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Augmentation of aromatase activity by FSH in ovaries of fetal and neonatal mice in organ culture
J. steroid &o&em. Vol. 20, No. 3, pp. 741-745, hinted in Great Britain. All rights reserved
0022-4731/84
1984
$3.00 + 0.00
Copyright C 1984Pergamon Press Ltd
AUGMENTATION OF AROMATASE ACTIVITY BY FSH IN OVARIES OF FETAL AND NEONATAL MICE IN ORGAN CULTURE N. TERADA, H. KVRODA*, M. NAMIKI~, Y. KITAMUBA and K. MATSUMOTO*$ Institute for Cancer Research, *Department of Pathology and FDepartment of Urology, Osaka University Medical School, Kita-ku, Osaka, 530 Japan (Received
7 June 1983)
Summary-The stimulative effect of FSH on aromatase activity was investigated in ovaries of fetal (on days 17 and 18 of gestation) and neonatal mice (on days 0, 3, 6 and 9 after birth). Two to six ovaries were cultured for 48 h in 2 ml of Medium 199 supplemented with insulin (5 pg/ml) and [la, 2c1,6a, 7n, fi-3H]4-androstene-3,17-dione (0.35 FM) in the presence or absence of porcine FSH (OS units/ml) and the mount of [3H]~stradiol-17~ and [‘Hjoestrone produced was estimated. In the presence of FSH, aromatase activity per ovary, which was found in all fetal and neonatal ovaries examined, increased with age. In the absence of FSH, however, the production of oestrogens could be demonstrated only in ovaries from 3- to Q-day old mice. FSH increased the aromatase activity by up to IO-fold. In spite of the stimulative effect of FSH on aromatase activity, FSH exerted no significant effect on DNA synthesis of the ovaries. The formation of primordial follicles could not be observed histologically in ovaries of fetal mice on day 17 of gestation, although the ovaries of 6- and 9-day old mice contained multilayered follicles. These results show that FSH stimulates the aromatase activity of the mouse ovary even before the fo~ation of primordial follicles and that the stimulative effect of FSH on ovarian aromatase is not due to the proliferation of ovarian cells.
MATERIALS AND METHODS
INTRODUCTION
The effect of FSH on oestrogen
Mice
containing
Fetal and neonatal female (WB x C57BL/6)F, mice were obtained by crossing female WB and male C57BL/6 mice in our laboratory. Female WB mice were induced to ovulate by injection of 5 I.U. pregnant mare serum gonadotropin (Serotropin, Teikoku Hormone, Japan) at 4p.m., followed 48 h later by 5 I.U. of human chorionic gonadotropin and the females were placed with male C57BL/6 mice at 4p.m. On the morning after mating (day 0 of gestation), all mice were examined for vaginal plugs. The female mice with vaginal plugs were caged separately. Delivery took place on days 2&22 of gestation. The day of birth was designated as day 0 after birth. All mice were kept at 25°C under light control (14 h light/l0 h darkness) and allowed free access to water and pellet food (Oriental Yeast Co., Ltd., Japan).
well-developed
synthesis in the ovary follicles has been reported
by several investigators [l-l 11. The results have shown that granulosa cells from well-developed follicles have FSH receptors [8-l 1] and that FSH stimulates CAMP formation and aromatase activity of the granulosa cells [l-8]. However, there are only a few papers on effect of FSH on aromatase activity in the ovary, which is composed only of follicles at very early stages of development. Furthermore, results obtained in the immature ovaries are not in good agreement [12, 131. In the ovary of neonatal rat, Funkenstein et al. showed that FSH enhances the aromatase activity of 4-day old rat ovaries which consist only of primordial follicles [12]. In contrast, Ellinwood et al. reported that gonadotropins containing both LH and FSH activity are unable to stimulate the production of CAMP in fetal rhesus monkey ovaries, which are composed only of primordial follicles (131. Therefore, we investigated the stimulative effect of FSH on aromatase activity and early development of follicles in fetal and neonatal mouse ovaries. We found that FSH stimulates the aromatase activity in these mouse ovaries including fetal ovaries having no follicles and that the stimulative effect of FSH on aromatase is not due to the proliferation of FSHresponsive ovarian cells. -.. $To whom correspondence
should be addressed.
Chemicals
[l~,2ff,6ff,7a,~-3H]~androstene-3,17~ione (82 Ci/ mmol) and [4-‘4C]oestrone (59 mCi/mmol) were obtained from the Radiochemical Center Amersham, U.S.A. and were purified by paper chromatography using the hexane-formamide or benzene-formamide system just before use[l4]. Medium 199 (Hanks’ salts) was obtained from Grand Island Biochemical Company, U.S.A. Tissue culture dishes were purchased from Becton Dickinson Overseas Incorporation, Japan. 4-Androstene-3,17-dione, oestrone, oestradiol-17/l, penicillin G and porcine FSH were 741
N.
742
TERADA
et al.
obtained from Sigma Chemical Company, U.S.A. The contamination of porcine LH is less than 0.03 Armour units/unit FSH. Human chorionic gonadotropin (HCG) was obtained from Teikoku Hormone, Japan. Crystalline porcine zinc insulin was a gift from Eli Lilly and Company, U.S.A. Streptomycin sulfate was obtained from Banyu Pharmaceutical Company, Japan. Protosol (tissue solubilizer) and [methyl-3H]thymidine (82.6 Ci/mmol) were purchased from New England Nuclear Corporation, U.S.A. All other reagents were of analytical grade.
and oestrone by paper chromatography in the benzene-formamide system [ 141. The formation of [3H]oestradiol-17P and [3H]oestrone was confirmed by recrystallization to constant specific activity, following addition of 15 mg of non-radioactive oestradiol-78 or oestrone. In each experiment, 0.1 PCi of [‘4C]oestrone was added to two medium samples for the estimation of procedure loss during extraction, separation of the neutral and phenolic fractions and chromatographic separation. Recovery of [‘4C]oestrone during the procedure was 7&75:/,, and the correction was made for the procedure loss.
Estimation of ovarian aromatase activity by organ culture
DNA synthesis by cultured ovaries
The ovaries were cultured according to the method reported by Topper et a[.[151 for mammary gland explant culture. The mice were killed by cervical dislocation and the ovaries of the fetal and neonatal mice were removed under aseptic conditions using a dissection microscope. Two to six ovaries were placed on siliconized lens paper, floated on 2 ml of Medium 199 supplemented with penicillin G (35 pg/ml), streptomycin sulfate (50 pg/ml), insulin (5 pg/ml) and [3H]4-androstene-3,17-dione (0.35 PM, specific activity, 10 pCi/O.7 nmol) in the presence or absence of various concentrations of FSH. Stock solutions of hormones were insulin, 5 mg/ml in 0.005 N HCI; [3H]4-androstene-3,17-dione, 0.35 gmol/ml in absolute ethanol; FSH, 50 units/ml in 0.45% (w/v) NaCl. The final concentration of ethanol in the medium was 0.2% (v/v) and the ethanol at this concentration exerted no significant effect on the aromatase activity of ovaries. A volume of 0.45% NaCl added to the medium was adjusted to 1% (v/v) in all experiments. The ovaries were cultured at 37°C under 100% humidity in air containing 0.8% (v/v) carbon dioxide to adjust the pH of the medium to 7.4. After culture, the medium added with 25 pg of 4-androstene-3,17-dione, oestradiol-17/I and oestrone, was collected and stored at -20°C. Steroids in the medium and ovarian tissue were extracted three times with 10 ml of ether
DNA synthesis by ovaries in culture was estimated according to the method described by Oka et a/.[171 with slight modifications. One ovary from a mouse was cultured for 48 h in Medium 199 (2 ml) contain4-androstene-3,17-dione ing insulin (5 p g/ml), (0.35 PM) and [methyl-3H]thymidine (0.5 pCi/ml, specific activity, 82.6 Ci/mmol). Another ovary from the same mouse was cultured for 48 h in Medium 199 (2 ml) containing insulin, 4-androstene-3,17-dione, [methyl-3H]thymidine plus FSH (0.5 units/ml). After culture, each ovary was placed on absorption paper (5 x 10mm) and put into cold 10% (v/v) trichloroacetic acid (TCA). The ovaries were washed twice with cold 5”/;, TCA, followed by washing with cold ethanol and acetone. The dried ovary was dissolved in 0.3 ml of Protosol (tissue solubilizer) and the incorporated radioactivity was measured after addition of 12 ml toluene-based scintillator (PPO, 20 g; POPOP. 500 mg; toluene, 5 L). RESULT
Assay of ovarian aromatase actid The aromatase activity of the ovary was estimated by organ culture. As shown in Table 1, ovaries of
T 2
Table I. Oestradiol-17/I and oestrone in the medium and ovarian tissue after 9-day old mouse ovaries were cultured with 4-androstene-3,17-dione Oestrogen (pmol/4 ovaries/48 h) Sample Medium Tissue
Oestradiol-17/I 15.3 1.2
Oestrone
Total
4.6 ND
19.9 (94.3%) 1.2(5.7%)
Four ovaries from 9.day old mice were cultured for 48 h in 2 ml of Medium 199 containing insulin (0.5 fig/ml), [3H]4-androstene3,17-dione (0.35 wM) and FSH (0.5 units/ml). After culture, the amount of oestradiol-17g and oestrone in both the medium and tissue was estimated. The numbers in parentheses indicate percentage
of total oestrogens
produced.
ND: not detected.
Culture
time (h)
Fig. 1. Time course of oestrogen production from 4-androstene-3,17-dione by ovaries of 9-day old mice in culture. Two ovaries from 9-dav old mice were cultured for the indicated time in 2 ml of Medium 199 containing insulin (5 ueiml). _ IgH14-androstene-3. 17-dione (0.35 u M) and FSH .._, _ (0.5 units/ml). After culture, the ambunt ‘of bestrogens (oestradiol-17p + oestrone) in the medium was estimated. Each point represents the mean k SE of four separate determinations.
Aromatase
Number
of
activity
in mouse
ovaries
2
ovarfes
143
&;Ot10”
Days
after
birth
2
Fig. 2. Oestrogen production from 4-androstene-3,17-dione by various numbers of ovaries of 9-day old mice. Two to six ovaries from 9-day old mice were cultured for 48 h in 2 ml containing (5 pg/ml), &4-~%~rkre-~,~?-dione (0.35 p M;““:: FSH (0.5 the amount of oestrogens units/ml). After culture, (oestradioL17P + oestrone) in the medium was estimated. Each point represents the mean + SE of at least four separate determinations.
9-day old mice produced both oestradiol-17P and oestrone during culture in the presence of FSH. OestradioL17P was the major oestrogen found in both medium and tissue. The findings shown in Table 1 clearly indicate that almost all the oestrogens produced from 4-androstene-3,17-dione were found in the medium, since only 5.7% of the oestrogens produced was present in the ovarian tissue. Therefore, the amount of oestrogens present only in the medium was determined in the following experiments. The amount of oestrogens (oestradiol-178 + oestrone) produced from 4-androstene-3,17-dione by ovaries of 9-d?y old mice increased almost linearly with culture time up to 72 h (Fig. 1). The amount of oestrogens produced was also proportional to the number of ovaries (Fig. 2). FSH stimulated oestrogen production dose-dependently and the maximum
;2 ii 4
DL ’ 0 0001
I 0.004
Concentrations
I 0 01 of
I 0.04 FSH
I
01
I 04
I I
(units/ml)
Fig. 3. Dose-dependent effect of FSH on oestrogen production from 4-androstene-3,17-dione by ovaries of 9-day old mice in culture. Two ovaries from 9-day old mice were cultured for 48 h in 2 ml of Medium 199 containing insulin (5 pg/ml), [‘HlCandrostene-3,17-dione (0.35 pM) and various concentrations of FSH. After culture, the amount of oestrogens (oestradiol-17B + oestrone) was estimated.
5
Fig. 4. Oestrogen production from 4-androstene-3,17-dione by ovaries of fetal or neonatal mice in the presence or absence of FSH. Ovaries of fetal or neonatal mice were cultured for 48 h in 2 ml of Medium 199 containing insulin (5 pg/ml) and [‘H]4-androstene-3,17-dione (0.35 PM) in the presence or absence of FSH (0.5 units/ml). After culture, the amount of oestrogens (oestradiol-17/I f oestrone) was estimated. The open and hatched bars indicate oestrogen production from 4-androstene-3,17-dione in the absence and presence of FSH, respectively. Each bar represents the mean k SE of at least four separate determinations.
effect was obtained at concentrations of more than 0.1 units/ml (Fig. 3). In the present study, therefore, aromatase activity was estimated as the sum of oestrogens produced from 4-androstene-3,17-dione by 2-6 ovaries cultured for 48 h in the presence or absence of 0.5 units/ml of FSH. The aromatase activity was expressed as pmol of oestrogens produced/ovary/48 h.
Aromatase activity in ovaries of fetal and neonatal mice The aromatase activity of ovaries from fetal and neonatal mice was determined in the presence or absence of FSH by cultivating the ovaries for 48 h with 4-androstene-3,17-dione (Fig. 4). In the absence of FSH, the aromatase activity of the ovary could not be detected at day 18 of gestation or at birth. Activity in the ovary was first detected at day 3 and the activity per ovary increased with age. In the presence of FSH, however, the aromatase activity of the ovary could be detected at day 18 of gestation and the activity per ovary increased thereafter. The FSHstimulated aromatase activity increased markedly after 6 days of age. The findings in Fig. 4 show that FSH stimulated the aromatase activity of ovaries from all ages of mice which were examined. Aromatase activity was also demonstrated in the ovaries of fetal mice at day 17 of gestation in the presence of FSH but not in the absence of FSH (Table 2). Similarly, the presence of 17b-hydroxysteroid dehydrogenase in the ovaries of fetal mice at day 17 of since both gestation was also demonstrated, oestradiol-17B and oestrone were produced from 4-androstene-3,17-dione. The increase in aromatase activity in the ovaries of fetal and neonatal mice by
744
N. TERADA et al.
Table 2. Oestrogen production from 4-androstene-3,17-dione cultured ovaries of fetal mice at day 17 of gestation
by
Oestrogen (pmol/ovary/48 h) Culture
Oestradiol- 178
oestrone
Total
FSH (-) FSH (+)
ND 0.28
ND 0.25
0.53
Six ovaries from fetal mice at day 17 of gestation were cultured for 48 h in 2 ml of Medium 199 containing insulin (0.5 pg/ml) and (3H]4-androstene-3,17-dione (0.35 PM) in the presence OI absence of FSH (0.5 units/ml). After culture, the amount of oestradiol-178 and oestrone in the medium was estimated. The amount of total oestrogens produced under the same conditions for 24 h was 0.24 pmol/ovary. ND: not detected.
FSH preparation was not attributed to LH contamination in the FSH preparation, since 5 I.U. of HCG added to the culture medium showed no significant effect on the aromatase activity of the neonatal mouse ovaries (data not shown). Histological examination revealed that there were a few single-layered primordial follicles in the ovaries at birth. An increase in formation of primordial follicles and the formation of multilayered follicles were observed in the ovaries of 3 to 9-day old mice. However, in spite of extensive examination of serial sections before and after culture, no primordial follicles were observed in the ovaries of fetal mice at day 17 of gestation. The present observations indicate that FSH stimulates the aromatase activity of mouse ovaries even before the formation of primordial follicles. Effect of FSH on DNA
synthesis
in ovaries
The effect of FSH on DNA synthesis in culture was investigated in ovaries from 0- and 9-day old mice (Table 3). DNA synthesis was found in the ovaries as shown in Table 3 in the presence of insulin and 4-androstene-3,17-dione. However, the addition of FSH exerted no significant effect on DNA synthesis. DISCUSSION
The present findings show that the organ culture method is reliable for the estimation of aromatase activity in very small mouse ovaries because the amount of oestrogens from produced 4-androstene-3,17-dione was proportional to the in-
cubation time and number of ovaries. The ovaries of fetal and neonatal mice are too small to be used for the estimation of aromatase activity by incubating the ovarian homogenate with the substrate. Therefore, the present organ culture method is a useful technique for the evaluation of aromatase activity in the ovaries of fetal and neonatal mice. FSH stimulated the aromatase activity of the fetal mouse ovary in which no primordial follicles were formed, In the rat ovary, it has been reported that FSH receptors exist in the granulosa cells of developed follicles but not in theta or interstitial cells [8,9, 10, 111. Moreover, the aromatase activity in granulosa cells has been shown to be stimulated by FSH [l-7]. We found FSH-responsive cells in fetal mouse ovary with no follicles. Therefore, it is suggested that the precursor cells of the granulosa cells (pregranulosa cells) contain aromatase activity and have the ability to respond to FSH before the formation of primordial follicles. The fetal mouse ovary produced both oestradiol17/j’ and oestrone from 4-androstene-3,17-dione in culture in the presence of FSH. This result indicates that the fetal mouse ovary has 17/I-hydroxysteroid dehydrogenase activity. In rat and hamster ovaries, it has been shown that 17/I-hydroxysteroid dehydrogenase activity, which is stimulated by FSH, is mostly localized in the granulosa cells of developed follicles [16, 18, 191, as has been reported for aromatase activity [ l-111. Therefore, it might be possible that the pregranulosa cells of fetal mouse ovary also possess 17b-hydroxysteroid dehydrogenase activity under the influence of FSH. The demonstration of FSH-responsiveness of the mouse ovary at birth, which is composed only of a few primordial follicles, is in good agreement with the findings reported by Funkenstein et af.[12]. They showed the FSH-responsiveness of 4-day old rat ovary, which consisted only of primordial follicles. However, Ellinwood et a[.[ 131 reported that the ovary of fetal rhesus monkey consisted only of primordial follicles which were unable to respond to FSH. In the present study and the study by Funkenstein et a1.[12], FSH-responsiveness was evaluated in terms of stimulation of aromatase activity by FSH during the culture period of 2448 h, whereas in the work by
Table 3. Effect of FSH on DNA synthesis in cultured ovaries of 0- and 9.day old mice DNA synthesis (cpm/ovary/48 h) Hormones Insulin + 4-androstene-3,17-dione Insulin + 4-androstene-3,17-dione FSH
+
Day 0
Day 9
5790 * 400
12,900 f 560
4770 k 460
10,900 f 1390
Ovaries from 0- and 9-day old mice were cultured for 48 h in 2 ml of Medium 199 containing insulin (5 pg/ml), 4-androstene-3,17-dione (0.35 PM) and [methyl‘Hlthymidine (0.5 pCi/ml) in the presence or absence of FSH (0.5 units/ml). After culture, the incorporation of [methyl-‘HJthymidine into DNA was measured. Each value represents the mean k SE of four separate determinations.
Aromatase activity in mouse ovaries Ellinwood et al., the FSH-responsiveness was evaluated in terms of CAMP production during incubation for 3 h. Therefore, the different results might be due to the different assay methods employed in these studies. The aromatase activity per ovary in the ovaries of neonatal mice increased with age and an abrupt increase was observed after 6 days of age. On one hand, follicular development proceeds with age and the number of follicles with more than 2 layers of granulosa cells rapidly increases around day 6 after birth [20]. Therefore, the increase of aromatase activity seems to reflect the follicular development. FSH did not stimulate DNA synthesis of the neonatal mouse ovary. This result is supported by the findings by Funkenstein et a1.[12], who showed that FSH did not stimulate the follicular development of neonatal rat ovary in culture. The absence of effect of FSH on DNA synthesis suggests that the stimulative effect of FSH on aromatase activity is not attained by the proliferation of FSH-responsive cells. Acknowledgements-This work was supported in part by Grants for Cancer Research from the Ministry of Education, Science and Culture and the Ministry of Health and Welfare. The authors are grateful to Eli Lilly and Company for the generous supply of crystalline porcine zinc insulin. We thank Mr D. Eiick for editing the manuscript.
REFERENCES
J. H. and Armstrong D. T.: I. Moon Y. S., Dorrington Stimulatory action of follicle-stimulating hormone on estradiol- 178 secretion by hypophysectomized rat ovaries in organ culture. Endocrinology 97 (1975) 244-247. 2. Armstrong D. T. and Papkoff H.: Stimulation of aromatization of exogenous and endogenous androgen in ovaries of hypophysectomized rats in vioo by folliclestimulating hormone. Endocrinology 99 (1976) 1144-1151. D. T.: Hormonal control 3. Fortune J. E. and Armstrong of 178-estradiol biosynthesis in proestrous rat follicles: Estradiol production by isolated theta versus granulosa. Endocrinology 102 (1978) 227-235. 4. Moon Y. S., Tsang B. K., Simpson C. and Armstrong D. T.: 17/j’-Estradiol biosynthesis in cultured granulosa and theta cells of human ovarian follicles: Stimulation by follicle-stimulating hormone. J. clin. Endocr. Metab. 47 (1978) 263-267. control of ovarian follicular 5. Richard J. S.: Hormonal
development: A 1978 prospective. Res. 35 (1979) 343-374.
745 Recent Prog. Horm.
6. Dorrington
J. H. and Armstrong D. T.: Effect of FSH on gonadal function. Recent Prog. Horm. Res. 35 (1979)
301-342. 7. Erickson G. F.: Primary cultures of ovarian cells in serum-free medium as models of hormone-dependent differentiation. Molec. Cell. Endocr. 29 (1983) 21-49. 8. Zelezink A. R., Midgley A. R. and Reichert L. E. Jr.: Granulosa cells maturation in the rat: increased binding of HCG following treatment with FSH in vitro. Endocrinology 95 (1974)8 18-825. 9. Richard J. S., Irland J. J., Rao M. C., Bernath G. A., Midgley A. R. Jr. and Reichert L. E. Jr.: Hormone receptor regulation by estradiol. Follicle-stimulating hormone and lutenizing hormone. Endocrinology 99 (1976) 1562-1570. 10. Louvet J. P. and Vaitukaitis J. L.: Induction of folhclestimulating hormone receptors in rat ovaries by estrogen priming. Endocrinology 99 (1976) 758-764. 11. Nimrod A., Erickson G. F. and Ryan K. J.: A specific FSH receptor in rat granulosa cells: properties of binding in vilro. Endocrinology 98 (1976) 56-64. B., Nimrod A. and Lindner H. R.: The 12. Funkenstein development of steroidogenic capability and responsiveness to gonadotropins in cultured neonatal rat ovaries. Endocrinology 106 (1980) 98-106. 13. Elhnwood W. E., McClellian M. C., Brenner R. M. and Resko J. A.: Estradiol synthesis by fetal monkey ovaries correlates with antral follicle formation. Biol. Reprod. 28 (1983) 505-516. of corti14. ZatTaroni A. and Burton R. B.: Identification costeroids of beef adrenal extract by paper chromatography. J. biol. Chem. 193 (1951) 749-767. B. K.: Tech15. Topper Y. J., Oka T. and Vonderhaar niques for studying development of normal mammary epithehal cells in organ culture. Merh. Enzym. 39 (I 975) 443-454. N., Sato B., Imori T. and Mat16. Fukuda S., Terakawa sumoto K.: Hormonal regulation of 17P-ol-dehydrogenase. aromatase and 4-ene-5a-reductase in immature rat ovaries. J. steroid Biochem. 11 (1979) 1421-1427. accumu17. Oka T. and Topper Y. J.: Hormone-dependent lation of rough endoplasmic reticulum in mouse mammary epithelial cells in vitro. J. biol. Chem. 246 (1971) 7701-7703. A. and Ryan K. J.: Progesterone, an18. Makris drostenedione, testosterone, estrone and estradiol synthesis in hamster ovarian follicle cells. Endocrinology 96 (1975) 694-701. Y., Fukuda S. and Matsumoto K.: 19. Aono T., Kitamura Localization of 4-ene-5a-reductase, I7a-ol-dehydrogenase and aromatase in immature rat ovary. J. steroid Biochem. 14 (1981) 1369-1377. of the mouse ovary from 20. Petres H.: The development birth to maturity. Acra endocr., Copenh. 62 (1969) 98-116.
0022-4731/84
1984
$3.00 + 0.00
Copyright C 1984Pergamon Press Ltd
AUGMENTATION OF AROMATASE ACTIVITY BY FSH IN OVARIES OF FETAL AND NEONATAL MICE IN ORGAN CULTURE N. TERADA, H. KVRODA*, M. NAMIKI~, Y. KITAMUBA and K. MATSUMOTO*$ Institute for Cancer Research, *Department of Pathology and FDepartment of Urology, Osaka University Medical School, Kita-ku, Osaka, 530 Japan (Received
7 June 1983)
Summary-The stimulative effect of FSH on aromatase activity was investigated in ovaries of fetal (on days 17 and 18 of gestation) and neonatal mice (on days 0, 3, 6 and 9 after birth). Two to six ovaries were cultured for 48 h in 2 ml of Medium 199 supplemented with insulin (5 pg/ml) and [la, 2c1,6a, 7n, fi-3H]4-androstene-3,17-dione (0.35 FM) in the presence or absence of porcine FSH (OS units/ml) and the mount of [3H]~stradiol-17~ and [‘Hjoestrone produced was estimated. In the presence of FSH, aromatase activity per ovary, which was found in all fetal and neonatal ovaries examined, increased with age. In the absence of FSH, however, the production of oestrogens could be demonstrated only in ovaries from 3- to Q-day old mice. FSH increased the aromatase activity by up to IO-fold. In spite of the stimulative effect of FSH on aromatase activity, FSH exerted no significant effect on DNA synthesis of the ovaries. The formation of primordial follicles could not be observed histologically in ovaries of fetal mice on day 17 of gestation, although the ovaries of 6- and 9-day old mice contained multilayered follicles. These results show that FSH stimulates the aromatase activity of the mouse ovary even before the fo~ation of primordial follicles and that the stimulative effect of FSH on ovarian aromatase is not due to the proliferation of ovarian cells.
MATERIALS AND METHODS
INTRODUCTION
The effect of FSH on oestrogen
Mice
containing
Fetal and neonatal female (WB x C57BL/6)F, mice were obtained by crossing female WB and male C57BL/6 mice in our laboratory. Female WB mice were induced to ovulate by injection of 5 I.U. pregnant mare serum gonadotropin (Serotropin, Teikoku Hormone, Japan) at 4p.m., followed 48 h later by 5 I.U. of human chorionic gonadotropin and the females were placed with male C57BL/6 mice at 4p.m. On the morning after mating (day 0 of gestation), all mice were examined for vaginal plugs. The female mice with vaginal plugs were caged separately. Delivery took place on days 2&22 of gestation. The day of birth was designated as day 0 after birth. All mice were kept at 25°C under light control (14 h light/l0 h darkness) and allowed free access to water and pellet food (Oriental Yeast Co., Ltd., Japan).
well-developed
synthesis in the ovary follicles has been reported
by several investigators [l-l 11. The results have shown that granulosa cells from well-developed follicles have FSH receptors [8-l 1] and that FSH stimulates CAMP formation and aromatase activity of the granulosa cells [l-8]. However, there are only a few papers on effect of FSH on aromatase activity in the ovary, which is composed only of follicles at very early stages of development. Furthermore, results obtained in the immature ovaries are not in good agreement [12, 131. In the ovary of neonatal rat, Funkenstein et al. showed that FSH enhances the aromatase activity of 4-day old rat ovaries which consist only of primordial follicles [12]. In contrast, Ellinwood et al. reported that gonadotropins containing both LH and FSH activity are unable to stimulate the production of CAMP in fetal rhesus monkey ovaries, which are composed only of primordial follicles (131. Therefore, we investigated the stimulative effect of FSH on aromatase activity and early development of follicles in fetal and neonatal mouse ovaries. We found that FSH stimulates the aromatase activity in these mouse ovaries including fetal ovaries having no follicles and that the stimulative effect of FSH on aromatase is not due to the proliferation of FSHresponsive ovarian cells. -.. $To whom correspondence
should be addressed.
Chemicals
[l~,2ff,6ff,7a,~-3H]~androstene-3,17~ione (82 Ci/ mmol) and [4-‘4C]oestrone (59 mCi/mmol) were obtained from the Radiochemical Center Amersham, U.S.A. and were purified by paper chromatography using the hexane-formamide or benzene-formamide system just before use[l4]. Medium 199 (Hanks’ salts) was obtained from Grand Island Biochemical Company, U.S.A. Tissue culture dishes were purchased from Becton Dickinson Overseas Incorporation, Japan. 4-Androstene-3,17-dione, oestrone, oestradiol-17/l, penicillin G and porcine FSH were 741
N.
742
TERADA
et al.
obtained from Sigma Chemical Company, U.S.A. The contamination of porcine LH is less than 0.03 Armour units/unit FSH. Human chorionic gonadotropin (HCG) was obtained from Teikoku Hormone, Japan. Crystalline porcine zinc insulin was a gift from Eli Lilly and Company, U.S.A. Streptomycin sulfate was obtained from Banyu Pharmaceutical Company, Japan. Protosol (tissue solubilizer) and [methyl-3H]thymidine (82.6 Ci/mmol) were purchased from New England Nuclear Corporation, U.S.A. All other reagents were of analytical grade.
and oestrone by paper chromatography in the benzene-formamide system [ 141. The formation of [3H]oestradiol-17P and [3H]oestrone was confirmed by recrystallization to constant specific activity, following addition of 15 mg of non-radioactive oestradiol-78 or oestrone. In each experiment, 0.1 PCi of [‘4C]oestrone was added to two medium samples for the estimation of procedure loss during extraction, separation of the neutral and phenolic fractions and chromatographic separation. Recovery of [‘4C]oestrone during the procedure was 7&75:/,, and the correction was made for the procedure loss.
Estimation of ovarian aromatase activity by organ culture
DNA synthesis by cultured ovaries
The ovaries were cultured according to the method reported by Topper et a[.[151 for mammary gland explant culture. The mice were killed by cervical dislocation and the ovaries of the fetal and neonatal mice were removed under aseptic conditions using a dissection microscope. Two to six ovaries were placed on siliconized lens paper, floated on 2 ml of Medium 199 supplemented with penicillin G (35 pg/ml), streptomycin sulfate (50 pg/ml), insulin (5 pg/ml) and [3H]4-androstene-3,17-dione (0.35 PM, specific activity, 10 pCi/O.7 nmol) in the presence or absence of various concentrations of FSH. Stock solutions of hormones were insulin, 5 mg/ml in 0.005 N HCI; [3H]4-androstene-3,17-dione, 0.35 gmol/ml in absolute ethanol; FSH, 50 units/ml in 0.45% (w/v) NaCl. The final concentration of ethanol in the medium was 0.2% (v/v) and the ethanol at this concentration exerted no significant effect on the aromatase activity of ovaries. A volume of 0.45% NaCl added to the medium was adjusted to 1% (v/v) in all experiments. The ovaries were cultured at 37°C under 100% humidity in air containing 0.8% (v/v) carbon dioxide to adjust the pH of the medium to 7.4. After culture, the medium added with 25 pg of 4-androstene-3,17-dione, oestradiol-17/I and oestrone, was collected and stored at -20°C. Steroids in the medium and ovarian tissue were extracted three times with 10 ml of ether
DNA synthesis by ovaries in culture was estimated according to the method described by Oka et a/.[171 with slight modifications. One ovary from a mouse was cultured for 48 h in Medium 199 (2 ml) contain4-androstene-3,17-dione ing insulin (5 p g/ml), (0.35 PM) and [methyl-3H]thymidine (0.5 pCi/ml, specific activity, 82.6 Ci/mmol). Another ovary from the same mouse was cultured for 48 h in Medium 199 (2 ml) containing insulin, 4-androstene-3,17-dione, [methyl-3H]thymidine plus FSH (0.5 units/ml). After culture, each ovary was placed on absorption paper (5 x 10mm) and put into cold 10% (v/v) trichloroacetic acid (TCA). The ovaries were washed twice with cold 5”/;, TCA, followed by washing with cold ethanol and acetone. The dried ovary was dissolved in 0.3 ml of Protosol (tissue solubilizer) and the incorporated radioactivity was measured after addition of 12 ml toluene-based scintillator (PPO, 20 g; POPOP. 500 mg; toluene, 5 L). RESULT
Assay of ovarian aromatase actid The aromatase activity of the ovary was estimated by organ culture. As shown in Table 1, ovaries of
T 2
Table I. Oestradiol-17/I and oestrone in the medium and ovarian tissue after 9-day old mouse ovaries were cultured with 4-androstene-3,17-dione Oestrogen (pmol/4 ovaries/48 h) Sample Medium Tissue
Oestradiol-17/I 15.3 1.2
Oestrone
Total
4.6 ND
19.9 (94.3%) 1.2(5.7%)
Four ovaries from 9.day old mice were cultured for 48 h in 2 ml of Medium 199 containing insulin (0.5 fig/ml), [3H]4-androstene3,17-dione (0.35 wM) and FSH (0.5 units/ml). After culture, the amount of oestradiol-17g and oestrone in both the medium and tissue was estimated. The numbers in parentheses indicate percentage
of total oestrogens
produced.
ND: not detected.
Culture
time (h)
Fig. 1. Time course of oestrogen production from 4-androstene-3,17-dione by ovaries of 9-day old mice in culture. Two ovaries from 9-dav old mice were cultured for the indicated time in 2 ml of Medium 199 containing insulin (5 ueiml). _ IgH14-androstene-3. 17-dione (0.35 u M) and FSH .._, _ (0.5 units/ml). After culture, the ambunt ‘of bestrogens (oestradiol-17p + oestrone) in the medium was estimated. Each point represents the mean k SE of four separate determinations.
Aromatase
Number
of
activity
in mouse
ovaries
2
ovarfes
143
&;Ot10”
Days
after
birth
2
Fig. 2. Oestrogen production from 4-androstene-3,17-dione by various numbers of ovaries of 9-day old mice. Two to six ovaries from 9-day old mice were cultured for 48 h in 2 ml containing (5 pg/ml), &4-~%~rkre-~,~?-dione (0.35 p M;““:: FSH (0.5 the amount of oestrogens units/ml). After culture, (oestradioL17P + oestrone) in the medium was estimated. Each point represents the mean + SE of at least four separate determinations.
9-day old mice produced both oestradiol-17P and oestrone during culture in the presence of FSH. OestradioL17P was the major oestrogen found in both medium and tissue. The findings shown in Table 1 clearly indicate that almost all the oestrogens produced from 4-androstene-3,17-dione were found in the medium, since only 5.7% of the oestrogens produced was present in the ovarian tissue. Therefore, the amount of oestrogens present only in the medium was determined in the following experiments. The amount of oestrogens (oestradiol-178 + oestrone) produced from 4-androstene-3,17-dione by ovaries of 9-d?y old mice increased almost linearly with culture time up to 72 h (Fig. 1). The amount of oestrogens produced was also proportional to the number of ovaries (Fig. 2). FSH stimulated oestrogen production dose-dependently and the maximum
;2 ii 4
DL ’ 0 0001
I 0.004
Concentrations
I 0 01 of
I 0.04 FSH
I
01
I 04
I I
(units/ml)
Fig. 3. Dose-dependent effect of FSH on oestrogen production from 4-androstene-3,17-dione by ovaries of 9-day old mice in culture. Two ovaries from 9-day old mice were cultured for 48 h in 2 ml of Medium 199 containing insulin (5 pg/ml), [‘HlCandrostene-3,17-dione (0.35 pM) and various concentrations of FSH. After culture, the amount of oestrogens (oestradiol-17B + oestrone) was estimated.
5
Fig. 4. Oestrogen production from 4-androstene-3,17-dione by ovaries of fetal or neonatal mice in the presence or absence of FSH. Ovaries of fetal or neonatal mice were cultured for 48 h in 2 ml of Medium 199 containing insulin (5 pg/ml) and [‘H]4-androstene-3,17-dione (0.35 PM) in the presence or absence of FSH (0.5 units/ml). After culture, the amount of oestrogens (oestradiol-17/I f oestrone) was estimated. The open and hatched bars indicate oestrogen production from 4-androstene-3,17-dione in the absence and presence of FSH, respectively. Each bar represents the mean k SE of at least four separate determinations.
effect was obtained at concentrations of more than 0.1 units/ml (Fig. 3). In the present study, therefore, aromatase activity was estimated as the sum of oestrogens produced from 4-androstene-3,17-dione by 2-6 ovaries cultured for 48 h in the presence or absence of 0.5 units/ml of FSH. The aromatase activity was expressed as pmol of oestrogens produced/ovary/48 h.
Aromatase activity in ovaries of fetal and neonatal mice The aromatase activity of ovaries from fetal and neonatal mice was determined in the presence or absence of FSH by cultivating the ovaries for 48 h with 4-androstene-3,17-dione (Fig. 4). In the absence of FSH, the aromatase activity of the ovary could not be detected at day 18 of gestation or at birth. Activity in the ovary was first detected at day 3 and the activity per ovary increased with age. In the presence of FSH, however, the aromatase activity of the ovary could be detected at day 18 of gestation and the activity per ovary increased thereafter. The FSHstimulated aromatase activity increased markedly after 6 days of age. The findings in Fig. 4 show that FSH stimulated the aromatase activity of ovaries from all ages of mice which were examined. Aromatase activity was also demonstrated in the ovaries of fetal mice at day 17 of gestation in the presence of FSH but not in the absence of FSH (Table 2). Similarly, the presence of 17b-hydroxysteroid dehydrogenase in the ovaries of fetal mice at day 17 of since both gestation was also demonstrated, oestradiol-17B and oestrone were produced from 4-androstene-3,17-dione. The increase in aromatase activity in the ovaries of fetal and neonatal mice by
744
N. TERADA et al.
Table 2. Oestrogen production from 4-androstene-3,17-dione cultured ovaries of fetal mice at day 17 of gestation
by
Oestrogen (pmol/ovary/48 h) Culture
Oestradiol- 178
oestrone
Total
FSH (-) FSH (+)
ND 0.28
ND 0.25
0.53
Six ovaries from fetal mice at day 17 of gestation were cultured for 48 h in 2 ml of Medium 199 containing insulin (0.5 pg/ml) and (3H]4-androstene-3,17-dione (0.35 PM) in the presence OI absence of FSH (0.5 units/ml). After culture, the amount of oestradiol-178 and oestrone in the medium was estimated. The amount of total oestrogens produced under the same conditions for 24 h was 0.24 pmol/ovary. ND: not detected.
FSH preparation was not attributed to LH contamination in the FSH preparation, since 5 I.U. of HCG added to the culture medium showed no significant effect on the aromatase activity of the neonatal mouse ovaries (data not shown). Histological examination revealed that there were a few single-layered primordial follicles in the ovaries at birth. An increase in formation of primordial follicles and the formation of multilayered follicles were observed in the ovaries of 3 to 9-day old mice. However, in spite of extensive examination of serial sections before and after culture, no primordial follicles were observed in the ovaries of fetal mice at day 17 of gestation. The present observations indicate that FSH stimulates the aromatase activity of mouse ovaries even before the formation of primordial follicles. Effect of FSH on DNA
synthesis
in ovaries
The effect of FSH on DNA synthesis in culture was investigated in ovaries from 0- and 9-day old mice (Table 3). DNA synthesis was found in the ovaries as shown in Table 3 in the presence of insulin and 4-androstene-3,17-dione. However, the addition of FSH exerted no significant effect on DNA synthesis. DISCUSSION
The present findings show that the organ culture method is reliable for the estimation of aromatase activity in very small mouse ovaries because the amount of oestrogens from produced 4-androstene-3,17-dione was proportional to the in-
cubation time and number of ovaries. The ovaries of fetal and neonatal mice are too small to be used for the estimation of aromatase activity by incubating the ovarian homogenate with the substrate. Therefore, the present organ culture method is a useful technique for the evaluation of aromatase activity in the ovaries of fetal and neonatal mice. FSH stimulated the aromatase activity of the fetal mouse ovary in which no primordial follicles were formed, In the rat ovary, it has been reported that FSH receptors exist in the granulosa cells of developed follicles but not in theta or interstitial cells [8,9, 10, 111. Moreover, the aromatase activity in granulosa cells has been shown to be stimulated by FSH [l-7]. We found FSH-responsive cells in fetal mouse ovary with no follicles. Therefore, it is suggested that the precursor cells of the granulosa cells (pregranulosa cells) contain aromatase activity and have the ability to respond to FSH before the formation of primordial follicles. The fetal mouse ovary produced both oestradiol17/j’ and oestrone from 4-androstene-3,17-dione in culture in the presence of FSH. This result indicates that the fetal mouse ovary has 17/I-hydroxysteroid dehydrogenase activity. In rat and hamster ovaries, it has been shown that 17/I-hydroxysteroid dehydrogenase activity, which is stimulated by FSH, is mostly localized in the granulosa cells of developed follicles [16, 18, 191, as has been reported for aromatase activity [ l-111. Therefore, it might be possible that the pregranulosa cells of fetal mouse ovary also possess 17b-hydroxysteroid dehydrogenase activity under the influence of FSH. The demonstration of FSH-responsiveness of the mouse ovary at birth, which is composed only of a few primordial follicles, is in good agreement with the findings reported by Funkenstein et af.[12]. They showed the FSH-responsiveness of 4-day old rat ovary, which consisted only of primordial follicles. However, Ellinwood et a[.[ 131 reported that the ovary of fetal rhesus monkey consisted only of primordial follicles which were unable to respond to FSH. In the present study and the study by Funkenstein et a1.[12], FSH-responsiveness was evaluated in terms of stimulation of aromatase activity by FSH during the culture period of 2448 h, whereas in the work by
Table 3. Effect of FSH on DNA synthesis in cultured ovaries of 0- and 9.day old mice DNA synthesis (cpm/ovary/48 h) Hormones Insulin + 4-androstene-3,17-dione Insulin + 4-androstene-3,17-dione FSH
+
Day 0
Day 9
5790 * 400
12,900 f 560
4770 k 460
10,900 f 1390
Ovaries from 0- and 9-day old mice were cultured for 48 h in 2 ml of Medium 199 containing insulin (5 pg/ml), 4-androstene-3,17-dione (0.35 PM) and [methyl‘Hlthymidine (0.5 pCi/ml) in the presence or absence of FSH (0.5 units/ml). After culture, the incorporation of [methyl-‘HJthymidine into DNA was measured. Each value represents the mean k SE of four separate determinations.
Aromatase activity in mouse ovaries Ellinwood et al., the FSH-responsiveness was evaluated in terms of CAMP production during incubation for 3 h. Therefore, the different results might be due to the different assay methods employed in these studies. The aromatase activity per ovary in the ovaries of neonatal mice increased with age and an abrupt increase was observed after 6 days of age. On one hand, follicular development proceeds with age and the number of follicles with more than 2 layers of granulosa cells rapidly increases around day 6 after birth [20]. Therefore, the increase of aromatase activity seems to reflect the follicular development. FSH did not stimulate DNA synthesis of the neonatal mouse ovary. This result is supported by the findings by Funkenstein et a1.[12], who showed that FSH did not stimulate the follicular development of neonatal rat ovary in culture. The absence of effect of FSH on DNA synthesis suggests that the stimulative effect of FSH on aromatase activity is not attained by the proliferation of FSH-responsive cells. Acknowledgements-This work was supported in part by Grants for Cancer Research from the Ministry of Education, Science and Culture and the Ministry of Health and Welfare. The authors are grateful to Eli Lilly and Company for the generous supply of crystalline porcine zinc insulin. We thank Mr D. Eiick for editing the manuscript.
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