Low molecular weight substance from rat ovary induces steroidogenesis in cultured granulosa cells

Molecular and Cellular Endocrinology, Elsevier Scientific Publishers Ireland.

141

36 (1984) 141- 155 Ltd.

MCE 01164

Low molecular

weight substance from rat ovary induces steroidogenesis cultured granulosa cells

P. Weinberger-Ohana, Department

R. Shoshani, Y. Farkash, N. Hershkovits, R. Epstein-Almog and J. Orly *

of Biological Chemrstry, Institute of Life Scrences, the Hebrew Uniuersrty (Received

Keywords;

progestins;

aromatase;

14 December

LH responsiveness:

1983, accepted

6 March

in

N.B. Goldring,

of Jerusalem,91904 Jerusalem (Israel) 1984)

CAMP.

Summary Ovaries from immature intact rats contain an apparently low molecular weight substance which mimics the action of follitropin (FSH) on ovarian granulosa cells in culture. Similar to FSH action, the ovarian substance (OS) induced cell-shape changes followed by intensive progestin production. Like FSH action, OS-induced steroidogenesis reversibly ceased upon washing the factor from the cultured cells, and could be blocked in the presence of cycloheximide or cr-amanitin. Although OS stimulated aromatase activity in granulosa cells, it failed to elicit LH responsiveness in the cultured cells. Androstenedione synergistically augmented OS-induced progestin production and aromatase activity. OS itself synergistically augmented FSH-induced progestin but did not have any effect on FSH-induced aromatase activity. In contrast to FSH action which is mediated via CAMP formation, OS doses which evoked extensive synthesis of progestin products failed to stimulate significant increases in intracellular CAMP accumulation. These results suggest the existence of a putative intraovarian hormone-like substance which can mimic some effects of the gonadotropins on the follicular granulosa cell differentiation and may facilitate FSH action at yet unknown stages of the follicular development.

In our search for possible intraovarian substances which might regulate the response of the follicular granulosa cells to follitropin (FSH), we have recently found an apparently low molecular weight substance which exists in the rat ovary and can mimic the action of FSH on granulosa cells in culture (Orly et al., 1982a, b). Similar to FSH response, this ovarian substance (OS) induced temporal morphological changes in the granulosa cell monolayer, occurring within minutes after the addition of OS. When incubation with OS continued for 48 h, substantial amounts of progestins * To whom all correspondence

should

be addressed.

could be detected in the culture medium. Unlike FSH, however, our preliminary studies indicated that OS does not induce accumulation of cyclic AMP (CAMP) in granulosa cells. The ovarian substance could not be CAMP, as its biological activity is not abolished by phosphodiesterase treatment. Neither is it a possible phosphodiesterase inhibitor, as it did not augment FSH-induced CAMP accumulation in granulosa cells. OS was found to be heat-stable, protease-resistant and relatively acid-stable, but rapidly lost its biological activity after alkali treatment. We have also provided evidence to exclude the possibility that OS may be one of various neurotransmitters or com-

142

mon prostaglandins which are known to participate in the ovarian function and physiology (Orly et al., 1982b). The present report describes extended studies to characterize the biological activity of the ovarian substance on the granulosa cytodifferentiation. Our results suggest that OS induces progestin production in a characteristic fashion which closely resembles that of the FSH-stimulated steroidogenesis. However, detailed dose-dependent responses of granulosa cells to OS and FSH agonists reinforce the notion that, unlike FSH, OS bypasses the need for CAMP to mediate the induction of steroidogenesis. In contrast to FSH, OS also failed to induce LH responsiveness but was found rather efficient in stimulating estrogen production. Surprisingly, under conditions which did not yield OS-induced progestin production, OS synergistically increased the FSH responsiveness in progestin synthesis. The possible physiological relevance of OS in the ovarian function and follicular development is discussed. Materials and Methods Agonist Ovine FSH (NIAMDD-oFSH-13) was a generous gift from the National Institute of Arthritis, Metabolism and Digestive Diseases. If not otherwise stated, FSH was used at a concentration of 100 ng/ml. Reagents cY-Amanitin (A-2263), cycloheximide (C-6255) and 3-isobutyl-1-methylxanthine (I-5879) as well as insulin (I-5500), transferrin (T-4515), hydrocortisone (H-4001), glucuronidase (G-3510) and sulfatase (S-9626), were obtained from Sigma Chemicals (St. Louis, MO). Fibronectin was prepared as previously described (Orly and Sato, 1979; Orly et al., 1980). Radiochemicals All radiochemicals were purchased England Nuclear (Boston, MA). Culture media The basic nutrient

cell culture

from

medium

New

con-

sisted of a 1 : 1 (v/v) mixture of Dulbecco’s modified Eagle’s medium (DME) (Grand Island Biological, NY) and Ham’s nutrient mixture F-12 (Grand Island Biological, NY), prepared as previously described (Orly and Sato, 1979). Serum-free medium (4F medium) consisted of a DME: F-12 mixture, supplemented by insulin, transferrin. hydrocortisone and fibronectin, as previously described (Orly and Sato, 1979; Orly et al., 1980). Animals Immature female rats (Wistar-derived strain, 22-25 days old) were neither hypophysectomized nor treated with diethylstilbestrol. Animals were sacrificed by cervical dislocation. Granulosa cell culture Granulosa cells were collected as previously described (Orly et al., 1982b). Briefly, the ovaries were removed and dissected free of non-ovarian tissue. The intercellular gap junctions of the granulosa cells were disrupted (Campbell, 1979) by incubation of the intact ovaries in 4F medium, containing 10 mM EGTA and 0.5 M sucrose. After 45 min at 37°C the ovaries were transferred to 4F medium for a second 45 min incubation at room temperature. The granulosa cells were expressed into warm 4F medium by puncturing the follicles with a 22-gauge sterile needle. The cells were washed twice in the above medium and counted in a hemocytometer, after Trypan Blue staining. About 5 X lo4 viable cells were plated into each well (2 cm*) of a 24 multiwell plate (Nunc, Denmark), containing 0.5 ml of medium. Insulin, transferrin and hydrocortisone were added immediately after inoculation of the cells, whereas purified human fibronectin (5 pg/well) was added to the wells 30 min before inoculation of the cells. Cell count

Cell number was determined counter, as previously described 1979).

using (Orly

a Coulter and Sato,

OS preparation The ovarian substance was released into the culture medium (conditioned medium) while puncturing the ovaries to express the granulosa cells, as described above. A standard preparation of OS

143

contained activity produced by puncturing 12 ovaries in 8 ml of 4F medium. OS-containing medium was ultrafiltered through PM-10 Diaflo membrane (Amicon stirring cell) and filter sterilized before storage at 4°C. Steroid measurements The 20 a-hydroxypregn-4-en-3-one (20a-OH-P), which is the main progesterone metabolite secreted into the culture medium by the responding granulosa cells, was measured by radioimmunoassay (RIA) as previously described (Orly et al., 1980). The data present the mean steroid content in duplicate or triplicate wells for each treatment. Each RIA sample was assayed in duplicate determinations. Anti-20a-OH-P was a generous gift from Dr. F. Kohen of the Weizmann Institute of Science, Rehovot, Israel. Aromatase assay Aromatase activity was determined by measuring the stereospecific release of tritium to produce 3H 2O when [lp,2/3- 3H]testosterone is aromatized to 17&estradiol (Thompson and Siiteri, 1974; Reed and Ohno, 1976). This assay was later used by Gore-Langton and his colleagues (Gore-Langton et al., 1980, 1981) as an alternative method for assessment of aromatizing activity in reproductive organs, instead of measuring estrogen formation by RIA. Briefly, the cells were primed for 2 days with FSH or OS preparation. After thorough washing with warm medium, the cells were further incubated in 0.2 ml 4F medium containing 150000 dpm of [lp,2P-3H]testosterone (0.25 PM). At the indicated times the culture medium was transferred into plastic RIA tube (10 x 75 mm) and the cell monolayers were washed twice with aliquots of 0.2 ml and 0.3 ml RIA buffer (Gore-Langton et al., 1980). The culture medium and the washing buffer were combined and cooled for 15 min at 0°C. The radiolabeled testosterone and estrogen were adsorbed on charcoal by adding 0.2 ml of charcoal (Fisher 170) paste (250 mg/ml in RIA buffer) to the tube. After vigorous vortexing, the tubes were incubated for 30 min at 0°C. The content of 3H,0 in the charcoal supernatant was measured after centrifugation (2000 X g for 5 min at 4°C) and decantation of the supernatant into a scintillation tube containing 3.5 ml of scintillation

liquid. The formation of ‘H,O determined by this assay was linear up to 3 h of incubation with and the affinity constant for [ 3HItestosterone, testosterone was found to be 0.13 PM (data not shown). These findings were in agreement with previous reports by Gore-Langton et al. (1980) and by Reed and Ohno (1976) for testicular and placental aromatase activities, respectively. Cyclic AMP determinations Accumulation of CAMP in intact cells was measured, after some modifications, as previously described (Schulster et al., 1978). Prior to addition of agonists, the cell monolayers were incubated in 0.2 ml of 4F medium containing 2 PCi of [3H]adenine (2 PM). After 2 h of incubation at 37°C the monolayers were washed twice with warm 4F medium and agonists were added at zero time. About 25% of the added [ 3H]adenine was incorporated into the cells (- lO’/well), with most of it converted to [3H]ATP (Schulster et al., 1978; Humes et al., 1969). After 50 min with agonist the cells were lysed by addition of 0.3 ml ethanol solution (70%) and 0.2 ml ‘stop solution’. The content of the stop solution and the procedure for determination of [ 3H]cAMP, using sequential chromatography on Dowex (BioRad Laboratories, Richmond, CA) AG 5OW-X4 and aluminum oxide columns, were as described in a previous report (Schulster et al., 1978). The counts corresponding to purified [ 3H]cAMP were normalized when expressed as percentage of conversion of tritium cpm to [ ‘HIcAMP (% of conversion). This radioactive measurement of CAMP accumulation in intact granulosa cells was the method of our choice because it is more accurate, highly sensitive, inexpensive and much less tedious than the one using RIA for CAMP determinations. Statistical analysis All experimental data are presented as the mean + SE of duplicate or triplicate cultures. Comparable results were obtained from 3 separate experiments. Treatment differences were tested by Student’s t-test and analysis of variance. Analysis of morphological changes Cells were incubated for 45 min at 37°C in the

144

presence of FSH or OS. Induction of cell-shape changes (‘rounding-up’) was monitored by light microscopy, after fixation with glutaraldehyde and staining with Toluidine Blue-O, as previously described (Lawrence et al., 1979; Orly et al., 1982b). The rounded-up cells are presented as the percentage of fraction of the total number of scored cells in each microscopic field (80-120 cells/field). 3-5 fields were scored in each culture well. Duplicate wells were scored in each treatment. Quantitative determinations of OS activity This bioassay was based on OS ability to cause rounding-up of granulosa cells, as described above. A series of OS dilutions were tested for their ability to cause morphological changes in 10-12day-old granulosa cell monolayers. Rounding-up was monitored after 30 min of incubation with 0.2 ml of diluted OS at 37°C. The last dilution of solution OS which still caused a significant rounding-up of the cells was determined to contain 1 unit activity per ml. Partial purification of OS Step 1: Chromatography on Bio-Gel P-2. Following ultrafiltration (Diaflo PM-10 membrane, nominal MW cutoff lOOOO), 200 ml of OS-conditioned medium were lyophilized and applied to gel permeation chromatography on a column (12 X 96 cm) (90 ml bed volume) of Bio-Gel P-2 (100&200 mesh, Bio-Rad Laboratories). The column was equilibrated with 1 mM Na+ phosphate buffer, pH 7.4, at 4°C. Chromatography was performed at 4°C at a constant flow rate of 5 ml/h. Fractions (1.4 ml) were collected into siliconized glass tubes. The fractions were assayed for OS activity by diluting the appropriate aliquot with concentrated phosphate saline buffer to maintain isotonic ionic strength. Ninhydrin-reactive material in each fraction was monitored as previously described (Orly et al., 1982b) but for phenylalanine which served to construct a standard curve (A,,,, = 20 OD/ p mole). Active fractions, after BioStep 2: Extraction. Gel P-2 chromatography, were pooled and lyophilized. The lyophilized powder (200 mg) was extracted with 10 ml of the organic phase of pyridine : n-butanol : acetic acid : H,O mixture

(30 : 50 : 0.1 : 110). The upper phase of the extracted lyophilizate was vacuum evaporated and redissolved in growth medium for further tests. Results Granulosa ceil responsiveness to OS during culture In our previous report (Orly et al., 1982b) we have demonstrated that graded doses of OS induced dihydroprogesterone (20cr-OH-P, a main progesterone metabolite) production in ll-day-old cultured rat granulosa cells. However, unlike such long-term cultured cells, in freshly inoculated cells the cell responsiveness to OS depended on the inoculum size at the time of seeding. As shown in Fig. 1, 20a-OH-P production in response to FSH markedly improved with increasing plating densities. This result was expected since gap-junction formation is facilitated at high plating densities. and it has recently been shown that intercellular junctions play an important role in gonadotropin-

0.6

22

1.8

cells x10’/ well Fig. 1. Inoculum-dependent response of granulosa cells to OS and FSH. Granulosa cells at various inoculi were seeded into triplicate wells (24 multiwell plate) as described in Materials 0) or OS (O0) was and Methods. FSH (0 ~ added to the cells 24 h after seeding, and 48 h later the culture media were removed and the cell number per well determined. 20~OH-P content in the culture medium was determined by RIA. as described in Materials and Methods. Note that the steroid content is normalized per 10s cells and is illustrated as a function of cell number present at the end of the experiment. Data are the mean*SE of duplicate wells in each treatment. Where the error bare is not indicated, the standard error is within the data point.

145

induced cytodifferentiation of granulosa cells (Amsterdam et al., 1981). In contrast, the granulosa cell responsiveness to OS surprisingly decreased as the inoculum size increased. The reason for this phenomenon is still obscure, although degradation of OS by the densely inoculated cultures should be considered. We have previously shown that during days 4-8 in culture the granulosa cells transiently lost their steroidogenic responsiveness to both FSH and OS (Orly et al., 1982b). Therefore, since this study consistently compares FSH with OS stimulatory effects, we chose to conduct most of the following experiments on 8%14-day-old granulosa monolayers which were maximally responsive to both agonists. In a few experiments we also compared the cellular responses to OS and FSH in freshly inoculated cells. OS-induced progestin production Fig. 2 demonstrates a time-dependent accumulation of 20a-OH-P in response to OS. After a 24 h lag period the granulosa cells responded to OS at a linear rate of 20a-OH-P production for an additional 48 h. FSH induced a very similar time-de-

pendent pattern of steroid biosynthesis. This pattern is in agreement with previous studies, indicating a typical latency period of 18-24 h until progestins are synthesized at a linear rate by the cultured rat granulosa cells (Nimrod, 1977; Knecht et al., 1982; Wang et al., 1982). An additional criterion by which OS mimics FSH induction of progestin production is presented in Fig. 3, showing that OS action is reversible. Upon washing the cells following 2 days of incubation with OS, the cells gradually ceased to produce 20a-OH-P. It can therefore be concluded that the continuous presence of OS is required to maintain the granulosa cells’ expression of their differentiated function. Similar studies, which demonstrated dedifferentiation of granulosa cells occurring upon FSH withdrawal from the culture medium, have previously been reported (Casper and Erickson, 1981). It has been well established that protein synthesis is required for the induction of steroid formation in both the gonads and the adrenal tissue (for review see Wicks, 1974). Most of the evidence implicating the involvement of de novo translation

loo-

80

-

Y z “0 7

60-

!? _ a & 40d N

10

30

50

70

HOURS Fig. 2. Time-dependent accumulation of ~OCY-OH-P in response lo OS or FSH. After 11 days in culture. cells (1.64~ 105/well) were incubated with 0.5 ml of 4F medium containing OS 0) or 100 ng/ml FSH (0 -0). At each time(Opoint, 50 ~1 aliquots were removed from 4 wells of each treatment and stored until 20c~-OH-P content was determined by RIA. Dashed line (- - -) represents control wells without added agonist. When the error bar is not shown, the standard error (duplicate wells) is within the data point.

20-

12

14

16 days

12 In culture

14

16

Fig. 3. Reversible action of OS on progestin biosynthesis. After 10 days in culture, duplicate wells (1.5~10~ cells/well) were incubated with OS (A) or 100 ng/ml FSH (B). Following 48 h of incubation, all the cells were thoroughly washed and further incubated in 4F medium for an additional 4 days, without the agonists. At each time-point the culture media were removed and stored until 20a-OH-P content was determined by RIA. At the end of the experiment the cells were counted. The data represent the mean of 20ol-OH-P production normalized per lo5 cells.

146 TABLE

1

CYCLOHEXIMIDE GENESIS

INHIBITS

OS-INDUCED

Treatment

Days in 20~OH-P culture (n&IO5 cells)

No additions FSH OS FSH f cycloheximide OS + cycloheximide FSH (inhibitor removed) OS (inhibitor removed)

13-15 13-15 13-15 13-15 13-15 15-17 15-17

0.93fO.lX 59.5 i-9.9 44 h8.3 1.3 10.3 1.7 i:O.26* 1X.8 i5.8 34.2 +- 5.8

STEROIDO-

% Inhibition

0 0 98 96 -

After 13 days in culture, cells (1.26 X 105/well) were treated as indicated with OS or 100 ng/ml FSH, in the presence or absence of 2 pM cycloheximide. After 4X h of incubation the cells were washed twice and further incubated for an additional 2 days without the inhibitor. At each time-point the culture medium from duplicate wells was removed and stored until steroid content was determined by RIA. Cycloheximide was toxic above 3 FM and showed a dose-dependent inhibition at the range of 0.1-0.2 FM (not shown). Data present the mean+ SE of triplicate determinations for each treatment. * P < 0.05 when compared with untreated cells.

TABLE

of messenger RNA in stimulated cells originated from observations that protein synthesis inhibitors blocked the stimulatory effects of corticotropin (Schulster et al., 1976) and gonadotropins (Marsh, 1975) on steroidogenesis in the respective organs. We therefore tested the effect of cycloheximide on progestin induced by OS. Table 1 shows that. like FHS action, OS-induced 20a-OH-P biosynthesis was completely blocked in the presence of 2 FM cycfoheximide. The cells resumed synthesizing progestins upon removal of the antibiotic from the culture medium, thus indicating that the inhibitory action of cycloheximide was not merely the result of non-specific toxic effects. In contrast to the genera1 agreement concerning the role of protein synthesis in the stimulation of steroidogenesis, the need for RNA synthesis in the action of hormone-induced steroid production is less certain. Again, most of the studies employed an antibiotic, actinomycin D, in attempts to demonstrate the inhibitory effect of the drug on steroidogenesis. The actinomycin D inhibitory ef-

2

a-AMANITIN

INHIBITS

OS-INDUCED

PROGESTIN

PRODUCTION 20(x-OH-P (ng/105 cells)

% inhibition

Treatment

Days in culture

No addition OS OS+ 5 pg/ml a-amanitin OS+ 15 pg/ml n-amanitin OS+ 30 pg/ml a-amanitin OS + 5 pg/ml cy-amanitin (inhibitor removed) OS+ 15 pg/ml a-amanitin (inhibitor removed) OS+ 30 pg/mt a-amanitin (inhibitor removed)

10-12 IO-12 10-12 10-12 10-12 12-14

5 + 73.3& 37 i 23 & 13 & 98 +

12-14

99

+12

0

12-14

90

+10

0

FSH FSH + 5 pg/ml ff-amanitin FSH + 5 pg/ml a-amanitin (inhibitor removed)

IO-12 10-12 12-14

1.2 7.9 0.3 0.7 2.3 ** 2.3

122 *27 36.52 1 68 k 8

_ 0

50 69 82 0

0 70

56

After 10 days in culture, cells (1.1 x105/well) expressed to OS or 100 ng/ml FSH in the absence or presence of graded of doses of a-amanitin. After 48 h of incubation the media were removed from duplicate wells and stored for determination of 20~OH-P content. The cefls were thoroughly washed to remove the inhibitor, and further incubated in the presence of OS or FSH for two additional days (days 12 .14). Data represent the mean f SE of duplicate determinations. * P < 0.02 when compared to untreated cells.

147

fects on progestin production in luteal tissues or Graafian follicles were found to be inconsistent (Marsh, 1975). However, Magoffin and Erickson (1982) recently reported the need for RNA synthesis for LH-induced androgen synthesis in interstitial cells. The need for newly transcribed mRNA was also demonstrated for the FSH-induced functions in undifferentiated cultured granulosa cells (Wang et al., 1982). Since in our experiments OS induces progestin production in a similar undifferentiated granulosa cell system, we questioned whether inhibitors of RNA synthesis could abolish the granulosa cell responsiveness to OS. For that purpose we chose a-amanitin, which selectively inhibits transcription of messenger RNA, without interfering with ribosomal or 5s RNA synthesis. Table 2 shows that cY-amanitin (5-30 pg/ml) inhibited up to 82% of 20(~-OH-P synthesis induced by OS. Comparable inhibitory effects were observed also for control monolayers of granulosa cells treated with FSH. The progestin responses returned to near normal after the inhibitor was removed from the culture medium. At a concentration of 50 pg/ml, cr-amanitin caused total death of the culture. Thus, the results are in agreement with previous reports suggesting the need for newly synthesized mRNA for hormonal induction of progestin formation. The obligatory role of transcription and translation for the OS-induced steroidogenesis rules out the possibility that OS may act as a putative steroid precursor which can be converted to progestins upon addition to the cultured cells. OS induces aromatase activity Besides progestin production, two other biochemical markers are associated with granulosa cell maturation, namely, acquisition of LH receptors (Nimrod et al., 1977; Knecht et al., 1981; Sanders and Midgley, 1983) and appearance of androgen-aromatizing enzyme (Dorrington et al., 1975; Dorrington and Armstrong, 1979). Both qualities are FSH-inducible. Fig. 4 shows a timedependent accumulation of 3H,0 which is generated as a result of aromatized [3H]testosterone fed into the culture medium (see Materials and Methods). This alternative method of measuring aromatase activity instead of RIA determination of estrogen has been widely employed before by

80

‘;b -

60

;

a 0

0

TN ‘I’

.o

1

2

3

4

HOURS Fig. 4. Induction of aromatase enzyme by OS. After 11 days in culture, cells (1.15X105/well) were primed with either 100 q/ml FSH (0). OS preparation (A) or the two agonists added together (0). Control cells (X---X) were not treated with either agonist. After priming for 48 h, the cells were thoroughly washed and further incubated with 60000 cpm of [l/3,2P-3H]testosterone (0.25 pM). At the indicated times the culture medium was removed and the content of 3H,0, generated as a result of aromatase action, was determined as described in Materials and Methods. Data are the mean*SE of triplicate wells for each time-interval. Background radioactivity (zero time) was not subtracted from the various time-points.

Gore-Langton et al. (1980). Clearly, a 2-day incubation with OS induced aromatase activity, although somewhat lower than that evoked by FSH. Addition of FSH together with OS did not yield a higher activity than addition of FSH alone. Androgen effects on OS- and FSH-induced aromatase Daniel and Armstrong (1980) recently reported that androgens enhance FSH-induced aromatase activity in cultured granulosa cells. We therefore

148

questioned whether androgen could also augment the OS stimulatory effect on aromatase activity. Fig. 5A shows that androstenedione indeed

80-

60'

increased OS stimulation of aromatase activity in granulosa cells primed after 3 days in culture. Androstenedione doubled the OS-inducible aromatase activity in a synergistic fashion, since priming with androgen alone did not yield any aromatizing activity. An almost identical synergistic effect was observed for the androgen effect on FSH action. We also compared the effect of androgen on the induction of aromatase by OS in 13-day-old cultured cells (Fig. 5B). Surprisingly,

250

1

A.

120

80

9

40

2 ‘0 \

c”

**

* i i

B.

3

U

F

A

F+A

Fig. 5. Effect of androstenedione on OS-induced aromatase. (A) After 3 days in culture, cells (105,/well) were incubated for 2 days in the absence (B = basal) or presence of either 0.2 PM androstenedione (A); 100 ng/ml FSH (F); FSH + androstenedione (F+A); OS preparation (OS); or the combined incubation with OS and androstenedione (OS + A). After thorough washing, the cells were further incubated with 50000 cpm of [lp,2/3-‘HJtestosterone (0.25 pM). After 3 h of incubation the culture medium was removed and the content of ‘Hz0 was determined as described in Materials and Methods. ** P < 0.02 when compared to OS treatment. (B) After 13 days in culture the cells (1.3 x105/well) were primed for 2 days with agonists and assayed for aromatase, as described in (A). Data are the mean f SE of triplicate wells. Background radioactivity (zero time) was not subtracted from the various time-points. Hatched histograms emphasize the combined treatment with androgen and FSH or OS. * P c 0.05 compared to FSH-treated cells.

200 -

100 -

B

A

F

F+A

OS

OS*A

Fig. 6. Effect of androstenedione on OS-induced progestin production. (A) After 24 h in culture, cells (7.8 X104/well) in duplicate wells were treated with either 0.2 PM androstenedione (A); 100 q/ml FSH (F); FSH + androstenedione (F + A): OS preparation (OS); or the combined addition of OS and androstenedione (OS+A). After 48 h of incubation the 20~ OH-P content in the culture medium was determined by RIA. (B) The experiment described in (A) was repeated using a second set of cells after 11 days of growth in culture. The data represent the mean k SE of duplicate wells. Hatched histograms emphasize the combined addition of androgen with FSH or OS. * P < 0.05 when compared to FSH treatment (F); ** P < 0.02 when compared to OS treatment.

149

androstenedione did not have any marked effect on OS- or FSH-induced aromatase activity in these cells. Androgen effects on OS- and FSH-induced progestin production Androgens have also been implicated in the regulation of progestin production. Both aromatizable and non-aromatizable androgenic steroids were shown to enhance the stimulation by FSH of progestin production (Schomberg et al., 1976; Nimrod and Lindner, 1976; Armstrong and Dorrington, 1976). To test the effect of androgen on OS-induced 20a-OH-P production, we incubated 2-day-old (Fig. 6A) or ll-day-old granulosa cells (Fig. 6B) with either FSH or OS in the presence of androstenedione. Clearly, androstenedione synergistically enhanced OS- and FSH-induced 20&-OHP production by 8-lo-fold compared to the agonist’s action in the absence of the androgen (Fig. 6A). In contrast, the ability of androstenedione to enhance OS and FSH action was much reduced, if the cells were treated after 11 days of growth in culture; no more than 40-50% increments in 20a-OH-P production could be observed for the combined actions of androstenedione and FSH or OS (Fig. 6B). A weakened effectiveness of androgen in modulating the steroidogenic pathways in long-term cultured granulosa cells is therefore consistent in both progestin production and estrogen synthesis.

OS enhancement of FSH-induced progestin production Searching for a possible role for OS in modulating FSH action on the cultured granulosa cells, we obviously tested its effects on FSH-induced progestin secretion. Fig. 7 demonstrates that graded doses of OS continuously augmented the FSH-induced 20a-OH-P production. The maximal available concentration of OS synergistically increased FSH activity to a similar extent achieved by the combined action of androgen and FSH. Moreover, OS augmented FSH-inducible progestin production to even higher levels when added to l-day-old cells which barely responded to OS alone (Fig. 8). Up to a 4-fold increase in steroidogenesis was obtained in the presence of both 100 ng/ml FSH

320 r t

OS+FSH

I O- o_i” 20

**

/y

,

1

60

I

I

100

A F A+F

condhoned medium % "/, Fig. 7. OS augments FSH-induced progestin production. After 11 days in culture, cells (1.3x105/well) were incubated with the indicated dilutions of OS-conditioned medium, in the absence (0) or presence of 100 ng/ml FSH (0). After 48 hours of incubation the 20~OH-P content in the culture medium was determined by RIA. The dashed line indicates the calculated sum of FSH and OS individual effects. Histograms present similar treatments with 100 ng/ml FSH (F) or FSH together with 0.2 pM androstenedione (A + F). The hatched histogram emphasizes the synergistic effect of androgen on FSH action under these culture conditions. The data represent the mean k SE of duplicate wells for each treatment. ** P < 0.02 when compared to FSH treatment.

and OS preparation over their additive when tested separately.

responses

OS induction of LH responsiveness The third biochemical marker we chose to compare OS and FSH actions on granulosa cells involves the induction of LH receptors. Rather than estimating the number of LH receptors by direct [‘*‘I]hCG binding, we preferred to measure the functionality of newly appeared receptors. We therefore primed the cells for 2 days with either OS preparation or FSH, during which time the induced cells were expected to express new LH receptors. Thereafter, the ability of these receptors was tested for either activating LH-responsive adenylate cyclase or triggering steroidogenesis. Fig. 9 shows that priming freshly inoculated cells for 2 days with FSH indeed led to expression of new

150

60

n_-n-O-+-O

0

20

40

cwdltloned

60 mdum

&I

IM) % X

0

20

I

I

40

60

FSH

assay : 80

1c0

ng/mt

Fig. 8. OS augments FSH action in freshly inoculated cells. After 24 h in culture, two sets of cells (1.35X105/well) were challenged with OS and FSH. (A) Cells in duplicate wells were incubated with the indicated dilutions of OS-conditioned medium, in the absence (0) or presence of 100 ng/ml FSH (0). After 48 h of incubation, 20a-OH-P content in the culture medium was determined by RIA. (B) Alternatively, cells in duplicate wells were similarly incubated to determine 20a-OH-P production in response to graded doses of FSH, in the absence (0) or presence of maximal concentrations of OS (0). The dashed line indicates the calculated sum of the individual effects of FSH and OS. Data are the meankSE. Where the error bar is not indicated, the standard error is within the data point.

LH responsiveness. Those cells produced a 140-fold increase of 20a-OH-P over unprimed cells. In contrast, OS-primed cells failed to respond to LH, indicating a lack of newly expressed receptors. However, since OS was found to be a rather poor agonist in freshly inoculated cells but highly potent in long-term cultured cells, we tested its ability to induce LH receptors also in 11-day-old granulosa cells. Fig. 10 indicates that in such cells LH responsiveness is still inducible by FSH treatment, whereas the ability of OS to do so is much less pronounced. Priming with OS resulted in no more than twice the activity of LH-coupled adenylate cyclase and a 50% (P < 0.05) increment of LH-responsive steroidogenesis (Fig. 10A). Apparent lack of CAMP involvement in the OS mechanism of action It is now accepted that hormonally induced steroid formation is mediated by CAMP as the intracellular second messenger (Kolena and Channing, 1972; Goff and Armstrong, 1977; Sala et al.,

priming :

B L c no add.

BL 4 FSH

B L 4 OS

Fig. 9. Effect of OS on induction of LH responsiveness in freshly inoculated cells. Following 24 h in culture. cells (7x 104/well) were primed for 48 h with either OS preparation or 100 ng/ml FSH. Control cultures (no additions) were not primed with either agonist. After thorough washing, the cells were further incubated for 48 h (assay) in the absence (B = basal) or presence of 100 ng/ml FSH (F) or LH (L). The 20a-OH-P content in the culture medium was determined by RIA. The hatched histogram emphasizes the LH treatments during the assay period. Data represent the meankSE of duplicate wells for each treatment.

1977). We confirmed this notion in our long-term cultured granulosa cells, since the ED,, values for the FSH-induced CAMP formation and steroid production were practically identical ( - 80 ng/ml) as shown in Fig. 11. However, in contrast to FSH action, we have previously presented preliminary evidence to show that conditioned medium containing OS activity did not cause measurable increases in intracellular CAMP levels (Orly et al., 1982b). To evaluate further the ability of OS to activate adenylate cyclase, highly concentrated preparations of OS material were needed. For that purpose we partially purified OS activity in two steps, including gel permeation chromatography followed by extraction into the organic phase of a pyridine : butanol : acetic acid : water mixture (see Materials and Methods). This procedure resulted in a 20-fold concentration of OS activity, as assessed by a quantitative bioassay described in Materials and Methods. Using this partially purified OS preparation we compared the dose-dependent responses of

i ISI x-

ln z

300 I

200

“Er \ c” a

50

g i R

25

I

I

J I

i

***

I

J/

assay : prlmlng :

ELF 4 no add.

B L FiH

I

4 8 L

O’s

Fig. 10. Effect of OS on LH-induced responsiveness in long-term cultured cells. After 11 days in culture, cells (1.2XlO’/well) were primed with either OS preparation or 100 ng/ml FSH. Control cultures (no additions) were incubated without agonists. After 48 h of incubation the agonists were thoroughly washed and the cells in two sets of wells were assayed for LH responsiveness. (A) Cells in duplicate wells were incubated for 48 h without (B = basal) or with 100 ng/ml LH (L) or FSH (F). The 20a-OH-P content in the culture medium was determined by RIA. * P -e 0.05 when compared with basal levels (OS + B). (B) Cells in duplicate wells were incubated for 2 h with [3H]adenine (2 pCi/ml, 2pM) and the gonadotropin-induced accumulation of CAMP was determined as described in Materials and Methods. All incubations were performed in the presence of 0.5 mM 3-isobutyl-1-methylxanthine to minimize degradation of intracellular CAMP by phosphodiesterase. The histograms represent the mean* SE of duplicate wells. Hatched histograms emphasize LH responses. *** P c 0.01 when compared with basal levels (OS + B).

granulosa cells in both steroidogenesis and CAMP formation. Fig. 12 clearly shows that OS concentrations which evoked maximal steroidogenic responses did not stimulate CAMP accumulation above basal levels. However, at higher concentrations OS caused significant increases in the in-

11111111 10

100 FSH

I I IIIIIII

,

1000

ng/mL

Fig. 11. Dose-dependent responsiveness of granulosa cells to FSH. After 13 days in culture, cells ( - 1.4X10s/well) were treated in two groups of wells with graded doses of FSH. (A) CAMP accumulation: prior to FSH treatment the cells were incubated with [3H]adenine (2 ~Ci/ml, 2 pM). [3H]cAMP accumulation was determined after 45 min of incubation with graded doses of FSH (A). CAMP accumulation is presented as percentage of conversion of tritium cpm to [ 3H]cAMP. The data points represent the means? SE of triplicate determinations. Inset presents double reciprocal plot, according to the method of Lineweaver and Burk. Abscissa = l/u (% conversion) _ ’ ; ordinate = l/s (ng/ml)-‘. (B) Steroidogenesis: following 48 h of incubation with graded doses of FSH (0) the 20~OH-P content in the culture medium was determined by RIA. Data are the mean k SE of duplicate determinations. Inset represents double reciprocal plot according to the method of Lineweaver and Burk. Abscissa = I/U (ng/105 cells))‘; ordinate = l/s (ng/ml)) ‘.

tracellular cyclic nucleotide content which was further augmented by the phosphodiesterase inhibitor MIX. The discrepancy between the ED,, values for OS action on CAMP accumulation (17 units/ ml) and OS-induced steroidogenesis (100 units/ml) may therefore suggest that, unlike FSH-mediated responses, CAMP is apparently not involved in the OS mechanism of action. In addition, we also examined the possibility that OS might have induced accumulation of intracellular cyclic GMP levels in granulosa cells. It

t

al., 1978). We therefore loaded the cells with [~Hlguanine and conducted a similar assay for cyclic GMP accumulation as described for the cyclic adenine nucleotide. No increase in the cGMP content could be detected in the granulosa cells induced either with OS or FSH (not shown).

A.

Discussion

8

[5

10 OS

100

F

u/ml

Fig. 12. Dose-dependent responsiveness of granulosa cells to partially purified OS. After 14 days in culture, cells ( - 1.2~ 105/well) were treated in two groups of wells with graded doses of partially purified OS preparation, The quantitative determination of OS concentration (units/ml) was as described in Materials and Methods. (A) CAMP accumulation: prior to OS treatment the cells were incubated with [“Hladenine (2 gCi/mI, 2 PM). 13HjcAMP accumulation was determined after 45 min of incubation with graded doses of OS in the absence (A) or presence of 0.5 mM MIX (A). Control wells were incubated with 100 ng/ml FSH (F) in the absence or presence of MIX (F+I). cAMP accumulation is presented as percentage of conversion of tritium cpm to [ 3H]cAMP (see Materials and Methods). The data points represent the mean + SE of triplicate determinations. Inset presents double reciprocal plot according to Lineweaver and Burk. Abscissa = l/o (% conversion)-‘; ordinate = l/s (OS units/ml)) ‘. * P < 0.05 when compared with FSH treatment. (B) Steroidogenesis: following 48 h of incubation with graded doses of OS (o), the 20a-OH-P content in the culture medium was determined by RIA. Control cells were treated with 100 ng/ml FSH Data are the mean rf: SE of duplicate determinations. Inset represents double reciprocal plot according to the method of Lineweaver and Burk. Abscissa = l/o fng/105 cells)--‘; ordinate = I/s (OS units/ml)-“.

has been suggested that cyclic GMP, rather than cyclic AMP, is the physiological mediator of adrenocorticotropic hormone (ACTH)-induced steroidogenesis in the adrenal gland (Perchellet et

For the purposes of this study we used a defined medium to maintain the rat granulosa cells in long-term cultures. This medium (4F medium) consisted of a 1 : 1 (v/v) mixture of DME and Ham’s F-12 media, supplemented with insulin, transferrin, hydrocortisone and fibronectin (Orly and Sato, 1979; Orly et al., 1980). The various supplements were essential for the maintenance of the cells in culture for periods of weeks, as well as for expressing their cellular responses to gonadotropin, which were markedly suppressed by the addition of serum to the culture medium (Orly et al., 1980). This report characterizes the biological activities of a low molecular weight substance which is released from rat ovaries during processing to express their granulosa cells into culture. Such ovaries, freed from granulosa cells, continuously conditioned the culture medium with OS activity while bathing in 4F medium for more than 2 weeks. OS activity could not be detected in the conditioned medium of the cultured granulosa cells themselves, not even after 48 h of exposure to FSH. We may thus conciude that probably ovarian cells, other than granulosa, produce the OS activi ty. As large quantities of purified OS are not yet available, we have characterized its biological activity by using crude conditioned medium which had been freed from large proteins by ultrafiltration. To elucidate the effects of OS on the granulosa cells, we repetitively compared OS action with various well-known FSH-induced phenomena such as synthesis of progestins, development of estrogen-producing aromatase enzyme and acquisition of new LH receptors. These three FSH-induced biochemical markers have been demonstrated both in vivo (Channing, 1970; Zeleznik et al., 1974; Armstrong and Papkoff, 1976) and in vitro (for review see Erickson, 1983).

153

Regarding OS-induced synthesis of progestins, OS practically mimicked the action of FSH on long-term cultured granulosa cells: like FSH, the cells responded to OS in progestin production after a typical 18-24 h lag period; the cellular responsiveness to OS was reversible and ceased upon removal of OS from the culture medium; progestin production in response to both OS and FSH was dependent upon newly transcribed mRNA and protein synthesis, since cu-amanitin and cycloheximide inhibited steroidogenesis in the cultured cells. We may therefore conclude that OS effect on progestin production in granulosa cells meets several criteria known for hormonal stimulation of steroidogenesis in the gonads. OS also mimicked the stimulatory effects of FSH on estrogen production (Fig. 4) which indicates that aromatase enzyme as well as the mitochondrial enzyme, cytochrome P-450-cholesterol side-chain cleavage, is inducible under OS stimulus. Moreover, OS inductions of both enzymatic activities were synergistically augmented in the presence of androgen in the culture medium (Figs. 6 and 7) Comparable synergistic effects of androgens on FSH inductions of progestin and estrogen synthesis have been well documented, using similar granulosa cell cultures (Schomberg et al., 1976; Nimrod and Lindner, 1976; Daniel and Armstrong, 1980). In contrast to the above steroidogenic activities, OS failed to induce biologically significant responsiveness to LH in cultured granulosa cells (Figs. 9 and 10). Hence, this inability of OS to cause new expression of LH responsiveness remains the one and only FSH-inducible function which could not be mimicked by OS. OS may thus provide a useful tool to distinguish between two putative mechanisms which cannot be resolved under FSH stimulus: one leading to the induction of steroidogenesis and the other expressing LH receptors. When OS responsiveness was studied using freshly inoculated cells, a marked difference was revealed between OS and FSH induction of progestin synthesis. As the inoculum size decreased, the cell responsiveness to OS surprisingly increased. The fact that FSH responsiveness contrastingly increased with higher cell densities may provide a clue concerning the physiological target

cell of OS action in the ovary. If the analogy between an in vitro culture of sparse granulosa cells is allowed to be made with a single layer of granulosa cells comprising the unilamellar primordial follicle in vivo, it can be hypothesized that OS is meant to act on the primitive follicular cells which may not respond to FSH. Furthermore, although OS in culture seemed poorly active in high-density inoculi (Fig. l), it still augmented synergistically FSH-induced steroidogenesis (Figs. 7 and 8). Analogously, if the high-density cells in culture presumably represent a stratified granulosa epithelium in the growing primary follicle, OS is therefore suggested to substitute or augment FSH action as a trophic hormone during the early stages of follicular development when the hypophyseal gonadotropic hormone levels in the circulation are very low and ineffective. For that purpose, ovaries from neonatal rat are plausible experimental systems which we are currently investigating in attempts to elucidate the physiological relevance of OS existence in the ovary. Numerous studies have recently accumulated using 8-Br-CAMP and (Bu),-CAMP to support a unifying concept concerning the obligatory role of CAMP in FSH induction of progestins (Sala et al., 1977; Wang et al., 1982) and LH receptors (Nimrod, 1981; Knecht et al., 1981; Sanders and Midgley, 1983). Our data are consistent with this view, since the ED,, values for FSH-induced steroidogenesis and the gonadotropin-dependent CAMP formation are almost identical (Fig. 11). In contrast, OS seems to activate the granulosa cells in a fashion more like ACTH acts in the adrenal. Buckley and Ramachandran (1981) as well as previous reports (Mackie et al., 1972; Nakamura et al., 1972; .Lee et al., 1980) have shown that physiological concentrations of ACTH which stimulated maximal steroidogenesis in suspended adrenal cells elicited only marginal increases in CAMP levels. Similarly, using a partially purified concentrated preparation of OS, we revealed that the ED,, value for OS stimulation of steroidogenesis was 5 times lower than the half-maximal concentration needed for CAMP formation. Furthermore, at the maximally effective concentration, OS evoked only a fifth of CAMP accumulation when compared to FSH action. Therefore, we may conclude that although OS may be considered as a putative

154

hormone, it apparently does not stimulate progestin production via a CAMP-mediated mechanism. In the light of these findings we also examined the possibility that OS may induce steroidogenesis via a mechanism involving Ca2+ mobilization or altering the turnover rate of phosphatidylinositides in the cultured granulosa cell membranes. Studies using Ca2 + -free medium, Ca2+ ionophore or CaCl Z did not reveal any alterations of OS-induced effects (not shown). Hence, calcium mobilization did not seem to correlate with OS action. We also verified that OS did not act via induction of prostaglandin synthesis, since indomethacine (l-5 PM) was unable to block OS activity (not shown). Consequently, we are currently seeking for mechanisms that may provide an explanation for OS action independently of CAMP formation. Activation of a protein kinase resulting from the interaction of cells with OS is one such possibility. In summary, at this preliminary stage, we have characterized the biological activities of a low molecular weight substance which is released in vitro from ovaries of immature rats. Although in the absence of purified OS we cannot exclude the possibility that OS represents a multitude of effects by more than one factor, partially purified preparations of OS (presented in Fig. 12) showed an identical activity as compared to the crude conditioned medium. In the light of the intriguing effects of OS on the cultured granulosa cells, our future objective should thus be focused on the purification of large amounts of OS, which will allow us to elucidate the chemical nature of OS and reveal its biological role in the ovarian physiology. Acknowledgements We wish to thank Dr. F. Kohen from the Department of Hormone Research, The Weizmann Institute of Science, Rehovot, Israel, for providing us with the anti-20cr-OH-P serum. We are also grateful to Mrs. E. Dicker for her excellent editorial and secretarial assistance. J.O. is an incumbent for the Charles H. Revson Career Development Chair. This work was supported by the United States-Israel Binational Science Foundation, Grant No. 2656/81, and by the

Bat-Sheva de Rothschild Fund for the Encouragement of Science and Technology.

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