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JOURNAL OF EXPERIMENTAL ZOOLOGY 284:91–99 (1999)
Evidence for Two-Cell Model of Steroidogenesis in
Four Species of Amphibian
RYUN S. AHN, MYUNG S. YOO, AND HYUK B. KWON*
Hormone Research Center and Department of Biology, Chonnam National
University, Kwangju 500-757, Republic of Korea
ABSTRACT
Previously, based on studies conducted using Rana nigromaculata, a two-cell model
involving the theca and granulosa cells was proposed to account for the steroidogenic activity of
amphibian ovarian follicles. Experiments were carried out to ascertain whether the model was
applicable to four other frog species with different reproductive cycles (R. dybowskii, R. rugosa, R.
catesbeiana, and Bombina orientalis). Ovarian follicles were collected from each species and manually microdissected to obtain various follicular components: theca-epithelium (THEP) and granulosa cell-enclosed oocyte (GCEO). Subsequent to collection, equal numbers of intact follicles and
various follicular components were cultured for 6 hr in the presence of known inducers of steroidogenesis (frog pituitary homogenate [FPH] or 3-iso-butyl-1-methylxanthine [IBMX] + forskolin) or
various steroids that serve as substrates for specific steroidogenic enzymes. Following incubation,
culture medium was collected and analyzed by radioimmunoassay (RIA). Both FPH and IBMX +
forskolin consistently stimulated secretion of androstenedione (AD), testosterone (T), and estradiol (E2) from intact follicles obtained from all four frog species. Additionally, in R. dybowskii,
these treatments stimulated secretion of progesterone (P4) and 17α-hydroxyprogesterone (17αOHP4) into the culture medium. Intact follicles obtained from all species readily converted pregnenolone (P5), P4, and 17α-OHP4 to AD, T, and E2. In contrast GCEO converted P5, P4, and 17α-OHP4
to AD and E2, but not to T. However, AD, but not P5, P4, or 17α-OHP4, was converted to T when
cultured in the presence of isolated THEP. The microdissection procedure was also modified to
isolate THEP without contaminating granulosa cells. The steroidogenic capacities of “impure” THEP
and “pure” theca-epithelium (P-THEP) were then compared. Basal amounts of P4 were produced
when P5 was added to P-THEP, whereas significantly higher amounts were produced in the presence of impure THEP. No significant conversion of P5 or P4 to 17α-OHP4 occurred following culture
with pure or impure THEP layer. Results suggest that the enzyme activity necessary to metabolize AD → T is localized in the THEP, whereas the metabolic capacities to convert P5 → AD and T
→ E2 are present in the granulosa cell. Furthermore, the data show that the two-cell model is
applicable to other frog species. J. Exp. Zool. 284:91–99, 1999. © 1999 Wiley-Liss, Inc.
Amphibian ovarian follicles consist of a prophase-arrested oocyte and somatic components,
which are considered to be the site of steroid production (Schuetz, ’74; Maller, ’85). Among the steroids produced in response to gonadotropin by
somatic components of ovarian follicles, estradiol
(E2) and progesterone (P4) play key roles in oocyte growth and maturation (Wallace and Bergink,
’74; Masui and Clarke, ’79; Schuetz, ’85; Wallace,
’85). Somatic components of the amphibian follicle
consist of three major cell layers: a surface epithelium, an outer theca layer, and an inner granulosa cell layer (Masui, ’67; Schuetz, ’74). Although
the relative roles of theca and granulosa cells on
steroidogenesis are well established in mammals
(reviewed by Gore-Langton and Armstrong, ’88),
chickens (Bahr et al., ’83; Wang and Bahr, ’83),
© 1999 WILEY-LISS, INC.
and fish (Young et al., ’86; Nagahama, ’87), only
limited information is available concerning the involvement of these cell layers with steroidogenesis in amphibian follicles. In Rana pipiens
follicles, granulosa cells were shown to produce
P4 in response to gonadotropin and convert 25OH-cholesterol to P4 (Schuetz and Lessman, ’82;
Petrino and Schuetz, ’87).
We subsequently determined in R. nigromaculata that theca and granulosa cells had differ-
Grant sponsor: Korea Science and Engineering Foundation; Grant
number: HRC-96-0101; Grant sponsor: Ministry of Education of Korea; Grant number: BSRI-95-4425.
*Correspondence to: Dr. Hyuk B. Kwon, Hormone Research Center
and Department of Biology, Chonnam National University, Kwangju
500-757, Republic of Korea.
Received 6 April 1998; Accepted 28 September 1998.
92
R.S. AHN ET AL.
ent roles in follicular steroidogenesis and proposed
a two-cell type model to explain our results. In
this model, granulosa cells are the main cellular
source for P4, 17α-hydroxyprogesterone (17αOHP4), androstenedione (AD), or E2 whereas the
theca/epithelium (THEP) layers produce testosterone (T; Kwon and Ahn, ’94).
However, this two-cell type model was based on
data from one species of Rana (R. nigromaculata).
Moreover, it was unclear what role the theca layers played in conversion of pregnenolone (P5) to
17α-OHP4, because we subsequently observed that
variable numbers of granulosa cells remained attached to isolated THEP during the procedure for
collecting the THEP. Thus, experiments reported
here were carried out to ascertain whether the
two-cell type model was applicable to other frog
species. Additionally, the follicular microdissection
procedure was modified to eliminate granulosa cell
contamination of the THEP and used to assess
whether “impure” and “pure” THEP preparations
differ in their steroidogenic capacity to convert P5
to 17α-OHP4.
MATERIALS AND METHODS
Animals
Most frogs (R. dybowskii, R. rugosa, R. catesbeiana, and Bombina orientalis) were collected
from fields, ponds, or streams in the Chonnam
area, a southwestern region of the Korean peninsula. Full-grown follicles were obtained from various frog species collected during different periods:
R. dybowskii from October to February (1.5–1.8
mm in diameter), R. rugosa and B. orientalis from
May to July (1.4–1.5 mm and 1.8–2.0 mm, respectively), and R. catesbeiana from June to July (1.2–
1.3 mm). Medium-sized follicles were also utilized
for our experiments, since it was previously demonstrated they secreted E2 in response to gonadotropin stimulation (Kwon et al., ’91, ’93). The
seasons for collecting medium-sized follicles varied with species. Such follicles were collected from
R. dybowskii from August to September and from
R. rugosa, R. catesbeiana, and B. orientalis from
July to August. Frogs were kept in plastic boxes
containing tap water, maintained at room temperature, and used for experiments within three
days of collection.
Culture of follicular components
After animals were killed by decapitation, ovaries were removed immediately and divided into
fragments in amphibian Ringer’s solution (AR).
Intact follicles (IFs), granulosa cell-enclosed oocytes (GCEOs), THEP layers, and granulosa cell
free pure THEP layer (P-THEP) were separated
from ovarian fragments by manual dissection under a dissecting microscope. The detailed microdissection technique was described by Schuetz and
Lessman (’82). Using fine watchmaker’s forceps,
the outer THEP layer with blood vessels was
peeled from the ovarian follicle. This procedure
produced GCEOs that were simultaneously separated from the THEP layers. Most granulosa cells
usually remain attached to the oocyte membrane
rather than to the THEP layer. However, a small
number of granulosa cells were found in THEP
layers. Thus, to obtain pure THEP layers free of
granulosa cells, the outside of the basal laminar
layer was peeled from the follicle. The presence
of the basal laminar in isolated GCEOs was
readily detected, since the surface of GCEOs with
the basal laminar has a shiny appearance under
a stereomicroscope while that of GCEOs without
the laminar appears rough.
Equal numbers of the different types of follicular
components were cultured for 6 hr in AR in the
presence or absence of frog pituitary homogenate
(FPH; 0.05 gland/mL), various steroid precursors
(P5, P4, 17α-OHP4, AD or T; 100 ng/mL each) or 3iso-butyl-1-methylxanthine (IBMX, 0.27 mM) +
forskolin (9 µM). Steroid production by isolated follicular tissues of R. rugosa was stimulated by treatment with IBMX (0.27 mM) + forskolin (9 µM)
instead of FPH because R. rugosa ovarian follicles
produced steroids in response to IBMX (0.27 mM)
+ forskolin (9 µM) better than FPH (Kwon et al.,
’90). The FPH was prepared from female frogs collected during the corresponding experimental periods (Kwon et al., ’91). All steroids were purchased
from Sigma Chemical Co. (St. Louis, MO). The duration of follicle culture and the doses of FPH or
exogenously added steroids were chosen on the basis of previous data from several species of Rana
(Ahn et al., ’93; Kwon et al., ’93; Kwon and Ahn,
’94). Different types of follicular components were
distributed into 24 well tissue culture dishes (Nunc,
Roskilde, Denmark) in each experiment. Each culture well contained 10 components in 1 mL of AR.
Culture dishes were placed in a shaking incubator
(24°C) at 80 oscillations per minute for 6 hr. After
culture, media were saved and kept in a deep freezer
(–40°C) for steroid radioimmunoassay (RIA).
Steroid RIA
Amounts of steroids (P4, 17α-OHP4, AD, T, and
E2) contained in the culture medium were mea-
TWO-CELL TYPE MODEL OF STEROIDOGENESIS IN AMPHIBIAN
sured by RIA. General assay procedures were
adapted from those described by Fortune (’83) and
utilized in previous studies (Kwon et al., ’91; Kwon
and Ahn, ’94). Labeled P4 ([1,2,6,7-3H]-progesterone; 99 Ci/mmol), 17α-OHP4 ([1,2,6,7-3H]-hydroxy
progesterone; 58.5 Ci/mmol), T ([1,2,6,7-3H]-testosterone; 98 Ci/mmol), and E2 ([2,4,6,7-3H]-estradiol; 108 Ci/mmol) were obtained from Amersham
(Buckinghamshire, England). Labeled AD ([1,2,
6,7-3H] -androstenedione; 86.1 Ci/mmol) was purchased from New England Nuclear (Boston, MA).
The steroid antisera were produced and evaluated
by Dr. Y.D. Yoon (Hanyang University, Seoul). To
validate the RIA procedure for measurement of
specific steroid in the presence of exogenous precursors, 100 ng/mL of various steroid precursors
was added to culture medium (AR) and aliquots
(100 µl) were analyzed for a specific steroid by
RIA. Results from such analysis thus provided a
means of assessing the effects of cross-reactivity
and served as an additional experimental control.
The nonspecific binding values obtained are represented in the figures as blanks. Cross reactivities of antisera with other steroids have been
described in a previous report (Kwon and Ahn,
’94). Each sample was quantified for tritium using a Packard Tri-Carb 1500 liquid scintillation
analyzer. Routinely, duplicate steroid standards
were included in each assay (P4: 12.5–2,000 pg,
17α-OHP4, AD, T, or E2: 2.5–500 pg). Steroid concentrations were calculated on a microcomputer
using SecuRIA software (Packard, Downners
Grove, IL). The between- and within-assay coefficients of variation (CVs) for P4 were 9.2% and
8.5%, respectively. The CVs for 17α-OHP4 were
8.2% and 6.7%; for T, 9.4% and 7.4%; for E2, 10.1%
and 8.7%; and for AD, 8.4% and 7.2%, respectively.
The lower limit of assay sensitivity for P4 was 12.5
pg, and for 17α-OHP4, T, E2, and AD, it was 5 pg.
93
obtained from R. dybowskii. FPH stimulated P4
production by IFs (725 pg/follicle) and GCEOs (436
pg/follicle) but failed to stimulate P4 production
by THEP layers (36 pg/follicle) (Fig. 1). Similar
amounts of P4 were produced by GCEOs (2,143
pg/follicle) and IFs (2,346 pg/follicle), whereas the
THEP layers produced the least amounts of P4
(495 pg/follicle) in the presence of exogenous P5
(Fig. 1) (P < 0.01, when compared with GCEOs).
Without addition of steroid precursors or FPH,
very low or nondetectable levels of P4 were produced by all the follicular components examined
(<20 pg/follicle). Clearly, GCEOs are much more
efficient than THEP layers in converting P5 to P4
(P < 0.01, by two-way ANOVA).
Production of 17a-OHP4 and P5 or P4
conversion to 17a-OHP4 by different types
of follicular components of
R. dybowskii follicles
The abilities of the different types of follicular
components to produce 17α-OHP4 and convert P5
or P4 to 17α-OHP4 were examined. The FPH alone
stimulated 17α-OHP4 production by GCEOs (394
pg/follicle) and by IFs (623 pg/follicle) but failed
Statistical analysis
Statistical analysis of data included one or
two-way analysis of variance (ANOVA) or Student’s t-test.
RESULTS
P4 production and P5 to P4 conversion by
different types of follicular components of
R. dybowskii follicles
Initially, experiments were carried out to monitor the capacity to produce P4 in response to FPH
and convert exogenously added P5 to P4 during in
vitro culture of the various follicular components
Fig. 1. Conversion of exogenous P5 to P4 by different
types of follicular components of R. dybowskii. The IFs,
GCEOs, and THEP layers were obtained from full-grown
follicles and cultured for 6 hr in the presence or absence of
P5 (100 ng/mL) or FPH (0.05 gland/mL). Concentrations of
P4 in the medium were measured by RIA. The P4 levels in
AR in the presence of exogenous P5 or FPH were also measured to check the cross reactivity of P4 antiserum (blank).
Each bar in the figure represents the average number of
picogram (mean ± SEM) of P4 per follicle (n = 9, three incubations per animal, three animals; n = 10, in blank). N.D.
indicates nondetectable. **P < 0.01, when compared with
GCEO or IF.
94
R.S. AHN ET AL.
to stimulate 17α-OHP 4 production by THEP
preparations (43 pg/follicle) (Fig. 2). Exogenous
P4 was converted in large quantities to 17α-OHP4
by GCEOs (1,118 pg/follicle) and IFs (1,333 pg/
follicle) and to a much smaller extent by THEP
layers (289 pg/follicle; Fig. 2; P < 0.01, when compared with GCEOs). Significant amounts of exogenously added P5 were converted to 17α-OHP4
by GCEOs (668 pg/follicle) and IFs (1,183 pg/follicle) but not by THEP layers (62 pg/follicle; Fig.
2). Without addition of steroid precursors or FPH,
low or nondetectable levels of 17α-OHP4 were produced by the follicular components examined (<20
pg/follicle). Thus, it is clear that GCEOs were considerably more efficient than THEP layers in mediating 17α-OHP4 production (P < 0.01).
Conversion of P5 to 17a-OHP4 by THEP and
P-THEP layer of R. dybowskii
THEP, and P-THEP layers from ovarian fragments
of R. dybowskii were cultured for 6 hr in the presence or absence of exogenous P5 or P4 (100 ng/mL
each). Addition of exogenous P5 resulted in a marked
increase in P4 levels by GCEOs (3,350 pg/follicle)
and IFs (3,477 pg/follicle) but lesser amounts by
THEP layers (672 pg/follicle). The P-THEP layer
produced 17α-OHP4 (134 pg/follicle) (Fig. 3A), an
amount significantly less than that produced by
THEP (P < 0.01). Following P4 addition, high levels
of 17α-OHP4 were present in cultures containing
GCEOs (844 pg/follicle) or IFs (901 pg/follicle), while
lesser amounts were produced by isolated THEP
layers (310 pg/follicle) or P-THEP layers (283 pg/
follicle; Fig. 3B). Likewise, P5 addition produced high
levels of 17α-OHP4 by IFs or GCEOS, but negligible levels by THEP or P-THEP.
An experiment was carried out using the granulosa-cell-free THEP layer (P-THEP) of R. dybowskii follicles to ascertain whether the THEP layer
contains the enzyme activities required to convert
of P5 or P4 to 17α-OHP4. Isolated IFs, GCEOs,
Fig. 2. Conversion of exogenous P4 to 17α-OHP4 by different types of follicular components of R. dybowskii. The
IFs, GCEOs, and THEP layers were obtained from full-grown
follicles and cultured for 6 hr in the presence or absence of
P5 or P4 (100 ng/mL) or FPH (0.05 gland/mL). The 17α-OHP4
levels in AR in the presence of exogenous P4, P5 or FPH
were measured to check the cross reactivity of 17α-OHP4
antiserum (blank). Each bar in the figure represents the
average number of picograms (mean ± SEM) of 17α-OHP4
per follicle (n = 9, three incubations per animal, three animals; n = 10, in blank). **P < 0.01, when compared with
GCEO or IF.
Fig. 3. Conversion of exogenous P5 to P4 by THEP and
P-THEP layers of R. dybowskii. The THEP and P-THEP were
obtained from full-grown follicles and cultured for 6 hr in
the presence of P5 (100 ng/mL). The P4 and 17α-OHP4 levels in AR in the presence of exogenous P4, P5 or FPH were
measured to check the cross reactivity of P4 or 17α-OHP4
antiserum (blank). Each bar in the figure represents the
average number of picograms (mean ± SEM) of steroid per
follicle (n = 9, three incubations per animal, three animals;
n = 10, in blank). *P < 0.01, when compared with THEP;
**P < 0.01, when compared with GCEO or IF.
TWO-CELL TYPE MODEL OF STEROIDOGENESIS IN AMPHIBIAN
95
Endogenous production of AD and
17a-OHP4 conversion to AD
Conversion of 17α-OHP4 to AD by the different
follicular components was also examined in the
four frog species. Isolated IFs, GCEOs, or THEP
layers from ovarian fragments (R. dybowskii, R.
rugosa, R. catesbeiana, and B. orientalis) were cultured for 6 hr in the presence or absence of exogenous steroid precursors (P5, P4, or 17α-OHP4, 100
ng/mL each), FPH (0.05 gland/mL), or IBMX (0.27
mM) + forskolin (9 µM).
Although the absolute amounts of AD produced
were different, FPH or IBMX + forskolin alone
stimulated AD production by GCEOs (110–394 pg/
follicle) and IFs (44–623 pg/follicle) but not by the
THEP layers in any of species examined (nondetectable; Fig. 4). In R. dybowskii, 17α-OHP4 was
converted, in varying amounts, to AD by GCEOs
(1,415 pg/follicle) or IFs (561 pg/follicle) but not at
all by the THEP layers (nondetectable; Fig. 4; P <
0.01, THEP vs. GCEOs). Additionally, considerable
amounts of AD were produced by GCEOs and IFs
(138–497 pg/follicle) in the presence of exogenous
P5 or P4 but not by THEP layers (nondetectable).
The amounts of endogenous AD secreted by the
follicular tissues of the three other frog species varied, and exogenously added 17α-OHP4 was converted to AD by GCEOs (220–1,725 pg/follicle) and
IFs (220–1,796 pg/follicle) but not THEP layers
(nondetectable). Likewise, P5 or P4 was converted
in significant amounts to AD by GCEOs (55–1,107
pg/follicle) and IFs (20–402 pg/follicle) but not the
THEP layers (nondetectable; Fig. 4). Thus, it is clear
that GCEOs and IFs were capable of synthesizing
AD endogenously or of converting 17α-OHP4 to AD,
whereas the THEP layers neither secreted AD nor
converted 17α-OHP4 to AD.
Endogenous production of T and
AD conversion to T
Use of FPH or IBMX + forskolin alone stimulated T production by IFs (250–963 pg/follicle) but
failed to stimulate T production by THEP layers
(nondetectable) or GCEOs (65 pg/follicle) in all
species examined (Fig. 5). The amounts of endogenous T produced varied with species but exogenously added AD was converted in large amounts
to T by THEP layers (1,975–6,263 pg/follicle) and
IFs (1,564–3,012 pg/follicle), and only to a limited
extent by the GCEOs (270–339 pg/follicle; Fig. 5;
P < 0.01, GCEO vs. THEP layers). Considerable
amounts of T were also produced by IFs in the
presence of P5, P4, or 17α-OHP4 (157–1,692 pg/
Fig. 4. Conversion of exogenous 17α-OHP4 to AD by different types of follicular components from four frog species.
The IFs, GCEOs, and THEP layers were obtained from fullgrown follicles and cultured for 6 hr in the presence or absence of 17α-OHP4 or other precursors (P5 or P4; each at 100
ng/mL), FPH (0.05 gland/mL), or IBMX (0.27 mM) + forskolin
(9 µM). The AD levels in AR in the presence of exogenous P5,
P4, 17α-OHP4, or FPH were also measured to check the cross
reactivity of AD antiserum (blank). Each bar in the figure
represents the average number of picograms (mean ± SEM)
of AD per follicle (n = 9, three incubations per animal, three
animals; n = 10, in blank). **P < 0.01, when compared with
GCEO or IF.
follicle), but very low or nondetectable levels of T
were produced by THEP layers or GCEOs (10–
100 pg/follicle; Fig. 5). These data demonstrate
that THEP layers failed to produce T in response
to FPH or IBMX + forskolin alone but were much
more efficient than GCEOs in converting exogenous AD to T (P < 0.01).
96
R.S. AHN ET AL.
Fig. 5. Conversion of exogenous AD to T by different
types of follicular components of four frog species. The IFs,
GCEOs, and THEP layers were obtained from full-grown
follicles and cultured for 6 hr in the presence or absence of
AD or other precursors (P5, P4 or 17α-OHP4; each at 100
ng/mL), FPH (0.05 gland/mL), or IBMX (0.27 mM) + forskolin
(9 µM). The T levels in AR in the presence of exogenous P5,
P4, 17α-OHP4, AD, or FPH were measured to check the cross
reactivity of T antiserum (blank). Each bar in the figure
represents the average number of picograms (mean ± SEM)
of T per follicle (n = 9, three incubations per animal, three
animals; n = 10, in blank). **P < 0.01, when compared with
THEP or IF.
Endogenous production of E2 and T
conversion to E2
In general, the amounts of endogenous E2 produced or converted by the follicular tissues from
R. dybowskii, R. rugosa, and B. orientalis were
much higher than those of R. catesbeiana (Fig.
6). Furthermore, FPH or IBMX + forskolin significantly stimulated E 2 secretion in GCEOs
(28–180 pg/follicle) and IFs (53–451 pg/follicle),
whereas nondetectable amounts of E2 were produced by THEP layers (Fig. 6). Addition of AD
Fig. 6. Conversion of exogenous T to E2 by different types
of follicular components of 4 frog species. The IFs, GCEOs,
and THEP layers were obtained from medium-sized follicles
and cultured for 6 hr in the presence or absence of steroid
precursors (AD or T; 100 ng/mL each), FPH (0.05 gland/mL),
or IBMX (0.27 mM) + forskolin (9 µM). The E2 levels in AR
in the presence of exogenous AD, T, or FPH were also measured to check the cross reactivity of E2 antiserum (blank).
Each bar in the figure represents the average number of picograms (mean ± SEM) of E2 per follicle (n = 9, three incubations per animal, three animals; n = 10, in blank). **P < 0.01,
when compared with GCEO or IF.
or T resulted in a marked increase in E2 levels
in the presence of GCEOs (147–551 pg/follicle)
and IFs (98–887 pg/follicle) but not THEP layers
(<20 pg/follicle) isolated from the four species (Fig.
6). In R. catesbeiana, although amounts of converted E2 were much lower than the other species, relatively higher levels of E2 were produced
by IFs (27 pg/follicle) and GCEOs (22 pg/follicle)
TWO-CELL TYPE MODEL OF STEROIDOGENESIS IN AMPHIBIAN
than by THEP layers (nondetectable; P < 0.01,
THEP vs. GCEOs). Likewise, addition of AD increased E2 levels by GCEOs (28–226 pg/follicle) and
IFs (104–627 pg/follicle) but not by THEP layers
(nondetectable; Fig. 6). Without addition of steroid
precursors or hormone, low levels or nondetectable
levels of E2 were produced by the follicular components of all species examined. Thus, it is clear that
GCEOs were more efficient than THEP layers in
production of E2 (P < 0.01).
DISCUSSION
The present data clearly show that granulosa
cells are the principal source of P4, 17α-OHP4, AD,
and E2, whereas the combined THEP layers are
the only cells capable of converting AD to T in
amphibian ovarian follicles. Moreover, we found
that the THEP layer does not contain the enzymatic capacity to convert P5 to 17α-OHP4 (3β-HSD
and 17α-hydroxylase).
If we assume that FPH induces de novo synthesis of steroid, precursor(s) for P4, such as P5
will be synthesized by granulosa cells rather than
THEP layer because high levels of P4 were produced by GCEOs but not by THEP layers in the
presence of FPH (Fig. 1). This indicates that
granulosa cell also carry the enzyme activity necessary for cholesterol metabolism to P5 (such as
cholesterol side chain cleavage). However, we
could not confirm whether granulosa cells contain
the enzyme activity necessary for cholesterol metabolism as evidenced by the conversion of cholesterol or its derivative (25-OH-cholesterol) to P4
(data not shown).
After addition of exogenous P5, high levels of
P4 were produced by GCEOs and IFs but not by
THEP layers (Fig. 1), whereas the actual amounts
of P4 produced by P-THEP layers were negligible
(Fig. 3A). This finding indicates that the granulosa cells exclusively contain the 3β-hydroxysteroid dehydrogenase (3β-HSD) enzyme. Moreover,
exogenously added steroid precursors (P5 or P4)
were readily converted to high levels of 17αOHP4 by GCEOs but not by THEP and P-THEP
layers (Figs. 2 and 3B), clearly indicating that
granulosa cells also contain 17α-hydroxylase activity. Although, the amounts of steroids produced varied with each species, exogenously
added steroid precursors elevated levels of AD
and E2 produced by GCEOs but not THEP layers (Figs. 4 and 6). These results indicate that
granulosa cells also contain the C17, 20-lyase and
aromatase activity. Strikingly, only the THEP
layer converted AD to T (Fig. 5) indicating that
97
the theca layer contains the 17β-hydroxysteroid
dehydrogenase (17β-HSD).
Amounts of AD and T produced by full-grown
follicles or their components obtained from different species of frogs varied markedly (Figs. 4 and
5) and appears to be related to differences in follicle sizes. As the size of full-grown follicles of four
species of frogs are in the range of 1.2–2.0 mm in
diameter, the number of granulosa and theca cells
may be different among different species of frogs.
However, in the case of E2, the amounts produced
by the follicles during culture are associated with
the stage of follicle development rather than follicle size (Fig. 6). Previously we found that E2 was
produced by follicles only at their early phase of
growth, just prior to reaching medium sized follicles (Kwon et al., ’91, ’93).
On the basis of present data, we propose a modified two-cell type model for amphibian follicular
steroidogenesis, which is depicted in Figure 7. This
two-cell type model is basically the same as the
previous model proposed with R. nigromaculata.
However, we can now conclude with considerable
confidence that the theca layer lacks the ability
to convert P5 to 17α-OHP4 because of the lack of
the appropriate enzymes (3b-HSD and 17α-hydroxylase). Thus, granulosa cells play a major role
Fig. 7. A modified two-cell type model for follicular steroidogenesis in frogs. Granulosa cells are main sites for
progesterone, 17α-hydroxyprogesterone, androstenedione, and
estradiol synthesis, whereas the theca cells are the primary
source for testosterone (androstenedione metabolism).
98
R.S. AHN ET AL.
in follicular steroid production and regulation in
frogs because granulosa cells contain many important enzymes (17α-hydroxylase, C17, 20-lyase, and
aromatase) that are responsible for the shift in steroidogenesis that occurs during follicular development in Rana (Ahn et al., ’93; Kwon et al., ’93).
Interestingly, the cooperative interaction between
the two types of cells in mediating follicular steroid
production is very similar to that observed in some
teleost fishes and mammals. Theca layers contribute to E2 production by synthesizing androgens (AD
and T) which are then aromatized by granulosa cells
to E2 in fishes (Kagawa et al., ’82; Nagahama and
Adachi, ’85) and mammals (reviewed by GoreLangton and Armstrong, ’88). Also, in some other
fish (Fundulus heteroclitus), the theca layer has a
negligible role in follicular steroidogenesis, while
granulosa cells alone can produce E2 and T following gonadotropin stimulation (Petrino et al., ’89).
However, our model is not consistent with the twocell type model for the elasmobranch, Squalus
acanthias, in which granulosa cells produce large
amounts of P4, T, and E2 while theca cells have a
very limited capacity to produce these steroids
(Tsang and Callard, ’92). Thus, it seems that the
relative roles of theca and granulosa cells differ
among the various species.
Likewise, in hens, granulosa cells were found
to produce progestins, which are converted to T
and eventually aromatized to E2 by theca cells
(Huang et al., ’79; Bahr et al., ’83). Other investigators have linked different steroidogenic enzyme
functions to theca externa and interna cells and
have proposed three- or multi-cell models for mediating steroidogenesis in avian follicles (Porter
et al., ’89; Nitta et al., ’91). In the multiple cell
type model, theca interna produce progestins and
androgens, whereas the theca externa synthesize
androgens and estrogens. Consequently, it appears
that granulosa and theca cells in avian ovarian
follicles carry out more complex functions than
we have observed in four frog species.
In summary, our data here show that granulosa cells contain the enzymatic machinery necessary to produce P4, 17α-OHP4, AD, and E2,
whereas the theca layer is mainly responsible for
T production in amphibian ovarian follicles. This
fact suggests that cooperation between the two
cell types is required for efficient production of T
and E2. Thus, the two-cell type model for follicular
steroidogenesis in amphibians previously proposed is also applicable to other species. Furthermore, our results suggest that the theca layer
does not contain the enzyme activities required
for conversion of P5 to 17α-OHP4.
ACKNOWLEDGMENTS
This work was supported by grants awarded to
Dr. H.B. Kwon by the Korea Science and Engineering Foundation through the Hormone Research Center (HRC-96-0101) and by the Ministry
of Education of Korea (BSRI-95-4425).
LITERATURE CITED
Ahn RS, Ko SK, Bai DG, Yoon YD, Kwon HB. 1993. Steroidogenic shift by cultured ovarian follicles of Rana
dybowskii at breeding season. J Exp Zool 267:275–282.
Bahr JM, Wang S-C, Huang MY, Calvo FO. 1983. Steroid
concentrations in isolated theca and granulosa layers of
preovulatory follicles during the ovulatory cycle of the domestic hen. Biol Reprod 29:326–334.
Fortune JE. 1983. Steroid production by Xenopus ovarian
follicles at different developmental stages. Dev Biol 99:
502–509.
Gore-Langton RE, Armstrong DT. 1988. Follicular steroidogenesis and its control. In: Knobil E, JD Neill, editors. The
physiology of reproduction, volume 1. New York: Raven
Press. p 331–385.
Huang ES-R, Kao KJ, Nalvandov AV. 1979. Synthesis of sex
steroids by cellular components of chicken follicles. Biol
Reprod 20:442–461.
Kagawa H, Young G, Adachi S, Nagahama Y. 1982. Estradiol-17β production in amago salmon (Oncorhynchus
rhodurus) ovarian follicles: role of theca and granulosa cells.
Gen Comp Endocrinol 47:440–448.
Kwon HB, Ahn RS. 1994. Relative roles of theca and granulosa cells in ovarian follicular steroidogenesis in the amphibian, Rana nigromaculata. Gen Comp Endocrinol
94:207–214.
Kwon HB, Kim JY, Ko SK. 1990. Progesterone production
and oocyte maturation of frog (Rana nigromaculata and
Rana rugosa) follicles in vitro. Korean J Zool 33:
175–182.
Kwon HB, Choi HH, Ahn RS, Yoon YD. 1991. Steroid
production by amphibian (Rana nigromaculata) ovarian follicles at different developmental stages. J Exp Zool
260:66–73.
Kwon HB, Ahn RS, Lee WK, Im WB, Lee CC, Kim K. 1993.
Changes in the activities of steroidogenic enzymes during
the development of ovarian follicles in Rana nigromaculata.
Gen Comp Endocrinol 92:225–232.
Maller JL. 1985. Regulation of amphibian oocyte maturation.
In: Browder L, editor. Developmental biology, volume 1. New
York: Plenum. p 289–311.
Masui Y. 1967. Relative role of the pituitary, follicle cells and
progesterone in induction of oocyte maturation in Rana
pipiens. J Exp Zool 167:365–376.
Masui Y, Clarke HJ. 1979. Oocyte maturation. Int Rev Cytol
57:185–281.
Nagahama Y. 1987. Gonadotropin action on gametogenesis
and steroidogenesis in teleost gonads. Zool Sci 4:209–222.
Nagahama Y, Adachi S. 1985. Identification of maturation
inducing steroid in teleost, the amago salmon (Oncorhynchus rhodurus). Dev Biol 109:428–435.
Nitta H, Osawa Y, Bahr JM. 1991. Multiple steroidogenic cell
populations in the theca layer of preovulatory follicles of
the chicken ovary. Endocrinology 129:2033–2040.
Petrino TR, Greeley MS Jr, Selman K, Lin YW-P, Wallace
TWO-CELL TYPE MODEL OF STEROIDOGENESIS IN AMPHIBIAN
RA. 1989. Steroidogenesis in Fundulus heteroclitus II: production of 17α, 20β-dihydroxyprogesterone, testosterone,
and 17β-estradiol by various components of the ovarian follicles. Gen Comp Endocrinol 76:230–240.
Petrino T, Schuetz AW. 1987. Cholesterol mediation of progesterone production and oocyte maturation in cultured amphibian (Rana pipiens) ovarian follicles. Biol Reprod
36:1219–1228.
Porter TE, Hargis BM, Silsby JL, El Halawani ME. 1989.
Differential steroid production between theca interna and
theca externa cells: A three-cell model for follicular steroidogenesis in avian species. Endocrinology 125:109–116.
Schuetz AW. 1974. Role of hormone in oocyte maturation. Biol
Reprod 10:150–178.
Schuetz AW. 1985. Local control mechanism during oogenesis and folliculogenesis. In: Browder L, editor. Developmental biology, volume 1. New York: Plenum. p 3–83.
Schuetz AW, Lessman C. 1982. Evidence for follicle wall
involvement in ovulation and progesterone production
99
by frog (Rana pipiens) follicles in vitro. Differentiation
22:79–84.
Tsang PCW, Callard IP. 1992. Regulation of ovarian steroidogenesis in vitro in the viviparous shark, Squalus acanthias.
J Exp Zool 261:97–104.
Wallace RA. 1985. Vitellogenesis and oocyte growth in
nonmammalian vertebrates. In: Browder L, editor. Developmental biology, volume 1. New York: Plenum. p 127–177.
Wallace RA, Bergink GW. 1974. Amphibian vitellogenin: properties, hormonal regulation of hepatic synthesis and ovarian uptake, and conversion of yolk protein. Am Zool
14:1159–1175.
Wang S-C, Bahr JM. 1983. Estradiol secretion by theca cells
of the domestic hen during the ovulatory cycle. Biol Reprod
28:618–624.
Young G, Adachi S, Nagahama Y. 1986. Role of ovarian theca and granulosa layer in gonadotropin-induced synthesis
of salmonoid maturation inducing substance (17α, 20βdihydroxy-4-pregenen-3-one). Dev Biol 118:1–8.
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