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Luteinizing Hormone Induces Mouse Vas Deferens Protein Expression in the Murine Ovary

Research Laboratories of Schering AG (E.B., M.P.-K., C.H.-H., M.L.), D-13342 Berlin, Germany; Max Delbrück Center for Molecular Medicine (V.B.), D-13125 Berlin, Germany; and WITA GmbH (E.B.), D-14513 Teltow, Germany

Address all correspondence and requests for reprints to: Dr. Monika Lessl, Research Laboratories of Schering AG, Female Health Care, Müllerstrasse 178, D-13342 Berlin. E-mail: monika.lessl{at}schering.de

	Abstract

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The aim of our study was to isolate and identify novel proteins that are involved in the process of ovulation. To achieve this goal we used the technique of proteome analysis. Comparison of ovary protein patterns, obtained by high resolution two-dimensional gel electrophoresis from recombinant FSH (rFSH)- and rFSH + human CG (hCG)-treated mice, showed significant differences in protein spot positions and intensities. Subsequent analysis of one of these proteins was performed by mass spectrometry, resulting in the identification of the mouse vas deferens protein (MVDP). MVDP, which was absent in the two-dimensional gel electrophoresis protein pattern of rFSH-primed mice and appeared 3 h after the hCG surge, is a member of the aldo-keto reductase superfamily and was originally identified in the mouse vas deferens. This is the first study describing MVDP expression and regulation by LH in the ovary. Northern blot analysis of female mice tissues showed that mvdp messenger RNA (mRNA) was only present in adrenal glands and in hCG-treated ovaries. In situ hybridization studies localized the mvdp mRNA unequivocally to ovarian thecal and interstitial cells with an expression profile starting already 1.5 h, and decreasing 24 h, after LH treatment. In the adrenal glands, mvdp mRNA was not regulated by LH and localized in the cells of the zona fasciculata. In murine adrenocortical cells, a recent study proposed a detoxifying role of MVDP. MVDP might fulfill the same function in the ovary; however, because of its strong and early transcriptional induction by LH, it is also possible that MVDP catalyses another important step during the cascade of events occurring at the time of ovulation.

	Introduction

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THE DEVELOPMENT OF ovarian follicles within the ovary is a highly integrated process. It involves a series of sequential events in which a recruited follicle acquires its structural and functional properties as it matures, ovulates, and becomes luteinized, under the control of a group of endocrine, autocrine, and paracrine factors (1). Mature ovarian follicles are susceptible to LH, which plays a central role in the control of ovulation. Three physiological processes are initiated within the follicle approximately at the time of the LH surge: 1) meiosis I resumes; 2) luteinization begins as the follicular granulosa cells switch to synthesis and secretion of progesterone (initiation of luteinization); and 3) ovulation itself, which leads to the release of fertilizable oocytes into the oviduct (2).

LH and FSH act through stimulatory G protein-coupled receptors expressed on target cells (3, 4) and transduce their signals, at least in part, by the activation of adenylyl cyclase and the production of the second-messenger cAMP (1, 5). It is suggested that FSH and LH can also act through signaling pathways that lead to the activation of protein kinase C (6, 7) or intracellular tyrosine kinases (7). However, a basic pattern of FSH stimulation and LH repression, during the cycle, is common to a number of genes expressed in the ovary, including the LH receptor (8), the FSH receptor (9), the inhibin - and ß-subunits (10), and aromatase (11).

Although progress has been made in understanding the mechanism of follicular development, ovulation, and the transformation of the follicle into the corpus luteum, major aspects of this pathway remain undefined. Relatively little is known about the molecular events after the preovulatory LH surge that triggers the biochemical and morphological changes, resulting in the ovulation of the oocyte and the transformation of the follicle into the corpus luteum. Thus, determining these mechanisms is important for understanding the normal control of the ovarian cycle and promises to lend insights into disruption of this control that might be associated with reproductive dysfunctions.

In this study, we used human CG (hCG) induction in the immature female FSH-primed mouse as a model system to characterize LH-regulated proteins. To identify the LH- regulated proteins, we analyzed the protein patterns from recombinant FSH (rFSH) and rFSH+hCG-treated mouse ovaries, by high-resolution two-dimensional gel electrophoresis (2-DE). 2-DE (12, 13) can be used to investigate complex protein mixtures (14, 15, 16). We describe, in detail, the identification of one of the highly LH-regulated variant protein spots, by tryptic in-gel digestion and subsequent mass spectrometry combined with computer analysis. To verify the protein data, we examined the messenger RNA (mRNA) expression level of this protein, by Northern blotting, in the ovary, as well as in various female mouse tissues. In situ hybridization studies were performed to localize the mRNA expression of the variant protein within the ovary.

	Materials and Methods

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Animals
Ovaries were obtained from immature female mice (C57BL/6J x DBA/2JF1, Charles River Laboratories, Inc., Sulzfeldt, Germany) weighing 13–16 g. Mice were kept under controlled temperature (20-22 C), light (lights on 0600–1800 h), and relative humidity (50–70%), with food and water ad libitum. They were injected ip with a single dose of 20 IU rFSH (Gonal-F; Serono Laboratories, Inc., Randolph, WA). Forty-eight hours later, one group was killed by cervical dislocation. FSH-primed mice in other groups were injected ip, after 48 h, with 10 IU hCG, and killed 3, 6, 14, 24, and 32 h later. The ovaries were removed and freed of extraneous tissue. All experiments on animals were conducted in accordance with the Guiding Principles for the Care and Use of Research Animals promulgated by the Endocrine Society.

Preparation of protein samples
Four mouse ovaries were immediately frozen, at -70 C, after preparation, and pulverized on liquid nitrogen using a glass pistill in an Eppendorf tube mortar. The pulverized ovaries were thawed and, during this procedure, rapidly mixed with protease inhibitor solutions (17). The final concentration of protease inhibitors, salts, and buffers in the protein sample was 1.4 µM pepstatin A, 1 mM phenylmethylsulfonylfluoride, 1 mM benzamidine, 2.1 µM leupeptin, 1 mM EDTA, 1 mM KCl, and 20 mM Tris/HCl (pH 7.1), according to Klose and Kobalz (17). Simultaneously, the pulverized mouse ovaries were rapidly mixed with urea (9 M final concentration), dithiothreitol (DTT; 70 mM final concentration), and then finally with 2.5% carrier ampholytes (Servalyt pH 2–4; Serva, Heidelberg, Germany). After 30 min of gentle stirring at room temperature, the samples were centrifuged in an ultracentrifuge at 100,000 x g for 25 min. The clear supernatant was frozen at -70 C. Then, 10–12 µl of these samples were loaded at the anodic side on an analytical gel, and 45–55 µl were applied on a micropreparative gel.

High-resolution 2-DE
The proteins were separated by a large-gel 2-DE technique employing carrier ampholytes for isoelectric focusing. The complete 2-DE equipment and gel solutions were purchased from WITA GmbH. The 20-cm isoelectric focusing rod gels (inner diameter analytical gels, 0.09 cm; and micropreparative gels, 0.15 cm) contained 3.5% acrylamide, 0.3% piperazine diacrylamide (Bio-Rad Laboratories, Inc., Munich, Germany) and a total of 4% (wt/vol) carrier ampholytes, pH 2–11 (WITA). The electrophoretic conditions of the rod gels during isoelectric focusing were according to Klose and Kobalz (17). After focusing, the rod gels were equilibrated for 10 min in a buffer containing 125 mM Tris/phosphate (pH 6.9), 40% glycerol, 70 mM DTT, and 3% SDS. The equilibrated rod gels were frozen at -70 C. After thawing, the isoelectric focusing rod gels were immediately applied to an SDS-PAGE, which contained 15% (wt/vol) acrylamide and 0.2% N,N'-methylenebisacrylamide. The SDS-PAGE system of Laemmli (18) was used, but the stacking gel was replaced by an equilibrated isoelectric focusing gel. The electrophoresis of the analytical SDS-PAGE (gel size, 30 x 23 x 0.075 cm) was performed using a two-step increase of current, starting with 15 min at 65 mA, followed by a run of 7–8 h at 85 mA, until the dye reached the end of the gel. The electrophoresis of the micropreparative SDS-PAGE (gel size, 30 x 23 x 0.15 cm) was performed using a two-step increase of current, starting with 15 min at 130 mA, followed by a run of 7–8 h at 170 mA, until the dye reached the end of the gel. For analytical gels, the proteins were silver-stained, based on the procedure of Klose and Kobalz (17). For micropreparative gels, the proteins were stained with Coomassie Blue, as described by Eckerskorn et al. (19).

Enzymatic in-gel digestion, extraction of the peptides from the gel, and concentration using reversed-phase material
For micropreparative investigations, Coomassie-stained protein spots were used from micropreparative gels. The enzymatic in-gel digestion with trypsin (Promega Corp., Madison, WI), the extraction of the generated peptides from the gel, desalting, and concentration by reversed-phase material (Serva) were performed according to Otto et al. (20).

Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS)
MALDI-MS was performed with a VG TOF Spec (Fisons, Manchester, England), equipped with a nitrogen laser (337 nm; pulse duration, 4 nsec) and a VAX 400 VLC station, using OPUS software, version 3.1. Spectra were obtained in the linear or reflectron mode by summing 20–50 laser shots. A thin layer of small homogeneous matrix crystals was prepared on the target by placing 1 µl of a saturated solution of -cyano-4-hydroxycinnamic acid in acetone and 1 µg/µl nitrocellulose, as matrix additive, on the target, and allowing the droplet to spread and dry. A droplet (0.5 µl) of the analyte, dissolved in 60% acetonitrile, 0.1% TFA, was placed on top of this matrix layer and allowed to dry at room temperature. The resulting matrix-analyte layer was washed twice with water, to remove sodium and potassium ions. Measurement took place under vacuum at 22 or 24 kV (21).

Electrospray ionization tandem mass spectrometry (ESI-MS/MS)
ESI-MS/MS experiments were performed with a Q-Tof (Micromass, Manchester, UK) equipped with a nanoflow Z-spray ion source. This was operated at a temperature of 30 C, with a nitrogen drying gas flow of about 180 liters/h. A potential of 1.4 kV was applied to the nanoflow borosilicate glass capillary. An aliquot of 1 µl was sufficient for one MS spectrum and five to six MS/MS experiments, employing a flow rate of 30 nl/min. The collision energy of 28–35 V, depending on the charge state of the parent ions, was applied; the gas pressure in the collision cell was regulated to 6.0 x 10^-5 mbar. The Q-Tof calibration was performed with [Glu]-fibrinopeptide (Sigma, Deisenhoven, Germany).

Protein identification by computer analysis
The peptide mass maps produced by MALDI-MS were searched using the program FRAGMOD (E.-C. Mueller, MDC, Berlin), a modified version of the FRAGFIT program (22) and MS-FIT (http://www.falcon.ludwig.ucl.ac.uk/ucsfhtml3.2/msfit.htm). For MS/MS data, the program Peptide Sequence Tag (http://www.mann.embl-heidelberg.de/ services/peptidesearch/fr_peptidepatternform.html) was used. Amino acid sequences were compared with the SWISS-PROT database and with the deduced amino acid sequence of the GenEMBL database. Interpretation of ESI MS/MS spectra (de novo sequencing) were performed manually, searching in protein, nucleotide, or combined databases by Fasta and TFasta algorithms (http://www.vega.crbm.cnrs-mop.fr/bin/fasta-guess.cgi).

Northern blot analysis
For Northern blot analysis, uterus, adrenal gland, spleen, liver, heart, intestine, brain, muscle, lung, kidney, and ovary were removed from 20 female mice, freed of extraneous tissue, frozen in liquid nitrogen, and stored at -70 C until use. Total RNA was isolated using the guanidine thiocyanate/CsCl gradient method (23). Purified RNA samples were separated in a 1% agarose-formaldehyde gel; 25 µg of total RNA was loaded on each lane. After electrophoresis, RNA was transferred to nylon membrane (Hybond-N+, Amersham Pharmacia Biotech, Arlington, IL). The membrane was incubated with prehybridization solution (QuickHyb; Stratagene, La Jolla, CA) for 30 min at 68 C and hybridized with ³²P-labeled probes, for 2 h, at 68 C, in the same solution. After hybridization, the filter was washed in 2 x SSC at room temperature for 30 min, followed by 0.1x SSC and 0.1% SDS, at 68 C, for 30 min. The autoradiogram was obtained by exposing the film, at -80 C, with an intensifying screen. The membrane was washed and rehybridized with a mouse gapdh probe, to compare the amounts of RNA loaded and transferred. To obtain an mvdp-specific complementary DNA (cDNA) probe, a subcloned 340-bp PCR-derived fragment (using oligoprimer pair 5'-GGT-CTT-CGA-CTT-CCA-GTT-GAG-3' and 5'-TGT-TCT-ATC-ATT-GTA-TCT-TC-3'), spanning the region of 843 bp to 1183 bp of the submitted mvdp sequence (accession number j05663) (24), was used for the radioactive labeling.

In situ hybridization
DIG-labeled mvdp sense and antisense RNA-probes were synthesized using linearized plasmid DNA templates. A PCR-generated cDNA fragment (using the oligoprimer pair 5'-GGT-CTT-CGA-CTT-CCA-GTT-GAG-3' and 5'-TGT-TCT-ATC-ATT-GTA-TCT-TC-3') of the mvdp, inserted at the multiple cloning site of pBluescript II KS plasmid, was used in template preparation. Synthesis of sense and antisense RNA probes was carried out by in vitro transcription using DIG RNA labeling mix of nucleotides (Roche Molecular Biochemicals, Mannheim, Germany) with either T3 or T7 RNA polymerase (Stratagene). Ovaries were removed and frozen in dry ice after embedding in Tissue Tek OCT compound (Miles, Elkhart, IN). Cryostat sections (5-µm) were thaw-mounted on glass slides and stored at -80 C. Before hybridization, the slides were air-dried for 1 h at RT and fixed in 4% paraformaldehyde in PBS for 10 min at RT. Tissue sections were acetylated with 0.25% acetic anhydride in 0.1 M triethanolamine and washed in PBS. After prehybridization in hybridization solution (50% formamide, 5x SSC, 5x Denhardts, 200 µg/ml bakers yeast transfer RNA (Sigma), 100 µg/ml hering sperm DNA (Sigma), for 4 h at RT, the sections were hybridized overnight at 72 C in a humidifying box with 300 ng of the DIG-labeled complementary RNA probe in hybridization solution (100 µl/slide). After hybridization, the sections were treated with ribonuclease A (40 µg/ml) in ribonuclease buffer (50 mM NaCL, 1 mM Tris-HCl, 0.1 mM EDTA, 1 mM DTT, pH 8.0), at 37 C for 1 h, to degrade unhybridized probe, and washed in 0.2x SSC for 1 h at 72 C. The hybridized digoxigenin-labeled probe was detected using alkaline phosphatase-conjugated anti-DIG antibody (Roche Molecular Biochemicals) and visualized with the chromogen combination 5-bromo-4-chloro-3-indolyl phosphate and NBT.

	Results

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Reproducible high-resolution 2-DE protein patterns were obtained from mouse ovarian tissues
As a model system for the identification of LH-regulated proteins and their corresponding genes in mouse ovaries, we used immature FSH-primed mice, treated or untreated with hCG. To see changes in the protein composition, we chose the technique of 2-DE combined with subsequent mass spectrometry. We obtained reproducible high-resolution 2-DE protein maps of mouse ovaries before and after the treatment. The gel patterns presented are representative of experiments performed 10 times and shared a resolution of approximately 5250 protein spots. An example of a gel pattern, obtained from protein extractions of rFSH-primed mouse ovaries without hCG-treatment, is shown in Fig. 1. Spots A, B, and C were excised from a gel and subsequently identified by peptide mass fingerprinting using MALDI-MS (MALDI-MS data not shown). For this mass spectrometric approach, spots were excised from a Coomassie-stained gel and digested with trypsin. Elution of the generated peptides and subsequent mass spectrometry resulted in several specific peptide mass peaks of a protein (peptide mass fingerprint). The evaluation of these mass data, combined with computer analysis, resulted in the identification of the proteins. It was shown that spot A is actin [42 kDa; isoelectric point (pI), 5.3], spot B is superoxide dismutase (Cu-Zn) (16 kDa, pI, 6.0), and spot C is glutathione S-transferase GT 8.7 (26 kDa; pI, 8.2). These spots were used as reference spots for molecular weight (Mr) and pI assignment in the gel.

View larger version (115K): [in this window] [in a new window]   Figure 1. 2-DE protein pattern of ovarian tissue obtained from rFSH-primed immature mice. The original gel size was 23 x 30 x 0.075 cm. Proteins were detected by silver staining. Molecular mass and pI calibration were performed with theoretical values for the identified protein spots A, B, and C. Spots A (actin; 42 kDa; pI, 5.3), B (superoxide dismutase [Cu-Zn]; 16 kDa; pI, 6.0), and C (glutathione S-transferase; 26 kDa; pI, 8.2) were identified by peptide mass fingerprinting using MALDI-MS.

Figure 2 shows the enlarged area around spot X from a mouse ovary gel pattern before hCG treatment, and mouse ovary gel patterns after 3, 6, and 14 h of hCG treatment.

View larger version (91K): [in this window] [in a new window]   Figure 2. Enlarged section of the framed area in the high-resolution 2-DE protein pattern of ovarian tissue obtained from rFSH-primed immature mice in Fig. 1 (A) and the corresponding enlarged sections of the protein patterns of mouse ovaries after 3 h (B), 6 h (C), and 14 h (D) after LH treatment. The altered protein spot is marked with the letter X.

View larger version (22K): [in this window] [in a new window]   Figure 3. Peptide mass fingerprinting of the LH-induced protein spot X. MALDI-MS of the tryptic digestion mixture. Six peaks matched peptides of MVDP using the FRAGMOD program. The corresponding peptide sequences for the mass peaks are shown in parenthesis: 950.6 Da (70–77), 1458.0 Da (305–315), 1646.6 Da (155–168), 2149.3 Da (177–194), 2532.3 Da (41–61), and 3215.8 Da (203–232).

View larger version (50K): [in this window] [in a new window]   Figure 4. Northern blot analysis of various mouse tissues using an mvdpspecific cDNA probe. Total RNA was extracted from tissues obtained from hCG-treated (14 h) animals (+LH) as indicated. Uterus, ovary, and adrenal glands were also prepared from untreated animals. Northern blot analysis was performed using a radiolabeled mvdp cDNA probe. Radiolabeled cDNA for gapdh was used to monitor loading. Densitometric units corresponding to mvdp mRNA are expressed relative to the gapdh mRNA signal. GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

View larger version (186K): [in this window] [in a new window]   Figure 5. In situ hybridization of mvdp mRNA before and after the LH treatment. Ovaries were collected from immature mice. Tissue sections were hybridized with an antisense mvdp probe and photographed at x100 magnification using bright field optics to show structures; before LH treatment (A); 1.5 h (B), 6 h (C), 14 h (D), 24 h (E), and 32 h (F) after the LH treatment.

In situ hybridization analysis of mvdp mRNA in adrenals of immature female mice
Northern blot analysis showed high levels of mvdp mRNA in adrenal glands of immature female mice (Fig. 4). To analyze the cell-specific expression of the mvdp mRNA, we performed in situ hybridization studies. The hybridization results of the digoxigenin-labeled antisense probe to the tissue sections revealed that mvdp expression was confined to the cells of the zona fasciculata (Fig. 6). No transcripts were detectable in the cells of the zona glomerulosa and the zona reticularis. The same expression pattern was obtained after staining of adrenal glands obtained from mature male and female animals (data not shown).

View larger version (142K): [in this window] [in a new window]   Figure 6. Localization of mvdp mRNA in sections of adrenals from immature female mice. Tissue sections were hybridized with an digoxigenin-labeled antisense probe. The hybridized probe was detected using alkaline phosphatase-conjugated anti-DIG antibody. Mvdp transcripts were localized in the cells of the zona fasciculata.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

We identified a highly regulated protein spot X, which was absent in rFSH-primed ovarian tissue and appeared 3 h after the hCG treatment. Our group was able to unequivocally characterize this differentially expressed protein spot using MALDI-MS and Nan-ESI-MS/MS as the MVDP. MVDP was initially described as a major androgen-dependent secretory component of the mouse vas deferens (25). Until now, the potential role of MVDP in reproductive function has not been understood.

MVDP belongs to the aldo-keto reductase (AKR) superfamily. This protein family of NAD(P)(H)-dependent oxidoreductases consists of at least 42 members, which are known to metabolize a wide range of substrates, including aliphatic and aromatic aldehydes, monosaccharides, steroids, PGs, and xenobiotics (27). Specific members of the AKR family are known to be expressed in the ovary. One example is the expression of aldose reductase in ovarian tissue and its possible hormonal regulation during the estrous cycle (28). Aldose reductase is closely related to MVDP and has been a focus of interest because of its potential role in the development of diabetic complications (29). However, the function of aldose reductase in the female reproductive system is not understood. Another AKR detected in the ovary is the fibroblast growth factor-regulated protein (FR-1) (30). MVDP shares 82% amino acid sequence identity to FR-1, and Seery et al. (31) postulated that MVDP and FR-1 form an own subgroup within the aldose-reductase clade of the AKR superfamily. Although both proteins are closely related, FR-1 expression in the ovary was in contrast with MVDP expression not inducible by LH (data not shown). This indicates that FR-1 might fulfill a distinct function from MVDP in ovarian cells.

Using in situ hybridization studies, we were able to specifically localize mvdp mRNA transcripts to the thecal and interstitial cells of the mouse ovary. Interestingly, Inazu et al. (32), and very recently Espey et al. (33), described a number of carbonyl reductases showing an expression pattern comparable with MVDP in the ovary. The four homologous carbonyl reductases analyzed by Espey et al. (33) were also found to be inducible by LH treatment. Sequence comparisons, however, revealed that MVDP is neither identical nor highly related to one of these four carbonyl reductases. Another enzyme with carbonyl reductase activity is the 20 ß- hydroxysteroid dehydrogenase (34). However, MVDP is neither closely related to this enzyme nor does it represent the mouse homolog of this enzyme that has been identified in pigs so far. It will be of great interest to elucidate the specific functions of these enzymes, especially with respect to the fact that carbonyl reductases are NAD(P)(H)-dependent oxidoreductases showing very similar substrate specifities to the AKRs.

Until now, MVDP expression in the ovary has not been investigated. This study describes, for the first time, the expression of MVDP in mouse ovarian tissue and its regulation by LH/hCG. By using in situ hybridization, we could clearly demonstrate a start of mvdp mRNA expression in mouse ovaries as early as 1.5 h after hCG treatment. This suggests that the observed increase in MVDP protein level is attributable to a transcriptional up-regulation of mvdp mRNA. The time course of the induction demonstrates an early transcriptional response to hCG. The amount of transcripts of the mvdp gene was fully induced in thecal, as well as in interstitial cells, 6 h after treatment. Mvdp mRNA levels did not increase in magnitude with longer exposures. By Northern blot analysis, we could demonstrate that the mvdp mRNA level was increased approximately 300-fold by hCG. Moreover, we were able to show that the expression of the mvdp gene was transient. According to the time course of mvdp gene expression revealed by in situ hybridization, the mRNA transcripts were undetectable in the ovary 32 h after hCG treatment. It is important to note that the thecal as well as the interstitial cells are known to express the LH-receptor (35). In different ovarian cell types, the LH response is mediated predominantly by increases in the intracellular cAMP level. The promoter region of the mvdp gene contains two sequences that are homologoues to the cAMP response element. Because mvdp mRNA expression starts already 1.5 h after hCG treatment, it might be assumed that cAMP directly mediates this effect. In the adrenal gland, cAMP has already been postulated as an inducer of MVDP expression. Aigueperse et al. (36) showed that, in murine and human adrenocortical cells, the MVDP expression is induced by cAMP at the mRNA and protein level. They postulate that ACTH might be the key regulator of MVDP expression in adrenal glands. In the vas deferens, however, androgens have been shown to be the primary regulators of mvdp gene transcription (37). It would be very interesting to analyze, in future studies, whether LH is also able to directly regulate MVDP expression in the vas deferens.

The results from proteome profiling, in situ hybridization, and Northern blot analysis might indicate that the AKR MVDP converts a substrate potentially important for ovulation. Because the mvdp gene is also strongly expressed in the adrenal glands, it is possible that this protein is associated with steroidogenic activity. Lately, Lefrancois-Martinez et al. (38) analyzed the enzymatic function of MVDP in murine adrenocortical cells. They described that, in adrenocortical cells, MVDP acts as a major reductase for isocaproaldehyde, which is formed during steroidogenesis. Isocaproaldehyde is a product of side-chain cleavage of cholesterol. It is also formed during the ovarian steroidogenesis; for example, when cholesterone is converted to testosterone in the thecal cells. It might be suggested that MVDP is responsible for detoxifying isocaproaldehyde not only in adrenocortical cells but also in ovarian cells. However, it is also possible that MVDP fulfills a different function in the ovary, considering that we were able to demonstrate that not all of the steroidogenically active cells of the ovary show mvdp expression. For example, there is no mvdp mRNA detectable in the corpus luteum. In summary, our data suggest that MVDP, because of its strong regulation by LH, plays an important at least, indirect role during the cascade of events occurring at the time of ovulation. However, further experiments have to be done to elucidate whether MVDP enzyme activity contributes in any direct way to the mechanisms of ovulation.

	Acknowledgments

 
We are grateful to Andrea Dubrow, Sabine Feller, and Andrea Seipp for their excellent technical assistance.

Received December 28, 1999.

	References

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