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Ovary |
Department of Biology,3 Trinity University, San Antonio, Texas 78212
Department of Obstetrics and Gynecology,4 Kumamoto University Medical School, Kumamoto 860-8556, Japan
Department of Molecular and Cellular Biology,5 Baylor College of Medicine, Houston, Texas 77030
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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corpus luteum, follicle, ovary, ovulation, progesterone
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In the present study, we used reverse transcription polymerase chain reaction (RT-PCR) differential display to discover a substantial amount of MT-1 mRNA in ovarian luteal tissue of immature rats that ovulated following treatment with gonadotropins. The mRNA for this metal-binding protein increased significantly in the ovary within 24 h after the ovulatory process was induced by hCG, and it remained elevated in the postovulatory corpora lutea for at least 12 days. The expression of ovarian mRNA for MT-1 was not affected by indomethacin or epostane, two inhibitors of ovulation that are known to block ovarian prostaglandin (PG) and progesterone (P4) synthesis, respectively. Because one of the hypothetical functions of MT is to regulate the activity of zinc-requiring enzymes such as metalloproteinases, we also explored ovarian MT-1 in relation to the expression of a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS-1) and a tissue inhibitor of metalloproteinase (TIMP-1). Both ADAMTS-1 and TIMP-1 expression, which were also detected in the ovary by RT-PCR differential display, reached a peak 12 h after induction of the ovulatory process and then declined during the onset of ovarian MT-1 expression, i.e., at the time when ruptured ovarian follicles begin to transform into steroid-secreting corpora lutea. Ovarian expression of ADAMTS-1 [8] and TIMP-1 [9, 10] has been reported previously, but this is the first evidence for induction of the MT-1 gene following gonadotropic stimulation of a mammalian ovary. Considering the evidence that ovulation is an inflammatory-like process [11, 12] and the substantial evidence that MT protects tissues against the oxidative stress generated by inflammatory reactions [13–16], we concluded that ovarian MT-1 might function to modulate the degenerative aspects of ovulation and to promote a local environment supporting the transition of a disintegrated follicle into a steroid-secreting corpus luteum.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Immature Wistar rats were induced to superovulate by injections of eCG and hCG as described previously [17]. Animals weighing 40–50 g (i.e., approx 24–25 days old) were administered 10 IU of eCG s.c. to induce folliculogenesis. Subsequently, 48 h later, the ovulatory process was initiated by injecting 10 IU of hCG s.c. This treatment resulted in a superovulation rate of 
60 ruptured follicles/rat, with onset of ovulation occurring at 12–14 h after administration of hCG [18, 19]. Indomethacin (Sigma Chemical Co., St. Louis, MO) and epostane (courtesy of SanofiSynthelab Research, Malvern, PA) were injected s.c. also as described previously [17]. These antiovulatory agents were administered at 3 h after hCG in doses of 1.0 mg and 5.0 mg, respectively. The ovulation rate in the various experimental animals was determined by a procedure that also has been described previously [17]. For the determination of ovulation rate and the extraction of ovarian RNA, rats were killed by exposure to CO2. The work was conducted in accord with accepted standards of humane animal care, and the animals were handled in compliance with the NIH Guide and with the approval of the institutional committee on animal care.
Ovarian PGE2 and P4 Analysis
Ovaries for PGE2 and P4 assay were extirpated at 8 h after hCG treatment because at this time during the ovulatory process rat ovaries synthesize maximal amounts of these two ovulation-related agents [8]. The ovaries were stored frozen in 1.0 ml acetate buffer (pH 4.5) until there was adequate time for extraction and assay. The ovaries were homogenized for 30 sec using a Tissue Tearor homogenizer (Biospec Products, Bartlesville, OK). The homogenate was centrifuged at 5000 rpm for 10 min, and the supernatant fluid was pipetted into a fresh tube. This simple aqueous extract was used to assay both PGE2 and P4 according to instructions in commercial RIA kits for the prostanoid (NEK020; PerkinElmer, Boston, MA) and the steroid (TKPG1; Diagnostic Products, Los Angeles, CA). A reagent kit (P5656; Sigma) was used to perform protein assays on aliquots of each RIA sample. The amount of eicosanoid or steroid per sample was expressed as nanograms per milligram of protein in each sample.
Differential Display Protocols That Led to Detection of MT-1
The steps of the differential display procedure were carried out as described previously [17]. Ovarian RNA was extracted initially at the periovulatory intervals of 0, 2, 4, 8, 12, and 24 h after hCG injection. These initial extracts of nucleic acid were used for differential display and for Northern blotting. In subsequent experiments, the RNA was extracted at 0, 4, 12, 24, 72, 144, and 288 h after hCG for more extensive Northern blotting. RNA was extracted by a standard guanidine isothiocyanate/cesium chloride procedure. RT-PCR was performed using primers from RNAimage Kits (G505 and G508; GenHunter Corporation, Nashville, TN). The specific primer set that yielded differentially expressed cDNA for MT-1 was 5'-HTTTTTTTTTA-3' and 5'-HGCTGCTC-3', where H represents a HindIII restriction site attached to the primers. The poly-T primer for both ADAMTS-1 and TIMP-1 was 5'-HTTTTTTTTTG-3', and the random primers were 5'-HTCGAATC-3' and 5‘-HCGACGCT-3’, respectively. The amplified populations of radiolabeled cDNAs were separated by PAGE. Upon detection of a differentially expressed cDNA in the parallel lanes of the polyacrylamide gels, the unique cDNA was extracted from the gel and reamplified by PCR. After extraction and reamplification of the differentially expressed cDNAs, standard Northern analyses were performed to confirm the ovulation-related expression of the parent mRNAs that were eventually identified as transcripts for MT-1, ADAMTS-1, and TIMP-1. Subsequently, these unique cDNA fragments were individually cloned using a pCR-TRAP Cloning System (P404; GenHunter), and cloning colonies containing each of the three different cDNAs were identified by secondary Northern analyses. Manual sequencing of the cDNAs was performed using a Sequenase 2.0 DNA Sequencing Kit (US70770; Amersham Pharmacia Biotech, Piscataway, NJ). In situ hybridization was performed as described previously [17]. At each time point, three ovaries from different animals were sectioned and placed on the same slide for in situ hybridization. Sense controls were performed on the ovarian sections that were used for in situ hybridization. Further details about the step-by-step protocols for RNA extraction, differential display, PAGE, Northern blotting, cloning, and sequencing are available at www.trinity.edu/lespey/ddisplay/dd.html.
Statistical Analysis
Densitometric analyses of the intensity of the signals from the Northern blots were performed by the NIH-image program as described previously [17]. All analyses were conducted on densitometric data from Northern blots that were probed with cDNA obtained from individual cloning colonies that were also used for sequencing. Numerical data are presented as means ± SEM. The significance of the differences among the principal time points of the various experimental groups was determined by Duncan multiple range tests after a completely randomized one-way ANOVA of the means of the groups. Differences were considered significant at P < 0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Following RT-PCR, the subpopulations of radioactively labeled cDNAs that were generated from RNA extracts at each of the 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 (which was eventually determined to be a segment of the gene for MT-1) that was more conspicuous in the lane of PCR products representing mRNA extracted at 24 h after hCG (Fig. 1). This uniquely expressed cDNA band was excised from the 24-h lane of the acrylamide gel and reamplified for use as a probe in Northern analysis.
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Northern Analysis of MT-1 mRNA Expression During the Periovulatory Period
A preliminary Northern analysis revealed a pattern of MT-1 mRNA expression during the periovulatory period that was similar to the pattern on the differential display autoradiograph. The peak in expression was at 24 h after hCG administration, and this time point was subsequent to the well-established time of 12 h after hCG when the follicles begin to rupture [18, 19]. Because the most intense Northern signal was after ovulation, fresh sets of ovaries were extracted to include groups of postovulatory follicles that were in more advanced stages of luteinization. This more comprehensive Northern analysis revealed that the signal for MT-1 mRNA became even stronger with the progression of luteinization (Fig. 2). Because densitometric analysis showed that the intensity of the signal from the 144-h lane was the strongest, this lane was arbitrarily set at 100%, and the densities at the other times during the periovulatory period were expressed as fractions of that maximum. Based on four Northern blots containing lanes of RNA extracted at 0, 4, 12, 24, 72, 144, and 288 h after hCG administration, the intensities of the signals for MT-1 mRNA were 3.8% ± 1.2%, 5.2% ± 1.5%, 8.3% ± 3.3%, 46.6% ± 13.8%, 66.0% ± 1.6%, 100%, and 76.4% ± 16.9%, respectively. The elevation in MT-1 mRNA persisted for at least 288 h after treatment of the animals with hCG to induce ovulation and luteinization.
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Comparison of ADAMTS-1 and TIMP-1 mRNA Expression with MT-1 mRNA Expression
In contrast to the results for MT-1 mRNA, Northern blotting revealed that the ovarian expression of ADAMTS-1 and TIMP-1 mRNA peaked at 12 h after the injection of hCG, i.e., at the time when mature ovarian follicles begin to rupture (Fig. 2). However, expression of these two genes, which are functionally associated with metalloproteinase activity, was decreasing by 24 h after hCG, i.e., at the time when MT-1 mRNA was increasing in the ovaries. During the given time points, the relative intensities of the signals for ADAMTS-1 mRNA were 0.8% ± 0.4%, 33.6% ± 5.3%, 100%, 26.3% ± 8.8%, 2.1% ± 1.5%, 1.2% ± 0.5%, and 1.9% ± 0.7%, and those for TIMP-1 mRNA were 19.9% ± 6.0%, 85.7% ± 13.2%, 100%, 80.8% ± 18.4%, 12.0% ± 7.3%, 2.4% ± 1.7%, and 2.7% ± 2.2%, respectively. Thus, ADAMTS-1 and TIMP-1 mRNAs were negligible in the samples collected from luteinized ovaries during most of the luteal phase of the experiment.
Sequences of cDNA Fragments for MT-1, ADAMTS-1, and TIMP-1
After the gonadotropin-induced expression of the MT-1, ADAMTS-1, and TIMP-1 genes had been confirmed by secondary Northern analyses, the cDNA fragments of these genes were cloned further and sequenced. The length of the sequence between the primers that yielded MT-1 was 215 base pairs (bp). The National Center for Biotechnology Information (NCBI) accession number for this MT-1 fragment is AF411318. This cDNA fragment is essentially identical to a segment of the same gene from various rat tissues that have been registered (M11797, M11794, M24327, and J00750) in the NCBI database four times previously. The 246-bp cDNA fragment for ADAMTS-1 (NCBI AF159096), which has been reported previously [8], is identical to two other entries for ADAMTS-1 (NM_024400 and AF149118) in the NCBI database. The 501-bp cDNA fragment for TIMP-1 (NCBI AF411319) is equivalent to sequences of the same inhibitor (L29512, L31883, and U06179) that have been recorded in the database three times previously.
Effects of Indomethacin and Epostane on MT-1 Gene Expression
For these tests, Northern blots were prepared from RNA that was extracted from control ovaries at 0 and 24 h after treatment of the animals with hCG or was extracted from experimental ovaries that were taken at 24 h after hCG from rats that had been treated 21 h earlier with ovulation-inhibiting doses of indomethacin or epostane [8]. As in the Northern blots for the temporal pattern of expression (Fig. 2), the signal density (normalized against ß-actin controls) of the 24-h control lane was arbitrarily set at 100% (Fig. 3). As observed previously, there was limited expression of MT-1 mRNA at 0 h but substantial expression at 24 h. In animals treated with the antiovulatory agent indomethacin, which blocks PG synthesis, the signal density for ovarian MT-1 mRNA was not significantly different from the 24-h control value. In animals treated with the antiovulatory agent epostane, which blocks P4 synthesis, there was also a nonsignificant difference in MT-1 mRNA at 24 h after hCG. Likewise, treatment of the animals with exogenous P4 to reverse the antiovulatory action of epostane did not affect the ovarian expression of MT-1 mRNA (Fig. 3). Specifically, the relative values for MT-1 mRNA in the 0-h control, 24-h control, 24-h indomethacin, 24-h epostane, and 24-h epostane plus exogenous P4 were 16.2% ± 2.0%, 100%, 89.6% ± 4.5%, 104.8% ± 9.2%, and 91.3% ± 5.5%, respectively.
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Effects of Indomethacin and Epostane on PGE2, P4, and Ovulation Rate
To confirm the anticipated effects of indomethacin and epostane on ovarian PGE2, P4, and ovulation rate, parallel groups of animals were treated with these ovulation-inhibiting agents at 3 h after hCG administration, and their ovaries were analyzed at the most appropriate intervals after hCG. Ovulation was significantly (P < 0.001) inhibited by both indomethacin and epostane (Fig. 3), but treatment of the epostane-treated animals with exogenous P4 resulted in a complete recovery of the ovulation rate. The mean values for ovulation rate in the 0-h control, 24-h control, 24-h indomethacin, 24-h epostane, and 24-h epostane plus exogenous P4 groups were 0 ± 0, 54.3 ± 7.6, 5.7 ± 0.6, 0 ± 0, and 55.1 ± 3.7, respectively. Indomethacin but not epostane significantly inhibited ovarian PGE2 (P < 0.001) (Fig. 3). The mean values for PGE2 in the 0-h control, 24-h control, 24-h indomethacin, 24-h epostane, and 24-h epostane plus exogenous P4 groups were 1.1 ± 0.2, 57.9 ± 7.6, 0.9 ± 0.2, 55.1 ± 10.2, and 53.1 ± 8.9, respectively. In contrast, epostane but not indomethacin significantly inhibited ovarian P4 (P < 0.001), and the treatment of animals with exogenous P4 resulted in a predictable increase in the measurable P4 in the ovaries (Fig. 3). The mean values for P4 in the 0-h control, 24-h control, 24-h indomethacin, 24-h epostane, and 24-h epostane plus exogenous P4 groups were 14.7 ± 2.5, 289.6 ± 21.4, 317.7 ± 27.6, 52.5 ± 7.8, and 171.2 ± 22.0, respectively.
Localization of MT-1 mRNA Expression by In Situ Hybridization
In situ hybridization confirmed the temporal pattern of MT-1 mRNA expression observed in the differential display autoradiograph and the Northern analysis. There was limited signal at 0–12 h after hCG but a substantial increase in signal from the granulosa area of the developing corpora lutea beginning at 24 h after hCG (Fig. 4). By 72 h after hCG, the corpora lutea were clearly delineated by the signal from the MT-1 mRNA. A closer examination of ovarian sections revealed that the limited signal from the ovaries taken at 0–4 h after hCG was emanating primarily from the thin layer of theca interna cells just outside the granulosa layer of the larger follicles (Fig. 5). In addition, MT-1 mRNA was expressed by the granulosa cells of a few of the largest follicles in the 4-h and 8-h ovaries (Fig. 4).
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Comparison of In Situ Hybridization of MT-1 with the Patterns for ADAMTS-1 and TIMP-1
The spatial pattern of expression of the two metalloproteinase-related mRNAs was quite different from that of MT-1 mRNA. Localization of ADAMTS-1 mRNA was primarily in the granulosa layer of the larger follicles (Fig. 6). However, localization of TIMP-1 mRNA was greatest in the collagenous connective tissue of the thecal shell of the follicles (Fig. 7). Some areas of the ovarian stromal tissue also displayed intense transcription of the TIMP-1 gene. In contrast, granulosa cells of both large and small follicles displayed minimal expression of TIMP-1 mRNA (Fig. 7).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Mammalian MTs are a group of low-molecular-weight proteins consisting of a single chain of 60–68 amino acid residues, including 20 highly conserved cysteines that together bind seven zinc atoms or bind other transition metals such as copper and cadmium in vivo [2–6, 20, 21]. The MT proteins are dumbbell-shaped molecules with two domains: one containing 9 cysteines that bind three zinc atoms and another containing 11 cysteines that bind four zinc atoms [22]. The physiological release of zinc from these domains may be regulated by MT-bound ATP and by glutathione, which probably affect conformational changes in the MT molecules [3]. These evolutionarily preserved metal-binding proteins have been found in life forms as simple as yeast and as complex as humans, with four varieties of MT identified in the mouse and as many as 17 isoforms in the human. The family of isometallothioneins has been divided into four principal subgroups: MT-1, MT-2, MT-3, and MT-4 [1, 3, 6, 21]. The closely related MT-1 and MT-2 isoforms, which are ubiquitous in mammalian tissues, have been studied extensively in the liver, pancreas, intestine, and kidney, whereas the expression of MT-3 and MT-4 is more characteristic of the brain and the keratinizing epithelium of the skin, respectively [6, 21].
The amount of MT in any given tissue is regulated mainly at the transcriptional level [4]. Transcription of MT genes is usually very rapid and can be induced by zinc itself and by growth factors, cytokines, tumor necrosis factors, and steroid hormones such as glucocorticoids and progestins [1, 4, 20]. On a broader scale, MTs are also expressed in response to cytotoxic metals, inflammatory agents, and virtually any physical or chemical condition that generates oxidative stress. The promoter regions of all the MT genes contain multiple copies of metal-responsive elements that are sensitive to zinc and other metals, antioxidant-responsive elements that are activated by H2O2 and other oxidative elements, and hormone-responsive elements that react with glucocorticoids, progestins, and possibly other steroid hormones [1, 5, 6, 23–25]. In addition, the MT promoters have binding sites for the Sp1, AP-1, and AP-2 transcription factors that mediate the effects of growth factors and protein kinases on transcription processes [4–6]. After transcription, the MT mRNA is, reportedly, rather short lived [6], but the translated protein usually persists for a day or more [5], with degradation taking place mainly in lysosomes [1].
It has been commonly assumed that proteins such as the MTs, which are based on the enduring evolution of so many genes and such a variety of transcription regulators, must have important functions in fundamental homeostatic processes of living organisms [1, 3]. However, to date no essential functions have been established. To the contrary, removal of MT genes from mice does not impair normal development and reproduction, suggesting that there might be parallel systems to compensate for the loss of MT [1, 5, 6, 20]. Nevertheless, there are a number of hypothetical roles for MT proteins. These possible functions include 1) the sequestration and release of zinc and other essential metals as required by homeostasis [1, 3, 5], 2) the regulation of zinc-dependent transcription factors and other metalloproteins [5, 6], 3) the scavenging of free radicals [1, 2, 5, 20], 4) the neutralization of hydroxy radicals [3, 7, 8], and 5) the protection of cells against metal toxification [1, 2, 5]. The MT proteins also have been linked to pathophysiological processes such as apoptosis, inflammation, suppression of the immune system, and tumorogenesis [1, 4, 7, 20, 26].
This background information leads to the question of the role(s) of MT-1 in the mammalian ovary during the periovulatory period. In an earlier study, based on immunostaining for MT-1 protein in reproductive tissues, the protein was localized in luteal tissue of adult rats [27]. However, those authors did not relate MT-1 to LH/hCG stimulation of follicular tissue nor did they detect any day-to-day differences in the amount of MT-1 during the estrous cycle. The conclusion was that MT-1 might be associated with cell proliferation and differentiation that occurs in reproductive tissues such as the mammary glands, uterus, vagina, and ovary [25]. However, such a deduction does not explain the persistent expression of MT-1 mRNA in the fully developed corpora lutea that were examined in the present study.
We recently reported an increase in early growth response protein 1 (Egr-1) gene expression in the ovary following treatment of the immature rat with the same doses of gonadotropins as administered in the present study [28]. Therefore, because Egr-1 is a zinc-finger transcription factor, there is the possibility that MT-1 could be involved in the regulation of this transcription factor. However, ovarian Egr-1 mRNA reaches a peak at only 4 h after initiating the ovulatory process by hCG, and transcription of this gene is already declining notably by 12 h after hCG, i.e., before ovarian MT-1 mRNA concentration begins to increase. Therefore, it seems unlikely that the persistent ovarian expression of MT-1 during the lengthy luteal phase of the pseudopregnant immature rat has any significant role in the regulation of Egr-1 during the periovulatory period.
It may be relevant that MT-1 mRNA expression begins to increase in the ovary shortly after ovulation (which occurs at 12–14 h after hCG treatment), when transcription of the ADAMTS-1 gene starts to subside. The MT-1 protein may function to divest the active ADAMTS-1 enzyme of the zinc cofactor that it needs to degrade follicles during ovulation and thereby may promote the postovulatory healing process in the ovary. However, as pointed out above with regard to Egr-1 gene expression, such a hypothetical role for ovarian MT-1 does not explain the prolonged expression of this MT gene for at least 11 days beyond the time of ADAMTS-1 expression and ovulation. Furthermore, it would seem more likely that TIMP-1 mRNA, which increases throughout the ovary concurrent with the expression of ADAMTS-1, would be more pertinent to the regulation of this zinc-dependent metalloproteinase.
There appears to be some association between ovarian steroid synthesis and the expression of MT-1 mRNA. The present data show that this gene is expressed in the thin theca interna layer of large follicles at 0–4 h after the administration of hCG in rats. This period of time is when the theca interna cells are actively synthesizing androgens that diffuse into the granulosa layer and are converted by cytochrome P450 aromatase into 17ß-estradiol [29]. However, by 8 h after hCG treatment of these rats, transcription of the genes for both cytochromes P450 aromatase [30, 31] and P450 17
[29] has ceased. On the other hand, by this time the granulosa layer has begun producing substantial amounts of progesterone in response to the induction of the genes for steroidogenic acute regulatory protein and cytochrome P450scc [32], and this elevation in steroid secretion persists through the postovulatory luteal phase. Therefore, the present in situ hybridization patterns of MT-1 mRNA expression in the theca interna at 0–4 h after hCG and in the granulosa at 12–24 h after hCG indicate that the MT-1 signal is localized in the steroid-secreting cells in the ovaries.
The ovulatory process has biophysical and biochemical features that are characteristic of acute inflammatory reactions [11, 12, 33]. Proinflammatory cytokines such as interleukin-1 (IL-1) and tumor necrosis factor (TNF) are integral components of the ovulatory process [34, 35]. Therefore, it may be highly relevant that MT-1 and MT-2 have been recognized for more than two decades as response elements to inflammatory stress [36, 37]. These antioxidant zinc proteins reduce the expression of proinflammatory cytokines such as IL-1ß, IL-6, and TNF and promote angiogenesis and stabilization of tissues that are subject to oxidative stress and apoptotic degradation [13–16, 38–40]. Therefore, it appears that postovulatory expression of ovarian MT-1 might be a protective response that downregulates the local inflammatory reaction, facilitates the healing of ruptured tissue, promotes angiogenesis in the developing lutein tissue, and minimizes apoptotic events for the life of the corpus luteum.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence. FAX: 210 999 7229; lespey{at}trinity.edu
Received: 17 November 2002.
First decision: 30 November 2002.
Accepted: 13 December 2002.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, cytochrome P450scc and 3-hydroxy-3-methyl CoA reductase in developing rat ovarian follicles and corpora lutea: correlation with theca cell steroidogenesis. Biol Reprod 1987 37:211-223[Abstract] and apoptotic cell death during experimental autoimmune encephalomyelitis (EAE). Exp Neurol 2001 170:1-14[CrossRef][Medline]This article has been cited by other articles:
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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]
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