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Regular Article |
a Institute for Hormone and Fertility Research, University of Hamburg, 22529 Hamburg, Germany
ABSTRACT
Lysophosphatidic acid (LPA) is a biologically active phospholipid recently introduced as a new marker for ovarian cancer. Because high concentrations of LPA have also been found in the follicular fluid from healthy subjects, one can presume that this biological mediator may have relevance for normal ovarian physiology as well. We have reported earlier that luteal cells possess specific binding sites for LPA. Using these cells as a model, we show now that LPA is able to modulate the morphological cell shape changes induced by LH in that it inhibits the formation of stellate processes induced by LH. This morphoregulatory effect of LPA is mimicked by cytotoxic necrotizing factor 1, a bacterial toxin known to activate small G-proteins from the Rho family. On the other hand, C3-exotransferase that acts mainly through the inhibition of Rho A mimics the effects of LH. Furthermore, we report here that the morphoregulatory effects of LPA are accompanied by the translocation of Rho proteins from the cytosol to cell membrane, an effect generally considered to be an indicator for the activation of Rho-GTPases. During the development and rescue of the corpus luteum, major morphoregulatory effects are exerted by LH that appear to be modulated by LPA via an activation of Rho proteins.
corpus luteum, growth factors, ovary, signal transducers, signal transduction
INTRODUCTION
Throughout the female reproductive cycle the corpus luteum exhibits regular periods of growth, differentiation, and regression [1, 2]. The molecular mechanisms regulating this process of tissue remodeling are still obscure. Luteal cells are known to be regulated mainly by either LH (during the ovarian cycle) or chorionic gonadotropins (during the pregnancy). In addition, there are reports [1–3] showing that a number of other hormones and growth factors can exert modulatory effects on the action of LH in luteal cells. It was of interest to investigate if lysophosphatidic acid (LPA) could be one such factor. Lysophosphatidic acid is a biologically active phospholipid [4, 5] known to be present in significant quantities in follicular fluid from healthy subjects [6] as well as in ascites fluid from patients suffering from ovarian cancer [7, 8]. We have reported previously that bovine luteal cells possess specific binding sites for LPA [9]. Therefore, it is conceivable that this biologically active phospholipid may have an important local relevance for ovarian cell biology both in a physiological and a pathological scenario.
Being the smallest and structurally simplest biologically active phospholipid, LPA may act as a paracrine or autocrine mediator having an apparently greater range of physiological activities than polypeptide growth factors [4, 5]. Freshly prepared platelet-rich blood plasma has been shown to contain high amounts of LPA, but its production and release are not restricted just to platelets [5, 10]. Recently, LPA has been introduced as a marker for ovarian cancer [7, 8, 11]. It has been reported that human follicular fluid contains a lysophospholipase D activity responsible for the local production of LPA [12]. The physiological basis of the effects exerted by LPA in a healthy ovary, however, has not been appreciated so far. In the present study, using luteal cells as a model, we have evaluated the effects of LPA on luteal cells and propose that LPA may have a modulatory role in the corpus luteum, antagonizing the morphoregulatory effects of LH. Rho proteins are proposed to play a role in this novel signaling mechanism.
MATERIALS AND METHODS
The sources of various chemicals were as follows: 1-oleoyl-LPA, the reagents for the May-Grünwald Giemsa staining, protease inhibitors, and monoclonal anti-actin antibody were from Sigma-Aldrich (Deisenhofen, Germany). Anti-Rho antibody was from Upstate Biotechnologies (provided by Biomol, Hamburg, Germany), and Clostridum botulinum C3-exotransferase was from Biomol (Plymouth, PA). Cy3-, Cy2-, and peroxidase-conjugated secondary antibodies were from Jackson Immunochemicals (provided by Dianova, Hamburg, Germany). The peroxidase-based Western-blot detection system was from Pierce (provided by KMF, Sankt Augustin, Germany). Vitrogen 100 collagen was from Celtrix Laboratories (Palo Alto, CA) and collagen CSB from Cellon (Strassen, Luxemburg). Bovine LH was a gift from the National Institute of Arthritis, Diabetes and Digestive and Kidney Diseases (NIADDK, Bethesda, MD), and 8-bromo-cAMP (8Br-cAMP) was from Boehringer Mannheim (Mannheim, Germany). Amicon Centricon filters were from Amicon-Millipore (Eschborn, Germany). All other reagents were obtained from commercial sources and were of the highest purity grade.
Isolation and Culture of Ovarian Cells
The methods for the isolation and purification of bovine luteal and theca cells have been published elsewhere [9, 13, 14]. Briefly, bovine ovaries were obtained from the local abattoir, and corpora lutea (stage II according to Ireland) [15] were dissected out and used for the preparation of luteal cells and for setting up the primary culture [9, 14]. For isolation of theca cells, healthy preovulatory bovine follicles were used as described [13]. The luteal cells were maintained in Dulbecco minimum essential medium/Ham F-12 medium (including 2 mM glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin, and 2% heat-inactivated fetal calf serum) on Vitrogen-coated cell culture plates (using either Vitrogen 100 collagen or collagen CBS, according to the protocol provided by the manufacturer; both products are prepared from pepsin-treated bovine dermis and contain 95% type I collagen and 5% type III collagen). On the third day, the medium was replaced by fresh medium supplemented with 5 µg/ml BSA, 5 µg/ml insulin, 5 µg/ml transferrin, 5 ng/ml sodium selenite, and the cells were cultured further for 72 h before treatment. On Day 6, before stimulation, culture medium was removed from the confluent monolayers of cells, and the cells were washed with fresh serum-free medium supplemented with 5 µg/ml BSA. For the induction of luteinization in vitro, theca cells were incubated in the presence of 2 µM forskolin for the first 2 culture days and then cultured further as above. The morphology, diameter, basal, and stimulated progesterone production were comparable to that of small luteal cells.
Treatments of Cells in Culture
Unless otherwise stated, the cells were treated with either 100 ng/ml LH or 1 mM 8Br-cAMP in the presence and absence of LPA (2–20 µM) for 180–300 min (or as indicated). Because LPA might be air and light sensitive, stock solutions were made up in ethanol, and shortly before stimulation aliquots were diluted in albumin-containing serum-free medium.
Preparation of Particulate Membrane Fractions
For membrane preparation, cell pellets were resuspended in 1 ml of 10 mM Tris-HCl (pH 7.4) containing 10 µg/ml leupeptin, 1 µg/ml pepstatin, 1 µg/ml amastatin, and 1 mM PMSF (Tris buffer A). This mixture was homogenized and sonicated before sucrose solution was added to a final concentration of 0.25 M [16]. The homogenate was centrifuged at 200 x g for 15 min at 4°C. The supernatant was recentrifuged at 100 000 x g for 60 min at 4°C and was used as a cytosolic fraction. The 100 000 x g pellet was resuspended in Tris buffer A and used as a particulate membrane fraction. For some experiments, the membrane fraction was further purified as described below. Protein content was determined according to Bradford [17].
Preparation of Nuclear and Nonnuclear Membrane Fractions
For the preparation of nuclear membrane fractions the cells were resuspended in isotonic Tris sucrose buffer A (10 mM Tris/HCl, pH 7.4, including 0.25 M sucrose containing 10 µg/ml leupeptin, 1 µg/ml pepstatin, 1 µg/ml amastatin, and 1 mM PMSF (Tris-sucrose buffer). The cells were homogenized using an all-glass Dounce-type homogenizer. The homogenate was centrifuged first at 100 x g at 4°C/15 min. Supernatant 1 was further separated as described below; pellet 1 (nuclear fractions) was homogenized in Tris buffer A containing 0.5% Nonidet P-40 and centrifuged first at 200 x g and then at 100 000 x g for 60 min at 4°C; the resulting pellet 2 (nuclear membrane fractions) was resuspended in Tris-buffer; and the supernatant (nucleoplasm) was concentrated using an Amicon filter (with 10 000 cutoff). Supernatant 1 was subjected to centrifugation at 100 000 x g for 60 min at 4°C. Pellet 3 (nonnuclear membrane fractions) was resuspended in Tris-buffer A, and supernatant 3 was recentrifuged (at 100 000 x g for 60 min) to obtain pure cytosolic fractions. The protein content of the various cell subfractions was determined as above [17].
Expression/Translocation of Rho Proteins
For investigation of the expression of Rho proteins, the cytosolic and particulate fractions from stimulated cells were separated by SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membranes. After blocking, the membranes were hybridized overnight at 4°C with polyclonal antibodies recognizing Rho (A, B, C) 24 kDa and two additional nonspecific bands at 27 kDa and 50 kDa. The antibody was raised against a peptide corresponding to amino acids 73–94 of Rho proteins (SYPDTDVILMCFSIDSPDSLENKK) with two C-terminal lysines added to increase peptide solubility. The immunocomplex was detected by chemiluminescence using a peroxidase-based luminol chemiluminescence system after incubation with peroxidase-conjugated secondary antibody. Control blots were run with nonimmune serum and secondary antibody. Unfortunately, the commercially available antibodies that recognize only the RhoA isoform did not show any immunoreactivity when using bovine material (data not shown).
Immunohistochemistry/Cell Staining
Cells were grown on chamber slides coated with Vitrogen under the culture conditions described above. After stimulation, the cells were washed with PBS and fixed with 3% paraformaldehyde for 30 min at room temperature. Before blocking the nonspecific binding by treatment with 5% nonimmune serum (species of the secondary antibody) for 60 min, the cells were permeabilized with 0.5% Triton X-100 for 4 min. Incubation with the primary antibody (i.e., anti-actin, anti-Rho) continued for an additional 60 min. The cells were then washed and incubated with Cy3- (or Cy2-) conjugated secondary antibody at room temperature in the dark chamber. The conjugate was washed several times, and the chamber slides were kept in the dark at 4°C overnight before viewing with a Nikon fluorescence microscope. Micrographs were taken on Kodak EPY 64 T Film. Additionally the cells were routinely stained using the May-Grünwald-Giemsa technique. Briefly, cells grown either on chamber slides or on 24-well cell culture plates were stimulated as described above. After stimulation, the cells were washed with PBS and fixed with methanol. The fixed cells were stained for 20 min with May-Grünwald solution followed by a 10-min incubation with Giemsa stain. After extensive washing, the plates were air dried and analyzed with a Nikon microscope using a brightfield technique. The morphological changes were routinely monitored and analyzed for each individual preparation.
Data Analysis
Each experiment was performed three to eight times with similar results (as indicated). The morphological changes were routinely monitored and analyzed using cells stained with either May-Grünwald-Giemsa or actin stain. Cells were scored as morphologically changed on the basis of the existence of cell-associated processes and increased cytoplasmic staining. Each morphological data set shows results from an individual representative experiment. In Western-blot experiments, blots and films were scanned, and specific bands were analyzed densitometrically using National Institutes of Health (NIH) Scion Image Software (Scion Corporation, Bethesda, MD). The integrated optical density of the bands was quantitated using a computer-assisted analysis system. The statistical analysis was performed using Graph PAD data analysis software (San Diego, CA).
RESULTS
Lysophosphatidic Acid Reverses Morphological Changes Induced by LH in Luteal Cells In Vitro
When cultured in the presence of gonadotropins, ovarian cells undergo morphological changes that are accompanied by a stellate appearance characterized by neurite-like outgrowth processes. This induction of star-like, branching pseudopodia-like processes has been morphologically well characterized for FSH-stimulated rat granulosa cells [18–20]. Similar morphological effects, referred to as stellation processes, have also been described [10, 21] for nonovarian cells.
Using cultured bovine luteal cells, in Figure 1 we show that the addition of LH leads to a stellate formation. Similar morphological transition was described for FSH-stimulated undifferentiated granulosa cells. Figure 1 shows that while addition of LPA alone (Fig. 1, compare B for LPA with A for medium only) does not affect the cell shape, addition of LH in contrast resulted in a marked change in the shape of luteal cells to a polygonal appearance with multiple branching stellate processes (Fig. 1, compare C with A). Such effects of LH could be completely abolished by the addition of 2 µM LPA (compare D with C in Fig. 1). Interestingly, similar morphoregulatory effects of LPA were also observed in cultured bovine theca cells (data not shown).
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From our preliminary results, it appeared that although the LH-induced process of the stellate formation occurs rapidly (within 60–90 min), longer incubation periods were necessary for a complete reversal of this process by LPA. Closer examination of individual cells revealed that not all of the stellate structures vanished in the presence of LPA, if the incubation duration was less than 120 min. Therefore, for further analysis a treatment period of at least 180 min was used.
Previously, reports showed [18–20] that the FSH-induced stellate formation in granulosa cells was mediated by cAMP. In luteal cells as well, the morphoregulatory effects of LH could be mimicked by the cAMP analogue 8Br-cAMP (compare Fig. 2, C with D). Furthermore, as shown in this figure, addition of LPA to either LH- or cAMP-stimulated cells reversed the morphological changes, with a rearrangement of the actin cytoskeleton and disappearance of the stellate processes (compare Fig. 2, C with E for cells treated with LH alone versus with LH plus LPA and Fig. 2, D with F for cells treated with 8Br-cAMP versus 8Br-cAMP plus LPA). Statistical analysis revealed 84% ± 6% inhibition of stellate-containing cells after stimulation with LPA (±SEM, cells counted per chamber, n = 8).
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Possible Involvement of Rho GTPases in the Mechanism of Action of LPA in Luteal Cells
In nonovarian cells, the action of LPA has been reported [21, 22] to be mimicked by activated RhoA, while its effect could be blocked by Rho-inactivating Clostridium botulinum C3-exotransferase [22, 23]. This toxin is a useful tool for the study of mechanisms mediated by Rho proteins because it has been shown to ADP-ribosylate and inactivate this class of proteins selectively, impairing their function [24, 25]. To investigate if the modulation of Rho function could also affect the LPA-induced effects in the corpus luteum, we have introduced C3-exotransferase to cell cultures and treated cells in the presence and absence of LPA. Interestingly, if luteal cells were exposed to C3-exotransferase alone, cells started the formation of bridges and junctions, indicating that C3-exotransferase mimicked the effects of LH and was able to induce the stellation process by itself (Fig. 3: A2, B2, for C3 exotoxin and A1/B1 for cells treated with medium only). It is likely that activated Rho (translocated to the membranes) might be more resistant to subsequent treatment with C3-exotransferase.
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However if the cells were stimulated with C3-exotransferase in the presence of LPA, only a minor inhibition (if any) of stellate formation could be observed (Fig. 3, A3). Therefore, we have pretreated cells with or without LPA (to induce a maximal Rho stimulation) and treated them further with C3-exotransferase (or with C3-exotransferase plus LPA) for 180 min (Fig. 3, B1–B3). Figure 3, B3, shows that LPA pretreatment prevents the formation of stellate processes induced by C3-exotransferase (compare Fig. 3, B3 with B2).
Transfection of the cells with a constitutively active mutated Rho may be an ideal experiment to discern if indeed Rho proteins are involved in the morphological effects induced by LPA. However, the primary cell cultures appear not to be a good system for such transfection experiments (<1% transfection efficiency). Alternatively, it is possible to use another Rho-directed toxin, CNF1 (Escherichia coli cytotoxic necrotizing factor 1), that has been shown to activate Rho irreversibly [26]. CNF1 has been widely used to determine the involvement of an activated Rho [24, 27]. Therefore, we have treated the cells with CNF1 to examine if it could influence the observed morphological changes in luteal cells. Figure 4 shows that the formation of the stellate processes in response to LH stimulation was impeded in luteal cells treated with either CNF1 or LPA. Compare Figure 4C that shows cells stimulated with LH alone with Figure 4D for cells stimulated with LH plus CNF1. The control panels show cells treated with LH plus LPA (Fig. 4E) or with either CNF1 or LPA alone (Fig. 4B for CNF1, Fig. 4F for LPA, and Fig. 4A for cells stimulated with medium only). Thus, treatment with either LPA or CNF1 resulted in a comparable inhibition of stellate formation induced by gonadotropin. Because LPA has been reported [21, 22, 25] to stimulate RhoA, it may be suggested that the observed morphoregulatory effects of LPA in the corpus luteum may in fact take place at the level of Rho GTPases.
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Lysophosphatidic Acid and CNF1 Induce Translocation of Rho Proteins in Luteal Cells
The translocation of Rho proteins from the cytosol to the membrane fraction is generally considered to be an indicator for the activation of Rho and has been used as a functional assay for its activity [28]. Therefore, it was of interest to examine if in fact, stimulation with either LPA or CNF1 may lead to cellular redistribution of Rho GTPases. In the first series of experiments, we have prepared cytosolic and plasma membrane fractions from luteal cells treated with either LPA or CNF1 and could observe that incubation with CNF1 resulted in a marked increase in the proportion of Rho protein associated with the membrane fractions (Fig. 5B, compare with Fig. 5A for the cytosolic fractions). When compared to the effects produced by CNF1, addition of LPA had only a subtle effect on the redistribution of Rho proteins (note the different scale for LPA and CNF1 in Fig. 5B). It may, however, be noted that CNF1 is a bacterial toxin displaying a strong Rho-activating effect, whereas effects of a biological ligand such as LPA may be considered to be much less prominent. Nevertheless, the effects of LPA were comparable to the published results using cell lines like 3T3 cells [28]. It may also be noted that the latter authors have used 100 µM LPA to produce a marked effect using a similar assay system. Interestingly, addition of either 8Br-cAMP (Fig. 5C) or LH (data not shown) redistributed the Rho proteins back to the cytosolic fraction.
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We could observe in additional control experiments that if the cells were stimulated for longer durations the effects of LPA on the translocation (activation) of Rho were considerably higher. Therefore, in order to increase the amplitude of the observed effects, we decided to use longer incubation periods instead of considerably higher concentration of LPA. The results in Figure 6 show that indeed, treatment of luteal cells with 2 µM LPA for 300 min resulted in a nearly 50% decrease in the amount of Rho proteins in the cytosolic fractions (Fig. 6A, lane 2 versus lane 1) with a concomitant increase in the amount of Rho protein associated with the cell membrane fraction (Fig. 6B, lane 2 versus lane 1). In addition, a marked increase in the amount of Rho proteins associated with nuclear membrane fractions could be observed (Fig. 6C, lane 2 versus 1). The stimulation of luteal cells with CNF1, as expected, also caused a comparatively stronger redistribution of the Rho proteins from the cytosol to the cellular membrane as well as to the nuclear membrane fractions (Fig. 6, compare lanes 3 in B and C with lane 3 in A). Addition of 8Br-cAMP had only a moderate modulatory effect on this translocation of Rho proteins (data not shown).
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DISCUSSION
As far as we are aware, this is the first report investigating the effects of physiological concentrations of LPA on a nonmalignant ovarian cell. At the outset, we have shown that the luteal cells when subjected to gonadotropic stimulation underwent a marked morphological change in cell shape accompanied by stellate formation in a manner analogous to the observation reported for FSH-stimulated granulosa cells [18]. In addition, we report here that the addition of LPA to these cells in culture resulted in a reversal of the effects of LH. The changes in the cell morphology induced in response to treatment with LH (or with cAMP) in luteal cells and its reversal by LPA appears to be mediated by Rho proteins. The morphoregulatory effect of LPA could be mimicked by CNF1, a bacterial toxin known to activate small G-proteins from the Rho family. On the other hand, C3-exotransferase, another toxin known to elicit its effects mainly through the inhibition of RhoA mimics the effects of LH and induces the formation of stellate processes by itself. This formation of stellation processes could be blocked by pretreating the cells with LPA. Furthermore, we observed that the morphoregulatory effects of LPA are accompanied by the translocation of Rho proteins from the cytosol to the membrane fractions (both plasma and nuclear membranes). Thus, it may be suggested that morphoregulatory mechanisms exist in luteal cells involving LPA, a fact that has not been hitherto recognized in any ovarian cells.
Although the major role played by LH in ovarian differentiation has been known for a long time, only recently a model has been developed to explain how LH pushes the balance from the proliferating to the differentiating phenotype in the early postovulatory phase [3, 29]. In cell culture, LH-induced initiation of the cell remodeling process can be monitored by a stellate appearance of neurite-like outgrowth processes. Such changes have also been shown previously for nonovarian cells as a result of cAMP stimulation [10, 21]. This has been well characterized using rat granulosa cells [18–20]. Grieshaber et al. [20] have recently demonstrated the effects of FSH on the formation of stellate structures (lamellipodia and filopodia) in a rat granulosa cell line. Thereby the authors have speculated, "that small molecular weight G proteins could be involved in this process" [20]. We here present evidence that in luteal cells this in fact may be the case.
It is likely, that stellate formation precedes the contact-dependent formation of specialized cell contacts like tight junctions or gap junctions. Detailed morphological analysis by transmission electron microscopy performed by other groups [30, 31] revealed existence of specialized cell contact structures such as gap junctions in bovine luteal cells in vitro. This suggests that these cells actively communicate with each other through these junctions. Furthermore, contact between the small and large luteal cells is required for maximal LH-induced steroid synthesis [32]
Using an in vitro luteal cell model system, we show here that the stellate-forming effects of LH may be modulated by LPA, a bioactive phospholipid found locally within the ovary. Furthermore, we present additional evidence that Rho proteins may be involved in the morphoregulatory effects induced by LPA. It may be noted that most studies concerning the effects of LPA have been performed on tumor cells, whereas its effects under normal physiological conditions have not been ascertained at all. Our findings support the concept that LPA could also play an important physiological role in control of the corpus luteum function. Although gonadotropins have been known for a long time as the driving force for the differentiation and rescue of the corpus luteum, the molecular mechanisms of the morphoregulatory effects are largely unknown. It is known that cAMP is involved in the morphological changes induced by LH in the corpus luteum [3]. Additional signal transduction pathways, mainly those activated by growth factors, have also been shown to interact or cross talk with the signaling pathways activated by gonadotropins/cAMP [33, 34]. Based on our results, it is possible to propose a mechanism in which Rho proteins could play a central role in mediating the morphoregulatory effects of LPA in LH-stimulated luteal cells. The present observation has been made in cells cultured in vitro and therefore, it will be interesting to extend these studies to the corpus luteum in vivo.
It is still unknown how cAMP modulates Rho GTPases. Rho has been identified as a substrate for protein kinase A, while on the other hand it has also been shown to mediate the rearrangements of actin cytoskeleton in cells stimulated with LPA [21–23]. In neuronal cells, a rapid increase in the amount of F-actin after the addition of cAMP has been shown to be accompanied by stellate formation [35]. Cells of neuronal origin appear to be the most investigated model where axonal growth is driven by actin polymerization within the growth cone, a highly dynamic structure at the tip of the axon consisting of filopodial and lamellipodial protrusions [35]. Also in either astrocytes [25] or in melanoma cells [36], an overexpression of RhoA protein results in inhibition of the stellation process induced by either cAMP or C3-exotransferase. Though these mechanisms appear to be physiologically far removed from the developing corpus luteum, the underlying biochemical cause seems to be similar. Based on the results presented here and the experimental evidence from other cell systems it is, therefore, possible to propose that Rho may control luteal cell morphology via a mechanism involving LPA-induced reversal of stellation. This action of LPA is mediated through an active Rho, whereas an inactivation of Rho (possibly in response to LH/cAMP) in itself is sufficient to induce stellation. Thus although the molecular mechanisms that regulate morphoregulatory changes in the corpus luteum remain poorly understood, the available information may indicate that the signal transduction pathways regulating the LPA-induced cell shape changes involve novel signaling mechanisms [10, 21], including a Rho-dependent pathway (possibly involving Rho, Rac, and cdc42).
Interestingly, in luteal cells Rho proteins appeared to be targeted not only to the plasma membrane fraction but also redistributed toward the nucleus. There is some information in the literature that may explain the role of this nuclear signaling. Cdc 24p, another small GTPase from the Rho family known to regulate actin rearrangements, has been reported [37] to target to polarized growth sites as well as show similar nuclear localization. In Madin-Darby kidney cells a nuclear localization of RhoA (but not RhoB or ADP-ribosylation factor) could be demonstrated [38]. Small GTPases appear to be molecular switches that control signaling pathways critical for diverse cellular functions. Because multiple effector molecules can be activated by small GTPases, Lacal [39] has suggested that rather than a single linear pathway, an integration of complementary signals is required for the various events to occur between the membrane and nucleus. In fact cdc42, Rac, and Rho have been shown to cross talk with each other to control the early differentiation steps [35]. It appears therefore difficult at the moment to ascertain the relative importance of one specific member of the Rho family.
During development of the corpus luteum, a great deal of tissue remodeling occurs, exhibiting regular periods of growth, differentiation, and regression. An essential component of corpus luteum development is the recruitment of blood supply [40]. The highly vascular corpus luteum is thus under the continuous control of blood-derived factors like LPA that appears to be important for its normal growth and functioning. Alternatively, it is possible that LPA may be produced locally in the ovarian tissue through an interaction with growth factors. We have previously reported [41] that treatment of luteal cells with epidermal growth factor (EGF) can lead to an activation of phospholipase D in luteal cells that may be responsible for generation of phosphatidic acid. It is tempting to speculate that LPA may arise from phosphatidic acid generated in response to EGF, although we have no evidence yet to show that this indeed may occur in the corpus luteum. Secretory type II phospholipase A2 (sPLA2 type II) was the first enzyme shown to be able to generate LPA directly from phosphatidic acid [42]. Gene expression as well as secretion of a sPLA2 protein has been demonstrated [43] in the rat ovary. A metal-ion-dependent lysophospholipase D, present both in serum and in follicular fluid samples from patients undergoing an in vitro fertilization stimulation protocol [12, 44] appear also to be capable of generating LPA. We have taken an important step forward, to demonstrate here that small molecular weight GTPases from the Rho family belong to the targets that could be used by LPA to fulfill its morphoregulatory effects on LH-stimulated luteal cells. However, a lot more research will be necessary to resolve the exact mechanism responsible for the generation of LPA in ovarian tissue, the way it is degraded, and the mechanism by which signal is transduced from LPA receptor to the activation of Rho proteins.
ACKNOWLEDGMENTS
We thank Prof. K. Aktories and Dr. G. Schmidt (Insititut für Pharmakologie der Albert-Ludwigs-Universität, Freiburg, Germany) for their generous gift of CNF1 and for their useful comments, NIADDK for the bovine LH standard, and Ms. U. Steuber for technical assistance.
FOOTNOTES
First decision: 5 January 2001.
1 Presented in part as an oral presentation at the 32nd Annual Meeting of the Society for Study of Reproduction, 1999. 
2 Correspondence: L.T. Budnik, Institute for Hormone and Fertility Research, University of Hamburg, Grandweg 64, 22529 Hamburg, Germany. FAX: 4940 56190864; budnik{at}ihf.de Reprint requests: A.K. Mukhopadhyay, Institute for Hormone and Fertility Research, University of Hamburg, Grandweg 64, 22529 Hamburg, Germany. FAX: 4940 56190864; amal{at}ihf.de
Accepted: March 5, 2001.
Received: December 7, 2000.
REFERENCES
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 |
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|
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|
 |
|
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|
|
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|
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|
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|
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|
|
 |
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|
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|
|
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|
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