Introduction

ABSTRACT

An increase in metallothionein-1 (MT-1) mRNA was detected in the ovaries of immature Wistar rats that were primed with 10 IU eCG, sc, followed 48 h later by 10 IU hCG, sc, to initiate the ovulatory process. Ovarian RNA was extracted at 0, 2, 4, 8, 12, 24, 72, 144, and 288 h after the primed animals were injected with hCG. These extracts were used for RT-PCR differential display and Northern analyses that yielded complimentary gene fragments for MT-1. Expression of MT-1 mRNA increased significantly by 24 h after hCG treatment of the animals and reached a peak at 144 h after hCG. In contrast, a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS-1) and a tissue inhibitor of metalloproteinase-1 (TIMP-1), which were also detected by the RT-PCR differential display procedure, reached a peak at 12 h after hCG and returned to control levels in the ovaries by 72 h after hCG. In situ hybridization indicated that the spatial pattern of MT-1 mRNA expression occurred in the vicinity of ovarian steroid synthesis in ovulatory follicles and post-ovulatory corpora lutea.

INTRODUCTION

Zinc, an essential component of cellular metabolism, is not readily available as a “free” ion. Instead, it is firmly attached to thousands of different proteins, usually by metal-thiolate bonding to various cysteine residues [1, 2]. Such bonding contributes to the structural configuration and the catalytic activity of zinc enzymes. To regulate the availability of zinc (and other metals) in cellular metabolism, there are a number of binding proteins that act transiently to chelate and release heavy metals during the enzymatic regulation of cellular homeostasis. Metallothionein (MT) is one such zinc-binding thiol that oftentimes represents the single most abundant intracellular protein thiol [3].

MT was first identified in 1957 as a non-enzymic cadmium-binding protein in the equine kidney [3, 4]. This small (6 to 7 kDA) metal-binding protein is present in essentially all eukaryotes, and has been found in some prokaryotes. However, in spite of more than four decades of investigation, and after more than 5,000 publications that discuss MT, the precise biological role(s) of this thiol compound remains uncertain [1, 5-7].

In the present study, we report the discovery by RT-PCR differential display of a substantial amount of MT-1 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 post-ovulatory corpora lutea for at least 12 days. The expression of ovarian mRNA for MT-1 was not affected by epostane or indomethacin, two inhibitors of ovulation that are known to block ovarian progesterone and prostaglandin synthesis, respectively. Since one of the hypothetical functions of MT is to regulate the activity of zinc-requiring enzymes such as metalloproteinases, this report also considers 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 at 12 h after induction of the ovulatory process and then declined during the onset of ovarian MT-1 expression. 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. The results indicate that MT-1 can serve as a ‘marker’ for ovarian luteal tissue, and that the luteinization process might be a useful model for assessment of the elusive function(s) of MT-1 in mammalian tissues.

MATERIALS AND METHODS

Animal Tissue and Animal Injections

Immature Wistar rats were induced to superovulate by injections of eCG and hCG as described previously [11]. Ovarian RNA was extracted initially at the peri-ovulatory intervals of 0, 2, 4, 8, 12, and 24 h after hCG. 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. Epostane (courtesy of Sanofi~Synthelab Research, Malvern, PA) and indomethacin (Sigma Chemical Company, St. Louis, MO) were injected s.c., also as described previously [11]. These anti-ovulatory agents were administered at 3 h after hCG in doses of 5.0 mg and 1.0 mg, respectively. The ovulation rate in the various experimental animals was determined by a procedure that also has been described previously [11]. 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.

Differential Display Protocols That Led to Detection of MT-1

The steps of the differential display procedure were carried out as described previously [11]. In brief, 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. After extraction and re-amplification of the differentially expressed cDNAs, standard Northern analyses were performed to confirm the ovulation-related expression of the parent mRNAs for MT-1, ADAMTS-1, and TIMP-1. Subsequently, the 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 Version 2.0 DNA Sequencing Kit (US70770, Amersham Pharmacia Biotech, Inc., Piscataway, NJ). In situ hybridization was performed as described previously [11].

Statistical Analysis

Densitometric analyses of the intensity of the signals from the Northern blots were performed by the NIH-image program, as described previously [11]. Numerical data are presented as means ± SEM. The significance of the differences among the principal time points of the various experimental groups were determined by Duncan's multiple range tests after a completely randomized one-way analysis of variance of the means of the groups. The probability value used as the cutoff between "significant" and "not significant" was P = 0.05.

RESULTS

Differential Display of MT–1 cDNA during the Peri-Ovulatory Period

Following RT-PCR, the sub-populations of radioactively labeled cDNAs that were generated from RNA extracts at each of the stages of the peri-ovulatory 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 (Figure 1). This uniquely-expressed cDNA band was excised from the 24-h lane of the acrylamide gel and re-amplified for use as a probe in Northern analysis.

Northern analysis of MT-1 mRNA expression during the peri-ovulatory period

A preliminary Northern analysis revealed a pattern of mRNA expression during the peri-ovulatory period that was similar to the pattern on the differential display autoradiograph (Figure 2). The strongest signal from the Northern blot was from hybridization with mRNA that had been extracted at 24 h after hCG. The NIH-image program was used to digitize all of the bands on the Northerns, and the ratio of the density of each experimental band to its corresponding b-actin control band was calculated for each lane. Since the most intense signal was from the 24-h RNA extract, this lane was arbitrarily set at 100%, and the densities at the other points during the peri-ovulatory period were expressed as fractions of that maximum. At 0, 2, 4, 8, 12, and 24 h after hCG, the relative intensities of the signals from this preliminary Northern were 38.9%, 11.1%, 17.1%, 24.5% 30.6% and 100%, respectively. Therefore, MT-1 gene expression was substantially elevated at 24 h after hCG treatment, and this interval was subsequent to the well-established time of 12 h after hCG when the follicles begin to rupture in the immature rat model.

Northern analysis of MT-1 mRNA expression in comparison to ADAMTS-1 and TIMP-1

More extensive Northern analyses revealed that the signal for MT-1 mRNA became even stronger during progression of the luteinization process that was induced by treatment of the immature rats with gonadotropin. Based on four Northern blots containing lanes of RNA extracted at 0, 4, 12, 24, 72, 144, 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 (Figure 3). Thus, the elevation in MT-1 mRNA persisted for at least 288 h after treatment of the animals with hCG to induce ovulation and luteinization. In contrast, the results from four Northern blots each revealed that the ovarian expression of ADAMTS-1 and TIMP-1 mRNA crested at 12 h after the injection of hCG, i.e., at the time when mature ovarian follicles begin to rupture (Figure 3). However, expression of these two metalloproteinase-related genes was decreasing by 24 h after hCG, 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 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 the cDNA fragments for MT-1, ADAMTS-1 and TIMP-1

After the gonadotropin-induced expressions of the MT-1, ADAMTS-1 and TIMP-1 genes had been confirmed by Northern analyses, the cDNA fragments of these genes were cloned and sequenced. The length of the sequence between the primers that yielded MT-1 was 215 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 (accession numbers M11797, M11794, M24327, and J00750) in the NCBI database four times, previously. The 246 bp cDNA fragment for ADAMTS-1 (NCBI accession number AF159096), which has been reported previously by our laboratory [8], is identical to two other entries for ADAMTS-1 (accession numbers NM_024400 and AF149118) in the NCBI database. The 501 bp cDNA fragment for TIMP-1 (NCBI accession AF411319) is equivalent to sequences of the same inhibitor (accession numbers 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 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 Northerns for the temporal pattern of expression (Figure 2), the signal density (normalized against b-actin controls) of the 24-h control lane was arbitrarily set at 100% (Figure 4). As observed previously, there was minimal expression of MT-1 mRNA at 0 h, but substantial expression at 24 h. In animals treated with the anti-ovulatory agent indomethacin, which blocks prostaglandin synthesis, the signal density for ovarian MT-1 mRNA was not significantly different from the 24-h control value. In animals treated with the anti-ovulatory agent epostane, which blocks progesterone synthesis, there was a slight, but statistically significant, elevation in MT-1 mRNA at 24 h after hCG. However, treatment of the animals with exogenous progesterone, to reverse the anti-ovulatory action of epostane, lowered the ovarian expression of MT-1 mRNA back to a level that was not significantly different from the 24-h control value (Figure 4). 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 progesterone were 10.8% ± 1.3%, 100%, 99.1% ± 5.7%, 132.4% ± 8.1%, and 108.4% ± 2.7%, respectively.

Localization of MT-1 mRNA expression by in situ hybridization

In situ hybridization confirmed the temporal pattern of MT-1 mRNA expression that was observed in the differential display autoradiograph and the Northern analysis at 0-24 h after hCG was administered to the animals. 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 at 24 h after hCG (Figure 5). A closer examination of the 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 (Figure 6). In addition, the closer view showed that MT-1 mRNA was also expressed in the cumulus cells around the oocyte by 12 h after hCG, when the follicles are about to enter the lutein phase.

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 genes was quite different from that of MT-1 mRNA. Transcription of the ADAMTS-1 gene was limited primarily to the granulosa layer of the larger follicles (Figure 7). However, transcription of the TIMP-1 gene occurred throughout the ovary, with greatest expression in the collagenous connective tissue that comprises the thecal layers of the follicles and some of the ovarian stromal tissue, while there was minimal expression in the granulosa cells (Figure 8).

DISCUSSION

Mammalian MTs are a group of low-molecular-weight proteins consisting of a single chain of 60 to 68 amino acid residues, including 20 highly conserved cysteines that together bind 7 zinc atoms, or bind other transition metals such as copper and cadmium, in vivo [2-6, 12, 13]. The MT proteins are dumb-bell-shaped molecules with two domains—one containing nine cysteines that bind 3 zinc atoms and another containing 11 cysteines that bind 4 zinc atoms [14]. It has been suggested that the physiological release of zinc from these domains is regulated by MT-bound ATP and by glutathione, which probably affect conformational changes in the MT molecules [2]. 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, namely MT-1, MT-2, MT-3, and MT-4 [2, 3, 6, 13]. 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, while the expression of MT-3 and MT-4 is more characteristic of the brain and the keratinizing epithelium of the skin, respectively [6, 13].

The amount of MT in any given tissue is regulated mainly at the transcriptional level [1]. 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, 3, 12]. 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 (MREs) that are sensitive to zinc and other metals, antioxidant-responsive elements (AREs) that are activated by H₂O₂ and other oxidative elements, and hormone-responsive elements (HREs) that react with glucocorticoids, progestins, and possibly other steroid hormones [3, 5, 6, 15-17]. 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 [1, 5, 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 [3].

It has been commonly assumed that proteins like 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 [2, 3]. However, to date, not a single essential function has 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 [3, 5, 6, 12]. Nevertheless, there are a number of hypothetical roles for MT proteins. These possible functions include (i) the sequestration and release of zinc and other essential metals as required by homeostasis [2, 3, 5], (ii) the regulation of zinc-dependent transcription factors and other metalloproteins [5, 6], (iii) the scavenging of free radicals [3-5, 12], (iv) the neutralization of hydroxy radicals [3, 7, 17], and (v) the protection of cells against metal toxification [3-5]. In addition, the MT proteins have been related to pathophysiological processes such as apoptosis, inflammation, suppression of the immune system, and tumorogenesis [1, 3, 7, 12, 18].

This background information leads to the question of the role(s) of MT-1 in the mammalian ovary during the peri-ovulatory period. We have 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 in the present study [19]. Therefore, since 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 expression 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 peri-ovulatory 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. It is possible that the MT-1 protein functions to divest the active ADAMTS-1 enzyme of the zinc cofactor that it needs to degrade follicles during ovulation and thereby promotes the post-ovulatory 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 to the rats. This is a period of time when it is known that the theca interna cells are actively synthesizing androgens that diffuse into the granulosa layer and are converted by cytochrome P450 aromatase into b-estradiol [20]. However, by 8 h after hCG treatment of the rats, transcription of the genes for both cytochromes P450 aromatase [21, 22] and P450 17a [20] 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 steroidogenesis associated regulatory protein and cytochrome P450scc [23], and this elevation in steroid secretion persists through the post-ovulatory 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.

Lastly, it has been established that the ovulatory process has biophysical and biochemical features that are characteristic of acute inflammatory reactions [24, 25]. Therefore, since MT-1 gene expression has been associated with inflamed tissues [26-32], the present evidence that MT-1 mRNA is transcribed in the mammalian ovary during the peri-ovulatory period is further evidence that ovulation and luteinization involve an inflammatory reaction. Furthermore, since luteal tissue has been likened to granuloma tissue that forms in association with chronically inflamed tissue [24, 33], it would be interesting to determine whether the MT-1 gene is also expressed in other models of granuloma tissue. In any event, the present study shows that MT-1 mRNA expression can serve as a useful marker to identify luteal tissue in the ovary, and that the corpus luteum, itself, is a novel experimental tissue that might be useful in the search for the elusive function(s) of MT-1.

ACKNOWLEDGEMENT

We appreciate the reliable assistance of Claire Lo in performing the in situ hybridization.

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LEGENDS

FIG. 1. Autoradiograph of differentially displayed MT-1 cDNA (arrow). Note that the cDNA band is more pronounced in the 24-h lane.

FIG. 2. Amount of ovarian MT-1 mRNA at six intervals of the peri-ovulatory period, as determined by a single, preliminary Northern analysis of the cDNA that was detected by differential display. The signal density at 24 h was arbitrarily set at 100%. The actual Northern blot of the MT-1 cDNA, along with its b-actin control, is shown below the linear graph. Note that the pattern of signal intensity is comparable to the RT-PCR amplification shown in FIG. 1.

FIG. 3. Comparison of signal for MT-1 mRNA with signals for ADAMTS-1 mRNA and TIMP-1 mRNA over the extended interval of 288 h after hCG administration. Each point on the linear graph represents the mean value tabulated from densitometric analyses of four Northerns prepared from two separate extractions of ovarian RNA obtained from pools of seven pairs of ovaries taken at each of the different time intervals. (Actual means and SEM values are specified in the Results section.)

FIG. 4. Comparison of the MT-1 mRNA signal from Northern blots with data on ovulation rate in parallel groups of animals that were treated with either 1 mg indomethacin (Indo) or 5 mg epostane (Epo), administered at 3 h after hCG. An additional group of animals was treated with epostane plus progesterone (E + P). Ovarian RNA was extracted at 24 h after hCG treatment because this was the first time that MT-1 mRNA was significantly elevated, and it was the shortest interval after treating the animals with the anti-ovulatory agents. The bar graphs that quantitate Northern blot data are based on NIH Image analyses of four different Northern blots that were prepared from one RNA extraction from experimental groups consisting of seven rats each. The signal from the 24-h control lane was arbitrarily set at 100% OD to compare the intensities of signals from the four different Northern blots. In parallel groups of rats, the ovulation rate was determined at the optimal time of 24 h after hCG by counting ova in the oviducts. "a", significantly (p < 0.001) different from 0-h control; "b", significantly (p < 0.05) different from the 24 h control.

FIG. 5. Change in intensity of the in situ hybridization signal for MT-1 mRNA during the first six peri-ovulatory intervals after hCG administration. Lightfield micrographs on the left show the histology of ovarian sections stained with hematoxylin and eosin, whereas the darkfield micrographs of the same sections show the localization of MT-1 mRNA as detected by hybridization of a ³⁵S-labeled antisense probe derived from the MT-1 cDNA. Note that one large follicle at 4 h and two large follicles at 8 h have begun ‘premature’ expression of the MT-1 gene. By 12 h after hCG, the MT-1 signal begins to appear faintly in the granulosa layer of the follicles. By 24 h, it is strong in the luteinizing granulosa layer. Magnification, ~X7.

FIG. 6. Closer view of the distribution of MT-1 mRNA probe in the ovary during the first 24 h after hCG administration. Note that the signal at 0-4 h is mostly from the thin layer of theca interna cells (arrows pointed right) that circumscribe the granulosa layer. The signal is dissipating from the theca interna by 8 h, but it becomes visible in the granulosa layer and cumulus cells (arrow pointed left) by 12-24 h after hCG. Magnification, ~X35.

FIG. 7. Spatial distribution of ADAMTS-1 mRNA that is localized in the granulosa layer of the larger antral follicles. Magnification, ~X50.

FIG. 8. Spatial distribution of TIMP-1 mRNA, emanating primarily from the thecal and stromal layers of the ovary. Note the absence of signal from the antral cavities of several large follicles and from the lumen of a cluster of ovarian blood vessels in the lower-right corner of the section. Magnification, ~X50.