Ovarian Carbonyl Reductase Research Articles

Teleost ovarian carbonyl reductase-like 20beta-hydroxysteroid dehydrogenase: potential role in the production of maturation-inducing hormone during final oocyte maturation.

17alpha,20beta-Dihydroxy-4-pregnen-3-one is the major oocyte maturation-inducing hormone of several teleost species. Gonadotropin-induced increase in ovarian 20beta-hydroxysteroid dehydrogenase activity is essential for the synthesis of maturation-inducing hormone. Cloning and expression studies suggest that ayu (Plecoglossus altivelis) ovarian carbonyl reductase can function as 20beta-hydroxysteroid dehydrogenase. The amino acid sequence deduced from the isolated cDNA had 276 amino acid residues and shared approximately 60% homology with mammalian and teleostean carbonyl reductases. The sequence data search showed that the ayu cDNA clone belongs to the short-chain dehydrogenase/reductase family. The clear lysate prepared from Escherichia coli harboring the cDNA catalyzed the production of maturation-inducing hormone. Its identification was confirmed by two-dimensional, thin-layer chromatography followed by recrystallization. Purification of the E. coli-expressed cDNA product revealed that it possessed both carbonyl reductase and steroid dehydrogenase activities, and 17alpha-hydroxyprogesterone, the endogenous immediate precursor of maturation-inducing hormone, was one of the preferred substrates. Furthermore, Northern blot analysis denoted that the transcripts are present both in fully grown, immature ovarian follicles and at higher levels in mature ovarian follicles. These results demonstrate that the carbonyl reductase of ayu ovary is involved in the production of maturation-inducing hormone, and they provide evidence for a novel physiological role of this enzyme in the final maturation of oocytes. Based on its functional properties, the enzyme can be referred to as carbonyl reductase-like 20beta-hydroxysteroid dehydrogenase.

Characterization of ovarian carbonyl reductase gene expression during ovulation in the gonadotropin-primed immature Rat.

In this differential-display polymerase chain reaction-based study, four different primer sets generated cDNA fragments of ovarian carbonyl reductase genes that were uniquely expressed during the ovulatory process in eCG-primed immature rats. The temporal pattern of expression of this aldo-keto reductase gene was delineated by extracting ovarian RNA at 0, 2, 4, 8, 12, and 24 h after induction of ovulation via injection of the primed animals with hCG. The results showed that at least four homologous forms of this gene were transcribed during ovulation. Northern blot analyses indicated a 14-fold increase in ovarian mRNA for carbonyl reductase, with expression reaching a peak at 8 h after hCG treatment and then declining to negligible levels during the next 16 h. In situ hybridization revealed that most of the transcription was in the thecal connective tissue of the ovary and was absent from the granulosa layer of ovarian follicles. Treatment of the animals with ovulation-blocking doses of epostane (an inhibitor of progesterone synthesis) or indomethacin (an inhibitor of prostanoid synthesis) did not reduce the expression of ovarian carbonyl reductase. Nevertheless, the temporal pattern of expression of carbonyl reductase after the induction of ovulation suggests that this enzyme activity is at least indirectly associated with the ovulatory process.

Ovarian carbonyl reductase activity in rats at persistent oestrus.

Age-related changes and effects of gonadotrophins on ovarian carbonyl reductase (CR) of virgin female Wistar-Imamichi rats were investigated. In mature rats ovarian CR is an LH-dependent oxidoreductase and is closely involved in the periovulatory process. After 8 months of age, all of the rats exhibited persistent oestrus and the ovarian mass was significantly decreased, whereas 4- and 5-month-old rats showed the regular 4 day oestrous cycles and ovulation occurred. The ovarian CR activity towards four substrates declined steeply consistent with persistent oestrus and the enzyme activity for each substrate at 15 months of age was 1.4-53.7% lower than at 4 months of age. The ovarian CR concentration measured by western blot analysis was also lower at 8 months of age. Equine chorionic gonadotrophin (eCG) and hCG were administered to 10- and 15-month-old rats at persistent oestrus to examine whether the ovarian CR in rats at persistent oestrus is influenced by gonadotrophins. Treatment with hCG not only increased ovarian CR activity and its concentration but also caused ovulation at both ages. The immunoreactivity to anti-ovarian CR antibody in the theca cells of the hCG-treated ovary was greater than that of the control ovary. Equine(e) CG did not stimulate ovarian CR activity in rats at persistent oestrus, although ovarian mass of 10-month-old rats was increased after administration of 50 iu of eCG. These results indicate that changes in ovarian CR with ageing are related to the transition of regular oestrous cyclicity to acyclicity and that the expression of ovarian CR markedly decreased in aged rats is stimulated by hCG.

P-775 - Differences in the response of ovarian carbonyl reductase to gonadotropins.

P-775 - Differences in the response of ovarian carbonyl reductase to gonadotropins.

P1-72 - Effects of nicardipine on ovarian carbonyl reductase in rats

P1-72 - Effects of nicardipine on ovarian carbonyl reductase in rats

Ovarian Carbonyl Reductase Delayed Onset of Persistent Estrus in the Offspring of Parathyroidectomized Mother Rats

We investigated the effects of maternal parathyroidectomy on day 5 of pregnancy on the ovarian carbonyl reductase (CR) in the offspring of rats. Changes in the ovarian CR of the offspring were examined at 4 and 8 months of age. In 8-month-old female offspring, the ovarian weight and the ovarian CR activity were significantly higher in rats from parathyroidectomized mothers than in rats from sham-operated mothers, and the offspring of the parathyroidectomized mothers showed the regular 4-day estrous cycles at 8 months of age, while the offspring of the sham-operated mothers were in the state of persistent estrus. Furthermore, intense immunostaining was found in the theca interna cells and the interstitial gland cells in the ovary of rats from the parathyroidectomized mothers, whereas in the ovary of the age-matched normal rats, immunostaining was faint. These results suggest that the maternal parathyroidectomy affect the activity and localization of CR in the ovary of female offspring.

Activation by human chorionic gonadotropin of ovarian carbonyl reductase in mature rats exposed in vivo to estrogens

We investigated the effects of exogenous estrogens and human chorionic gonadotropin (hCG) on the activity, content, and immunohistochemical localization of ovarian carbonyl reductase (CR) in mature cycling rats. Estrogens, estradiol, hexestrol (HEX) and diethylstilbestrol (DES) were given s.c. to rats daily for 3 days from the first day of diestrus, and hCG was given s.c. at 3:00 p.m. on the day of expected proestrus. The ovaries were isolated on the day of expected estrus. Ovarian CR activity was measured by using two substrates that reflect the activity of the enzyme in rats, and the enzyme content was determined by western blot analysis. Ovarian CR activity and content were decreased by estrogens as well as by inhibition of ovulation; hCG restored both the activity and the content decreased by estrogens to levels produced by nGC alone. Nevertheless, the number of ova in the oviduct when ovulation was decreased or blocked by estrogens was not restored completely by hCG treatment. Faint immunostaining in the interstitial gland cells of HEX-treated rat ovaries was observed. These results suggest that (i) although hCG activates ovarian CR in estrogen-treated rats, this increase in both enzyme activity and content may not be an obligatory event in the ovulatory process, and (ii) exogenous estrogens may predominantly influence the ovarian CR in the interstitial gland cells in mature rats by inhibiting luteinizing hormone release from the pituitary.

P-390 - Age-related changes in ovarian carbonyl reductase of hypercalcitoninemia rats.

P-390 - Age-related changes in ovarian carbonyl reductase of hypercalcitoninemia rats.

Interindividual variability of carbonyl reductase levels in human livers

Interindividual variability of carbonyl reductase levels in human livers (N = 11) was examined by measuring reductase activity toward various substrates and by western blot analysis using anti-rat ovarian carbonyl reductase CR2 antibody. The carbonyl reductase activity toward p-nitrobenzaldehyde (PNBA) ( 58.1 ± 5.4 nmol mg protein/min, mean ± SE) was highest among the substrates examined, followed by 4-benzoylpyridine (4BP) ( 14.4 ± 2.0 nmol mg protein/min and p-nitroacetophenone (PNAP) ( 2.00 ± 0.37 nmol mg protein/min). The reductase activity ( 6.33 ± 0.56 nmol mg protein/min) toward 13, 14-dihydro-15-keto-prostaglandin F 2α (ISKD-PGF 2α), which is a diagnostic substrate for rat ovarian carbonyl reductases, was relatively high compared to that in other species. Western blot analysis revealed that each human liver contained several immunoreactive proteins to anti-CR2 antibody. The activities toward 15KD-PGF 2α ( r = 0.85, P < 0.01) and 4BP ( r = 0.84, P < 0.01), but not PNBA ( r = 0.53, not significant) or PNAP ( r = 0.52, not significant), were closely correlated with the relative amounts of the high molecular weight immunoreactive proteins determined with a densitometer. Thus, the major carbonyl reductases in human liver are similar to those of rat ovarian enzymes.

Gonadotropin-induced ovarian carbonyl reductase in mice and hamsters: Comparison with carbonyl reductase in rats

We investigated the effects of pregnant mare serum gonadotropin (PMSG) and human chorionic gonadotropin (hCG) on ovarian carbonyl reductase activities towards 13, 14-dihydro-15-ketoprostaglandin F 2α (15KD-PGF 2α), p-nitroacetophenone (PNAP) and p-nitrobenzaldehyde (PNBA) in mice and hamsters, and compared with their effects on those we observed previously in rats. The treatment with PMSG and hCG caused a significant increase in ovarian weights and superovulation in both mice and hamsters. Hamster ovary possessed appreciable carbonyl reductase activities towards all three substrates. whereas the activities were lower than those in rat ovary. The reductase activities were not increased by the treatment with gonadotropins, differing from rat ovarian carbonyl reductase. In untreated mice, carbonyl reductase activity towards 15KD-PGF 2α was not detected, whereas the activities towards PNAP and PNBA were detected, which activities were lower than those in rats and hamsters. The PNAP and PNBA reductase activities in mouse ovary were significantly increased up to 7.1- and 1.7-fold, respectively, by the treatment with gonadotropins. These results show that there are species differences in ovarian carbonyl reductase and response of the enzyme to gonadotropins.

Effects of Age and Calcium Ion on Testis Carbonyl Reductase in Rats

To examine the role of carbonyl reductase (CR) in the development and function of testis, age-related changes in CR and the effect of exogenous calcium on CR in rat testis were studied. Testicular CR activity was the highest at 3 weeks of age when the enzyme activity was measured at 2, 3, 4, 8 and 20 weeks of age. The intensity of positive protein bands in testicular cytosol was similar among these age groups in Western blot analysis with anti-rat ovarian CR antibody. In 3-week-old rats, intravenous administration of 4 mg/kg calcium markedly suppressed the activity to about 50% of the control level 1 min after the treatment. Pretreatment with nicardipine prevented the inhibitory effect of exogenous calcium on the testicular CR activity in 3-week-old rats, indicating that the suppressing action of calcium on the enzyme activity was mediated by the entry of extracellular calcium via calcium channels. There were no significant changes in the CR activity following the treatment with calcium and nicardipine in the other 3 age groups examined. These findings suggest that testicular CR plays an important role in the functional development of the testis during the infantile period in rats.

Human chorionic gonadotropin causes an estrogen-mediated induction of rat ovarian carbonyl reductase

We earlier reported that human chorionic gonadotropin (hCG) stimulates rat ovarian carbonyl reductase (CR) activity and content, and that estrogen enhances the stimulatory effect. The present study was performed to determine the mode of action of the gonadotropin. Cycloheximide (CHX) and actinomycin D (AD) were given to estradiol-pretreated immature rats 6 h before hCG treatment. The enzyme activity was measured with three substrates, and the enzyme content was determined by the method of Westernblot analysis using anti-rat ovarian CR antiserum as the first antibody. Both protein inhibitors significantly prevented hCG from increasing the enzyme activity and content in estradiol-pretreated ovary. These results indicate that rat ovarian CR is induced by LH via the action of estrogen.

Consecutive Treatment of Disulfiram Inhibits Ovarian Carbonyl Reductase Activity in Rats

We investigated the effects of disulfiram (DS) on ovarian carbonyl reductase activity in rats to determine the influence of DS on female reproductive function. Three consecutive treatments with DS significantly inhibited ovarian carbonyl reductase activity as well as ovulation, dose-dependently. Single treatment with DS had no effect on ovarian carbonyl reductase activity. Our observations indicate that consecutive treatment with DS has an inhibitory action on female reproductive function, although DS is well-known to inhibit aldehyde dehydrogenase and dopamine β-hydroxylase.

Consecutive treatment of disulfiram inhibits ovarian carbonyl reductase activity in rats.

We investigated the effects of disulfiram (DS) on ovarian carbonyl reductase activity in rats to determine the influence of DS on female reproductive function. Three consecutive treatments with DS significantly inhibited ovarian carbonyl reductase activity as well as ovulation, dose-dependently. Single treatment with DS had no effect on ovarian carbonyl reductase activity. Our observations indicate that consecutive treatment with DS has an inhibitory action on female reproductive function, although DS is well-known to inhibit aldehyde dehydrogenase and dopamine beta-hydroxylase.

Regulation of ovarian carbonyl reductase mediated by estrogen receptor in immature rats

In the present study, the enhancing effect of synthetic estrogen on ovarian carbonyl reductase, a new prostaglandin (PG)-metabolizing enzyme, was investigated, and the antagonistic effect of antiestrogen on this enhancement was examined in immature rats. Ovarian carbonyl reductase activity towards 13,14-dihydro-15-keto-PGF 2α (15KD-PGF 2α), 4-benzoylpyridine (4BP) and menadione was determined photometrically and radiochemically, and quantitation of ovarian carbonyl reductase content was performed by Western blot analysis. Diethylstilbestrol (DES) and hexestrol (HX) enhanced the increasing effect of human chorionic gonadotropin (hCG) on ovarian carbonyl reductase activity and content when these synthetic estrogens (0.2 mg/kg) were administered for 3 days from 26 days of age, before hCG treatment. On the other hand, tamoxifen, which inhibits the binding of estradiol to the estrogen receptor, significantly prevented estradiol (E 2) from enhancing the effect of hCG on ovarian carbonyl reductase. Furthermore, the ovarian carbonyl reductase activities towards the three substrates correlated well with the ovarian carbonyl reductase content. These results indicate that ovarian carbonyl reductase in immature rats may be regulated by a specific increase in the ovarian response to luteinizing hormone mediated by estrogen receptor.

Changes and Localization of Ovarian Carbonyl Reductase during Pseudopregnancy and Pregnancy in Rats

The present study investigates changes in the activity and enzyme content of ovarian carbonyl reductase (CR), which catalyzes the reduction of 9-keto and 15-ketoprostaglandins in rats during pseudopregnancy and pregnancy. The activity of ovarian CR decreased from the onset of pseudopregnancy and pregnancy, reaching 20-30% of the Day 1 value by Day 12 of pseudopregnancy and 50-60% of the Day 1 value by Day 14 of pregnancy. In the case of pregnant rats, the enzyme activity maintained a minimal level between Day 14 of pregnancy and Day 22 of parturition. An acute increase of the enzyme activity was found on the morning after parturition. The CR content in the ovary maintained a constant level from Day 1 to Day 12 of pseudopregnancy and to Day 18 of pregnancy. In pregnant rats, there was a gradual decrease after 18 days and then a surge during parturition. CR was primarily localized in interstitial gland cells and in theca interna cells but was not found in corpora lutea cells in the ovary during the estrous cycle. Additional immunostaining was also observed in corpora lutea cells during pseudopregnancy and pregnancy. The changes in ovarian CR activity, i.e. the rapid decrease with progressing pseudopregnancy and pregnancy, correlated with the increase in progesterone and the decrease in LH. These results indicate that rat ovarian CR may be regulated via the hypothalamo-pituitary-ovarian axis and may also be involved in luteal function.

Immunological and Enzymological Localization of Carbonyl Reductase in Ovary and Liver of Various Species

The tissue distribution of carbonyl reductase in ovary and liver of various animal species was investigated by measuring the reduction of 13,14-dihydro-15-keto-prostaglandin F2a, a specific substrate for rat ovarian carbonyl reductases, and by means of Western blotting analysis using anti-rat ovarian carbonyl reductase antibody. The highest ovarian carbonyl reductase activity towards 13,14-dihydro-15-keto-PGF2a was found in rat among ten animal species tested, followed by hamster and monkey. The immunoreactive protein was detected in hamster and monkey ovaries. Although carbonyl reductase activity towards 13,14-dihydro-15-keto-PGF2a was not detectable in non-pregnant rabbit ovary, pregnant rabbit ovary showed not only moderate activity but also immunoreactivity with anti-rat ovarian carbonyl reductase antibody. On the other hand, carbonyl reductase activity towards 13,14-dihydro-15-keto-PGF2a was detected in hepatic tissue of all the species tested, except for rat and left-eye flounder. Immunoreactive proteins were present in hepatic tissue of various species that exhibited measurable carbonyl reductase activity towards 13,14-dihydro-15-keto-PGF2a.

Immunohistochemical Localization and Physiological Regulation of Carbonyl Reductase in Immature Rat Ovary

The present study was performed to clarify the role of the ovarian carbonyl reductase (OCR) in ovarian function in immature rats. The OCR activities towards three specific substrates, 13,14-dihydro-PGF2α, 4-benzoylpyridine and menadione, were photometrically and radiochemically determined in the 9,000 × g supernatants of ovaries, and OCR content was measured by Western-blot-peroxidase anti-peroxidase (PAP) analysis. Immunohistochemical locafization of the enzyme in the ovary was performed by the avidin-biotin-complex (ABC) method for paraffin sections. Positive immunoreactivity with OCR antibody was observed for the theca cells and interstitial gland cells at 72 hr after pregnant mare serum gonadotropin (PMSG) treatment when ovulation was confirmed, and the granulosa cells were consistently negatively stained. The OCR activity was significantly increased by PMSG, human chorionic gonadotropin (hCG) and PMSG-hCG treatments, but estradiol and tamoxifen overcame the effect of PMSG on the enzyme activity. Moreover, estradiol enhanced the effect of hCG, but tamoxifen did not. Changes in the OCR activity well-correlated with those in the OCR content. These findings indicate that the OCR is regulated by gonadotropin and estrogen and that metabolites formed by the enzyme could be closely involved in ovarian cell function.

Inhibitory effect of glucocorticoid and stimulatory effect of human chorionic gonadotropin on ovarian carbonyl reductase in rats

Effects of three glucocorticoids on ovulation, and on content and activity of ovarian carbonyl reductase in rats were investigated. Glucocorticoid treatment caused both significant decrease in ovarian and uterine weights, blockade of ovulation and marked decrease in content and activity of ovarian carbonyl reductase. Furthermore, the weak immunoreactivity to antibody against ovarian carbonyl reductase was shown in the theca cells and the interstitial gland cells in the glucocorticoid-treated ovary. The treatment with hCG (LH activity specific) restored the ovarian weight, and both the content and activity of ovarian carbonyl reductase, which were inhibited by glucocorticoids, to control level as well as ovulation. These results indicate that the ovarian carbonyl reductase may be closely involved in ovarian function, especially ovulation, and may be an LH-dependent enzyme, because it is well known that glucocorticoids inhibit both LH surge and ovulation in rats.

Changes in Rat Ovarian Carbonyl Reductase Activity and Content during the Estrous Cycle, and Localization1

Carbonyl reductase 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 enzyme activity increased after the preovulatory surge of luteinizing hormone (LH) on proestrus, and the enzyme content began to increase prior to the LH surge. Although the enzyme content reached the highest level at 2000 h and remained at a plateau for 8 h, the enzyme activity increased linearly until it reached the highest level at 0800 h on the morning of estrus. At their maximum, enzyme activity and content were approximately 1.5-fold and 2-fold greater, respectively, then basal diestrus values. The enzyme protein amounted to 1-4% of the ovarian cytosolic protein. An immunohistochemical study revealed that the enzyme was primarily localized in interstitial gland cells and theca interna cells of secondary and Graafian follicles as well as atretic follicles.