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Endocrinology Vol. 143, No. 11 4375-4384
Copyright © 2002 by The Endocrine Society

ARTICLE

Induction of Hyaluronan Synthase 2 by Human Chorionic Gonadotropin in Mural Granulosa Cells of Equine Preovulatory Follicles

Angelika E. Stock, Nadine Bouchard, Kristy Brown, Andrew P. Spicer, Charles B. Underhill, Monique Doré and Jean Sirois

Centre de Recherche en Reproduction Animale (A.E.S., N.B., K.B., J.S.) and Département de Pathologie et Microbiologie (M.D.), Faculté de Médecine Vétérinaire, Université de Montréal, Saint-Hyacinthe, Québec J2S 7C6, Canada; Center for Extracellular Matrix Biology (A.P.S.), Texas A&M University System Health Science Center, Institute of Biosciences and Technology, Houston, Texas 77030; and Department of Oncology and Cell Biology (C.B.U.), Georgetown Medical Center, Georgetown University, Washington, DC 20007

Address all correspondence and requests for reprints to: Dr. Jean Sirois, Faculté de Médecine Vétérinaire, Université de Montréal, 3200 Sicotte, Saint-Hyacinthe, Québec, Canada J2S 7C6. E-mail: siroisje{at}medvet.umontreal.ca.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
In contrast to other species, the preovulatory rise in gonadotropins in mares causes a remarkable expansion of the entire granulosa cell layer in vivo, suggesting that hyaluronan (HA) synthesis may be regulated in mural granulosa cells in this species. The objectives of this study were to clone and characterize equine hyaluronan synthase 2 (HAS2) and investigate the regulation of its transcript and of HA synthesis in equine follicles during human chorionic gonadotropin (hCG)- induced ovulation. Results showed that the equine HAS2 cDNA contains a 5'-untranslated region of 436 bp, an open reading frame of 1659 bp, and a 3'-untranslated region of 707 bp. The open reading frame encodes a 552-amino acid protein that is highly conserved (98–99% identity), compared with other known mammalian homologs. The regulation of HAS2 mRNA was studied in equine follicles isolated during estrus between 0 and 39 h after an ovulatory dose of hCG and in corpora lutea obtained on d 8 of the estrous cycle. Results from semiquantitative RT-PCR/Southern blotting analyses revealed a transient induction of HAS2 during the ovulatory process. Levels of HAS2 transcripts were undetectable in follicles before hCG treatment (0 h), increased markedly after gonadotropin treatment (P < 0.05), but returned to undetectable levels in corpora lutea. Analyses performed on isolated preparations of theca interna and granulosa cells showed that the granulosa cell layer was the predominant site of HAS2 expression. An immunohistochemical approach showed that this induction of HAS2 transcript was accompanied by a dramatic increase in HA production after hCG treatment. The isolation and characterization of a 1.8-kb fragment of genomic sequence located immediately upstream of equine HAS2, and comparison with corresponding human and mouse genomic regions identified several conserved putative cis-acting elements. Thus, this study describes the primary structure of equine HAS2, demonstrates for the first time the regulation of HAS2 in mural granulosa cells during the ovulatory process in vivo and identifies a valuable model in which to study the molecular control of HAS2 gene expression.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
HYALURONAN (HA), FORMERLY called hyaluronic acid, is a linear high-molecular-weight glycosaminoglycan composed of disaccharide repeats (up to 10,000 or more) of glucuronic acid and N-acetylglucosamine (1, 2, 3). This large polysaccharide forms unique entangled molecular networks that provide remarkable viscosity and elasticity to solutions (4). HA is a major component of the extracellular matrix and is present in virtually every tissue in vertebrates (1, 2, 3, 4). Although considered originally as an inert structural molecule, HA is now recognized as a functional ligand involved in the control of dynamic biological functions such as cell migration, proliferation, and differentiation through interactions with specific binding proteins, referred to as hyaladherins (2, 5, 6, 7, 8). HA also plays an important role in wound healing and inflammation, and altered HA production has been associated with the pathogenesis of various diseases such as rheumatoid arthritis, renal injury, atherosclerosis, and several human cancers (9, 10, 11, 12, 13, 14, 15). HA is synthesized at the inner face of the plasma membrane and is extruded through the membrane outside the cell as it is produced (1). Hyaluronan synthase (HAS) is the membrane-bound enzyme responsible for the polymerization of HA. Thus far, three distinct HASs (HAS1, HAS2, and HAS3) have been isolated and characterized in mammalian cells (1, 2, 16). They have different enzymatic properties, they are encoded by distinct genes located on different chromosomes, and their expression is regulated in a tissue- and agonist-specific manner (16, 17, 18, 19, 20, 21).

The ovulatory process is accompanied by a dramatic induction of extracellular matrix synthesis and cumulus cells, which leads to the characteristic volumetric expansion of the cumulus-oocyte complex (20- to 40-fold expansion) (3, 22). The highly viscoelastic extracellular matrix is composed predominantly of hyaluronan as well as other factors essential for matrix assembly, including proteoglycans, link protein, TNF-stimulated gene-6 (TSG-6), inter{alpha}-trypsin inhibitor, and serum-derived HA-associated protein (3, 22). Ultimately, studies in vivo and in vitro clearly showed that the induction of HA synthesis in mouse and pig cumulus cells results from an induction of HAS2 mRNA (23, 24).

Preovulatory follicular development and ovulation in mares possess several distinctive characteristics; the diameter of the ovulatory follicle is large (40–45 mm), the ovulatory process is relatively long (39–42 h), and follicular rupture is restricted to the ovulatory fossa (25, 26, 27, 28). One unique and remarkable cellular modification in equine follicles during gonadotropin-induced ovulation is the extensive expansion of the entire granulosa cell layer (29). In contrast to other species in which this process appears limited to the cumulus-oocyte complex, the preovulatory rise in gonadotropins in mares causes the expansion of mural granulosa cells, and this process is associated with an abundant accumulation of extracellular matrix (29). Although the biological significance of these observations is not known, the resulting entrapment of equine granulosa cells in the matrix renders their isolation particularly difficult for in vitro studies (28, 30, 31). The appearance and viscoelastic nature of the extracellular matrix led us to hypothesize a role for HAS2 induction in equine mural granulosa cells during the ovulatory process. The specific objectives of this study were to clone and characterize the structure of equine HAS2, determine the regulation of its transcript in equine follicles during human chorionic gonadotropin (hCG)-induced ovulation, and establish the relative contribution of theca interna and granulosa cells in follicular HAS2 expression.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
The QuikHyb hybridization solution, ExAssist/SOLR system and equine genomic library were purchased from Stratagene Cloning Systems (La Jolla, CA); the Access RT-PCR kit, Prime-a-Gene labeling system, and pGEM-T easy Vector System I were obtained from Promega Corp. (Madison, WI); [{alpha}-32P]dCTP was purchased from Mandel Scientific-NEN Life Science Products (Mississauga, Ontario, Canada); TRIzol total RNA isolation reagent, 1-kb DNA ladder, and synthetic oligonucleotides were obtained from Invitrogen Canada Inc. (Burlington, Ontario, Canada); Biotrans nylon membranes (0.2 µm) were purchased from ICN Pharmaceuticals, Inc. (Montréal, Québec, Canada); the Vectastain AEC kit was obtained from Vector Laboratories (Burlingame, CA); hyaluronan and hyaluronidase type 1-S were purchased from Sigma (Oakville, Ontario, Canada); electrophoretic reagents were obtained from Bio-Rad Laboratories, Inc. (Richmond, CA); hCG was purchased from The Buttler Co. (Columbus, OH).

Cloning of the equine HAS2 cDNA
To clone the equine HAS2 cDNA, an equine cDNA library prepared with mRNA extracted from a preovulatory follicle obtained 36 h post hCG (30) was screened with a human HAS2 cDNA (32). The probe was labeled with [{alpha}-32P]dCTP using the Prime-a-Gene labeling system (Promega Corp.) to a final specific activity greater than 1 x 108 cpm/µg DNA. Approximately 125,000 phage plaques were screened, and hybridization was performed at 55 C with QuikHyb hybridization solution (Stratagene). Positive clones were plaque purified through secondary and tertiary screenings, and pBluescript phagemids containing the cloned DNA inserts were excised in vivo with the Ex-Assist/SOLR system (Stratagene). DNA sequencing was performed commercially (Université Laval, Québec, Canada).

To provide a control gene for RT-PCR/Southern blot analysis, a 0.5-kb fragment of equine ribosomal protein L7a (rpL7a) was generated by RT-PCR using sense (5'-ACAGGACAT CCAGCCCAAACG-3') and antisense (5'-GCTCCTTTGTCTTCCGAGTTG-3') primers designed from the published sequence (GenBank accession no. AF508309), and the Access RT-PCR System (Promega Corp.) according to the manufacturer’s protocol. The reaction was performed using 100 ng RNA isolated from granulosa cells, and cycling conditions were one cycle of 48 C for 45 min and 94 C for 2 min, followed by 40 cycles of 94 C for 30 sec, 55 C for 1 min, and 68 C for 2 min. The PCR product was isolated after electrophoresis, subcloned into pGEM-T easy Vector, and sequenced to confirm its identity.

Isolation of the equine, mouse, and human HAS2 5'-flanking genomic DNA
To obtain a fragment of the 5'-flanking genomic region of equine HAS2, an equine genomic library (Stratagene) was screened following the manufacturer’s protocol with a 5'-fragment of the equine HAS2 cDNA labeled with [{alpha}-32P]dCTP. Two positive clones (clones 1–1 and 1–10) were identified and purified from an initial screen of 300,000 phage plaques. Using a walk-in approach with specific oligonucleotide primers (Invitrogen Canada Inc.), a 1.8-kb fragment of genomic DNA located immediately upstream of the cDNA sequence was characterized by sequencing. P1 genomic clones (129Sv/J mouse strain) containing the mouse HAS2 gene were obtained as previously described (33). Restriction fragments were subcloned into pBluescript vectors (Stratagene), and sequences upstream of the first exon of mouse HAS2 were determined using automated sequencing with synthetic oligonucleotides. The equivalent region of the human HAS2 gene was identified from the public domain database of the human genome (NCBI Human Genome sequence NT 023705). Sequence alignment of the equine, mouse, and human HAS2 presumptive promoter regions was performed using OMIGA software.

Equine tissues and RNA extraction
Testicular tissues were obtained from the Large Animal Hospital of the Faculté de médecine vétérinaire (Université de Montréal) following a routine castration, whereas other nonovarian tissues were collected at a local slaughterhouse. Equine preovulatory follicles and corpora lutea were isolated at specific stages of the estrous cycle from Standardbred and Thoroughbred mares as previously described (28). Briefly, when preovulatory follicles reached 35 mm in diameter during estrus, the ovulatory process was induced by injection of hCG (2500 IU, iv) and ovariectomies were performed via colpotomy using a chain écraseur at 0, 12, 24, 30, 33, 36, or 39 h post hCG (n = 4–6 mares/time point) (28). Follicles were dissected into preparations of follicle wall (theca interna with attached granulosa cells) or further dissected into separate isolates of granulosa cells and theca interna. Ovariectomies were also performed on d 8 of the estrous cycle (d 0, day of ovulation) to obtain corpora lutea (n = 3 mares). All animal procedures were approved by the institutional animal use and care committee. Total RNA was isolated from tissues with TRIzol reagent (Invitrogen Canada Inc.), according to manufacturer’s instructions using a Kinematica PT 1200C Polytron Homogenizer (Fisher Scientific, Montréal, Canada).

Semiquantitative RT-PCR and Southern analysis
The Access RT-PCR System (Promega Corp.) was used for semiquantitative analysis of HAS2 and rpL7a mRNA levels in equine tissues. Reactions were performed as directed by the manufacturer, using sense (5'-AGAGAAGTCATGTACACGGCCTTC-3') and antisense (5'-GGTCTGCTGGTTTAACCATCTGAG-3') primers specific for equine HAS2, and sense (5'- ACAGGACATCCAGCCCAAACG-3') and antisense (5'-GCTCCTTTGTCTTCCGAGTTG-3') primers specific for equine rpL7a. These reactions resulted in the production of HAS2 and rpL7A DNA fragments of 486 and 516 bp, respectively. Each reaction was performed using 100 ng total RNA, and cycling conditions were one cycle of 48 C for 45 min and 94 C for 2 min, followed by a variable number of cycles of 94 C for 30 sec, 55 C for 1 min, and 68 C for 2 min. The number of cycles used was optimized for each gene to fall within the linear range of PCR amplification and were 18 cycles for both HAS2 and rpL7a. Following PCR amplification, samples were electrophoresed on 2% TAE-agarose gels, transferred to nylon membranes, and hybridized with corresponding radiolabeled HAS2 and rpL7A cDNA fragments using QuikHyb hybridization solution (Stratagene). Membranes were exposed to a phosphor screen, and signals were quantified on a Storm imaging system using the ImageQuant software version 1.1 (Molecular Dynamics, Amersham Biosciences, Sunnyvale, CA).

Immunohistochemical localization of HA
The immunohistochemical localization of HA in equine follicular tissues was studied using a biotinylated HA-binding fragment of aggrecan (HA-binding probe), according to a technique previously described with minor modifications (34). Briefly, pieces of preovulatory follicles isolated prior or after hCG treatment were formalin fixed and paraffin embedded, and 3-µm-thick sections were deparaffinized through graded alcohol series. Endogenous peroxidase was quenched by incubating the slides in 0.3% hydrogen peroxide in methanol for 30 min. The sections were incubated with HA probe (10 µg/ml in 0.1 M phosphate buffer with 10% fetal bovine serum) for 1 h at room temperature. The HA probe-binding sites were revealed using the Vectastain complex (AEC kit, Vector Laboratories). Sections were washed in PBS for 10 min and incubated with the streptavidin-horseradish peroxidase conjugate reagent for 45 min at room temperature. After a 5-min PBS wash, the reaction was revealed using 3-amino-9-ethylcarbazole as the chromogen. Sections were counterstained with Mayer’s hematoxylin and mounted. The specificity of the HA-binding probe was tested using two independent approaches: 1) preincubation of the probe (10 µg/ml) with HA (100 µg/ml in PBS containing 10% FBS) for 60 min at room temperature before application to control sections; and 2) preincubation of control sections with hyaluronidase (4000 U/ml in PBS) overnight at 37 C before HA staining.

Statistical analysis
One-way ANOVA was used to test the effect of time after hCG on levels of HAS2 mRNA in samples of follicle wall, corpora lutea, theca interna, and granulosa cells. HAS2 mRNA levels were normalized with the control gene rpL7a. When ANOVAs indicated significant differences (P < 0.05), Dunnett’s test was used for multiple comparisons of individual means. Statistical analyses were performed using JMP software (SAS Institute, Inc., Cary, NC).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Characterization of the equine HAS2 cDNA
Twenty-three clones were obtained from an initial screen of 125,000 plaques of an equine follicular cDNA library with a human HAS2 cDNA probe. Four of these clones (clones 1, 2, 5, and 12) were purified and used for characterization by DNA sequencing. Results revealed the complete coding region of equine HAS2 as well as portions of the 5'- and 3'-untranslated regions. The longest clone (clone 12) contained a 5'-untranslated region of 436 bp, an open reading frame of 1659 bp, and a 3'-untranslated region of 707 bp (Fig. 1Go). The coding region of equine HAS2 encodes a 552-aminoacid protein that is highly conserved, compared with human HAS2, with a 99% identity at the amino acid level and a 94% identity at the nucleic acid level (Fig. 2Go). All putative structural and functional domains implicated in HAS function appear to be present in the equine enzyme (Fig. 2Go). The 5'- and 3'-untranslated regions are also remarkably conserved between equine and human HAS2, with a corresponding 75% and 85% identity, respectively.



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  		Figure 1. Nucleotide sequence of the equine HAS2 cDNA. The equine HAS2 cDNA was cloned by library screening, as described in Materials and Methods. The equine HAS2 cDNA is composed of a 5'-untranslated region of 436 bp (lowercase letters), an open reading frame of 1659 bp (uppercase letters), and a 3'-untranslated region of 707 bp (lowercase letters). The translation initiation (ATG) and stop (TGA) codons are shown in bold type, and numbers on the right refer to the last nucleotide on that line. The nucleotide sequence was submitted to GenBank (accession no. AY056582).

 


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  		Figure 2. Deduced amino acid sequence of equine HAS2 and comparison with the human homolog. The amino acid sequence of equine (equ) HAS2 is aligned with human (hum) HAS2. Identical residues are indicated by a printed period, putative transmembrane domains are overlined, conserved residues involved in N-acetylglucosaminyltransferase activity (67 ) are bold and marked with an asterisk, and consensus B(X7)B HA binding motifs (68 ) are underlined. Numbers on the right refer to the last amino acid on that line.

 
Tissue distribution of equine HAS2
To study the tissue distribution of equine HAS2, various equine tissues were obtained and the expression of HAS2 mRNA was studied by RT-PCR/Southern blot. Results showed that levels of HAS2 transcripts varied across tissues (Fig. 3Go). Levels of HAS2 mRNA were highest in a preovulatory follicle isolated 36 h after an ovulatory dose of hCG (i.e. approximately 3–6 h before ovulation); moderate in lung and thymus; low in brain, uterus, testis, and skin; and very low or undetectable in other tissues tested. In contrast, levels of the control gene rpL7a remained constant in these tissues.



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  		Figure 3. Expression of HAS2 mRNA in equine tissues. Total RNA was extracted from various equine tissues, and samples (100 ng) were analyzed for HAS2 and rpL7a (control gene) content by a semiquantitative RT-PCR/Southern blotting technique, as described in Materials and Methods. A, Expression of HAS2 mRNA in equine tissues. B, Expression of rpL7a mRNA in equine tissues. Numbers of PCR cycles for each gene were within the linear range of amplification, and they represented 18 cycles for both HAS2 and rpL7a. Numbers on the right indicate the size of the PCR fragment.

 
Regulation of HAS2 follicles and corpora lutea
To study the regulation of HAS2 mRNA in equine follicles before ovulation, preovulatory follicles were isolated during estrus at 0, 12, 24, and 36 h after the administration of an ovulatory dose of hCG. Total RNA was extracted from the follicle wall (theca interna with attached granulosa cells) and analyzed by semiquantitative RT-PCR/Southern blot. In addition, RNA samples from three corpora lutea obtained on d 8 of the estrous cycle were included in the analysis. Results showed a marked regulation of HAS2 transcripts during the ovulatory process and after ovulation (Fig. 4Go). Levels of HAS2 mRNA were undetectable in equine preovulatory follicles before hCG treatment (0 h) but were clearly induced at 12, 24, at 36 h post hCG (Fig. 4AGo). However, this induction appeared transient as evidenced by the disappearance of the transcript in a d 8 corpus luteum (Fig. 4AGo). When results from multiple follicles and corpora lutea were expressed as ratios of HAS2 to rpL7A, a significant increase in HAS2 mRNA levels was observed only in follicles at 12, 24, and 36 h post hCG (P < 0.05; Fig. 4CGo). Also, levels of HAS2 transcript at 36 h were higher than those present at 12 and 24 h post hCG (P < 0.05). The gonadotropin treatment had no effect on rpL7A transcript at any time point in preparations of follicle wall and corpora lutea.



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  		Figure 4. Regulation of HAS2 mRNA by hCG in equine preovulatory follicles. Preparations of follicle wall were obtained from preovulatory follicles isolated between 0 and 36 h after hCG and of corpora lutea isolated on d 8 of the estrous cycle. Samples (100 ng) of total RNA were analyzed for HAS2 and rpL7a content by a semiquantitative RT-PCR/Southern blotting technique, as described in Materials and Methods. A, Regulation of HAS2 mRNA in equine follicles (one representative follicle per time point). B, Constitutive expression of rpL7a mRNA in the same follicles. Numbers on the right indicate the size of the PCR fragment. C, Relative changes in HAS2 transcripts in equine follicles after hCG treatment. The intensity of HAS2 signal was normalized with the control gene rpL7a [n = 4, 5, 5, and 5 distinct follicles (i.e. animals) per time point at 0, 12, 24, and 36 h post hCG, respectively; n = 3 corpora lutea on d 8 (CL; D8)]. Bars marked with an asterisk are significantly different from 0 h post hCG (P < 0.05).

 
To help determine the cellular localization of HAS2 mRNA expression within the equine follicle wall, isolated preparations of granulosa cells and theca interna were obtained from follicles isolated between 0 and 39 h after hCG treatment (Fig. 5Go). Results indicated that the granulosa cell layer was the predominant site of HAS2 mRNA expression in the follicle, with a marked and significant induction observed between 12 and 39 h post hCG (P < 0.05; Fig. 5AGo). Also, levels of HAS2 transcript observed at 33, 36, and 39 h were significantly higher than those at 24 and 30 h post hCG (P < 0.05). In contrast, levels of HAS2 mRNA remained very low in theca interna with no significant changes between 0 and 33 h post hCG, and a very modest increase in HAS2 was observed at 36 and 39 h after hCG treatment (Fig. 5BGo). The gonadotropin treatment had no effect on rpL7A transcript in preparations of theca interna and granulosa cells at any time point.



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  		Figure 5. Cellular localization of HAS2 mRNA in equine preovulatory follicles. Preparations of granulosa cells (A) and theca interna (B) were isolated from equine preovulatory follicles between 0 and 39 h post hCG, and samples (100 ng) of total RNA were analyzed for HAS2 and rpL7a content by a semiquantitative RT-PCR/Southern blotting technique, as described in Materials and Methods. The HAS2 signal was normalized with the control gene rpL7a, and results are presented as a ratio of HAS2 to rpL7a [mean ± SEM; n = 4 samples (i.e. mares) per time point]. Bars marked with an asterisk are significantly different from 0 h post hCG (P < 0.05). Insets show representative results of HAS2 and rpL7a mRNA levels from one sample per time point.

 
Production of hyaluronan in equine follicles
The gonadotropin-dependent induction of HA production in equine preovulatory follicles was studied by immunohistochemistry in follicles isolated between 0 and 39 h post hCG using a biotinylated HA-binding probe. Results showed a dramatic increase in HA production after hCG treatment (Fig. 6Go). Follicles isolated before gonadotropin treatment (0 h post hCG) were characterized by a very compact granulosa cell layer and by the absence of HA (Fig. 6AGo). The administration of hCG caused a progressive loosening (i.e. expansion) of the granulosa cell layer that was associated with an increase in accumulation of HA (Fig. 6Go, B–E). At 24 h post hCG, granulosa cell expansion and HA production appeared relatively modest (Fig. 6BGo) but increased markedly at 30–39 h post hCG (Fig. 6Go, C–E). Control sections of follicles isolated 39 h post hCG showed no HA staining when the HA-binding probe was preincubated with excess HA before tissue staining (Fig. 6FGo) or when sections were incubated with hyaluronidase before applying the probe (data not shown). Likewise, no HA staining was observed when the HA-binding probe was omitted (data not shown).



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  		Figure 6. Immunohistochemical localization of HA in equine preovulatory follicles. The presence of HA in sections of preovulatory follicles isolated between 0 and 39 h after hCG treatment was studied by immunohistochemistry using a biotinylated HA-binding probe, as described in Materials and Methods. Results showed the absence of HA in a preovulatory follicle obtained 0 h post hCG (A), but the presence of HA staining at 24 (B), 30 (C), 36 (D), and 39 h post hCG (E). F, No staining was detected in an adjacent section of the follicle shown in E (39 h post hCG) when the HA-binding probe was preincubated with excess HA before tissue staining. Magnification, x200 (A–C) and x600 (D–F).

 
Characterization of the 5'-flanking region of equine, human, and mouse HAS2
The structure and molecular control of the HAS2 promoter have not been characterized in any mammalian species. To provide a preliminary characterization, 1.8-kb fragments of 5'-flanking DNA were obtained from equine and mouse genomic clones, and the corresponding region in humans was identified from the public domain database of the human genome. A common putative TATA signal and a predicted region of transcription initiation were identified in all three species using the Hamming-clustering method for TATA signal prediction in eukaryotic genes (www.itba. mi.cnr.it/webgene) and the Neural Network Promoter Prediction program (www.fruitfly.org) (Fig. 7Go). The proximal putative promoters of the equine, human, and mouse HAS2 were highly conserved, especially in the region lying 200 bp immediately upstream of the putative TATA box sequence (Fig. 7Go). Homology searches with the transcription factor database Tfsites (www.ifti.org) revealed the presence of numerous consensus cis-acting elements in the putative HAS2 promoters (Fig. 7Go). Some of the potential regulatory elements present were conserved in all three species, whereas others were detected only in the equine sequence (Fig. 7Go). They included SP1, p300, CAAT-box, AP2, MyoD, NFkB, and CACA sites that were either in the sense or antisense orientation (Fig. 7Go).



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  		Figure 7. Homology analysis of the 5'-flanking genomic regions of equine, human, and mouse HAS2. DNA fragments located immediately upstream of equine and mouse HAS2 were isolated from genomic clones, as described in Materials and Methods, and the human sequence was identified from the public domain database of the human genome. The nucleic acid sequence of the equine (equ) putative promoter is aligned with the corresponding human (hum) and mouse (mou) regions. The sequence of the equine genomic DNA fragment is shown in uppercase letters, whereas the beginning of the equine HAS2 cDNA clone (Fig. 1Go) is depicted in lowercase letters. Identical residues in the human and mouse sequence are indicated by a printed period; gaps in nucleic acid sequences created to optimize alignment are indicated by hyphens. Selected potential cis-acting promoter elements are shown in boxed regions. The predicted region of the transcription initiation identified by Neural Network Promoter Prediction program is shown in bold type; the region includes a central adenosine as the primary predicted start site as well as the three upstream and downstream nucleotides as alternative sites. The equine and mouse nucleotide sequences have been deposited in GenBank (accession nos. AF508308 and AY115483, respectively).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
This is the first study to report the presence of a gonadotropin-dependent induction of HAS2 and HA in vivo in mural granulosa cells during the ovulatory process. Whereas previous investigations in mice and pigs have documented the induction of HAS2 in cumulus cells during expansion of the cumulus-oocyte complex (23, 24), the present study clearly establishes that a similar phenomenon occurs in equine mural granulosa cells before ovulation. The preovulatory rise in LH in mares causes a remarkable expansion of the cumulus-oocyte complex, in keeping with a similar effect in other species (3, 22, 35). However, in contrast to other species, there is a marked expansion of the entire granulosa cell layer in equine follicles before ovulation, and this expansion is accompanied by an abundant production of viscoelastic extracellular matrix within the layer (29). It is now evident that this extracellular matrix is composed, at least in part, of HA, and involves the induction of HAS2 transcripts. The hCG- dependent induction of HAS2 in equine granulosa cells is a relatively early event, being first detected within the first 12 h of an ovulatory process that lasts 39–42 h in mares (27, 28). By comparisons, other key regulatory enzymes and proteins involved in ovulation and steroidogenesis are induced much later, with cyclooygenase-2 (COX-2), steroidogenic acute regulatory protein (StAR) and P450 cholesterol side-chain cleavage being up-regulated in granulosa cells only 30–39 h after hCG treatment (28, 30, 36, 37).

The gonadotropin-dependent induction of HAS2 and HA in the cumulus-oocyte complex has been ascribed to various potential functions during ovulation and fertilization (3, 22, 38). The HA-containing viscoelastic matrix may help cumulus cells to remain associated with the oocyte, facilitate extrusion of the detached complex from the follicle during ovulation, and ease its capture by, and transport in, the oviduct (3, 39). The matrix may also provide a physical barrier for the selection of highly functional and motile spermatozoa (40, 41). Besides these more mechanical functions, HA may act as a ligand involved in oocyte maturation and cumulus/granulosa cell functions through interactions with specific cell surface HA receptors (CD44) present on these cells (24, 42, 43). However, the precise reasons for HAS2 induction and the unique expansion of the entire granulosa cell layer in equine follicles before ovulation remain unknown. It is tempting to speculate that the resulting entrapment of mural granulosa cells within the HA-enriched extracellular matrix may help maintain cells inside the equine follicle during the rapid evacuation of follicular fluid at ovulation (44, 45). This may be particularly important in mares, considering the large diameter of the ovulatory follicle (40–45 mm) and the substantial volume of fluid (30–45 ml) exiting the follicle at the time of rupture, and that only granulosa cells, but not theca interna cells, are thought to contribute to the corpus luteum in this species (26, 46). Functional roles of HA through activation of CD44-mediated intracellular signaling pathways are also likely present in equine granulosa cells and could potentially include a putative antiapoptotic function as reported in human cells (47).

The molecular control of HAS2 induction in equine mural granulosa cells remains to be characterized, but the absence of HAS2 mRNA before hCG treatment and the marked induction thereafter suggest a transcription activation event, as proposed in mice cumulus cells (23). Elegant studies in vitro have shown that, in addition to gonadotropins, a soluble factor release by the oocyte is essential for induction of HA synthesis and cumulus-oocyte complex expansion in mice (48, 49, 50), and GDF-9 was recently identified as the putative factor (51). The limited diffusion of the factor from the oocyte was proposed to explain the absence or very little synthesis of HA by mural granulosa cells in vivo in mice (52). It is possible that, in contrast to mice, only gonadotropins are required for induction of HA synthesis in the equine preovulatory follicle, as observed in pigs and cows (53, 54, 55, 56). Further studies will be required to resolve this issue.

The COX-2/prostaglandin E2/PGE receptor type 2 (EP2) signaling pathway has also been implicated in gonadotropin- dependent regulation of HA synthesis and cumulus expansion in mice (22, 57, 58, 59). The timing of COX-2 induction in equine mural granulosa cells (30 h post hCG) (28, 30) follows that of HAS2 (12 h post hCG), which does not support a role for prostaglandins in triggering matrix deposition. However, it is interesting to note that the apparent increase in HAS2 mRNA expression after 30 h of hCG treatment does coincide with the induction of COX-2, thereby supporting the concept that prostaglandins may play a role in enhancing HA synthesis (57, 58).

The preovulatory follicle isolated 36 h post hCG was by far the tissue with the strongest expression of HAS2 mRNA among the various tissues tested. Considering that the ovulatory process shares many features of a classic acute inflammatory reaction (60), the involvement of HA in the inflammatory response is not excluded. Within the follicle wall, the granulosa cell layer was clearly the predominant site of HAS2 expression. The modest increase in HAS2 mRNA levels in theca interna at 36 and 39 h post hCG should be interpreted with caution, considering that the copious amounts of extracellular matrix at these time points rendered separation of granulosa and theca interna cells very difficult. The presence of a few contaminating granulosa cells in the theca interna preparations may have been sufficient to produce low detectable levels of HAS2 transcripts by RT-PCR/Southern blot. Levels of HAS2 mRNA in the thymus were relatively high, compared with other nonovarian tissues, in keeping with the ability of thymic epithelial cell to produce HA (61). Likewise, the detection of HAS2 transcripts in equine lung, brain, testis, and uterus agrees with previous studies in other species reporting either the presence of HAS2 or a role of HA in these tissues (32, 62, 63).

Lastly, this is the first report to describe the cloning and characterization of equine HAS2. Our sequencing results underscore the remarkable similarities in the primary structure of the enzyme across species. The length of the equine enzyme, 552 amino acids, is identical with that of other mammalian species characterized thus far (23, 32, 64, 65) and one amino acid longer that the Xenopus laevis homolog (66). More importantly, comparative analyses indicate that the amino acid sequence of equine HAS2 is almost identical with that of other animals, being 98–99% identical with human (64), mouse (23), rat (GenBank accession no. AF008201), rabbit (65), and bovine HAS2 (GenBank accession no. AJ004951), and very well conserved when compared with Gallus domesticus (93% identity; GenBank accession no. AF106940) and Xenopus HAS2 (88% identity) (66). All putative structural and functional domains are conserved in the equine protein, including the multiple transmembrane domains and HA binding sites (1, 2, 32, 64). The present report also provides a preliminary characterization of the genomic sequences located immediately upstream of equine, human, and mouse HAS2. Surprisingly, despite the role and regulation of HAS2 in development, physiology, and various pathologies (9, 10, 11, 12, 13, 14, 15), the HAS2 promoter has not been characterized in any species. Inspection of the equine, human, and mouse HAS2 5'-flanking regions suggests the presence of numerous putative cis-acting elements. However, future effort should focus on determining experimentally the precise transcription start site (s) of the equine HAS2 gene and on characterizing the molecular determinants involved in HAS2 promoter activity.

In summary, this is the first study to demonstrate the regulation of HAS2 in mural granulosa cells during the ovulatory process in vivo. The dramatic induction of HAS2 transcripts in equine mural granulosa cells is associated with increased HA synthesis and appears directly related to the unique and remarkable expansion of the granulosa cell layer in this species. A tissue survey revealed that the levels of HAS2 mRNA induced by hCG in equine preovulatory follicles are very high, compared with those present in other equine tissues. This study also provides the primary structure of the equine HAS2 transcript and deduced protein, as well as a 1.8-kb upstream genomic DNA fragment potentially involved in HAS2 promoter activity. Considering the large size of the equine preovulatory follicle and the remarkable level of HAS2 induction observed following gonadotropin treatment, we propose that mural equine granulosa cells could serve as a valuable model to study the molecular control of HAS2 gene expression.


   Acknowledgments
 


   Footnotes
 
This work was supported by Natural Sciences and Engineering Research Council of Canada Grant OPG0171135 (to J.S.) and by a Canadian Institutes of Health Research Investigator Award (to J.S.).

The nucleotide sequences reported in this paper have been submitted to GenBank with accession nos. AY056582, AF508308, and AY115483.

Abbreviations: COX-2, Cyclooygenase-2; HA, hyaluronan; HAS2, hyaluronan synthase 2; hCG, human chorionic gonadotropin; rpL7a, ribosomal protein L7a.

Received May 29, 2002.

Accepted for publication July 25, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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