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Regular Article |
a Department of Biology, Trinity University, San Antonio, Texas 78212
b Department of Cell Biology, Baylor College of Medicine, Houston, Texas 77030
c Department of Obstetrics and Gynecology, Kumamoto University School of Medicine, Kumamoto, Japan
ABSTRACT
The ovulatory process in mammals begins when an endogenous surge in LH 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 eCG s.c., and 48 h later the 12-h ovulatory process was initiated by 10 IU hCG (a homolog of LH) 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 reverse transcription-polymerase chain reaction differential display to detect gene expression in the stimulated ovarian tissue. Two of the amplified cDNAs that were upregulated 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.
follicle, granulosa cells, hCG, hormone action, LH, ovulation, signal transduction
INTRODUCTION
Mammalian ovulation is initiated by the coupling of a gonadotropic hormone such as LH 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 lasts 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 
as well as their ß
subunits [6–11]. The binding of hormone to receptor leads to a conformational change in the receptor that induces a dissociation of GDP from the subunit. In rapid succession, the subunit binds a GTP and separates from the ß heterodimer. Subsequently, both the and the ß 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 subunit of an activated G-protein [6–11]. This event causes the de-energized subunit to reunite with the ß 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 recoupling 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. Gene transcription of RGS2 was detected by reverse transcription-polymerase chain reaction (RT-PCR) differential display of ovarian RNA that was extracted at six intervals during the periovulatory 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 hCG, a homolog of LH.
MATERIALS AND METHODS
Animal Tissue and Animal Injections
Immature Wistar rats were induced to superovulate by eCG and hCG treatment as described previously [12]. Ovarian RNA was extracted at the periovulatory intervals of 0, 2, 4, 8, 12, and 24 h after hCG. These nucleic acid extracts were used for differential display and for Northern blotting. Epostane and indomethacin were injected s.c., also as described previously [12]. These antiovulatory 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. The 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 reamplification 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 ANOVA 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 subpopulations of radioactively labeled cDNAs that were generated from RNA extracts at each of the six stages of the periovulatory 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 (Fig. 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) (Fig. 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 reamplified for use as probes in Northern analyses.
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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 (Fig. 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 periovulatory 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 ß-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 eightfold 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.
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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 (Fig. 3). The NCBI accession number for the larger fragment that was isolated by primer set A72 is no. AF233441. The shorter RGS2 cDNA that was amplified by primer set G72 was identical to a 262-base pair (bp) segment of the 861-bp longer fragment. The random primer (i.e., primer no. 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 no. NM_009061.1) and from human (NCBI accession no. NM_002923.1).
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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 ß-actin control) of the 8-h control lane was arbitrarily set at 100% (Fig. 4). There was minimal expression of RGS2 mRNA at 0 h but substantial expression at 8 h. In animals treated with the antiovulatory agent epostane that 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 antiovulatory agent indomethacin that 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 (Fig. 4). These results indicate that the ovulation-related increase in RGS2 gene expression is not dependent on ovarian P4 or ovarian PG levels.
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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 (Fig. 5). Hybridization was localized in the granulosa layer of the larger follicles (Fig. 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 (Fig. 6).
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DISCUSSION
When GTP initially binds to an subunit and promotes the dissociation of a ß 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 subunit [6, 10]. The significance of RGS proteins is that they stimulate a 100- to 1000-fold increase in the GTPase activity of their target 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 subunit to facilitate GTPase activity and promote the hydrolysis of -GTP to -GDP and reformation of the ß-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 the 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 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 upregulated in ovarian follicles that have been stimulated by LH to rupture acutely and transform rapidly 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 Gq more than other G-proteins such as Gs or Gi [18]. Therefore, because Gq 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 Ca2+ [6, 10]. Because 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.
FOOTNOTES
1 This work was supported by NSF grant 9870793 (L.L.E.), by a grant to support T.U. as a Research Fellow of The Lalor Foundation, Providence, Rhode Island (L.L.E.), and by NIH grant HD-16229 (J.S.R.). 
2 Correspondence. FAX: 210 999 7229; lespey{at}trinity.edu
Accepted: July 5, 2000.
Received: April 14, 2000.
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