days 21 and 70. On postnatal day 21, the serum concentration of LH was 270 6 15 pg/mL in control rats and 1,990 6 964 pg/mL in G21 rats (Fig. 4A); the difference between the two groups was not statistically significant because of a wide variation among individual G21 rats (range, 157–5,990 pg/mL versus 241–332 pg/mL in control animals). Similarly, there were no statistically significant differences in the serum concentration of LH between G70 or Ex-G rats and control rats. The serum concentrations of FSH in G21 and G70 or Ex-G rats were not different from those of their respective controls (Fig. 4B). Serum E2 and progesterone levels are shown in Figures 4C and 4D, respectively. Estradiol and progesterone concentrations in G21 rats were drastically reduced (E2: 3.9 6 1.7 pg/mL; progesterone: 1.2 6 0.6 ng/mL) compared with those in their controls (E2: 36.6 6 4.1 pg/mL; progesterone: 12.8 6 1.5 ng/mL). However, such reductions in E2 and progesterone levels were not evident in G70 or Ex-G rats. 443
FIGURE 5 Ovaries of 70-day-old rats exposed to genistein in utero and then throughout life (G70; A) or in utero and then for the first 21 days of life (Ex-G; B), and of a control rat (C). Aggregates of secondary interstitial cells (arrowheads) are conspicuous in genistein-exposed animals. Whereas numerous corpora lutea are present in control animals, fewer are seen in genisteinexposed animals. Hematoxylin and eosin; original magnification, 350.
Vaginal Opening and Cyclicity There was no statistically significant difference in the age at which vaginal opening occurred between G70 (35.60 6 0.07 days) or Ex-G (35.30 6 0.02 days) rats and their controls (36.60 6 0.03 days). Evaluation of vaginal smears taken daily for 3 weeks between postnatal days 50 and 70 revealed that 3 (27%) of 11 G70 rats had marked irregularity in the length of the estrous cycle ranging from 7–9 days, mostly due to prolonged estrus that lasted 2– 4 days. Two (25%) of 9 Ex-G rats had prolonged estrus and/or diestrus, whereas only 1 (12%) of 8 control rats had prolonged proestrus.
Histologic Evaluation of the Ovary and Uterus Numerous antral follicles were observed in both genistein-fed and control animals by day 21 (data not shown). Follicular atresia characterized by fragmented ovum and apoptotic morphologic changes in granulosa cells was conspicuous in G21 animals. Follicular atresia also was evident in control animals, but to a much lesser extent. The 444
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theca cells of the atretic follicles had begun to transform into secondary interstitial cells; aggregates of these cells were a common occurrence in G21 animals. In addition to this change in ovarian architecture, the lining epithelium of the rete ovarii was hyperplastic and somewhat hypertrophic in two of five G21 animals compared with the simple cuboidal appearance in the controls (data not shown). Although not quantified by morphometric means, it was apparent that follicular atresia and aggregation of secondary interstitial cells were more frequent in G70 and Ex-G animals than in control animals (Fig. 5). Similarly, there were fewer functional corpora lutea in the first two groups of animals. The lining epithelium of the rete ovarii was hyperplastic and hypertrophic in three of five G70 animals. In the remaining two G70 animals, the rete ovarii was dilated and the epithelium was flattened (Fig. 6A), presumably as a result of fluid accumulating in the cysts and compressing the lining epithelium. In the Ex-G group, one animal had hypertrophied epithelium and another had cysts in the rete ovarii (Fig. 6B). In Vol. 70, No. 3, September 1998
FIGURE 6 Rete ovarii of 70-day-old rats exposed to genistein in utero and then throughout life (G70; A) or in utero and then for the first 21 days of life (Ex-G; B), and of a control rat (C). In genistein-exposed animals, the rete ovarii was dilated and the epithelium was flattened. Evidently, fluid accumulated in the cysts compressed the lining epithelium. The cystic appearance is more conspicuous in A. In the control animal, the rete ovarii had relatively narrow cross-sectional profiles, as would be expected normally, and the lining epithelium was simple cuboidal or low columnar (C). Stain, hematoxylin and eosin; original magnification, 3125.
70-day control animals, the rete ovarii had relatively narrow cross-sectional profiles, as would be expected normally, and the lining epithelium remained simple cuboidal or low columnar (Fig. 6C). No conspicuous changes were observed in the histologic features, particularly in the lining epithelium, of the reproductive tract of genistein-treated animals.
DISCUSSION The results of this study demonstrate that in utero and neonatal exposure to dietary genistein exerts marked structural and functional changes in female rats even before they attain sexual maturity. The fact that the ovarian changes were identical in G70 and Ex-G animals suggests that the damage occurred early in life, which probably is a critical period in the process of reproductive development and functional differentiation. In this study, pregnant rats were exposed to genistein in the diet at concentrations that mimic possible human expoFERTILITY & STERILITYt
sure. These concentrations did not appear to affect adversely their pregnancy or parturition, or the survival of their offspring. In G21 (prepubertal) animals, estrogen and progesterone concentrations were significantly reduced but gonadotropin concentrations were not different from control values. The apparent lack of a statistically significant difference between the mean concentrations of gonadotropins of treated and control animals was the result of a wide variation among individual G21 animals. Because of this, we cannot be certain whether this variation was treatment-related or reflective of the maturational state of the ovarian follicles at day 21 (possibly as a result of incomplete maturation of the hypothalamic-pituitary axis). However, evidence exists to suggest that the marked reduction in E2 and progesterone concentrations may be due to the effect of genistein on granulosa cell growth and maturation. For example, previous studies in monkeys 445
(28, 29) have shown that estrogen can reduce granulosa cell viability and follicular steroid content. Therefore, it is possible that in utero and neonatal exposure to genistein could delay granulosa cell viability and follicular steroid content, affecting the amount of androgen that is available for aromatization to E2. Genistein could have exerted this effect also by acting on enzymes involved in estrogen synthesis and metabolism. It has been reported that isoflavones are capable of potently inhibiting estrogen synthetase (30) and the estrogen-specific isozymes of type 1 17b-hydroxysteroid dehydrogenase– catalyzed estrone reduction (31, 32), and 3a-hydroxysteroid dehydrogenase (32). The basis of this inhibition has been attributed to the structural resemblance of isoflavones to that of estrogen. Atretic changes in developing follicles and the formation of secondary interstitial glands were observed more commonly in genistein-exposed rats than in control rats. The apparent increased rate of follicular atresia in these animals may account for the reduction in ovarian weight in G21 rats. It is known that secondary interstitial cells are derived from the theca cells of atretic follicles, and that they have the capacity to synthesize androgen throughout reproductive life and even into postreproductive life (33). We speculate that the formation of these androgen-secreting cellular aggregates may contribute to the deranged ovarian cyclicity we observed in the genistein-exposed animals. Similarly, polycystic ovary syndrome is known to occur in women who have high levels of intra-ovarian androgen and deranged follicular development. The observation of normal levels of sex steroids and gonadotropins in G70 and Ex-G animals would not necessarily mitigate against this possibility. For example, the possibility that the functionality of secondary interstitial cells had been fully established by day 70 might explain the replenishment of steroid hormones to control levels at this age. Although there were no noticeable histologic changes in the uterine tube, uterus, cervix, or vagina, the lesions observed in the rete ovarii are marked and could be associated with abnormalities of reproductive health. Reproductive failure associated with cystic rete ovarii has been reported in animals as well as humans (34 –36). It has been observed that this rather obscure structure may have more significance than previously recognized (37). In fact, Rutgers and Scully (36) described 16 cases of cysts (cystadenomas), two solid adenomas, and one adenocarcinoma of rete ovarii origin in women. Cysts in these cases showed many similarities to microscopic dilations of the rete. In summary, our results indicate that in utero and neonatal exposure to genistein may adversely affect reproductive processes in adult female rats. Our present observations argue for further studies investigating the impact of prolonged 446
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fetal, neonatal, or adult exposure to estrogens on reproductive function.
Acknowledgments: The authors appreciate the technical assistance of Jennifer Palmer, MS and Carol Moeller, MS in histologic and photographic preparations.
References 1. Harborne JB. Flavonoid pigments. In: Rosenthal GA, Janzen DH, editors. Herbivores: their interaction with secondary plant chemicals. New York: Academic Press, 1979:649 –55. 2. Verdeal K, Ryan DS. Naturally occurring estrogens in plant foodstuff—a review. J Food Protection 1979;42:577– 83. 3. Whitten PL, Russell E, Naftolin F. Effects of normal, human-concentration, phytoestrogen diet on rat uterine growth. Steroids 1992;57:98 –106. 4. Murphy PA. Phytoestrogen content of processed soybean products. Food Technol 1982;36:62– 4. 5. Xu X, Wang H, Murphy PA, Cook L, Hendrich S. Diadzein is more bioavailable soymilk isoflavone than is genistein in adult women. J Nutr 1994;124:825–32. 6. Adlercreutz H, Fotsis T, Heikkinen R, Dwyer JT, Woods M, Goldin BR, et al. Excretion of the ligands enterolactone and enterodiol and of equol in omnivorous and vegetarian women and in women with breast cancer. Lancet 1982;2:1295–9. 7. Axelson M, Sjovall J, Gustafsson BE, Setchell KDR. Soya—a dietary source of the non-steroidal oestrogen equol in man and animals. J Endocrinol 1984;102:29 –56. 8. McLachlan JA, Newbold RR. Estrogens and development. Environ Health Perspect 1987;75:25–7. 9. Matossian MK. Poisons of the past. New Haven (CT): Yale University Press, 1989. 10. Kitts WD, Newsome FE, Runeckles VC. The estrogenic and antiestrogenic effects of coumestrol and zerananol on the immature rat uterus. Can J Anim Sci 1983;63:823– 4. 11. Burroughs CD, Mills KT, Bern HA. Long-term genital tract changes in female mice treated neonatally with coumestrol. Reprod Toxicol 1990; 4:127–35. 12. Fredricks GR, Kincaid RL, Bondioli KR, Wright RW Jr. Ovulation rates and embryo degeneracy in female mice fed the phytoestrogen, coumestrol. Proc Soc Exp Biol Med 1981;167:237– 41. 13. Medlock KL, Branham WS, Sheehan DM. The effects of phytoestrogens on neonatal rat uterine growth and development. Proc Soc Exp Biol Med 1995;208:307–13. 14. Newsome FE, Kitts WD. Action of phytoestrogens coumestrol and genistein on cytosolic and nuclear oestradiol-17b receptors in immature rat uterus. Anim Reprod Sci 1980;3:233– 45. 15. Thrain RI. Bovine infertility caused by subterranean clover: further report and herd histories. Aust Vet J 1966;42:199 –203. 16. Lindsay DR, Kelly RW. The metabolism of phytoestrogens in sheep. Aust Vet J 1970;46:219 –22. 17. Adams NR, Martin GB. Effects of oestradiol on plasma concentrations of luteinizing hormone in ovariectomized ewes with clover disease. Aust J Biol Sci 1983;30:223–30. 18. Adams NR, Oldham CM, Reydon RA. Ovulation rate and oocyte numbers in ewes after exposure to oestrogenic pasture. J Reprod Fertil 1979;55:88 –9. 19. Mathieson RA, Kitts WD. Binding of phytoestrogens and oestradiol17b by cytoplasmic receptors in the pituitary gland and hypothalamus of the ewe. J Endocrinol 1980;85:317–25. 20. Adams NR. A changed responsiveness to oestrogen in ewes with clover disease. J Reprod Fertil Suppl 1981;30:223–30. 21. Faber KA, Hughes CL Jr. Dose-response characteristics of neonatal exposure to genistein on pituitary responsiveness to gonadotropin releasing hormone and volume of the sexually dimorphic nucleus of the preoptic area (SDN-POA) in postpubertal castrated female rats. Reprod Toxicol 1993;7:35–9. 22. Pierce JG, Parsons TF. Glycoprotein hormones: structure and function. Annu Rev Biochem 1981;50:465–95. 23. Barnes S. Effect of genistein on in vitro and in vivo models of cancer. J Nutr 1995;125:777S– 83S. 24. Steele VE, Pereira MA, Sigman CC, Keloff GJ. Cancer chemopreventive agent development strategies for genistein. J Nutr 1995;125:713S– 16S.
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25. Manson JM, Kang YJ. Test for assessing female reproductive and developmental toxicology. In: Hayes AW, editor. Principles and methods of toxicology. 2nd ed. New York: Raven Press, 1989:328. 26. L’Hernite M, Niswender GD, Reichert LE, Midgley AR. Serum follicle stimulating hormone in sheep as measured by radioimmunoassay. Biol Reprod 1972;6:325–32. 27. Scheffe´ H. The analysis of variance. New York: Wiley and Sons, 1959. 28. Hutz RJ, Dierschke DJ, Wolf RC. Role of estradiol in regulating ovarian follicle atresia in rhesus monkeys. A review. J Med Primatol 1990;19:553–71. 29. Dierschke DJ, Hutz RJ, Wolf RC. Induced follicular atresia in rhesus monkeys: strength-duration relationships of the estrogen stimulus. Endocrinology 1985;117:1397– 403. 30. Adlercruetz H, Bannwart C, Wahala K, Makela T, Brunow G, Hase T, et al. Inhibition of human aromatase by mammalian lignans and isoflavonoid phytoestrogens. J Steroid Biochem Mol Biol 1993;44:147–53. 31. Makela S, Poutanen M, Lehtimake J, Kostian ML, Santti R, Vihko R. Estrogen-specific 17 beta-hydroxysteroid oxidoreductase type 1 (E.C.
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32.
33. 34. 35. 36. 37.
1.1.162) as possible target for the action of phytoestrogens. Proc Soc Exp Biol Med 1995;208:51–9. Hasebe K, Hara A, Nakayama T, Hayashibara M, Inoue Y, Sawada H. Inhibition of hepatic 17 beta- and 3 alpha-hydroxysteroid dehydrogenase by antiinflammatory drugs and nonsteroidal estrogens. Enzyme 1987;37:109 –14. Gore-Langton RE, Armstrong DT. Follicular steroidogenesis and its control. In: Knobil E, Neil J, editors. The physiology of reproduction. New York: Raven Press, 1988:331– 85. Keller LS, Griffith JW, Lang CM. Reproductive failure associated with cystic rete ovarii in guinea pigs. Vet Pathol 1987;24:335–9. Gelberg HB, McEntee K, Heath EH. Feline cystic rete ovarii. Vet Pathol 1984;21:304 –7. Rutgers JL, Scully RE. Cysts (cystadenomas) and tumors of the rete ovarii. Int J Gynecol Pathol 1988;7:330 – 42. Odend’hal S, Wenzel JG, Player EC. The rete ovarii of cattle and deer communicates with the uterine tube. Anat Rec 1986;216:40 –3.
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Reproductive sequelae in female rats after in utero and neonatal exposure to the phytoestrogen genistein Caleb A. Awoniyi, Ph.D.,* Dawn Roberts, M.S.,* D. N. Rao Veeramachaneni, D.V.M., Ph.D.,† Bradley S. Hurst, M.D.,* Kathleen E. Tucker, Ph.D.,* and William D. Schlaff, M.D.* University of Colorado Health Sciences Center, Denver, and Colorado State University, Fort Collins, Colorado
Objective: To determine reproductive sequelae in female rats after in utero and lactational dietary exposure to genistein. Design: Experimental animal study. Setting: University laboratory. Animal(s): Sprague Dawley rats. Intervention(s): Pregnant rats were fed control rat chow or rat chow incorporated with genistein (approximately 50 mg/d) beginning on day 17 of gestation and continuing until the end of lactation (postpartum day 21). Genistein-exposed female pups were divided into two groups on day 21. One group continued to receive a genistein-added diet (G70); the other group was changed to a control diet (Ex-G). At necropsy (days 21 and 70), blood and reproductive tissues were collected. Main Outcome Measure(s): Serum levels of gonadotropins and gonadal steroids and histopathologic examination of the ovaries. Received December 18, 1997; revised and accepted April 29, 1998. Supported in part by grant DK48520 from the National Institutes of Health, Bethesda, Maryland, and the Academic Enrichment Fund from the Department of Obstetrics and Gynecology, University of Colorado Health Sciences Center, Denver, Colorado. Reprint requests: Caleb A. Awoniyi, Ph.D., Division of Reproductive Endocrinology, Department of Obstetrics and Gynecology, University of Colorado Health Sciences Center, Denver, Colorado 80262 (FAX: 303-315-8889; E-mail: CALEB.AWONIYI @UCHSC.EDU). * Division of Reproductive Endocrinology, Department of Obstetrics and Gynecology, University of Colorado Health Sciences Center. † Animal Reproduction and Biotechnology Laboratory, Colorado State University. 0015-0282/98/$19.00 PII S0015-0282(98)00185-X
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Result(s): The weight of the ovaries and uterus and serum levels of E2 and progesterone in genistein-exposed rats on day 21 (G21) were significantly reduced compared with control rats. On day 70, serum levels of E2, progesterone, LH, and FSH were similar in all groups. Atretic follicles and secondary interstitial glands were more common in G70 and Ex-G rats compared with control rats. Cystic rete ovarii was observed in some G70 and Ex-G rats. Conclusion(s): Our data indicate that in utero and lactational exposure to dietary genistein adversely affects reproductive processes in the adult female rat. (Fertil Sterilt 1998;70:440 –7. ©1998 by American Society for Reproductive Medicine.) Key Words: Phytoestrogen, genistein, estrous cyclicity, ovary, uterus, gonadotropins, estrogen, progesterone
Isoflavonoids are present in plants and resemble steroidal estrogens and mimic many of their actions (1–3). However, the biologic roles played by isoflavonoids when they are ingested by animals are not fully understood. Genistein and diadzein, the major isoflavones in soybeans (4, 5), are found in high concentrations in the urine of Asian women, a population known to be resistant to breast cancer. Conversely, they are found in low concentrations in patients with breast cancer, suggesting that they may have a role in the prevention of estrogen-dependent carcinoma (6, 7). Although the possible role of isoflavonoids as anticancer agents is being considered, there also is increasing concern that naturally occurring and xenobiotic environmental agents may exert some adverse effects as well (8). In fact, a recent review suggested that the ingestion of
a fungal estrogen may have contributed significantly to the decline of the rural 19th and 20th century European population (9). Given the widespread consumption of these phytoestrogens by humans and the perceived potential for developing therapeutic, anticancer treatments with them, it is imperative to assess the potential impact of these isoflavonoids on reproductive function. Phytoestrogens have been shown to cause both direct and indirect toxicologic effects on the reproductive tract of animals (3, 10 –13). These effects include persistent vaginal cornification, hemorrhagic ovarian follicles, and premature vaginal opening (11, 14). Although it has been known for years that grazing in certain clover fields causes significant infertility in sheep and cattle (15, 16), only recently has it been discussed that the phytoestrogens
contained in clover produce alterations in the hypothalamicpituitary-ovarian axis by binding to hypothalamic estrogen receptors (17–20). To date, the effects of phytoestrogens on sexual development and on the development of the neuroendocrine system are not well understood.
FIGURE 1 Schematic diagram of the experimental design. GD 5 gestation day; PND 5 postnatal day.
Recently, Faber and Hughes (21) observed an increase in the size of the sexually dimorphic nucleus of the preoptic area of the hypothalamus in adult rats after neonatal exposure to various doses of genistein. They also found that low doses of genistein resulted in increased pituitary responsiveness to GnRH, whereas high doses resulted in decreased responsiveness. Available animal studies on phytoestrogens provide little insight into the consequences of the long-term low-dose exposure that is likely to be characteristic of the natural situation in humans. The objective of this study was to examine reproductive sequelae at critical periods of reproductive development in rats after dietary exposure to a phytoestrogen, genistein, at doses relevant to possible human exposure.
MATERIALS AND METHODS Animals Pregnant Sprague Dawley rats (Sasco, Omaha, NE) were purchased at day 10 of gestation. Animals were housed individually in a room maintained at 22°C with a constant light-dark cycle (14 hours of light and 10 hours of darkness), and were provided isoflavonoid-free rat chow and water ad libitum. These studies were performed in accordance with the Guidelines for the Care and Use of Experimental Animals and were approved by the Animal Care Committee of the University of Colorado Health Sciences Center.
Administration of Genistein Because commercial rodent chow contains some isoflavonoid (22), all rats were fed the AIN semipurified rat-mouse diet (Bioserve, Frenchtown, NJ), which is devoid of isoflavonoid and contains casein-high nitrogen (20%), DL-methionine (0.3%), cornstarch (15%), sucrose (50%), fiber-celfil (5%), mineral mixture (3.5%), vitamin mixture (1%), and chlorine bitartrate (0.2%), until day 17 of gestation. We chose to administer genistein in the diet at a dose of 5 mg/kg of feed based on the data of Barnes (23) and of Steele et al. (24). Barnes estimated that human intake of genistein is approximately 50 mg/d. Steele et al. reported that rats given a diet containing 150 mg of genistein per kilogram of feed had a daily intake of 10.45 mg/kg of body weight. On the basis of these observations, we estimated that the required concentration of genistein in the diet is about 2.5 mg/kg of feed to provide 50 mg/d to a 300-g rat. However, because of variations in weight and dietary intake that are certain to occur, we elected to supply a dose of 5 mg/kg of feed to ensure that all animals consumed at least 50 mg of genistein per day. FERTILITY & STERILITYt
Experimental Regimen Because the critical period of steroid-mediated development of the reproductive system in the rat begins around day 17 of gestation, we began dietary genistein exposure (5 mg/kg of feed) in 12 rats on day 17 of gestation. Eight pregnant rats were fed the semipurified control diet. Animals were weighed weekly throughout the experiment. Daily feed consumption was determined by weighing feed daily at 11 AM. Pups were weaned on day 21 and only female rats were used in this study. Pups from four litters in the control (C21) and genisteinexposed (G21) groups were euthanized on day 21 (Fig. 1); their hormones and reproductive organs were evaluated. On postnatal day 21, four litters of genistein-treated rats were given a control diet (Ex-G) and the remaining four continued to receive the genistein-added diet until necropsy was performed on approximately postnatal day 70 (G70). Four litters that had been receiving the control diet continued to receive the same diet and served as controls (C70) for the day 70 groups. The time of vaginal opening was determined, and vaginal smears were taken daily to evaluate estrous cyclicity. All rats were euthanized in proestrus. Trunk blood and reproductive organs were collected. Serum was separated and stored at 220°C for subsequent determination of concentrations of E2, progesterone, LH, and FSH by RIA. Weights of the ovaries and reproductive tracts were recorded, and tissues were processed for histopathologic examination. 441
FIGURE 2 Daily genistein intake in rats fed a genistein-added diet. Values are means 6 SEM.
Briefly, the status of follicular development; the normalcy of ovarian structures such as the surface epithelium, rete ovarii, interstitial glands, and corpora lutea; and the status of the general architecture and the lining epithelium of the oviducts, uterus, cervix, and vagina were evaluated qualitatively.
Statistical Analysis One-way analysis of variance was used to detect significant treatment effects between groups. Scheffe´’s test was used to identify differences between groups (27). The level of statistical significance was P,0.05. The data are reported as means 6 SEM.
RESULTS Genistein Intake The daily mean (6 SEM) consumption of genistein per rat was 52.8 6 1.0 mg during the first week after weaning and 53.0 6 3.0 mg during the second week. This amount of
Evaluation of the Estrous Cycle The vaginal vault was washed with 1–2 drops of sterile phosphate-buffered saline with the use of an eyedropper (Pasteur pipette), and a smear of vaginal cells from the wash was made on a microscopic slide. The smears were air-dried and stained by the Papanicolaou method. Proestrus was identified when the vaginal smear showed nucleated epithelial cells, leukocytes, and occasional cornified cells; estrus when large squamous cornified cells predominated; metestrus when there was a large number of leukocytes and cornified cells, caseous detritus, and large, flat nucleated cells; and diestrus when there were mostly leukocytes (25).
FIGURE 3 The effect of early exposure to genistein on the weight of the ovaries (A) and the uterus (B) at 21 and 70 days of life. Open bars represent the control group, stippled bars represent the genistein-exposed group, and lined bars represent the exgenistein group. For each graph, bars with asterisks are significantly different (P,0.05) from controls.
Radioimmunoassays Serum concentrations of E2 and progesterone were determined in duplicate samples (100 mL/tube) with the use of RIA kits obtained from Diagnostic Products Corporation (Los Angeles, CA), according to the protocol provided with each kit. The sensitivities of hormone detected per assay tube were 8.0 pg/mL and 0.02 ng/mL for E2 and progesterone, respectively. Serum concentrations of gonadotropins (LH and FSH) were measured with the use of a double-antibody RIA as previously described by L’Hernite et al. (26). The samples were measured at one time. The intra-assay coefficients of variation for E2, progesterone, LH, and FSH were 6.5%, 4.4%, 4.7%, and 5.3%, respectively.
Processing and Evaluation of Tissues Tissues from the ovary, oviduct, uterus, cervix, and vagina were immerse-fixed in Bouin’s fluid for 24 –36 hours, dehydrated in a graded series of alcohol, and embedded in paraffin. Five-micron–thick sections were cut, stained with hematoxylin and eosin or periodic acid-Schiff reaction and hematoxylin, and examined with the use of a Nikon Microphot-FXA light microscope (Nikon Inc, Melville, NY). 442
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FIGURE 4 Serum concentrations of LH (A), FSH (B), E2 (C), and progesterone (D) of rats exposed neonatally to genistein on days 21 and 70 of life. Open bars represent the control group, stippled bars represent the genistein-exposed group, and lined bars represent the ex-genistein group. For each graph, bars with asterisks are significantly different (P,0.05) from controls.
genistein approximates our intended dosage. However, on average, these rats consumed more food during the remaining duration of the experiment and hence their genistein intake was higher (Fig. 2). At these dose levels, no adverse effects on the length of gestation, the size of the litter, or the survival of the offspring were observed.
Body and Organ Weights There was a statistically significant (P,0.04) reduction in the body weight of G21 rats (54 6 1 g) compared with that of control rats (58 6 1 g). Similarly, the body weight of G70 rats (215 6 3 g) was significantly reduced compared with that of control rats (240 6 5 g), whereas that of Ex-G rats (281 6 6 g) was significantly higher than that of control rats. The weights of the ovaries and uterus on postnatal days 21 and 70 are presented in Figure 3. Whereas there were significant reductions in the weights of both the ovaries (Fig. 3A) and the uterus (Fig. 3B) of G21 rats, no such reductions were observed in G70 and Ex-G rats.
Serum Hormone Levels Figure 4 shows the effect of in utero exposure to genistein FERTILITY & STERILITYt
on hormone levels on postnatal days 21 and 70. On postnatal day 21, the serum concentration of LH was 270 6 15 pg/mL in control rats and 1,990 6 964 pg/mL in G21 rats (Fig. 4A); the difference between the two groups was not statistically significant because of a wide variation among individual G21 rats (range, 157–5,990 pg/mL versus 241–332 pg/mL in control animals). Similarly, there were no statistically significant differences in the serum concentration of LH between G70 or Ex-G rats and control rats. The serum concentrations of FSH in G21 and G70 or Ex-G rats were not different from those of their respective controls (Fig. 4B). Serum E2 and progesterone levels are shown in Figures 4C and 4D, respectively. Estradiol and progesterone concentrations in G21 rats were drastically reduced (E2: 3.9 6 1.7 pg/mL; progesterone: 1.2 6 0.6 ng/mL) compared with those in their controls (E2: 36.6 6 4.1 pg/mL; progesterone: 12.8 6 1.5 ng/mL). However, such reductions in E2 and progesterone levels were not evident in G70 or Ex-G rats. 443
FIGURE 5 Ovaries of 70-day-old rats exposed to genistein in utero and then throughout life (G70; A) or in utero and then for the first 21 days of life (Ex-G; B), and of a control rat (C). Aggregates of secondary interstitial cells (arrowheads) are conspicuous in genistein-exposed animals. Whereas numerous corpora lutea are present in control animals, fewer are seen in genisteinexposed animals. Hematoxylin and eosin; original magnification, 350.
Vaginal Opening and Cyclicity There was no statistically significant difference in the age at which vaginal opening occurred between G70 (35.60 6 0.07 days) or Ex-G (35.30 6 0.02 days) rats and their controls (36.60 6 0.03 days). Evaluation of vaginal smears taken daily for 3 weeks between postnatal days 50 and 70 revealed that 3 (27%) of 11 G70 rats had marked irregularity in the length of the estrous cycle ranging from 7–9 days, mostly due to prolonged estrus that lasted 2– 4 days. Two (25%) of 9 Ex-G rats had prolonged estrus and/or diestrus, whereas only 1 (12%) of 8 control rats had prolonged proestrus.
Histologic Evaluation of the Ovary and Uterus Numerous antral follicles were observed in both genistein-fed and control animals by day 21 (data not shown). Follicular atresia characterized by fragmented ovum and apoptotic morphologic changes in granulosa cells was conspicuous in G21 animals. Follicular atresia also was evident in control animals, but to a much lesser extent. The 444
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theca cells of the atretic follicles had begun to transform into secondary interstitial cells; aggregates of these cells were a common occurrence in G21 animals. In addition to this change in ovarian architecture, the lining epithelium of the rete ovarii was hyperplastic and somewhat hypertrophic in two of five G21 animals compared with the simple cuboidal appearance in the controls (data not shown). Although not quantified by morphometric means, it was apparent that follicular atresia and aggregation of secondary interstitial cells were more frequent in G70 and Ex-G animals than in control animals (Fig. 5). Similarly, there were fewer functional corpora lutea in the first two groups of animals. The lining epithelium of the rete ovarii was hyperplastic and hypertrophic in three of five G70 animals. In the remaining two G70 animals, the rete ovarii was dilated and the epithelium was flattened (Fig. 6A), presumably as a result of fluid accumulating in the cysts and compressing the lining epithelium. In the Ex-G group, one animal had hypertrophied epithelium and another had cysts in the rete ovarii (Fig. 6B). In Vol. 70, No. 3, September 1998
FIGURE 6 Rete ovarii of 70-day-old rats exposed to genistein in utero and then throughout life (G70; A) or in utero and then for the first 21 days of life (Ex-G; B), and of a control rat (C). In genistein-exposed animals, the rete ovarii was dilated and the epithelium was flattened. Evidently, fluid accumulated in the cysts compressed the lining epithelium. The cystic appearance is more conspicuous in A. In the control animal, the rete ovarii had relatively narrow cross-sectional profiles, as would be expected normally, and the lining epithelium was simple cuboidal or low columnar (C). Stain, hematoxylin and eosin; original magnification, 3125.
70-day control animals, the rete ovarii had relatively narrow cross-sectional profiles, as would be expected normally, and the lining epithelium remained simple cuboidal or low columnar (Fig. 6C). No conspicuous changes were observed in the histologic features, particularly in the lining epithelium, of the reproductive tract of genistein-treated animals.
DISCUSSION The results of this study demonstrate that in utero and neonatal exposure to dietary genistein exerts marked structural and functional changes in female rats even before they attain sexual maturity. The fact that the ovarian changes were identical in G70 and Ex-G animals suggests that the damage occurred early in life, which probably is a critical period in the process of reproductive development and functional differentiation. In this study, pregnant rats were exposed to genistein in the diet at concentrations that mimic possible human expoFERTILITY & STERILITYt
sure. These concentrations did not appear to affect adversely their pregnancy or parturition, or the survival of their offspring. In G21 (prepubertal) animals, estrogen and progesterone concentrations were significantly reduced but gonadotropin concentrations were not different from control values. The apparent lack of a statistically significant difference between the mean concentrations of gonadotropins of treated and control animals was the result of a wide variation among individual G21 animals. Because of this, we cannot be certain whether this variation was treatment-related or reflective of the maturational state of the ovarian follicles at day 21 (possibly as a result of incomplete maturation of the hypothalamic-pituitary axis). However, evidence exists to suggest that the marked reduction in E2 and progesterone concentrations may be due to the effect of genistein on granulosa cell growth and maturation. For example, previous studies in monkeys 445
(28, 29) have shown that estrogen can reduce granulosa cell viability and follicular steroid content. Therefore, it is possible that in utero and neonatal exposure to genistein could delay granulosa cell viability and follicular steroid content, affecting the amount of androgen that is available for aromatization to E2. Genistein could have exerted this effect also by acting on enzymes involved in estrogen synthesis and metabolism. It has been reported that isoflavones are capable of potently inhibiting estrogen synthetase (30) and the estrogen-specific isozymes of type 1 17b-hydroxysteroid dehydrogenase– catalyzed estrone reduction (31, 32), and 3a-hydroxysteroid dehydrogenase (32). The basis of this inhibition has been attributed to the structural resemblance of isoflavones to that of estrogen. Atretic changes in developing follicles and the formation of secondary interstitial glands were observed more commonly in genistein-exposed rats than in control rats. The apparent increased rate of follicular atresia in these animals may account for the reduction in ovarian weight in G21 rats. It is known that secondary interstitial cells are derived from the theca cells of atretic follicles, and that they have the capacity to synthesize androgen throughout reproductive life and even into postreproductive life (33). We speculate that the formation of these androgen-secreting cellular aggregates may contribute to the deranged ovarian cyclicity we observed in the genistein-exposed animals. Similarly, polycystic ovary syndrome is known to occur in women who have high levels of intra-ovarian androgen and deranged follicular development. The observation of normal levels of sex steroids and gonadotropins in G70 and Ex-G animals would not necessarily mitigate against this possibility. For example, the possibility that the functionality of secondary interstitial cells had been fully established by day 70 might explain the replenishment of steroid hormones to control levels at this age. Although there were no noticeable histologic changes in the uterine tube, uterus, cervix, or vagina, the lesions observed in the rete ovarii are marked and could be associated with abnormalities of reproductive health. Reproductive failure associated with cystic rete ovarii has been reported in animals as well as humans (34 –36). It has been observed that this rather obscure structure may have more significance than previously recognized (37). In fact, Rutgers and Scully (36) described 16 cases of cysts (cystadenomas), two solid adenomas, and one adenocarcinoma of rete ovarii origin in women. Cysts in these cases showed many similarities to microscopic dilations of the rete. In summary, our results indicate that in utero and neonatal exposure to genistein may adversely affect reproductive processes in adult female rats. Our present observations argue for further studies investigating the impact of prolonged 446
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fetal, neonatal, or adult exposure to estrogens on reproductive function.
Acknowledgments: The authors appreciate the technical assistance of Jennifer Palmer, MS and Carol Moeller, MS in histologic and photographic preparations.
References 1. Harborne JB. Flavonoid pigments. In: Rosenthal GA, Janzen DH, editors. Herbivores: their interaction with secondary plant chemicals. New York: Academic Press, 1979:649 –55. 2. Verdeal K, Ryan DS. Naturally occurring estrogens in plant foodstuff—a review. J Food Protection 1979;42:577– 83. 3. Whitten PL, Russell E, Naftolin F. Effects of normal, human-concentration, phytoestrogen diet on rat uterine growth. Steroids 1992;57:98 –106. 4. Murphy PA. Phytoestrogen content of processed soybean products. Food Technol 1982;36:62– 4. 5. Xu X, Wang H, Murphy PA, Cook L, Hendrich S. Diadzein is more bioavailable soymilk isoflavone than is genistein in adult women. J Nutr 1994;124:825–32. 6. Adlercreutz H, Fotsis T, Heikkinen R, Dwyer JT, Woods M, Goldin BR, et al. Excretion of the ligands enterolactone and enterodiol and of equol in omnivorous and vegetarian women and in women with breast cancer. Lancet 1982;2:1295–9. 7. Axelson M, Sjovall J, Gustafsson BE, Setchell KDR. Soya—a dietary source of the non-steroidal oestrogen equol in man and animals. J Endocrinol 1984;102:29 –56. 8. McLachlan JA, Newbold RR. Estrogens and development. Environ Health Perspect 1987;75:25–7. 9. Matossian MK. Poisons of the past. New Haven (CT): Yale University Press, 1989. 10. Kitts WD, Newsome FE, Runeckles VC. The estrogenic and antiestrogenic effects of coumestrol and zerananol on the immature rat uterus. Can J Anim Sci 1983;63:823– 4. 11. Burroughs CD, Mills KT, Bern HA. Long-term genital tract changes in female mice treated neonatally with coumestrol. Reprod Toxicol 1990; 4:127–35. 12. Fredricks GR, Kincaid RL, Bondioli KR, Wright RW Jr. Ovulation rates and embryo degeneracy in female mice fed the phytoestrogen, coumestrol. Proc Soc Exp Biol Med 1981;167:237– 41. 13. Medlock KL, Branham WS, Sheehan DM. The effects of phytoestrogens on neonatal rat uterine growth and development. Proc Soc Exp Biol Med 1995;208:307–13. 14. Newsome FE, Kitts WD. Action of phytoestrogens coumestrol and genistein on cytosolic and nuclear oestradiol-17b receptors in immature rat uterus. Anim Reprod Sci 1980;3:233– 45. 15. Thrain RI. Bovine infertility caused by subterranean clover: further report and herd histories. Aust Vet J 1966;42:199 –203. 16. Lindsay DR, Kelly RW. The metabolism of phytoestrogens in sheep. Aust Vet J 1970;46:219 –22. 17. Adams NR, Martin GB. Effects of oestradiol on plasma concentrations of luteinizing hormone in ovariectomized ewes with clover disease. Aust J Biol Sci 1983;30:223–30. 18. Adams NR, Oldham CM, Reydon RA. Ovulation rate and oocyte numbers in ewes after exposure to oestrogenic pasture. J Reprod Fertil 1979;55:88 –9. 19. Mathieson RA, Kitts WD. Binding of phytoestrogens and oestradiol17b by cytoplasmic receptors in the pituitary gland and hypothalamus of the ewe. J Endocrinol 1980;85:317–25. 20. Adams NR. A changed responsiveness to oestrogen in ewes with clover disease. J Reprod Fertil Suppl 1981;30:223–30. 21. Faber KA, Hughes CL Jr. Dose-response characteristics of neonatal exposure to genistein on pituitary responsiveness to gonadotropin releasing hormone and volume of the sexually dimorphic nucleus of the preoptic area (SDN-POA) in postpubertal castrated female rats. Reprod Toxicol 1993;7:35–9. 22. Pierce JG, Parsons TF. Glycoprotein hormones: structure and function. Annu Rev Biochem 1981;50:465–95. 23. Barnes S. Effect of genistein on in vitro and in vivo models of cancer. J Nutr 1995;125:777S– 83S. 24. Steele VE, Pereira MA, Sigman CC, Keloff GJ. Cancer chemopreventive agent development strategies for genistein. J Nutr 1995;125:713S– 16S.
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25. Manson JM, Kang YJ. Test for assessing female reproductive and developmental toxicology. In: Hayes AW, editor. Principles and methods of toxicology. 2nd ed. New York: Raven Press, 1989:328. 26. L’Hernite M, Niswender GD, Reichert LE, Midgley AR. Serum follicle stimulating hormone in sheep as measured by radioimmunoassay. Biol Reprod 1972;6:325–32. 27. Scheffe´ H. The analysis of variance. New York: Wiley and Sons, 1959. 28. Hutz RJ, Dierschke DJ, Wolf RC. Role of estradiol in regulating ovarian follicle atresia in rhesus monkeys. A review. J Med Primatol 1990;19:553–71. 29. Dierschke DJ, Hutz RJ, Wolf RC. Induced follicular atresia in rhesus monkeys: strength-duration relationships of the estrogen stimulus. Endocrinology 1985;117:1397– 403. 30. Adlercruetz H, Bannwart C, Wahala K, Makela T, Brunow G, Hase T, et al. Inhibition of human aromatase by mammalian lignans and isoflavonoid phytoestrogens. J Steroid Biochem Mol Biol 1993;44:147–53. 31. Makela S, Poutanen M, Lehtimake J, Kostian ML, Santti R, Vihko R. Estrogen-specific 17 beta-hydroxysteroid oxidoreductase type 1 (E.C.
FERTILITY & STERILITYt
32.
33. 34. 35. 36. 37.
1.1.162) as possible target for the action of phytoestrogens. Proc Soc Exp Biol Med 1995;208:51–9. Hasebe K, Hara A, Nakayama T, Hayashibara M, Inoue Y, Sawada H. Inhibition of hepatic 17 beta- and 3 alpha-hydroxysteroid dehydrogenase by antiinflammatory drugs and nonsteroidal estrogens. Enzyme 1987;37:109 –14. Gore-Langton RE, Armstrong DT. Follicular steroidogenesis and its control. In: Knobil E, Neil J, editors. The physiology of reproduction. New York: Raven Press, 1988:331– 85. Keller LS, Griffith JW, Lang CM. Reproductive failure associated with cystic rete ovarii in guinea pigs. Vet Pathol 1987;24:335–9. Gelberg HB, McEntee K, Heath EH. Feline cystic rete ovarii. Vet Pathol 1984;21:304 –7. Rutgers JL, Scully RE. Cysts (cystadenomas) and tumors of the rete ovarii. Int J Gynecol Pathol 1988;7:330 – 42. Odend’hal S, Wenzel JG, Player EC. The rete ovarii of cattle and deer communicates with the uterine tube. Anat Rec 1986;216:40 –3.
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