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Endocrinology Vol. 142, No. 6 2499-2507
Copyright © 2001 by The Endocrine Society

ARTICLES

The Androgen-Conjugating Uridine Diphosphoglucuronosyltransferase-2B Enzymes Are Differentially Expressed Temporally and Spatially in the Monkey Follicle throughout the Menstrual Cycle1

Olivier Barbier2, Caroline Girard2, Louise Berger, Mohamed El Alfy, Alain Bélanger and Dean W. Hum

Oncology and Molecular Endocrinology Research Center (O.B., C.G., L.B., M.E.A., A.B., D.W.H.) and Medical Research Council Group in Molecular Endocrinology (A.B.), CHUL Research Center, Laval University, Québec, Canada G1V 4G2

Address all correspondence and requests for reprints to: Alain Bélanger, Ph.D., Laboratory of Molecular Endocrinology and Oncology, CHUL Research Center, 2705 Laurier Boulevard, Sainte-Foy, Québec, Canada G1V 4G2. E-mail: alain.belanger{at}crchul.ulaval.ca


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
UDP-glucuronosyltransferase (UGT) enzymes enhance the polarity of steroid hormones by catalyzing their conjugation with the sugar group from UDP-glucuronic acid. Previous results have shown that the monkey is a suitable animal model to study steroid glucuronidation in steroid target tissues. In humans, as in the monkey, the main androgen metabolites found in the circulation are 5{alpha}-androstane-3{alpha},17ß-diol-glucuronide and androsterone glucuronide, and high levels of androsterone glucuronide were also measured in human follicular fluid. Ovarian androgens play a significant role as precursors for estrogens and may modulate the recruitment and growth of follicles. To analyze the expression pattern of UGT2B enzymes involved in androgen metabolism throughout the menstrual cycle, cynomolgus monkey ovaries were collected during the mid and late follicular and luteal phases. Microsomal proteins and total RNA were analyzed for UGT2B expression in the whole ovary. Western blot and specific RT-PCR analyses demonstrated no significant changes in the expression of UGT2B protein or transcripts during the menstrual cycle. Immunocytochemistry analysis showed that UGT2B proteins are expressed in the cytoplasm of thecal and granulosa cells of growing follicles. Interestingly, the thecal cells of secondary follicles and of corpus luteum were extensively stained, whereas luteal granulosa cells were not labeled. These results suggest an important regulation of cell type-specific UGT2B expression during follicular development. Previous results demonstrated similar changes in the expression of the androgen receptor. The colocalization of the androgen receptor and UGT2B enzymes in the same cell types of the ovary provide evidence for a potential role of glucuronidation as a modulator of the intracellular androgen response during follicular development.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
STEROID HORMONES are specific ligands of steroid nuclear receptors that regulate the expression of many genes to control physiological processes. It is clear that enzymes involved in the synthesis of steroids play an important role in determining the level in a given tissue (1). However, it is apparent that enzymes, such as sulfotransferases and UDP-glucuronosyltransferases, involved in the metabolism of these hormones are also important for determining steroid levels and influencing the steroid response. In mammals, androgens play many important physiological roles, including the regulation of sex organ development during the embryonic stage and the maintenance of homeostasis in adult life. The ovaries secrete sex hormones, namely estrogens, that regulate the menstrual cycle throughout the female reproductive years. In this organ, androgens (C19 steroids) produced by thecal cells serve as a substrate for P450 aromatase-catalyzed estrogen synthesis in granulosa cells. Although the synthesis of C19 steroids in thecal cells is under LH stimulation, the formation of estrogens in granulosa cells is induced by FSH (2). As follicular development progresses, thecal androgen production gradually increases and promotes the FSH-stimulated steroidogenesis of granulosa cells by increasing cAMP levels (3). In addition, it was demonstrated that androgens increase follicle cell proliferation and suppress granulosa cell apoptosis (4, 5). These data clearly demonstrate that androgens act as precursors for ovarian estrogen synthesis, but also have a fundamental trophic role in follicular development. This mode of androgen utilization may be regulated by several factors, such as the rate of androgen production and the relative level of androgen receptor (AR) and P450 aromatase expression (3). On the other hand, deregulation of steroidogenesis within the adrenal gland and gonads that lead to elevated androgen levels could induce hyperandrogenism implicated in pathologies such as polycystic ovary syndrome (PCOS) (3), where the increased androgen production is suggested to result from abnormal steroid production in thecal cells (6, 7). Although the role of enzymes involved in steroid synthesis in the normal and pathological actions of androgen is well studied, the enzymes involved in steroid metabolism have received less attention.

Glucuronidation is an important pathway of cellular detoxification and consists of the transfer of glucuronic acid from uridine diphosphoglucuronic acid (UDPGA) to numerous compounds. The products of glucuronidation are generally more polar, less toxic, and more easily excreted from the body through the bile or urine. In addition to drugs or xenobiotics, several classes of endogenous compounds, such as bile acids and thyroid and steroid hormones, are glucuronidated (8, 9). Steroid glucuronidation is catalyzed by a family of membrane proteins located in the endoplasmic reticulum, named UDP-glucuronosyltransferase (UGT) enzymes (EC 2.4.1.17) (10). To date, more than 60 different UGT enzymes have been cloned in several mammalian species and on the basis of sequence similarities have been divided into 2 families, UGT1 and UGT2 (9). The primary structures of proteins of the UGT family are more than 45% identical, and they are more than 60% identical when they are within the subfamilies of UGT1A or UGT2B (9). In humans, the UGT1 gene family is located on chromosome 2q37 where the gene locus contains 12 different exons 1 (including 3 pseudogenes) and 4 common exons 2–5 (11, 12). UGT2 proteins are further categorized into 3 subfamilies, UGT2A, UGT2B, and UGT2C (9). The human UGT2B proteins are encoded by separate genes, which share the same organization of 6 exons and 5 introns. The human UGT2B4, UGT2B15, and UGT2B17 genes have been located on chromosome 4q13–4q21.1 (13, 14, 15, 16).

In humans, the plasma levels of 5{alpha}-reduced C19 steroid glucuronides, androsterone glucuronide (ADT-G) and 5{alpha}-androstane-3{alpha},17ß-diol-G (3{alpha}-Diol-G), reflect the peripheral tissue conversion of adrenal and gonadal precursor C19 steroids to active androgens (17). Castration in the adult leads to a 50% decrease in circulating testosterone, which is reflected by a similar decrease in circulating ADT-G and 3{alpha}-Diol-G (18). In addition, the plasma levels of steroid glucuronides are increased in some hyperandrogenic pathologies, such as acne or hirsutism, which are related to the increased plasma concentration of 5{alpha}-reduced C19 steroids (19). The monkey also has high plasma levels of androgen glucuronides, which indicates that simians represent a relevant animal model to study in vivo steroid glucuronidation (20). The similarities of steroid glucuronidation between humans and monkeys are further supported by the high sequence homology and similar biochemical characteristics between the human enzymes and the six UGT1A and five UGT2B simian enzymes cloned to date (21, 22, 23, 24, 25). Although both monkey UGT1A and UGT2B enzymes conjugate various steroid hormones, it is interesting to note that, as in humans, simian UGT2B isoforms are more specific for androgens and 5{alpha}-reduced C19 steroid metabolites. In contrast, UGT1A enzymes glucuronidate preferentially C18 steroids, such as estrogens and catechol estrogens (26, 27).

Measurement of unconjugated and glucuronidated C19 steroids in human follicular fluid demonstrated that the levels of 3{alpha}-Diol-G, ADT-G, and dihydrotestosterone (DHT) glucuronide are 2- to 6-fold higher than their unconjugated forms (28). In addition, determination of ADT, 3{alpha}-Diol, 4- hydroxyestradiol, and eugenol glucuronidation by microsomal proteins from more than 20 monkey tissues demonstrated that androgen-glucuronidating activity is higher in monkey ovaries than in other tissues (26). Due to the major role that UGT2B enzymes play in the glucuronidation of 5{alpha}-reduced C19 steroid metabolites, it was of interest to analyze the expression of these enzymes in the ovarian follicle during the menstrual cycle.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
Restriction enzymes and other molecular biology reagents were purchased from Stratagene (La Jolla, CA), Pharmacia LKB (Milwaukee, WI), Life Technologies, Inc. (Ontario, Canada), and Roche Molecular Biochemicals (Indianapolis, IN). Protein assay reagents were obtained from Bio-Rad Laboratories, Inc. (Richmond, CA). [{alpha}-32P]Deoxy-CTP (3000 Ci/mmol) and [3H]recombinant UTP were obtained from NEN Life Science Products-DuPont (Boston, MA). The Riboprobe R Gemini II kit was obtained from Promega Corp. (Madison, WI), and the immunohistochemical kit (Vectastain ABC kit) was purchased from Vector Laboratories, Inc. (Burlingame, CA). The UGT2B complementary DNAs (cDNAs) and stable cell lines were obtained as previously described (21, 22, 23, 24, 25).

Tissue preparation
Adult ovary tissues were taken from 11 female cynomolgus monkey by bilateral ovariectomy (Table 1Go). Immediately after ovariectomy, one or two ovaries were fixed by immersion in 2% glutaraldehyde, 4% formaldehyde, and 3% dextran in 0.05 M phosphate buffer (pH 7.4). After 4 h, the specimens were processed and embedded in paraffin until in situ hybridization or immunohistochemistry analysis. The remaining ovary was frozen by immersion in liquid nitrogen and keep at -80 C until RNA or microsomal protein isolation. The ovariectomies were scheduled according to the end of the last menstruation of the female monkey (TableGo 1).


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  		Table 1. Schedule of monkey ovariectomies and treatments of ovaries

Eleven cynomolgus female monkeys were ovariectomized at dates scheduled in accordance to the end of their last menstruation. Some tissues were fixated in 2% glutaraldehyde, 4% formaldehyde, and 3% dextran in 0.05 M phosphate buffer (pH 7.4). After 4 h, the specimens were processed and embedded in paraffin until in situ hybridization or immunohistochemistry analyses. Remaining ovaries were frozen by immersion in liquid nitrogen, and kept at -80 C until RNA or microsomal protein isolation. ISH, In situ hybridization analyses; IHC, immunohistochemical analyses; RNA, total RNA isolation; microsomes, microsomal protein extraction.

 
Total RNA and microsomal protein isolation
Total RNA was isolated from monkey ovaries (no. 125–431R, 125–906R, 125–900R, 125–395L, and 125–684L), monkey liver, and HK293 cells according to the Tri-Reagent acid-phenol protocol as specified by the supplier (Molecular Research Center, Inc., Cincinnati, OH). Quantitation was made by OD at 260 nm. For microsomal protein isolation, ovaries (no. 125–388L, 125–391L, 114–311R, and M873R) and transfected or untransfected HK293 cells (8 x 106) were homogenized in 5 ml homogenization buffer [0.1 M K2HPO4, 0.1 M KH2PO4 (pH 7.4), 20% glycerol, 1 mM EDTA, 1 mM dithiothreitol, 2.5 µg/ml pepstatin, and 0.5 µg/ml leupeptin] using a Potter-Glass-col (Terre Haute, IN)-type homogenizer with a Teflon pestle at 4 C and centrifuged at 12,000 x g for 20 min at 4 C. The supernatant was centrifuged at 105,000 x g for 1 h at 4 C. The microsome pellets were resuspended in 0.5 ml homogenization buffer. The protein concentration was determined using the Bradford method.

Subcloning of the UGT2B probe and Southern blot analysis
Sequencing of numerous mammalian UGT2B proteins demonstrated that their amino-terminal domain (amino acids 291–530) is highly conserved. This amino acid identity reflects the high homology of the 3'-end translated region of UGT2B cDNAs. Based on this characteristic, the conserved EcoRI-SacI fragment (445 bp) of the UGT2B20 cDNA was subcloned in the corresponding sites of phagemid pBluescript (Stratagene). This fragment was used for complementary RNA probes synthesis, with a predicted binding domain composed of coding nucleic acids from 912-1357 of all monkey cDNAs. To analyze the ability of this fragment to bind UGT2B cDNAs, they were electrophoresed on a 1% agarose gel and transferred onto a nylon membrane for Southern blot analysis. The blot was prehybridized in 50% formamide, 5 x Denhardt’s solution, 6 x SSC (standard saline citrate), 50 mM Tris (pH 8.0), 1% SDS, and 100 µg/ml salmon sperm DNA for 5 h at 42 C. Hybridization was performed with 1.5 x 106 cpm/ml of the UGT2B probe in the same buffer as prehybridization for 16 h at 42 C. The blot was washed twice in 0.1% SDS and 2 x SSC at 65 C for 30 min and exposed on XAR5 film with an intensifying screen (Eastman Kodak Co., Rochester, NY) for 16 h.

Northern blot analysis
To determine the ability of the UGT2B probe to bind messenger RNA (mRNA) extracted from monkey tissues and to analyze the level of UGT2B mRNAs in monkey liver and ovary, a Northern blot analysis was performed. In this experiment, 5 µg total RNA from five different monkey ovaries (Table 1Go) were pooled, and the total of 25 µg was electrophoresed on a 1% agarose gel. Five micrograms of total RNA from monkey liver and 25 µg from HK293 cells were also electrophoresed as positive and negative controls, respectively. The gel was transferred onto a nylon membrane (Amersham Pharmacia Biotech, Oakville, Canada), which was prehybridized in 50% formamide, 5 x Denhardt’s solution, 6 x SSC, 50 mM Tris (pH 8.0), 1% SDS, and 100 µg/ml salmon sperm DNA for 5 h at 42 C. Hybridization was performed with 1.5 x 106 cpm/ml of the UGT2B probe in the same buffer as prehybridization for 18 h at 42 C. The blot was washed twice in 0.1% SDS and 2 x SSC at 65 C for 30 min and exposed on XAR5 film with an intensifying screen (Eastman Kodak Co.) for 15 days.

RT-PCR analysis
The expression of each UGT2B transcript in the whole ovary during menstrual cycle was achieved using a RT-PCR technique as previously reported (23). Five micrograms of total RNA from cynomolgus monkey ovaries (Table 1Go) and liver were used for reverse transcriptase reactions, with incubation in presence of 500 pmol of an oligodeoxythymidine primer and of 200 U SuperScript II reverse transcriptase, according to the manufacturer’s instructions (Life Technologies, Inc., Ontario, Canada). The PCR reaction was carried out in presence of 1/10th of RT products, 100 ng of each specific oligonucleotides sense and antisense primer (Table 2Go) using AmpliTaq DNA polymerase. The PCR reactions were performed for 30 cycles (1 min 20 sec at 95 C, 1 min 20 sec at annealing temperature, and 1 min 20 sec at 72 C; Table 2Go). One fifth of the PCR products were electrophoresed on an ethidium bromide-stained 1% agarose gel. PCR reactions were controlled using sense and antisense glyceraldehyde-3-phosphate dehydrogenase primers. The identity of all PCR products was verified by direct sequencing (29).


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  		Table 2. Specific oligonucleotides and annealing temperature for RT-PCR experiments

 
Western blot analysis
Microsomal proteins of monkey ovaries (25 µg) and liver (5 µg), untransfected HK293 cells (25 µg), and HK293 cells stably expressing UGT2B9, UGT2B18, UGT2B19, UGT2B20, and UGT2B23 (10 µg) were separated on a 10% SDS-polyacrylamide gel, transferred onto nitrocellulose membrane, and probed with the EL-93 anti-UGT2B antisera (1:3000 dilution). An antirabbit IgG antibody conjugated with peroxidase (Amersham Pharmacia Biotech, Ontario, Canada) was used as the second antibody, and the resulting immunocomplexes were visualized using a chemiluminescence kit (Renaissance, Quebec, Canada) and exposed on Hyperfilm for 2 min (Kodak).

Immunohistochemistry analysis
Ovaries fixed in 4% formaldehyde (0.05 M phosphate buffer) and embedded in paraffin were cut in 4-µm sections. Sections were mounted, deparaffined using toluene, and then rehydrated. Immunostaining was performed using the anti-UGT2B EL-93 antisera diluted 1:300 in Tris saline, pH 7.6, for 1 h at room temperature. After incubation, sections were washed with PBS and incubated with a biotin-labeled goat antirabbit {gamma}-globulin diluted 1:1500 for 10 min. Then, the sections were treated with streptavidin coupled with peroxidase, and the diaminobenzidine was used as the chromogen to visualize the biotin streptavidin-peroxidase complex with exposition for 3 min (Vectastain ABC kit, Vector Laboratories, Inc., Burlingame, CA). Endogenous peroxidase activity was eliminated by preincubation with 3% H2O2 for 20 min. The intensity of the staining was controlled under the microscope. The sections were then counterstained with hematoxylin. Control experiments were performed on adjacent sections by substituting preimmune rabbit serum (1:100).

Riboprobe synthesis and in situ hybridization analysis
Riboprobes were generated by in vitro transcription from the pBluescript phagemid containing the UGT2B fragment. Using T3 and T7 RNA polymerase, respectively, the sense and antisense complementary RNA were synthesized in presence of [3H]UTP (NEN Life Science Products DuPont) with the Riboprobe R Gemini II as indicated by the supplier (Promega Corp.). Thick sections (20 µm) of ovary tissues (Table 1Go) were cut and deparaffined in toluene. The sections were subsequently rehydrated, postfixed in 2% glutaraldehyde, 4% formaldehyde, and 3% dextran in 0.05 M phosphate buffer, and washed in the same buffer containing 7.5% glycine. Hybridization of the floating sections was performed overnight at 40 C with [3H]UTP riboprobes. After hybridization, they were postfixed in osmium tetroxide, flat-embedded in Epon, and cut at 0.7 µm with an ultramicrotome. Sections were coated with liquid photographic emulsion (Kodak NTB2) and developed after 8 weeks of exposure.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Expression of steroid conjugating UGT enzymes in the monkey ovary during the menstrual cycle
Expression of UGT transcripts. To obtain a cDNA probe that hybridizes to monkey UGT2B transcripts in Northern blot analyses, a 445-bp EcoRI-SacI cDNA fragment from UGT2B20 was demonstrated to bind all UGT2B sequences as shown by Southern blot analysis (Fig. 1AGo). To ascertain the expression of UGT2B transcript in the ovary, total RNA was isolated and pooled from five monkey ovaries harvested at different stages of the menstrual cycle (Table 1Go). The UGT2B20 cDNA probe hybridized to transcripts of between 1.6–1.8 kb observed in monkey liver and ovaries, whereas no UGT2B transcripts were detected in HK293 cells, which were used as a negative control (Fig. 1BGo). However, it was clear that there was a lower level of UGT2B transcript expression in the ovary compared with liver.



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  		Figure 1. A, Southern blot analysis of monkey UGT2B cDNAs hybridized with the in situ hybridization UGT2B probe. B, Northern blot analysis of UGT2B transcripts in monkey liver and ovary. A, The cDNAs encoding five monkey UGT2B enzymes were Southern blotted and shown to hybridize with a radiolabeled 445-bp UGT2B cDNA probe. B, Five micrograms of total RNA purified from five monkey ovaries (no. 125–431R, 125–906R, 125–900R, 125–395L, and 125–684L) were pooled for a total of 25 µg and were electrophoresed on a 1% agarose gel. Ten micrograms of total RNA from monkey liver and 25 µg from HK293 cells were also electrophoresed as positive and negative controls, respectively. The blot was transferred onto a nylon membrane and hybridized with the UGT2B probe. A significant level of UGT2B transcripts is expressed in simian ovaries.

 
Monkey and human UGT2B enzymes are encoded by highly homologous transcripts, which have nucleotide sequence identities of up to 98%. Therefore, it is very probable that UGT2B cDNA probes would hybridize to multiple transcripts in Northern blot analysis, which is consistent with the relatively broad band observed. To analyze the expression of specific UGT2B transcripts expressed in the ovary during different phases of the menstrual cycle, RT-PCR experiments were designed to specifically amplify each monkey UGT2B transcript known to date from RNA isolated from monkey ovaries. The ovaries were collected at different stages of the menstrual cycle relative to the last menstruation and corresponded to the midfollicular phase (ovary 125–431R), the late follicular phase (ovary 125–900R), the midluteal phase (ovary 114–395L), and the late luteal phase (ovary 125–684L). The integrity of each RNA sample was verified by PCR amplification of the glyceraldehyde-3-phosphate dehydrogenase transcript using specific oligonucleotides (data not shown). Results demonstrated that all UGT2B transcripts were expressed at all stages of the menstrual cycle in monkey ovaries (Fig. 2Go). The levels of amplification products corresponding to UGT2B19, UGT2B20, and UGT2B23 were similar in all four ovaries analyzed. Although the RT-PCR is only semiquantitative, the lower level of UGT2B9 and UGT2B18 PCR products suggests a lower level of these transcripts in the ovary harvested during the late follicular phase than in those corresponding to other stages of the menstrual cycle (Fig. 2Go).



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  		Figure 2. Ovarian expression of monkey UGT2B transcripts during the menstrual cycle analyzed by specific RT-PCR experiments. The expression of UGT transcripts in total RNA from cynomolgus monkey liver and whole ovaries from the midfollicular phase (ovary 125–431R), the late follicular phase (ovary 125–900R), the midluteal phase (ovary 114–395L), and the late luteal phase (ovary 125–684L) was assessed by RT-PCR analyses using specific oligonucleotides, as shown in Table 2Go. All of the monkey UGT transcripts tested are expressed in the whole monkey ovaries.

 
Expression of UGT2B proteins. To determine the ability of an antibody (EL-93) raised against human UGT2B15 to recognize the monkey UGT2B proteins, a Western blot analysis was performed on microsomal proteins from HK293 cells stably expressing the monkey UGT2B proteins (Fig. 3AGo). All of the simian UGT2B proteins, which have an apparent molecular mass of 51–55 kDa, were recognized by the EL-93 antibody. The slower migrating protein observed in HK293 cells expressing UGT2B20 corresponds to the glycosylated form of this protein, as previously described (25, 30).



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  		Figure 3. Immunoblot analyses of UGT2B protein expression. A, Microsomal preparations from HK293 cells stably expressing UGT2B9, UGT2B18, UGT2B19, UGT2B20, and UGT2B23 (10 µg protein) were separated by SDS-PAGE. Using an anti-UGT2B antibody, the UGT2B proteins were demonstrated in all derived cell lines. B, Microsomal preparations from monkey liver (5 µg), ovaries (25 µg), and HK293 cells (25 µg) were chromatographed by SDS-PAGE. The resulting Western blot was probed with an anti-UGT2B polyclonal antibody. Although the expression of UGT2B proteins is lower relative to that in liver, significant levels are found throughout the menstrual cycle (Table 1Go) in whole ovaries. As expected untransfected HK293 cells do not express detectable levels of UGT2B proteins.

 
To further demonstrate the expression of steroid-conjugating UDP glucuronosyltransferases in the monkey ovary, the presence of UGT2B proteins was assessed by Western blot analyses performed on microsomal proteins extracted from monkey ovaries harvested at different stages of the menstrual cycle (Table 1Go). UGT2B protein expression was found in every stage of the menstrual cycle (Fig. 3BGo). Interestingly, a slower migrating UGT2B protein, which could correspond to a glycosylated UGT2B protein, was found in the late luteal phase ovary (M873R). Two different immunocomplexes of approximately 51 and 55 kDa were also detected in monkey liver, which may correspond to different glycosylated forms of UGT2B proteins, as previously described (25). In fact, previous studies have shown that endoglycosidase H treatment of microsomal proteins from monkey liver and ovary led to faster migrating UGT2B proteins (25). A recent study has demonstrated that of the monkey proteins isolated to date only UGT2B20 is glycosylated (30). Thus, it is reasonable to speculate that the slower migrating UGT2B protein observed in ovary M873R could correspond to UGT2B20; however, it remains possible that it corresponds to a novel uncharacterized glycosylated UGT2B protein. These results suggest significant changes in the pattern of UGT2B expression in the whole ovary during the menstrual cycle. However, these changes could also be associated with interindividual variations in the expression of these enzymes.

Follicular expression of androgen-conjugating UGT2B enzymes in the ovary throughout the menstrual cycle
Cellular expression of UGT2B transcripts in monkey ovary. To further determine the cell type-specific expression of UGT2B proteins during the menstrual cycle, in situ hybridization analyses were performed using a [3H]UTP-labeled UGT2B complementary RNA probe. Although no follicles were found in the analyzed sections of ovaries numbered 125–431L and K270R, in the antral follicle (ovary M853R) all granulosa and thecal cells were significantly labeled (Fig. 4AGo). In contrast, when hybridization was performed using the control 3H-labeled sense riboprobe, only a few silver grains were detected in the follicle, demonstrating the specificity of the antisense hybridization (Fig. 4BGo). In corpus luteum (ovary 114–311L), an intense labeling was observed with the UGT2B antisense riboprobe, with a majority of silver grains found in thecal lutein cells (Fig. 4CGo), whereas only a few silver grains were found with the sense negative control probe (Fig. 4DGo).



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  		Figure 4. Cell type-specific expression of UGT2B transcripts in the monkey ovary determined by in situ hybridization. A, Semithin Epon sections (0.7 µm thick) of growing follicles (ovary M853R) hybridized with the antisense probe. Granulosa and thecal cells of the follicles are labeled (x300). B, Similar area from the same ovary hybridized with the sense probe as negative control. Only scattered silver grains can be detected (x300). C, Hybridization of corpus luteum (ovary 114–311L) with the antisense probe, demonstrating the UGT2B transcript expression in thecal lutein cells (x300). D, Corpus luteum from the same section hybridized with the sense probe as a negative control (x300).

 
Expression of UGT2B proteins in the different cell types of the monkey ovary. After demonstrating UGT2B expression in the growing follicle and in corpus luteum by in situ hybridization, the pattern of UGT2B protein expression in ovaries 125–388R, M853L, 125–395R, and K270L was estimated by immunostaining using a polyclonal anti-UGT2B antibody (EL-93). All ovaries demonstrated the same pattern of UGT2B protein expression. In the primary follicles, staining was found only in granulosa cells, whereas thecal cells were unstained (Fig. 5AGo). In secondary and growing follicles, characterized by the presence and development of the antrum, the antiserum reacted with UGT2B proteins expressed in granulosa and thecal cells and in the ovum (Fig. 5Go, B and C). In the periovulatory follicle, staining was significantly higher in thecal cells than in granulosa cells (Fig. 5DGo). Using preimmune serum, no staining was observed, indicating the specificity of the EL-93 immune serum (Fig. 5FGo). Interestingly, the most intense staining was obtained in thecal lutein cells of the corpus luteum, in contrast to granulosa lutein cells, which were not labeled (Fig. 5EGo).



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  		Figure 5. Cell type-specific expression of UGT2B proteins in the primary and secondary follicles of monkey ovary determined by immunohistochemistry with the EL-93 anti-UGT2B antibody (1:300). A, Expression of UGT2B proteins is found in granulosa cells of primary follicles, whereas thecal cells do not expressed these proteins (x100). B and C, Expression found in granulosa cells and thecal cells of growing follicles. Ovum are also significantly stained (x100). D, Thecal cells of periovulatory follicles are significantly more stained than granulosa cells (x100). E, An intense staining is found specifically in thecal lutein cells (->), whereas granulosa cells of corpus luteum are not stained ({blacktriangleright}; x250). F, The same section as E probed with the preimmune serum (1:100), which shows no staining, indicating the specificity of the reaction when using the immune serum. G, Granulosa layer; T, thecal cells; S, stroma; A, antrum; O, ovum.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The level of steroid hormones, which serve as specific ligands of nuclear receptors in steroid target tissues, is regulated by enzymes involved in steroid biosynthesis and metabolism. The role as well as the cell type-specific expression of steroidogenic enzymes and hormone receptors in the mammalian ovary are well known (31). In the ovary, androgens have important functions as substrates for estrogen synthesis and as enhancers of follicular growth (3, 5). It was demonstrated that an excess of ovarian androgen could have deleterious effects and lead to PCOS (6, 32). Therefore, the regulation of ovarian androgen levels is important for follicular function. In addition to enzymes involved in steroid synthesis, it is clear that enzymes that catalyze steroid metabolism can also play a key role in determining steroid levels and the steroid response. To ascertain the potential physiological role of UGT2B enzymes in the regulation of ovarian androgen levels, we have studied the expression of steroid-conjugating UGT2B enzymes throughout the menstrual cycle in the whole monkey ovary and the follicles.

To date, five simian UGT2B enzymes, UGT2B9, UGT2B18, UGT2B19, UGT2B20, and UGT2B23, have been cloned and could be divided into three categories according to their stereospecificity for androgen glucuronidation. UGT2B9 catalyzes the glucuronidation of both the 3{alpha}- and 17ß-hydroxy positions on the androgen molecule (22). UGT2B18 and UGT2B23 are more specific for glucuronidation of 3{alpha}-hydroxyandrogens (21, 25), whereas UGT2B19 and UGT2B20 glucuronidate androgens more specifically at the 17ß-hydroxy position (23, 24). Thus, the presence of these UGT2B transcripts in the ovary demonstrates that this tissue can strongly metabolize precursor androgens (testosterone) in addition to active androgens (DHT) and their 5{alpha}-reduced metabolites (3{alpha}-Diol and ADT). The expression of UGT2B mRNAs and proteins is also consistent with the elevated concentrations of C19 steroid glucuronides measured in the follicular fluid (28). Interestingly, in a recent study Albert et al. measured the ability of different cynomolgus monkey tissues to glucuronidate androgen and estrogen metabolites compared with a common UGT substrate, eugenol (26). They found that monkey ovaries were unique in having higher conjugation activity toward ADT, 3{alpha}-Diol, and 4-hydroxyestrone compared with eugenol. In addition, the calculated ratios of steroid glucuronides/eugenol glucuronide were higher in ovaries than in any other tissue, thus indicating the predominant role of UGT enzymes in steroid metabolism in the ovary.

Ovarian steroidogenesis is regulated by enzymes involved in each specific step of hormone synthesis leading to estrogens and androgens, which includes cytochromes P450 side-chain cleavage (P450scc), P450c17, P450 aromatase, 17ß- hydroxysteroid dehydrogenase (17ßHSD) and 3ßHSD (33). The distribution of these enzymes in the ovary during the menstrual cycle is well characterized in humans. For example, the P450scc enzyme was shown to be expressed in all granulosa and thecal cells of follicles and corpus luteum, whereas the P450c17 protein was found only in thecal cells. The estrogen-synthesizing P450 aromatase enzyme is expressed specifically in granulosa cells of follicles during both follicular and luteal phases. In the case of hydroxysteroid dehydrogenase enzymes, 17ßHSD type 5 expression was shown in granulosa as well as thecal cells of corpora lutea, whereas 17ßHSD type 3 is not expressed in human ovary (34). 3ßHSD has been demonstrated to be expressed in thecal cells of antral follicle and in thecal and granulosa cells of preovulatory follicles and corpus luteum (33, 35). These results show that follicular cells express all of the steroidogenic enzymes necessary for hormone synthesis. The results obtained in the present study demonstrate that in addition to the above-mentioned steroidogenic enzymes, follicular cells express UGT2B proteins that are involved in hormone inactivation and elimination. Thus, these cells possess all of the the enzymatic activities required for active hormone synthesis and metabolism.

The differential cell type-specific expression of UGT2B proteins during the maturation and degradation of follicles suggest that these enzymes could have different physiological roles throughout this process. The actions of steroids are determined by binding to their related nuclear receptor, such as DHT to the AR, and it is well characterized that androgens are synthesized in thecal cells before their translocation to granulosa cells where they exert their actions (7). It is interesting to note that AR expression varies during follicle development, as demonstrated in human and monkey ovary, where it was localized principally in granulosa cells of growing follicles (3, 33, 35, 36, 37), and in thecal cells of the corpus luteum (Fig. 6Go) (36, 37). The localization of androgen-conjugating UGT2B enzymes in granulosa cells of primary follicles illustrates their potential role in the metabolism of androgens received from thecal cells, where these enzymes can increase androgen elimination from these cells and may influence the androgen response (Fig. 6Go). In contrast, the expression of UGT2B proteins in thecal cells of growing and preovulatory follicles suggests that locally produced C19 steroids are metabolized in cells in which they are produced (Fig. 6Go). In light of recent findings of in vivo studies, which demonstrate the positive correlation between AR expression and granulosa proliferation (5), it is interesting to speculate that androgen inactivation by way of glucuronidation is involved in the regulation of granulosa growth. Moreover, it has been shown that the ovary is the principal source of androgen excess in women with PCOS (6, 38), where impaired ovulatory function is correlated with an excess of small growing follicles (4). Although the implication of androgen-conjugating enzymes in this pathology is still unknown, it is possible that UGT2B enzymes found in granulosa cells serve to prevent the accumulation of DHT, leading to this abnormal follicular growth.



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  		Figure 6. Variation in cell type expression of UGT2B and AR proteins during the follicular and luteal phases.

 
The expression of both UGT2B mRNA and protein observed by in situ hybridization and immunohistochemistry indicates a follicular expression that is cell type specific, which may explain the apparently low levels of transcript and protein when ascertained by Northern and Western blot analyses. Thus, important information can be overlooked when assessing the expression level of UGT2B transcripts in the whole tissue. UGT enzymes may have important physiological functions in specific cell types of tissues that demonstrate low overall expression. The results of the present study suggest that UGT2B enzyme expression in follicular cells of the simian ovary plays a role in the regulation of androgen trophic effects. Further experiments will be needed for a better understanding of the hormonal mechanisms involved in the regulation of UGT2B expression and to assess the role of UGT2B enzymes in ovarian diseases associated with androgen excess, such as PCOS.


   Acknowledgments
 
We thank Dr. Pei Min Rong for excellent technical assistance with the Western blot, Dr. Jim Gourdon for monkey ovariectomies, and Hélène Lapointe for help with in situ hybridization studies. We are grateful to Dr. Chantal Guillemette for critical reading of the manuscript.


   Footnotes
 
1 This work was supported by the Medical Research Council of Canada, the Fonds de la Recherche en Santé du Québec, and Endorecherche. Back

2 O.B. and C.G. contributed equally to this work. Back

Received June 9, 2000.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Hum DW, Bélanger A, Lévesque E, Barbier O, Beaulieu M, Albert C, Vallée M, Guillemette C, Tchernoff A, Turgeon D, Dubois S 1999 Characterization of UDP-glucuronosyltransferase active on steroid hormones. J Steroid Biochem Mol Biol 28:413–423
  2. Hillier SG, Whitelaw PF, Smyth CD 1994 Follicular oestrogen synthesis: the "two cell, two-gonadotropin" model revisited. Mol Cell Endocrinol 10:51–54
  3. Tetsuka M, Hillier SG 1997 Differential regulation of aromatase and androgen receptor in granulosa cells. J Steroid Biochem Mol Biol 61:233–239[CrossRef][Medline]
  4. Weil S, Vendola K, Zhou J, Bondy CA 1999 Androgen and follicle-stimulating hormone interactions in primate ovarian follicle development. J Clin Endocrinol Metab 84:2951–2956[Abstract/Free Full Text]
  5. Vendola KA, Zhou J, Adesanya OO, Weil SJ, Bondy CA 1998 Androgens stimulate early stages of follicular growth in the primate ovary. J Clin Invest 101:2622–2629[Medline]
  6. Nelson VL, Legro RS, Strauss III JF, McAllister JM 1999 Augmented androgen production is a stable steroidogenic phenotype of propagated theca cells from polycystic ovaries. Mol Endocrinol 13:946–957[Abstract/Free Full Text]
  7. Gilling-Smith C, Storey H, Rogers V, Franck S 1997 Evidence for a primary abnormality in theca cells steroidogenesis in the polycystic ovary syndrome. Clin Endocrinol (Oxf) 47:93–99[CrossRef][Medline]
  8. Dutton GJ 1980 Glucuronidation of Drugs and Other Compounds. CRC Press, Boca Raton
  9. Mackenzie PI, Owens IS, Burchell B, Bock KW, Bairoch A, Bélanger A, Fournel-Gigleux S, Green M, Hum DW, Iyanagi T, Lancet D, Louisot P, Magdalou J, Chowdhury JR, Ritter JK, Schachter H, Tephly TR, Tipton KF, Nebert DW 1997 The UDP glycosyltransferase gene superfamily: recommended nomenclature update based on evolutionary divergence. Pharmacogenetics 7:255–269[Medline]
  10. Meech R, Mackenzie PI 1997 Structure and function of uridine diphosphate glucuronosyltransferases. Clin Exp Pharmacol Physiol 24:907–915[Medline]
  11. Burchell B, Brierley CH, Monaghan G, Clarke DJ 1998 The structure and function of the UDP-glucuronosyltransferase gene family. Adv Pharmacol 42:335–338
  12. Owens IS, Ritter JK 1995 Gene structure at the human UGT1 locus creates diversity in isozyme structure, substrate specificity, and regulation. Prog Nucleic Acids Res Mol Biol 51:305–338[Medline]
  13. Turgeon D, Carrier JS, Lévesque É, Beatty BG, Hum DW, Bélanger A 2000 Isolation and characterization of the human UGT2B15 gene, localized within a cluster of UGT2B genes and pseudogenes on chromosome 4. J Mol Biol 285:486–504
  14. Beaulieu M, Lévesque É, Tchernof A, Beatty BG, Bélanger A, Hum DW 1997 Chromosomal localization, structure, and regulation of the UGT2B17 gene, encoding a C19 steroid metabolizing enzyme. DNA Cell Biol 16:1143–1154[Medline]
  15. Monaghan G, Clarke DJ, Povey S, See CG, Boxer M, Burchell B 1994 Isolation of a human YAC contig encompassing a cluster of UGT2 genes and its regional localization to chromosome 4q13. Genomics 23:496–499[CrossRef][Medline]
  16. Monaghan G, Burchell B, Boxer M 1997 Structure of the human UGT2B4 gene encoding a bile acid UDP-glucuronosyltransferase. Mamm Genome 8:692–694[CrossRef][Medline]
  17. Bélanger A, Brochu M, Lacoste D, Noel C, Labrie F, Dupont A, Cusan L, Caron S, Couture J 1991 Steroid glucuronides: human circulatory levels and formation by LNCaP cells. J Steroid Biochem Mol Biol 40:593–598[CrossRef][Medline]
  18. Bélanger A, Brochu M, Cliche J 1986 Levels of plasma steroid glucuronides in intact and castrated men with prostatic cancer. J Clin Endocrinol Metab 62:812–815[Abstract]
  19. Brochu M, Bélanger A, Tremblay RR 1987 Plasma levels of C19 steroids and 5{alpha}-reduced steroid glucuronides in hyperandrogenic and idiopathic hirsute women. Fertil Steril 48:948–953[Medline]
  20. Guillemette C, Hum DW, Bélanger A 1996 Levels of plasma C19 steroids and 5{alpha}-reduced C19 steroid glucuronides in primates, rodents, and domestic animals. Am J Physiol 271: E348–E353
  21. Beaulieu M, Lévesque É, Barbier O, Turgeon D, Bélanger G, Hum DW, Bélanger A 1998 Isolation and characterization of a simian UDP-glucuronosyltransferase UGT2B18 active on 3-hydroxyandrogens. J Mol Biol 275:785–794[CrossRef][Medline]
  22. Bélanger G, Beaulieu M, Lévesque É, Hum D, Bélanger A 1997 Expression and characterization of a novel UDP-glucuronosyltransferase, UGT2B9, from cynomolgus monkey. DNA Cell Biol 16:1195–1205[Medline]
  23. Bélanger G, Barbier O, Hum DW, Bélanger A 1999 Molecular cloning, expression and characterization of a monkey steroid UDP-glucuronosyltransferase, UGT2B19, that conjugates testosterone. Eur J Biochem 260:701–708[Medline]
  24. Barbier O, Bélanger A, Hum DW 1999 Cloning and characterization of simian UDP-glucuronosyltransferase enzyme UGT2B20, a novel C19 steroid-conjugating protein. Biochem J 337:567–574
  25. Barbier O, Lévesque É, Bélanger A, Hum DW 1999 UGT2B23, a novel uridine-diphosphate-glucuronosyltransferase enzyme expressed in steroid target tissues that conjugates androgen and estrogen metabolites. Endocrinology 140:5538–5548[Abstract/Free Full Text]
  26. Albert C, Barbier O, Vallée M, Beaudry G, Bélanger A, Hum DW 2000 Distribution of UDP-glucuronosyltransferase (UGT) expression and activity in the cynomolgus monkey tissues: evidence for a differential expression of steroid conjugating UGT enzymes in steroid target tissues. Endocrinology 141:2472–2480[Abstract/Free Full Text]
  27. Albert C, Vallée M, Beaudry G, Bélanger A, Hum DW 1999 The monkey and human uridine diphosphate-glucuronosyltransferase UGT1A9, expressed in steroid target tissues, are estrogen-conjugating enzymes. Endocrinology 140:3292–302[Abstract/Free Full Text]
  28. Prévost J, Brochu M, Bélanger A, Lambert R 1987 Conjugated and unconjugated C-21, C-19 and C-18 steroid concentrations in human follicular fluid from hyperstimulated follicles. Gynecol Endocrinol 1:331–338[Medline]
  29. Rhéaume E, Sirois I, Labrie F, Simard J 1991 Codon 377 polymorphism of the human type I 3ß-hydroxysteroid dehydrogenase/isomerase gene (HS5DB3). Nucleic Acids Res 19:6060–6068[Free Full Text]
  30. Barbier O, Girard C, Breton R, Bélanger A, Hum DW 2000 N-Glycosylation and residue 96 are involved in the functional properties of UDP-glucuronosyltransferase enzymes. Biochemistry 39:11540–11552[CrossRef][Medline]
  31. Suzuki T, Sasano H, Sasaki H, Fukaya T, Nagura H 1994 Quantitation of P450 aromatase immunoreactivity in human ovary during the menstrual cycle: relationship between the enzyme activity and immunointensity. J Histochem Cytochem 42:1565–1573[Abstract]
  32. Qin K, Rosenfield RL 1998 Role of cytochrome P450c17 in polycystic ovary syndrome. Mol Cell Endocrinol 145:111–121[CrossRef][Medline]
  33. Takayama K, Fukaya T, Sasano H, Funayama Y, Suzuki T, Takaya R, Wada Y, Yajima A 1996 Immunohistochemical study of steroidogenesis and cell proliferation in polycystic ovarian syndrome. Hum Reprod 11:1387–1392[Abstract/Free Full Text]
  34. Pelletier G, Luu-The V, Tétu B, Labrie F 1999 Immunocytochemical localization of type 5 17ß-hydroxysteroid dehydrogenase in human reproductive tissues. J Histochem Cytochem 47:731–738[Abstract/Free Full Text]
  35. Suzuki T, Sasano H, Kimura N, Tamura M, Fukaya T, Yajima A, Nagura H 1994 Immunohistochemical distribution of progesterone, androgen and oestrogen receptors in the human ovary during the menstrual cycle: relationship to expression of steroidogenic enzymes. Hum Reprod 9:1589–1595[Abstract/Free Full Text]
  36. Hild-Petito S, West NB, Brenner RM, Stouffer RL 1991 Localization of androgen receptor in the follicle and corpus luteum of the primate ovary during the menstrual cycle. Biol Reprod 44:561–568[Abstract]
  37. Weil SJ, Vendola K, Zhou J, Adesanya OO, Wang J, Okafor J, Bondy CA 1998 Androgen receptor gene expression in the primate ovary: cellular localization, regulation, and functional correlations. J Clin Endocrinol Metab 83:2479–2485[Abstract/Free Full Text]
  38. Azziz R, Black V, Hines GA, Fox LM, Boots LR 1998 Adrenal androgen excess in the polycystic ovary syndrome: swensitivity and responsivity of the hypothalamic-pituitary-adrenal axis. J Clin Endocrinol Metab 83:2317–2323[Abstract/Free Full Text]



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