Expression of Regulator of G-Protein Signaling Protein-2 (RGS2) Gene

in the Rat Ovary at the Time of Ovulation¹

Takeshi Ujioka,² Darryl L. Russell,³ Hitoshi Okamura,⁴ JoAnne S. Richards,³ Lawrence L. Espey^2,5

Department of Biology,² Trinity University, San Antonio, Texas 78212; Department of Cell Biology,³ Baylor College of Medicine, Houston, Texas 77030; Department of Obstetrics and Gynecology,⁴ Kumamoto University School of Medicine, Kumamoto, Japan

Short Title: RGS2 expression in ovulation

¹Grant Support: This work was supported by NSF Grant #9870793 (L.L.E.), by a Grant to support T. Ujioka as a Research Fellow of The Lalor Foundation, Providence, Rhode Island (L.L.E.), and by NIH Grant HD-16229 (J.S.R.)

⁵Correspondence: Lawrence Espey, Ph.D.

Department of Biology

Trinity University

San Antonio, TX 78212

tel: (210) 999-7237

fax: (210) 999-7229

e-mail: lespey@trinity.edu

ABSTRACT

The ovulatory process in mammals begins when an endogenous surge in luteinizing hormone circulates to the ovary and couples with receptors in the plasma membranes of granulosa cells in mature ovarian follicles. This study provides evidence that the ovulatory stimulus includes induction of the gene for regulator of G-protein signaling protein-2 (RGS2). Immature Wistar rats were primed with 10 IU equine chorionic gonadotropin (eCG) s.c., and 48 h later the 12-h ovulatory process was initiated by 10 IU human chorionic gonadotropin (hCG, a homolog of luteinizing hormone) s.c.. Ovarian RNA was extracted at 0, 2, 4, 8, 12, and 24 h after injecting the animals with hCG. The RNA extracts were used for RT-PCR differential display to detect gene expression in the stimulated ovarian tissue. Two of the amplified cDNAs that were up-regulated within 2 h after the ovaries had been stimulated by hCG were homologous to segments of the mouse gene for RGS2. In situ hybridization indicated that the RGS2 mRNA was expressed in the granulosa layer of mature follicles. In conclusion, the gene for RGS2, which is known to regulate membrane signaling pathways, is transcribed in ovarian follicles in response to an ovulatory dose of gonadotropin.

INTRODUCTION

Mammalian ovulation is initiated by the coupling of a gonadotropic hormone such as luteinizing hormone to serpentine receptors that span the plasma membranes of follicular granulosa cells seven times [1-3]. Within moments after coupling of ligand to receptor, membrane-based signaling events initiate a cascade of biochemical reactions that last a species-specific number of hours and culminates in eruption of follicles at the time of ovulation. This ovulatory process involves a number of cellular activities that are initiated in association with the common zinc-finger transcription factor known as early growth response protein-1 (Egr-1) [4]. The target-cell response includes a marked elevation in progesterone (P₄) synthesis by the follicle [1] and the eventual expression of metalloproteinase(s) that degrade the follicular connective tissue and cause it to rupture [5].

It is well established that receptors containing seven membrane-spanning segments are coupled to heterotrimeric G-proteins that mediate signal transduction via their a as well as their bg subunits [6-11]. The binding of hormone to receptor leads to a conformational change in the receptor that induces a dissociation of guanosine diphosphate (GDP) from the a subunit.In rapid succession, the a subunit binds a GTP and separates from the bg heterodimer. Subsequently, both the a and the bg entities activate downstream components of the signal transduction process. However, the elicited response cannot proceed indefinitely. There must also be cellular components that diminish the signaling process. Recently, a superfamily of evolutionarily conserved regulators of G-protein signaling (RGS) have been characterized as GTPase-activating proteins (GAPs) that hydrolyze GTP to GDP on the a subunit of an activated G-protein [6-11]. This event causes the de-energized a subunit to reunite with the bg subunit and thereby terminate signaling by both of these G-protein entities. Thus, RGS proteins are now recognized as an important superfamily that regulates the duration and intensity of signal transduction processes. In addition, the RGS proteins serve not only as attenuators of G-protein signaling, it is now thought that they also orchestrate signaling pathways beyond the re-coupling of G-protein subunits [7, 10, 11]. That is to say, some RGS proteins may influence a multitude of other signaling pathways.

This report provides evidence that one member of the RGS superfamily, namely RGS2, is expressed in mature ovarian follicles of the rat in response to hormonal stimulation of the ovulatory process. RGS2 gene transcription was detected by RT-PCR differential display of ovarian RNA that was extracted at six intervals during the peri-ovulatory period. The temporal pattern of RGS2 mRNA expression was characterized by Northern blotting. The effects of ovarian P₄ and prostaglandin (PG) synthesis on RGS2 production were also assessed. In addition, the spatial distribution of ovarian RGS2 mRNA was determined by in situ hybridization. The results demonstrate a substantial increase in RGS2 gene expression in the granulosa layer of mature ovarian follicles in response to an ovulatory dose of human chorionic gonadotropin (hCG), a homolog of LH.

MATERIALS AND METHODS

Animal Tissue and Animal Injections

Immature Wistar rats were induced to superovulate by equine chorionic gonadotropin (eCG) and hCG treatment as described previously [12]. Ovarian RNA was extracted at the peri-ovulatory intervals of 0, 2, 4, 8, 12, and 24 h after hCG. These nucleic acid extracts were used for differential display and for Northern blotting. Epostane and indomethacin were injected s.c., also as described previously [12]. 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 [12]. For the determination of ovulation rate and the extraction of ovarian RNA, rats were killed by exposure to CO₂. The animals were acquired, retained, and used in compliance with the NIH Guide and with the approval of the institutional animal care review committee.

Differential Display Protocols That Lead to Detection of RGS2

The steps of the differential display were carried out as described previously [12]. In brief, RNA was extracted by a standard guanidine isothiocyanate/cesium chloride procedure. RT- PCR was performed using primers from an RNAimage Kit (G509, GenHunter Corporation, Nashville, TN). Two different primer sets yielded differentially expressed fragments of cDNAs for RGS2. One set was comprised of the poly-T primer 5'-HTTTTTTTTTG-3' and the random primer 5'-HTCAAAGA-3' (i.e., primer set G72), while the other set consisted of 5'-HTTTTTTTTTA-3' and 5'-HTCAAAGA-3' (i.e., primer set A72), with "H" representing the attachment of a HindIII restriction site to the primers. (Note that the random primers are the same, but the poly-T primers are different.) After extraction and re-amplification of the differentially expressed cDNAs, standard Northern analyses were performed to confirm the ovulation-specific expression of the parent mRNA for RGS2. The unique cDNA fragments were subcloned using a pCR-TRAP Cloning System (P404, GenHunter), and cloning colonies containing the RGS2 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 [12].

Statistical Analysis

The intensity of the signals from the Northern blots was analyzed by the NIH-image densitometry program, as described previously [12]. Numerical data are presented as means ± SEM. The significance of the differences among the six principal time points of 0, 2, 4, 8, 12, and 24 h after hCG was 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 RGS2 cDNAs During the Ovulatory Process

Following RT-PCR, the sub-populations of radioactively labeled cDNAs that were generated from RNA extracts at each of the six stages of the peri-ovulatory period were separated from one another by electrophoresis on polyacrylamide gels. The autoradiograph of these PAGE results revealed differentially expressed cDNA bands that were most evident at 4 and 8 h after hCG, but were minimal at 0 and 24 h into the ovulatory process (Figure 1). One set of primers (i.e., A72) amplified a differentially displayed cDNA that was relatively long (@ 900 bp), while another primer set (i.e., G72) amplified a smaller cDNA fragment (@ 270 bp) (Figure 1). In each case, the most intense cDNA band (i.e., the band in the 4-h lane) was excised from the two different acrylamide gels and re-amplified for use as probes in Northern analyses.

Northern Analyses of RGS2 mRNA Expression During Ovulation

The Northerns revealed an expression of mRNA (eventually identified as RGS2) during ovulation that was comparable to the expression of cDNA on the differential display autoradiograph (Figure 2). In order to compare the intensity of the signals from the Northern blots with other data on gene expression during ovulation, the intensity of the signal from the 8-h lane was arbitrarily set at 100%, and the densities at the other times during the peri-ovulatory period were expressed as fractions of that maximum. Accordingly, 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. Means (± SEM) of the signal densities at 0, 2, 4, 8, 12, and 24 h after hCG were 15.3% ± 4.1%, 55.0% ± 1.3%, 124.0% ± 9.6%, 100%, 55.4% ± 6.0%, and 16.8% ± 4.6%, respectively. Thus, RGS2 gene expression increased 8-fold within 4 h after initiation of the ovulatory process by injecting hCG into the animals. Subsequently, at 24 h after hCG (i.e., during early luteal development), RGS2 gene expression declined to a level that was not significantly different from the 0-h control value.

Sequence of the cDNA Fragments for RGS2

After the hCG-induced expression of the RGS2 gene was confirmed by Northern analyses, the cDNA fragments of this gene were subcloned and sequenced (Figure 3). The NCBI accession number for the larger fragment, which was isolated by primer set A72, is #AF233441. The shorter RGS2 cDNA, which was amplified by primer set G72, was identical to a 262-bp segment of the 861-bp longer fragment. The random primer (i.e., primer #72) annealed to the same site at the upstream ends of both the short and the long cDNA fragments. These cDNA fragments are highly homologous to segments of RGS2 genes that have been cloned from mouse (NCBI accession #NM_009061.1) and from human (NCBI accession #NM_002923.1).

Effects of Epostane and Indomethacin on RGS2 Gene Expression

For these tests, Northern blots were prepared from RNA that was extracted from control ovaries at 0 and 8 h into the ovulatory process, or extracted from experimental ovaries that were taken at 8 h after hCG from rats that had been treated 5 h earlier with ovulation-inhibiting doses of epostane or indomethacin. (These experimental intervals were selected in order to compare the present data with related studies using epostane and indomethacin [5, 12]). The signal density (normalized against the b-actin control) of the 8-h control lane was arbitrarily set at 100% (Figure 4). There was minimal expression of RGS2 mRNA at 0 h, but substantial expression at 8 h. In animals treated with the anti-ovulatory agent epostane, which blocks P₄ synthesis [4, 13-15], the signal density of 127.7% ± 18.7% was not significantly different from the 8-h control value. Animals treated with the anti-ovulatory agent indomethacin, which blocks PG synthesis [15, 16], had a signal density that was 73.1% ± 13.3% of the 8-h control value. Although this was significantly lower than in the ovaries of animals treated with epostane, it was not significantly different from the 8-h control value. The ovulation rates in parallel groups of animals treated with indomethacin and epostane were significantly inhibited (Figure 4). These results indicate that the ovulation-related increase in RGS2 gene expression is not dependent on ovarian P₄ or ovarian PG levels.

Localization of RGS2 mRNA Expression by In Situ Hybridization

In situ hybridization confirmed the temporal pattern of RGS2 mRNA expression that was observed in the differential display autoradiographs and the Northern analyses. There was minimal signal from the 0-h control ovaries, a strong signal at 4 h after hCG treatment, a declining signal at 8-12 h, and negligible signal at 24 h (Figure 5). Hybridization was localized in the granulosa layer of the larger follicles (Figure 6). As RGS2 expression began to decline at approximately 8 h after hCG, there still appeared to be a relatively strong signal from the innermost layer of granulosa cells, as well as from the cumulus cells that surround the oocytes. By 12 h after hCG, most of the lingering signal was from cumulus cells (Figure 6).

DISCUSSION

When GTP initially binds to an a subunit and promotes the dissociation of a bg dimer, the eventual termination of signaling by these two separate G-protein entities can occur naturally, albeit slowly, by GTPase activity that is intrinsic to the a subunit [6, 10]. The significance of RGS proteins is that they stimulate a 100-1000-fold increase in the GTPase activity of their target a subunits [10]. This kinetic value of the RGS family of proteins has been recognized by a number of investigators during the past decade [7, 11]. To date, the growing family of mammalian RGS proteins consists of approximately 30 highly diverse proteins that share a homologous 120-125 amino acid domain (sometimes referred to as the "RGS box") by which the RGS protein reacts directly with the GTP-bound a subunit to facilitate GTPase activity and promote the hydrolysis of a-GTP to a-GDP and reformation of the abg-GDP trimer to attenuate signaling [7, 10, 11]. These diverse RGS proteins can be subdivided into a group of smaller proteins (160-217 residues) and a group of larger proteins (372-1387 residues), with 211-residue RGS2 belonging to the smaller group [7]. Regardless of their size, virtually all of the RGS proteins are thought to be cytosolic proteins that are attracted to the plasma membrane by GTP-activated a subunits [10].

The array of RGS proteins reportedly attenuate signal transduction initiated by a wide variety of mitogens and morphogens, including hormones that bind to G-protein-coupled receptors to stimulate cell proliferation and differentiation [7, 9-11]. Therefore, it is not surprising that RGS2 expression is acutely up-regulated in ovarian follicles that have been stimulated by LH to acutely rupture and rapidly transform into steroid-secreting lutein tissue [1, 3]. The present Northern analyses and in situ hybridization data show that ovarian RGS2 mRNA increases significantly within 2 h after the ovaries were stimulated by hCG. This relatively rapid induction is consistent with other evidence that RGS2 (but not RGS4 or RGS7) is strongly induced in PC12 cells following 1 h of treatment with forskolin [17]. In the context of this rapid induction, it is important to point out that the RGS2 gene has binding sites for the zinc-finger transcription factor Egr-1 [11]. This observation is especially interesting because there is recent evidence that expression of the Egr-1 gene increases significantly in the rat ovary within 1 h after initiating the ovulatory process by hCG [4]. Therefore, it is possible that Egr-1 is involved in the regulation of ovarian RGS2 gene expression during ovulation.

It is also evident from the present data that ovarian RGS2 mRNA expression reaches a peak at some time close to 4 h after stimulating the ovarian follicles with hCG. This early peak in RGS2 occurs 4-8 h before the well-known ovulatory peak in ovarian P₄ and PG production at approximately 8-10 h after hCG administration in the rat model [13-16]. Thus, it appears that expression of ovulation-dependent genes such as cytochrome P450scc and PG synthetase-II occurs during the ovulatory process at a stage that is downstream from ovarian RGS2 expression. This temporal pattern of expression of RGS2 in relation to P₄ and PG might explain why the inhibition of ovulation by the inhibition of P₄ or PG synthesis did not significantly affect the earlier expression of RGS2.

The precise function(s) of RGS2 in ovulation remains to be determined. There is evidence that RGS2 selectively binds G_qa more than other Ga-proteins such as G_sa or G_ia [18]. Therefore, since G_qa normally activates phospholipase C (PLC) [11], it would appear that one function of RGS2 might be to attenuate PLC-mediated signaling of second messengers such as inositol triphosphate and Ca⁺⁺ [6, 10]. Since PLC activation has also been associated with K⁺ conductance, it has been suggested that RGS proteins speed the deactivation of inward-rectifying K⁺ channels [7]. This correlation of RGS to ion-gating and membrane conductance suggests that RGS proteins are potentially important in the regulation of neuronal signaling, and there is growing evidence to support this idea [7, 11]. In fact, RGS2 reportedly is unique in that it is rapidly induced in response to an increase of neuronal activity in the central nervous system [19]. Thus, it is feasible that RGS2 modulates the expression of action potentials generated by acute stimulation of cells that are targeted by hormones and neurotransmitters. Such an effect is relevant to the current discussion of ovulatory follicles because it has been proposed that the syncytial-like network of granulosa cells that comprise the innermost layer of a mature follicle may express action potentials comparable to neuronal spikes [20]. Collectively, the circumstantial evidence leaves open the possibility that the elevation of RGS2, reported here, may be a target-cell response to an intense ovulatory association between gonadotropin and G-protein-coupled receptors in the plasma membranes of depolarizing granulosa cells.

It is clear from the in situ hybridization data that ovarian RGS2 is localized in the granulosa layer of mature follicles, with maximum expression at approximately 4 h into the ovulatory process. It also appears that, as expression wanes, RGS2 mRNA persists for a longer period of time within the innermost portion of the stratum granulosum (see 8-h ovary in Fig. 5). Furthemore, the longest expression is within the cumulus cells that surround the oocytes (see 12-h ovary in Fig. 5). While it is presumed that the function of ovarian RGS2 is to attenuate G-protein signaling, it remains to be determined whether this GAP has additional signaling functions in the ovulatory process. Any future efforts to decipher the specific role(s) of RGS2 in ovarian physiology must also consider the growing evidence that RGS proteins may link signal transduction to other signaling pathways that are not yet firmly established. [7, 9, 11]. In any event, it is evident that hormonal stimulation of the ovulatory process induces a significant increase in ovarian RGS2, and it is likely this signaling modulator has important function(s) in the mechanism of ovulation.

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FIGURE LEGENDS

Figure 1. Autoradiographs of differentially displayed RGS2 cDNAs (arrows). Primer set A72 amplified a long cDNA that was located near the top of the electrophoresis gel, whereas primer set G72 amplified a shorter fragment that migrated substantially further down another gel. Note that these cDNAs for RGS2 are not visible in the lane containing the 0-h PCR product.

Figure 2. Intensity of Northern blot signals at the six intervals of the peri-ovulatory period following hCG administration. The signal density at 8 h was arbitrarily set at 100%, and the other points on the graph represent the mean values of the Northerns from two each of the long (A72) and the short (G72) cDNAs that were complimentary to the RGS2 gene. The actual Northerns of the two RGS2 cDNAs, along with a b-actin control, are shown below the linear graph. Note that the greatest intensity is at 4 h after hCG.

Figure 3. Sequence of the 861 bp long cDNA that was isolated by primer set A72. Primers are shown in brackets. The 262 bp shorter cDNA that was amplified by primer set G72 is underlined in bold print. Note that the polyT-G primer that amplified the shorter cDNA failed to amplify a "C" that was present in the longer sequence.

Figure 4. Comparison of the % of signal from Northern blots containing RNA extracted at 8 h after hCG from animals that were also treated with either 5 mg of epostane (Epo), or 1 mg of indomethacin (Indo) administered at 3 h after hCG. Bar graphs are based on NIH-image analyses of four different Northerns probed with the shorter cDNA that was amplified by primer set G72. The signal from the 8-h control lane (Ctrl) was arbitrarily set at 100% OD. In parallel groups of rats, the ovulation rate was determined at 24 h after hCG.

Figure 5. Change in intensity of the in situ hybridization signal during the six peri-ovulatory intervals following hCG administration. Light-field micrographs on the left show the histology of ovarian sections stained with hematoxylin and eosin (H & E), while the dark-field micrographs of the same sections show the localization of RGS2 mRNA as detected by hybridization of a ³⁵S-labeled anti-sense probe derived from the RGS2 cDNA.

Figure 6. Closer view of the distribution of probe in the inner layer of granulosa cells of ovarian follicles. White arrows in the follicular antra of the 12-h dark-field micrograph mark regions of the cumuli oopheri where the RGS2 probe mainly hybridized at this stage of the ovulatory process. The clear area inside two of the cumulus masses represents the oocytes inside these follicles.