LINK: biolreprod.org/content/53/6/1265.full.pdf
ABSTRACT
3a-Hydroxysteroid dehydrogenase (3a-HSD) activity and content in the rat ovary were measured at various stages of the estrous cycle,
and the enzyme protein in the ovary was localized by immunohistochemistry. The cyclic change of ovarian 3a-HSD activity towards 5adihydrotestosterone
(5a-DHT) as a substrate was characterized by two peaks. The first peak occurred at 0800 h on proestrus; then the
reductase activity decreased and reached its minimum at 2000 h on proestrus. Thereafter, it gradually increased, reaching the second peak
(170% of the value at 2000 h on proestrus) at noon of estrus. Quantitative analysis by immunoblotting revealed that the alteration in 3a-
HSD content in the rat ovary during the estrous cycle was essentially similar to that in 5a-DHT reductase activity. Changes in the reductase
activities towards 5a-androstane-3,17-dione and 5a-DHT and in the dehydrogenase activitytowards androsterone in the ovary were entirely
different from those in the 5a-DHT reductase activity and 3a-HSD content; on the other hand, the change in carbonyl reductase activity
towards p-nitroacetophenone was similar to changes in 5a-DHT reductase activity and 3a-HSD content. Therefore, p-nitroacetophenone
may be a useful substrate, instead of 5a-DHT, for detection of 3a-HSD activity at a high sensitivity, since the p-nitroacetophenone reductase
activity was 10-fold higher than the 5a-DHT reductase activity. The enzyme was primarily localized in the granulosa cells and CL cells. At
2000 h on proestrus, however, the overall intensity of immunostaining in the granulosa cells of the Graafian follicles was markedly
diminished. In addition, immunoreactivity in the CL cells at 0800 h on estrus was observed only in the cells outlining the CL in some cases.
INTRODUCTION
3a-Hydroxysteroid dehydrogenase (3a-HSD) [EC
1.1.1.50], a member of the NADPH-dependent oxidoreductase
family, catalyzes interconversion of 3-hydroxy and 3-
carbonyl groups of steroids in a positionally and sterically
specific manner [1-8]. 3a-HSD purified from livers of rats
[1-4], mice [5], hamsters [6], rabbits [7], and cattle [8] shows
carbonyl reductase activities towards a number of xenobiotic
carbonyl compounds, such as aromatic aldehydes
and ketones, and is also involved in detoxification of metabolites
of polycyclic aromatic hydrocarbons as a dihydrodiol
dehydrogenase [1, 3, 4, 8-10]. Recently, rat 3a-HSD
cDNA has been sequenced, and the results suggest that the
enzyme belongs to the aldo-keto reductase superfamily
[11, 12]. Thus, 3a-HSD also functions as one of the enzymes
that metabolize drugs that are not steroids. The enzyme is
ubiquitously distributed in tissues [10] and may catalyze the
interconversion of 5a-dihydrotestosterone (5a-DHT) or 5aandrostane-
3,17-dione (5a-androstanedione) to 5a-androstane-
3a,17p-diol or 5a-androstan-3a-ol-17-one (androsterone)
to regulate androgen actions in steroid-producing or
target tissues such as ovary.
Ovarian functions are tightly controlled by various steroid
hormones as well as peptide hormones [13, 14]. Since
androgens are synthesized from progestins and metabolized
to estrogens or 5a-reduced androgens, and are essen-
Accepted July 24, 1995.
Received April 17, 1995.
1Correspondence: Department of Forensic Medicine, Tokyo Medical College, 6-1-1
Shinjuku, Shinjuku-
On the other hand, although p-nitroacetophenone
and p-nitrobenzaldehyde are typical substrates
for hydroxysteroid dehydrogenases or prostaglandin ketoreductases
and dehydrogenases as well as carbonyl reductase
[2-8, 21-23], the alteration in p-nitroacetophenone reductase
activity during the estrous cycle was essentially
similar to the changes in 5a-DHT reductase activity and 3a-
HSD content. Therefore, 3a-HSD appears to be responsible
for the reductase activity towards p-nitroacetophenone in
the rat ovary. In addition, 3a-HSD activity may be detected
with high sensitivity when p-nitroacetophenone is used as
a substrate instead of 5a-DHT, since the p-nitroacetophenone
reductase activity was 10-fold higher than the 5a-DHT
reductase activity.Jarrell and Robaire [24] previously reported that ovarian
3a-HSD activity towards 5a-DHT was the highest during
proestrus within the 4-day estrous cycle in rats (in their article,
the time at which the ovaries were removed each day
during the estrous cycle was not specified). In this study,
we examined in detail the time-dependent changes in ovarian
3a-HSD activity, protein content, and localization during
the 4-day estrous cycle. The determination of protein content
using a specific antibody to 3a-HSD gave results consistent
with the change in the enzyme activity. The cyclic
change in ovarian 3a-HSD level in the present study was
characterized by two peaks, at 0800 h on proestrus and at
noon of estrus. In addition, the reduction in overall intensity
of immunostaining of the granulosa cells in the Graafian
follicles is consistent with the decrease in 3a-HSD level at
2000 h on proestrus, representing the valley between the
two peaks. Therefore, we conclude that 3a-HSD exhibits at
least two peaks during the estrous cycle.