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-Hydroxysteroid Dehydrogenase Enzymes in Immature, Mature, and Pregnant Rats1
Biocenter Oulu and the Department of Clinical Chemistry, University of Oulu, FIN-90220 Oulu, Finland
Address all correspondence and requests for reprints to: Prof. Pirkko Vihko, Biocenter Oulu and Department of Clinical Chemistry, University of Oulu, FIN-90220 Oulu, Finland.
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Abstract |
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-hydroxysteroid
dehydrogenase (20HSD) in mature and pregnant rats. Immunohistochemical
analysis of rat 17HSD type 1 showed that the enzyme is exclusively
expressed in the granulosa cells of developing, healthy, primary,
secondary, and tertiary follicles at all stages of the estrous cycle
and pregnancy, and is not detected in the corpora lutea. The data
showed that the amount of the enzyme expressed in the follicle
increases as follicular maturation progresses and is highest in
tertiary and Graafian follicles. However, Northern blot analysis of
total RNA from whole ovaries showed a rather constitutive expression of
the 17HSD type 1 enzyme. It is evident that compared with P450arom,
17HSD type 1 is more widely expressed in the follicles during the
various maturational stages of folliculogenesis. Hence, the data
indicate distinct localization, expression, and regulation patterns for
17HSD type 1 and P450arom during the rat estrous cycle and pregnancy.
Furthermore, compared with the two estradiol biosynthetic enzymes, a
different expression pattern was detected for 20HSD messenger RNA.
During the estrous cycle the enzyme was detected in the ovaries
throughout the cycle, and in the ovaries of pregnant animals the enzyme
showed an expression pattern the opposite of that observed for
P450arom. Rat 17HSD type 2, not detected in the ovaries, was
constitutively expressed in both female and male liver and small
intestine in 21-day-old fetuses up to 6-week-old mature animals.
Similarly, in these tissues the enzyme was constitutively expressed in
normal cycling and pregnant animals, but it showed increasing
expression in the placenta as pregnancy advanced. The relatively
constitutive expression of the enzyme at all physiological stages of
the animals suggests a general role for the enzyme in the inactivation
of circulating sex steroids. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In addition to its 17HSD activity, human 17HSD type 2 also
possesses 20-hydroxysteroid dehydrogenase (20HSD) activity (13),
interconverting 20-hydroxyprogesterone (20OHP) and progesterone
(P). In addition to P, a high concentration of 20OHP is secreted
from the rat ovary (15). 20OH-P is mainly formed via the activity of
20HSD, which is widely distributed in several steroid (adrenal,
ovarian, placental, and uterine) and nonsteroid-producing cells
(16, 17, 18, 19). As P is a necessary hormone during mammalian pregnancy,
enzymes with 20HSD activity could play a pivotal role in the
establishment and maintenance of pregnancy. The present study was
carried out to study the expression and regulation of 17HSD type 1 and
type 2, P450arom, and 20HSD enzymes in immature, mature, and pregnant
rats, especially during the estrous cycle and pregnancy.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-32P]Deoxy-CTP (3000 Ci/mmol) and
[-32S]deoxy-ATP (800 Ci/mmol) were purchased from
Amersham International (Aylesbury, UK). Guanidine isothiocyanate was
obtained from Fluka (Buchs, Switzerland). Cesium chloride and
oligo(deoxythymidine)-cellulose were purchased from Boehringer Mannheim
(Mannheim, Germany). Agarose was obtained from FCM Bioproducts
(Rockland, ME), and RNA mol wt markers (RNA ladder) were purchased from
BRL (Gaithersburg, MD). Restriction enzymes were obtained from New
England Biolabs (Beverley, MA) or Pharmacia (Piscataway, NJ), and T4
DNA ligase was obtained from Boehringer Mannheim. Pfu DNA polymerase
was purchased from Stratagene (La Jolla, CA). Other reagents not
mentioned in the text were purchased from Sigma Chemical Co. (St.
Louis, MO) or Merck (Darmstadt, Germany) and were of the highest purity
grade available.
Animals and histological techniques
The ovaries, liver, and small intestine were excised from adult
female regularly cycling Sprague-Dawley rats at proestrus, estrus,
metestrus, and diestrus and from pregnant rats on various days of
gestation. The stages of the estrous cycle were determined by light
microscopic examination of vaginal fluids of the animals. In addition,
placental tissues from the pregnant rats were analyzed. The liver and
small intestine were also removed for analysis from 21-day-old fetuses
and from immature male and female rats. All tissue specimens, except
those used for immunohistochemistry, were immediately frozen in liquid
nitrogen and stored at -70 C before processing. For
immunohistochemistry, ovarian tissues were excised and immediately
placed in 4% paraformaldehyde-PBS for 1 h at 4 C. The tissues
were further fixed in 4% paraformaldehyde-5% sucrose-PBS for 1 h
at 4 C. Thereafter, the ovaries were incubated overnight in 20%
sucrose-PBS at 4 C. The fixed tissues were washed in PBS and frozen in
isopentane cooled with liquid nitrogen. For immunohistochemical
staining, 7-µm cryosections were cut and mounted onto
polylysine-coated slides.
Immunohistochemistry
Immunohistochemical staining for rat 17HSD type 1 was carried
out as described previously (6, 10). Nonspecific binding of the
antibodies was blocked by incubating the slides in 10% goat serum-PBS
for 30 min at room temperature. Thereafter, the slides were further
incubated overnight at 4 C with a 1:200 dilution (in 10% goat
serum-PBS) of a rabbit antiserum raised against human 17HSD type 1
protein. All subsequent steps were carried out at room temperature. The
slides were washed three times with PBS for 5 min each time and
incubated for 2 h in a 1:50 dilution of biotinylated goat
antirabbit antibody (Dakopatts, Copenhagen, Denmark) in 10% goat
serum-PBS. The slides were washed and treated for 1 h in a 1:50
dilution of streptavidin-conjugated fluorescein isothiocyanate
(Dakopatts) in 10% goat serum-PBS. The slides were then washed five
times with PBS, for 5 min each time, and mounted with a drop of
Immu-mount (Shandon, Pittsburgh, CA).
Northern analysis
Total RNA was extracted from liver, ovary, placenta, and small
intestine by homogenization in guanidium isothiocyanate buffer,
followed by centrifugation through a cesium chloride density gradient.
Twenty micrograms of total RNA were subjected to electrophoresis in 1%
(wt/vol) agarose-formaldehyde gel, transferred overnight onto a Hybond
nylon membrane (Amersham), and fixed by UV irradiation. The membranes
were prehybridized for 2 h at 46 C in 5 x SSPE (1 x
SSPE = 0.15 M NaCl, 10 mM PBS, and 0.1
mM EDTA, pH 7.4) with formamide (50%), BSA (0.1%), Ficoll
(0.1%), polyvinylpyrrolidone (0.1%), SDS (0.5%), and 20 mg salmon
sperm DNA/liter. Hybridization was carried out overnight at 46 C with
32P-labeled complementary DNAs (cDNAs) of rat 17HSD type 1
[1.0-kilobase (kb) EcoRI-SacI fragment) (6), rat 17HSD type
2 (1.3-kb full-length EcoRI fragment) (7), rat P450
aromatase (1.2-kb EcoRI fragment; provided by Prof. JoAnne
Richards, Department of Cell Biology, Baylor College of Medicine,
Houston, TX), and rat 20HSD (981-bp full-length ApaI
fragment). After hybridization, the membranes were washed twice at 46 C
in 2 x SSPE-0.1% SDS and once in 1 x SSPE-0.1% SDS for 15
min each time and exposed to Kodak X-AR films (Eastman Kodak,
Rochester, NY) for 6–96 h. In between the different hybridizations,
the bound probes were removed by treating the membrane with 0.1% SDS
at 95 C. The amounts of 17HSD type 1, P450arom, and 20HSD messenger
RNAs (mRNAs) expressed in the pregnant rat ovary were analyzed by laser
densitometric scanning of the autoradiographs (Molecular Dynamics,
Sunnyvale, CA) after 18 h of exposure and were normalized against
the values obtained for 
-actin. The normalized values were used to
analyze the relative expression of the mRNAs. The experiments by
Northern analysis and immunohistochemistry were repeated several times
with identical results, and the data presented in this manuscript are
typical examples.
The 20HSD cDNA used was cloned by RT-PCR using polyadenylated RNA from adult rat ovaries. Oligo(deoxythymidine)-primed cDNA synthesis was carried out using a ZAP cDNA synthesis kit (Stratagene, La Jolla, CA). A PCR reaction was then carried out using pfu DNA polymerase (Stratagene) together with primers corresponding to nucleotides -3 to 18 of the open reading frame and 954–978 of the complimentary strand (18).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). The amount of the enzyme
expressed in the follicle increases as follicular maturation progresses
and is highest in tertiary and Graafian follicles. Northern blot
analysis of RNA extracted from whole ovaries, however, showed
constitutive expression of 17HSD type 1 mRNA at proestrus, estrus,
metestrus, and diestrus (Fig. 2). Compared with 17HSD
type 1, P450arom was more efficiently regulated during the estrous
cycle. The greatest expression of P450arom mRNAs was detected at
proestrus; this was lower but still significant at metestrus and
diestrus, but was barely detectable at estrus (Fig. 2). Among the
analyzed mRNAs, 20HSD mRNA showed the strongest expression throughout
the estrous cycle, with a gradual decline from proestrus to diestrus
(Fig. 2). In diethylstilbestrol (DES)-treated immature rat ovaries, no
significant expression of P450arom or 20HSD mRNAs was detected,
indicating that of the enzymes analyzed, only 17HSD type 1 is expressed
in the granulosa cells at the early stages of follicular maturation
induced by DES.
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). Similarly, no
variation in the amount of the enzyme was detected during rat pregnancy
or at any age of the animal from late fetal life up to 6 weeks of age,
regardless of sex (data not shown). This indicates constitutive
expression of 17HSD type 2 enzyme in the liver and small intestine
throughout the life span of the animals. In contrast, the expression of
17HSD type 2 in the placenta increased from a very low amount on day 8
to a relatively strong expression detected at the end of pregnancy
(Fig. 3).
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). Interestingly,
immunohistochemical analysis of rat 17HSD type 1 expression throughout
pregnancy showed strong expression of the enzyme only in the granulosa
cells of developing ovarian follicles, similar to that found during the
estrous cycle (Fig. 4). No significant expression of the
enzyme was detected in the CL at any stage of gestation. Constitutive
expression of the two 17HSD type 1 mRNAs in the ovary, with a slight
increase from day 5 to day 15, was confirmed by Northern blot analysis
(Fig. 3). Furthermore, our results using Northern analysis agree with
previous reports of strong up-regulation of ovarian P450arom expression
starting on day 5 and reaching a plateau on day 12, lasting until day
18 of rat pregnancy. Thereafter, the amount of enzyme decreased
precipitously to very low levels on day 21. Hence, 17HSD type 1
expression does not follow the pattern of P450arom regulation in
pregnant rat ovaries. Furthermore, 17HSD type 1 is present in
developing follicles, whereas P450arom is expressed in the CL, and the
relative concentration of P450arom mRNA is about 30-fold higher than
that of 17HSD type 1 on days 15–18 of pregnancy (Fig. 5).
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). The high amount
of mRNA expression detected throughout the estrous cycle continued on
days 1 and 2 of pregnancy. Thereafter, a gradual decrease in the mRNA
was observed, reaching a nadir on days 15–18. However, on day 21
a dramatic rise in the amount of 20HSD mRNA expression was observed,
resulting in expression comparable to that detected on days 1 and 2 of
gestation (Fig. 3). |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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OHP (15), a
progesterone metabolite formed by the activity of the 20HSD enzyme.
Previous reports have also shown that the peripheral plasma
concentrations of 20OHP are variable, highest at proestrus and
lowest at diestrus, probably mirroring similar variations in plasma
progesterone concentrations (15). These findings agree with our present
data that show a high level of expression of 20HSD mRNA during the
whole estrous cycle with a gradual decline from proestrus to
diestrus. Prolongation of luteal function in the rat and, hence, maintenance of pregnancy during the first week of gestation are modulated by pituitary PRL and LH (24). During the latter half of pregnancy, however, gestation is maintained by the placental production of rat placental lactogen and androgens, and by E2 and P produced by the CL (2, 25, 26). During pregnancy, the size of the CL increases rapidly in parallel to its capacity to secrete E2 and P to sustain normal pregnancy in the rat. Our data, however, indicate 17HSD type 1 expression only in the granulosa cells of the growing follicles in pregnant rat ovaries, whereas no significant amount of the enzyme was detected in the CL. Hence, the expression pattern of 17HSD type 1 during rat pregnancy appears to be identical to that found during the normal estrous cycle. These results suggest that once 17HSD type 1 expression is turned on in the granulosa cells by FSH and/or paracrine factors (6, 10, 11), it is maintained in the growing follicles and down-regulated in the CL by luteinization and perhaps by all hormonal factors that sustain CL functions during pregnancy. Whether the relatively low expression of 17HSD type 1 is sufficient for E2 production in pregnant rat ovaries from the placental androgenic precursor, androstenedione, remains to be clarified. Similar to previous reports, our results show strong up-regulation of P450arom on days 5–18 of rat pregnancy. These effects have been attributed to the luteotropic effects of PRL and rat placental lactogen during the first and second weeks of pregnancy in rats, respectively (2, 24). Hence, it is evident that these hormones are not similarly involved in the regulation of 17HSD type 1 in the pregnant rat.
The luteotropic effects of E2 secreted by the CL
are necessary in the latter half of pregnancy to sustain the functions
of the CL and thus for continued P production (2, 27). The mechanism of
regulation of 20HSD has yet to be clarified, but the enzyme activity is
significantly up-regulated in immature rats treated with equine CG/hCG
(18). The expression and regulation of 20HSD (which inactivates P to
20OHP) in the ovary might also determine P secretion and its
activities during rat gestation. Our results as well as those of
previous reports (16) show a significant drop in the amount of 20HSD
mRNA in the rat ovary from day 8 to day 18 of pregnancy. This period is
also associated with undetectable 20HSD activity in the
mesometrial/visceral yolk sac and spontaneous fetal loss in the rat
(16). This might support the hypothesis that 20HSD activity protects
the fetuses from the cytotoxic effects of P by inactivating it to
20OHP. Interestingly, the patterns of expression of P450arom and
20HSD during rat gestation appear to be opposite ones, suggestively
maintaining an optimal E2/P ratio at each stage
of gestation.
The significance of the high degree of expression of 17HSD type 2 mRNA noted in the rat placenta, especially toward the end of pregnancy, remains to be clarified further. However, previous results have shown that the type 2 enzyme has a predominant oxidative 17HSD activity (7). This together with the absence of 17HSD type 1 and P450arom enzymes in the rat placenta is in line with the fact that the rat placenta does not contribute significantly to E2 biosynthesis (28, 29, 30, 31). It is, therefore, hypothesized that 17HSD type 2 in the rat placenta could also protect the fetuses by inactivating the high activity 17ß-hydroxy forms of estrogens and androgens to their less active 17-keto forms. In addition, the significance, if any, of the putative 20HSD activity of the rat 17HSD type 2 enzyme in relation to progesterone metabolism during rat gestation remains to be clarified. To our surprise, the data suggest that in the liver and small intestine the enzyme is constitutively expressed throughout the life span of the animal, regardless of sex. The expression of rat 17HSD type 2 in the liver and small intestine does not appear to correlate with any of the changes in the circulating sex hormone concentrations in the animals. Thus, the enzyme is suggested to have a general constitutive role in the metabolic inactivation of potent systemic sex hormones to their less active forms.
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Acknowledgments |
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Footnotes |
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2 Recipient of Training Grant M8/181/4/A229 from the WHO Special
Program of Research, Development, and Research Training in Human
Reproduction.
Received January 30, 1997.
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References |
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), P450arom (
), and 20HSD (
) mRNAs in pregnant rat
ovaries on days 1, 3, 5, 8, 12, 15, 18, and 21 of gestation. Northern
hybridization signals were quantified and normalized against