HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) All Versions of this Article: 69/5/1506    most recent biolreprod.102.013714v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Madan, P. Articles by MacCalman, C. D. Search for Related Content PubMed PubMed Citation Articles by Madan, P. Articles by MacCalman, C. D. Agricola Articles by Madan, P. Articles by MacCalman, C. D.

Expression of Messenger RNA for ADAMTS Subtypes Changes in the Periovulatory Follicle after the Gonadotropin Surge and During Luteal Development and Regression in Cattle¹

Department of Obstetrics and Gynecology⁴ Faculty of Agricultural Sciences,⁵ University of British Columbia, Vancouver, British Columbia, Canada V5Z 4H4 Department of Biomedical Sciences,⁶ Cornell University, Ithaca, New York 14853

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Extensive remodeling of the extracellular matrix occurs in the ovary during the periovulatory period. Matrix metalloproteinases and their endogenous inhibitors, tissue inhibitors of metalloproteinases, are believed to play integral roles in this highly regulated series of cellular events, but their specific roles remain unclear. Recent cloning studies have identified a novel family of metalloproteinases, the ADAMTS (A Disintegrin And Metalloproteinase with ThromboSpondin motifs) family. The regulated expression of distinct ADAMTS subtypes has been shown to be required for tissue morphogenesis during embryonic development and for maintaining the integrity of tissues in the adult. In the present studies, we have determined that multiple ADAMTS subtypes are present in the bovine ovary using a reverse transcription-polymerase chain reaction strategy. In particular, ADAMTS-1, -2, -3, -4, -5 (also known as ADAMTS-11), -7, -8, and -9, but not ADAMTS-6, -10, or -12, mRNA transcripts were detected in granulosa cells of nonatretic ovarian follicles and corpora lutea. The levels of mRNA for these ovarian ADAMTS were up- or down-regulated or remained unchanged in the granulosa and/or theca cells of the dominant follicle following the preovulatory surge of gonadotropins, depending on the subtype and/or the cell compartment, and in the corpus luteum during the luteal phase of the estrous cycle. The complex expression patterns observed for the distinct ADAMTS subtypes in the granulosa and theca cells of the periovulatory follicle and in the luteal tissues of the bovine ovary suggest that these novel proteases mediate, at least in part, the remodeling events underlying folliculogenesis and ovulation and the formation, maintenance, and regression of the corpus luteum.

corpus luteum, follicle, granulosa cells, ovary, theca cells

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
The preovulatory surge of gonadotropins triggers a myriad of morphological and biochemical changes within the dominant follicle that culminate in ovulation and subsequent formation of the corpus luteum (CL). This highly regulated series of interrelated developmental processes is mediated, at least in part, by the extensive remodeling of the ovarian extracellular matrix (ECM). In particular, ovulation is associated with the degradation of the follicular basement membrane and the fragmentation of the ECM at the apex of the follicle wall, resulting in release of the oocyte [1, 2]. The ovarian ECM is subject to further remodeling, both deposition and degradation, during the formation, maintenance, and regression of the CL, and in turn, this remodeling modulates proliferation, differentiation, and/or apoptosis in the distinct cell populations that constitute this dynamic tissue [2, 3].

Matrix metalloproteinases (MMPs) and their endogenous inhibitors, tissue inhibitors of metalloproteinases (TIMPs), are believed to play integral roles in the degradation of the follicular ECM during ovulation and to be operative in CL tissues throughout the luteal phase of the estrous cycle [4–6]. However, the specific roles of MMPs and TIMPs in these developmental processes remain poorly defined. Furthermore, the spatiotemporal expression of distinct MMPs and TIMPs in the ovary appears to vary among species [4–6]. In the bovine ovary, MMP-2 and -14 have been detected in the periovulatory follicle, whereas MMP-2 and -9 are expressed primarily in the CL [7, 8]. The activity of these ovarian MMP subtypes is regulated by the constitutive expression of TIMP-1 and -2 in the follicle and CL [7–10].

Recent cloning studies have identified a rapidly expanding family of novel metalloproteinases known as ADAMTS (A Disintegrin And Metalloproteinase with ThromboSpondin motifs) [11, 12]. The ADAMTS proteinases are characterized by four structural and functional domains: an amino terminal prodomain, a catalytic domain, a disintegrin-like domain, and an ECM-binding domain (composed of a central thrombospondin type 1 motif, a spacer region, and a variable number of thrombospondin-like motifs) at the carboxyl terminal of the mature protein. To date, 20 members of the ADAMTS family have been identified in vertebrates [13]. However, the majority of these ADAMTS subtypes have been characterized only at the structural level. Consequently, the biological functions of many of these novel proteases remain poorly understood.

Gene-knockout studies have provided useful insight regarding the important biological roles that members of the ADAMTS gene family play in embryonic development, tissue morphogenesis, and reproduction. Mice that are null-mutant for ADAMTS-1 exhibited growth retardation and aberrant development of the kidneys, adrenals, and urogenital tract [14]. In addition, fewer mature follicles were formed in the ovaries of these mice, supporting previous observations that suggested a role for ADAMTS-1 in folliculogenesis [15, 16]. Similarly, ADAMTS-2 gene-knockout mice developed structural defects in their skin [17]. Although folliculogenesis was normal in these mice, testicular function was compromised, suggesting that ADAMTS-2 plays a key role in spermatogenesis. To date, the expression and function of other members of the ADAMTS gene family in the ECM remodeling events that occur in the ovary during each estrous cycle have not been characterized. In view of these observations, we have identified the ADAMTS subtypes present in the granulosa cells of small and large bovine follicles, in the theca and granulosa cells of the dominant follicle before and after the preovulatory surge of gonadotropins, and in CL tissues obtained at different stages of the luteal phase.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Collection of Bovine Follicles and CL Tissues

Ovaries were collected from Holstein cows at a local abattoir within 10–20 min of exsanguination and transported to the laboratory in sterile saline at 32–37°C.

Small (3–6 mm) or large (12–16 mm) nonatretic follicles, identified using the criteria described by Kruip and Dieleman [18], were aspirated through an 18-gauge needle into a 10-ml syringe containing follicular aspiration medium (PBS supplemented with 0.3% BSA and 50 µg/ml of gentamicin). The aspirates containing granulosa cells and the oocyte-cumulus complex were then centrifuged at 200 x g for 10 min and washed with follicular aspiration medium. This process was repeated twice before the ovarian cell pellets were finally collected for total RNA extraction.

The CL tissues were classified as early (stage I), mid (stage II), or late (stage III) luteal phase by macroscopic examination of the ovaries and the corresponding uterine tissues [19]. The CL were excised from the ovaries, cut in half through the papilla, and sliced into pieces representative of this complex tissue (1 g wet wt). The tissues were then snap-frozen for later extraction of total RNA.

Production of Bovine Embryos In Vitro

Small pools of bovine embryos (n = 25–30) were produced using a previously described in vitro fertilization protocol [20]. Briefly, cumulus-oocyte complexes were aspirated from small follicles and cultured in Tissue Culture Media 199 (TCM 199) (Sigma Aldrich, St. Louis, MO) containing 5% Superovulated Cow Serum (SCS) (Sigma) and supplemented with FSH (0.01 µg) and antibiotics (50 µg/ml of gentamicin) at 38.5°C for 24 h. Approximately 30 of these matured oocytes were then incubated with 100 µl of bull semen, diluted to a final concentration of 5 x 10⁶ sperm/ml with Brackett and Oliphant medium, for a further 16–18 h [21]. The fertilized oocytes were washed twice with culture medium to remove excess sperm; placed in a four-well culture dish containing TCM 199 supplemented with 5% SCS, 5 µg/ml of insulin, and 50 µg/ml of gentamicin; and allowed to undergo development into morulae or blastocysts before being harvested for total RNA extraction.

Isolation of Bovine Granulosa and Theca Cells from Periovulatory Follicles

Holstein heifers with regular estrous cycles were used in accordance with procedures approved by Cornell University's Animal Care and Use Committee. An experimental protocol that we have described and validated previously [22] was used to obtain follicles before or after the preovulatory gonadotropin surge. Briefly, heifers (n = 2–3/group) were injected with prostaglandin F₂ (25 mg of Lutalyse; Pharmacia and Upjohn Co, Kalamazoo, MI) on the evening of Day 6 of the estrous cycle (Day 0 = day of estrus) to regress the CL and thereby induce the follicular phase and further differentiation of the dominant follicle of the first follicular wave of the cycle. Injection of a GnRH analogue (100 µg of Cystorelin i.m.; Sanofi Animal Health, Inc., Overland Park, KS) 36 h later induces an LH/FSH surge, and ovulation follows at approximately 29 h after GnRH [22]. In the present study, the ovary bearing the preovulatory follicle was removed by colpotomy 36 h after injection of prostaglandin F₂ (i.e., time 0 after GnRH injection) or 24 h after injection of GnRH (before ovulation). The ovaries were examined daily by transrectal ultrasonography, and blood samples were collected before and during the experimental protocol to verify the progression of luteal regression and follicular development in response to the treatments.

The ovary was transported to the lab (10 min), where the preovulatory follicle was dissected from the ovary and the follicular fluid was removed by aspiration. In some experiments, the follicle wall (i.e., theca interna + attached granulosa cells) was isolated and cut into small pieces. In other experiments, theca and granulosa cells were separated by dissection, as described previously [23]. The theca interna was cut into small pieces, and the granulosa cells were collected by centrifugation. Pieces of follicle wall and theca interna and the granulosa cell pellet were snap-frozen for later extraction of total RNA.

Isolation of Total RNA and Generation of First-Strand cDNA

Total RNA was extracted from the ovarian tissues and cells and from bovine embryos at timed stages of development using the phenol-chloroform method of Chomczynski and Sacchi [24]. The total RNA extracts were then treated with deoxyribonuclease-1 to eliminate possible contamination with genomic DNA. To verify the integrity of the RNA, aliquots of the total RNA extracts were electrophoresed in a 1% (w/v) denaturing agarose gel containing 3.7% formaldehyde, and the 28S and 18S rRNA subunits were visualized by ethidium bromide staining. The purity and concentration of the total RNA present in each of the extracts were determined by optical densitometry (260/280 nm) using a Du-64 ultraviolet-spectrophotometer (Beckman Coulter, Mississauga, ON, Canada).

Aliquots (1 µg) of the total RNA extracts prepared from the ovarian tissues and cells or the preimplantation embryos were reverse transcribed into cDNA using a First-Strand cDNA Synthesis Kit according to a protocol recommended by the manufacturer (Amersham Pharmacia Biotech, Oakville, ON, Canada).

Primer Design and Preparation of cDNA Probes

Nucleotide sequences specific for human ADAMTS-1 through -12, which were determined to be conserved in the mouse and/or rat homologues deposited in GenBank, were identified using the BLAST (Basic Local Alignment Search Tool) computer program (National Center for Biotechnology Information, Bethesda, MD). Forward and reverse oligonucleotide primers corresponding to these DNA sequences and primers specific for the bovine 18S rRNA subunit, which served as an internal control for the present studies, were synthesized at the NAPS Unit, University of British Columbia. The specific nucleotide sequences of these primers, the optimized polymerase chain reaction (PCR) conditions for each of these primer sets, and the expected sizes of the PCR products are listed in Table 1.

To confirm the specificity of the primers, PCR was performed on three separate occasions using the specific ADAMTS primer sets and first-strand cDNA generated from total RNA extracts (n = 6) prepared from small or large ovarian follicles. Total RNA extracts (n = 6) prepared from the small pools of bovine embryos were used as positive controls for the present studies, because in other species, the levels of mRNA for the distinct ADAMTS subtypes have been found to be higher in embryos than in normal adult tissues [25, 26]. The resultant PCR products were subcloned into the PCR II vector by blunt-end ligation (Invitrogen, Carlsbad, CA) and subjected to nucleotide sequence analysis using an automated DNA sequence analyzer (Applied Biosystems, Foster City, CA) employing Taq DiDeoxy reagents (Perkin Elmer Life and Analytical Sciences Inc., Boston, MA). These clones were subsequently used to generate cDNA probes specific for each of the bovine ADAMTS subtypes identified in the follicles and/or preimplantation embryos and the 18S rRNA subunit using standard molecular biology techniques.

Semiquantitative PCR

Semiquantitative PCR was performed using the primer sets specific for ADAMTS-1 through -12 or the 18S rRNA subunit, and template cDNA generated from the total RNA extracts prepared from the tissues and cells obtained from the dominant follicle, before and after the preovulatory surge of gonadotropins or CL tissues obtained during the early, mid, or late luteal phase of the estrous cycle. The PCR cycles were repeated 20–40 times to determine a linear relationship between the yield of PCR products from representative samples of these ovarian tissues and cells and the number of cycles performed. The optimized numbers of cycles used to amplify the distinct ADAMTS subtypes and the 18S rRNA subunit from these ovarian cells and tissues are listed in Table 1.

Each experimental PCR reaction was performed on three separate occasions. Also, PCR was performed using the primer sets specific for the distinct ADAMTS subtypes and aliquots of total RNA extracts prepared from the ovarian tissues or cells (i.e., nontranscribed RNA) or diethyl pyrocarbonate-treated water under the same conditions as described above. These PCR reactions, which served as negative controls, did not yield any PCR products, confirming the purity of the total RNA extracts used in the present studies (data not shown).

Southern Blot Analysis

Aliquots (20 µl) of the PCR products generated from the ovarian tissue samples and cells or bovine embryos were separated by electrophoresis in a 1.2% agarose gel and visualized by ethidium bromide staining. The gels were then denatured with 0.5 M NaOH for 5 min, neutralized with 1 M Tris-HCl for 5 min, and transferred onto a charged nylon membrane (Hybond⁺, Amersham Canada Ltd., Oakville, ON).

The Southern blots were probed with a radiolabeled cDNA specific for each of the distinct ADAMTS subtypes or 18S rRNA according to the methods of MacCalman et al. [27]. The blots were then washed twice with 2x SSPE (20x SSPE consists of 0.2 M sodium phosphate, pH 7.4, containing 25 mM EDTA and 3 M NaCl) at room temperature, twice with 2x SSPE containing 0.1% SDS at 55°C, and twice with 0.2x SSPE at room temperature. The blots were subjected to autoradiography to detect the hybridization of the radiolabeled probes to the PCR products. The resultant autoradiograms were then scanned using a laser densitometer (Scion Corporation, Frederick, MD), and the absorbance values obtained for each of the distinct ADAMTS PCR products normalized relative to the corresponding 18S rRNA absorbance value.

Statistical Analysis

The results are presented as the relative absorbance value (mean ± SEM) obtained using the PCR products generated from the ovarian tissues or cells harvested from different animals (n = 2–3), with each PCR determination being replicated on three separate occasions. Statistical differences between stages of follicular or luteal development were assessed by ANOVA. Significant differences between the means were determined using the Fisher protected least significant difference test. Differences were considered to be significant at P 0.05.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Characterization of the ADAMTS Subtypes Present in Bovine Ovarian Follicles

In the total RNA extracts prepared from the granulosa cells of small and large ovarian follicles, ADAMTS-1, -2, -3, -4, -5 (also known as ADAMTS-11), -7, -8, and -9 mRNA transcripts were detected (Fig. 1). In contrast, ADAMTS-6 and -10 mRNA transcripts were not identified in the bovine granulosa cells but were detected in the total RNA extracts prepared from our positive control, preimplantation bovine embryos (Fig. 1). In the bovine ovarian cells or embryos, ADAMTS-12 mRNA transcripts were not detected by reverse transcription-PCR using multiple sets of primers (data not shown), including those used to detect this ADAMTS subtype in human and murine tissues and cells [26]. Nucleotide sequence analysis of the PCR products generated from the ovarian follicles and/or bovine embryos demonstrated that these cDNA fragments exhibited high sequence homology (86–100%) to the human and/or murine homologues deposited in GenBank.

View larger version (39K): [in this window] [in a new window]   FIG. 1. Characterization of the ADAMTS mRNA transcripts present in bovine ovarian follicles. Shown are representative autoradiograms of Southern blots containing PCR products generated using template cDNA synthesized from total RNA extracted from small or large ovarian follicles (lanes SF and LF, respectively) and primers specific for ADAMTS-1 through-11 or the 18S rRNA subunit. Template cDNA synthesized from bovine morulae or blastocysts served as positive controls for the present studies, and mRNA transcripts for ADAMTS-6 or -10, which were not detected in the follicles, were readily detectable in these tissues. The sizes of the distinct PCR products are shown to the left. bp, Base pairs

Effects of the Gonadotropin Surge on Levels of mRNA for ADAMTS Subtypes in Preovulatory Follicles

The repertoire of ADAMTS subtypes identified in the granulosa cells of small and large follicles was maintained in the follicle wall samples obtained from the dominant follicle (granulosa and theca cells) before and after the preovulatory surge of gonadotropins induced by the administration of GnRH (Fig. 2). Significant increases were detected in ADAMTS-1, -2, -3, and -9, and concomitant decreases in ADAMTS-5, -7, and -8, mRNA levels in the dominant follicle 24 h after the administration of GnRH (Figs. 2 and 3). In contrast to the other ADAMTS subtypes, ADAMTS-4 mRNA levels in the periovulatory follicle remained relatively constant, at least during the time points examined in the present studies.

View larger version (38K): [in this window] [in a new window]   FIG. 2. Characterization of the ADAMTS subtypes present in the preovulatory follicle. Shown are representative autoradiograms of Southern blots containing PCR products generated using template cDNA synthesized from total RNA extracted from the preovulatory follicle at 0 or 24 h after the administration of GnRH and primers specific for ADAMTS-1, -2, -3, -4, -5, -7, -8, or -9 (A–H, respectively) or 18S rRNA (I). The sizes of the distinct PCR products are shown to the left. bp, Base pairs

View larger version (22K): [in this window] [in a new window]   FIG. 3. Relative levels of the distinct ADAMTS mRNA transcripts in periovulatory follicles. Autoradiograms of Southern blots containing PCR products generated using template cDNA synthesized from total RNA extracts prepared from the preovulatory follicle at 0 or 24 h after GnRH was administered to induce the preovulatory gonadotropin surge and primers specific for the ADAMTS subtypes identified in these ovarian tissues or the 18S rRNA subunit were scanned using a laser densitometer. The absorbance values obtained for the distinct ADAMTS subtypes were then normalized to the absorbance value obtained for the 18S rRNA. The results are presented as the mean ± SEM (n = 2–3 follicles). Asterisks indicate significant differences between the means (P 0.05)

Effects of the Gonadotropin Surge on Levels of mRNA for ADAMTS Subtypes in the Granulosa and Theca Cells of Preovulatory Follicles

The preceding results using tissue samples of the follicle wall indicated that the gonadotropin surge induces specific changes in the levels of mRNA for most of the ADAMTS subtypes examined in the present studies. Therefore, the levels of the mRNA transcripts encoding these ovarian ADAMTS subtypes were next examined in granulosa and theca cells to localize these changes to specific cellular compartments.

The ADAMTS subtypes identified in the ovarian follicles were found to be present in both granulosa and theca cells isolated from the dominant follicle during the periovulatory period (Fig. 4). In agreement with our findings using tissue samples of the follicle wall, the preovulatory surge of gonadotropins significantly increased ADAMTS-1 mRNA levels in granulosa and theca cells isolated from the dominant follicle but decreased the levels of the mRNA transcripts encoding ADAMTS-7 and -8 in both of these ovarian cell types (Figs. 4 and 5). Similarly, GnRH had no significant effect on ADAMTS-4 mRNA levels in granulosa or theca cells, at least at the time points examined in the present studies. However, levels of ADAMTS-2 and -5 mRNA levels were higher in granulosa cells 24 h after GnRH, whereas levels of ADAMTS-2 were unchanged and levels of ADAMTS-5 had decreased in theca cells at 24 h post-GnRH. In contrast, the preovulatory surge of gonadotropins had no significant effect on ADAMTS-3 mRNA levels in granulosa cells but significantly increased the levels of this mRNA transcript in theca cells. Finally, a decrease was observed in ADAMTS-9 mRNA levels in granulosa cells, and a concomitant increase in the levels of the mRNA transcript encoding this ADAMTS subtype in theca cells, 24 h after the administration of GnRH.

View larger version (67K): [in this window] [in a new window]   FIG. 4. ADAMTS mRNA levels in granulosa and theca cells isolated from periovulatory follicles. Shown are representative autoradiograms of Southern blots containing PCR products generated using template cDNA synthesized from total RNA extracted from granulosa cells (GC) or theca cells (TC) isolated from the preovulatory follicle at 0 or 24 h after the administration of GnRH and primers specific for ADAMTS-1, -2, -3, -4, -5, -7, -8, or -9 (A–H, respectively) or the 18S rRNA subunit (I). The sizes of the distinct PCR products are shown to the left. bp, Base pairs

View larger version (28K): [in this window] [in a new window]   FIG. 5. Relative levels of the distinct ADAMTS mRNA transcripts present in granulosa and theca cells isolated from preovulatory follicles. Autoradiograms of Southern blots containing PCR products generated using template cDNA synthesized from total RNA extracts prepared from granulosa cells (GC) or theca cells (TC) isolated from the preovulatory follicle at 0 or 24 h after the administration of GnRH and primers specific for the ADAMTS subtypes identified in these two cell types or the 18S rRNA subunit were scanned using a laser densitometer. The absorbance values obtained for the distinct ADAMTS subtypes were then normalized to the absorbance value obtained for the 18S rRNA. The results are presented as the mean ± SEM (n = 2–3 follicles). Asterisks indicate significant differences between the means (P 0.05)

Effects of the Stage of the Luteal Phase on Levels of mRNA for ADAMTS in CL Tissue

In CL tissues, ADAMTS-1, -2, -3, -4, -5, -7, -8, and -9, but not ADAMTS-6, -10, or -12, mRNA transcripts were detected at all stages of the luteal phase (Fig. 6). Levels of ADAMTS-1, -7, and -8 mRNA were highest in CL tissues obtained during the early stage of the luteal phase (Figs. 6 and 7). A significant and progressive decline was observed in the levels of these mRNA transcripts in CL tissues as the estrous cycle entered the mid and late stages of the luteal phase. In contrast, ADAMTS-3 and -4 mRNA levels in CL tissues increased as the luteal phase progressed, with maximum levels being observed in the late-stage CL. Levels of both ADAMTS-5 and -9 mRNA were observed to increase between the early and mid stages of the luteal phase. However, levels of the mRNA transcripts encoding these two ADAMTS subtypes in CL tissues subsequently declined as the estrous cycle entered the late luteal phase. In contrast to the other ADAMTS subtypes, only small fluctuations in the levels of ADAMTS-2 mRNA transcripts were observed in CL tissues obtained at different stages of the luteal phase.

View larger version (61K): [in this window] [in a new window]   FIG. 6. Characterization of the ADAMTS subtypes present in CL tissues. Shown are representative autoradiograms of Southern blots containing PCR products generated using template cDNA synthesized from total RNA extracted from CL tissues obtained during the early (stage I), mid (stage II), or late (stage III) luteal phase and primers specific for ADAMTS-1, -2, -3, -4, -5, -7, -8, or -9 (A–H, respectively) or the 18S rRNA subunit (I). The sizes of the distinct PCR products are shown to the left. bp, Base pairs

View larger version (25K): [in this window] [in a new window]   FIG. 7. Relative levels of the distinct ADAMTS mRNA transcripts present in CL tissues obtained at different stages of the luteal phase of the estrous cycle. Autoradiograms of Southern blots containing PCR products generated using template cDNA synthesized from total RNA extracted from CL tissues obtained during the early (stage I), mid (stage II), or late (stage III) luteal phase and primers specific for the ADAMTS subtypes identified in these ovarian tissues or the 18S rRNA subunit were scanned using a laser densitometer. The absorbance values obtained for the distinct ADAMTS subtypes were then normalized to the absorbance value obtained for the 18S rRNA. The results are presented as the mean ± SEM (n = 3 CL/stage). Superscript letters indicate significant differences between the means (P 0.05)

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Multiple ADAMTS subtypes were detected in bovine ovarian follicles. This repertoire of ADAMTS subtypes was further localized to both the granulosa and theca cells of the large periovulatory follicle and was found to be maintained in CL tissues throughout the luteal phase. In agreement with our observations, ADAMTS-1 and -2, but not ADAMTS-12, mRNA transcripts have been previously detected in total RNA extracts prepared from adult human, mouse, and/or rat ovaries [15, 17, 26]. In addition to these ADAMTS subtypes, we have determined that mRNA transcripts encoding ADAMTS-3, -4, -5, -7, -8, and -9 are present in the bovine ovary. In contrast, ADAMTS-6 or -10 mRNA transcripts were not detected in any of the bovine ovarian tissues or cells examined in the present studies but were readily detectable in our positive control, preimplantation bovine embryos. To our knowledge, the presence of these ADAMTS subtypes in the ovaries of other species has not been determined. In addition, the present results show that levels of the mRNA transcripts encoding these ovarian ADAMTS subtypes are up- or down-regulated or remain unchanged in the dominant follicle after the gonadotropin surge or in CL tissues obtained at different stages of the luteal phase.

The biological significance of the distinct expression patterns of the ADAMTS subtypes observed in the bovine ovary remains to be elucidated. However, the preovulatory surge of gonadotropins is believed to decrease the expression of ovarian genes involved in folliculogenesis and, simultaneously, to increase the levels of those involved in ovulation and luteinization [1–3, 28]. In view of these observations, it is tempting to speculate that the ADAMTS subtypes identified in the ovarian follicles play distinct roles in folliculogenesis and ovulation. In particular, the presence of ADAMTS-5, -7, -8, or -9 mRNA in small and large follicles and the subsequent decline in the levels of these mRNA transcripts in granulosa and/or theca cells following the preovulatory surge of gonadotropins suggests that these ADAMTS subtypes may be involved in the ECM remodeling events underlying the structural and functional maturation of the dominant follicle. In contrast, the increase in ADAMTS-1, -2, and -5 mRNA levels in the granulosa cells and of ADAMTS-1, -3, and -9 mRNA levels in the theca cells of the dominant follicle following the preovulatory surge of gonadotropins suggests that these ADAMTS may mediate, at least in part, the degradation of the follicle wall during ovulation and/or the dissolution of the granulosa cell basement membrane, a necessary prelude to formation of the CL. However, a similar increase in ovarian ADAMTS-1 mRNA levels was observed in GnRH-primed rats treated with indomethacin, an anti-inflammatory agent capable of inhibiting ovulation [15], and mice that are null-mutant for the ADAMTS-1 gene are capable of ovulating [14]. Similarly, female mice that are null-mutant for ADAMTS-2 have normal ovarian function [17]. Taken together, these observations suggest that an increase in the expression of ADAMTS-1 or -2 in the dominant follicle during the periovulatory period is neither necessary nor sufficient to mediate ovulation and that more than one ADAMTS subtype may serve the same function, at least in the ovary [17]. Such redundancy in the regulation of reproductive processes is common. The biological significance of the increase in ADAMTS-3, -5, and -9 mRNA levels in the granulosa or theca cell layers of the periovulatory bovine follicle remains unclear, but their up-regulation after the gonadotropin surge suggests potential roles for these ADAMTS subtypes, either alone or in combination with ADAMTS-1 and -2, in the ovarian ECM remodeling events underlying ovulation.

Changes in levels of the mRNA for ADAMTS subtypes were also detected in bovine CL tissues obtained at different stages of the luteal phase, suggesting that these novel proteases may also play key integral roles in the formation and organization of this dynamic tissue. Remodeling of the ECM in CL tissues throughout the luteal phase not only modulates the biochemical differentiation of luteal cells but also promotes the formation and organization of a complex vascular network [5–7]. After ovulation, extensive growth of blood vessels occurs in bovine CL tissues, and this growth peaks at the midstage of the luteal phase [29, 30]. These blood vessels subsequently undergo regression during luteolysis. Several of the ADAMTS subtypes identified in the bovine CL have been shown to have angioinhibitory and/or angiogenic activity both in vivo and in vitro. In particular, ADAMTS-1 and -8 inhibit endothelial cell proliferation and are capable of reducing growth factor-induced vascularization of tissues in vitro [31, 32]. However, ADAMTS-1 appears to be necessary for development of the adrenomedullary capillary network in vivo [14], suggesting that ADAMTS subtypes may have differential effects on angiogenesis that are tissue-specific. The decrease in ADAMTS-1 and -8 mRNA levels in the CL during progression of the luteal phase suggests that these two ADAMTS subtypes have antiangiogenic activities in this dynamic tissue. Increased ADAMTS-5 expression has also been detected in the cells surrounding blood vessels in osteoarthritic synovium and is believed to be responsible for the extensive degradation of the surrounding ECM associated with the onset of this disease [33], whereas ADAMTS-4 expression has been associated with the metabolism of vascular proteoglycans during endothelial tube formation in vitro [34]. Although the increase in ADAMTS-4 mRNA levels in CL tissues during the late luteal phase suggests a role for this ADAMTS subtype in luteolysis, the angiogenic activity of bovine luteal tissues in vitro has been shown to increase with the age of the CL [35]. To date, it is not clear whether the ADAMTS subtypes identified in the bovine CL exhibit angiogenic and/or angioinhibitory activities in this highly vascularized tissue at different stages of the luteal phase. However, the present results provide information on the pattern of expression of mRNA for the ADAMTS subtypes that can serve as the basis for future studies of their localization and functions in the CL.

The preovulatory surge of gonadotropins regulated the levels of the distinct ADAMTS mRNA transcripts present in the granulosa and theca cells in either a coordinate or noncoordinate manner. Levels of the mRNA transcripts encoding some of the ADAMTS subtypes increased (ADAMTS-1) or decreased (ADAMTS-7 or -8) in both cell types after the gonadotropin surge, whereas other subtypes increased in one cell compartment but, in the other, either decreased (ADAMTS-5 or -9) or remained constant (ADAMTS-2 or -3). These observations suggest that the regulation of ADAMTS subtypes in the ovary is complex and involves the activation/inhibition of local regulatory factors that may act in an autocrine and/or paracrine manner. To date, the factors capable of regulating ADAMTS expression in mammalian tissues and cells remain poorly characterized. Transforming growth factor ß₁ has been shown to increase ADAMTS-12 mRNA levels in human fetal fibroblasts [26] and the secretion of active ADAMTS-2 in human osteosarcoma cells in vitro [36]. In addition, interleukin-1 is capable of increasing ADAMTS-4 mRNA levels in primary cultures of human tendon cells [37], whereas lipopolysaccharides increase levels of the mRNA transcript encoding ADAMTS-1 in renal and cardiac tissues of adult mice in vivo [38]. The ability of the progestin synthesis inhibitor, epostane, to inhibit the preovulatory increase in ADAMTS-1 mRNA levels in rat follicles [15], which was also not observed in mice that are null-mutant for the progesterone receptor [16], has provided indirect evidence that progesterone is a key regulator of this ADAMTS subtype in the ovary. A similar increase in ADAMTS-1 mRNA levels was observed in the preovulatory bovine follicle. Interestingly, the levels of the mRNA transcript encoding this ADAMTS subtype were found to be high in early stage CL tissues, when progesterone levels are low, and, subsequently, to decline during the midluteal phase of the estrous cycle, when the circulating levels of this gonadal steroid are high [39], suggesting that other factors are involved in the regulation of ADAMTS-1 mRNA levels, at least in the CL.

The ECM of the preovulatory follicle undergoes extensive remodeling, both degradation and deposition, during the periovulatory period and the subsequent formation and organization of the CL. In particular, these two interrelated developmental processes are associated with the loss of collagen type IV and laminin and with concomitant increases in collagen type I and fibronectin 4 [40]. To date, the specific substrates of many ADAMTS subtypes have not been identified. However, ADAMTS-1, -4, and -5 have been shown to degrade the large chondroitin-sulfate glycoproteins, aggrecan, brevican, and versican [41–43]. Although aggrecan and brevican have not been detected in bovine ovarian follicles, versican has been localized to the thecal layers, particularly in areas adjacent to the follicular basal lamina [44]. Procollagen I and II have also been identified as substrates for ADAMTS-1 and -2 and for ADAMTS-2 and -3, respectively [45, 46]. The accumulation of collagen type I in the CL that occurs during the luteal phase [4] may thus be attributed, at least in part, to the progressive decline in ADAMTS-1 and -2 mRNA levels observed in this tissue during the luteal phase.

In summary, we have determined that mRNAs for multiple ADAMTS subtypes are present in the bovine ovary. Although two of these subtypes (ADAMTS-1 and -2) have been detected previously in rodent ovaries, to our knowledge this is the first report to provide evidence for the expression of mRNA for ADAMTS-3, -4, -5, -7, -8, and -9 in mammalian ovaries. Because the levels of the mRNA transcripts encoding these distinct ADAMTS subtypes are differentially expressed in the granulosa and theca cell layers of the dominant follicle following the administration of GnRH and in CL tissues during the luteal phase, it is tempting to speculate that members of this novel family of proteases are involved in the ECM remodeling events required for ovulation and/or the formation, maintenance, and regression of the bovine CL. Elucidation of the specific roles and regulation of the various ADAMTS subtypes in bovine and other mammalian follicles and corpora lutea will require further experimentation.

	ACKNOWLEDGMENTS

 
C.D.M. is a career investigator of the BC Research Institute for Children's and Women's Health.

	FOOTNOTES

 
¹ Supported by a grant from the NIH (HD41592 to J.E.F.) and a Lalor Foundation fellowship (P.J.B.).

² Correspondence: Colin D. MacCalman, BC Research Institute for Children's and Women's Health, Room I3091-950, West 28th Avenue, Vancouver, BC, Canada V5Z 4H4. FAX: 604 875 3120; cdmaccalman{at}hotmail.com

³ Current address: Department of Animal Science, Iowa State University, 2356F Kildee Hall, Ames, IA 50011-3150

Received: 20 November 2002.

First decision: 18 December 2002.

Accepted: 8 May 2003.

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 

1. Richards JS, Russell DL, Robker RL, Dajee M, Alliston TN. Molecular mechanisms of ovulation and luteinization. Mol Cell Endocrinol 1998 145:47-54[CrossRef][Medline]
2. Richards JS, Robker RL, Russell D, Sharma CS, Espey LE, Lyndon J, O'Malley BS. Ovulation, a multi-gene, multi-step process. Steroids 2000 65:559-570[CrossRef][Medline]
3. Richards JS. The ovarian follicle—a perspective in 2001. Endocrinology 2001; 142:2184–2193
4. Silvester LM, Luck MR. Distribution of extracellular matrix components in the developing ruminant corpus luteum: a wound repair hypothesis for luteinization. J Reprod Fertil 1999 116:187-198[Abstract]
5. Smith MF, McIntush EW, Ricke WA, Kojima FN, Smith GW. Regulation of ovarian extracellular matrix remodeling by metalloproteinases and their tissue inhibitors: effects on follicular development, ovulation and luteal function. J Reprod Fertil Suppl 1999 54:367-381[Medline]
6. Fata JE, Ho TV, Leco KJ, Moorehead RA, Khokha R. Cellular turnover and extracellular matrix remodeling in female reproductive tissues: functions of metalloproteinases and their inhibitors. Cell Mol Life Sci 2000 57:77-95[CrossRef][Medline]
7. Curry TE Jr, Osteen KG. Cyclic changes in the matrix metalloproteinase system in the ovary and uterus. Biol Reprod 2001 64:1285-1296[Abstract/Free Full Text]
8. Bakke LJ, Dow MP, Cassar CA, Peters MW, Pusley JR, Smith GW. Effect of the preovulatory gonadotropin surge on matrix metalloproteinase (MMP)-14, MMP-2, and tissue inhibitor of metalloproteinases-2 expression within bovine preovulatory follicular and luteal tissue. Biol Reprod 2002 66:1627-1634[Abstract/Free Full Text]
9. Goldberg MJ, Moses MA, Tsang PCW. Identification of matrix metalloproteinases and metalloproteinase inhibitors in bovine corpora lutea and their variation during the estrous cycle. J Anim Sci 1996 74:849-857[Abstract]
10. Smith GW, Juengel JL, McIntush EW, Youngquist RS, Garverick HA, Smith MF. Ontogenies of messenger RNA encoding tissue inhibitor of metalloproteinases 1 and 2 within bovine periovulatory follicles and luteal tissue. Domest Anim Endocrinol 1996 13:151-160[CrossRef][Medline]
11. Tang BL, Hong W. ADAMTS: a novel family of proteases with an ADAM protease domain and thrombospondin 1 repeats. FEBS Lett 1999 445:223-225[CrossRef][Medline]
12. Tang BL. ADAMTS: a novel family of extracellular matrix proteases. Int J Biochem Cell Biol 2001 33:33-44[CrossRef][Medline]
13. Cal S, Obaya AJ, Llamazares M, Garabaya C, Quesada V, Lopez-Otin C. Cloning, expression analysis, and structural characterization of seven novel human ADAMTSs, a family of metalloproteinases with disintegrin and thrombospondin-1 domains. Gene 2002 283:49-62[CrossRef][Medline]
14. Shindo T, Kurihara H, Kuno K, Yokoyama H, Wada T, Kurihara Y, Imai T, Wang Y, Ogata M, Nishimatsu H, Moriyama N, Oh-hashi Y, Morita H, Ishikawa T, Nagai R, Yazaki Y, Matsushima K. ADAMTS-1: a metalloproteinase-disintegrin essential for normal growth, fertility, and organ morphology and function. J Clin Invest 2000 105:1345-1352[Medline]
15. Espey LL, Yoshioka S, Russell DL, Robker RL, Fujii S, Richards JS. Ovarian expression of a disintegrin and metalloproteinase with thrombospondin motifs during ovulation in the gonadotropin-primed immature rat. Biol Reprod 2000 62:1090-1095[Abstract/Free Full Text]
16. Robker RL, Russell DL, Espey LL, Lydon JP, O'Malley BW, Richards JS. Progesterone-regulated genes in the ovulation process: ADAMTS-1 and cathepsin L proteases. Proc Natl Acad Sci U S A 2000 97:4689-4694[Abstract/Free Full Text]
17. Li SW, Arita M, Fertala A, Bao Y, Kopen GC, Langsjo TK, Hyttinen MM, Helminen HJ, Prockop DJ. Transgenic mice with inactive alleles for procollagen N-proteinase (ADAMTS-2) develop fragile skin and male sterility. Biochem J 2001 355:271-278[CrossRef][Medline]
18. Kruip TAM, Dieleman SJ. Macroscopic classification of bovine follicles and its validation by micromorphological and steroid biochemical procedures. Reprod Nutr Develop 1982 22:465-473
19. Ireland JJ, Murphee RL, Coulson PB. Accuracy of predicting stages of bovine estrous cycle by gross appearance of the corpus luteum. J Dairy Sci 1980 63:155-160
20. Giritharan G, Rajamahendran R. In vitro embryo production using ovaries removed from culled cows. Can J Anim Sci 2001 81:589-591
21. Brackett BG, Oliphant G. Capacitation of rabbit spermatozoa in vitro. Biol Reprod 1975 12:260-274[Abstract]
22. Komar CM, Brendston AK, Evans AC, Fortune JE. Decline in circulating estradiol during the periovulatory period is correlated with decreases in estradiol and androgen, and in messenger RNA for P450 aromatase and P450 17-hydroxylase, in bovine periovulatory follicles. Biol Reprod 2001 64:1797-1805[Abstract/Free Full Text]
23. Fortune JE, Hansel W. The effects of 17ß-estradiol on progesterone secretion by bovine theca and granulosa cells. Endocrinology 1979 104:1834-1838[Medline]
24. Chomczynski P, Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 1987 162:156-159[Medline]
25. Hurskainen TL, Hirohata S, Seldin MF, Apte SS. ADAMTS-5, ADAMTS-6, and ADAMTS-7, novel members of new family of zinc metalloproteases. General features and genomic distribution of the ADAM-TS family. J Biol Chem 1999 274:25555-25563[Abstract/Free Full Text]
26. Cal S, Arguelles JM, Fernandez PL, Lopez-Otin C. Identification, characterization, and intracellular processing of ADAMTS12, a novel human disintegrin with a complex structural organization involving multiple thrombospondin-1 repeats. J Biol Chem 2001 276:17932-17940[Abstract/Free Full Text]
27. MacCalman CD, Bardeesy N, Holland PC, Blaschuk OW. Noncoordinate developmental regulation of N-cadherin, N-CAM, integrin, and fibronectin mRNA levels during myoblast terminal differentiation. Dev Dynam 1992 195:127-132[Medline]
28. Espey LL, Richards JS. Temporal and spatial patterns of ovarian gene transcription following an ovulatory dose of gonadotropin in rat. Biol Reprod 2002 67:1662-1670[Abstract/Free Full Text]
29. Zheng J, Redmer DA, Reynolds LP. Vascular development and heparin-binding growth factors in the bovine corpus luteum at several stages of the estrous cycle. Biol Reprod 1993 49:1177-1181[Abstract]
30. Modlich U, Kaup FJ, Augustin HG. Cyclic angiogenesis and blood vessel regression in the ovary: blood vessel regression during luteolysis involves endothelial cell detachment and vessel occlusion. Lab Invest 1996 74:771-780[Medline]
31. Vazquez F, Hastings G, Ortega MA, Lane TF, Oikemus S, Lombardo M, Iruela-Arispe ML. METH-1, a human ortholog of ADAMTS-1, and METH-2 are members of a new family of proteins with angio-inhibitory activity. J Biol Chem 1999 274:23349-23357[Abstract/Free Full Text]
32. Carpizo D, Iruela-Arispe ML. Endogenous regulators of angiogenesis-emphasis on proteins with thrombospondin-type I motifs. Cancer Metastasis Rev 2000 19:159-165[CrossRef][Medline]
33. Vankemmelbeke MN, Holen I, Wilson AG, Ilic MZ, Handley CJ, Kelner GS, Clark M, Liu C, Maki RA, Burnett D, Buttle DJ. Expression and activity of ADAMTS-5 in synovium. Eur J Biochem 2001 268:1259-1268[Medline]
34. Kahn J, Mehraban F, Ingle G, Xin X, Bryant JE, Vehar G, Schoenfeld J, Grimaldi CJ, Peale F, Draksharapu A, Lewin DA, Gerritsen ME. Gene expression profiling in an in vitro model of angiogenesis. Am J Pathol 2000 156:1887-1900[Abstract/Free Full Text]
35. Redmer DA, Grazul AT, Kirsch JD, Reynolds LP. Angiogenic activity of bovine corpora lutea at several stages of luteal development. J Reprod Fertil 1988 82:627-634[Abstract]
36. Wang WM, Lee S, Steiglitz BM, Scott IC, Lebares CC, Allen ML, Brenner MC, Takahara K, Greenspan DS. Transforming growth factor-beta induces secretion of activated ADAMTS-2: a procollagen III N-proteinase. J Biol Chem 2003 278:19549-19557[Abstract/Free Full Text]
37. Tsuzaki M, Guyton G, Garrett W, Archambault JM, Herzog W, Almekinders L, Bynum D, Yang X, Banes AJ. IL-1ß induces COX2, MMP-1, -3 and -13, ADAMTS-4, IL-ß and IL-6 in human tendon cells. J Orthop Res 2003 21:256-264[CrossRef][Medline]
38. Kuno K, Terashima Y, Matsushima K. ADAMTS-1 is an active metalloproteinase associated with the extracellular matrix. J Biol Chem 1999 274:18821-18826[Abstract/Free Full Text]
39. Tsang PCW, Poff JP, Boulton EP, Condon WA. Four-day-old bovine corpus luteum: progesterone production and identification of matrix metalloproteinase activity in vitro. Biol Reprod 1995 53:1160-1168[Abstract]
40. Bortolussi M, Zanchetta R, Doliana R, Castellani I, Bressan GM, Lauria A. Changes in the organization of the extracellular matrix in ovarian follicles during the preovulatory phase and atresia. An immunofluorescence study. Basic Appl Histochem 1989 33:31-38[Medline]
41. Tortorella M, Pratta M, Liu RQ, Abbaszade I, Ross H, Burn T, Arner E. The thrombospondin motif of aggrecanase-1 (ADAMTS-4) is critical for aggrecan substrate recognition and cleavage. J Biol Chem 2000 275:25791-25797[Abstract/Free Full Text]
42. Tortorella M, Malfait AM, Decicco CP, Arner E. The role of the ADAM-TS4 (aggrecanase-1) and ADAM-TS5 (aggrecanase-2) in a model of cartilage degradation. Osteoarthritis Cartilage 2001 6:539-552
43. Sandy JD, Westling J, Kenagy RD, Iruela-Arispe ML, Verscharen C, Rodriguez-Mazaneque JC, Zimmermann DR, Lemire JM, Fischer JW, Wight TN, Clowes AW. Versican V1 proteolysis in human aorta in vivo occurs at the Glu441-Ala442 bond, a site that is cleaved by recombinant ADAMTS-1 and ADAMTS-4. J Biol Chem 2001 276:13372-13378[Abstract/Free Full Text]
44. McArthur ME, Irving-Rodgers HF, Byers S, Rodger RJ. Identification and immunolocalization of decorin, versican, perlecan, nidogen, and chondroitin sulfate proteoglycans in bovine small antral follicles. Biol Reprod 2000 63:913-924[Abstract/Free Full Text]
45. Prockop DJ, Sieron AL, Li SW. Procollagen N-proteinase and procollagen C-proteinase. Two unusual metalloproteinases that are essential for procollagen processing probably have important roles in development and cell signaling. Matrix Biol 1998 7:399-408
46. Fernandes RJ, Hirohata S, Engle JM, Colige A, Cohn DH, Eyre DR, Apte SS. Procollagen II amino propeptide processing by ADAMTS-3: insights into dermatosporaxis. J Biol Chem 2001 276:31502-31509[Abstract/Free Full Text]

This article has been cited by other articles:

				K. R. Dunning, M. Lane, H. M. Brown, C. Yeo, R. L. Robker, and D. L. Russell Altered composition of the cumulus-oocyte complex matrix during in vitro maturation of oocytes Hum. Reprod., November 1, 2007; 22(11): 2842 - 2850. [Abstract] [Full Text] [PDF]

				S. Gao, C. De Geyter, K. Kossowska, and H. Zhang FSH stimulates the expression of the ADAMTS-16 protease in mature human ovarian follicles Mol. Hum. Reprod., July 1, 2007; 13(7): 465 - 471. [Abstract] [Full Text] [PDF]

				D. L. Russell and R. L. Robker Molecular mechanisms of ovulation: co-ordination through the cumulus complex Hum. Reprod. Update, May 1, 2007; 13(3): 289 - 312. [Abstract] [Full Text] [PDF]

				H. Zhu, P.C.K. Leung, and C.D. MacCalman Expression of ADAMTS-5/implantin in human decidual stromal cells: regulatory effects of cytokines Hum. Reprod., January 1, 2007; 22(1): 63 - 74. [Abstract] [Full Text] [PDF]

				J. Wen, H. Zhu, S. Murakami, P. C. K. Leung, and C. D. MacCalman Regulation of A Disintegrin And Metalloproteinase with ThromboSpondin Repeats-1 Expression in Human Endometrial Stromal Cells by Gonadal Steroids Involves Progestins, Androgens, and Estrogens J. Clin. Endocrinol. Metab., December 1, 2006; 91(12): 4825 - 4835. [Abstract] [Full Text] [PDF]

				P. J. Bridges, C. M. Komar, and J. E. Fortune Gonadotropin-Induced Expression of Messenger Ribonucleic Acid for Cyclooxygenase-2 and Production of Prostaglandins E and F2{alpha} in Bovine Preovulatory Follicles Are Regulated by the Progesterone Receptor Endocrinology, October 1, 2006; 147(10): 4713 - 4722. [Abstract] [Full Text] [PDF]

				M Shozu, N Minami, H Yokoyama, M Inoue, H Kurihara, K Matsushima, and K Kuno ADAMTS-1 is involved in normal follicular development, ovulatory process and organization of the medullary vascular network in the ovary J. Mol. Endocrinol., October 1, 2005; 35(2): 343 - 355. [Abstract] [Full Text] [PDF]

				J. S. Richards, I. Hernandez-Gonzalez, I. Gonzalez-Robayna, E. Teuling, Y. Lo, D. Boerboom, A. E. Falender, K. H. Doyle, R. G. LeBaron, V. Thompson, et al. Regulated Expression of ADAMTS Family Members in Follicles and Cumulus Oocyte Complexes: Evidence for Specific and Redundant Patterns During Ovulation Biol Reprod, May 1, 2005; 72(5): 1241 - 1255. [Abstract] [Full Text] [PDF]

				M. Shimada, M. Nishibori, Y. Yamashita, J. Ito, T. Mori, and J. S. Richards Down-Regulated Expression of A Disintegrin and Metalloproteinase with Thrombospondin-Like Repeats-1 by Progesterone Receptor Antagonist Is Associated with Impaired Expansion of Porcine Cumulus-Oocyte Complexes Endocrinology, October 1, 2004; 145(10): 4603 - 4614. [Abstract] [Full Text] [PDF]

				K. A. Young, B. Tumlinson, and R. L. Stouffer ADAMTS-1/METH-1 and TIMP-3 expression in the primate corpus luteum: divergent patterns and stage-dependent regulation during the natural menstrual cycle Mol. Hum. Reprod., August 1, 2004; 10(8): 559 - 565. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) All Versions of this Article: 69/5/1506    most recent biolreprod.102.013714v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal