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a Division of Reproductive Biology, Department of Gynecology and Obstetrics, Stanford University School of Medicine, Stanford, California 94305
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONREFERENCES |
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cyclic adenosine monophosphate, follicle, granulosa cells, mechanisms of hormone action, ovary
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONREFERENCES |
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Cyclic Nucleotide Signaling: New Insights
A simple linear scheme of the steps involved in the cAMP cascade depicts the occupied gonadotropin receptors coupled to heterotrimeric G proteins (Fig. 1), which in turn regulate the effector adenylyl cyclase [1]. Changes in cAMP synthesis are the outcome of the activation of this membrane transduction machinery. Three classes of intracellular effectors are activated via binding to cAMP. These include the newly discovered class of cAMP-activated guanine nucleotide exchange factors (cAMP-GEF or EPAC) [2, 3], cyclic nucleotide-gated channels (CNGC) [4], and cAMP-dependent protein kinases (PKAs) [5], the latter being the best-characterized effectors in granulosa cells. It is established that cAMP binds to the regulatory subunit of PKA, thereby promoting the dissociation and activation of the C subunit. The activated C either phosphorylates cytoplasmic substrates or, once released from the regulatory subunit anchor, is translocated to the nucleus to phosphorylate transacting factors, thus controlling gene expression. The cAMP-regulatory element-binding protein CREB is the prototype transcription factor activated by PKA [6], although several other transcription factors are involved in cyclic nucleotide-regulated gene expression. It is well established that phosphorylation of serine 133 in the KID domain of CREB is obligatory for transcriptional activation of CREB and its association with the CREB-binding protein, CBP/p300, a transcription coactivator. Several reviews have covered the details of the activation of PKA as well as other cyclic nucleotide receptors and transacting factors [7–10].
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This linear signaling cascade is, however, insufficient to fully explain the divergent outcomes of cAMP activation in granulosa cells observed in vitro as well as the distinct patterns of gene expression during granulosa cell differentiation that are activated by the two gonadotropins [11]. Whereas granulosa cells from preantral follicles express only the FSH receptors (FSHR), mature granulosa cells express FSHR and LH receptors, both of which use cAMP as the primary signaling pathway (see below). FSH promotes massive growth of granulosa cells in the preantral follicle by regulation or induction of genes involved in cell cycle control and stimulates androgen aromatization via activation of the cAMP-signaling cascade [12, 13]. Conversely, through activation of seemingly identical cAMP signaling in the mature follicle, LH promotes the exit from the cell cycle and the suppression of aromatase expression and androgen aromatization [12, 13], as well as the induction of genes related to ovulation and luteinization.
Several explanations can be put forward to account for divergent outcomes from the occupancy of two structurally related receptors that activate cAMP signaling in granulosa cells. It is possible that, in addition to cAMP, the FSH and LH receptors are coupled to other signaling pathways and that branching of the signaling at the membrane accounts for these divergent outcomes. Furthermore, it is possible that the properties and propagation of the cAMP signal itself account for divergent effects when FSH or LH stimulates granulosa cells. Finally, this specificity in gonadotropin signaling may be dependent on the cell context (i.e., the state of granulosa cell differentiation) and on the compartmentalization of cAMP within granulosa cells. Thus an identical initial cAMP signal may be distributed to different downstream pathways that include different kinase cascades and transcription factors and ultimately different patterns of gene expression.
Divergent effects of an apparently identical cAMP signal are not a peculiarity of granulosa cells, as several other cells have been found where occupancy of two receptors coupled to cyclase produces two different outcomes. For instance, PGE1 and ß adrenergic receptors activate cAMP signaling in cardiac myocytes but have clearly distinct effects on cardiac functions, including contractility that suggests specificity and compartmentalization of signaling [14].
FSH and LH Receptor Coupling to Gs and Adenylyl Cyclase and to Other G Proteins and Effectors
Both FSH and LH receptors belong to the large superfamily of G protein-coupled receptors (GPCRs) with the distinctive structural characteristics of a large leucine-rich extracellular domain [15, 16]. The leucine-rich repeats are thought to be important for glycoprotein hormone binding. Because of this unique feature, the TSH, LH, and FSHR are segregated into a subfamily of the so-called leucine-rich repeat-containing G protein-coupled receptors (LGRs), which includes the more distantly related LGR4-8 [17, 18]. Given their seven transmembrane topology, both LH receptors and FSHRs signal by coupling to heterotrimeric G proteins. Countless in vivo and in vitro studies have demonstrated that occupancy of the native and recombinant gonadotropin receptors produces an increase in intracellular cAMP and that this is the major signal arising from these receptors [19] (Fig. 1). A coupling to Gs, and therefore to adenylyl cyclase, has been directly observed in membranes from porcine follicles, in bovine corpora lutea, and in reconstituted systems [20–22]. In these studies, however, it was observed that gonadotropin receptors may interact with other G proteins, namely Gi and Gq/11. Several reports have further established that gonadotropin binding may promote coupling of these receptors to G proteins other than Gs, thus activating additional signaling pathways (see below). In the case of granulosa cells and follicle development and maturation, one could therefore envisage that the FSHR predominantly activates the cAMP signaling pathway, whereas the LH receptor utilizes additional second messengers (i.e., Ca2+ and phospholipids), and the LH divergent effects are the result of the activation of the two pathways [13].
That the two gonadotropin receptors may have distinct and nonredundant functions in mature granulosa cell signaling is a matter of debate and is inconsistent with experiments administering recombinant FSH devoid of LH bioactivity. In hypophysectomized rats, a bolus of FSH induces oocyte maturation and ovulation [23]. Similar studies performed in monkeys show that oocyte maturation and granulosa cell luteinization is induced by FSH with the same efficiency as hCG [24]. However, the LH receptor knockout (LuRKO) mouse suggests different functions for the two receptors. The loss of signaling from the LH receptors causes an ovarian phenotype characterized by the absence of ovulatory follicles and CLs [25], a phenocopy of the effect of LH receptor mutations observed in humans [26]. Although the interpretation of the results may be complicated by a 50% reduction in ovarian estrogen content consequent to a decreased androgen production by theca cells, several findings suggest that FSH and its receptor cannot entirely compensate for the loss of the LH receptor. A 3- to 4-fold increase in circulating FSH levels was observed in these mice, and recombinant FSH or eCG were unable to induce follicle maturation (I.T. Huhtaniemi, personal communication). Thus the phenotype of this mouse suggests that signaling from the FSHR cannot entirely substitute for the LH receptor in the final stages of follicle growth and ovulation [25].
Data recently reported by Bebia et al. [27] on the expression of LH receptors in immature granulosa cells are also consistent with the view that occupancy of the two gonadotropin receptors produces distinct effects. These authors have attempted to circumvent a problem inherent to comparing the effects of FSH and LH, i.e., that immature granulosa cells express only FSHR, and LH responses can be measured only following FSH priming. To address this issue, LH receptors were expressed in immature rat granulosa cells using adenovirus vectors. Interestingly, under conditions where similar amounts of cAMP are generated in these cells, the endogenous FSH and LH receptor activation produces quantitatively different responses; most notably that estrogen production was considerably higher after FSH stimulation [27]. In agreement with this finding, FSH stimulated the expression of p450 aromatase mRNA more than a constitutively active LH receptor, whereas inhibin 
and 3ß-HSD mRNA induction were comparable [27]. With the caveat that the overexpression of the LH receptor in experimental conditions may produce spurious coupling, these results argue that the differences in signaling from the two gonadotropin receptors are intrinsic to the receptors themselves and are independent of the cell context, i.e., the state of differentiation of granulosa cells. Previous studies with endogenous FSH and VIP receptors revealed a similar phenomenon [28].
Early studies in rat and bovine ovaries had shown that LH stimulates phosphoinositide (PI) turnover in intact granulosa cells in culture [29, 30]. In addition, an association between LH receptor activation and an increase in intracellular Ca2+ in granulosa cells has been observed [31, 32]. More recently, studies using recombinant receptors showed that overexpression of LH receptors in the Xenopus oocyte leads to an LH-dependent increase in Ca2+-activated Cl- current, as measured by the two-microelectrode voltage-clamp method [33]. This indicates that Ca2+ mobilization from intracellular stores occurs in this reconstitution system, probably as a consequence of LHR receptor coupling to Gq and phospholipase (PLC) (Fig. 2). Given the high concentration of LH required to elicit these currents, it was speculated that this coupling may be important only at the time of the LH surge. Consistent with this observation, it has been shown countless times that LH receptors overexpressed in HEK293 and other cells are coupled to both cAMP and IP3 signaling [16, 22, 34–36]. However, differences were observed between the behavior of the LH receptors and FSHRs coupling to IP3 formation, with LHR being more effective than FSHR [36]. Data obtained by overexpression of receptors need to be taken with some caution because "spurious" or "promiscuous" coupling is consistently observed [37]. It should also be pointed out that stimulation of IP3 formation has often proven difficult to measure with endogenous LH receptors [38] or FSHRs [39, 40], in spite of the initial positive reports. Furthermore, a consistent finding of the studies is that the concentration of LH required for the activation of PLC is 10- to 100-fold higher than that required for the stimulation of adenylyl cyclase.
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This LHR coupling to PI turnover may indicate that during the terminal differentiation of granulosa cells, occupancy of this receptor activates two parallel signaling cascades. Indeed, it has been proposed that PLC and protein kinase C (PKC) activation are required for granulosa cell luteinization. The PKC activator phorbol 12-myristate-13-acetate induces morphological signs and a pattern of gene expression characteristic of luteinization in granulosa cells in vitro, and inhibitors of PKC block luteinization [41]. It should be noted, however, that species differences may be present as porcine granulosa cells are inhibited by PKC activation [42].
Regulation of Ca2+ levels is not unique to the LH receptor, as occupancy of the FSHR itself may be coupled to an influx of Ca2+. An increase in intracellular Ca2+ was observed in swine, rat, and avian granulosa cells stimulated with FSH [32, 43]. This conclusion is strengthened by similar findings reported for FSHR in Sertoli cells, the FSH responsive cells of the male gonad [44, 45]. The biochemical mechanisms causing this FSH-dependent Ca2+ influx are unclear. Inositol phosphate measurements in both granulosa and Sertoli cells also have been inconclusive. These conflicting results should not be construed as proof that Ca2+ signaling is irrelevant for granulosa cells. Several reports, including the knockout of a CaM kinase IV [46], clearly demonstrate that this pathway is critical for the function of these cells.
An interesting twist to this issue comes from a recent observation that alternate splicing variants of the FSHR may be expressed in ovaries and testes [47]. One of these variants, the FSHR3, is structurally distinct from the GPCR because it exhibits a growth factor type I receptor motif. Expression of this receptor in HEK293 cells leads to an FSH-stimulated Ca2+ entry and activation of the mitogen-activated protein kinase (MAPK) pathway. Although this provocative observation needs to be confirmed in other laboratories, it may explain some discrepancies between the effects of occupancy of the native FSHR and findings with reconstitution systems using full-length recombinant receptors.
Regulation of intracellular calcium concentration in granulosa cells by gonadotropins may not be directly dependent on the coupling of the receptors to a G protein that activates PLC (Fig. 2). For instance, it is possible that ß
subunits released from the activation of the heterotrimeric Gs may interact and activate PLCß [48]. In addition, accumulation of Ca2+ in granulosa cells may be distal to cAMP accumulation and dependent on the expression of other PLC isoenzymes regulated by other effectors (see below).
In summary, there is ample evidence in the literature that the two gonadotropin receptors may be coupled to different G proteins, particularly when using reconstitution systems. Nevertheless, it remains unclear whether coupling of the two receptors to different pathways indeed occurs in vivo under physiological conditions. Moreover, given the fact that occupancy of both receptors regulates intracellular Ca2+, perhaps by different mechanisms, it is unclear whether distinct signaling pathways are activated by the two receptors in immature and mature granulosa cells. Finally, more work needs to be done to determine whether these additional signaling pathways specifically impact gene expression in vivo during the growth and differentiation of the follicle.
Properties of the cAMP Signal Itself May Specify Distal Gonadotropin Effects: The Role of Phosphodiesterases
An alternative possibility to consider is that the cAMP signal itself encodes all the necessary information for the divergent effects of activation of this pathway during granulosa cell differentiation. According to this hypothesis, different patterns of gene expression and steroidogenesis are the result of a combination of subtle differences in the intensity, duration, and subcellular compartmentalization of the cAMP signal [49]. Such a possibility clearly has been established for Ca2+ signaling [50]. The most striking example of this is the finding that variations in the frequency and duration of Ca2+ spikes lead to divergent patterns of gene transcription and cytokine production in T cells [51]. In addition, the subcellular localization of PKA to discrete subcellular compartments and the presence of scaffold proteins support the view that cAMP signaling may be highly compartmentalized in the cell [52].
A survey of the data on the pattern of cAMP accumulation in granulosa cells indeed shows that the time course after FSH and LH stimulation differs in several characteristics. FSH stimulation of granulosa cells in vitro and possibly in vivo is biphasic [53, 54]. A first rapid and transient cAMP response is followed by a second slow rise that occurs between 24 and 48 h after FSH stimulation. Conversely, LH stimulation causes only a large transient increase that is followed by a profound desensitization of the cAMP-signaling pathway. In addition, it is commonly held that a modest but tonic FSH stimulation needs to be followed by a rapid, large, and transient LH spike in order to maximize follicular maturation and ovulation. Consistent with the requirement for a low but sustained FSH action, treatment with low continuous FSH doses is effective in inducing aromatase, whereas a single, surge dose produces ovulation and luteinization [23].
The temporal and spatial pattern of cAMP concentration in a cell is dependent on both the rate of synthesis by adenylyl cyclases and the rate of degradation by phosphodiesterases (PDEs). Although they have received little attention over the years, recent data have demonstrated that PDEs play a critical role in signaling and determining the cAMP response. It is therefore possible that expression and regulation of PDE activity may be critical in shaping the cAMP signal in granulosa cells and in contributing to the specificity of the gonadotropin responses.
Of the 11 families of PDEs identified in mammalian cells, members of at least four families are expressed in the follicle. The expression of PDE1, PDEs regulated by Ca2+ and calmodulin, has been identified by biochemical means in rat granulosa cells [54]. The cAMP-specific PDE4s are expressed in granulosa and theca cells but not in oocytes [55]. In addition, tissue surveys of the expression of novel PDE family members that belong to families 7 and 8 show mRNA expression in the ovary and in granulosa cells [56, 57]. Whereas the function and regulation of the newly discovered PDE7 and PDE8 are largely unknown and only are recently being explored, more is known about the properties of PDE4s. This family of PDEs is composed of four genes, and numerous splicing variants have been identified [58]. The PDE4D gene is expressed in mural granulosa cells, whereas expression of PDE4B is more prominent in theca cells [55]. At present, no data are available on whether PDE4A or PDE4C are expressed in mural granulosa cells or in cumulus cells.
Studies on endocrine and nonendocrine cells have shown that PDE4s are part of a feedback control of cAMP levels and are subject to both short-term and long-term regulation [59]. Activation of cAMP signaling causes an increase in PKA activity, which in turn phosphorylates and activates the long splicing variants of PDE4s [60, 61]. A more sustained cAMP accumulation instead causes activation of a cAMP-regulated promoter in PDE4B and PDE4D genes and accumulation of the short PDE4 variants [59]. More importantly, PKA and PDE4D are part of a signaling complex coordinated by the scaffold proteins termed A kinase anchoring proteins [62, 63]. The assembly of these complexes is probably critical for generating compartmentalized signals in the cell.
A first indication that PDE4s play a role in defining the cyclic nucleotide signal in granulosa cells comes from the observation that the activity of these enzymes is regulated by gonadotropins. Early studies by Schmidtke et al. [64] reported that the activity of PDE4 is regulated during the ovarian cycle in vivo. Moreover, stimulation of cultured rat granulosa cells with FSH causes a significant increase in PDE4 activity [54]. More recent studies from our laboratory have demonstrated that the PDE4D mRNA is induced by both FSH in preantral follicles and by LH in the periovulatory follicle in vivo (F. Richard, personal communication). As shown for other endocrine cells, only the mRNA coding for the short forms of PDE4D is induced by gonadotropins. Conversely, the mRNA coding for the long forms is unaffected or perhaps decreased. These data are similar to the regulation of PDE4D by FSH that has been extensively characterized in Sertoli cells [65], and demonstrate that PDE4D should be considered a bona fide gonadotropin-regulated gene. It should be noted that during the LH surge, the PDE4D mRNA levels increase rapidly, reach a maximum within 1 h, and decline thereafter. Thus this increase in PDE4D may be responsible for the transient nature of the cAMP signal induced by the LH surge.
Studies conducted in cell lines indicate that PDE4 regulation plays a critical role in defining the duration and intensity of the cAMP signal [58]. A major question that needs to be addressed is whether changes in the time, concentration, and spatial dimensions of this signal produce only quantitative changes in distal responses, including gene expression, or whether qualitative changes are induced. An initial evaluation of this issue comes from the analysis of the PDE4D knockout mice. A PDE4 role in female reproduction was first implied by studies on the "dunce" gene in Drosophila [66]. This gene codes for a PDE with all the features of a PDE4, and mutations of this gene in Drosophila produce sterile flies [67]. In the PDE4D-deficient mouse, pharmacological induction of ovulation by eCG-hCG treatment demonstrated a 70%–90% reduction in the rate of ovulation, depending on the age of the mice [68]. A reduction in the number of oocytes recovered in the ampulla was present whether mice were matched by age or weight, ruling out the possibility that a delay in puberty is the cause of the impaired ovulation. eCG-hCG treatment of adult PDE4D null females also uncovered a significant reduction in ovulation rate. Histological examination of adult ovaries from PDE4D null mice did not show major alterations, and all stages of follicle development and corpora lutea were present. In several instances, however, a predominance of small follicles was observed in PDE4D-/- ovaries [68]. Superovulation induction in immature PDE4D-/- females demonstrated the presence of numerous luteinized follicles with entrapped, often degenerating, oocytes, suggesting a defect in follicle rupture or a disruption of the synchronization between oocyte maturation and ovulation. This defect in the terminal differentiation of the follicle was confirmed by an in situ hybridization study to examine the pattern of gene expression at the transition between follicle maturation and luteinization. Although P27kip1 was correctly expressed at the appropriate times during follicle maturation or luteinization, expression of the P450scc steroidogenic enzyme was altered in the PDE4D null ovary, with precocious expression in preantral follicles [68] (F. Richard, personal communication). This observation suggests that the program of gene expression during follicle maturation is disrupted after inactivation of a PDE gene.
An additional assessment of the function of granulosa cells was obtained by in vitro stimulation for 1 h of antral granulosa cells derived from wild-type and PDE4D knockout (KO) mice. Granulosa cells from the PDE4D KO mice displayed a significant reduction in cAMP accumulation in response to FSH or hCG [68]. Conversely, the response to forskolin was normal or elevated when compared to granulosa cells from wild-type ovaries [68]. A significant increase in cAMP accumulation under basal conditions was observed in all experiments performed, a finding consistent with the increase in basal cAMP in lung and brain tissue of the PDE4D-/- mice. On the basis of these findings, it is hypothesized that a disruption of cAMP signaling in granulosa cells (increased basal/decreased responsiveness to hormone) is responsible for an altered pattern of gene expression and the impaired ovulation observed in the PDE4D null mice.
In spite of the fact that these data are preliminary and the phenotype of the PDE4D-/- mouse needs to be investigated further, these findings suggest that the intensity and duration of the cAMP signal may indeed be critical for the optimal FSH and LH signaling. The disruption in P450scc expression observed in the PDE4D-/- granulosa cells would be consistent with the idea that an altered FSH signal induces premature expression of genes that are normally activated later during the periovulatory period. It should be pointed out that treatment of granulosa cells with rolipram, a PDE4 inhibitor, has not uncovered major changes in gene expression [69]. However, the author has observed that in vivo administration of rolipram induces precocious luteinization.
Although the phenomenon of gonadotropin desensitization was first described in granulosa and luteal cells, its exact role in follicle maturation and luteinization remains unclear [70, 71]. It has been amply demonstrated that the LH surge causes a profound desensitization of the granulosa cells. Data from cell cultures and reconstitution systems show that this desensitization involves either LH receptor phosphorylation or activation of a small G protein Arf6, uncoupling from Gs, and recruitment of arrestins [72, 73]. Desensitization also involves steps distal to the membrane signal transduction pathway. For instance, a truncated dominant negative regulator of CREB-mediated transcription is rapidly induced during the LH surge [74]. This induction is bound to affect several cAMP-regulated genes, including inhibin . The gonadotropin-regulated expression of PDE4D may contribute to desensitization in granulosa cells, and it is possible that some of the granulosa cell phenotypes observed in the PDE4D null granulosa cells are due to disruption of this desensitization process.
Distinct Kinase Cascades Downstream of an Identical cAMP Signal Are Activated by the Two Gonadotropin Receptors
The last scenario that may account for the divergent effects of FSH and LH on granulosa cells is that the same cAMP signal activates distinct downstream signaling cascades in immature and mature granulosa cells. According to this view, the initial signal is common to both receptors, whereas the cellular context in which this signal develops is different, and different signaling cascades downstream of cAMP produce distinct patterns of transcription-factor phosphorylation and gene expression. It should be pointed out that the presence of these signaling networks may contribute to the specificity of cAMP signaling, and that PDEs may play a role controlling diffusion of cAMP, and therefore the access to different effectors. Several reports have indicated that the cAMP pathway in granulosa cells is connected to other signaling pathways, and therefore branching of the cAMP signal may dictate the ultimate outcome of the gonadotropin stimulation (Fig. 3).
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As previously discussed, it is possible that the Ca2+ influx measured in granulosa cells exposed to FSH or LH is distal to an increase in intracellular cAMP. Although this pathway has not been clearly charted in granulosa cells, a novel mode of Ca2+ regulation is mediated by cAMP and the Rap GTPase in HEK923 cells [75] (Fig. 2). This pathway is not mediated by PKA phosphorylation but by cAMP activation of the cAMP-GEF [75]. Rap and EPAC are indeed expressed in granulosa cells at least at the level of mRNA [76]. It remains to be determined whether the Rap2B-activated PLC-
is indeed expressed and whether this circuit is functioning in granulosa cells.
Following the development of sensitive immunological probes to investigate the state of activation of the MAPK pathway, it has been demonstrated that extracellular signal-regulated kinase (ERK) phosphorylation and activation follow gonadotropin activation of granulosa cells. Thus FSH rapidly stimulates ERK phosphorylation and activity 2- to 5-fold in the rat [77] and pig [78]. This stimulation is dependent on cAMP synthesis and PKA activation because forskolin mimics the FSH effects and the PKA inhibitor H89 blocks FSH stimulation. This activation may play a role in the regulation of granulosa cell replication. Less clear is the role in steroidogenesis, as it has been shown that in a granulosa cell line MAPK activation inhibits steroidogenesis [79].
The p38 mitogen-activated protein kinase (p38MAPK), an additional kinase cascade distinct from the RAF-MEK-ERK [80], is also activated by FSH [76, 81] (Fig. 3). There is a question, however, as to whether PKA is involved in this gonadotropin-dependent regulation, as in one report p38MAPK phosphorylation was sensitive to H89, whereas in a later report an increase in p38 phosphorylation, rather than a decrease, was observed [76, 81]. Although some caution needs to be exerted in using data with this not-entirely-selective PKA inhibitor, the different times at which the phosphorylation was measured (10 min vs. 1 h) may indicate that multiple pathways contribute to the regulation of this kinase. As for the significance of this regulation, it has been shown that p38MAPK inhibitors block the FSH-induced changes in the morphology of granulosa cells [81], thus suggesting that this pathway may be involved in the cytoskeleton rearrangements. The effects of LH on this pathway have also been reported [82].
An additional pathway that apparently is activated by gonadotropins via stimulation of cAMP production is the PI-3 kinase-signaling pathway (Fig. 3). Phosphorylation of several components of this pathway that are activated by insulin/IGF-1 and other growth factors is induced by FSH in rat granulosa cells in culture. PKB/Akt phosphorylation is increased several fold 30 min after FSH stimulation, and the activation of this kinase is inferred by an increased phosphorylation of GSK-3ß in granulosa cells [76]. Although this activation is reproduced by treating the cells with forskolin, PKA activation is not required because an even more robust phosphorylation of PKB is observed in the presence of the PKA inhibitor H89 [76]. These findings suggest that these gonadotropin effects are mediated by cAMP-GEF/EPACs, probably via regulation of the small G protein RAP1/2. The expression of cAMP-GEF in granulosa cells has been detected at the mRNA level; obviously much needs to be done to determine the exact role of this novel cAMP-binding protein in gonadotropin signaling. As mentioned above, this pathway may also be involved in Ca2+ regulation of granulosa cells via activation of a PLC. It is likely that activation of this pathway plays a role in granulosa cell replication and survival.
In addition to activating PKB/Akt, FSH regulates the expression and phosphorylation of a related kinase, the serum- and glucocorticoid-inducible kinase (SGK), an additional gonadotropin-inducible gene. SGK is one of the transcriptionally inducible, immediate-early genes expressed in epithelial cells and in fibroblasts [83, 84]. FSH regulates the expression of this kinase in a biphasic manner with rapid induction at 1–2 h followed by a decrease, then a sustained increase at 24–48 h. Although this kinase has properties and regulations similar to PKB/Akt [85] and has been implicated in aldosterone function [86, 87], its exact role in gonadotropin signaling remains unclear.
The above-described findings clearly demonstrate that activation of the cAMP pathway by gonadotropins branches into a myriad of signaling cascades that can potentially be linked to the regulation of gene expression. The challenge now is to determine whether these pathways indeed are responsible for the divergent effects of gonadotropins on the expression of different genes.
Concluding Remarks
The regulation of granulosa cell replication and differentiation by the gonadotropins FSH and LH provides an interesting model in which to study the specificity of cAMP signaling. Although most of the components of this signaling pathway have been characterized in great detail, a major unanswered question is how the two gonadotropin receptors that are coupled to a seemingly identical cAMP signal produce such a variety of responses. In this review, several hypotheses that may account for these divergent effects of gonadotropin receptor activation of cAMP signaling have been discussed. One possibility is that the two gonadotropin receptors are coupled to different heterotrimeric G proteins and therefore to more than one signaling pathway. Second, the specificity of cAMP signaling may reside in the spatial and temporal dimensions of the cAMP response. Finally, the cAMP signal originating from the two receptors may activate signaling cascades that are specific for the state of differentiation of granulosa cells. It is also possible that all three scenarios contribute to the specificity of the gonadotropin-induced cAMP signaling in these cells. Ultimately, where the specificity of gonadotropin signaling resides will be determined by connecting these multiple pathways to the regulation of genes expressed during follicle maturation and ovulation in vivo.
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FOOTNOTES |
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Received: 25 February 2002.
First decision: 12 April 2002.
Accepted: 19 June 2002.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONREFERENCES |
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