From the Department of Internal Medicine, Division of Endocrinology and Metabolism, Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, Texas 75390-8857
Received for publication, November 25, 2002, and in revised form, January 6, 2003
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ABSTRACT |
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Progesterone has long been considered the primary
mediator of Xenopus oocyte maturation. We have recently
shown, however, that androgens, which are equal or more potent
promoters of maturation and are present at higher levels in ovulating
frogs, may also be playing an important physiologic role in mediating
maturation. Here, we examined the role of CYP17, a key enzyme mediating
sex steroid synthesis, in Xenopus ovarian androgen
production. We found that the 17,20-lyase activities of
Xenopus CYP17 exceeded the 17 The phenomenon of steroid-induced maturation of Xenopus
oocytes has served as a model for studying cell cycle and nongenomic steroid signaling for several decades (1-5). During this time, progesterone has been considered the primary physiologic mediator of
oocyte maturation, perhaps through interactions with classical progesterone receptors expressed in the oocyte (6, 7). We have recently
shown, however, that testosterone, rather than progesterone, may be the
primary physiologic mediator of Xenopus oocyte maturation (8). This dominant role of testosterone is evidenced by the observation
that testosterone is a substantially more potent activator of oocyte
maturation than progesterone, and that testosterone levels are
significantly higher than progesterone (which is nearly undetectable) in the serum and ovaries of
The high levels of ovarian testosterone in the absence of detectable
progesterone imply that Xenopus ovaries are extremely efficient at metabolizing progesterone. Additionally, the ability of
isolated oocytes to rapidly convert progesterone to androgens suggests
that the oocytes themselves may be important contributors to
progesterone metabolism in the ovary. To explain the lack of in
vivo progesterone accumulation, as well as to characterize the
role of oocytes in ovarian androgen production, we separated oocytes
from surrounding follicular cells and examined the ability of both cell
types to metabolize progesterone and other androgen precursors. We
focused on the function of the cytochrome P450 enzyme CYP17 in ovarian
androgen production, as it is known to play a pivotal role in the
synthesis of androgens (Fig. 1). CYP17, an endoplasmic reticulum membrane-bound multifunctional enzyme (9-11),
exhibits two enzymatic activities. Its 17-hydroxylase activities in
both the
4 and
5 pathways; thus, Xenopus CYP17
rapidly converted pregnenolone and progesterone to
dehydroepiandrosterone (DHEA) and androstenedione, respectively. This remarkably robust activity exceeds that of CYP17 from most higher vertebrates, and likely explains why virtually no progesterone is detected in ovulating frogs. Additionally, ovarian CYP17 activity was present exclusively in oocytes, although all other enzymes involved
in sex steroid production were expressed almost entirely in surrounding
follicular cells. This compartmentalization suggests a "two-cell"
model whereby Xenopus ovarian androgen production requires
both follicular cells and oocytes themselves. The requirement of
oocytes for ovarian androgen production further introduces the unusual
paradigm whereby germ cells may be responsible for producing important
steroids used to mediate their own maturation.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-HCG1-stimulated frogs. In
addition, we have shown that, in vitro, isolated oocytes
rapidly convert exogenously added progesterone to the androgen
androstenedione (AD), which is an equally potent promoter of maturation
when compared with progesterone. These data suggest that both AD and
progesterone are likely inducing maturation under in vitro
conditions typical for "progesterone-mediated" maturation.
-hydroxylase activity
converts pregnenolone and progesterone into their respective 17
-hydroxylated products 17
-hydroxypregnenolone (17OHPreg)
and 17
-hydroxyprogesterone (17OHProg). The second 17,20-lyase
activity cleaves the steroid side chains of 17OHPreg and 17OHProg to
yield dehydroepiandrosterone (DHEA) and AD, respectively. CYP17 is
expressed in several steroidogenic tissues (12, 13), including adrenal cortex, ovary, and testis. Whereas earlier work implicates the presence
of CYP17 in oocytes (8), the kinetic characteristics of the
Xenopus CYP17 (XeCYP17), as well as its expression levels in
other ovarian cell types, is not known. Furthermore, expression levels
of other important steroidogenic enzymes in various ovarian cell types
have not been examined.
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Fig. 1.
Ovarian steroid biosynthesis. The sex
steroid synthesis pathway, including several key enzymes, is shown.
CYP17 is highlighted with a box, and the 17 -hydroxylase
and 17,20-lyase activities are indicated on the right. The
5 and
4 pathways are indicated on the top, with the
5 steroids on the left and
4 steroids on the
right.
We show here that XeCYP17 has high 17-hydroxylase, and even more
pronounced 17,20-lyase, activities in both the
5 (pregenenolone) and
4 (progesterone) pathways (Fig. 1). This differs from most known
CYP17 isoforms, which generally favor one pathway over the other, and
rarely have 17,20-lyase activity that rivals their 17
-hydroxylase
activity. Furthermore, we show that ovarian CYP17 activity is present
exclusively in oocytes, whereas other important steroidogenic enzymes,
including 3
-HSD and 17
-HSD, are located primarily in the
surrounding follicular cells. Finally, we propose a "two-cell"
model for androgen synthesis in the Xenopus ovary that
involves both oocytes and follicular cells. This model implies that
germ cells themselves are critical for Xenopus ovarian
androgen production, which in turn may play an important physiologic
role in promoting their own maturation.
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EXPERIMENTAL PROCEDURES |
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Oocyte and Follicular Cell Preparation-- Ovaries were harvested from Xenopus laevis (Nasco, Fort Atkinson, WI) and oocytes were isolated by incubation with 1 mg/ml collagenase A (Roche Molecular Biochemicals) at room temperature for 4 h in modified Barth's solution (MBSH) as previously described (14, 15). Oocytes were then washed and incubated overnight at 16 °C in MBSH with 1 mg/ml bovine serum albumin, 1 mg/ml Ficoll, 100 units/ml penicillin, and 0.1 mg/ml streptomycin. Stage V-VI oocytes were selected and examined microscopically to confirm the absence of follicular cells.
Follicular cells were isolated by incubating ovaries in collagenase as above for 1 h. Oocytes were allowed to settle by gravity for ~5 min, and the supernatant containing follicular cells was then centrifuged at 800 × g for 5 min. The pelleted cells were washed 3 times with MBSH, and any remaining oocytes were removed manually under a dissecting microscope. Similar numbers of follicular cells were used in all of the metabolism experiments. In addition, nearly identical results were obtained using follicular cells removed from oocytes after up to 4 h of treatment with collagenase, or using follicular cells separated from oocytes by incubation with trypsin (16).
Preparation of Oocyte Membranes--
Crude oocyte membranes were
prepared as previously described (17). In short, stage V-VI oocytes
were homogenized in membrane buffer (83 mM NaCl, 1 mM MgCl2, 1 mM phenylmethylsulfonyl
fluoride, 0.5 µg/ml aprotinin, 0.5 µg/ml leupeptin, 0.5 µg/ml
pepstatin, 10 mM Hepes, pH 7.6) at 4 °C. The homogenate
was centrifuged at 800 × g for 5 min and the
supernatant containing the membranes was removed and centrifuged two
more times at 800 × g. The supernatant was then
centrifuged at 15,000 × g for 15 min, and the membrane pellet was resuspended in membrane buffer. Membranes were centrifuged two more times at 15,000 × g and resuspended in MBSH.
Protein concentrations were then measured using the BCA kit (Pierce), and samples were frozen at 80 °C until needed. We saw no
significant drop in CYP17 activity after a single freeze/thaw cycle of
the membranes.
CYP17 Enzyme Assays in Oocyte Membranes--
17-Hydroxylase
and 17,20-lyase activities in oocyte membranes were performed as
previously described (18). Membranes were assayed under initial rate
kinetics by incubation in 50 mM potassium phosphate buffer
(pH 7.4) with 0.03-1 µM steroid (Sigma and Steraloids, Newport, RI) and 1 mM of the cofactor NADPH in a 500-µl
total volume at 16 °C for 30 min. Each reaction contained 50 µg of
membranes and 50,000 cpm of [7-3H]pregnenolone
(19), [3H]17
-hydroxypregnenolone,
[1,2,6,7-3H]progesterone, or
[1,2,6,7-3H]17
-hydroxyprogesterone (PerkinElmer Life
Sciences). Steroids were extracted with 3 ml of 3:2 ethyl
acetate:hexane. Amounts of radioactivity were measured using a
scintillation counter, with >90% recovery from medium. Steroids were
concentrated under nitrogen and separated by TLC using 3:1
chloroform:ethyl acetate. Quantitation of steroids was measured by
cutting out the steroid spots on the TLC plate and measuring
radioactivity using liquid scintillation (20). Kinetic behavior was
approximated as a Michaelis-Menten system for data analysis. The
identities of all steroids in these and the other metabolism
experiments were confirmed by high performance liquid chromatography.
CYP17 Enzyme Assays in Transfected HEK-293 Cells--
HEK-293
cells were grown in complete medium consisting of Dulbecco's modified
Eagle's medium, 10% fetal bovine serum, 100 units/ml penicillin, and
0.1 mg/ml streptomycin (Invitrogen). Experiments were performed
on 12-well plates. Cells were transfected by calcium phosphate
precipitation (8, 21) with either pcDNA3.1 (mock) or pcDNA3.1
containing cDNA encoding the Xenopus or human CYP17
protein (8). After 48 h, cells were placed in fresh complete medium containing 5% charcoal-stripped fetal bovine serum and 60,000 cpm/well of radiolabeled progesterone, 17-hydroxyprogesterone, pregnenolone, or 17
-hydroxypregnenolone at the indicated
concentrations. Cells were incubated at 37 °C, and medium was
removed at the specified times. Steroids were extracted with 5 ml of
3:2 ethyl acetate:hexane, the organic layer was concentrated, and TLC
followed by autoradiography and quantitation was performed as described above.
Steroid Metabolism Assays in Ovaries, Oocytes, and Follicular Cells-- Ovaries (~500 mg/sample), isolated oocytes (30 oocytes per sample), follicular cells, or recombined follicular cells and oocytes (20 oocytes per sample) were incubated with ~500,000 cpm of [1,2,6,7-3H]progesterone, [7-3H]pregnenolone, [1,2,6,7-3H]androstenedione, or [1,2,6,7-3H]DHEA in 1 ml of MBSH for 4 h at 16 °C. Medium was removed from the ovaries, oocytes, or follicular cells, and steroids were extracted with 3:2 ethyl acetate:hexane and treated as above.
-HCG-mediated Maturation of Oocyte in Ovarian
Fragments--
Ovarian fragments of ~100-200 mg were washed in
MBSH and treated for 1 h in 2 ml of MBSH with either ethanol or
100 nM VN/85-1 (a gift from A. Brodie, University of
Maryland). Ethanol concentrations were kept constant.
-HCG was then
added at a concentration of 100 units/ml, and the ovarian fragments
were incubated at 16 °C for ~12 h. The MBSH was removed, and
steroids were extracted and analyzed by radioimmunoassay (8). Oocytes
were manually removed from the ovarian fragments and maturation was
determined by visualization of a white spot on the animal pole.
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RESULTS |
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Sex Steroid Precursors Pregnenolone and Progesterone Are Rapidly
Metabolized to Testosterone by Xenopus Ovaries--
Previous work in
our laboratory demonstrated that -HCG or pregnant mare serum
gonadotropin (PMSG) stimulation of Xenopus ovaries in vivo and in vitro produced high levels
of testosterone and small to moderate amounts of androstenedione. In
contrast, little to no androgen precursors, including pregnenolone,
17OHPreg, progesterone, and 17OHProg, were detected (8). To determine whether Xenopus ovaries could convert sex steroid precursors
to androgens in the absence of gonadotropins, we examined pregnenolone and progesterone metabolism by incubating ovarian tissue with the
respective radiolabeled steroids. Both pregnenolone and progesterone were rapidly converted to testosterone (Fig.
2), with more than 90% loss of each
steroid by 2 h. Virtually no intermediates or further metabolites
of testosterone (e.g. estrogen or dihydrotestosterone) were
detected in both the pregnenolone- and progesterone-treated ovaries
(<10% of total counts at all time points). These data suggest that
Xenopus ovaries contain all of the enzymatic machinery necessary for the conversion of sex steroid precursors to testosterone independent of gonadotropin stimulation. Furthermore, this ability to
rapidly metabolize pregnenolone and progesterone likely explains why
both steroids are nearly undetectable in gonadotropin-stimulated ovaries both in vivo and in vitro (8).
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CYP17 in Xenopus Oocyte Membranes Possesses High 17-Hydroxylase
and 17,20-Lyase Activity in Both the
5 and
4 Pathways--
The
rapid conversion of progesterone and pregenenolone to androgens implies
that the Xenopus ovary contains CYP17, the first key enzyme
in the conversion of these sex steroid precursors to androgens (22,
23). Indeed, we previously cloned the XeCYP17 enzyme from an oocyte
cDNA library and demonstrated its ability to catalyze two enzymatic
reactions: the hydroxylation of progesterone to 17OHProg, and the
conversion of 17OHProg to AD (8). To further characterize the XeCYP17
enzyme, and to determine its role in Xenopus ovarian
androgen production, we used membrane preparations from isolated
oocytes to measure the kinetic characteristics of XeCYP17.
Representative Lineweaver-Burk plots for the 17
-hydroxylase and
17,20-lyase activities in both the
4 and
5 pathways are shown in
Fig. 3, with the calculated average
Km and Vmax values for five
separate experiments shown in Table I.
The Km values for the 17
-hydroxylase and
17,20-lyase activities in both the
4 and
5 pathways ranged
between 26 and 117 nM. The maximal velocities
(Vmax) for 17
-hydroxylase activity in both
the
4 and
5 pathways were similar in magnitude, ranging from 1.3 to 5.2 pmol/mg of protein/min. Surprisingly, XeCYP17 contained
remarkably high 17,20-lyase activity in both the
4 and
5
pathways, with Vmax = 2.3 pmol/mg of protein/min
for the conversion of 17OHPreg to DHEA (
5 pathway), and
Vmax = 12.3 pmol/mg of protein/min for the
conversion of 17OHProg to AD (
4 pathway). This relatively high
17,20-lyase activity could be seen more clearly by examining the ratio
of Vmax values for the 17,20-lyase
versus 17
-hydroxylase reactions in both pathways (Table
I). The lyase/hydroxylase values were 1.8 in the
5 pathway, and 2.5 in the
4 pathway, indicating that XeCYP17 catalyzed the 17,20-lyase
reaction even more quickly than the 17
-hydroxylase reaction in both
pathways. Furthermore, these results suggest that, in the
Xenopus ovary, androgens can be rapidly and efficiently
produced via both the
4 or
5 pathways.
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Xenopus CYP17 Expressed in HEK-293 Cells Completely Metabolizes Sex
Steroid Precursors in Both the 4 and
5 Pathways, whereas Human
CYP17 Favors the
5 Pathway--
The high 17,20-lyase activity of
XeCYP17 in both the
4 and
5 pathways is rather unusual, as most
CYP17 isoforms favor one pathway over the other (12, 18, 24). For
example, the human CYP17 (HuCYP17) has little to no 17,20-lyase
activity in the
4 pathway; thus, human sex steroid synthesis is felt
to involve primarily the
5 pathway (18). To directly compare the
kinetic profiles of the Xenopus and human CYP17 enzymes, and
to confirm that the observed enzymatic activities in the
Xenopus oocyte membranes were in fact mediated by the cloned
XeCYP17 enzyme, we expressed the human and Xenopus CYP17
proteins individually in HEK-293 cells. Fig.
4, A and B, shows
that XeCYP17 converted progesterone to 17OHProg, and then to AD (40%
of total counts by 8 h), confirming that both 17
-hydroxylase
and 17,20-lyase activities are carried by this protein. In contrast,
HuCYP17 contained high 17
-hydroxylase, but very little 17,20-lyase,
activity in the
4 pathway (Fig. 4, A and C,
6% conversion to AD by 8 h). Calculated Km values for HuCYP17-mediated 17
-hydroxylase reactions in the
4 and
5 pathways were ~0.54 and 0.28 µM, respectively
(data not shown), which correlate well with published values (18). This confirms that the enzyme is functioning as expected in our HEK-293 expression system.
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Comparison of the calculated lyase/hydroxylase
Vmax ratios in HEK-293 cells confirmed that
XeCYP17 had high 17,20-lyase activity in both pathways, with
lyase/hydroxylase Vmax ratios of approximately unity (Fig. 4D). In contrast, the 17-hydroxylase reaction
was dominant in both pathways for the HuCYP17, with ratios of ~0.3 in
the
5 pathway and <0.05 in the
4 pathway. The lower
lyase/hydroxylase ratios of XeCYP17 expressed in the HEK-293 cells when
compared with the oocyte membranes could be because of many factors,
including the availability of important cofactors such as NADPH and the flavoprotein reductase(s), or the species compatibility of the 17,20-lyase cofactor cytochrome b5 in human
versus Xenopus tissues. The 17,20-lyase activity
in HEK-293 cells was still relatively high, however, thus these
experiments appear to confirm that the cloned XeCYP17 enzyme is indeed
responsible for the potent 17,20-lyase activity seen in oocyte
membranes. Notably, similar results were qualitatively seen in COS
cells; however, the presence of endogenous CYP17 in these cells
precluded their use for quantitative studies.
Xenopus Oocytes Possess High CYP17 Activity, but Little to No
3-HSD or 17
-HSD Activity--
Having established that
Xenopus CYP17 can metabolize sex steroid precursors equally
well in both the
4 and
5 pathways, we next determined which cells
within the ovary contained CYP17 activity. As mentioned, we had
previously shown that isolated Xenopus oocytes possess high
CYP17 activity in the
4 pathway (8). To confirm the presence of
CYP17 in Xenopus oocytes, we separated oocytes from
surrounding follicular cells and performed steroid metabolism experiments. Notably, isolated oocytes were examined very carefully both under the dissecting microscope and by staining of oocyte sections
to exclude the presence of follicular cell contamination in our
preparations. Fig. 5 represents one of
over 50 different hematoxylin/eosin-stained oocyte sections, with no
detectable follicular cell contamination.
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As expected, isolated oocytes contained high CYP17 activity in both the
5 and
4 pathways, as radiolabeled pregnenolone and progesterone
were rapidly metabolized to only DHEA and AD, respectively (Fig.
6A). Interestingly, the
isolated oocytes did not appear to significantly express any of the
other enzymes responsible for the various stages in the production of
sex steroids. For example, 3
-HSD is responsible for the conversion
of
5 to
4 steroids (Fig. 1). The rapid conversion of radiolabeled
pregnenolone to DHEA in the absence of detectable
4 steroid
production (Fig. 6A, left panel) argues strongly
that oocytes lack significant 3
-HSD activity. Likewise, 17
-HSD is
necessary for the conversion of DHEA to androstenediol, and of
androstenedione to testosterone (Fig. 1). The lack of detectable
androstenediol in the pregnenolone-treated cells, as well as the near
absence of testosterone in progesterone-treated cells (<10% of the
total counts at all time points measured), suggests that oocytes
contain little 17
-HSD activity (Fig. 6A). Finally,
stimulation of isolated oocytes with
-HCG did not promote significant steroid production by radioimmunoassay (data not shown), suggesting that they contain little CYP11A1 or steroidogenic acute regulatory protein (stAR) activity, both of which are necessary to augment formation of pregnenolone (Fig. 1).
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Xenopus Ovarian Follicular Cells Contain 3-HSD and 17
-HSD
Activities, but No CYP17 Activity--
If oocytes have no significant
3
-HSD and 17
-HSD activity, then these enzymes must be present in
the surrounding ovarian follicular cells to complete ovarian androgen
synthesis. In support of this hypothesis, small amounts of radiolabeled
pregnenolone were converted to progesterone by isolated ovarian
follicular cells, indicating the presence of 3
-HSD in these cells
(Fig. 6B, left panel). Similar numbers of
follicular cells converted radiolabeled DHEA to AD at a significantly
higher rate (Fig. 6B, middle panel), thus
confirming the presence of 3
-HSD and suggesting that DHEA may be
preferred over progesterone as a substrate for Xenopus
ovarian 3
-HSD. Finally, radiolabeled AD was very efficiently converted to testosterone (Fig. 6B, middle and
right panels), thereby demonstrating the presence of
17
-HSD in the follicular cells as well. Surprisingly, follicular
cells did not hydroxylate progesterone or pregnenolone at all (Fig.
6B, left panel, and data not shown), indicating
that ovarian CYP17 activity is contained exclusively in the oocytes themselves.
Because separation of oocytes and follicular cells revealed differential expression of the steroidogenic enzymes, the isolated cell types were recombined to determine whether the complete steroidogenic pathway from pregnenolone to testosterone could be reconstituted. The recombination of follicular cells and oocytes resulted in testosterone production (Fig. 6C), confirming that these two populations of cells were indeed sufficient to mediate complete steroidogenesis. Several intermediate steroids were also produced, including progesterone, DHEA, and AD, suggesting that the reconstituted system was less efficient than the intact ovary in producing testosterone.
Blockade of Androgen Production Attenuates -HCG-stimulated
Maturation of Xenopus Oocytes in Intact Ovarian Follicles--
To
confirm the physiologic importance of CYP17-mediated androgen
production in Xenopus oocyte maturation, intact ovarian
follicles were stimulated with
-HCG alone or in combination with the
potent CYP17 inhibitor VN/85-1. The maximum amount of oocyte maturation induced by 100 units/ml
-HCG over 12 h was 47% (Table
II, ethanol), which is consistent with
more than 8 similar studies using identical conditions (data not
shown). This amount of maturation is also similar to that seen when
ovarian follicles were treated with 500 nM AD (Table
II),2 suggesting that ~50%
maturation is likely the maximum attainable under these conditions.
Inhibition of CYP17 with VN/85-1 significantly decreased
-HCG-mediated oocyte maturation by 28%. As expected, VN/85-1 also
decreased, but did not eliminate, both testosterone and AD production.
Interestingly, although progesterone production increased slightly in
the presence of VN/85-1, progesterone levels still remained quite low,
suggesting that very little was being produced in vivo, even
the presence of a CYP17 inhibitor. This result confirms the data in
Fig. 6, where pregnenolone was shown to be inefficiently converted to
progesterone by follicular cells. Together, these data suggest that
CYP17-mediated androgen production may indeed be important for
maturation in vivo. Incomplete inhibition of maturation by
VN/85-1 was likely because of induction by the remaining AD and
testosterone (which is 10-fold more potent than AD and progesterone
(8)), as well as perhaps by the slightly higher amounts of
progesterone. Addition of 500 nM AD rescued the inhibitory
effects of VN/85-1 by increasing AD and testosterone levels (Table II).
Addition of exogenous progesterone also rescued the effects of VN/85-1
by increasing progesterone levels while androgen levels remained low.
These data are consistent with earlier work where progesterone promoted
maturation of isolated oocytes treated with ketoconazole (8), and
suggest that, although low endogenous progesterone production precludes
its importance relative to androgens in vivo, exogenous
progesterone is still capable of promoting maturation in
vitro.
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DISCUSSION |
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Ovarian sex steroid production is essential for follicular growth and subsequent ovulation in nearly every animal (25-27). In frogs and fish, these steroids also appear to be critical regulators of oocyte maturation (28, 29), which is defined as the resumption of meiosis from prophase I to metaphase II. Fish oocyte maturation is regulated by various hydroxylated progesterone metabolites (30, 31), whereas testosterone may be an important physiologic mediator of Xenopus oocyte maturation (8). Sex steroids may be involved in higher vertebrate oocyte maturation as well; however, evidence for or against such a role is still minimal at this point in time.
Sex steroid production requires the cytochrome P450 enzyme CYP17 to
catalyze both its 17-hydroxylase and 17,20-lyase reactions, respectively. Whereas all mammalian CYP17 isoforms have relatively equal 17
-hydroxylase activities in both the
4 (progesterone) and
5 (pregnenolone) pathways, most have preferential 17,20-lyase activity for one of the two pathways. For example, in small mammals, such as rats (32), mice (33), hamsters (34), and guinea pigs (35),
CYP17 is more active in the
4 pathway. In contrast, human and
primate CYP17s prefer the
5 pathway (18, 20). We have found that
XeCYP17 has high 17,20-lyase activities in both the
4 (progesterone)
and
5 (pregnenolone) pathways (Table I), suggesting that, unlike
most mammals, Xenopus frog ovaries can utilize both pathways
to produce sex steroids. Furthermore, the rate of the XeCYP17-mediated
17,20-lyase reaction equals (HEK-293 cells, Fig. 4D) or
exceeds (oocyte membranes, Fig. 3 and Table I) that of the
17
-hydroxylase reaction in both pathways. These high lyase
activities dramatically contrast with nearly every mammalian isoform of
CYP17, where the 17
-hydroxylase activities markedly exceed the
17,20-lyase activities (18, 36) (Fig. 5D). Interestingly,
the eel, fish, and shark CYP17 enzymes, which are most closely related
to XeCYP17 by phylogenetic analysis (Fig. 7), appear to have robust 17,20-lyase
activities in both pathways as well (37, 38), although no exact
measurements of 17,20-lyase activity have been reported.
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Taken together, these data lead us to speculate that the level of
CYP17-mediated 17,20-lyase activity, as well the preferential use of
one or both (4 and
5) of the steroidogenic pathways, may reflect
the need of the enzyme to produce reproductive steroids versus cortisol in various organisms. In humans, for
example, cortisol is essential for normal growth and development.
Cortisol is derived primarily from the 21-hydroxylation of
17
-hydroxyprogesterone; thus, the relatively low 17,20-lyase
activities of human CYP17, which leads to the accumulation of
17
-hydroxylated steroids, may be necessary to permit cortisol
production in human adrenal glands. In contrast, frogs, as well as
sharks, eels, and most fish, do not appear to need cortisol; instead,
they rely on corticosterone or corticosterone metabolites as their
primary glucocorticoids (39-41). CYP17 is therefore not required for
glucocorticoid production in these lower vertebrates; its sole function
appears to be to generate sex steroids. In short, CYP17 may have
evolved from favoring primarily sex steroid production in lower
vertebrates, which do not require cortisol, to promoting both cortisol
and sex steroid production in higher vertebrates. Accordingly, the
lower vertebrate CYP17s appear to form a closely related phylogenetic
family by sequence homology, whereas the more evolved higher vertebrate CYP17s appear to be part of a distinctly separate family of proteins (Fig. 7).
The mechanism behind the unusually high 17,20-lyase activity of XeCYP17 relative to other CYP17 isoforms is an intriguing issue. One clearly important factor that regulates 17,20-lyase activity of human CYP17 is the presence of the co-factor protein cytochrome b5 (18, 42). Perhaps the high 17,20-lyase activity of XeCYP17 is because of differences in the influence of or dependence on cytochrome b5 as a cofactor for this reaction. Alternatively, Xenopus CYP17 may contain sequences that enhance its ability to bind to 17-hydroxylated steroids or other important cofactors. Given its relatively high 17,20-lyase activity, the Xenopus isoform may serve as a useful tool in teasing apart the mechanisms controlling the two enzymatic activities of the CYP17 enzyme.
The exclusive expression of XeCYP17 in oocytes suggests an unusual
mechanism of sex steroid production driving oocyte maturation in the
frog ovary. Taking into account the results reported in this study, we
propose a model for steroid biosynthesis and steroid-induced maturation
of oocytes (Fig. 8). In this model,
pregnenolone would be produced in the follicular cells. Because
pregnenolone is inefficiently converted to progesterone (Fig. 6), even
in the presence of a CYP17 inhibitor (Table II), very little
progesterone is likely being produced by 3-HSD at any time. Because
the follicular cells do not express CYP17, pregnenolone must then enter
the surrounding oocytes to be converted to DHEA. Additionally, because
CYP17 is equally active in the
4 pathway, any small amounts of
progesterone that are produced by the follicular cells would be rapidly
converted to AD in the oocyte, thus further preventing significant
accumulation of progesterone. DHEA and AD would then be transported
back to the follicular cells, where 17
-HSD and 3
-HSD would
complete testosterone synthesis. Finally, testosterone from the
follicular cells would re-enter the oocyte to promote its maturation.
Because AD is also capable of promoting maturation, one cannot rule out the possibility that AD also plays a role in oocyte maturation in
vivo; however, given the significantly higher potency and ovarian concentrations of testosterone relative to AD (8), testosterone is most
likely the primary physiologic mediator of maturation in
Xenopus oocytes. Although progesterone appears capable of
promoting maturation in vitro, the lack of significant
progesterone production at all times by
-HCG-stimulated frog
ovaries, even in the presence of a CYP17 inhibitor, argues against a
major role for progesterone in oocyte maturation in
vivo.
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Our model bears some similarity to the two-cell models put forth
to explain sex steroid biosynthesis in other systems (43, 44). In this
case, we do not actually know how many different cell types exist
within our follicular fraction; however, it is quite clear that these
cells contain all of the important steroid synthetic enzymes except
CYP17, whereas oocytes contain CYP17 but no other relevant activities.
It is still possible that our oocyte preparations contain a small
population of follicular cells that contain all of the detected CYP17
activity; however, this explanation seems less likely for the following
reasons. First, this population of cells would have to be so tightly
associated with oocytes that it was completely resistant to separation
by both collagenase and trypsin, as the follicular cell preparations contained no detectable CYP17. Second, this contaminating population of
cells would have to be very small, as it was undetectable by both
stereoscopic and histologic examination. Third, in order for such a
small contamination to be mediating the high velocities recorded in
Table I, which are very similar to the velocities of other CYP17
enzymes overexpressed in fibroblast or COS cells, as well as with those
seen in cultured thecal cells (45-47), the turnover rate of the
Xenopus CYP17 would have to be extremely high. We know that
this is not the case, as comparison of the human and Xenopus
CYP17 17-hydroxylase activities in HEK cells (Fig. 5), and recently
in yeast microsomes (data not shown), reveals that they have nearly
identical turnover rates (~6 min
1). Finally,
progesterone injected directly into oocytes is immediately converted to
AD (8), arguing that the enzymatic activity is within the oocyte itself.
We are left with the intriguing notion that, in the frog ovary, germ cells, or oocytes, play a critical role in the production of the steroid used for their own maturation. To our knowledge, this is the first example of germ cells being directly involved in steroid production, although it has not been carefully examined in other lower vertebrates, such as fish. Because oocytes make up >90% of the ovarian volume in frogs and fish, one could speculate that such lower vertebrate animals might require their oocytes to contribute to ovarian sex steroid production. In contrast, higher vertebrates, in which the ovarian volume primarily consists of follicular cells, may no longer need oocytes to subserve this function.
Interestingly, this concept of oocytes actively participating in their own maturation (in our case through the production of androgens) is consistent with the recently described work in mammalian systems, where oocytes have been shown to communicate with surrounding somatic cells to promote granulosa cell proliferation and differentiation (48). Oocytes may therefore utilize many different mechanisms to assist in orchestrating proper follicular development and subsequent ovulation.
Finally, these studies further explain and support earlier work
suggesting that, although progesterone is capable of promoting maturation in vitro, androgens may be the primary mediators
of maturation in vivo, where very little progesterone is
ever produced. Steroid-induced maturation of Xenopus oocytes
has been a puzzling field of investigation for many years, as it
appears to occur independent of transcription and may involve signaling
via classical steroid receptors acting outside of the nucleus (6-8,
49). Further studies of Xenopus oocyte maturation by
androgens, in addition to progesterone, may aid in finally determining
the details behind this complex and fascinating process.
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ACKNOWLEDGEMENT |
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We thank Dr. Richard Auchus for valuable advice and input.
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FOOTNOTES |
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* This work was supported in part by National Institutes of Health Grant DK59913 and Welch Foundation Grant I-1506.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
W. W. Caruth, Jr. Endowed Scholar in Biomedical Research. To
whom correspondence should be addressed. Tel.: 214-648-4793; Fax:
214-648-7934; E-mail: stephen.hammes@utsouthwestern.edu.
Published, JBC Papers in Press, January 9, 2003, DOI 10.1074/jbc.M212027200
2 W-H. Yang, L. B. Lutz, and S. R. Hammes, unpublished data.
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ABBREVIATIONS |
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The abbreviations used are:
-HCG,
-human
chorionic gonadotropin;
AD, androstenedione;
DHEA, dehydroepiandrosterone;
XeCYP17, Xenopus CYP17;
HuCYP17, human CYP17;
HSD, hydroxysteroid dehydrogenase;
17OHPreg, 17-hydroxypregnenolone;
17OHProg, 17-hydroxyprogesterone;
HEK, human
embryonic kidney;
MBSH, modified Barth's solution.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Smith, L. D., and Ecker, R. E. (1969) Dev. Biol. 19, 281-309[Medline] [Order article via Infotrieve] |
2. | Smith, L. D., and Ecker, R. E. (1971) Dev. Biol. 25, 232-247[Medline] [Order article via Infotrieve] |
3. | Maller, J. L., and Krebs, E. G. (1977) J. Biol. Chem. 252, 1712-1718[Abstract] |
4. | Maller, J. L., and Krebs, E. G. (1980) Curr. Top. Cell. Regul. 16, 271-311[Medline] [Order article via Infotrieve] |
5. | Newport, J. W., and Kirschner, M. W. (1984) Cell 37, 731-742[Medline] [Order article via Infotrieve] |
6. |
Bayaa, M.,
Booth, R. A.,
Sheng, Y.,
and Liu, X. J.
(2000)
Proc. Natl. Acad. Sci. U. S. A.
97,
12607-12612 |
7. |
Tian, J.,
Kim, S.,
Heilig, E.,
and Ruderman, J. V.
(2000)
Proc. Natl. Acad. Sci. U. S. A.
97,
14358-14363 |
8. |
Lutz, L. B.,
Cole, L. M.,
Gupta, M. K.,
Kwist, K. W.,
Auchus, R. J.,
and Hammes, S. R.
(2001)
Proc. Natl. Acad. Sci. U. S. A.
98,
13728-13733 |
9. |
Zuber, M. X.,
John, M. E.,
Okamura, T.,
Simpson, E. R.,
and Waterman, M. R.
(1986)
J. Biol. Chem.
261,
2475-2482 |
10. | Zuber, M. X., Simpson, E. R., and Waterman, M. R. (1986) Science 234, 1258-1261[Medline] [Order article via Infotrieve] |
11. |
Lin, D.,
Harikrishna, J. A.,
Moore, C. C.,
Jones, K. L.,
and Miller, W. L.
(1991)
J. Biol. Chem.
266,
15992-15998 |
12. | Chung, B. C., Picado-Leonard, J., Haniu, M., Bienkowski, M., Hall, P. F., Shively, J. E., and Miller, W. L. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 407-411[Abstract] |
13. | Miller, W. L. (1988) Endocr. Rev. 9, 295-318[Medline] [Order article via Infotrieve] |
14. | Vu, T.-K. H., Hung, D. T., Wheaton, V. I., and Coughlin, S. R. (1991) Cell 64, 1057-1068[Medline] [Order article via Infotrieve] |
15. | Williams, J. A., McChesney, D. J., Calayag, M. C., Lingappa, V. R., and Logsdon, C. D. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 4939-4943[Abstract] |
16. | Arellano, R. O., and Miledi, R. (1995) J. Physiol. 488, 351-357[Abstract] |
17. |
Lutz, L. B.,
Kim, B.,
Jahani, D.,
and Hammes, S. R.
(2000)
J. Biol. Chem.
275,
41512-41520 |
18. |
Auchus, R. J.,
Lee, T. C.,
and Miller, W. L.
(1998)
J. Biol. Chem.
273,
3158-3165 |
19. | Aaronson, S. A., and Todaro, G. J. (1968) J. Cell Physiol. 72, 141-148[Medline] [Order article via Infotrieve] |
20. | Lin, D., Black, S. M., Nagahama, Y., and Miller, W. L. (1993) Endocrinology 132, 2498-2506[Abstract] |
21. | Gao, T., Marcelli, M., and McPhaul, M. J. (1996) J. Steroid Biochem. Mol. Biol. 59, 9-20[CrossRef][Medline] [Order article via Infotrieve] |
22. | Picado-Leonard, J., and Miller, W. L. (1987) DNA (N. Y.) 6, 439-448[Medline] [Order article via Infotrieve] |
23. | Sparkes, R. S., Klisak, I., and Miller, W. L. (1991) DNA Cell Biol. 10, 359-365[Medline] [Order article via Infotrieve] |
24. | Higuchi, A., Kominami, S., and Takemori, S. (1991) Biochim. Biophys. Acta 1084, 240-246[Medline] [Order article via Infotrieve] |
25. | Nillius, S. J., and Wide, L. (1975) Br. Med. J. 3, 405-408[Medline] [Order article via Infotrieve] |
26. | Yding Andersen, C., Westergaard, L. G., Figenschau, Y., Bertheussen, K., and Forsdahl, F. (1993) Hum. Reprod. 8, 840-843[Abstract] |
27. | Haresign, W., Foxcroft, G. R., and Lamming, G. E. (1983) J. Reprod. Fertil. 69, 383-395[Abstract] |
28. | Antila, E. (1977) Differentiation 8, 71-77[Medline] [Order article via Infotrieve] |
29. | Skoblina, M. N., Shmerling, ZhG., and Kondratieva, O. T. (1981) Gen. Comp. Endocrinol. 44, 470-475[Medline] [Order article via Infotrieve] |
30. | Canario, A. V., Scott, A. P., and Flint, A. P. (1989) Gen. Comp. Endocrinol. 74, 77-84[Medline] [Order article via Infotrieve] |
31. | Ponthier, J. L., Shackleton, C. H., and Trant, J. M. (1998) Gen. Comp. Endocrinol. 111, 141-155[CrossRef][Medline] [Order article via Infotrieve] |
32. |
Koh, Y.,
Buczko, E.,
and Dufau, M. L.
(1993)
J. Biol. Chem.
268,
18267-18271 |
33. | Youngblood, G. L., and Payne, A. H. (1992) Mol. Endocrinol. 6, 927-934[Abstract] |
34. | Cloutier, M., Fleury, A., Courtemanche, J., Ducharme, L., Mason, J. I., and Lehoux, J. G. (1997) DNA Cell Biol. 16, 357-368[Medline] [Order article via Infotrieve] |
35. | Tremblay, Y., Belanger, A., Fleury, A., Beaudoin, C., Provost, P., and Martineau, I. (1995) Endocr. Res. 21, 495-507[Medline] [Order article via Infotrieve] |
36. | Brock, B. J., and Waterman, M. R. (1999) Biochemistry 38, 1598-1606[CrossRef][Medline] [Order article via Infotrieve] |
37. | Trant, J. M. (1996) Arch. Biochem. Biophys. 326, 8-14[CrossRef][Medline] [Order article via Infotrieve] |
38. | Kazeto, Y., Ijiri, S., Todo, T., Adachi, S., and Yamauchi, K. (2000) Gen. Comp. Endocrinol. 118, 123-133[CrossRef][Medline] [Order article via Infotrieve] |
39. | Sandor, T., Chan, S. W., Phillips, J. G., Ensor, D., Henderson, I. W., and Jones, I. C. (1970) Can. J. Biochem. 48, 553-558[Medline] [Order article via Infotrieve] |
40. | Simpson, T. H., and Wright, R. S. (1970) J. Endocrinol. 46, 261-268[Medline] [Order article via Infotrieve] |
41. | Armour, K. J., O'Toole, L. B., and Hazon, N. (1993) J. Mol. Endocrinol. 10, 235-244[Abstract] |
42. | Lee-Robichaud, P., Wright, J. N., Akhtar, M. E., and Akhtar, M. (1995) Biochem. J. 308, 901-908[Medline] [Order article via Infotrieve] |
43. | Kagawa, H., Young, G., Adachi, S., and Nagahama, Y. (1982) Gen. Comp. Endocrinol. 47, 440-448[Medline] [Order article via Infotrieve] |
44. | Dorrington, J. H., and Armstrong, D. T. (1979) Recent Prog. Horm. Res. 35, 301-342[Medline] [Order article via Infotrieve] |
45. |
Martens, J. W.,
Geller, D. H.,
Arlt, W.,
Auchus, R. J.,
Ossovskaya, V. S.,
Rodriguez, H.,
Dunaif, A.,
and Miller, W. L.
(2000)
J. Clin. Endocrinol. Metab.
85,
4338-4346 |
46. | Demeter-Arlotto, M., Rainey, W. E., and Simpson, E. R. (1993) Endocrinology 132, 1353-1358[Abstract] |
47. |
Swart, A. C.,
Kolar, N. W.,
Lombard, N.,
Mason, J. I.,
and Swart, P.
(2002)
Eur. J. Biochem.
269,
5608-5616 |
48. |
Matzuk, M. M.,
Burns, K. H.,
Viveiros, M. M.,
and Eppig, J. J.
(2002)
Science
296,
2178-2180 |
49. |
Sadler, S. E.,
and Maller, J. L.
(1982)
J. Biol. Chem.
257,
355-361 |