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Steroid productions by Co-cultures of granulosa cells with inner and outer theca cells in preovulatory follicles of gonadotropin stimulated calves
J. Steroid Biochem. Molec. Biol. Vol. 62, No. 2/3, pp. 213-221, 1997 © 1997 Elsevier Science Ltd. All rights reserved Printed in Great Britain PII: S0960-0760(97)00030-7 0960-0760/97 $17.00 + 0.00
Pergamon
S t e r o i d P r o d u c t i o n s by C o - c u l t u r e s of G r a n u l o s a Cells with Inner and O u t e r T h e c a Cells in P r e o v u l a t o r y Follicles of G o n a d o t r o p i n S t i m u l a t e d Calves M. J. Bosc* and Agn s Nicolle Laboratoire de Physiologie de la Reproduction des Mammifires Domestiques, Institut National de la Recherche Agronomique, Centre de Tours-Nouzilly, 37380 Nouzilly, France
G r a n u l o s a , i n t e r n a a n d e x t e r n a t h e c a cells w e r e isolated f r o m large follicles o f e q u i n e - c h o r i o n i c g o n a d o t r o p i n ( e C G ) - p r i m e d calves a n d c o - c u l t u r e d d u r i n g 3 days in the a b s e n c e o r in t he p r e s e n c e o f d e h y d r o e p i a n d r o s t e r o n e ( D H E A ) . C o - c u l t u r e s w ere p e r f o r m e d by a d d i n g d e f i n e d n u m b e r s o f t h e c a a n d / o r granulos.a cells w hi c h r e p r e s e n t e d 0, 10, 20, 50 or 100% o f total cells p e r well. S e c r e t i o n o f oestradiol-17fl (E2), a n d r o s t e n e d i o n e (A4) a n d p r o g e s t e r o n e (P4) d e p e n d e d on t he type o f t h e c a cells ( P < 0.001), on t he p e r c e n t a g e o f s eeded g r a n u l o s a cells ( P < 0.001) a n d on the d a y o f c u l t u r e ( P < 0.001). D H E A i n c r e a s e d ( P < 0.001) Ee a n d A4, b u t n o t P4 (P > 0.05) p r o d u c t i o n s . I n t e r a c t i o n s existed b e t w e e n t he s e f a c t o r s ( P < 0.01). O n day 1, A4 p r o d u c t i o n was nil in g r a n u l o s a cells alone. E2 p r o d u c t i o n was negligible in t h e c a cells alone b u t it i n c r e a s e d w h e n g r a n u l o s a cells w ere a d d e d . Ez a n d A4 v a r i e d in an opposite m a n n e r a c c o r d i n g to t he p e r c e n t a g e o f g r a u u l o s a ceils a n d with the type o f t h e c a cells. O n d a y 3, w i t h o u t D H E A , Ez a n d A4 w ere low. O n day 3 with D H E A , E2 p r o d u c t i o n was m a i n t a i n e d in g r a n u l o s a cells alone b u t not with any c o m b i n a t i o n o f t h e c a cells. In these c o n d i t i o n s , A4 p r o d u c t i o n was m a i n t a i n e d in t he p r e s e n c e o f t h e c a ceils b u t n o t in g r a n u l o s a ceils alone. G r a n u l o s a cells alone s e c r e t e d m o r e P4 t h a n t h e c a cells. P4 i n c r e a s e d as a f u n c t i o n o f the p e r c e n t a g e o f g r a n u l o s a in c o - c u l t u r e s with e x t e r n a b u t n o t i n t e r n a t h e c a cells with w h ic h it r e m a i n e d low. In conclusion, t h e c a cells in c u l t u r e have two effects in r e l a t i o n to t he g r a n u l o s a cells, w h ich differ a c c o r d i n g to the s t e r oi d c o n c e r n e d a n d to the cell c o m b i n a t i o n . B o t h types o f t h e c a ceils have an i n h i b i t o r y effect on E2 s e c r e t i o n w h e r e a s only i n t e r n a t h e c a ceils are able to al te r P4 p r o d u c t i o n . © 1997 E l s evi er Science Ltd.
J. Steroid Biochem. Molec. Biol., Vol. 62, No. 2/3, pp. 213-221, 1997
INTRODUCTION The ovarian follicle provides a structural and functional support to the oocyte and its surrounding cells. In this structure, granulosa and theca cells face each other through a basal membrane allowing for multiple cell interactions [1]. In the bovine as in other mammals, several interact:ions associate granulosa and theca cells. For example, androgens produced by theca cells serve as a substrate for oestrogen synthesis by granulosa cells [2, 3]. This cooperation in steroid production is in fact :more complex. Oestradiol-17fl *Correspondence to M. J. Bosc. Fax: +33 2 47 42 77 43; E-mail: [email protected]. Received 18 Nov. 1996; accepted 21 Feb. 1997. 213
(E2) can alter progesterone (P4) secretion by the theca cells [4-6], but granulosa cells can stimulate androstenedione (A4) synthesis [6, 7], probably by providing pregnenolone as a substrate [7]. Other interactions between the two types of cells have also been described. They concern the factors which are implicated in the stimulation or in the inhibition of follicular growth particularly during the late phases which precede ovulation [1,8]. In the bovine, granulosa cells proliferate in response to factors such as epidermal growth factor [9], insulin [10], insulin and FSH [11] or insulin-growth-factor-I [12, 13]. Theca cells can provide the transforming growth factors T G F~ [14] and TGF]~ [15], which have antagonist effects on growth and steroidogenesis of granulosa cells [15, 16]. All
214
M.J. Bosc and Agn+s Nicolle
these facts underline the importance of granulosatheca cell interactions in the mechanisms controlling follicular functions. T h e use of co-culture is one of the means used to study the interactions between different cell types. Few reports have been devoted to such a tool which allows an integration of different regulatory steps. In long-term co-cultures or in cultures with theca cell conditioned medium, it has thus been shown that theca cells promote granulosa cell proliferation [1719]. Such co-cultures have also demonstrated that theca and granulosa cells interact in a reciprocal modulation of their morphology and structure [19]. In co-cultures, it has been shown that E2 synthesis by granulosa cells is inhibited whereas A4 production is stimulated by theca cells [19]. In these preceding studies, the co-cultures were performed with granulosa and interna theca cells. T h e extema theca cells which are also in the vicinity of granulosa cells have not yet been used. Moreover, in these studies follicular cells were also issued from ovaries collected without reference to a m o m e n t of the oestrous cycle. Ovulation is associated with profound changes in steroid secretion by the ovary. At the onset of final maturation of the dominant follicle, the main steroid produced locally is E2 [20]. At the time of L H surge, thecal A4 synthesis is inhibited [20] depleting the granulosa cells of their precursor for E2 synthesis [21]. It is due to the loss of 17ct-hydroxylase activity and to a decrease in aromatase activity [22]. Therefore, we have studied steroid productions of granulosa and of intema or externa theca cells cultured alone or in co-cultures. A previous study [23] indicated that interna and externa theca cell preparations differ in their respective steroid production. Immature calves, which are potential donors of oocytes, were used after a gonadotropin stimulation and the cells were obtained just before the L H surge. This report indicates that the two types of theca cells can alter E2 synthesis by the granulosa cells, and they present functional differences with regard to P4 production.
chased from Sigma (Sigma Chemical Co., La Verpilli~re, France): bovine insulin, human transfertin, hepes, deoxyribonuclease I, pronase E, hyaluronidase (type 2) and percoll. Ascorbic acid was obtained from Prolabo (Paris, France), collagenase A from Boehringer-Mannheim (Meylan, France) and culture dishes from Costar (Cambridge, MA, U.S.A.). T h e unlabelled steroids (Sigma, France) used were progesterone (4-pregne-3,20-dione) (P4), dehydroepiandrosterone (5-androsten-3//-ol-17one) (DHEA), androstenedione (4-androsten-3,17-dione) (A4) and oestradiol-17// (1,3,5 oestratrien-3,17//-diol) (E2). T h e labelled steroids purchased from Amersham (Little Chalfont, U.K.), were used as tracers in radioimmunoassays (RIAs): (1,2,6,7, 3H) androst-ene 3,17 dione (specific activity: 98 Ci/mmol), (1,2,6,7,16,17, 3H) 4-pregne-3,20-dione (specific activity: 100130 Ci/mmol), (2,4,6,7,16,17 3H) oestradiol-17// (specific activity: 155Ci/mmol). M e d i u m A was Ham's F12 buffered with hepes and supplemented with gentamicin (20 mg/1) and nystatin (4 mg/l). For cell isolation (medium I), medium A was added 5% FCS. For cell culture (medium C), medium A was added 1% FCS and insulin (10mg/1), transferrin (10 mg/1) and ascorbic acid (17.6 mg/1). Animals
Female calves (Holstein or crossbred Holstein), suckled artificially, received 1500 UI of e C G by an intramuscular injection at approximately 3 months of age. T h e y were killed 96 h afterwards in order to obtain preovulatory follicles before the expected peaks of oestradiol [24] and L H [25]. T h e y had at slaughter a body weight of 140-190kg. Ovaries were transported to the laboratory in a physiological saline solution containing miconazole (0.4 g/l), streptomycin (2 g/l) and penicillin (400 000 UI/I). T h e y were kept in this solution at room temperature for 15-20 min and placed in the physiological saline solution. For each experiment, two to four calves were used and three experiments were necessary to complete the series. Cell isolation
MATERIALS AND METHODS
Reagents
For calf treatment, equine-chorionic-gonadotropin (eCG; P M S G ; Intervet, Angers, France) was used. T h e reagents and products were H a m ' s F12, fetal calf serum (FCS) and trypsin (Life Technology, CergyPontoise, France), heparin (Laboratoires Roche, Paris, France), miconazole (Laboratoires Janssen, Boulogne-Billancourt, France), nystatin (Seromed, Berlin, Germany), penicillin (Sarbach, Suresnes, France), gentamicin (Shering-Plough, LevalloisPerret, France), streptomycin (Laboratoires Diamant, Puteaux, France). T h e following products were pur-
Theca and granulosa cells were collected from follicles of more than 5 m m in diameter. Healthy follicles were retained according to the criteria of McNatty et al. [26], a healthy follicle having a vascular pink to red theca, without debris in the follicular fluid. Granulosa cells were obtained with a hypodermic needle using medium I containing heparin (10 UI/ml). Follicular walls were scraped with a loop to remove the adhering granulosa cells which were flushed with medium I and also retained. Cell suspensions were pooled, rinsed, resuspended in medium C, and counted with a haemocytometer. Theca cells were obtained after dissection of the follicular wall. Theca interna was isolated from the follicular wall
Steroids in Co-cultures of Granulosa and Theca Cells with a fine forceps under a microscope [26-28]. T h e r e m n a n t of the follicle wall was referred to as the theca externa [26] and was collected separately. T h e two tissues were washed and chopped into fine pieces before chemical dissoc!iation. Pieces of theca interna were put in m e d i u m I containing collagenase (0.4 mg/ ml) and deoxyribonuc]ease (5000 units/l). Pieces of theca extema were put in a solution adapted from Marcus et al. [29] constituted of m e d i u m I plus collagenase (1 mg/ml), pronase (1 mg/ml), hyaluronidase ( l m g / m l ) and deoxyribonuclease (5000 units/l). T h e s e two preparations were incubated twice for 20 rain at 37°C under gentle shaking. T h e solutions were recovered, filtered and centrifuged (300g for 7 min). T h e cells were resuspended in m e d i u m I, washed twice and resuspended in 1 ml of m e d i u m I. Cellular debris and most of the red blood cells were eliminated using a percoll separation. After recovery, cells were placed in m e d i u m C and counted.
Cell cultures Cells were distributed into 48-well plates in a total volume of 0.5 ml of me.dium C. Co-cultures were performed by adding defined numbers of thecal and/or granulosa cells to each well in order to have a total of 150 000 viable cells per well. T h e combinations performed were 0, 10, 20, 50 and 100% of granulosa cells or 100, 90, 80, 5(I and 0%, respectively, of theca interna or theca externa cells. In one experiment, the combination of 5% of granulosa cells was added. In each experiment, cells were cultured with or without D H E A which was used as a substrate for A4 or oestradiol-17/3 production (10 -7 M; 14.4 ng/well). T r e a t m e n t s were performed in triplicate. Spent media were taken daily and kept at - 2 0 ° C for steroid determinations. Cultures were performed at 37°C under a water-saturated gas phase of 95% air and 5% CO2 and were stopped after 3 days. Cells were detached by trypsination (2.5% trypsin in phosphate-buffered saline) and counted. T h e m e a n rates of recovered cells on day 3 were 44.5 and 35.8%, respectively, in co-cultures performed with externa and interna theca cells.
Radioimmunoassay Spent media were :assayed for A4, P4 and E2 by ILIA using specific arttisera. T h e methods used for culture m e d i u m were adapted from assays of plasma samples without extraction. Each sample was measured in duplicate. F o r A4 [30], the antiserum cross-reacted with testosterone (7%), 0.2% with dehydroepiandrosterone and less than 0.1% with other steroids tested. T h e limit of detection was 50 pg/ml and the intra-assay coefficients of variation were 11.6 and 15.0%, respectively, for the doses of 10 and 0.5 ng/ml. For P4 [31], the antiserum cross-reacted slightly with 20-hydroxyprogesterone (1.7%) and less than 1% with other steroids. T h e limit of detection
215
was 60 pg/ml and the intra-assay coefficients of variation were 18.0 and 15.0%, respectively, for the doses of 16 and 0.5 ng/ml. For E2 assays [32], the antiserum cross-reacted with 6-ketoestradiol (12.3%), 16-epiestriol (5%), 16-ketoestradiol (1.3%) and less than 1% with other steroids. T h e limit of detection was 60 pg/ ml and the intra-assay coefficients of variation were 8.6 and 9.4%, respectively, for the doses of 2 and 0.125 ng/ml. Values are expressed in picograms or in nanograms per 105 cells (+SD).
Statistical analysis T h e data were analysed by experiment according to the two-way analysis of variance [33]. T h e y were log transformed due to the heterogeneity of variances. T h e main factors taken into account were the steroid substrate and the day of culture per type of cells (granulosa with either interna or externa theca cells) or the steroid substrate and the type of cells (granulosa with either intema or externa theca cells) per day of culture. T h e N e w m a n - K e u l s test was used to c o m pare the means and a level of P < 5% was considered significant.
RESULTS
Oestradiol-17t~ production E2 production depended on the type of theca cells ( P < 0 . 0 0 1 ) , on the percentage of granulosa cells seeded at the beginning of culture ( P < 0.001), on the presence of D H E A (P < 0.001) and on the day of culture ( P < 0 . 0 0 1 ) . T h e r e were also interactions between these factors ( P < 0.01 or <0.001). Contamination of interna and e x t e m a theca cells by granulosa was estimated to be less than 1% when their E2 productions on day 1 were c o m p a r e d to the a m o u n t of E2 produced by the combination of 90% of theca cells and 10% of granulosa at the beginning of culture. In the absence of D H E A on day 1, E2 production was higher when granulosa cells were cultured with intema than with externa theca cells ( P < 0 . 0 0 1 ) . With interna theca cells, E2 levels were m a x i m u m with either 20 or 50% of granulosa cells (between 15 and 26 ng per 105 cells according to the experiment). With 10% of granulosa cells, the quantity of E2 was lower (P < 0.05) (Fig. 1 (a)). With externa theca cells, the m a x i m u m level of E2 was reached with either 10 or 20% of granulosa cells (between 1.2 and 2.5 ng per 105 cells according to the experiment) (Fig. l(b)). E2 was insignificant with either interna or extema theca cells alone. With granulosa cells alone, E2 levels (between 0.4 and 1.4 ng per 105 cells according to the experiment) were lower than with any other c o m bination of cells (P < 0.05). In the absence of D H E A on day 3, the E2 measured was m u c h lower than on day 1 ( P < 0.001)
216
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% o f granulosa at t h e b, g i n n i n g o f culture Fig. 1. A n d r o s t e n e d i o n e (A4) a n d oestradiol-17~ (E2) concentrations on day 1 in co-cultures o f granulosa cells with i n t e r n a (part a) o r externa (part b) t h e c a cells in a r e p r e s e n t a t i v e e x p e r i m e n t . The cells w e r e i s s u e d f r o m e C G - p r i m e d calf follicles; 10s cells w e r e s e e d e d p e r well with a n u m b e r o f granulosa cells r e p r e s e n t i n g 0, 5, 10, 20, 50 o r 100% o f the total. They w e r e c u l t u r e d i n t h e a b s e n c e o f D H E A . E a c h t r e a t m e n t w a s p e r f o r m e d in triplicate a n d the results a r e e x p r e s s e d in n a n o g r a m s p e r 10 s cells. Within a p a n e l a n d by steroid, m e a n s (+SD) with different superscripts differ ( P < 0.05; N e w m a n - K e u l s test). Note t h e d i f f e r e n t scales o f o r d i n a t e s i n the two panels.
(data not shown). Maximal E2 levels did not exceed 0.3 ng per 105 cells (50% of intema cells and 50% of granulosa cells at the beginning of culture). T h e production was negligible in the case of co-culture performed with extema theca cells. In the presence of D H E A on day 1, E2 production was higher ( P < 0.01) in all cases than in the absence of D H E A (Fig. 1 and Fig. 2). This production was m a x i m u m when 50% granulosa cells were seeded with both types of theca cells ( P < 0.05). E2 levels were decreased ( P < 0.05) when the proportion of granulosa cells was lower (10 or 5%). W h e n theca cells were cultured alone, E2 production was not modified by the addition of D H E A (P > 0.05). It is interesting to note that granulosa cells produced, in the presence of D H E A , significant amounts of E2 similar to the quantities observed in co-cultures having 20 or 50% of granulosa cells ( P > 0.05). In the presence of D H E A on day 3, E2 production by granulosa cells alone was maintained at levels similar to those observed on day 1 ( P > 0.05). T h e y were higher than levels observed in the different co-cultures ( P < 0.05) (Fig. 2). Granulosa cells cultured with externa theca cells produced more E2 than granulosa cells co-cultured with intema theca cells ( P < 0.05) but in all cases, E2 concentrations were low. With 50% of seeded granulosa cells the levels of E2 thus varied between 337 and 748 pg and between 760 and 2380 pg per 105 cells when co-cultured, respectively, with intema and with extema theca cells. In comparison, E2 levels were comprised between 7.4 and 43 ng per 105 cells when granulosa cells were cultured
alone. Figure 2 illustrates these differences of production observed between days 1 and 3 of culture in the presence of D H E A .
Androstenedione production With our system of co-culture, the production of A4 was a function of the type of theca cells ( P < 0.001) and of the percentage of granulosa cells seeded at the beginning of culture ( P < 0.001). It was also sharply modified by the addition of D H E A (P < 0.001) and was greatly affected by the day of culture ( P < 0.001). T h e r e were interactions between these factors ( P < 0 . 0 1 ) . Co-cultures done with intema theca cells thus produced m o r e A4 than cocultures performed with extema theca cells in the absence (Fig. l(a and b)) and the presence (Fig. 3(a and b)) of D H E A ( P < 0.01). In the absence of D H E A on day 1, A4 levels were higher when the percentages of seeded theca cells were the greatest (100 or 90%). With interna theca cells alone, A4 levels varied between 23 and 57 ng per 105 cells, and only between 0.7 and 2.7 ng for extema theca cells. Granulosa cells alone did not produce A4. Intermediate levels of A4 were produced when percentages of granulosa were of 20 or 50% (Fig. l(a and b)). In the absence of D H E A and on day 3, A4 levels were considerably lower by comparison to day 1 or day 2 (data not shown). T h e s e levels were always less than 1 ng per 105 cells and were sometimes undetectable particularly with extema theca cells. In the presence of D H E A , A4 levels were a function of the type of theca cells (interna theca
Steroids in Co-cultures of Granulosa and Theca Cells
217
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% of granulosa at the beginning of culture Fig. 2. Oestradiol-.17fl (E2) on days 1 and 3 in co-cultures of granulosa cells and of interna (part a) or externa (part b) theca cells in a representative experiment. The cells were issued from eCG-primed calf follicles; l0 s cells were seeded per well with a number of granulosa cells representing 0, 5, 10, 20, 50 or 100% of the total. They were cultured in the presence of DHEA. Each treatment was performed in triplicate and the results are expressed in nanograms per l0 s cells. Within a panel and by day, means (+SD) with different superscripts differ (P< 0.05; Newman-Keuls test).
cells > e x t e r n a t h e c a cells; P < 0.001) a n d o f the p e r c e n t a g e o f s e e d e d g r a n u l o s a cells (Fig. 3(a a n d b)). G r a n u l o s a cells a l o n e p r o d u c e d negligible A 4 levels t h r o u g h o u t t h e culture. O n d a y 1 a n d w i t h 5 0 % o f g r a n u l o s a cells, A 4 levels r e m a i n e d low w h e n s e e d e d w i t h e x t e r n a t h e c a cells; a n d t h e y were h i g h e r with i n t e r n a t h e c a cells (Fig. 3(a)). F o r i n t e m a t h e c a cells,
A 4 p r o d u c t i o n was h i g h e r w i t h 10 o r 2 0 % g r a n u l o s a cells t h a n with 5 0 % ( P < 0.05) a n d was c o m p a r a b l e to t h e levels o b t a i n e d w i t h t h e c a cells alone. O n d a y 3, D H E A m a i n t a i n e d significant levels o f A 4 w h i c h v a r i e d a c c o r d i n g to the e x p e r i m e n t a n d t h e t y p e o f t h e c a cells. F i g u r e 3(a) (theca i n t e r n a ) a n d Fig. 3(b) ( t h e c a e x t e r n a ) i l l u s t r a t e d the c h a n g e s o f t h e q u a n -
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% o f g r a n u l o s a at t h e b e g i n n i n g o f c u l t u r e Fig. 3. Androstenedione (A4) concentrations on days 1 and 3 in co-cultures of granulosa cells and of interna (part a) or externa (part b) theca cells in a representative experiment. The cells were issued from eCG-primed calf follicles; 105 cells were seeded per well with a number of granulosa cells representing 0, 5, 10, 20, 50 or 100% of the total. They were cultured in the presence of DHEA. Each treatment was performed in triplicate and the results are expressed in nanograms per 105 cells. Within a panel and by day, means (+SD) with different superscripts differ (P < 0.05; Newman-Keuls test). Note the different scales of ordinates in the two panels.
218
M.J. Bosc and Agn6s Nicolle
titles produced from day 1 to day 3 in a representative experiment.
Progesteroneproduction T h r e e main factors affected P4 production: the type of theca cells ( P < 0.001), the percentage of seeded granulosa cells ( P < 0 . 0 0 1 ) and the day of culture ( P < 0.001). For this last factor, the quantities of P4 recovered were greater on day 3 than on day 1 ( P < 0.001). T h e r e was a strong interaction between the type of theca cells and the percentage of granulosa cells ( P < 0.001). T h e addition of D H E A to the m e d ium had no or only a weak effect on P4 levels (P > 0.05). On day 1, m a x i m u m levels of P4 were observed with granulosa cells alone (P < 0.05 vs the other combinations). T h e respective quantities recovered in cultures of theca interna and extema cells alone represented on average 5.6 and 7.2% of the quantities measured in the culture of pure granulosa cells. In coculture with theca interna cells, P4 production was weak and its m a x i m u m (with the combination of 50% theca and granulosa cells) represented 16.8% of the granulosa cells alone. In co-culture with externa theca cells, the production of P4 was apparently proportional to the percentage of seeded granulosa cells. For 0, 10, 20, and 50% seeded granulosa cells, P4 levels represented on average 5.6, 17.1, 29.8 and 59.0% of the levels obtained with 100% of granulosa. These differences between the two types of co-culture are illustrated in Fig. 4(a).
On day 3, the greatest P4 production was always obtained with 100% granulosa ( P < 0.05 vs all other combinations) and the lowest with theca cells alone. On this day, the m e a n productions of externa and interna theca cells alone represented, respectively, 4.3 and 5.2% of that of granulosa cells. T h e interaction observed on day 1 was also seen on day 3 (Fig. 4(b)). With theca interna cells, the levels of P4 did not increase proportionally to the percentage of seeded granulosa cells. With 10, 20 or 50% seeded granulosa cells, m e a n P4 levels were, respectively, 9.0, 8.5 and 16.6% of the production of 100% granulosa. In comparison with externa theca cells, P4 production was markedly increased in the presence of granulosa cells. W h e n percentages of granulosa cells passed from 10 to 20 and to 50%, the m e a n rates of P4 were, respectively, 39.9, 71.4 and 87.7% of the P4 levels observed with granulosa cells alone.
DISCUSSION
T h e present study indicates that theca cells have two effects on the granulosa cells which differ according to the type of steroid production and the cell combination. Both types of theca cells have inhibited E 2 production after the first day of culture. By comparison with P4, the interna but not the externa theca cells have apparently inhibited its production by the granulosa as early as the first day of culture. Therefore the inner and outer parts of the theca have not only morphological but also functional differences. T h e s e results confirm the main features charac-
b- d a y 3
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Fig. 4. P r o g e s t e r o n e (P4) o n d a y s 1 ( p a r t a) a n d 3 ( p a r t b) i n c o - c u l t u r e s o f g r a n u l o s a cells w i t h i n t e r n a o r e x t e r n a t h e c a cells i n a r e p r e s e n t a t i v e e x p e r i m e n t . T h e ceils w e r e i s s u e d f r o m e C G - p r i m e d c a l f follicles; 10 s ceils w e r e s e e d e d p e r w e l l w i t h a n u m b e r o f g r a n u l o s a cells r e p r e s e n t i n g 0, 5, 10, 20, 50 o r 100% o f t h e total. T h e y w e r e c u l t u r e d in t h e a b s e n c e o f D H E A . E a c h t r e a t m e n t w a s p e r f o r m e d i n t r i p l i c a t e a n d t h e r e s u l t s a r e e x p r e s s e d i n n a n o g r a m s p e r l 0 s cells. W i t h i n a p a n e l a n d b y t y p e o f t h e c a cells, m e a n s ( + S D ) w i t h d i f f e r e n t s u p e r s c r i p t s d i f f e r ( P < 0.05; N e w m a n - K e u l s test). N o t e t h e d i f f e r e n t s c a l e s o f o r d i n a t e s i n t h e t w o p a n e l s .
Steroids in Co-cultures of Granulosa and Theca Cells terizing A4 and P4 productions by b o t h types of theca cells in vitro. T h e y showed a loss of 17~-hydroxylase activity and a persistence of the 3fl-hydroxy-steroiddeshydrogenase activity which are not modified by the addition of granulosa cells. With regard to E2 production, two points at least have to be considered in the light of the cooperation of the two types of cells. T h e first is related to theca and granulosa cells ctdtured alone. T h e c a cells did not produce E2, where, as granulosa cells produced it in significant amounts on days 1 and 3 of culture when they were in the presence of androgens. In the absence of androgens:, E2 decreased in 2 days to barely detectable levels. If the aromatizing capacity decreases in cultured granulosa cells [34-37], our results indicate that !it also depends on precursor availability, as was shown in previous studies [11, 38, 39]. T h e second point concerns the great differences of E2 levels observed between the beginning and the end of b o t h types of co-cultures. On day 1, granulosa cells produced E2 in significant but in different quantities according to the type of theca cells in co-cultures. T h e s e variations are easily explained by the differences of A4 production between the two types of theca cells; this production being greater in interna than in externa theca cells in vitro [21, 23]. T h e limiting factor of E2 production in our conditions was the androgen production of externa theca cells, and inversely, it was the n u m b e r of granulosa cells co-cultured with interna theca cells. Our results also stress the sensibility of granulosa and theca cells co-cultured with regard to E2 synthesis because only 5 or 10% of seeded granulosa cells are sufficient to get high levels of E2 on the first day. On day 3, in contrast, E2 levels were low in co-cultures with both types of theca cells, whereas they were maintained in granulosa cells alone in the presence of D H E A . This was particularly obvious when theca and granulosa cells were seeded in similar quantities. This apparent inhibition of E2 synthesis by granulosa cells raises several questions about the role(s) of theca cells. T h e first concerns the n u m b e r of granulosa cells which survived after 3 days. One m a y assume that their survival rate was not affected by the presence of theca cells. Indirect evidence of the presence of granulosa cells in our co-cultures is provided by the P4 concentrations observed in the co-cultures of granulosa and externa theca cells. P4 levels were m u c h higher in granulosa than in theca cell cultures. One m a y note that P4 concentrations were mostly related to the proportion of seeded granulosa cells rather than the n u m b e r of externa theca cells on day 1 as on day 3, indicating a good survival of granulosa cells in these conditions. T h e apparent inhibition of the aromatizing capacity observed in our systems is thus unlikely to be due to the absence of granulosa cells. A second question conceres the mechanism(s) which is (are) implicated in this inhibitory effect exerted by b o t h types of theca
219
cells. This effect is observed in the absence of any gonadotropin supplementation and in conditions which have allowed the secretion of large A4 quantities. T h e same results have also being obtained, after 8 days, on granulosa co-cultured with theca cells on the opposite sides of a collagen m e m b r a n e [19]. One may postulate that this inhibition of E2 synthesis is due to some agent(s) secreted by the theca cells. Preliminary results obtained with theca cell conditioned m e d i u m suggest such a possibility. H o w e v e r several observations have to be taken into account with regard to the possible mechanisms implicated in this interaction between granulosa and theca cells. In long-term co-culture on collagen m e m b r a n e , this inhibition ceases and E2 synthesis is stimulated by L H or by L H and F S H , whereas F S H alone has no effect [19]. In short-term culture on fibronectin, granulosa ceils maintain their aromatizing capacity for at least 4 days with appropriate doses of F S H and/or insulin [11]. In short-term cultures, a significant aromatizing activity can be observed in spite of low levels of m R N A encoding P450 aromatase in the presence or in the absence of F S H [39]. All these facts taken together indicate that, in vitro, the aromatizing capacity of the granulosa cells is regulated not only by the amounts of androgens produced by the theca cells but also by another type of cell interaction which could be counteracted by gonadotropins. In our conditions, P4 was secreted by all types of follicular cells, but the granulosa produced the largest amounts as observed previously [4, 6, 37]. T h e striking fact is the difference in P4 levels between the two types of theca cells in co-culture. As pointed out before, P4 production was a function of the proportion of seeded granulosa cells in co-cultures with theca extema cells. In these conditions, a large P4 increase was also noted from the beginning to the end of the experiments. In contrast, P4 levels remained low in co-cultures performed with interna theca cells. This p h e n o m e n o n was observed from the first day of culture at a time at which E2 levels were a function of seeded granulosa cells, the main contributor to P4 production. Therefore, it cannot be attributed to a rapid and a complete disappearance of granulosa cells. It also cannot be explained by an inhibition of P4 production by interna theca cells [4-6] because interna theca cells produced low amounts of P4 on the first day of culture [23]. Furthermore, it cannot be due to a transformation in A4 because its synthesis, which was not affected, is normally performed through the d 5 pathway [23, 40]. This inhibitory effect of granulosa P4 secretion, specific to interna theca cells, lasted 3 days in our conditions and it can be prolonged because it has been observed after 8 days in the absence or in the presence of gonadotropins [18, 19]. T h e reason for this inhibitory effect is unknown. In a previous study [23], we noted that after the addition of P4 as a substrate its recovery in
220
M . J. Bosc and Agn~s Nicolle
intema theca cell cultures was lower than the amount added to the medium. All these facts suggest that intema theca cells would be able to catabolize P4 or to secrete some P4 binding proteins. As pointed out before [23], the theca cells issued from eCG-primed calves behave in vitro as adult theca cells with regard to steroid production. T h e present results thus confirm that both theca cell preparations have a continuous functional 3fl-hydroxysteroid dehydrogenase activity and that they lose their 17~-hydroxylase activity in a 3 days culture system. This was illustrated by A4 levels obtained with or without D H E A . Three days of culture of theca cells with granulosa cells has not changed the functional characteristics of the theca cells. This result contrasts with long-term co-cultures, in which A4 production by theca cells was enhanced [19]. This discrepancy might, however, be explained by the differences in co-culture lengths. T h e cell preparations used in these experiments are certainly not entirely specific to the externa and the intema parts of the theca. However, these results illustrate that the different parts of the theca of a large follicle have not only morphological differences [41-43], but also functional differences with regard to their own steroid production [23] and to the local interactions which modulate the final follicular P4 production. In conclusion, the simple system of co-culture adopted in these experiments shows that E2 and P4 productions by granulosa cells can be altered in the presence of theca cells. Both types of theca cells have an inhibitory effect on E2 production whereas only the interna theca cells are able to alter P4 production. These results underline the fact that interna and externa theca cells from eCG-primed calves have functional differences with regard to steroid production by the granulosa cells; they show the necessity to take into account all the components of the follicle for the study of such local functions. Acknowledgements--We wish to thank the INRA institute which gave us the means to perform these studies. The antibodies raised against E2 and A4 were kindly provided by M. Terqui, and against P4 by the "Laboratoire des Dosages Hormonaux - - I.N.R.A ". We are indebted to J. L. Touzfi and his colleagues for animal care. We also thank D. Monniaux for her advice and her encouragement throughout the course of this work, and A. Y. Kermabon for her help in the preparation of the English version of this manuscript.
REFERENCES
1. Mariana J. C., Monniaux D., Driancourt M. A. and Maul~on P., Folliculogenesis. In Reproduction in Domestic Animals, Chap. 4, ed. P. T. Cupps. Academic Press, New York, 1991, pp. 119-171. 2. Fortune J. E., Ovarian follicular growth and development in mammals. Biology of Reproduction 50 (1994) 225-232. 3. Erickson G. F., Magoffin D. A., Dyer C. A. and Hofeditz, The ovarian androgen producing cells: a review of structure/function relationships. Endocrine Reviews 6 (1985) 371-399.
4. Fortune J. E. and Hansel W., The effects of 17fl-estradiol on progesterone secretion by bovine theca and granulosa cells. Endocrinology 104 (1979) 1834 1838. 5. Roberts A. J. and Skinner M. K., Estrogen regulation of theca cell steroidogenesis and differentiation: theca ceU-granulosa cell interactions. Endocrinology 127 (1990) 2918-2929. 6. Wrathall J. H. M. and Knight P. G., Effects of inhibin-related peptides and oestradiol on androstenedione and progesterone secretion by bovine theca cells in vitro. Journal of Endocrinology 145 (1995) 491-500. 7. Fortune J. E., Bovine theca and granulosa cells interact to promote androgen production. Biology of Reproduction 35 (1986) 292-299. 8. Lussier J. G., Matron P. and Dufour J. J., Growth rates of follicles in the ovary of the cow. Journal of Reproduction and Fertility 81 (1987) 301-307. 9. Gospodarowicz D., Ill C. R. and Birdwell R., Effects of fibroblast and epidermal growth factors on ovarian cell proliferation in vitro. II. Proliferative response of luteal cells to FGF but not EGF. Endocrinology 4 (1977) 1121-1128. 10. Savion N., Lui G.-M., Laherty R. and Gospodarowicz D., Factors controlling proliferation and progesterone production by bovine granulosa cells in serum-free medium. Endocrinology 109 (1981) 409-420. 11. Saumande J., Culture of bovine granulosa cells in a chemically defined serum-free medium: the effect of insulin and fibronectin on the response to FSH. Journal of Steroid Biochemistry and Molecular Biology 38 (1991) 189-196. 12. Gong J. G., Mcbride D., Bramley T. A. and Webb R., Effects of recombinant bovine somatotrophin, insulin-like growth factor-i and insulin on the proliferation of bovine granulosa cells in vitro. Journal of Endocrinology 139 (1993) 67-75. 13. Spicer L. J., Alpizar E. and Echternkamp S. E., Effects of insulin, insulin-like growth factor-i, and gonadotropins on bovine granulosa cell proliferation, progesterone production, estradiol production, and (or) insulin-like growth factor-i production in vitro. Journal of Animal Science 71 (1993) 1232-1241. 14. Lobb D. K., Kobrin M. S., Kudlow J. E. and Dorrington J. H., Transforming growth factor-alpha - - in the adult bovine ovary: identification in growing ovarian follicles. Biology of Reproduction 40 (1989) 1087-1093. 15. Skinner M. K., Keski-Oja J., Osteen K. G. and Moses H. L., Ovarian theca cells produce transforming growth factor-beta which can regulate granulosa cell growth. Endocrinology 121 (1987) 786-792. 16. Skinner M. K. and Coffey R. J., Regulation of ovarian cell growth through the local production of transforming growth factor-alpha by theca cells. Endocrinology 123 (1988) 26322638. 17. Bendell J. J., Lobb D. K., Chuma A., Gysler M. and Dorrington J. H., Bovine theca cells secrete factor(s) that promote granulosa cell proliferation. Biology of Reproduction 38 (1988) 790-797. 18. Kotsuji F., Kamitani N., Goto K. and Tominaga T., Bovine theca and granulosa cell interactions modulate their growth, morphology and function. Biology of Reproduction 43 (1990) 726-732. 19. Kotsuji F. and Tominaga T., The role of granulosa and theca cell interactions in ovarian structure and function. Microscopy Research Techniques27 (1994) 97-107. 20. Dieleman S. J., Kruip Th A. M., Fontijne P., de Jong W. H. R. and Van der Weyden G. C., Changes in oestradiol, progesterone and testosterone concentrations in follicular fluid and in the micromorphology of preovulatory bovine follicles relative to the peak of luteinizing hormone. Journal of Endocrinology 97 (1983) 31-42. 21. McNatty K. P., Heath D. A., Henderson K. M., Lun S., Hurst P. R., Ellis L. M., Montgomery G. W., Morrison L. and Thurley D. C., Some aspects of thecal and granulosa cell function during follicular development in the bovine ovary. Journal of Reproduction and Fertility 72 (1984) 39-53. 22. Rodgers R. J., Waterman M. R. and Simpson E. R., Cytochromes P-450scc, P-450-17fl, adrenotoxin, and reduced nicotinamide adenine dinucleotide phosphate cytochrome P450 reductase in bovine follicles and corpora lutea, changes in specific contents during the ovarian cycle. Endocrinology 118 (1986) 1366-1374.
Steroids in C o - c u l t u r e s o f G r a n u l o s a and T h e c a Cells 23. Bosc M. J. and Nicolle A., Androstenedione and progesterone productions in vitro by the inner and the outer theca cells in preovulatory follicles of gonadotropin stimulated calves. (in preparation). 24. Saumande J. and Testm't J., Plasma levels of oestradiol-17/3 in the pre-puhertal heifer treated to induce superovulation. Theriogenology 2 (1974) 121-129. 25. Testart J. and Kann G., Taux plasmatique de LH chez la G~nisse impub~re sournise fi un traitement de superovulation. Comptes Rendus de l'Academie des Sciences, Paris, Skrie D 277 (1973) 1181-1184. 26. McNatty K. P., Hillier ;~. G., Van den Boogaard A., TrimbosKemper T. C. M., Reichert L. E. Jr and van Hall E. V., Follicular development during the luteal phase of the human menstrual cycle. Journal of Clinical Endocrinology and Metabolism 56 (1983) 1022-1031. 27. Voss A. K. and Fortune J. E., Oxytocin stimulates progesterone production by bovine granulosa cells isolated before, but not after, the luteinizing hormone surge. Molecular and Cellular Endocrinology 78 (1991) 17-24. 28. Meidan R., Girsh E., BLum O. and Aberdam E., In vitro differentiation of bovine theca and granulosa cells into small and large luteal-like cells: morphological and functional characteristics. Biology of Reproduction 43 (1990) 913-921. 29. Marcus G. J., Connor L., Domingo M. T., Tsang B. K., Downey B. R. and Ainsworth L., Enzymatic dissociation of ovarian and uterine tissues. Endocrine Research 10 (1984) 151162. 30. Williams J. and de Reviers M., Variations in the plasma levels of luteinizing hormone and androstenedione and their relationship with the adult daily sperm output in cockerels raised under different photoschedules. Reproduction Nutrition Development 21 (1981) 1125-1135. 31. Saumande J., Tamboura D. and Chupin D., Changes in milk and plasma concentrations of progesterone in cows after treatment to induce superovulation and their relationships with number of ovulations znd of embryos collected. Theriogenology 23 (1985) 719-731. 32. Saumande J., Radioimraunoassy of estradiol-17/3 in unextracted ewe plasma. Steroids 38 (1981) 425-437. 33. Vessereau, A., M~thodes Statistiques en Biologie et en Agronomie. Tec and Doc, Lavoisier, J. B. Baill~re, Paris, 1988.
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34. Henderson K. M. and Swanston I. A., Androgen aromatization by luteinized bovine granulosa cells in tissue culture. Journal of Reproduction and Fertility 52 (1978) 131-134. 35. Luck M. R., Rodgers R. J. and Findlay J. K., Secretion and gene expression of inhibin, oxytocin and steroid hormones during the in vitro differentiation of bovine granulosa cells. Reproduction, Fertility and Development 2 (1980) 11-25. 36. Bao B., Thomas M. G., Griffith M. K., Burghardt R. C. and Williams G. L., Steroidogenic activity, insulin-like growth factor-i production, and proliferation of granulosa and theca cells obtained from dominant preovulatory and nonovulatory follicles during the bovine estrous cycle: effects of low-density and high-density lipoproteins. Biology of Reproduction 53 (1995) 1271-1279. 37. Berndtson A. K., Vincent S. E. and Fortune J. E., Effects of gonadotrophin concentration on hormone production by theca interna and granulosa cells from bovine preovulatory follicles. Journal of Reproduction and Fertility 49 (Suppl.) (1995) 527531. 38. Henderson K. M. and Moon Y. S., Luteinization of bovine granulosa cells and corpus luteum formation associated with loss of androgen-aromatizing ability. Journal of Reproduction and Fertility 56 (1979) 89-97. 39. Berndtson A. K., Weaver C. J. and Fortune J. E., Differential effects of oxytocin on steroid production by bovine granulosa cells. Molecular and Cellular Endocrinology 116 (1996) 191-198. 40. Lacroix E., Eechaute W. and Leusen I., The biosynthesis of estrogens by cow follicles. Steroids 23 (1974) 337-356. 41. Priedkalns J., Weber A. F. and Zemjanis R., Qualitative and quantitative morphologic studies of the cells of the membrana granulosa, theca interna and corpus luteum of the bovine ovary. Zeitschrift Zellforschung 85 (1968) 501-520. 42. Yamada O., Abe M., Takehana K., Iwasa K., Hiraga T. and Hiratsuka T., Microvasculature of mature bovine follicles and its changes with ovulation. Journal of Reproduction and Development 40 (1994) 307-315. 43. Yamada O., Abe M., Takehana K., Hiraga T., Iwasa K. and Hiratsuka T., Microvascular changes during the development of follicles in bovine ovaries: a study of corrosion casts by scanning electron microscopy. Archives of Histology and Cytology 58 (1995) 567-574.
Pergamon
S t e r o i d P r o d u c t i o n s by C o - c u l t u r e s of G r a n u l o s a Cells with Inner and O u t e r T h e c a Cells in P r e o v u l a t o r y Follicles of G o n a d o t r o p i n S t i m u l a t e d Calves M. J. Bosc* and Agn s Nicolle Laboratoire de Physiologie de la Reproduction des Mammifires Domestiques, Institut National de la Recherche Agronomique, Centre de Tours-Nouzilly, 37380 Nouzilly, France
G r a n u l o s a , i n t e r n a a n d e x t e r n a t h e c a cells w e r e isolated f r o m large follicles o f e q u i n e - c h o r i o n i c g o n a d o t r o p i n ( e C G ) - p r i m e d calves a n d c o - c u l t u r e d d u r i n g 3 days in the a b s e n c e o r in t he p r e s e n c e o f d e h y d r o e p i a n d r o s t e r o n e ( D H E A ) . C o - c u l t u r e s w ere p e r f o r m e d by a d d i n g d e f i n e d n u m b e r s o f t h e c a a n d / o r granulos.a cells w hi c h r e p r e s e n t e d 0, 10, 20, 50 or 100% o f total cells p e r well. S e c r e t i o n o f oestradiol-17fl (E2), a n d r o s t e n e d i o n e (A4) a n d p r o g e s t e r o n e (P4) d e p e n d e d on t he type o f t h e c a cells ( P < 0.001), on t he p e r c e n t a g e o f s eeded g r a n u l o s a cells ( P < 0.001) a n d on the d a y o f c u l t u r e ( P < 0.001). D H E A i n c r e a s e d ( P < 0.001) Ee a n d A4, b u t n o t P4 (P > 0.05) p r o d u c t i o n s . I n t e r a c t i o n s existed b e t w e e n t he s e f a c t o r s ( P < 0.01). O n day 1, A4 p r o d u c t i o n was nil in g r a n u l o s a cells alone. E2 p r o d u c t i o n was negligible in t h e c a cells alone b u t it i n c r e a s e d w h e n g r a n u l o s a cells w ere a d d e d . Ez a n d A4 v a r i e d in an opposite m a n n e r a c c o r d i n g to t he p e r c e n t a g e o f g r a u u l o s a ceils a n d with the type o f t h e c a cells. O n d a y 3, w i t h o u t D H E A , Ez a n d A4 w ere low. O n day 3 with D H E A , E2 p r o d u c t i o n was m a i n t a i n e d in g r a n u l o s a cells alone b u t not with any c o m b i n a t i o n o f t h e c a cells. In these c o n d i t i o n s , A4 p r o d u c t i o n was m a i n t a i n e d in t he p r e s e n c e o f t h e c a ceils b u t n o t in g r a n u l o s a ceils alone. G r a n u l o s a cells alone s e c r e t e d m o r e P4 t h a n t h e c a cells. P4 i n c r e a s e d as a f u n c t i o n o f the p e r c e n t a g e o f g r a n u l o s a in c o - c u l t u r e s with e x t e r n a b u t n o t i n t e r n a t h e c a cells with w h ic h it r e m a i n e d low. In conclusion, t h e c a cells in c u l t u r e have two effects in r e l a t i o n to t he g r a n u l o s a cells, w h ich differ a c c o r d i n g to the s t e r oi d c o n c e r n e d a n d to the cell c o m b i n a t i o n . B o t h types o f t h e c a ceils have an i n h i b i t o r y effect on E2 s e c r e t i o n w h e r e a s only i n t e r n a t h e c a ceils are able to al te r P4 p r o d u c t i o n . © 1997 E l s evi er Science Ltd.
J. Steroid Biochem. Molec. Biol., Vol. 62, No. 2/3, pp. 213-221, 1997
INTRODUCTION The ovarian follicle provides a structural and functional support to the oocyte and its surrounding cells. In this structure, granulosa and theca cells face each other through a basal membrane allowing for multiple cell interactions [1]. In the bovine as in other mammals, several interact:ions associate granulosa and theca cells. For example, androgens produced by theca cells serve as a substrate for oestrogen synthesis by granulosa cells [2, 3]. This cooperation in steroid production is in fact :more complex. Oestradiol-17fl *Correspondence to M. J. Bosc. Fax: +33 2 47 42 77 43; E-mail: [email protected]. Received 18 Nov. 1996; accepted 21 Feb. 1997. 213
(E2) can alter progesterone (P4) secretion by the theca cells [4-6], but granulosa cells can stimulate androstenedione (A4) synthesis [6, 7], probably by providing pregnenolone as a substrate [7]. Other interactions between the two types of cells have also been described. They concern the factors which are implicated in the stimulation or in the inhibition of follicular growth particularly during the late phases which precede ovulation [1,8]. In the bovine, granulosa cells proliferate in response to factors such as epidermal growth factor [9], insulin [10], insulin and FSH [11] or insulin-growth-factor-I [12, 13]. Theca cells can provide the transforming growth factors T G F~ [14] and TGF]~ [15], which have antagonist effects on growth and steroidogenesis of granulosa cells [15, 16]. All
214
M.J. Bosc and Agn+s Nicolle
these facts underline the importance of granulosatheca cell interactions in the mechanisms controlling follicular functions. T h e use of co-culture is one of the means used to study the interactions between different cell types. Few reports have been devoted to such a tool which allows an integration of different regulatory steps. In long-term co-cultures or in cultures with theca cell conditioned medium, it has thus been shown that theca cells promote granulosa cell proliferation [1719]. Such co-cultures have also demonstrated that theca and granulosa cells interact in a reciprocal modulation of their morphology and structure [19]. In co-cultures, it has been shown that E2 synthesis by granulosa cells is inhibited whereas A4 production is stimulated by theca cells [19]. In these preceding studies, the co-cultures were performed with granulosa and interna theca cells. T h e extema theca cells which are also in the vicinity of granulosa cells have not yet been used. Moreover, in these studies follicular cells were also issued from ovaries collected without reference to a m o m e n t of the oestrous cycle. Ovulation is associated with profound changes in steroid secretion by the ovary. At the onset of final maturation of the dominant follicle, the main steroid produced locally is E2 [20]. At the time of L H surge, thecal A4 synthesis is inhibited [20] depleting the granulosa cells of their precursor for E2 synthesis [21]. It is due to the loss of 17ct-hydroxylase activity and to a decrease in aromatase activity [22]. Therefore, we have studied steroid productions of granulosa and of intema or externa theca cells cultured alone or in co-cultures. A previous study [23] indicated that interna and externa theca cell preparations differ in their respective steroid production. Immature calves, which are potential donors of oocytes, were used after a gonadotropin stimulation and the cells were obtained just before the L H surge. This report indicates that the two types of theca cells can alter E2 synthesis by the granulosa cells, and they present functional differences with regard to P4 production.
chased from Sigma (Sigma Chemical Co., La Verpilli~re, France): bovine insulin, human transfertin, hepes, deoxyribonuclease I, pronase E, hyaluronidase (type 2) and percoll. Ascorbic acid was obtained from Prolabo (Paris, France), collagenase A from Boehringer-Mannheim (Meylan, France) and culture dishes from Costar (Cambridge, MA, U.S.A.). T h e unlabelled steroids (Sigma, France) used were progesterone (4-pregne-3,20-dione) (P4), dehydroepiandrosterone (5-androsten-3//-ol-17one) (DHEA), androstenedione (4-androsten-3,17-dione) (A4) and oestradiol-17// (1,3,5 oestratrien-3,17//-diol) (E2). T h e labelled steroids purchased from Amersham (Little Chalfont, U.K.), were used as tracers in radioimmunoassays (RIAs): (1,2,6,7, 3H) androst-ene 3,17 dione (specific activity: 98 Ci/mmol), (1,2,6,7,16,17, 3H) 4-pregne-3,20-dione (specific activity: 100130 Ci/mmol), (2,4,6,7,16,17 3H) oestradiol-17// (specific activity: 155Ci/mmol). M e d i u m A was Ham's F12 buffered with hepes and supplemented with gentamicin (20 mg/1) and nystatin (4 mg/l). For cell isolation (medium I), medium A was added 5% FCS. For cell culture (medium C), medium A was added 1% FCS and insulin (10mg/1), transferrin (10 mg/1) and ascorbic acid (17.6 mg/1). Animals
Female calves (Holstein or crossbred Holstein), suckled artificially, received 1500 UI of e C G by an intramuscular injection at approximately 3 months of age. T h e y were killed 96 h afterwards in order to obtain preovulatory follicles before the expected peaks of oestradiol [24] and L H [25]. T h e y had at slaughter a body weight of 140-190kg. Ovaries were transported to the laboratory in a physiological saline solution containing miconazole (0.4 g/l), streptomycin (2 g/l) and penicillin (400 000 UI/I). T h e y were kept in this solution at room temperature for 15-20 min and placed in the physiological saline solution. For each experiment, two to four calves were used and three experiments were necessary to complete the series. Cell isolation
MATERIALS AND METHODS
Reagents
For calf treatment, equine-chorionic-gonadotropin (eCG; P M S G ; Intervet, Angers, France) was used. T h e reagents and products were H a m ' s F12, fetal calf serum (FCS) and trypsin (Life Technology, CergyPontoise, France), heparin (Laboratoires Roche, Paris, France), miconazole (Laboratoires Janssen, Boulogne-Billancourt, France), nystatin (Seromed, Berlin, Germany), penicillin (Sarbach, Suresnes, France), gentamicin (Shering-Plough, LevalloisPerret, France), streptomycin (Laboratoires Diamant, Puteaux, France). T h e following products were pur-
Theca and granulosa cells were collected from follicles of more than 5 m m in diameter. Healthy follicles were retained according to the criteria of McNatty et al. [26], a healthy follicle having a vascular pink to red theca, without debris in the follicular fluid. Granulosa cells were obtained with a hypodermic needle using medium I containing heparin (10 UI/ml). Follicular walls were scraped with a loop to remove the adhering granulosa cells which were flushed with medium I and also retained. Cell suspensions were pooled, rinsed, resuspended in medium C, and counted with a haemocytometer. Theca cells were obtained after dissection of the follicular wall. Theca interna was isolated from the follicular wall
Steroids in Co-cultures of Granulosa and Theca Cells with a fine forceps under a microscope [26-28]. T h e r e m n a n t of the follicle wall was referred to as the theca externa [26] and was collected separately. T h e two tissues were washed and chopped into fine pieces before chemical dissoc!iation. Pieces of theca interna were put in m e d i u m I containing collagenase (0.4 mg/ ml) and deoxyribonuc]ease (5000 units/l). Pieces of theca extema were put in a solution adapted from Marcus et al. [29] constituted of m e d i u m I plus collagenase (1 mg/ml), pronase (1 mg/ml), hyaluronidase ( l m g / m l ) and deoxyribonuclease (5000 units/l). T h e s e two preparations were incubated twice for 20 rain at 37°C under gentle shaking. T h e solutions were recovered, filtered and centrifuged (300g for 7 min). T h e cells were resuspended in m e d i u m I, washed twice and resuspended in 1 ml of m e d i u m I. Cellular debris and most of the red blood cells were eliminated using a percoll separation. After recovery, cells were placed in m e d i u m C and counted.
Cell cultures Cells were distributed into 48-well plates in a total volume of 0.5 ml of me.dium C. Co-cultures were performed by adding defined numbers of thecal and/or granulosa cells to each well in order to have a total of 150 000 viable cells per well. T h e combinations performed were 0, 10, 20, 50 and 100% of granulosa cells or 100, 90, 80, 5(I and 0%, respectively, of theca interna or theca externa cells. In one experiment, the combination of 5% of granulosa cells was added. In each experiment, cells were cultured with or without D H E A which was used as a substrate for A4 or oestradiol-17/3 production (10 -7 M; 14.4 ng/well). T r e a t m e n t s were performed in triplicate. Spent media were taken daily and kept at - 2 0 ° C for steroid determinations. Cultures were performed at 37°C under a water-saturated gas phase of 95% air and 5% CO2 and were stopped after 3 days. Cells were detached by trypsination (2.5% trypsin in phosphate-buffered saline) and counted. T h e m e a n rates of recovered cells on day 3 were 44.5 and 35.8%, respectively, in co-cultures performed with externa and interna theca cells.
Radioimmunoassay Spent media were :assayed for A4, P4 and E2 by ILIA using specific arttisera. T h e methods used for culture m e d i u m were adapted from assays of plasma samples without extraction. Each sample was measured in duplicate. F o r A4 [30], the antiserum cross-reacted with testosterone (7%), 0.2% with dehydroepiandrosterone and less than 0.1% with other steroids tested. T h e limit of detection was 50 pg/ml and the intra-assay coefficients of variation were 11.6 and 15.0%, respectively, for the doses of 10 and 0.5 ng/ml. For P4 [31], the antiserum cross-reacted slightly with 20-hydroxyprogesterone (1.7%) and less than 1% with other steroids. T h e limit of detection
215
was 60 pg/ml and the intra-assay coefficients of variation were 18.0 and 15.0%, respectively, for the doses of 16 and 0.5 ng/ml. For E2 assays [32], the antiserum cross-reacted with 6-ketoestradiol (12.3%), 16-epiestriol (5%), 16-ketoestradiol (1.3%) and less than 1% with other steroids. T h e limit of detection was 60 pg/ ml and the intra-assay coefficients of variation were 8.6 and 9.4%, respectively, for the doses of 2 and 0.125 ng/ml. Values are expressed in picograms or in nanograms per 105 cells (+SD).
Statistical analysis T h e data were analysed by experiment according to the two-way analysis of variance [33]. T h e y were log transformed due to the heterogeneity of variances. T h e main factors taken into account were the steroid substrate and the day of culture per type of cells (granulosa with either interna or externa theca cells) or the steroid substrate and the type of cells (granulosa with either intema or externa theca cells) per day of culture. T h e N e w m a n - K e u l s test was used to c o m pare the means and a level of P < 5% was considered significant.
RESULTS
Oestradiol-17t~ production E2 production depended on the type of theca cells ( P < 0 . 0 0 1 ) , on the percentage of granulosa cells seeded at the beginning of culture ( P < 0.001), on the presence of D H E A (P < 0.001) and on the day of culture ( P < 0 . 0 0 1 ) . T h e r e were also interactions between these factors ( P < 0.01 or <0.001). Contamination of interna and e x t e m a theca cells by granulosa was estimated to be less than 1% when their E2 productions on day 1 were c o m p a r e d to the a m o u n t of E2 produced by the combination of 90% of theca cells and 10% of granulosa at the beginning of culture. In the absence of D H E A on day 1, E2 production was higher when granulosa cells were cultured with intema than with externa theca cells ( P < 0 . 0 0 1 ) . With interna theca cells, E2 levels were m a x i m u m with either 20 or 50% of granulosa cells (between 15 and 26 ng per 105 cells according to the experiment). With 10% of granulosa cells, the quantity of E2 was lower (P < 0.05) (Fig. 1 (a)). With externa theca cells, the m a x i m u m level of E2 was reached with either 10 or 20% of granulosa cells (between 1.2 and 2.5 ng per 105 cells according to the experiment) (Fig. l(b)). E2 was insignificant with either interna or extema theca cells alone. With granulosa cells alone, E2 levels (between 0.4 and 1.4 ng per 105 cells according to the experiment) were lower than with any other c o m bination of cells (P < 0.05). In the absence of D H E A on day 3, the E2 measured was m u c h lower than on day 1 ( P < 0.001)
216
M.J. Bosc and Agn& Nicolle a-int. --O-E2
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% o f granulosa at t h e b, g i n n i n g o f culture Fig. 1. A n d r o s t e n e d i o n e (A4) a n d oestradiol-17~ (E2) concentrations on day 1 in co-cultures o f granulosa cells with i n t e r n a (part a) o r externa (part b) t h e c a cells in a r e p r e s e n t a t i v e e x p e r i m e n t . The cells w e r e i s s u e d f r o m e C G - p r i m e d calf follicles; 10s cells w e r e s e e d e d p e r well with a n u m b e r o f granulosa cells r e p r e s e n t i n g 0, 5, 10, 20, 50 o r 100% o f the total. They w e r e c u l t u r e d i n t h e a b s e n c e o f D H E A . E a c h t r e a t m e n t w a s p e r f o r m e d in triplicate a n d the results a r e e x p r e s s e d in n a n o g r a m s p e r 10 s cells. Within a p a n e l a n d by steroid, m e a n s (+SD) with different superscripts differ ( P < 0.05; N e w m a n - K e u l s test). Note t h e d i f f e r e n t scales o f o r d i n a t e s i n the two panels.
(data not shown). Maximal E2 levels did not exceed 0.3 ng per 105 cells (50% of intema cells and 50% of granulosa cells at the beginning of culture). T h e production was negligible in the case of co-culture performed with extema theca cells. In the presence of D H E A on day 1, E2 production was higher ( P < 0.01) in all cases than in the absence of D H E A (Fig. 1 and Fig. 2). This production was m a x i m u m when 50% granulosa cells were seeded with both types of theca cells ( P < 0.05). E2 levels were decreased ( P < 0.05) when the proportion of granulosa cells was lower (10 or 5%). W h e n theca cells were cultured alone, E2 production was not modified by the addition of D H E A (P > 0.05). It is interesting to note that granulosa cells produced, in the presence of D H E A , significant amounts of E2 similar to the quantities observed in co-cultures having 20 or 50% of granulosa cells ( P > 0.05). In the presence of D H E A on day 3, E2 production by granulosa cells alone was maintained at levels similar to those observed on day 1 ( P > 0.05). T h e y were higher than levels observed in the different co-cultures ( P < 0.05) (Fig. 2). Granulosa cells cultured with externa theca cells produced more E2 than granulosa cells co-cultured with intema theca cells ( P < 0.05) but in all cases, E2 concentrations were low. With 50% of seeded granulosa cells the levels of E2 thus varied between 337 and 748 pg and between 760 and 2380 pg per 105 cells when co-cultured, respectively, with intema and with extema theca cells. In comparison, E2 levels were comprised between 7.4 and 43 ng per 105 cells when granulosa cells were cultured
alone. Figure 2 illustrates these differences of production observed between days 1 and 3 of culture in the presence of D H E A .
Androstenedione production With our system of co-culture, the production of A4 was a function of the type of theca cells ( P < 0.001) and of the percentage of granulosa cells seeded at the beginning of culture ( P < 0.001). It was also sharply modified by the addition of D H E A (P < 0.001) and was greatly affected by the day of culture ( P < 0.001). T h e r e were interactions between these factors ( P < 0 . 0 1 ) . Co-cultures done with intema theca cells thus produced m o r e A4 than cocultures performed with extema theca cells in the absence (Fig. l(a and b)) and the presence (Fig. 3(a and b)) of D H E A ( P < 0.01). In the absence of D H E A on day 1, A4 levels were higher when the percentages of seeded theca cells were the greatest (100 or 90%). With interna theca cells alone, A4 levels varied between 23 and 57 ng per 105 cells, and only between 0.7 and 2.7 ng for extema theca cells. Granulosa cells alone did not produce A4. Intermediate levels of A4 were produced when percentages of granulosa were of 20 or 50% (Fig. l(a and b)). In the absence of D H E A and on day 3, A4 levels were considerably lower by comparison to day 1 or day 2 (data not shown). T h e s e levels were always less than 1 ng per 105 cells and were sometimes undetectable particularly with extema theca cells. In the presence of D H E A , A4 levels were a function of the type of theca cells (interna theca
Steroids in Co-cultures of Granulosa and Theca Cells
217
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% of granulosa at the beginning of culture Fig. 2. Oestradiol-.17fl (E2) on days 1 and 3 in co-cultures of granulosa cells and of interna (part a) or externa (part b) theca cells in a representative experiment. The cells were issued from eCG-primed calf follicles; l0 s cells were seeded per well with a number of granulosa cells representing 0, 5, 10, 20, 50 or 100% of the total. They were cultured in the presence of DHEA. Each treatment was performed in triplicate and the results are expressed in nanograms per l0 s cells. Within a panel and by day, means (+SD) with different superscripts differ (P< 0.05; Newman-Keuls test).
cells > e x t e r n a t h e c a cells; P < 0.001) a n d o f the p e r c e n t a g e o f s e e d e d g r a n u l o s a cells (Fig. 3(a a n d b)). G r a n u l o s a cells a l o n e p r o d u c e d negligible A 4 levels t h r o u g h o u t t h e culture. O n d a y 1 a n d w i t h 5 0 % o f g r a n u l o s a cells, A 4 levels r e m a i n e d low w h e n s e e d e d w i t h e x t e r n a t h e c a cells; a n d t h e y were h i g h e r with i n t e r n a t h e c a cells (Fig. 3(a)). F o r i n t e m a t h e c a cells,
A 4 p r o d u c t i o n was h i g h e r w i t h 10 o r 2 0 % g r a n u l o s a cells t h a n with 5 0 % ( P < 0.05) a n d was c o m p a r a b l e to t h e levels o b t a i n e d w i t h t h e c a cells alone. O n d a y 3, D H E A m a i n t a i n e d significant levels o f A 4 w h i c h v a r i e d a c c o r d i n g to the e x p e r i m e n t a n d t h e t y p e o f t h e c a cells. F i g u r e 3(a) (theca i n t e r n a ) a n d Fig. 3(b) ( t h e c a e x t e r n a ) i l l u s t r a t e d the c h a n g e s o f t h e q u a n -
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% o f g r a n u l o s a at t h e b e g i n n i n g o f c u l t u r e Fig. 3. Androstenedione (A4) concentrations on days 1 and 3 in co-cultures of granulosa cells and of interna (part a) or externa (part b) theca cells in a representative experiment. The cells were issued from eCG-primed calf follicles; 105 cells were seeded per well with a number of granulosa cells representing 0, 5, 10, 20, 50 or 100% of the total. They were cultured in the presence of DHEA. Each treatment was performed in triplicate and the results are expressed in nanograms per 105 cells. Within a panel and by day, means (+SD) with different superscripts differ (P < 0.05; Newman-Keuls test). Note the different scales of ordinates in the two panels.
218
M.J. Bosc and Agn6s Nicolle
titles produced from day 1 to day 3 in a representative experiment.
Progesteroneproduction T h r e e main factors affected P4 production: the type of theca cells ( P < 0.001), the percentage of seeded granulosa cells ( P < 0 . 0 0 1 ) and the day of culture ( P < 0.001). For this last factor, the quantities of P4 recovered were greater on day 3 than on day 1 ( P < 0.001). T h e r e was a strong interaction between the type of theca cells and the percentage of granulosa cells ( P < 0.001). T h e addition of D H E A to the m e d ium had no or only a weak effect on P4 levels (P > 0.05). On day 1, m a x i m u m levels of P4 were observed with granulosa cells alone (P < 0.05 vs the other combinations). T h e respective quantities recovered in cultures of theca interna and extema cells alone represented on average 5.6 and 7.2% of the quantities measured in the culture of pure granulosa cells. In coculture with theca interna cells, P4 production was weak and its m a x i m u m (with the combination of 50% theca and granulosa cells) represented 16.8% of the granulosa cells alone. In co-culture with externa theca cells, the production of P4 was apparently proportional to the percentage of seeded granulosa cells. For 0, 10, 20, and 50% seeded granulosa cells, P4 levels represented on average 5.6, 17.1, 29.8 and 59.0% of the levels obtained with 100% of granulosa. These differences between the two types of co-culture are illustrated in Fig. 4(a).
On day 3, the greatest P4 production was always obtained with 100% granulosa ( P < 0.05 vs all other combinations) and the lowest with theca cells alone. On this day, the m e a n productions of externa and interna theca cells alone represented, respectively, 4.3 and 5.2% of that of granulosa cells. T h e interaction observed on day 1 was also seen on day 3 (Fig. 4(b)). With theca interna cells, the levels of P4 did not increase proportionally to the percentage of seeded granulosa cells. With 10, 20 or 50% seeded granulosa cells, m e a n P4 levels were, respectively, 9.0, 8.5 and 16.6% of the production of 100% granulosa. In comparison with externa theca cells, P4 production was markedly increased in the presence of granulosa cells. W h e n percentages of granulosa cells passed from 10 to 20 and to 50%, the m e a n rates of P4 were, respectively, 39.9, 71.4 and 87.7% of the P4 levels observed with granulosa cells alone.
DISCUSSION
T h e present study indicates that theca cells have two effects on the granulosa cells which differ according to the type of steroid production and the cell combination. Both types of theca cells have inhibited E 2 production after the first day of culture. By comparison with P4, the interna but not the externa theca cells have apparently inhibited its production by the granulosa as early as the first day of culture. Therefore the inner and outer parts of the theca have not only morphological but also functional differences. T h e s e results confirm the main features charac-
b- d a y 3
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Fig. 4. P r o g e s t e r o n e (P4) o n d a y s 1 ( p a r t a) a n d 3 ( p a r t b) i n c o - c u l t u r e s o f g r a n u l o s a cells w i t h i n t e r n a o r e x t e r n a t h e c a cells i n a r e p r e s e n t a t i v e e x p e r i m e n t . T h e ceils w e r e i s s u e d f r o m e C G - p r i m e d c a l f follicles; 10 s ceils w e r e s e e d e d p e r w e l l w i t h a n u m b e r o f g r a n u l o s a cells r e p r e s e n t i n g 0, 5, 10, 20, 50 o r 100% o f t h e total. T h e y w e r e c u l t u r e d in t h e a b s e n c e o f D H E A . E a c h t r e a t m e n t w a s p e r f o r m e d i n t r i p l i c a t e a n d t h e r e s u l t s a r e e x p r e s s e d i n n a n o g r a m s p e r l 0 s cells. W i t h i n a p a n e l a n d b y t y p e o f t h e c a cells, m e a n s ( + S D ) w i t h d i f f e r e n t s u p e r s c r i p t s d i f f e r ( P < 0.05; N e w m a n - K e u l s test). N o t e t h e d i f f e r e n t s c a l e s o f o r d i n a t e s i n t h e t w o p a n e l s .
Steroids in Co-cultures of Granulosa and Theca Cells terizing A4 and P4 productions by b o t h types of theca cells in vitro. T h e y showed a loss of 17~-hydroxylase activity and a persistence of the 3fl-hydroxy-steroiddeshydrogenase activity which are not modified by the addition of granulosa cells. With regard to E2 production, two points at least have to be considered in the light of the cooperation of the two types of cells. T h e first is related to theca and granulosa cells ctdtured alone. T h e c a cells did not produce E2, where, as granulosa cells produced it in significant amounts on days 1 and 3 of culture when they were in the presence of androgens. In the absence of androgens:, E2 decreased in 2 days to barely detectable levels. If the aromatizing capacity decreases in cultured granulosa cells [34-37], our results indicate that !it also depends on precursor availability, as was shown in previous studies [11, 38, 39]. T h e second point concerns the great differences of E2 levels observed between the beginning and the end of b o t h types of co-cultures. On day 1, granulosa cells produced E2 in significant but in different quantities according to the type of theca cells in co-cultures. T h e s e variations are easily explained by the differences of A4 production between the two types of theca cells; this production being greater in interna than in externa theca cells in vitro [21, 23]. T h e limiting factor of E2 production in our conditions was the androgen production of externa theca cells, and inversely, it was the n u m b e r of granulosa cells co-cultured with interna theca cells. Our results also stress the sensibility of granulosa and theca cells co-cultured with regard to E2 synthesis because only 5 or 10% of seeded granulosa cells are sufficient to get high levels of E2 on the first day. On day 3, in contrast, E2 levels were low in co-cultures with both types of theca cells, whereas they were maintained in granulosa cells alone in the presence of D H E A . This was particularly obvious when theca and granulosa cells were seeded in similar quantities. This apparent inhibition of E2 synthesis by granulosa cells raises several questions about the role(s) of theca cells. T h e first concerns the n u m b e r of granulosa cells which survived after 3 days. One m a y assume that their survival rate was not affected by the presence of theca cells. Indirect evidence of the presence of granulosa cells in our co-cultures is provided by the P4 concentrations observed in the co-cultures of granulosa and externa theca cells. P4 levels were m u c h higher in granulosa than in theca cell cultures. One m a y note that P4 concentrations were mostly related to the proportion of seeded granulosa cells rather than the n u m b e r of externa theca cells on day 1 as on day 3, indicating a good survival of granulosa cells in these conditions. T h e apparent inhibition of the aromatizing capacity observed in our systems is thus unlikely to be due to the absence of granulosa cells. A second question conceres the mechanism(s) which is (are) implicated in this inhibitory effect exerted by b o t h types of theca
219
cells. This effect is observed in the absence of any gonadotropin supplementation and in conditions which have allowed the secretion of large A4 quantities. T h e same results have also being obtained, after 8 days, on granulosa co-cultured with theca cells on the opposite sides of a collagen m e m b r a n e [19]. One may postulate that this inhibition of E2 synthesis is due to some agent(s) secreted by the theca cells. Preliminary results obtained with theca cell conditioned m e d i u m suggest such a possibility. H o w e v e r several observations have to be taken into account with regard to the possible mechanisms implicated in this interaction between granulosa and theca cells. In long-term co-culture on collagen m e m b r a n e , this inhibition ceases and E2 synthesis is stimulated by L H or by L H and F S H , whereas F S H alone has no effect [19]. In short-term culture on fibronectin, granulosa ceils maintain their aromatizing capacity for at least 4 days with appropriate doses of F S H and/or insulin [11]. In short-term cultures, a significant aromatizing activity can be observed in spite of low levels of m R N A encoding P450 aromatase in the presence or in the absence of F S H [39]. All these facts taken together indicate that, in vitro, the aromatizing capacity of the granulosa cells is regulated not only by the amounts of androgens produced by the theca cells but also by another type of cell interaction which could be counteracted by gonadotropins. In our conditions, P4 was secreted by all types of follicular cells, but the granulosa produced the largest amounts as observed previously [4, 6, 37]. T h e striking fact is the difference in P4 levels between the two types of theca cells in co-culture. As pointed out before, P4 production was a function of the proportion of seeded granulosa cells in co-cultures with theca extema cells. In these conditions, a large P4 increase was also noted from the beginning to the end of the experiments. In contrast, P4 levels remained low in co-cultures performed with interna theca cells. This p h e n o m e n o n was observed from the first day of culture at a time at which E2 levels were a function of seeded granulosa cells, the main contributor to P4 production. Therefore, it cannot be attributed to a rapid and a complete disappearance of granulosa cells. It also cannot be explained by an inhibition of P4 production by interna theca cells [4-6] because interna theca cells produced low amounts of P4 on the first day of culture [23]. Furthermore, it cannot be due to a transformation in A4 because its synthesis, which was not affected, is normally performed through the d 5 pathway [23, 40]. This inhibitory effect of granulosa P4 secretion, specific to interna theca cells, lasted 3 days in our conditions and it can be prolonged because it has been observed after 8 days in the absence or in the presence of gonadotropins [18, 19]. T h e reason for this inhibitory effect is unknown. In a previous study [23], we noted that after the addition of P4 as a substrate its recovery in
220
M . J. Bosc and Agn~s Nicolle
intema theca cell cultures was lower than the amount added to the medium. All these facts suggest that intema theca cells would be able to catabolize P4 or to secrete some P4 binding proteins. As pointed out before [23], the theca cells issued from eCG-primed calves behave in vitro as adult theca cells with regard to steroid production. T h e present results thus confirm that both theca cell preparations have a continuous functional 3fl-hydroxysteroid dehydrogenase activity and that they lose their 17~-hydroxylase activity in a 3 days culture system. This was illustrated by A4 levels obtained with or without D H E A . Three days of culture of theca cells with granulosa cells has not changed the functional characteristics of the theca cells. This result contrasts with long-term co-cultures, in which A4 production by theca cells was enhanced [19]. This discrepancy might, however, be explained by the differences in co-culture lengths. T h e cell preparations used in these experiments are certainly not entirely specific to the externa and the intema parts of the theca. However, these results illustrate that the different parts of the theca of a large follicle have not only morphological differences [41-43], but also functional differences with regard to their own steroid production [23] and to the local interactions which modulate the final follicular P4 production. In conclusion, the simple system of co-culture adopted in these experiments shows that E2 and P4 productions by granulosa cells can be altered in the presence of theca cells. Both types of theca cells have an inhibitory effect on E2 production whereas only the interna theca cells are able to alter P4 production. These results underline the fact that interna and externa theca cells from eCG-primed calves have functional differences with regard to steroid production by the granulosa cells; they show the necessity to take into account all the components of the follicle for the study of such local functions. Acknowledgements--We wish to thank the INRA institute which gave us the means to perform these studies. The antibodies raised against E2 and A4 were kindly provided by M. Terqui, and against P4 by the "Laboratoire des Dosages Hormonaux - - I.N.R.A ". We are indebted to J. L. Touzfi and his colleagues for animal care. We also thank D. Monniaux for her advice and her encouragement throughout the course of this work, and A. Y. Kermabon for her help in the preparation of the English version of this manuscript.
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