Oestradiol Exerts Disparate Effects on Ultrastructure and Steroidogenesis of Ovarian Theca from Preovulatory Hamster Follicles(Endocrinology)

8 Pages·1991·5.5 MB·English

by  Patrick H Kitzman

ZOOLOGICAL SCIENCE 8: 767-774 (1991) i lv»iM ZoologicalSocietyofJapan
Oestradiol Exerts Disparate Effects on Ultrastructure and
Steroidogenesis of Ovarian Theca from
Preovulatory Hamster Follicles
Patrick H. Kitzman1 and Rcinhold J. Hutz
Department of Biological Sciences, I 'nive.-yity of Wisconsin
Milwaukee, WI, USA
ABSTRACT—Oestrogens can induce atresia in vivo in mammals; and can diminish oestradiol-17/9
(OE:) secretion from granulosa cells in vitro though apparently not at the level of aromatase. We
therefore investigatedwhetherOE:exertsthelattereffect byinhibitingoestrogenprecursor(androgen)
synthesis at the level ofthe theca. Thecal shells were explained from ovarian follicles ofhamsters and
wereculturedin mediumalone (control)orin medium pluseitherOE2(1 /*g/ml)orOE;(1 //g/ml) plus
the OE:-antagonist. CI-628 (IX10 4M). OE2 induced a 48rr increase in lipid droplet number and a
4rir; decrease in mitochondrial numberascompared tocontrol. Although ultrastructural modifications
were observed, OE; did not alter progesterone (P4) or androstenedione (A4) secretion; unexpectedly,
CI-628 enhanced both. The OE:-induced increase in lipid droplet number in thecal shells iscorrelated
with atresia luteinization. Although mitochondrial number was reduced, the effect may not be
physiologically relevant since thecal P4 secretion was unchanged. The lack of effect on smooth
endoplasmic reticulumcorrelated with the unchanged thecal A4secretion, i.e.. substrate foraromati/a-
tion was unaltered. We therefore believe that the atretogenic effects ofOE: that we and others have
observed with respect to hamster ovarian follicles in vitro and in vivo, are not exerted at the level oi
thecal A4 production.
the ovary. In rat ovaries, the implantation of
INTRODUCTION
Silastic capsules containing oestrogen reduced the
Oestrogen is produced in the granulosa layer of number of ovulations in the treated ovary but had
the developing ovarian follicle and is necessary for no effect on the contralateral ovary [8J. In the
the maintenance of granulosa cells (GC) and fol- hamster, OE2 has been shown to alter morpholog)
licular follicle-stimulating hormone (FSH). ofthe apical follicular epithelium as observed with
luteinizing hormone (LH). and oestradiol-17,? SEM [9J. Hutz et al. [10] showed that OE:-
(OE:) receptors [I. 2]. A lack of OE2 induces containing Silastic implants induced atresia of the
atresia [3]. In the hypophysectomized. immature dominant follicle in monkeys. Analysis of the
female rat. oestrogen diminishes follicular atresia follicular contents showed a decrease in GC, dimi-
[4] and increases ovarian weight [1]. In granulosa nished steroidogenic capacity of GC, and reduced
cells, oestrogen has been shown to enhance cell- levels Ol both oestrogen and progesterone (P,) in
cell communication by increasing intercellular pro- follicular fluid. Hutzetal. [11. 12| demonstrated
cesses and gap junctions [5\. These effects plus that oestrogen exerted a negative elled on oes-
increases in FSH-induced adenylate cyclase and trogen secretion In hamster granulosa cells, but
aromatase [6], and cAMP-dependent protein that this effect was not at the level ol aromatase
kinase [7] point to folliculotropic effect(s) of ( )[ Oestrogen ma) affect granulosa cell viability 01 it
on GC. Paradoxically, it has also been shown that may exert its elfeet on an earlier step in the
trogen exerts a number of inhibitory effects on steroidogenic pathway, possibly causing a decrease
Accepted April 8, 1991 m the availability ol androgen precursors (<
Received November 20. 1990 androstenedione [AJ) needed for estrogen pro-
1 Present address: The Ohio State University. duction 113. 141.
768 P. H. KlTZMAN AND R. J. HlJTZ
Since the theca is the site of androgen produc- cated to the GC may be reduced.
tion and androgens are used as precursors for
aromatization to oestrogens, Silavin and Green-
MATERIALS AND METHODS
wald [15] hypothesized that the theca may be the
site at which atresia is initiated by OE2. Tonettaet
al. [16, 17] showed that OE2 inhibited P4 produc- Animals
tion at 36hr onward in culture. Leung and Arm- On the morning of prooestrus, 10 mature (3-6
strong [18] showed that in the rat ovary, direct months of age) female golden hamsters (Meso-
injection of OE2 decreased testosterone and cricetus auratus) were sacrificed under ether anes-
androstenedione accumulation while increasing thesia. Ovaries were removed and the 4-5 large,
alternate progestin metabolite accumulation (e.g., preovulatory follicles perovary were excised. The
5a-pregnanedione and 3a-OH-pregnan-20-one). follicles were cut in half and the granulosa cells
These researchers hypothesized that OE2 may act were gently scraped from the follicle. The thecal
at the level of the 17a-hydroxylase/Cn_20 lyase shells were then prepared for culture.
enzyme complex; thereby diverting C2rsubstrates
through alternate metabolic pathways leading to Cultures
accumulation of 5«-pregnane compounds. The thecal shells were placed in Falcon organ
In healthyratherthanatreticfollicles, thereisan culture dishes (VWR, Chicago, II) and randomly
inverse relationship between intraovarian concen- allocated to four groups. Each of the cultures
trations of androgens and oestrogens. High ratios containedfourthecalshells. Thethecalshellswere
of androgens to oestrogens in follicular fluid have incubatedfor72hrat37°C and5% C02inair; this
been associated with nonovulatory and atretic duration of incubation was based on our previous
follicles [19]. Terranova etal [20] showed that in studies [9, 11, 12]. Culture medium contained the
hamster follicles which are undergoing induced following constituents: Dulbecco's Modified
atresia, there is a shift from follicular OE2 to P4 Eagle's medium (DMEM) mixed one to one with
production. This shift in this OE2:P4 ratio may Ham's F-12 (Gibco, Grand Island, NY) sup-
resultin the loss ofavailable androgensfor aroma- plemented with heparin (1 U/ml), gentamycin
tizationandthusmaybeonefactorwhichmaylead (lOOng/ml), HEPES buffer (12.5 mM), hFSH
to the onset ofatresia. Since OE2 has been shown (lOOng/ml), and hamster serum (10%) sterilized
to alter thecal steroidogenesis it may cause this with a Millipore Filter (VWR Scientific, Chicago,
alteration by changing morphology of the struc- IL). The first group, which served as a control,
tures involved in steroidogensis it may cause this was cultured in medium alone. The second group
alteration by changing morphology of the struc- was cultured in medium plus oestradiol-17/?(OE2;
tures involved in steroidogenesis (e.g., lipid drop- Sigma, St. Louis, MO.; 1 ^g/ml). This concentra-
lets, mitochondria, and smooth endoplasmic re- tion has been shown by previous studies to have
ticulum). atretogenic effects on whole follicles. The third
Since OE2 does not appear to induce atresia at group was cultured in medium and OE2 (1 //g/ml;
the level of the granulosa cell, the focus of the 3.4X10~6M) plusCI-628(Sigma, St. Louis, MO.;
present study was to determine whether OE2 an OE2-receptor antagonist; lXlO~4M). This
causedanymorphologicchangesinthethecaatthe 30-fold excess of the antagonist was chosen in
TEM levelwhichmaybeindicativeofatresia. The orderto sufficiently block the effects ofOE2. OE2
TEM analysis focused on changes in thecal and CI-628 were dissolved in 100% ethanol, to
mitochondria, lipid droplets, and smooth endo- produce a final concentration in medium of
plasmic reticulum. All of these structures are <0.5% ethanoi. At the end of 72hr, the thecal
involved in steroid production. We also wished to shells were removed and processed for TEM (see
A
correlate changes in thecal morphology with below) or protein determination (see below).
changes in P4 and A4 production, since previous fourth group of thecal shells that was not incu-
studies suggest that oestrogen precursors translo- bated,wasalsoprocessedfortransmissionelectron
Thecal Alterations with Oestrogen 7fcQ
microscopy immediately along with control and (0. 1 5, 5, 7.5. 10. 20 ug) o( bovine
treatment groups. This fourth group served as albumin of known concentration (25 fig). Each
another control (time 0) to determine whether concentration was brought to a final volume of800
some component of the medium caused the mor- pA using a 0.99S NaCl solution. The thecal tissue
phologicchanges and not the OE:. The incubation was placed into a glass mortal containing 200/A in
medium was analyzed by radioimmunoassay for 0Mr( NaCl solution and homogenized with a
progesterone (P4) and androstenedione (A4) to Teflon pestle until completely dispersed. This
determine changes in steroid output (see below). process was conducted with the mortar immersed
in an ice bath. The contents of the mortar were
Transmission electron microscopy pipetted and placed into a standard test tube. Into
For TEM analysis, tissue was placed in 3rr each test tube 200//I oi Bio-Rad dye were added.
M
glutaraldehyde in 0.05 potassium phosphate. Each tube was then VOItexed and allowed to stand
pH 7.3 [21). The tissue was fixed for 1 1 2 hours at for at least 15 min to allow complete protein-dye
room temperature in corked shell vials. The tissue binding. The protein content of each tube was
was then postfixed for 1 hour at room temperature determined spcctrophotometrically (Coleman; us-
in 2rr osmium tetroxide (Os04) in 0.05 M potas- ing 1.6 ml microcuvettes [Fisher Scientific, Spring-
sium phosphate buffer. pH 7.3. Next, the tissue field, NJ.]). The lowest level of sensitivity of the
was dehydrated in a series of graded alcohols assay was 1.0/ug/ml. The interassay coefficient of
(10%-100% ethanol). After dehydration, the variation among five assays was 11.489?
.
tissue was embedded in Spurr's low-viscosity
epo\\ resin [22] and heated to 70°C for24 hr. The Radioimmunoassay
sample blocks were formed into truncated pyra- Aliquots ofincubation medium were fro/en and
mids and sectioned on a LKB ultramicrotome. subsequently analyzed for P4. A4 and OE:. Analy-
with glass knives made on a LKB knife breaker. ses for P4, A4, and OE: were carried out using
Thick sections were stained with \7c methylene rapid P4, A4. and OE: assay kits (Diagnostic
blue and NaOH. These thick sections allowed for Products. Corp., [Los Angeles, CA]). and a Pack-
orientation of the tissue for thin sectioning. Thin ard Multi-Prias autogamma spectrometer (Cour-
sections were placed on cleaned. 300-mesh copper tesy of Dr. W. Wehrenberg). The human P4. A4.
grids. The gridswere then exposed to95% alcohol and OE2 assays were validated for the hamstei
vapours for 1 hour and stained with uranyl acetate demonstrating parallelism between samples in se-
and lead citrate [23]. Finally, the grids were rial dilution vs. the standard curve. Interassay
examined in a Hitachi H-600 electron microscope variation of the P4. A4. and OE2 assa\s measured
at a magnification of 12.000x This magnification 4.2%, 5.2%, and 6.02% respectively. Interassa)
.
was chosen because all of the structures which variation for the A, assa\ was 9.1%. The lowest
were to be analyzed could be easily distinguished level o\ sensitivity of the P4 and A., assa\s was 0. 10
and measured at this magnification. Five random ng ml. Thesentitivit) oftheOE2assay was20pg
micrographs per follicle were taken. Pooled ml. P4. A1. and OE2 values were expressed as ng
micrographs for each of the groups were analyzed mg protein.
for the structures mentioned previously. The area
comprising the SER was analyzed by a Jandel Statistical analyses
Scientific digitizer (Courtesy of Dr. M. Boraas). The thecal shells from both ovaries were com-
Preliminary micrographs substantiated the purity bined and randoml) assigned to one of the four
of the thecal shells. treatment groups. Comparisons ol mitochondrial
Proiein determination cnoummpbleert.edP4,usainndg Aon•. ea-mwoanjg atnhaelytsoeusr gorlouvpasriWaenrcee
Because of the small amount of tissue used, the 1 \M)\ \i Groups were compared using Shi
Bradford microassay (24) was used. A standard dent-Newman-Keuls with p- 0.05 considered to
curve was developed using various concentrations be significant [25J. Comparison of lipid droplet
770 P. H. KlTZMAN AND R. J. HUTZ
number for the four groups was conducted after no difference existed in lipid droplet number be-
the data were transformed using log (X+1) trans- tween theca which were not cultured and theca
formation. The transformeddatawere analyzed in cultured in medium only (1.8+0.5 [12] vs 1.8+0.5
a manner similar to that for the analysis of [20]). OE2 induced a 48% decrease and a 46%
mitochondrial number. increase inmitochondrialnumberandlipiddroplet
number, respectively, as compared with control
RESULTS (Table 1). There was no difference in the amount
of SER between the OE2 and control groups.
Electron microscopy
Thecal shells treated with CI-628 started to de-
Both the control and OE2-treated thecal cells velop disruption of cell membranes and of cyto-
contained mitochondria with lamelliform cristae plasmic organelles; this disruption made quantita-
(compare Fig. 1A and B). The number of tive analysis of the organelles unfeasible in this
mitochondriain thecal shells thatwere immediate- group.
ly excised from the ovaries and processed, and in
the theca cultured in medium only for 72hr were Radioimmunoassay
not significantly different (18.9+2.0 [12] vs 19.4+ OE2 had no effect on the absolute secretion of
1.7 [20]; x number/63.5 cm2 of a standard micro- P4 as compared with control. Superimposition
graph+1 S.E.M. [n micrographs analyzed; each a upon OE2 of30-fold excess ofCI-628 increased P4
mean of3-5 determinations]; p<0.05). Similarly, secretion 180% over that ofboth control and OE2
p M
1 IB
Fig. 1. A) Transmission electron micrograph of a preovulatory hamster follicle incubated for 72hr in control
medium. Note the mitochondria (M) with lamelliform cristae and lipid droplets (L).
B)Transmissionelectronmicrographofapreovulatoryhamsterfollicleincubatedfor72hrinmediumcontaining
1jugOE2/ml. Notetheapparentincreaseinlipiddroplet(L)numberanddecreaseinmitochondrial(M)number.
Table 1. The effects of oestradiol-17/? on ovarian thecal ultrastructure
Area of Smooth
Treatment Mitochondrial Number1 Lipid droplet number1 Endoplasmic
Reticulum (jum2 1
)
Control 20.9+1.3 [20]a 0.3+0.1 [20]a 0.12+0.03 [4]a
OE2 10.9+1.1 [20]b 0.6+0.1 [20]b 0.12+0.01 [4]a
1 Mean number/63.2cm2 ofa standard micrograph+1 SEM (n micrographs, 3-5 random determinations
per follicle).
Different superscripts within a column denote significance (p<0.05).
~
Thecal Alterations with Oestrogen 771
With an augmented source of steroid precursor
(cholesterol) for conversion to the sex steroids
[26]. However, since OK: induced a decrease in
mitochondrial number, there were fewer o( the
organelles necessary for the conversion of
cholesterol to pregnenolone (a precursor for prog-
esterone); whether this has physiologic relevance
to Steroid synthesis in the present study in un-
known. In addition to mitochondrial number.
morphology is also important. Luteinizing cells oi
the rat follicle manifest a transformation of
1.000-- B
mitochondrial cristae from lamellilorm to the
0.900I tubular form [27]. In the hamster, mitochondria]
0.800
0.700f cristae are largely lamellilorm [28], while in the
0.600 human and bovine, mitochondrial cristae of luteal
0.500 cells are tubular [29]. Since the mitochondrion is
II 0.4O0-- the structure involved in side-chain cleavage [30],
0.300
an alteration of cristae morphology may change
0.200
the conversion ofcholesterol to pregnenolone. In
0.100|
0.000 the present study, however, both control and
OE
Fig. a2.ntagAo)niTsth.e CefIf-e6c2t8s.ofonOEp2roagnedstoefrothneeOsEec2r-erteicoenptboyr ther2e-ftorreeatwede etxhpeeccatctohnattatihneerde lwaomuelldlibleornmoccrhiastnagee;
explanted ovarian thecal shells from prooestrous in pregnenolone production. However, we did not
hamsters. OE2 (1 //g/ml); OE2 (1 //g/ml) plus CI- examine changes in pregnenolone secretion in this
628 (1X10 4M). study. Progesterone (the end product of 3,?-
h Different superscripts denote significance (p< hydroxysteroid dehydrogenase action on pregne-
0.05).
aBn)taTgohneiste.ffeCcIt-s62o8f,OoEn2Aa4ndsecorfettihoen ObyE2e-xrpelcaenptteodr nboeltowneee)nwcaosntarnoallyazneddOanEd2 tthreeraetmweanstnoondtihfefcearlencPe,
ovarian thecal shells from prooestrous hamsters. secretion. Previous studies have shown in the rat
Different superscipts denote significance (p< that OE increased 3/3-hydroxysteroid dehydro-
2
0.05). genase and decreased 17a-hydrox\lase in vitro,
thus increasing P, and decreasing A4 secretion
(p<0.05; Fig. 2). OE:. similarly, exerted noeffect [31]. In the present study. Ol exerted no effect
on absolute secretion of A4 as compared with upon either P, or A, secretion, presumably no!
control. Superimposition upon OE: of 30-fold affecting either aforementioned enzyme in ex-
excess of CI-628 increased A4 secretion 190Of and planted hamster theca. I his is corroborated In the
B19E over that of control and OE:. respectively (p overall attenuated effect OE2 has on hamster as
- 0.05; Rg. 2). opposed to rat ovarian cells [32].
Because of the apparent cellular destruction in
the CI-628-treated group, we were not able to
DISCUSSION
quantitate changes in subcellular structures here
This is the first studs to examine the effects of Since this was not a kinetic Study, W€ do not know
OE; on thecal shells ofexplanted hamsters ovaries the time-course Ol cellular destruction during the
at both ultrastructural and endocrine levels simul- 72-hr culture period. In previous studies. CI-628
taneously. In the present Study, OEj increased has been shown to be an Ol -rcceptoi antagonist
lipid droplet number but decreased mitochondrial whose antioestrogenic action results from deple-
number in theca. The increase in the concentra- tion of cytosol receptor sites for Ol (preSUIfl
tion of lipid droplets has been shown to correlate unoccupied nuclear receptors), and not from its
772 P. H. KlTZMAN AND R. J. HUTZ
OE OE
ability to block specific 2 neclear-receptorbind- thesis of 2 inducing atresia at the level ofthecal
A
ing [33]. Other studes have shown that CI-628 4 production. If CI-628 antagonized the known
depresses cytosol receptor levels and prolongs inhibitory effects of OE2 on the 17a-hydroxylase/
uterine insensitivity to oestrogen in immature rats C17_20 lyase complex [18, 31], then we might
[34]. The antioestrogens, as a group, also appear expect to see an increase in A4. In our study we
A
to bind to an additional antioestrogen binding site did observe a large increase in 4 secretion with
(AEBS) [35]. CI-628, but we did not see the characteristic de-
Theca were incubated with CI-628 and OE2 in crease in A4 substrates with OE2 as shown for the
combination as a control to determine whether rat [31]. This again points to radical differences in
these two compounds might unexpectedly exhibit OE2 responsiveness by hamster vs rat [32]. Alter-
an additive effect (where CI-628wouldagonistical- natively, as we have mentioned, CI-628 may actas
ly enhance the effects ofOE2 at the same receptor an agonist in our model and synergize or act
site) or synergistic effect (acting at AEBS) on additively wth OE2 on increasing A4 secretion.
steroidproduction. Thepresentstudyshowedthat The following is an attempt to reconcile the
the superimposition upon OE2 of30-fold excess of presentexperimentwithourpreviousstudies. Pre-
CI-628 enhanced both P4 and A4 secretion above viousstudieshaveshownthatOE2/diethylstilboes-
that of the theca incubated with OE2. A severe trol enhanced P4, while depressing OE2 secretion
augmentation of A4 might saturate the active from hamster granulosa cell cultures [11, 12]; but
site(s) on the aromatase enzyme and thus produce thatthedecreasein OE2secretionwasnotduetoa
a build up of androgens with atresia as a possible direct effect at the level of aromatase. We there-
consequence. Conversely, sinceHutzera/. [11, 12] fore hypothesized that synthesis of OE2 may de-
have shown that OE2 does not affect hamster GC crease 17a-hydroxylaseorlyase enzymes (reducing
aromatase activity directly, it remains to be seen androgen precursors) while increasing 3/?-
whether the androgen oestrogen ratio would in- hydroxysteroiddehydrogenase activity (and there-
:
crease (if the enzymes were saturated) or remain by increasing P4); this has been substantiated by
the same in light of enhanced androgen secretion others [31]. Since androgens and their precursors
from the theca. Carson et al. [19] found a shift in are produced by the theca for aromatization by
the androgen :oestrogen ratio in hamster follicle granulosa cells, we wished in the present study to
undergoinginduced atresia. Although atresiamay assess the effect of OE2 on thecal P4 and A4
be initiated in the theca [15], it is apparent from secretion. Since OE2 had no effect on these
our study that OE2 does not exert an atretogenic endocrinologic endpoints, we believe that OE2-
A
effect at the level ofthecal 4 secretion, although induced atresia is not operative atthese levels; but
ultrastructural elements may be altered. maybe operational at the level of 17«-hydroxylase
From these preliminary results, it appears that orCi7_20lyase. ThechangesobservedattheTEM
OE2 increased lipid droplets (which are correlated level remained unexplained. A distinct possibility
with cholesterol concentrations [36]). In previous is that OE2 compromises granulosa cell and/or
studies, an increase in lipid droplet concentration thecal viability; however, certainly the present
has been correlated with atresia and luteinization studyshowed thatbasal P4andA4wereunaffected
[37, 27]. The decrease in mitochondrial number by OE2, andwe have shown byvital dye exclusion
may not be physiologically relevant since P4 secre- that the viability of granulosa cells in culture was
tion was not changed. Since P4 was unaltered, not reduced with OE2. A further test of this
sufficient substrate for androgen synthesis was hypothesis is the evaluation ofthe abilityofgranu-
available. Additionally, since SER concentration losa cells and theca to respond to a LH/hCG
did not change, this would also explain the lack of stimulation challenge with normal increases in P4,
increase in A4 secretion, as SER area and A4 are OE2 and A4, respectively.
highlycorrelated (r=0.92; p<0.01; unpubl. data). Future studieswillinvolve evaluatingconcentra-
Since OE2 did not increase A4 secretion (which is tions of pregnenolone and 17a-hydroxypro-
characteristic of atresia), this negates our original gesterone (and their metabolizing enzymes), to
Thecal Alterations with Oestrogen 773
further Investigate the possible locus (loci) at tions in adult rats; Direct action on the ovary. Biol.
which OE may affect theca. Additional studies Reprod.. 29: 114^-1154.
:
will utilize eo-incubation oi theca and granulosa 9 Hut/. R. J.. Schaller, M.. Kit/man. P. H. and
cells, and the use of4-hydrox\-tamoxifen (a highly Bejvan, s. M. (1991) Estradiol-17£minimally alters
specific OE:-receptor antagonist), to compare re- ovivaabriiliatn) faolnldiclpersoginesvtietrroo.neZoaoclc.umSucil.a.ti8:on81ol8h8.amster
sults achieved with CI-628. 10 Hut/. R. .1. Dierschke. D. J. and Wolf. R. C.(1986)
Markers o\ atresia in ovarian follicular components
from rhesus monkeys treated with estradiol-17/9.
ACKNOWLEDGMENTS
Biol. Reprod., 34: 65-70.
The authors with to thank Dr. S. Raiti for supplying 11 Hut/. R J . Cold. D. A. and Dierschke. D .1
the hFSH: Dr. Martin Black oiWarner Lambert Parke- (1987) Diminished steroidogenic response ol hams
Davis Pharmaceutical Co.. Ann Arbor. MI. for the ter granulosa cells to estrogen in vitro. Cell TlSS.
CI-62S: toMs. LeslieQuandt forassistancewithradioim- Res.. 248: 531-534.
munoassay and to Ms. Dorothy Perry and Ms. Alnita 12 Hutz. R. J.. Kruegcr. (i. S.. Mcllcr. P A . Sholl. S
Allen for preparation of this manuscript. Support was A. and Dierschke. D. J. (1987) FSH-mduccd aro
provided in part, by an Endocrine Society Fellowship to matasc activity in hamstergranulosa cells: Effectsof
P.H.K.. Sigma \i. and the UWM Graduate School and estradiol-17,ym vitro. CellTiss. Res. 250: 101-104.
College of Letters and Science. Portions of this paper 13 Hunter. M. G. and Armstrong, I) I. (1987) Oes-
were presented at the American Endocrine Societ) trogens inhibit steroid production by dispersed por-
Annul Meeting, June 20-23. L990, Atlanta. GA. USA. cine thecal cells. Mol. Cell. Endocrinol., 50: 165-
170.
14 Tsang. B. K.. Leung. P. C. K and and Armstrong,
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