Ovarian Expression of a Disintegrin and Metalloproteinase with Thrombospondin Motifs During Ovulation in the Gonadotropin-Primed Immature Rat 1

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1 BIOLOGY OF REPRODUCTION 62, (2000) Ovarian Expression of a Disintegrin and Metalloproteinase with Thrombospondin Motifs During Ovulation in the Gonadotropin-Primed Immature Rat 1 Lawrence L. Espey, 2,3 Shinya Yoshioka, 3 Darryl L. Russell, 4 Rebecca L. Robker, 4 Shingo Fujii, 5 and JoAnne S. Richards 4 Department of Biology, 3 Trinity University, San Antonio, Texas Department of Cell Biology, 4 Baylor College of Medicine, Houston, Texas Department of Gynecology and Obstetrics, 5 Kyoto University Faculty of Medicine, Kyoto, Japan ABSTRACT Mammalian ovulation is a dynamic process that requires degradation of the collagenous connective tissue in the thecal layers of a mature follicle. In this reverse transcription-polymerase chain reaction differential display study, gonadotropin-primed immature rats were used to detect ovarian expression of a relatively new type of disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS-1) that is known to cleave extracellular matrix in acutely inflamed tissues. Immature Wistar rats were primed with 10 IU ecg s.c., and the temporal pattern of expression of the ADAMTS-1 gene was delineated by extracting ovarian RNA at 0, 2, 4, 8, 12, and 24 h after induction of ovulation by injecting the primed animals with 10 IU hcg s.c. The differential display data, Northern analyses, and in situ hybridization micrographs all showed significant up-regulation of ADAMTS-1 gene expression by 8 h after hcg administration. The in situ data indicated that the ADAMTS-1 mrna was in the granulosa layer of mature follicles. Expression reached a peak at 12 h and remained elevated at 24 h after hcg. ADAMTS-1 gene expression was impaired by the antiprogesterone agent epostane, but this inhibition could be overcome by exogenous progesterone. ADAMTS-1 expression was not affected when ovulation was blocked by treatment of the animals with the antieicosanoid agent indomethacin. In conclusion, the temporal pattern of expression of this gene, and its apparent regulation by progesterone, suggests that ADAMTS-1 has a significant role in the inflammatory events of the ovulatory process. INTRODUCTION Mammalian ovulation requires the degradation and rupture of healthy tissue at the surface of the ovary. During the first decades of the 20th century, several investigators suggested that a proteolytic enzyme might exert some digestive action on the resisting tissues to cause rupture of the follicle [1, 2]. In 1964, several other investigators used widely divergent methods to demonstrate that rabbit follicles undergo some kind of degeneration that leads to an increase in the distensibility of the follicle wall during the ovulatory process [3, 4]. These data suggested that ovulation involves degradation of the two contiguous layers of 1 This work was presented at the 32nd Annual Meeting of the Society for the Study of Reproduction held at Pullman, Washington, during the summer of Supported by NSF Grant # (L.L.E.), by a Grant from The Lalor Foundation, Providence, Rhode Island (L.L.E.), and by NIH Grant HD (J.S.R.). 3 Correspondence. FAX: ; lespey@trinity.edu Received: 13 September First decision: 13 October Accepted: 11 November by the Society for the Study of Reproduction, Inc. ISSN: collagenous connective tissue, namely the tunica albuginea and the theca externa, at the apex of mature follicles. To test this idea, microliter quantities of the earliest preparations of collagenolytic enzymes were injected into the antrum of mature rabbit follicles, and the results demonstrated that such injections could cause follicles to rupture in as little as 1% of the usual amount of time that is required for normal ovulation [5]. Subsequent tests with collagenase and several other protease preparations revealed that such enzyme activity could significantly reduce the tensile strength of the sow follicle wall [6]. Collectively, these observations led to the collagenolytic enzyme theory of ovulation. In the past three decades, there has been substantial progress in collagenase research, and this new science has led to the classification of a number of different collagenolytic metalloproteinases. As more and more members of this family of enzymes have been discovered, there has been an increase in the effort to assess their potential involvement in the degradation of collagenous connective tissue in ovulatory follicles [7 25]. The present study provides information about a rather different type of metalloproteinase that is a novel candidate in the search for a cardinal collagenase in the mechanism of ovulation. A disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS- 1) was isolated by the random reverse transcription (RT)- polymerase chain reaction (PCR) method known as differential display. This recently discovered [26] member of the ADAM family of enzymes is uniquely expressed in follicular granulosa cells at the time of ovulation. MATERIALS AND METHODS Animal Tissue and Animal Injections Immature Wistar rats (selected from litters in our breeding colony) were induced to superovulate by ecg and hcg treatment as described previously [27]. Ovarian mrna was extracted from these animals at the periovulatory intervals of 0, 2, 4, 8, 12, and 24 h after hcg. These nucleic acid extracts were used for differential display and for Northern blotting. Epostane and indomethacin were also injected s.c., as described previously [27]. These antiovulatory agents were administered at 3 h after hcg in doses of 5.0 mg and 1.0 mg, respectively. To overcome the inhibitory effect of epostane, progesterone was administered s.c. in two separate doses of 5 mg each at 3 h before and 2 h after hcg. The ovulation rate in the various experimental animals was also determined by a procedure that has been described previously [27]. Differential Display Protocols The steps of the differential display were carried out as described previously [27]. In brief, RNA was extracted by

2 EXPRESSION OF ADAMTS-1 IN OVULATION 1091 FIG. 1. Autoradiograph of differentially displayed ADAMTS-1 cdna (arrows). Note that the cdna is not visible in the 0- and 2-h RT-PCR product, and the greatest amplification was at 12 h. a standard guanidine isothiocyanate/cesium chloride procedure. RT and PCR amplification were performed using an RNAimage Kit (G507; GenHunter Corporation, Nashville, TN). The specific primer set that yielded differentially expressed cdna for ADAMTS-1 was 5 -HTTTTTTTTTG- 3 and 5 -HTCGAATC-3, in which H represents a HindIII restriction site attached to the primers. After extraction and reamplification of the differentially expressed cdna, a standard Northern analysis was performed to confirm the ovulation-specific expression of the parent mrna for ADAMTS-1. The unique cdna fragment was cloned using a pcr-trap Cloning System (P404; GenHunter), and a cloning colony containing the ADAMTS-1 cdna was identified by secondary Northern analysis. Manual sequencing of the cdna was performed using a Sequenase Version 2.0 DNA Sequencing Kit (US70770; Amersham Pharmacia Biotech, Inc., Piscataway, NJ). In situ hybridization was performed as described previously [27]. Statistical Analysis Densitometric analysis of the intensity of the signals from the Northern blots were analyzed by the National Institutes of Health (NIH) image program as described previously [27]. Numerical data are presented as means SE. The significances of the differences were determined by Duncan s multiple-range tests after a completely randomized one-way ANOVA of the means of the groups. The probability value used to determine significance was P RESULTS Differential Display of ADAMTS-1 cdna During the Ovulatory Process After RT-PCR, the subpopulations of radioactively labeled cdnas that were generated from RNA extracts at FIG. 2. Intensity of Northern blot signals at the six intervals of the periovulatory period following hcg administration. The signal density at 8 h was arbitrarily set at 100%, and the other points on the graph represent the mean values from five different Northern analyses. An actual Northern analysis of the ADAMTS-1 cdna, along with its -actin control, is shown below the graph. Note that the greatest intensity was at 12 h after hcg. each of the six stages of the periovulatory period were separated from one another by electrophoresis on a polyacrylamide gel. The autoradiograph of these PAGE results revealed differentially expressed cdna bands that were conspicuous at 8, 12, and 24 h after hcg but were not visible at 0, 2, or 4 h into the ovulatory process (Fig. 1). Therefore, the most intense cdna band (i.e., the band in the 12-h lane) was excised from the acrylamide gel and reamplified for use as a probe in Northern analysis. Northern Analysis of ADAMTS-1 mrna Expression During Ovulation The Northern blot analysis revealed a pattern of mrna expression during ovulation that was essentially identical to the differential display autoradiograph (Fig. 2). By a discretionary precedent in our laboratory, the intensity of the signal from the 8-h lane was arbitrarily set at 100%, and the densities at the other points during the periovulatory period were expressed as fractions of that maximum. Accordingly, the NIH image program was used to digitize all of the bands on the Northern blots, and the ratio of the density of each experimental band to its corresponding actin control band was calculated for each lane. On the basis of five Northern blots, the signal densities at 0, 2, 4, 8, 12, and 24 h after hcg were 0%, 4.4% 1.4%, 19.0% 2.4%, 100%, 129.3% 9.2%, and 71.7% 6.4%, respectively. Thus, ADAMTS-1 gene expression was at a maximum at 12 h into the ovulatory process (when follicles first begin to rupture), and this expression declined significantly during early luteal formation. Sequence of the cdna Fragment for ADAMTS-1 After the ovulation-specific expression of the ADAMTS- 1 gene had been confirmed by Northern analysis, the cdna

3 1092 ESPEY ET AL. FIG. 3. Comparison of the percentage of signal from Northern blots containing RNA extracted at 8 h after hcg from animals that were also treated, 3 h after hcg, with either 5 mg of epostane (Epo) or 1 mg of indomethacin (Indo). One group of rats received epostane plus progesterone (E P 4 ) as described in the Materials and Methods. Bar graphs are based on NIH image analyses of four different Northern analyses. The signal from the 8-h control lane was arbitrarily set at 100% optical density. In parallel groups of rats, the ovulation rate was determined at 24 h after hcg. fragment of this gene was cloned and sequenced. The National Center for Biotechnology Information (NCBI) accession number for this fragment is #AF The cdna fragment is identical to a segment of a gene (NCBI accession #AF149118) that has been cloned recently from ischemic cerebral tissue in the rat. Also, the fragment is homologous with a gene (NCBI accession #AB001735) cloned from mouse. FIG. 4. Changes in intensity of the in situ hybridization ADAMTS-1 signal during the six periovulatory intervals following hcg administration. Lightfield micrographs on the left show the histology of ovarian sections stained with hematoxylin and eosin, while the darkfield micrographs of the same sections show the localization of ADAMTS-1 mrna as detected by hybridization of an 35 S-labeled antisense probe derived from the ADAMTS-1 cdna. Original magnification approximately 8 (published at 76%). Effects of Epostane and Indomethacin on ADAMTS-1 Gene Expression For these tests, Northern blots were prepared from RNA that was extracted from control ovaries at 0 and 8 h into the ovulatory process, or extracted from experimental ovaries that were taken at 8 h after hcg from rats that had been treated 5 h earlier with ovulation-inhibiting doses of epostane or indomethacin. As in the Northern blotting tests at the six different intervals during ovulation, the signal density (normalized against the -actin control) of the 8-h lane was arbitrarily set at 100% (Fig. 3). There was no detectable expression of ADAMTS-1 mrna at 0 h, but substantial expression at 8 h. In animals treated with the antiovulatory agent epostane, which blocks progesterone synthesis, the signal density of % was significantly below the 8-h control value. However, when 10 mg of exogenous progesterone was administered to the animals before epostane administration, the expression of ADAMTS-1 mrna recovered to % of the control value, and the ovulation rate in a parallel group of animals was close to normal (Fig. 3). In contrast, animals treated with the antiovulatory agent indomethacin, which blocks prostanoid synthesis, had an unimpaired ADAMTS- 1 mrna level of %, which was not significantly different from the 8-h control level. Localization of ADAMTS-1 mrna Expression by In Situ Hybridization In situ hybridization confirmed the temporal pattern of ADAMTS-1 mrna expression that was observed in the differential display autoradiograph and the Northern analysis. There was negligible signal at 0 2 h into the ovulatory process, a strong signal at 8 12 h after hcg, and a substantial, but waning, signal at 24 h (Fig. 4). Hybridization

4 EXPRESSION OF ADAMTS-1 IN OVULATION 1093 FIG. 5. Closer view of the distribution of probe in the thecal connective tissue of the ovary. Black arrows pointing to the left in the 8-h lightfield hematoxylin and eosin micrograph indicate atretic-like follicles. The black arrows projecting from the hub point to a number of small follicles that did not express ADAMTS-1 mrna. The white arrows pointing to the right in the 8- h in situ darkfield micrograph show strong ADAMTS-1 signal in the granulosa layer of large follicles on the ovarian periphery. Original magnification approximately 18 (published at 69%). was localized in the granulosa layer of the follicles (Fig. 5). Large, atretic-like follicles exhibited negligible signal. Likewise, a number of smaller follicles located mainly in the center of the ovaries did not show any ADAMTS-1 mrna expression. Most of the other smaller-looking follicles that did exhibit hybridization had thicker granulosa layers indicating that these probably were large mature follicles that happened not to be sectioned on a plane close to their maximum diameter. DISCUSSION The ADAM family of metalloproteinases forms a large group of cell surface proteins that combine features of both cell surface adhesion molecules and proteases [28 30]. Members of the family share a metalloproteinase domain, a disintegrin domain, a cysteine-rich region, and an epidermal growth factor repeat. There are at least 23 full-length ADAM sequences of clones from mammals, frogs, worms, and flies that are now registered in public databases [29]. The proposed functions of these proteases include matrix degradation, cell migration, and localized shedding of various proteins from membrane-anchored precursors. The most intensively studied shedding event is the release of tumor necrosis factor- (TNF- ) from its membrane-bound precursor during a variety of inflammatory reactions that can cause severe local damage [29, 31, 32]. In this case, the specific metalloproteinase was initially called TNF- converting enzyme (TACE), but now it is commonly referred to as ADAM-17. ADAMTS-1 is a newly discovered species of metalloproteinase that is quite unlike the original ADAM family proteins [26, 33 37]. In addition to metalloproteinase and disintegrin domains, it possesses three thrombospondin type-1 (TS-1) motifs toward its carboxyl terminal. Furthermore, it does not have a transmembrane segment at the carboxyl terminal to anchor it to the cell membrane. Instead, it is secreted from cells, and its TS-1 motifs bind firmly to the extracellular matrix through interaction with sulfated glycosaminoglycans such as heparan sulfate [34]. Also, the gene for ADAMTS-1 is distinct from previously identified ADAM genes in that it is located in region C3- C5 of chromosome 16, and its exon/intron organization is atypical [33]. On the other hand, this gene shares one significant characteristic with other ADAM genes it is expressed in acute inflammatory processes that are induced by interleukin-1 in vitro and by i.v. administration of lipopolysaccharide in vivo [26, 30, 33, 34]. The evidence that ADAMTS-1 mediates the degradative events of inflammation is of special interest because the ovulatory process has been likened to an acute inflammatory reaction [38]. In addition, the temporal pattern of expression of this gene supports the idea that it has a significant role in ovulation. Maximum transcription was at 12 h after hcg, which coincides with the time that follicles first begin to rupture in the immature rat model. The lingering expression of the gene during the early hours of luteinization is not surprising, since the enzyme could contribute to the tissue remodeling that occurs during the rapid transformation of a follicle into a corpus luteum. It has been firmly established that follicular rupture requires the synthesis of ovarian progesterone [39], as well as the presence of progesterone receptors [40]. Therefore, the dependency of ADAMTS-1 gene expression on ovarian progesterone synthesis is further evidence that the enzyme from this gene is important in the mechanism of ovulation. On the other hand, the failure of indomethacin to block ADAMTS-1 gene expression suggests that ovarian prostanoid synthesis is not a requirement for this enzyme activity. This finding indicates that ovarian ADAMTS-1 is not regulated in a manner comparable to that of other metalloproteinases reported to be involved in ovulation [13, 19]. Also, the ADAM proteases are not inhibited by tissue inhibitor of metalloproteinase-1 (TIMP-1) [29, 41]. Therefore, if it turns out that ADAMTS-1 is also unaffected by TIMP-1, this will serve as additional evidence this enzyme activity is regulated in a different way than the other TIMP-sensi-

5 1094 ESPEY ET AL. tive metalloproteinases that heretofore have been associated with ovulation [10, 11, 13, 23 25]. The present data show (in Fig. 3) that the ADAMTS-1 gene is expressed normally in rats that have been treated with indomethacin, an anti-inflammatory agent that is known for its ability to block ovarian prostaglandin synthesis and ovulation [39]. Thus, it is clear that the ADAMTS-1 enzyme, by itself, cannot cause ovulation. Additional protease activity and/or certain biophysical events in the ovary must also contribute to the mechanism of follicular rupture. One example of a biophysical requirement that could be affected by indomethacin is the intrafollicular pressure that serves as an essential hydrostatic force to cause ballooning of a proteolytically degraded follicle wall [39]. Since intrafollicular pressure is dependent on capillary hydrostatic pressure in the vicinity of a follicle, and since prostaglandin E is a well-known vasodilatory agent that affects local blood flow, and since indomethacin is a wellknown inhibitor of prostaglandin synthesis, it is feasible that indomethacin might inhibit ovulation by impairing the vascular supply to ovarian follicles. In other words, regardless of the extent to which a follicle wall is degraded by proteolytic activity such as that of ADAMTS-1, normal ovarian blood flow and intrafollicular pressure are essential for rupture of a follicle [39]. In the superovulated rats in this study, the in situ hybridization data shows that the ADAMTS-1 gene is expressed primarily in the granulosa layer of the larger follicles around the periphery of the ovarian mass. In contrast, the gene was not expressed in smaller follicles located toward the center of the ovaries, or in atretic-looking follicles. Manifestation of ADAMTS-1 gene expression in the granulosa layer indicates that the translated metalloproteinase is not comparable to any of a number of previously reported metalloproteinases in ovarian thecal tissue [13, 15, 20 22]. Nevertheless, production of ADAMTS-1 in the granulosa layer of a follicle can have a significant effect on the collagenous connective tissue in the outer layers of the follicle because there is ample evidence that small amounts of proteases that are injected into the follicular antrum can degrade the thecal layers and induce follicular rupture in a matter of minutes [5]. In conclusion, the gene for ADAMTS-1 is uniquely expressed in follicular granulosa cells at the time of ovulation. The temporal pattern of expression of this gene, along with its regulation by progesterone, suggests that the metalloproteinase from this gene could have an important role in the mechanism of ovulation. 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