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Regular Article |
-Hydroxysteroid Dehydrogenase Messenger RNA Transcription in the Immature Rat Ovary in Response to an Ovulatory Dose of Gonadotropin1
a Department of Biology, Trinity University, San Antonio, Texas 78212
b Department of Obstetrics and Gynecology, Kyoto University School of Medicine, Kyoto 606-8507, Japan
c Department of Cell Biology, Baylor College of Medicine, Houston, Texas 77030
ABSTRACT
The ovulatory process in mammals involves a substantial increase in the metabolism of steroids and eicosanoids in response to a surge in LH or to an injection of hCG into experimental animals. This study provides evidence that the ovulatory stimulus causes induction of the gene for 3
-hydroxysteroid dehydrogenase (3-HSD), an enzyme that belongs to several oxidoreductase superfamilies that affect steroid and eicosanoid metabolism. Immature Wistar rats were primed with 10 IU eCG s.c., and 48 h later the 12-h ovulatory process was initiated by 10 IU hCG s.c. Ovarian RNA was extracted at 0, 2, 4, 8, 12, and 24 h after injecting the animals with hCG. The RNA extracts were used for reverse transcription-polymerase chain reaction (PCR) differential display to detect gene expression in the stimulated ovarian tissue. One of the PCR primer sets differentially amplified a cDNA fragment that is 52.3% homologous with a 3-HSD gene in rat liver. Northern analyses revealed that maximum transcription was at 8 h after the animals had been treated with hCG. The Northerns also indicated that the 3-HSD cDNA probe cross-hybridized with as many as six different bands of mRNA on the blots. In situ hybridization localized 3-HSD mRNA in the granulosa and thecal layers of mature follicles and in newly formed corpora lutea at 24 h after the ovulatory stimulus. In conclusion, gene(s) for 3-HSD are transcribed in ovarian follicles in response to an ovulatory dose of gonadotropin. A possible function of the oxidoreductase enzyme that is translated from the 3-HSD mRNA may be to reduce the toxic aldehyde and ketone components of the steroids and eicosanoids that accumulate in the mammalian ovary at the time of ovulation.
follicle, granulosa cells, mechanisms of hormone action, ovary, ovulation
INTRODUCTION
Mammalian 3-hydroxysteroid dehydrogenase (3-HSD) operates in conjunction with NADPH to catalyze the oxidoreduction of steroids and other compounds that possess aldehyde and ketone functional groups [1–3]. It is a rather ubiquitous reducing agent that belongs to the aldo-keto reductase and the short-chain dehydrogenase reductase superfamilies that encompass more than 60 enzymes in mammals, amphibians, insects, and bacteria [3–5]. Genetic expression of the enzyme has been reported in a wide variety of tissues, with steady-state levels in liver and intestine, and limited expression in stomach, lung, testis, and ovary [1].
The amino acid sequence of 3-HSD is quite different from 3ß-HSD [6], which is well known for its anabolic role in the synthesis of ovarian progesterone at the beginning of the ovulatory process in mammals [7]. In contrast to 3ß-HSD, 3-HSD has been associated with the catabolism of progesterone—converting this ovulation-dependent steroid into biologically inactive, or less active, products [3, 8]. In addition to its action on progesterone and other steroids, this multifunctional carbonyl reductase is known to catalyze polycyclic aromatic hydrocarbons and prostaglandins [1, 9].
Evidence for 3-HSD activity in ovarian tissue was first detected two decades ago by assessing the conversion of dihydrotestosterone to other steroids in the rat ovary [10]. Subsequently, there have been several brief reports of 3-HSD activity in avian [8] and rat [11] granulosa cells. More recently, there has been a study of ovarian 3-HSD activity at various stages of the estrous cycle of the adult rat [4]. However, the latter report concluded that the enzyme activity was minimal at the time of ovulation. Thus, none of these ovarian studies associated 3-HSD with the biochemical events of ovulation.
In the present reverse transcription-polymerase chain reaction (RT-PCR) differential display study, expression of mRNA for 3-HSD was discovered in the ovaries of immature rats that were primed with gonadotropins to induce ovulation. The cDNA fragment that was differentially expressed on an acrylamide gel hybridized at multiple sites on a Northern blot, suggesting that additional forms (or lengths) of the 3-HSD transcript reported here are expressed in the ovary at the time of ovulation. The results from in situ hybridization show that ovarian expression of 3-HSD mRNA occurs primarily in the granulosa layer and to a lesser extent in the thecal tissue. This transcription is not affected by ovulation-inhibiting doses of epostane or indomethacin, indicating that ovarian 3-HSD expression is not dependent on the ovulatory increase in ovarian progesterone or prostanoid synthesis. The oxidoreductase enzyme that is translated from the 3-HSD mRNA may function to reduce the toxic aldehyde and ketone components of the steroids and eicosanoids that accumulate in the mammalian ovary at the time of ovulation.
MATERIALS AND METHODS
Animal Tissue and Animal Injections, Ovulation Rate, and Prostaglandin Assay
Immature Wistar rats were induced to superovulate by treatment with eCG and hCG as described previously [12]. Ovarian RNA was extracted at the periovulatory intervals of 0, 2, 4, 8, 12, and 24 h after hCG. These nucleic acid extracts were used for differential display and for Northern blotting. Epostane and indomethacin were injected s.c., also as described previously [12]. These antiovulatory agents were administered at 3 h after hCG in doses of 5.0 mg and 1.0 mg, respectively. The ovulation rate in the various experimental animals was determined by a procedure that also has been described previously [12]. Ovarian prostaglandin E2 (PGE2) was assayed by a procedure that has also been described previously [13]. For the determination of ovulation rate, the assay of ovarian PGE2, and the extraction of ovarian RNA, rats were killed by exposure to CO2. The animals were acquired, retained, and used in compliance with the National Institutes of Health (NIH) Guide and with the approval of the institutional animal care review committee.
Differential Display Protocols That Lead to Detectionof 3-HSD
The steps of the differential display were carried out as described previously [12]. In brief, RNA was extracted by a standard guanidine isothiocyanate/cesium chloride procedure. RT-PCR was performed using primers from an RNAimage Kit (G503; GenHunter Corporation, Nashville, TN). The primer set that yielded differentially expressed cDNA for 3-HSD was comprised of the poly-T primer 5'-HTTTTTTTTTC-3' and the random primer 5'-HGGCTATG-3', with H representing the point of attachment of a HindIII restriction site to each primer. After extraction and reamplification of the differentially expressed cDNA, standard Northern analyses were performed as described previously [12] to confirm the ovulation-specific expression of the parent mRNA for 3-HSD. Lengths of the 3-HSD mRNAs were determined by including a lane in the Northern blots that contained RNA markers (G3191; Promega Corporation, Madison, WI). Subsequently, the unique cDNA fragment was subcloned using a pCR-TRAP Cloning System (P404; GenHunter), and cloning colonies containing the 3-HSD cDNA were identified by secondary Northern analyses. Manual sequencing of the cDNA was performed using a Sequenase Version 2.0 DNA Sequencing Kit (US70140; Amersham Pharmacia Biotech, Inc., Piscataway, NJ). In situ hybridization was performed as described previously [12].
Statistical Analysis
The intensity of the signals from the Northern blots was analyzed by the NIH-image densitometry program, as described previously [12]. Numerical data are presented as means ± SEM. The significance of the differences among the six principal time points of 0, 2, 4, 8, 12, and 24 h after hCG and the four groups in the epostane and indomethacin study were determined by Duncan multiple range tests after a completely randomized one-way analysis of variance of the means of the groups. The probability value used as the cutoff between significant and not significant was P = 0.05.
RESULTS
Temporal Pattern of Ova Accumulation in the Oviduct During Ovulation
The established convention to tabulate ovulation rate is to count the number of ova in the oviduct. In this study, the oviducts were examined from 10 rats each at 0, 2, 4, 6, 8, 10, 12, 14, 16, 20, and 24 h after hCG administration to the animals. In these groups of animals, the numbers of ova were 0, 0, 0, 0, 0, 0, 0.7 ± 0.4, 5.3 ± 2.3, 28.3 ± 7.2, 64.6 ± 6.9, and 72.3 ± 3.2, respectively (Fig. 1). Thus, ova first began to appear at 12 h after hCG administration, and they continued to accumulate during the next 8 h. Therefore, the data show that mature follicles begin to rupture at approximately 12 h after induction of the ovulatory process, but some follicles do not release ova until some hours later.
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Differential Display of 3-HSD cDNA During the Ovulatory Process
Following RT-PCR, the subpopulations of radioactively labeled cDNAs that were generated from RNA extracts at each of the six stages of the periovulatory period were separated from one another by electrophoresis on polyacrylamide gels. The autoradiograph of these PAGE results revealed a relatively weak signal for a differentially expressed cDNA band that was most evident at 8 h after hCG but was negligible in the 0-h control lane that represented the amount of gene expression just prior to initiation of the ovulatory process by hCG (Fig. 2). Therefore, this most intense cDNA band (i.e., the band in the 8-h lane) was excised from the acrylamide gel and reamplified for use as a probe in Northern analyses.
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Northern Analysis of 3-HSD mRNA Expression During Ovulation
The Northerns revealed an expression of mRNA (eventually identified as the message for 3-HSD) during ovulation that was comparable to the pattern of expression of cDNA on the differential display autoradiograph (Fig. 3). In order to compare the intensity of the signals from the Northern blots with other data on gene expression during ovulation, the amount of signal from the 8-h lane was arbitrarily set at 100%, and the densities at the other times during the periovulatory period were expressed as fractions of that maximum. Accordingly, the NIH-image program was used to digitize all of the bands on the Northerns, and the ratio of the density of each experimental band to its corresponding ß-actin control band was calculated for each lane. Means (±SEM) of the signal densities at 0, 2, 4, 8, 12, and 24 h after hCG were 4.7% ± 1.0%, 18.3% ± 5.8%, 55.1% ± 11.3, 100%, 68.5% ± 2.9%, and 20.7% ± 4.6%, respectively. Thus, 3-HSD gene expression increased 21-fold within 8 h after initiation of the ovulatory process by injecting hCG into the animals. Subsequently, by 12 h after hCG (i.e., at the approximate time that follicles begin to rupture in this experimental animal), 3-HSD gene expression had declined significantly.
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Temporal Pattern of Ovarian PGE2 Production During Ovulation
Ovarian PGE2 was assayed at 0, 2, 4, 8, 10, 12, 14, 16, 20, and 24 h after hCG treatment of the animals in order to correlate the pattern of synthesis of this major prostanoid with the pattern of expression of 3-HSD mRNA. The mean values of PGE2 in groups of 10 rats at each of the experimental intervals after hCG were 0.53 ± 0.10, 0.65 ± 0.11, 16.58 ± 3.39, 29.95 ± 5.05, 30.13 ± 5.38, 30.56 ± 3.21, 20.80 ± 3.57, 11.64 ± 1.18, 8.70 ± 1.55, 5.33 ± 0.85, and 2.43 ± 0.30 ng PGE2 per mg protein in the ovarian extracts (Fig. 4). Thus, the results show that the biphasic pattern of PGE2 production during ovulation is similar to the pattern of 3-HSD mRNA expression (Fig. 3).
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Expression of Multiple mRNAs for 3-HSD
The cDNA for 3-HSD actually hybridized to multiple bands of mRNA on the Northern blots. These multiple bands were elucidated more vividly by performing an NIH-image densitometric analysis of the 0-h control lane and the 8-h lane (Fig. 5). This optical density analysis was performed on one of the Northerns from the epostane and indomethacin experiment (see Fig. 6), because this particular Northern exhibited the finest resolution of the bands. The results indicated that the 3-HSD cDNA hybridized with two ovarian mRNAs of approximately 3.6 and 1.8 kilobases (kb) that were constitutively expressed before and during the ovulatory process. However, by 8 h after hCG, there were an additional four hybridization bands of approximately 5.0, 3.0, 2.5, and 1.4 kb, representing mRNAs that were uniquely expressed during ovulation.
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Effects of Epostane and Indomethacin on 3-HSD Gene Expression
For these tests, Northern blots were prepared from RNA that was extracted from control ovaries at 0 and 8 h into the ovulatory process or extracted from experimental ovaries that were taken at 8 h after hCG from rats that had been treated 5 h earlier with ovulation-inhibiting doses of epostane or indomethacin. These experimental intervals were selected in order to compare the present data with related studies using epostane and indomethacin [12, 14, 15]. The signal density (normalized against the ß-actin control) of the 8-h control lane was arbitrarily set at 100% (Fig. 6). There was minimal expression of 3-HSD mRNA at 0 h but substantial expression at 8 h. In animals treated with the antiovulatory agent epostane that blocks progesterone synthesis [15–17], the signal density of 101.3% ± 6.3% was not significantly different from the 8-h control value. Animals treated with the antiovulatory agent indomethacin that blocks prostanoid synthesis [15, 18] had a signal density that was 105.1% ± 5.4% of the 8-h control value and was not significantly different from the 8-h control value. On the other hand, the ovulation rates in parallel groups of animals treated with epostane and indomethacin were significantly inhibited (Fig. 6). These results indicate that the ovulation-related increase in 3-HSD gene expression is not dependent on ovarian progesterone or ovarian prostaglandin levels.
Sequence of the cDNA Fragment for 3-HSD
After the hCG-induced expression of the 3-HSD gene was confirmed by Northern analysis, the cDNA fragment of this gene was subcloned and sequenced. The NCBI accession number for this 501-base pair (bp) fragment is #AF159099. This fragment of cDNA is homologous to segments of a 3-HSD gene that has been cloned from rat liver (NCBI accession #M64393 [1] and NCBI accession #D17310 [9]). In comparison, the 501-bp ovarian cDNA that was isolated in the present study using RT-PCR differential display is 52.3% homologous (at the nucleotide level) with one of the rat liver clones (i.e., NCBI accession #M64393). The ovarian sequence contains four different segments, comprising 286 of the 501-bp total, that collectively are 91.6% homologous to complementary segments of the rat liver gene for 3-HSD (Fig. 7). (The sequence that was identified above as NCBI accession #D17310 is essentially identical to #M64393, except the segment of this liver mRNA that was submitted to the database did not include the last 21 bp at the poly-A end of the transcript.)
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Localization of 3-HSD mRNA Expression by In Situ Hybridization
In situ hybridization confirmed the temporal pattern of 3-HSD mRNA expression that was observed in the differential display autoradiographs and the Northern analyses. There was minimal signal from the 0-h control ovaries, a substantial increase in signal between 4 and 8 h after hCG treatment, a declining signal between 8 and 12 h, and limited signal at 24 h (Fig. 8). Hybridization was localized primarily in the granulosa of the larger follicles in ovaries staged at 4–12 h. In addition, a noticeable amount of 3-HSD mRNA was visible in the thecal tissue, and this expression was the most obvious at 4 h after hCG was administered to the animals (Fig. 9). Also, there was conspicuous signal from a limited number of newly formed corpora lutea in ovaries taken at 24 h after hCG treatment (Fig. 9).
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DISCUSSION
There are more than 60 related enzymes that exhibit aldo-keto reductase activity [5]. In the present study, the cDNA fragment of a gene that encodes such an enzyme is most homologous to rat liver 3-HSD [1]. This gene for rat liver 3-HSD transcribes a 2.4-kb message that encodes a 322-residue protein [1]. Northern analyses reveal that transcripts of this and related genes cross-hybridize with several different sizes of mRNA on Northern blots prepared from diverse tissues, including the ovary [1, 9]. However, these Northern studies report inconsistent sizes for the mRNAs that hybridize with hepatic 3-HSD. In one study, two bands of 3-HSD were identified at 3.6 and 1.8 kb on Northern blots of total RNA from rat ovary [1], while in another investigation four bands at 5.0, 3.0, 2.5, and 1.4 kb were detected on Northern blots from rat ovary [9]. It is difficult to explain the discrepancy between these reports. However, in both cases the experimental tissues were taken at random from adult rats, and therefore the ovaries could have been at different stages of the sexual cycle. It is also possible that, in the former report, the two bands represented constitutively expressed 3-HSD mRNA that is transcribed at steady-state levels in the ovary, while in the latter study the four bands may have resulted from transcription in response to gonadotropin action during the periovulatory period. Such an explanation would be congruous with the present observation of what appears to be two housekeeping mRNAs that are approximately 3.6 and 1.8 kb in length, and four gonadotropin-induced mRNAs of approximately 5.0, 3.0, 2.5, and 1.4 kb that were expressed at 4–12 h after treatment of the immature rats with hCG to induce ovulation. The limited expression of two housekeeping genes might explain the modest amount of 3-HSD mRNA that was detected in 0-h control tissue. In any case, the data indicate that multiple mRNAs for 3-HSD-like activity are transcribed in the rat following treatment of the animals with gonadotropin doses that induce ovulation.
It is relevant that members of the oxidoreductase families to which 3-HSD belongs are homologous to a number of other enzymes that exhibit dehydrogenase, or reductase, activity on prostanoids, steroids, and other polycyclic aromatic hydrocarbons. The ensemble of related enzymes includes 20-HSD [2], aldose reductase [1–3], chlordecone reductase [2], rho-crystallin [3], and prostaglandin F2 synthase [1–3, 6]. Prostaglandin F2 synthase is of particular interest in a study of ovarian function because this enzyme regulates the metabolism of prostaglandins that have been frequently implicated in the mechanism of ovulation during the past three decades [7, 19]. With regard to its action on steroid substrates, 3-HSD is especially known for its reduction of progesterone to 4-pregnen-3-ol,20-one [3, 8] and for its interconversion of 5-dihydrotestosterone or 5-androstane-3,17-dione to 5-androstane-3,17ß-diol or 5-androstane-3-ol,17-one [4]. Yet, its principal substrate(s) and product(s) in the ovary at the time of ovulation remain to be determined.
Regarding prostaglandin metabolism, 3-HSD (which is also known as dihydrodiol dehydrogenase) can function as a 9-, 11-, and 15-hydroxyprostaglandin dehydrogenase, while PGF2 synthase reduces PGE2 to PGF2 [6]. It is especially interesting to note that both of these enzymes are considered to be targets for nonsteroidal anti-inflammatory drugs (NSAIDs) [6]. Furthermore, NSAIDs are well known for their ability to block mammalian ovulation, and this inhibitory action has been attributed previously to interference with ovarian cyclooxygenase activity [7, 19, 20] and/or to inhibition of ovarian lipoxygenase activity [18] during the ovulatory process. Now, in view of the present evidence of transcription of 3-HSD mRNA in ovulatory follicles, it is important in the future to consider the possibility that at least part of the antiovulatory action of NSAIDs may be the result of inhibition of ovarian 3-HSD. Furthermore, it may be relevant that the temporal pattern of ovarian 3-HSD expression coincides with the transient increase in ovarian PGE2 and PGF2 synthesis [16, 17], along with 12- and 15-hydroxyeicosatetraenoic acids [17], during the ovulatory process. This correlation between ovarian 3-HSD mRNA expression and prostanoid synthesis can be observed by comparing the data in Figures 3 and 4. Collectively, this information suggests that 3-HSD may be another factor in the metabolism of ovarian eicosanoids in response to gonadotropic stimulation of the ovary.
As indicated above, 3-HSD enzyme activity is inhibited by NSAIDs [6]. However, based on the present findings, gonadotropin-induced transcription of 3-HSD mRNA is not affected by the potent NSAID, indomethacin. Similarly, epostane, which is a potent inhibitor of progesterone synthesis, did not affect the increase in ovarian 3-HSD. These results show that the ovulatory increase in 3-HSD mRNA occurs independently of ovarian prostanoid and progestin synthesis.
Several earlier studies on ovarian 3-HSD did not relate this oxidoreductase activity to the process of ovulation [4, 8, 10, 11]. It should be noted that the initial study assessed 3-HSD activity in adult rat ovaries by measuring the conversion of dihydrotestosterone to 5-androstane-3, 17ß-diol [10]. In this case, it was reported that peak ovarian activity was on the day of proestrus, while the nadir was on the day of estrus. However, the precise time of day that the ovaries were extracted for 3-HSD activity was not recorded. In contrast, the other major study of ovarian 3-HSD activity was based on immunohistochemical localization of the enzyme protein [4]. In this latter report, the intensity of immunostaining (in granulosa and luteal cells) was highest at 0800 h on the day of proestrus and at noon on the day of estrus. Thus, the former study concluded that ovarian 3-HSD activity was lowest at estrus, while the latter study found the most activity on this same day. Quite oppositely, the current investigation of mRNA shows that expression of the transcript is minimal in the 0-h control ovaries of gonadotropin-treated immature rats, and such a 0-h control is equivalent to an ovary that is taken from an adult rat at 0800–1200 h on the day of proestrus (i.e., before the usual endogenous surge of gonadotropin at approximately 1300–1400 h). Furthermore, the time of 2000 h on the day of proestrus (i.e., the reported time of the nadir for immunostaining of ovarian 3-HSD [4]) is comparable to 6–7 h after hCG injection to eCG-primed immature rats (i.e., the approximate time of the peak for 3-HSD mRNA expression that was observed in the present study). Therefore, because mRNA transcription and enzyme translation usually coincide with one another, the present findings on the temporal pattern of ovarian 3-HSD mRNA expression are not compatible with previous estimates of ovarian 3-HSD enzyme levels. The reason for this incongruity is not clear. However, it should be emphasized that the temporal data from the differential display autoradiograph, the Northern analysis, and the in situ hybridization experiments of the present study all show that ovarian 3-HSD mRNA expression is minimal at the beginning of the ovulatory process and maximal at 8 h after inducing the process by an injection of hCG. Future studies on the temporal pattern of expression of the 3-HSD protein and/or its enzymatic activity should clarify the discrepancy between the previous reports on 3-HSD enzymatic activity and the current data on the mRNA for this enzymatic activity.
Although the protein that is translated from ovarian 3-HSD mRNA is most likely a reductase, the nucleotide sequence of the parent mRNA is not homologous to the polymorphic sequence of an ovarian carbonyl reductase gene that we have recently reported as being upregulated following gonadotropic stimulation [12]. Furthermore, the message for carbonyl reductase is expressed in the thecal layers of ovarian interstitial tissue, whereas 3-HSD mRNA is expressed primarily in the granulosa layer of mature follicles and in the differentiated granulosa of some corpora lutea. It was noted that 3-HSD mRNA is not uniformly expressed among the numerous corpora lutea on the superovulated ovaries. This irregularity in the luteal distribution of 3-HSD could be related to the fact that all of the follicles on a given rat ovary do not ovulate at the same time. The first follicles begin to rupture at approximately 12 h after hCG administration, but they continue to be released for the next 8 h. Therefore, the follicles that are slower to rupture may be the ones that contain the greater amount of lingering 3-HSD mRNA that was observed by in situ hybridization at 24 h after hCG administration.
In summary, 3-HSD mRNA is expressed in substantial amounts in ovarian follicles that have been stimulated by an ovulatory dose of gonadotropin. The expression of this oxidoreductase message occurs simultaneously with the well-known increase in ovarian eicosanoids and progesterone. Therefore, because it has been established that 3-HSD reduces prostanoids, steroids, and a variety of other polycyclic aromatic hydrocarbons [3, 9], it has been suggested that one possible function of such reductase enzymes is to detoxify aldehydes and ketones that sometimes accumulate in tissues that are undergoing considerable metabolic differentiation during processes such as follicular-luteal transition [8, 12]. However, it should be noted that the reductase gene fragment that was isolated in the present study does not share any significant homology with a mouse vas deferens protein gene that has been reported recently as having "a detoxifying role" in murine ovaries that have been stimulated by LH/hCG [21]. It has also been suggested that oxidoreductase enzymes like 3-HSD naturally influence the life-span of bioactive eicosanoids and steroids by moderating the duration of their interaction with nuclear and membrane-bound receptors [3]. In any event, the significant increase in 3-HSD mRNA in the granulosa layer of ovulatory follicles suggests that the translated enzyme has some relevant purpose in the biochemical events of ovulation.
ACKNOWLEDGMENTS
We appreciate the excellent assistance of Mrs. Claire Lo in preparing the in situ hybridization data.
FOOTNOTES
First decision: 23 October 2000.
1 This work was supported by National Science Foundation grant #9870793 (L.L.E.), by a grant to support T.U. as a Research Fellow of The Lalor Foundation, Providence, Rhode Island (L.L.E.), and by NIH grant HD-16229 (J.S.R.) 
2 Correspondence. FAX: 210 999 7229; lespey{at}trinity.edu
Accepted: February 9, 2001.
Received: September 19, 2000.
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