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Significance of the Δ5 and Δ4 steroidogenic pathways in the hamster preovulatory follicle
3199
641 SIGNIFICANCE OF THE A5 AND A4 STEROIDOGENIC PATHWAYSIN THE HAMSTERPREOVDIATORYFOLLICLE Anastasia Makris, Dian Olsen and Kenneth J. Ryan Laboratory of Human Reproduction and Reproductive Biology Harvard Medical School Boston, Massachusetts 02115 Received 12-19-83 ABSTRACT
Isolated hamster granulosa cells and theta from preovulatory follicles were incubated in vitro for 2 and 6 h in the absence/or presence of LH and steroidyubstrates. The purpose of the experiments was to determine, in theta, the relative activities of the A5 and A4 pathways under controlled conditions, and to compare the ability of' granulosa cells form progesterone from exogenous and theta to pregnenolone. The results of the experiments show that the Aspathway in theta predominates before and up to 2 h after LH stimulation. The delayed effect of LH after 2 h is a switch from A3 to A4 as the major Progesterone formation from exogenous pregnenolone metabolic pathway. is 7 to 10 times greater in unstimulated granulosa cells than in theta. Acute effects of LH lead to increased conversion of exogenous pregnenolone to progesterone in granulosa cells but not theta. LH does, however, acutely stimulate the thecal conversion of DHEA to androstenedione. The longer term effect of LH in both cell types is to increase pregnenolone conversion to progesterone. INTRODUCTION We
have
previously
(4-pregnene-3,20-dione) the
following
progesterone
isolated after by
initial
studies (O-5
the h)
is
inhibition.
cells
follicle first not The
progesterone synthesis
(1).
in
appears
sensitive
In
(2,3).
LH stimulated
acute
either
In
prevent
The temporal
characteristics
these
studies
are parallel
to the acute
TmEOXDI
the
intact
or
by LH-stimulated 4-6 h
synthesis
10 h time
progesterone
5 h.
S
in
progesterone
after
Volume 42, Number 6
has acute
by RIA until
puromycin
however,
tissue
CAMP-stimulated
synthesis
addition,
progesterone
follicular
seen only
biphasic
to
inhibitors,
is
or
vitro
is not measurable
(1).
phase of
LH-
progesterone
or theta
--in
hamster
15 min)
2)
LH stimulation
intact
I)
(with in
follicle
that
by the
characteristics.
granulosa
the
synthesis
synthesis
recombined
reported
or
course
synthesis
cycloheximide
accumulation of follicular
of
progesterone response
December 1963
to
Lli
and
granulosa are to
the
cells
or
dependent the
delayed
on using
in
various
investigators
respective (5-14)
result
of
by
metabolized
finally
for
up
an
to
steroidogenesis
the
h after
than
theta
from
the
acute the
the in
the
A5pathway; LH
surge
term
3) are,
synergism
(or
--in
A4
granulosa
a
ideas:
1)
vitro
LH
steroidogenic which
follicle
to progesterone LH
is
report
cells
on a per
of
this
pregnenolone
pregnenolone effect
the
follicular
in
A5 than of
of
and
following
surge
synthesis
LH
conve,,rsion
the
LH
to
observations
presented
test
active
converting
longer a A5to
cells
particular
that
data
to
a more
via
4-6
partial
The
after
androgens
granulosa
in
theory
designed
LH stimulates
prior
synthesis
pa th*ay s
and,
the
cells.
have
efficient 4)
granulosa
theta
to
and
steroidogenic
test
just
(3S-hydroxy-5-pregnen-ZO-one)
immediately the
2)
to
experiments
and
stimulation),
us
observations
isolated
The reported
ovarian,
pregnenolone
from
to
pathway;
led
isolated
progesterone
theta
synthesis.
in
These
tissue
follicle
between
terone
systems
thecal
.derived
prior
more
hamster
when
(4).
concerning
have
progesterone
and
of
seen
LH (1).
follicle surge
progea
enzyme
tissue
with
preovulatory
response
acute
synthesis
stimulated
gonadotropin
a synergistic
resulting
are
are
characteristics
implies
to
theta
preovulatory The
progesterone
is
to
direct
is
before basis, and thecal
a A4 pathway.
MATERIALS
AND METHODS
Adult LAK:LVG (SYR) hamsters (Lakeview Hamster Colony) were sacrificed at proestrus prior to the preovulatory LH surge (4). Reproductive cycles were established by vaginal smears (5). Preovulatory follicles were dissected (average 8/pair of ovaries), cleaned and pooled. The follicles were used intact or dissected as previously described (1) to obtain isolated theta and granulosa cells. Each incubation vessel in all the experiments contained either 1 follicle, theta from 1 follicle, or granulosa cells from 1 follicle (approximately 50,000 cells).
The tissue was incubated in 12 x 75 mm tubes containing 1 ml of McCoy’s medium (Grand Island Biological), 1% w:v BSA (Sigma). The tubes were placed in a 37’C Dubnoff water bath operating at 60 cycles/min. The incubation time varied with the experiment. Specific experiments called for addition to the incubation medium of LH and/or radioactive or non-radioactive steroids. The LH was human LH LER (960) kindly donated by Dr. Leo Reichert, National Institutes of Health. Non-radioactive steroids used in the incubations were pregnenolone and DHEA (30-hydroxy-5.-androsten-17-one). They were obtained from Sigma Chemical an9 purified prior to use by celite chromatography. Pregnenolone [7- H(N)] (lo-25 Ci/mmol) was obtained from New England Nuclear, purified prior to use by celite chromatography and diluted with non-radioactive pregnenolone to specific activities that were dependent on the dose added to the incubation vessels. At the end of the incubation period, the incubates were sonicated, and frozen at -2O’C until extracted for steroids. Non-radioactive steroids were extracted from the medium and tissue by ether and analyzed quantitatively by radioimmunoassay after celite chromatography. The extraction, separation, and radioimmunoassay of progesterone and androstenedione (4-androstene-3,17-dione) have been previously described (1). Separatio;i’ of pregnenolone and DHEA by celite chromatography was done by applying the samples in 1 ml trimethylpentane (TMP) to the column. Pregnenolone is eluted with progesterone using 4.5 ml TMP. DHEA eluted with 4 ml 5% ethylacetate in TMP (after an additional 5 ml TMP was added and the eluate discarded). Pregnenolone was measured by radioimmunoassay using Ab #ll developed by Ms. Roberta Todd at the LHRRB RIA Core facility. This antibody has a cross reaction of 14% with progesterone. The quantitative determination of both progesterone and pregnenolone in the same fraction was adjusted to account for cross reactivities of the respective antibodies. DHEA was measured by RIA using Ab 1111 also developed by the RIA core facility. This antibody has 0% cross reactivity with testosterone (17g-hydroxy-4-androsten -3-one) which elutes with DHEA off the celite column. 3 H-pregnenolone the remaining When was used as a substrate, radioactive substrate and radioactive products, progesterone (PI, A4-A, DHEA , DHT (17R-hydroxy-5 u-androstan-3-one) and testosterone (T), were separated using the following protocol. The samples were extracted with ethyl dried and reconstituted with a 1:l methylene ether, chloride:ethanol mixture. Samples were applied to scored silica gel plates (Analab) along with standards of the 6 steroids (on channels separate from the unknowns). The plates were run 2X in a 9:l chloroform:ether mixture. The channels with the standards were sprayed with 0.001% Direct Yellow 59 (Sigma) and the steroid zones located under uv. The silica gel: chloroform:ether sys tern isolated progesterone A4-A and T. Pregnenolone, DHT and DHEA co-eluted. This zone of co-eluted steroids was scraped into test tubes containing ethanol. The steroid mixture was separated from the silica gel by centrifugation and applied to aluminium oxide plates (Analab). The plates were run 2X in a 1: 1 cyclohexane:ethyl acetate system which separated the three steroids. The respective steroid zones were located using standards applied to separate channels, which were sprayed with 0.001% Direct Yellow 59 and inspected under UV light. The zones containing the isolated steroid fractions were scraped off into scintillation vials containing 8 ml HP
The amount of radioactivity was converted to Liquifluor (Beckman). since the specific activity of each dose of the nanograms 0 steroid, f substrate ( H-pregnenolone) used was different. Where applicable the data were analyzed statistically using ANOVA. RESULTS De
Production
Novo
Theta
and Granulosa Isolated
without in
of
plus
detectable 0.001).
the
other
LH (Table
groups
Table 1. progesterone cells --in vitro
showed
Control LH (100
that levels
progesterone produced
Preg
was
theta
were
Analysis only of
incubated
by the
RIA
DHEA by
for
of
the
Isolated
in any
(P<
of
the
pregnenolone
Prog
+ 240
2 h with
content
theta
produced
0.001)
groups,
and
DHEA
and none .
or DHEA.
(pg)
and
steroid
LH-stimulated
pregnenolone,
found
detectable
(pg)
ND1 13390
rig/ml)
Granulosa
and
De novo synthesis of pregnenolone (Fog>, and DHEA by isolated theta and in 2 h incubations + LH, n = 5.
Theta Control LH (100
and 1).
significant
No
Progesterone
2 LH in Two Hours cells
tissue
and
(P<
Cells
granulosa
100 rig/ml
media
Pregnenolone,
(preg), granulosa
DHEA (pg)
ND ND
ND *382
ND ND
ND ND
+ 46
cells ND ND
rig/ml)
Each incubate consisted of either isolated granulosa cells (50,000 cells) or theta from one preovulatory follicle. The steroids were measured by RIA and the results analyzed by ANOVA. The steroid measurement indicated by an asterisk (*> is fignificantly different from the respective control (P< 0.01). ND = non-detectable levels Pregnenolone
Conversion
to
Progesterone
by
Isolated
Theta
7Cells The conversion
of
exogenous
non-radioactive
pregnenolone
and
Granulosa
of
s (doses
of
10 to
1000
of
isolated
incubations LH was added
rig/ml)
at
To
to
To
to
the
2
at
to
progesterone
theta
added
(Fig.
both
the
h
645
TDEOXDI
was
1A)
2 and
incubations
and
determined
granulosa
in cells
6 h incubations. and
at
To
2 and (Fig.
6 h 1B).
Substrate
plus
4 h to
was
the
6 h
incubations. Thecal plus/minus seen
conversion LH
by
was
control
pregnenolone
measurable of
stimulate
progesterone 1B)
but
incubations
significantly
(Fig.
of
in
the
(Fig.
approximately
granulosa thecal
2 h incubations
IA)
cells
1A)
but
2 h
7-10X
lower
(Fig.
conversion
(Fig.
in
of did
so in
1B).
incubations than LH did
that not
pregnenolone granulosa
to cells
(P< 0.05).
25
I
IO
Fig.
1.
I
20
I
I
I
I
50 100 250 500 Pregnenolone nghnl
I
1000
Dose-response conversion of exogenous pregnenolone rig/ml) to progesterone in the absence or presence rig/ml) in 2 and 6 ,h incubations. Control: 2h0, 6ha 2h@ , 6h n . n=A. A: theta; B: granulosa cells.
(10-1000 of LH (100 . LH:
S Theta higher
that
(P<
had
0.01)
progesterone
all
was essentially
incubations
+ LH (Fig.
To
10 to TO
1000
plus
taken
conversion
of
in
the
index DHEA
of LH to
6
same as
at
significantly
To plus
4 h)
to
6
h
1A). granulosa
that
seen
cells in
the
in
respective
2 h
2 and 6 H Incubations for
degree
in
both
stimulate
2 and
DHEA was added
at
To
of 2 h
the
DHEA and
to
the
doses
of
200
LH added
ranging
2 h incubations,
amount
of
A4-A
A"
LH
6 h incubations
loo
Dose-response conversion of A4 -A by isolated theta in rig/ml). Control: 2h0, 6h 0.
at
conversion.
accumulation
60
6 h + 100 rig/ml
added
The
DHEA
2.
(Fig.
h incubations.
of
20
Fig.
(added
by
incubated
DHEA was
to
6 h showed
2B).
were
rig/ml.
an
10 rig/ml
Non-radioactive
2).
4 h as
ability
theta
(Fig
pregnenolone
the
DHEA by Theta
Isolated at
above
LPI for
dose-response
incubations
of
with
of
doses
pregnenolone
Conversion
incubated
conversion
at
The
been
?FBEOXDrn
6ClCl
and
-A
fo&d
at was
stimulated (P<
from
from
the
0.05).
DHEA at
The
2 h is
COO
rig/ml
exogenous DHEA (10-1000 2 and 6 h incubations LH: 2h0, 6hm.
rig/ml) to + LH (100
in
contrast
theta
to the unchanged
(Fig.
This
Dose-response
experiment
of
in Thecal
of
the A5
into
and A'
3H-pregnenolone
were
(2200 used.
ratios.
2 summarizes The amounts
translated
to
use d .
substrate essentially It
total
ratio
in
both
7.61,
indicating
the A5 rather
6
0.07
to
significantly towards
theA
h
than through
pathway,
calculated
specific
theta
groups
10 rig/ml (2200 at
were
(11,000 CPM/rig)
To and To +
DHEA/progesterone T and DHT found were
activity data
varied (not
levels.
A4 pathway
groups
direction
are
not
in DHEA,A4 -A, T,
of
was over 1 (1.3
3H-pregnenolone
the
to
through
.
had DHEA/progesterone
that
increasing
shown)
dose
The DHEA/progesterone groups
metabolism
the
with
2 h incubates.
and LH-stimulated
indicating the
3H-pregnenolone
rig/ml
was added
incubation
a preferential
change
1000
the
relative
and LH-stimulated
DHEA, A4 -A,
progesterone
2 h control
the
Isolated
LH at 2 h caused an increase
affecting
0.24
and
and the
from the control
The 6 h LH-stimulated from
data
because
Control
can be seen that
and DHT without
from
whether
respectively.
progesterone,
ng,
different
of
Doses of
The substrate
the
of
groups.
CPM/rig)
4 h to the 2 and 6 h incubates Table
determine
formed
2 h control
groups,
rig/ml
to
function
(a
pathways)
and LH-stimulated
100
,
order
of time of LH exposure.
4 treatment
and 6 h control
in
products
would change as a function .
CPM/rig)
in LPI-stimulated
Tissue
was performed
DHEA/progesterone
activities
divided
accumulation
1 and Table 1).
a-Pregnenolone
ratio
progesterone
longer of
term
ratios LH effect
3H-pregnenolone
the accumulation
ranging was to
metabolism
of progesterone.
Conversion Table 2. androstenedione (A4-A), DHEA by isolated theta.
Substrate n=5
P (rig/ml)
3H-pregnenolone to progesterone (PI, of testosterone (T), dihydrotestosterone (DHT), and CONTROL(2 H) A4 -A
DIBA
T
DHT
DHgAf P
ng
10
0.15 0.11
0.24 0.15
0.06 0.17
0.05 0.06
0.33 0.13
1.54 1.33
100
0.65 0.62
1.73 2.38
1.57 3.50
0.73 1.64
3.70 1.16
2.66 3.83
1.90 11.90 5.14 14.70 3.36 10.10 8.80 16.10 ____-------------------------------------------------------------LH (2 HI 10 0.16 0.44 0.27 0.28 0.22 0.38 0.34 0.15
17.36 11.00
6.26 3.00
0.44 0.57
2.68 1.72
1000
100
0.78 1.61
3.10 4.80
12.70 6.10
2.10 3.34
1000
3.82 5.84
29.00 25.90
15.80 30.00
37.10 94.10
17.80 16.70
7.59 4.43
10
2.12 2.10
0.25 0.15
0.63 0.42
0.19 0.19
0.12 0.07
100
7.50 9.04
1.23 2.21
9.49 4.69
1.23 2.22
' 0.16 0.24
82.30 6.77 175.59 103.87 113.31 13.66 45.58 103.62 -------------------------------------------------------------------
8.21 3.30
0.08 0.12
LH (6 HI 0.52 0.69 9.39 22.60
1000
lost 10s t
3.97 2.98
.
Incubation of'the tissue was 2 or 6 h. LH was added at T . The substrate was added at T and T + 4 h to the 2 h and tf?e 6 h incubations, respectively. ' The radioactivity found was transposed to reight of steroid product (ng). Specific activities of the H-pregnenolone were 11,000 CPM/rig for 10 ngfml, and 2200 CPM/rig for 100 and 1000 rig/ml. Each dose/group consisted of only 2 incubation samples. The data derived from each sample is shown.
DISCUSSION The results that
phase 1
(acute)
of
the
experiments
synergistic
reported
progesterone
here synthesis
support
the
theory
by the hamster
follicle
depends
granulosa
cells.
Table
on thecal
1 shows
novo pregnenolone incubations. the
that
The probability
Both
de nova --
granulosa
non-stimulated
preferentially
with
synthesized
cells)
is
theta
and
prolonged
effect
from
which
0.07
resulted
to
0.24
in
ratios
progesterone
(1).
A5
(in
pathway
the data
2
h to
greater
the
synthesis
LH
DHEA
as
of
in Table of
2.
with
than 1.
activity
ratios
de nova --
be
shown in Table
for
significant
DHEA/progesterone
and significant
under
will
of values
2 h
of androgens
3H-pregnenolone
induce
LH was to
in
-de
in
pregnenolone
incubated
theta
exogenous
by the DHEA/progesterone
thecal
by
synthesize
of progesterone
thecal
the
indicated
indicated
pathway,
LH will
no detectable
through
metabolized
of
to progesterone
showed an accumulation
metabolized
of
with
no accumulation
conditions
that
conversion
stimulated
studies
same experimental
absence
theta
and DHEA with
Earlier
preferentially
pregnenolone
The
the A4 2 ranging
progesterone
by 4 to 6 h (1). Whether. the hamster synthesis
novo
oxidoreductase higher evidence
that
a
whether for
the
that
(13).
amount of cellular
A5
A4 pathway
the
associated associated
with with
(16).
existing newly active
than
DHEA is
not
has an altered known.
a pregnenolone-specific
e
-al.
found
cholesterol They
microsomal
structures
LH-stimulated
There
can be found that
that
LH can
and the
in
bovine
redirect
tissue
the A5
mitochondrial
is
and a separate
in follicular
found
due to -de
3l3-hydroxysteroid
enzyme system
oxidoreductase
Dimino
significant t0
existing
both
3l3-hydroxysteroid
tissue
becomes more significant
pregnenolone-specific
pregnenolone
indicates
DHEA-specific ovarian
of or
affinity
A4 pathway
from the
pathway
A4 pathway structures.
a
was was
Evidence
of
LH-stimulated
oxidoreductase
has
investigators rabbit
The and
undergoes
also
3P-hydroxy
a A5
was
stimulate
to not
thecal
stimulatory
effect
incubated
the
with
has
have
same acute
exogenous
cell
sys terns.
in theta,
and
of
post-coital
theta
6
(Fig.
incubations
was
conversion
of
seen to
LH
in
to progesterone
on theta h
1A
respective
effect
acute
effect
(Fig.
the
conversion
the
the
groups
the
in
The
however,
DHEA and
that
differences
pregnenolone
LH on
several
(21).
increase the
by
shown
LH on granulosa
conversion of
also
shift
tase
3g-hydroxysteroid
tissue
possible
oxidoreduc
LH did
1B).
ovarian
A4
of
indicates
cells
in
to
effect
steroid
granulosa
did
shown YoungLai
differential
1B)
(Fig.
been
(17-20).
ovary
associated
mitochondrial
when
1A) but An
acute
theta .
were
A4 -A measured
(Fig.
2).
Comparison and
granulosa
shows
than
(Fig.
seen
rabbit
relative
by
during where
thecal
significantly
of
to
convert
by
granulosa
theta.
between
LH stimulation
was increased
ability
1B)
granulosa
differences
6 h incubations
relative
accumulation
that
pre-coital
the
cells
progesterone
higher
after
of
and
theta
cells found
theta
and
conversion
but of
(Fig.
to
the
(Fig.
1A)
progesterone,
an order
(21).
granulosa
2 h incubations
(P< 0.01)
theta
pregnenolone
YoungLai
cells
unstimulated
of magnitude
same differences In
our
cells were
pregnenolone
studies, were
not
in
maintained
evident to
the
in
the
progesterone
1A and 1B).
ACKNOWLEDGEMENTS
This
work
was supported
by a grant
from
the
National
Institutes
of
Child Health and Human Development, ROl HD 14709-0181. thank the LHRRB RIA core facility, under the direction Todd for providing RIA materials and facilities.
We
would like to of Ms. Roberta
REFERENCES 1. Makris, A. and Ryan, K.J., Endocr. Res. Comm. 4(3+4), 233 (1977). 2. Makris, A. and Ryan, K.J., Abstr. # 29, 6th International Congr. on Hormonal Steroids, Jerusalem, Israel, 5-10 Sept., 1982. 3. Makris, A. and Ryan, K.J., (19831, submitted to Endocr. Res. Comm.. 4. Turgeon, J.and Greenwald, G.S., Endocrinology 90, 657 (1972). 5. Gougeon, A., Ann. Biol. Anim. Biochem. xophys. 1087 17(6), (1977). 6. Pupkin, M., Bratt, H., Weisz, J., Lloyd, C.W. and Balogh, K., J. Endocrinology 79, 316 (1976). 7. Bjersing, L., sstochemie l0, 295 (1967). 8. Ikonen, M., Niemi, H. Pesonen, S., and Timonen, S., Acta Endocr. (Kbh) 38, 293 (1961). 9. Rubin, B.L., Hamilton, J.A. and Driks, E.C., Endocrinology 2, 924 (1963). 10. Blaha, G.C. and Leavitt, W.W., Biol. Reprod. 2, 362 (1970). 11. Ryan, K.J. and Smith, O.T., Rec. Prog. Hormone Res. 21, 285 (1965). 12. Somma, M., Sander, T. and Lauthier, A., J. Clin. Endocrinol. Metab. 29, 457 (1969). 13. xbb, W. and Hagerman, D.D., Steroids 28, 31 (1976). 14. Mahajan, D..K. and Samuels, L.T., Sterxds 24, 713 (1974). 15. Ward, M.C., Anat. Rec. 94, 139 (1946). 16. Dimino, M.J., Elfont, E.A. and Berman, S.K., Adv. Exp. Med. Biol. 112, 505 (1978). 17. Westwerdt, W., Mullet, 0. and Brandau, H., Adv. Exp. Med. Biol. 112, 123 (1978). 18. Crisp, T.M. and Channing, C.P., Biol. Reprod. 2, 55 (1972). 19. Peluso, J.J., Steger, R.W. and Hatz, E.S.E., Biol. Reprod. 16, 600 (1977). 20. Shuchner, E.B. and Stockert, J.C., Protoplasma 76, 133 (1973). 21. YoungLai, E.V., Acta Endocrinol. (Kbh) 74, 775 (1973).
641 SIGNIFICANCE OF THE A5 AND A4 STEROIDOGENIC PATHWAYSIN THE HAMSTERPREOVDIATORYFOLLICLE Anastasia Makris, Dian Olsen and Kenneth J. Ryan Laboratory of Human Reproduction and Reproductive Biology Harvard Medical School Boston, Massachusetts 02115 Received 12-19-83 ABSTRACT
Isolated hamster granulosa cells and theta from preovulatory follicles were incubated in vitro for 2 and 6 h in the absence/or presence of LH and steroidyubstrates. The purpose of the experiments was to determine, in theta, the relative activities of the A5 and A4 pathways under controlled conditions, and to compare the ability of' granulosa cells form progesterone from exogenous and theta to pregnenolone. The results of the experiments show that the Aspathway in theta predominates before and up to 2 h after LH stimulation. The delayed effect of LH after 2 h is a switch from A3 to A4 as the major Progesterone formation from exogenous pregnenolone metabolic pathway. is 7 to 10 times greater in unstimulated granulosa cells than in theta. Acute effects of LH lead to increased conversion of exogenous pregnenolone to progesterone in granulosa cells but not theta. LH does, however, acutely stimulate the thecal conversion of DHEA to androstenedione. The longer term effect of LH in both cell types is to increase pregnenolone conversion to progesterone. INTRODUCTION We
have
previously
(4-pregnene-3,20-dione) the
following
progesterone
isolated after by
initial
studies (O-5
the h)
is
inhibition.
cells
follicle first not The
progesterone synthesis
(1).
in
appears
sensitive
In
(2,3).
LH stimulated
acute
either
In
prevent
The temporal
characteristics
these
studies
are parallel
to the acute
TmEOXDI
the
intact
or
by LH-stimulated 4-6 h
synthesis
10 h time
progesterone
5 h.
S
in
progesterone
after
Volume 42, Number 6
has acute
by RIA until
puromycin
however,
tissue
CAMP-stimulated
synthesis
addition,
progesterone
follicular
seen only
biphasic
to
inhibitors,
is
or
vitro
is not measurable
(1).
phase of
LH-
progesterone
or theta
--in
hamster
15 min)
2)
LH stimulation
intact
I)
(with in
follicle
that
by the
characteristics.
granulosa
the
synthesis
synthesis
recombined
reported
or
course
synthesis
cycloheximide
accumulation of follicular
of
progesterone response
December 1963
to
Lli
and
granulosa are to
the
cells
or
dependent the
delayed
on using
in
various
investigators
respective (5-14)
result
of
by
metabolized
finally
for
up
an
to
steroidogenesis
the
h after
than
theta
from
the
acute the
the in
the
A5pathway; LH
surge
term
3) are,
synergism
(or
--in
A4
granulosa
a
ideas:
1)
vitro
LH
steroidogenic which
follicle
to progesterone LH
is
report
cells
on a per
of
this
pregnenolone
pregnenolone effect
the
follicular
in
A5 than of
of
and
following
surge
synthesis
LH
conve,,rsion
the
LH
to
observations
presented
test
active
converting
longer a A5to
cells
particular
that
data
to
a more
via
4-6
partial
The
after
androgens
granulosa
in
theory
designed
LH stimulates
prior
synthesis
pa th*ay s
and,
the
cells.
have
efficient 4)
granulosa
theta
to
and
steroidogenic
test
just
(3S-hydroxy-5-pregnen-ZO-one)
immediately the
2)
to
experiments
and
stimulation),
us
observations
isolated
The reported
ovarian,
pregnenolone
from
to
pathway;
led
isolated
progesterone
theta
synthesis.
in
These
tissue
follicle
between
terone
systems
thecal
.derived
prior
more
hamster
when
(4).
concerning
have
progesterone
and
of
seen
LH (1).
follicle surge
progea
enzyme
tissue
with
preovulatory
response
acute
synthesis
stimulated
gonadotropin
a synergistic
resulting
are
are
characteristics
implies
to
theta
preovulatory The
progesterone
is
to
direct
is
before basis, and thecal
a A4 pathway.
MATERIALS
AND METHODS
Adult LAK:LVG (SYR) hamsters (Lakeview Hamster Colony) were sacrificed at proestrus prior to the preovulatory LH surge (4). Reproductive cycles were established by vaginal smears (5). Preovulatory follicles were dissected (average 8/pair of ovaries), cleaned and pooled. The follicles were used intact or dissected as previously described (1) to obtain isolated theta and granulosa cells. Each incubation vessel in all the experiments contained either 1 follicle, theta from 1 follicle, or granulosa cells from 1 follicle (approximately 50,000 cells).
The tissue was incubated in 12 x 75 mm tubes containing 1 ml of McCoy’s medium (Grand Island Biological), 1% w:v BSA (Sigma). The tubes were placed in a 37’C Dubnoff water bath operating at 60 cycles/min. The incubation time varied with the experiment. Specific experiments called for addition to the incubation medium of LH and/or radioactive or non-radioactive steroids. The LH was human LH LER (960) kindly donated by Dr. Leo Reichert, National Institutes of Health. Non-radioactive steroids used in the incubations were pregnenolone and DHEA (30-hydroxy-5.-androsten-17-one). They were obtained from Sigma Chemical an9 purified prior to use by celite chromatography. Pregnenolone [7- H(N)] (lo-25 Ci/mmol) was obtained from New England Nuclear, purified prior to use by celite chromatography and diluted with non-radioactive pregnenolone to specific activities that were dependent on the dose added to the incubation vessels. At the end of the incubation period, the incubates were sonicated, and frozen at -2O’C until extracted for steroids. Non-radioactive steroids were extracted from the medium and tissue by ether and analyzed quantitatively by radioimmunoassay after celite chromatography. The extraction, separation, and radioimmunoassay of progesterone and androstenedione (4-androstene-3,17-dione) have been previously described (1). Separatio;i’ of pregnenolone and DHEA by celite chromatography was done by applying the samples in 1 ml trimethylpentane (TMP) to the column. Pregnenolone is eluted with progesterone using 4.5 ml TMP. DHEA eluted with 4 ml 5% ethylacetate in TMP (after an additional 5 ml TMP was added and the eluate discarded). Pregnenolone was measured by radioimmunoassay using Ab #ll developed by Ms. Roberta Todd at the LHRRB RIA Core facility. This antibody has a cross reaction of 14% with progesterone. The quantitative determination of both progesterone and pregnenolone in the same fraction was adjusted to account for cross reactivities of the respective antibodies. DHEA was measured by RIA using Ab 1111 also developed by the RIA core facility. This antibody has 0% cross reactivity with testosterone (17g-hydroxy-4-androsten -3-one) which elutes with DHEA off the celite column. 3 H-pregnenolone the remaining When was used as a substrate, radioactive substrate and radioactive products, progesterone (PI, A4-A, DHEA , DHT (17R-hydroxy-5 u-androstan-3-one) and testosterone (T), were separated using the following protocol. The samples were extracted with ethyl dried and reconstituted with a 1:l methylene ether, chloride:ethanol mixture. Samples were applied to scored silica gel plates (Analab) along with standards of the 6 steroids (on channels separate from the unknowns). The plates were run 2X in a 9:l chloroform:ether mixture. The channels with the standards were sprayed with 0.001% Direct Yellow 59 (Sigma) and the steroid zones located under uv. The silica gel: chloroform:ether sys tern isolated progesterone A4-A and T. Pregnenolone, DHT and DHEA co-eluted. This zone of co-eluted steroids was scraped into test tubes containing ethanol. The steroid mixture was separated from the silica gel by centrifugation and applied to aluminium oxide plates (Analab). The plates were run 2X in a 1: 1 cyclohexane:ethyl acetate system which separated the three steroids. The respective steroid zones were located using standards applied to separate channels, which were sprayed with 0.001% Direct Yellow 59 and inspected under UV light. The zones containing the isolated steroid fractions were scraped off into scintillation vials containing 8 ml HP
The amount of radioactivity was converted to Liquifluor (Beckman). since the specific activity of each dose of the nanograms 0 steroid, f substrate ( H-pregnenolone) used was different. Where applicable the data were analyzed statistically using ANOVA. RESULTS De
Production
Novo
Theta
and Granulosa Isolated
without in
of
plus
detectable 0.001).
the
other
LH (Table
groups
Table 1. progesterone cells --in vitro
showed
Control LH (100
that levels
progesterone produced
Preg
was
theta
were
Analysis only of
incubated
by the
RIA
DHEA by
for
of
the
Isolated
in any
(P<
of
the
pregnenolone
Prog
+ 240
2 h with
content
theta
produced
0.001)
groups,
and
DHEA
and none .
or DHEA.
(pg)
and
steroid
LH-stimulated
pregnenolone,
found
detectable
(pg)
ND1 13390
rig/ml)
Granulosa
and
De novo synthesis of pregnenolone (Fog>, and DHEA by isolated theta and in 2 h incubations + LH, n = 5.
Theta Control LH (100
and 1).
significant
No
Progesterone
2 LH in Two Hours cells
tissue
and
(P<
Cells
granulosa
100 rig/ml
media
Pregnenolone,
(preg), granulosa
DHEA (pg)
ND ND
ND *382
ND ND
ND ND
+ 46
cells ND ND
rig/ml)
Each incubate consisted of either isolated granulosa cells (50,000 cells) or theta from one preovulatory follicle. The steroids were measured by RIA and the results analyzed by ANOVA. The steroid measurement indicated by an asterisk (*> is fignificantly different from the respective control (P< 0.01). ND = non-detectable levels Pregnenolone
Conversion
to
Progesterone
by
Isolated
Theta
7Cells The conversion
of
exogenous
non-radioactive
pregnenolone
and
Granulosa
of
s (doses
of
10 to
1000
of
isolated
incubations LH was added
rig/ml)
at
To
to
To
to
the
2
at
to
progesterone
theta
added
(Fig.
both
the
h
645
TDEOXDI
was
1A)
2 and
incubations
and
determined
granulosa
in cells
6 h incubations. and
at
To
2 and (Fig.
6 h 1B).
Substrate
plus
4 h to
was
the
6 h
incubations. Thecal plus/minus seen
conversion LH
by
was
control
pregnenolone
measurable of
stimulate
progesterone 1B)
but
incubations
significantly
(Fig.
of
in
the
(Fig.
approximately
granulosa thecal
2 h incubations
IA)
cells
1A)
but
2 h
7-10X
lower
(Fig.
conversion
(Fig.
in
of did
so in
1B).
incubations than LH did
that not
pregnenolone granulosa
to cells
(P< 0.05).
25
I
IO
Fig.
1.
I
20
I
I
I
I
50 100 250 500 Pregnenolone nghnl
I
1000
Dose-response conversion of exogenous pregnenolone rig/ml) to progesterone in the absence or presence rig/ml) in 2 and 6 ,h incubations. Control: 2h0, 6ha 2h@ , 6h n . n=A. A: theta; B: granulosa cells.
(10-1000 of LH (100 . LH:
S Theta higher
that
(P<
had
0.01)
progesterone
all
was essentially
incubations
+ LH (Fig.
To
10 to TO
1000
plus
taken
conversion
of
in
the
index DHEA
of LH to
6
same as
at
significantly
To plus
4 h)
to
6
h
1A). granulosa
that
seen
cells in
the
in
respective
2 h
2 and 6 H Incubations for
degree
in
both
stimulate
2 and
DHEA was added
at
To
of 2 h
the
DHEA and
to
the
doses
of
200
LH added
ranging
2 h incubations,
amount
of
A4-A
A"
LH
6 h incubations
loo
Dose-response conversion of A4 -A by isolated theta in rig/ml). Control: 2h0, 6h 0.
at
conversion.
accumulation
60
6 h + 100 rig/ml
added
The
DHEA
2.
(Fig.
h incubations.
of
20
Fig.
(added
by
incubated
DHEA was
to
6 h showed
2B).
were
rig/ml.
an
10 rig/ml
Non-radioactive
2).
4 h as
ability
theta
(Fig
pregnenolone
the
DHEA by Theta
Isolated at
above
LPI for
dose-response
incubations
of
with
of
doses
pregnenolone
Conversion
incubated
conversion
at
The
been
?FBEOXDrn
6ClCl
and
-A
fo&d
at was
stimulated (P<
from
from
the
0.05).
DHEA at
The
2 h is
COO
rig/ml
exogenous DHEA (10-1000 2 and 6 h incubations LH: 2h0, 6hm.
rig/ml) to + LH (100
in
contrast
theta
to the unchanged
(Fig.
This
Dose-response
experiment
of
in Thecal
of
the A5
into
and A'
3H-pregnenolone
were
(2200 used.
ratios.
2 summarizes The amounts
translated
to
use d .
substrate essentially It
total
ratio
in
both
7.61,
indicating
the A5 rather
6
0.07
to
significantly towards
theA
h
than through
pathway,
calculated
specific
theta
groups
10 rig/ml (2200 at
were
(11,000 CPM/rig)
To and To +
DHEA/progesterone T and DHT found were
activity data
varied (not
levels.
A4 pathway
groups
direction
are
not
in DHEA,A4 -A, T,
of
was over 1 (1.3
3H-pregnenolone
the
to
through
.
had DHEA/progesterone
that
increasing
shown)
dose
The DHEA/progesterone groups
metabolism
the
with
2 h incubates.
and LH-stimulated
indicating the
3H-pregnenolone
rig/ml
was added
incubation
a preferential
change
1000
the
relative
and LH-stimulated
DHEA, A4 -A,
progesterone
2 h control
the
Isolated
LH at 2 h caused an increase
affecting
0.24
and
and the
from the control
The 6 h LH-stimulated from
data
because
Control
can be seen that
and DHT without
from
whether
respectively.
progesterone,
ng,
different
of
Doses of
The substrate
the
of
groups.
CPM/rig)
4 h to the 2 and 6 h incubates Table
determine
formed
2 h control
groups,
rig/ml
to
function
(a
pathways)
and LH-stimulated
100
,
order
of time of LH exposure.
4 treatment
and 6 h control
in
products
would change as a function .
CPM/rig)
in LPI-stimulated
Tissue
was performed
DHEA/progesterone
activities
divided
accumulation
1 and Table 1).
a-Pregnenolone
ratio
progesterone
longer of
term
ratios LH effect
3H-pregnenolone
the accumulation
ranging was to
metabolism
of progesterone.
Conversion Table 2. androstenedione (A4-A), DHEA by isolated theta.
Substrate n=5
P (rig/ml)
3H-pregnenolone to progesterone (PI, of testosterone (T), dihydrotestosterone (DHT), and CONTROL(2 H) A4 -A
DIBA
T
DHT
DHgAf P
ng
10
0.15 0.11
0.24 0.15
0.06 0.17
0.05 0.06
0.33 0.13
1.54 1.33
100
0.65 0.62
1.73 2.38
1.57 3.50
0.73 1.64
3.70 1.16
2.66 3.83
1.90 11.90 5.14 14.70 3.36 10.10 8.80 16.10 ____-------------------------------------------------------------LH (2 HI 10 0.16 0.44 0.27 0.28 0.22 0.38 0.34 0.15
17.36 11.00
6.26 3.00
0.44 0.57
2.68 1.72
1000
100
0.78 1.61
3.10 4.80
12.70 6.10
2.10 3.34
1000
3.82 5.84
29.00 25.90
15.80 30.00
37.10 94.10
17.80 16.70
7.59 4.43
10
2.12 2.10
0.25 0.15
0.63 0.42
0.19 0.19
0.12 0.07
100
7.50 9.04
1.23 2.21
9.49 4.69
1.23 2.22
' 0.16 0.24
82.30 6.77 175.59 103.87 113.31 13.66 45.58 103.62 -------------------------------------------------------------------
8.21 3.30
0.08 0.12
LH (6 HI 0.52 0.69 9.39 22.60
1000
lost 10s t
3.97 2.98
.
Incubation of'the tissue was 2 or 6 h. LH was added at T . The substrate was added at T and T + 4 h to the 2 h and tf?e 6 h incubations, respectively. ' The radioactivity found was transposed to reight of steroid product (ng). Specific activities of the H-pregnenolone were 11,000 CPM/rig for 10 ngfml, and 2200 CPM/rig for 100 and 1000 rig/ml. Each dose/group consisted of only 2 incubation samples. The data derived from each sample is shown.
DISCUSSION The results that
phase 1
(acute)
of
the
experiments
synergistic
reported
progesterone
here synthesis
support
the
theory
by the hamster
follicle
depends
granulosa
cells.
Table
on thecal
1 shows
novo pregnenolone incubations. the
that
The probability
Both
de nova --
granulosa
non-stimulated
preferentially
with
synthesized
cells)
is
theta
and
prolonged
effect
from
which
0.07
resulted
to
0.24
in
ratios
progesterone
(1).
A5
(in
pathway
the data
2
h to
greater
the
synthesis
LH
DHEA
as
of
in Table of
2.
with
than 1.
activity
ratios
de nova --
be
shown in Table
for
significant
DHEA/progesterone
and significant
under
will
of values
2 h
of androgens
3H-pregnenolone
induce
LH was to
in
-de
in
pregnenolone
incubated
theta
exogenous
by the DHEA/progesterone
thecal
by
synthesize
of progesterone
thecal
the
indicated
indicated
pathway,
LH will
no detectable
through
metabolized
of
to progesterone
showed an accumulation
metabolized
of
with
no accumulation
conditions
that
conversion
stimulated
studies
same experimental
absence
theta
and DHEA with
Earlier
preferentially
pregnenolone
The
the A4 2 ranging
progesterone
by 4 to 6 h (1). Whether. the hamster synthesis
novo
oxidoreductase higher evidence
that
a
whether for
the
that
(13).
amount of cellular
A5
A4 pathway
the
associated associated
with with
(16).
existing newly active
than
DHEA is
not
has an altered known.
a pregnenolone-specific
e
-al.
found
cholesterol They
microsomal
structures
LH-stimulated
There
can be found that
that
LH can
and the
in
bovine
redirect
tissue
the A5
mitochondrial
is
and a separate
in follicular
found
due to -de
3l3-hydroxysteroid
enzyme system
oxidoreductase
Dimino
significant t0
existing
both
3l3-hydroxysteroid
tissue
becomes more significant
pregnenolone-specific
pregnenolone
indicates
DHEA-specific ovarian
of or
affinity
A4 pathway
from the
pathway
A4 pathway structures.
a
was was
Evidence
of
LH-stimulated
oxidoreductase
has
investigators rabbit
The and
undergoes
also
3P-hydroxy
a A5
was
stimulate
to not
thecal
stimulatory
effect
incubated
the
with
has
have
same acute
exogenous
cell
sys terns.
in theta,
and
of
post-coital
theta
6
(Fig.
incubations
was
conversion
of
seen to
LH
in
to progesterone
on theta h
1A
respective
effect
acute
effect
(Fig.
the
conversion
the
the
groups
the
in
The
however,
DHEA and
that
differences
pregnenolone
LH on
several
(21).
increase the
by
shown
LH on granulosa
conversion of
also
shift
tase
3g-hydroxysteroid
tissue
possible
oxidoreduc
LH did
1B).
ovarian
A4
of
indicates
cells
in
to
effect
steroid
granulosa
did
shown YoungLai
differential
1B)
(Fig.
been
(17-20).
ovary
associated
mitochondrial
when
1A) but An
acute
theta .
were
A4 -A measured
(Fig.
2).
Comparison and
granulosa
shows
than
(Fig.
seen
rabbit
relative
by
during where
thecal
significantly
of
to
convert
by
granulosa
theta.
between
LH stimulation
was increased
ability
1B)
granulosa
differences
6 h incubations
relative
accumulation
that
pre-coital
the
cells
progesterone
higher
after
of
and
theta
cells found
theta
and
conversion
but of
(Fig.
to
the
(Fig.
1A)
progesterone,
an order
(21).
granulosa
2 h incubations
(P< 0.01)
theta
pregnenolone
YoungLai
cells
unstimulated
of magnitude
same differences In
our
cells were
pregnenolone
studies, were
not
in
maintained
evident to
the
in
the
progesterone
1A and 1B).
ACKNOWLEDGEMENTS
This
work
was supported
by a grant
from
the
National
Institutes
of
Child Health and Human Development, ROl HD 14709-0181. thank the LHRRB RIA core facility, under the direction Todd for providing RIA materials and facilities.
We
would like to of Ms. Roberta
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