0013-7227/92/1301-0139$03.00/0
Endocrinology Copyright 0 1992
Vol. by
The Endocrine Society
In Viuo Action System
Printed
of Activin-A
MASAKI DOI, MASAO IGARASHI, HIROSHIRO SHIBAI, TOYOHIKO
130, No. 1
in U.S.A.
on Pituitary-Gonadal
YOSHIHISA HASEGAWA, YUZURU MIURA, AND YOSHITO IBUKI
ETO,
Department of Obstetrics and Gynecology (M.D., M.I., Y.H., Y.Z.) and Institute of Experimental Animal Research (T.M.), Gunmu University School of Medicine, Maebashi 371, Japan; and Central Research Laboratories (Y.E., H.S.), Ajinomoto Company, Inc., Kawasaki 210, Japan
ABSTRACT. Activin, a dimer of the P-subunits of inhibin, has been found to stimulate FSH secretion from the cultured pituitary cells. However, in uiuo action of activin is poorly elucidated. Daily SCinjections of 40 pg activin-A over a period of 1-3 days to intact immature female rats caused a significant increase in serum FSH, inhibin, estradiol, uterine weight, and ovarian FSH receptors. Daily SC injections of 5 pg or 20 pg activin-A for 6 days caused a marked increase in ovarian weight and the development of large ovarian follicles. However, daily SCinjections of 20 pg activin-A to hypophysectomized immature female rats for 3 days induced no significant changes in ovarian and uterine weight, serum inhibin, estradiol, and progesterone
levels. Simultaneous injections of both activin-A and 5 IU PMSG induced a significant increase in ovarian and uterine weight, serum inhibin, and estradiol levels, compared to simultaneous injections of both vehicle and PMSG in the hypophysectomized immature female rata. These results demonstrate that activin-A induces not only an increase of FSH secretion from the pituitary but also a direct autocrine or paracrine ovarian stimulation resulting in an increase of the number of ovarian FSH receptors and ovarian and uterine weight, as well as an increase in the level of inhibin and e&radio1 secretion from the ovary. (Endocrinology 130: 139-144, 1991)
A
ctivin, a stimulator of FSH secretion from the cultured pituitary cells, has been isolated from the mammalian follicular fluids and found to be a dimer of ,&subunits of inhibin (l-3). Since there are two types of P-subunit of inhibin, three types of activin are classified: activin-A (PA@* dimer), activin-AB (P&s dimer), and activin-B (/3&a dimer) (4). However, a polypeptide factor which can induce the differentiation of mouse Friend erythroleukemia cells has recently been isolated from the conditioned medium of a human leukemia cell line (5). This polypeptide, named erythroid differentiation factor (EDF), has been found to have exactly the same chemical structure as activinA (6) and also stimulates FSH secretion from the pituitary cells in uitro (7). Because of this similarity, an in vitro study using EDF instead of activin-A was carried out. It was found that EDF not only stimulated FSH secretion from the cultured pituitary cells but also induced gonadotropin receptors on the rat granulosa cells (8,9). It is clear that activin shows various physiological actions in vitro, but the effect of exogenous activin has
been poorly reported. This is because the amount of recombinant activin obtainable is very small and valued. Here we report on the in vivo action of exogenous activinA using EDF, especially in relation to the reproductive function. Materials
and Methods
Animals
Immature (21-day-old) female Wistar-Imamichi rata, weighing approximately 40 g, were housed in a temperature- and light-controlled room with a 14 h light:10 h dark light cycle (lights on at 0500 h), and provided Experimental
food and water ad lib&m.
treatments
Activin A was obtained from the culture medium of human leukemia cell THP-1 as previously described (5), dissolved in a 70 mM sodium acetate buffer containing 0.1% BSA (wt/vol). The diluted materials were stored in sterile containers at 4 C for the duration of the experiment. The vehicle consisted of a 70-mM sodium acetate buffer containing 0.1% BSA (wt/vol). Exp 1: effect of activin-A
injections
over a period of l-3 days
Twenty micrograms of activin-A per 500~~1 vehicle was SC injected twice per day (0800 and 2000 h) into the abovemen-
Received July 16, 1991. Address all correspondence and requests for reprints to: Dr. Yoshihisa Hasegawa, Department of Obstetrics and Gynecology, Gunma University School of Medicine, Maebashi 371, Japan.
tioned rats. Only the vehicle was injected to the control rats for 3 days. In the activin-A
l-day
injected
group,
139
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only the
140
ACTIVIN-A
TABLE 1. Time course of effects of activin-A weight in intact immature female rats Parameter
Vehicle control
1
dav
ACTION
ON PITUITARY-GONADAL
Exp 3: effects of activin-A female rats
Activin-A
The first and secondgroups hypophysectomizedat 21 days old had been treated twice per day (0800 and 2000 h) with SC injections (500~1vol) of vehicle and activin-A (10 rglinjection), respectively, for 3 days, starting the day after the hypophysectomy. Six hours after the last injections, these animals were killed. The third and fourth groups were injected with the vehicle and activin-A, respectively, for 3 days, and on the last day the final injections of the vehicle and activin-A were combined with 5 IU PMSG. These animals were killed 48 h later. The ovaries and uteri were weighed,and blood samples were collected aspreviously described.
2 davs
3 davs
vehicle wasinjected for 2 days,whereason the third day activinA wasinjected. In the activin-A 2&y injected group, only the vehicle wasinjected on the first day and activin-A was injected for the remaining 2 days. In the last group only activin-A was injected for the total 3 days. All the animals were killed 6 h after the final injection. The ovaries and uteri were removed, freed from extraneous tissue,and weighed.The ovaries were frozen immediately with dry ice, stored at -80 C until assay of FSH receptors. Blood sampleswere also collected at the time of autopsy, and sera separated by centrifugation at 2000 x g for 15 min, stored frozen at -30 C until assayed. injections
Endo. 1992 Vol 130. No 1
on ovarian and uterine
Ovarian wt (mg/pair) 13.7 f 1.0 11.7 + 0.7 13.0 + 1.1 12.4 k 1.2 Uterine wt (mg) 108.2 + 3.8132.3 & 4.7” 132.7 + 4.8” 131.7 + 5.2” Immature female rats hadbeentreatedtwicedaily with the vehicle or activin-A (20 pg/injection) for 1 day (two injections), 2 days (four injections),or 3 days(six injections).Six hoursafter the lastinjections, the animalswerekilled.Shownarethe mean+ SEM (n = 8 or 9 animals per time point). ’ P c 0.01compared to vehicle.
Exp 2: effect of activin-A
SYSTEM
over a period of 6 days
Activin-A (2.5 or 10 pg) per 500-/11vehicle was SCinjected twice per day (0800and 2000 h) for 6 days. The control group wasinjected with only the vehicle for 6 days. The animals were killed at 0900 h on the seventh day. The ovaries and uteri were removed and fixed in Bouin’s solution after being weighed.Blood sampleswere also collected at the time of autopsy as describedpreviously.
Histological
in hypophysectomized
immature
analysis
The ovaries were fixed in Bouin’s solution, embeddedin paraffin, sectionedserially at 10 pm, and stained with hematoxylin and eosin. Every section of each ovary was scanned under the microscopeat 40x, and those follicles greater than 250pm in diameter (including theta) showingthe ovum nucleus were measuredwith an ocular micrometer. To avoid measuring the samefollicle twice, the section containing the nucleolusof the oocyte was measured.Classification of follicular size into three groups(250-349 pm, 350-449 Km, and 450 pm or larger) and the lowest limit of developing follicle (250 pm) were determined arbitrarily. The uteri of each of the rats were also sectionedtransversely at the maximum diameter. RIA
Serum FSH concentrations were determined in duplicate by a doubleantibody RIA, usingmaterialssuppliedby the NIDDK. Rat FSH-RP-1 was used as a reference standard. Serum concentrations of inhibin (lo), estradiol (ll), and progesterone (12) were also determined in duplicate by RIA as previously described.
Serum
Estradlol
FIG. 1. Time course of effects of activin-
A on bloodhormonelevelsin intact immaturefemalerats. The animalswere treated as described in Table 1. Shown are the mean + SEM.
0 6
n 100 ng/.e
0
Serum
w/m
lnhfbln
I
4
Control
Activin 1 Day
Activln
Activin
2 Days
3 Days
0
** Ilit&
Serum
Progesterone
T
IntroI
Actlvin
1 Day
Activin
Act&n
2 Days
3 Days
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ACTIVIN-A
ACTION
SYSTEM
ON PITUITARY-GONADAL
141
C0ntd
Actwn
Kd 0.55nM Bmax 81 fM/mg
0.2-
.OJprotan
0 ,,;
Bcid
FSH
.g:ig
Actwn
protein
2 Days
0.83nM Bmax 132 fM/mg Kd
0 2IA
0
2
0
Bound
on ovarian and
Activin-A
Parameter
Vehicle control
Ovarian wt (mg/pair) Uterine wt (mg)
13.6 f 0.9 75.0 + 5.8
2.5 rg 15.1 + 0.7 102.0 f 10.5’
10 Pg 18.0 + 0.9“ 115.9 + 8.4’
Immature female rats had been treated twice daily for 6 days with the vehicle or activin-A (2.5 or 10 fig/injection). The animals were killed at 1200 h on the seventh day. Shown are the mean f SEM (n = 10 animalspergroup). a P < 0.01 compared to vehicle. * P < 0.05 compared to vehicle. ’ P c 0.001 compared to vehicle.
* 2 &Ia - m Ed
B ii
folllcular s,*e S : 250-349flm M : 350-449Nm L : 450-pm
***
PCO.05 PC0
001
I
“5 Control
FSH
I 4 ng/mg
’
,
-I protein
4 FSH
6
ng/mg
protein
3 Days
Actwn Kd 0.75 Bmax
LL ;ii
6
protan protem
Bound
0.3-
I
I
2
0
2. Effects of 6-day injections of activin-A uterine weight in intact immaturefemalerats
protein
O.I-
O.l-
TABLE
1 Day
Kd I .09 nM Bmax 112 fM/mg
2
b. m’
FIG. 2. Tie course of effects of activinA on FSH receptors in intact immature female rats. The animals were treated as described in Table 1. Shown are the scatchard plots of FSH binding to ovaries.
.
2
0
Bound
nM 169 fM/mg
4 FSH
ng/mg
protein
6 protein
Assay of FSH receptors The ovarieswere homogenizedin a teflon-glasshomogenizer with ice-cold Tris-MgCl, buffer (40 mM Tris-HCl, 5 mM MgCl,, pH 7.5) at a concentration of 200mg ovarian tissue/ml, filtered through a singlelayer of nylon gauze,and centrifuged at 30,000 x g for 20 min at 4 C. The precipitates were then resuspended in the samebuffer at a concentration of 75 mg equivalent to the ovarian tissue per milliliter and used for binding studies. Appropriate aliquots were taken for protein determinations using the method of Lowry et al. (13). A constant amount of [Yjrat FSH (NIDDK rat FSH I-6, 20,000 cpm/tube; 16 &i/pg) and various concentrations of unlabeled FSH were added to 100 ~1 homogenate, and the mixture was incubated at 37 C for 90 min by constant shaking at 90 cycles/min (final incubation vol; 300 ~1). In order to estimatenonspecificbinding of [‘251]ratFSH, 100~1Tris-MgCh buffer containing 50 IU PMSG wasaddedinstead of the standard hormonesolution. The reaction
was stopped
by adding
500 (~1 ice-cold
Tris-
MgClz buffer, the pellet was separated by centrifugation at 30,000 x g for 20 min, and the bound radioactivity
was deter-
mined by y-spectometry. Statistical analysis Data were compared by analysis of variance followed by Scheffe’s F test. When only two groups were compared, Stu-
dent’s t test wasused. 0
Results S
M
L
Control
T
S
M
L
Actlwn
2.5jlghJ
T A
S
M Activr
L
T A
lO!-LhJ
FIG. 3. Effects of 6-day injections of activin-A on follicular development in intact immature female rats. The animals were treated as described Table 2. Shown are the mean number of follicles per ovary + SEM.
Exp 1: time course of effects of activin-A in intact immature female rats The effects of activin-A on ovarian and uterine weight are shown in Table 1. A significant increase in uterine weight occurred in the activin-treated groups, even in
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142
ACTIVIN-A
ACTION
ON PITUITARY-GONADAL
Enda-
SYSTEM Vol130
l
No 1
FIG. 4. Cross-sections of whole ovaries (A, B, and C) and uteri (D, E, and F) sectioned at 10 pm and stained with hematoxylin and eosin. A and D, Treated with vehicle; B and E, treated with activin-A, 2.5 pg/injection; C and F, treated with activin-A, 10 *g/injection.
the group injected for only 1 day. But there was no significant increase in ovarian weight. Blood hormone levels are shown in Fig. 1. Serum FSH, inhibin, and estradiol levels were significantly increased in the activin-treated groups, with the maximum response in the group injected for 2 days. Serum estradiol levels especially were markedly increased even in the
group injected for only 1 day, which can account for the marked uterine growth. On the other hand, serum progesterone levels were inhibited in the activin-treated groups, in good agreement with in uitro studies (14). The Scatchard analysis of ovarian FSH receptors is shown in Fig. 2. Although the dissociation constant value did not change significantly among the four groups, a
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ACTIVIN-A
ACTION
ON PITUITARY-GONADAL
SYSTEM
143
TABLE 3. Effects of activin-A in hypophysectomized immature female rats Vehicle + Activin-A + PMSG PMSG Ovarian wt (mg/pair) 8.3 k 0.4 8.8 I? 0.5 14.0 + 0.8 17.0 t 0.5 Uterine wt (mg) 20.4 + 1.1 21.5 -c 1.0 29.4 + 2.4 47.3 * 3.1* Serum inhibin (U/ml) 30.1 f 2.4 25.4 k 2.2 17.7 f 1.5 25.1 + 3.0’ Serum estradiol (pg/ml) 1.7 zk 0.7 1.6 -e 0.6 6.4 f 1.4 19.0 + 4.5 Serum progesterone (rig/ml) 0.4 f 0.1 0.4 rt 0.1 0.7 + 0.2 0.6 k 0.1 ___-.. Hypophysectomized immature female rats had been treated twice daily with vehicle or activin-A (10 fig/injection) for 3 days. Six hours after the last injections, the animals were killed. Moreover, the last injections were combined with PMSG, 5 IU; 48 h later the animals were killed. Shown are the mean k SEM (n = 9-14 animals per group). a P < 0.01 compared to vehicle + PMSG treated. * P < 0.001 compared to vehicle + PMSG treated. ’ P < 0.05 compared to vehicle + PMSG treated. Parameter
Vehicle
significant increase in maximum binding capacity occurred in the groups injected with activin-A over the period of 2 or 3 days (P < 0.05, P < 0.01, respectively). Exp 2: effects of activin-A injections over a period of 6 days in intact immature female rats
Ovarian and uterine weight are shown in Table 2. A significant increase in both ovarian and uterine weight occurred in the activin-treated groups. In addition, as shown in Fig. 3, injections of activin-A significantly increased not only the number of follicles, but also their size. Cross-sections of whole ovaries and uteri are shown in Fig. 4. A marked follicular and uterine development is seen in activin-treated groups. The 6-day injection period of activin-A increased not only uterine weight but also ovarian weight; however, the RIA results revealed no significant increase in serum FSH, inhibin, or estradiol levels (data not shown). Exp 3: effects of activin-A immature female rats
in hypophysectomized
All the results are shown in Table 3. Injections of activin-A induced no significant change in ovarian and uterine weight, serum inhibin, estradiol, and progesterone levels. However, the effects of PMSG upon ovarian and uterine weight, serum inhibin, and estradiol levels were significantly augmented by the antecedent and simultaneous injections of activin-A. Discussion
Schwa11 et al. (15) have reported recently that daily SC injections of 10 pg or 40 pg recombinant human activinA for 3 days to immature female rats caused a marked increase in serum FSH levels but did not induce any effect on ovarian weight. In the present experiments, we have demonstrated that ovarian weight gain requires activin-A injections over a period of 6 days, whereas uterine weight gain requires injections only for 1 day. The increase of uterine weight can be explained by the
Activin-A
increase of serum estradiol, but the increase of estradiol in the group injected for 1 day cannot be explained by FSH, because FSH level did not increase. These results suggest the possibility that activin-A may act directly on the ovary. In the present research, the number of ovarian FSH receptors was demonstrated to be increased in activin-A-treated groups. To confirm this hypothesis, we used hypophysectomized immature female rats. Although ovarian weight, uterine weight, and serum estradiol levels were not affected by treatment with activin alone, simultaneous administration of activin-A and PMSG induced a significant increase in ovarian and uterine weight, serum inhibin, and estradiol levels. These data clearly demonstrate that activin stimulates the action of gonadotropins at the level of the ovary. However, one anomaly exists in our data. It is the fact that there was no significant change of blood hormone levels at the end of the experiment of the 6-day period of injections. The reason for this as yet cannot be accounted for, but there are some speculations. One possibility is that the injected activin-A might increase the intrafollicular activin binding protein (16), which in turn could neutralize the action of injected activin-A. But further studies will be required to explain these results. Recently, Woodruff et al. (17) have reported using ovarian intrabursal injection that inhibin acts as a follicular maturation factor and activin acts as a follicular maturation inhibitor. It is not clear why discrepancies occur between their results and ours, but intrabursally injected 1 pg activin locally administrated seems to be an unphysiological overdose, and the results were observed after only 24 h. Further studies are required to clarify these discrepancies. In conclusion, we have shown that systemic administration of activin-A can increase ovarian and uterine weight and follicular development. It also can stimulate not only the secretion of FSH at the pituitary level, but increase the level of inhibin, estradiol, and ovarian FSH receptor expression at the ovarian level. Moreover, in
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144
ACTIVIN-A
ACTION ON PITUITARY-GONADAL
hypophysectomized immature female rats, activin-A enhances the actions of PMSG to increase ovarian and uterine weight, as well as increase inhibin and estradiol secretion from the ovary. These data demonstrate that activin-A can act on not only the pituitary level, but also the ovarian level, to stimulate follicular development. Acknowledgments We wish to thank NIDDK for providing the rat FSH RIA kit.
References 1. Vale W, Rivier J, Vaughan J, McCIintoch R, Corrigan A, Woo W, Karr D, Spiess J 1986 Purification and characterization of an FSH releasing protein from porcine ovarian foliicuhrr fluid. Nature 321:776-779 2. Lmg N, Ying S-Y, Ueno N, Shimazaki S, Esch F, Hotta M, Guihemin R 1986 Pituitary FSH is released by a heterodimer of the @-subunita tram the two forms of inhibin. Nature 321:779-782 3. Ling N, Yii S-Y, Ueno N, Shimasaki S, Esch F, Hotta M, GuiIIemin R 1986 A homodimer of the fl-subunite of inhibin A stimuIatas the secretion of pituitary follicle stimulating hormone. Bioc~m Biophys Res Commun 138:1129-1137 4. Burger HG, Igarashi M 1988 Inhibin: definition and nomenclature, including reIat.ed substances. Endocrinology 122:1701-1702 5. Et.o Y, Tsuji T, Takesawa M, Takano S, Yokogawa Y, Shibai H 1987 Purification and characterization of erythroid differentiation fatter (EDF) isolated from human leukemia cell line THP-1. Biochem Biophys Res Commun 142:1095-1103 6. Murata M, Et0 Y, Shibai H, Sakai M, Muramatsu M 1988 Erythroid differentiation factor is encoded by the same mRNA as that
SYSTEM
of the inhibin I% chain. Proc Nat1 Acad Sci USA 85~2434-2438 7. Kitaoka M, Yamashita N, Eto Y, Shibai H, Ogata E 1987 Stimulation of FSH release by erythroid differentiation factor (EDF). Biochem Biophys Res Commun 146:1382-1385 8. Sugino H, Nakamura T, Hasegawa Y, Miyamoto K, Abe Y, Igarashi M, Eta Y. Shibai H, Titani K 1988 Ervthroid differentiation factor can modulate follicular granulosa cell-functions. Biochem Biophys Res Commun 153:281-288 9. Hasegawa Y, Miyamoto K, Abe Y, Nakamura T, Sugino H, Eto Y, Shibai H, Igarashi M 1988 Induction of follicle stimulating hormone receptor by erythroid differentiation factor on rat granulosa cell. Biochem Biophys Res Commun 156668-674 10. Hasegawa Y, Miyamoto K, Igarashi M 1988 Changes in serum concentrations of immunoreactive inhibin during the oestrous cycle of the rat. J Endocrinol121:91-100 11. Haeegawa Y, Miyamoto K, Iwamura S, Igarashi M 1988 Changes in serum concentrations of inhibin in cycling pigs. J Endocrinol 118:211-219 12. Takahashi Y, Hasegawa Y, Yazaki C, Igarashi M 1985 Direct radioimmunoassay of progesterone in rat serum. Endocrinol Jpn 32661-672 13. Lowry OH, Rosenhrough NJ, Farr AL, Randali RJ 1951 Protein measurement with the folin phenol reagant. J Biol Chem 193:265275 14. Shukovsky L, Findiay JK 1990 Activin-A inhibits oxytocin and progesterone production by preovrdatory bovine granulosa cells in vitro. Endocrinology126:2222-2224 15. SchwaIl R, Schmelser CH, Matsuyama E, Mason AJ 1989 Multiple actions of recombinant activin-A in vim Endocrinology 125:14201423 16. Nakamura T, Takio K, Eto Y, Shibai H, Titani K, Sugino H 1990 Activin-biidine nrotein from rat ovarv is follistatin. Science 247:036-838 - 17. Woodruff TK, Lyon FLJ, Hansen SE, Rice GC, Mather JP 1990 Inhibin and activin locally regulate rat ovarian foIIiculogenesis. Endocrinology 127:3196-3205
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 19 November 2015. at 02:46 For personal use only. No other uses without permission. . All rights reserved.
Endocrinology Copyright 0 1992
Vol. by
The Endocrine Society
In Viuo Action System
Printed
of Activin-A
MASAKI DOI, MASAO IGARASHI, HIROSHIRO SHIBAI, TOYOHIKO
130, No. 1
in U.S.A.
on Pituitary-Gonadal
YOSHIHISA HASEGAWA, YUZURU MIURA, AND YOSHITO IBUKI
ETO,
Department of Obstetrics and Gynecology (M.D., M.I., Y.H., Y.Z.) and Institute of Experimental Animal Research (T.M.), Gunmu University School of Medicine, Maebashi 371, Japan; and Central Research Laboratories (Y.E., H.S.), Ajinomoto Company, Inc., Kawasaki 210, Japan
ABSTRACT. Activin, a dimer of the P-subunits of inhibin, has been found to stimulate FSH secretion from the cultured pituitary cells. However, in uiuo action of activin is poorly elucidated. Daily SCinjections of 40 pg activin-A over a period of 1-3 days to intact immature female rats caused a significant increase in serum FSH, inhibin, estradiol, uterine weight, and ovarian FSH receptors. Daily SC injections of 5 pg or 20 pg activin-A for 6 days caused a marked increase in ovarian weight and the development of large ovarian follicles. However, daily SCinjections of 20 pg activin-A to hypophysectomized immature female rats for 3 days induced no significant changes in ovarian and uterine weight, serum inhibin, estradiol, and progesterone
levels. Simultaneous injections of both activin-A and 5 IU PMSG induced a significant increase in ovarian and uterine weight, serum inhibin, and estradiol levels, compared to simultaneous injections of both vehicle and PMSG in the hypophysectomized immature female rata. These results demonstrate that activin-A induces not only an increase of FSH secretion from the pituitary but also a direct autocrine or paracrine ovarian stimulation resulting in an increase of the number of ovarian FSH receptors and ovarian and uterine weight, as well as an increase in the level of inhibin and e&radio1 secretion from the ovary. (Endocrinology 130: 139-144, 1991)
A
ctivin, a stimulator of FSH secretion from the cultured pituitary cells, has been isolated from the mammalian follicular fluids and found to be a dimer of ,&subunits of inhibin (l-3). Since there are two types of P-subunit of inhibin, three types of activin are classified: activin-A (PA@* dimer), activin-AB (P&s dimer), and activin-B (/3&a dimer) (4). However, a polypeptide factor which can induce the differentiation of mouse Friend erythroleukemia cells has recently been isolated from the conditioned medium of a human leukemia cell line (5). This polypeptide, named erythroid differentiation factor (EDF), has been found to have exactly the same chemical structure as activinA (6) and also stimulates FSH secretion from the pituitary cells in uitro (7). Because of this similarity, an in vitro study using EDF instead of activin-A was carried out. It was found that EDF not only stimulated FSH secretion from the cultured pituitary cells but also induced gonadotropin receptors on the rat granulosa cells (8,9). It is clear that activin shows various physiological actions in vitro, but the effect of exogenous activin has
been poorly reported. This is because the amount of recombinant activin obtainable is very small and valued. Here we report on the in vivo action of exogenous activinA using EDF, especially in relation to the reproductive function. Materials
and Methods
Animals
Immature (21-day-old) female Wistar-Imamichi rata, weighing approximately 40 g, were housed in a temperature- and light-controlled room with a 14 h light:10 h dark light cycle (lights on at 0500 h), and provided Experimental
food and water ad lib&m.
treatments
Activin A was obtained from the culture medium of human leukemia cell THP-1 as previously described (5), dissolved in a 70 mM sodium acetate buffer containing 0.1% BSA (wt/vol). The diluted materials were stored in sterile containers at 4 C for the duration of the experiment. The vehicle consisted of a 70-mM sodium acetate buffer containing 0.1% BSA (wt/vol). Exp 1: effect of activin-A
injections
over a period of l-3 days
Twenty micrograms of activin-A per 500~~1 vehicle was SC injected twice per day (0800 and 2000 h) into the abovemen-
Received July 16, 1991. Address all correspondence and requests for reprints to: Dr. Yoshihisa Hasegawa, Department of Obstetrics and Gynecology, Gunma University School of Medicine, Maebashi 371, Japan.
tioned rats. Only the vehicle was injected to the control rats for 3 days. In the activin-A
l-day
injected
group,
139
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 19 November 2015. at 02:46 For personal use only. No other uses without permission. . All rights reserved.
only the
140
ACTIVIN-A
TABLE 1. Time course of effects of activin-A weight in intact immature female rats Parameter
Vehicle control
1
dav
ACTION
ON PITUITARY-GONADAL
Exp 3: effects of activin-A female rats
Activin-A
The first and secondgroups hypophysectomizedat 21 days old had been treated twice per day (0800 and 2000 h) with SC injections (500~1vol) of vehicle and activin-A (10 rglinjection), respectively, for 3 days, starting the day after the hypophysectomy. Six hours after the last injections, these animals were killed. The third and fourth groups were injected with the vehicle and activin-A, respectively, for 3 days, and on the last day the final injections of the vehicle and activin-A were combined with 5 IU PMSG. These animals were killed 48 h later. The ovaries and uteri were weighed,and blood samples were collected aspreviously described.
2 davs
3 davs
vehicle wasinjected for 2 days,whereason the third day activinA wasinjected. In the activin-A 2&y injected group, only the vehicle wasinjected on the first day and activin-A was injected for the remaining 2 days. In the last group only activin-A was injected for the total 3 days. All the animals were killed 6 h after the final injection. The ovaries and uteri were removed, freed from extraneous tissue,and weighed.The ovaries were frozen immediately with dry ice, stored at -80 C until assay of FSH receptors. Blood sampleswere also collected at the time of autopsy, and sera separated by centrifugation at 2000 x g for 15 min, stored frozen at -30 C until assayed. injections
Endo. 1992 Vol 130. No 1
on ovarian and uterine
Ovarian wt (mg/pair) 13.7 f 1.0 11.7 + 0.7 13.0 + 1.1 12.4 k 1.2 Uterine wt (mg) 108.2 + 3.8132.3 & 4.7” 132.7 + 4.8” 131.7 + 5.2” Immature female rats hadbeentreatedtwicedaily with the vehicle or activin-A (20 pg/injection) for 1 day (two injections), 2 days (four injections),or 3 days(six injections).Six hoursafter the lastinjections, the animalswerekilled.Shownarethe mean+ SEM (n = 8 or 9 animals per time point). ’ P c 0.01compared to vehicle.
Exp 2: effect of activin-A
SYSTEM
over a period of 6 days
Activin-A (2.5 or 10 pg) per 500-/11vehicle was SCinjected twice per day (0800and 2000 h) for 6 days. The control group wasinjected with only the vehicle for 6 days. The animals were killed at 0900 h on the seventh day. The ovaries and uteri were removed and fixed in Bouin’s solution after being weighed.Blood sampleswere also collected at the time of autopsy as describedpreviously.
Histological
in hypophysectomized
immature
analysis
The ovaries were fixed in Bouin’s solution, embeddedin paraffin, sectionedserially at 10 pm, and stained with hematoxylin and eosin. Every section of each ovary was scanned under the microscopeat 40x, and those follicles greater than 250pm in diameter (including theta) showingthe ovum nucleus were measuredwith an ocular micrometer. To avoid measuring the samefollicle twice, the section containing the nucleolusof the oocyte was measured.Classification of follicular size into three groups(250-349 pm, 350-449 Km, and 450 pm or larger) and the lowest limit of developing follicle (250 pm) were determined arbitrarily. The uteri of each of the rats were also sectionedtransversely at the maximum diameter. RIA
Serum FSH concentrations were determined in duplicate by a doubleantibody RIA, usingmaterialssuppliedby the NIDDK. Rat FSH-RP-1 was used as a reference standard. Serum concentrations of inhibin (lo), estradiol (ll), and progesterone (12) were also determined in duplicate by RIA as previously described.
Serum
Estradlol
FIG. 1. Time course of effects of activin-
A on bloodhormonelevelsin intact immaturefemalerats. The animalswere treated as described in Table 1. Shown are the mean + SEM.
0 6
n 100 ng/.e
0
Serum
w/m
lnhfbln
I
4
Control
Activin 1 Day
Activln
Activin
2 Days
3 Days
0
** Ilit&
Serum
Progesterone
T
IntroI
Actlvin
1 Day
Activin
Act&n
2 Days
3 Days
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ACTIVIN-A
ACTION
SYSTEM
ON PITUITARY-GONADAL
141
C0ntd
Actwn
Kd 0.55nM Bmax 81 fM/mg
0.2-
.OJprotan
0 ,,;
Bcid
FSH
.g:ig
Actwn
protein
2 Days
0.83nM Bmax 132 fM/mg Kd
0 2IA
0
2
0
Bound
on ovarian and
Activin-A
Parameter
Vehicle control
Ovarian wt (mg/pair) Uterine wt (mg)
13.6 f 0.9 75.0 + 5.8
2.5 rg 15.1 + 0.7 102.0 f 10.5’
10 Pg 18.0 + 0.9“ 115.9 + 8.4’
Immature female rats had been treated twice daily for 6 days with the vehicle or activin-A (2.5 or 10 fig/injection). The animals were killed at 1200 h on the seventh day. Shown are the mean f SEM (n = 10 animalspergroup). a P < 0.01 compared to vehicle. * P < 0.05 compared to vehicle. ’ P c 0.001 compared to vehicle.
* 2 &Ia - m Ed
B ii
folllcular s,*e S : 250-349flm M : 350-449Nm L : 450-pm
***
PCO.05 PC0
001
I
“5 Control
FSH
I 4 ng/mg
’
,
-I protein
4 FSH
6
ng/mg
protein
3 Days
Actwn Kd 0.75 Bmax
LL ;ii
6
protan protem
Bound
0.3-
I
I
2
0
2. Effects of 6-day injections of activin-A uterine weight in intact immaturefemalerats
protein
O.I-
O.l-
TABLE
1 Day
Kd I .09 nM Bmax 112 fM/mg
2
b. m’
FIG. 2. Tie course of effects of activinA on FSH receptors in intact immature female rats. The animals were treated as described in Table 1. Shown are the scatchard plots of FSH binding to ovaries.
.
2
0
Bound
nM 169 fM/mg
4 FSH
ng/mg
protein
6 protein
Assay of FSH receptors The ovarieswere homogenizedin a teflon-glasshomogenizer with ice-cold Tris-MgCl, buffer (40 mM Tris-HCl, 5 mM MgCl,, pH 7.5) at a concentration of 200mg ovarian tissue/ml, filtered through a singlelayer of nylon gauze,and centrifuged at 30,000 x g for 20 min at 4 C. The precipitates were then resuspended in the samebuffer at a concentration of 75 mg equivalent to the ovarian tissue per milliliter and used for binding studies. Appropriate aliquots were taken for protein determinations using the method of Lowry et al. (13). A constant amount of [Yjrat FSH (NIDDK rat FSH I-6, 20,000 cpm/tube; 16 &i/pg) and various concentrations of unlabeled FSH were added to 100 ~1 homogenate, and the mixture was incubated at 37 C for 90 min by constant shaking at 90 cycles/min (final incubation vol; 300 ~1). In order to estimatenonspecificbinding of [‘251]ratFSH, 100~1Tris-MgCh buffer containing 50 IU PMSG wasaddedinstead of the standard hormonesolution. The reaction
was stopped
by adding
500 (~1 ice-cold
Tris-
MgClz buffer, the pellet was separated by centrifugation at 30,000 x g for 20 min, and the bound radioactivity
was deter-
mined by y-spectometry. Statistical analysis Data were compared by analysis of variance followed by Scheffe’s F test. When only two groups were compared, Stu-
dent’s t test wasused. 0
Results S
M
L
Control
T
S
M
L
Actlwn
2.5jlghJ
T A
S
M Activr
L
T A
lO!-LhJ
FIG. 3. Effects of 6-day injections of activin-A on follicular development in intact immature female rats. The animals were treated as described Table 2. Shown are the mean number of follicles per ovary + SEM.
Exp 1: time course of effects of activin-A in intact immature female rats The effects of activin-A on ovarian and uterine weight are shown in Table 1. A significant increase in uterine weight occurred in the activin-treated groups, even in
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142
ACTIVIN-A
ACTION
ON PITUITARY-GONADAL
Enda-
SYSTEM Vol130
l
No 1
FIG. 4. Cross-sections of whole ovaries (A, B, and C) and uteri (D, E, and F) sectioned at 10 pm and stained with hematoxylin and eosin. A and D, Treated with vehicle; B and E, treated with activin-A, 2.5 pg/injection; C and F, treated with activin-A, 10 *g/injection.
the group injected for only 1 day. But there was no significant increase in ovarian weight. Blood hormone levels are shown in Fig. 1. Serum FSH, inhibin, and estradiol levels were significantly increased in the activin-treated groups, with the maximum response in the group injected for 2 days. Serum estradiol levels especially were markedly increased even in the
group injected for only 1 day, which can account for the marked uterine growth. On the other hand, serum progesterone levels were inhibited in the activin-treated groups, in good agreement with in uitro studies (14). The Scatchard analysis of ovarian FSH receptors is shown in Fig. 2. Although the dissociation constant value did not change significantly among the four groups, a
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ACTIVIN-A
ACTION
ON PITUITARY-GONADAL
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143
TABLE 3. Effects of activin-A in hypophysectomized immature female rats Vehicle + Activin-A + PMSG PMSG Ovarian wt (mg/pair) 8.3 k 0.4 8.8 I? 0.5 14.0 + 0.8 17.0 t 0.5 Uterine wt (mg) 20.4 + 1.1 21.5 -c 1.0 29.4 + 2.4 47.3 * 3.1* Serum inhibin (U/ml) 30.1 f 2.4 25.4 k 2.2 17.7 f 1.5 25.1 + 3.0’ Serum estradiol (pg/ml) 1.7 zk 0.7 1.6 -e 0.6 6.4 f 1.4 19.0 + 4.5 Serum progesterone (rig/ml) 0.4 f 0.1 0.4 rt 0.1 0.7 + 0.2 0.6 k 0.1 ___-.. Hypophysectomized immature female rats had been treated twice daily with vehicle or activin-A (10 fig/injection) for 3 days. Six hours after the last injections, the animals were killed. Moreover, the last injections were combined with PMSG, 5 IU; 48 h later the animals were killed. Shown are the mean k SEM (n = 9-14 animals per group). a P < 0.01 compared to vehicle + PMSG treated. * P < 0.001 compared to vehicle + PMSG treated. ’ P < 0.05 compared to vehicle + PMSG treated. Parameter
Vehicle
significant increase in maximum binding capacity occurred in the groups injected with activin-A over the period of 2 or 3 days (P < 0.05, P < 0.01, respectively). Exp 2: effects of activin-A injections over a period of 6 days in intact immature female rats
Ovarian and uterine weight are shown in Table 2. A significant increase in both ovarian and uterine weight occurred in the activin-treated groups. In addition, as shown in Fig. 3, injections of activin-A significantly increased not only the number of follicles, but also their size. Cross-sections of whole ovaries and uteri are shown in Fig. 4. A marked follicular and uterine development is seen in activin-treated groups. The 6-day injection period of activin-A increased not only uterine weight but also ovarian weight; however, the RIA results revealed no significant increase in serum FSH, inhibin, or estradiol levels (data not shown). Exp 3: effects of activin-A immature female rats
in hypophysectomized
All the results are shown in Table 3. Injections of activin-A induced no significant change in ovarian and uterine weight, serum inhibin, estradiol, and progesterone levels. However, the effects of PMSG upon ovarian and uterine weight, serum inhibin, and estradiol levels were significantly augmented by the antecedent and simultaneous injections of activin-A. Discussion
Schwa11 et al. (15) have reported recently that daily SC injections of 10 pg or 40 pg recombinant human activinA for 3 days to immature female rats caused a marked increase in serum FSH levels but did not induce any effect on ovarian weight. In the present experiments, we have demonstrated that ovarian weight gain requires activin-A injections over a period of 6 days, whereas uterine weight gain requires injections only for 1 day. The increase of uterine weight can be explained by the
Activin-A
increase of serum estradiol, but the increase of estradiol in the group injected for 1 day cannot be explained by FSH, because FSH level did not increase. These results suggest the possibility that activin-A may act directly on the ovary. In the present research, the number of ovarian FSH receptors was demonstrated to be increased in activin-A-treated groups. To confirm this hypothesis, we used hypophysectomized immature female rats. Although ovarian weight, uterine weight, and serum estradiol levels were not affected by treatment with activin alone, simultaneous administration of activin-A and PMSG induced a significant increase in ovarian and uterine weight, serum inhibin, and estradiol levels. These data clearly demonstrate that activin stimulates the action of gonadotropins at the level of the ovary. However, one anomaly exists in our data. It is the fact that there was no significant change of blood hormone levels at the end of the experiment of the 6-day period of injections. The reason for this as yet cannot be accounted for, but there are some speculations. One possibility is that the injected activin-A might increase the intrafollicular activin binding protein (16), which in turn could neutralize the action of injected activin-A. But further studies will be required to explain these results. Recently, Woodruff et al. (17) have reported using ovarian intrabursal injection that inhibin acts as a follicular maturation factor and activin acts as a follicular maturation inhibitor. It is not clear why discrepancies occur between their results and ours, but intrabursally injected 1 pg activin locally administrated seems to be an unphysiological overdose, and the results were observed after only 24 h. Further studies are required to clarify these discrepancies. In conclusion, we have shown that systemic administration of activin-A can increase ovarian and uterine weight and follicular development. It also can stimulate not only the secretion of FSH at the pituitary level, but increase the level of inhibin, estradiol, and ovarian FSH receptor expression at the ovarian level. Moreover, in
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144
ACTIVIN-A
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hypophysectomized immature female rats, activin-A enhances the actions of PMSG to increase ovarian and uterine weight, as well as increase inhibin and estradiol secretion from the ovary. These data demonstrate that activin-A can act on not only the pituitary level, but also the ovarian level, to stimulate follicular development. Acknowledgments We wish to thank NIDDK for providing the rat FSH RIA kit.
References 1. Vale W, Rivier J, Vaughan J, McCIintoch R, Corrigan A, Woo W, Karr D, Spiess J 1986 Purification and characterization of an FSH releasing protein from porcine ovarian foliicuhrr fluid. Nature 321:776-779 2. Lmg N, Ying S-Y, Ueno N, Shimazaki S, Esch F, Hotta M, Guihemin R 1986 Pituitary FSH is released by a heterodimer of the @-subunita tram the two forms of inhibin. Nature 321:779-782 3. Ling N, Yii S-Y, Ueno N, Shimasaki S, Esch F, Hotta M, GuiIIemin R 1986 A homodimer of the fl-subunite of inhibin A stimuIatas the secretion of pituitary follicle stimulating hormone. Bioc~m Biophys Res Commun 138:1129-1137 4. Burger HG, Igarashi M 1988 Inhibin: definition and nomenclature, including reIat.ed substances. Endocrinology 122:1701-1702 5. Et.o Y, Tsuji T, Takesawa M, Takano S, Yokogawa Y, Shibai H 1987 Purification and characterization of erythroid differentiation fatter (EDF) isolated from human leukemia cell line THP-1. Biochem Biophys Res Commun 142:1095-1103 6. Murata M, Et0 Y, Shibai H, Sakai M, Muramatsu M 1988 Erythroid differentiation factor is encoded by the same mRNA as that
SYSTEM
of the inhibin I% chain. Proc Nat1 Acad Sci USA 85~2434-2438 7. Kitaoka M, Yamashita N, Eto Y, Shibai H, Ogata E 1987 Stimulation of FSH release by erythroid differentiation factor (EDF). Biochem Biophys Res Commun 146:1382-1385 8. Sugino H, Nakamura T, Hasegawa Y, Miyamoto K, Abe Y, Igarashi M, Eta Y. Shibai H, Titani K 1988 Ervthroid differentiation factor can modulate follicular granulosa cell-functions. Biochem Biophys Res Commun 153:281-288 9. Hasegawa Y, Miyamoto K, Abe Y, Nakamura T, Sugino H, Eto Y, Shibai H, Igarashi M 1988 Induction of follicle stimulating hormone receptor by erythroid differentiation factor on rat granulosa cell. Biochem Biophys Res Commun 156668-674 10. Hasegawa Y, Miyamoto K, Igarashi M 1988 Changes in serum concentrations of immunoreactive inhibin during the oestrous cycle of the rat. J Endocrinol121:91-100 11. Haeegawa Y, Miyamoto K, Iwamura S, Igarashi M 1988 Changes in serum concentrations of inhibin in cycling pigs. J Endocrinol 118:211-219 12. Takahashi Y, Hasegawa Y, Yazaki C, Igarashi M 1985 Direct radioimmunoassay of progesterone in rat serum. Endocrinol Jpn 32661-672 13. Lowry OH, Rosenhrough NJ, Farr AL, Randali RJ 1951 Protein measurement with the folin phenol reagant. J Biol Chem 193:265275 14. Shukovsky L, Findiay JK 1990 Activin-A inhibits oxytocin and progesterone production by preovrdatory bovine granulosa cells in vitro. Endocrinology126:2222-2224 15. SchwaIl R, Schmelser CH, Matsuyama E, Mason AJ 1989 Multiple actions of recombinant activin-A in vim Endocrinology 125:14201423 16. Nakamura T, Takio K, Eto Y, Shibai H, Titani K, Sugino H 1990 Activin-biidine nrotein from rat ovarv is follistatin. Science 247:036-838 - 17. Woodruff TK, Lyon FLJ, Hansen SE, Rice GC, Mather JP 1990 Inhibin and activin locally regulate rat ovarian foIIiculogenesis. Endocrinology 127:3196-3205
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