Intracellular signaling in the gonads.

0163-769X/92/1303-0476$03.00/0 Endocrine Reviews Copyright © 1992 by The Endocrine Society

Vol. 13, No. 3 Printed in U.S.A.

Intracellular Signaling in the Gonads* PETER C. K. LEUNG AND GILLIAN L. STEELE Department of Obstetrics and Gynecology, University of British Columbia, Vancouver, British Columbia, Canada V6H3V5

I. Introduction II. Gonadotropin Receptors in the Gonads A. LH receptors in the gonads B. FSH receptors in the gonads III. Intracellular Signaling: Adenylate Cyclase-cAMP Pathway A. LH-stimulated adenylate cyclase activity B. FSH-stimulated adenylate cyclase activity C. Other factors stimulating adenylate cyclase in the gonads 1. Adenosine 2. Vasoactive intestinal peptide (VIP) 3. GRF IV. PLC Pathway: Generation of Multiple Second Messengers A. GnRH stimulation of PLC 1. Stimulation of the PLC pathway in the ovary 2. Stimulation of the PLC pathway in the testis B. PGF2a stimulation of PLC C. LH stimulation of PLC 1. Stimulation of the PLC pathway in the ovary 2. Stimulation of the PLC pathway in the testis D. Other potential activators of the PLC pathway 1. Ang II 2. Tumor necrosis factor-a (TNFa) 3. CRF V. PLA2 Signaling Pathway A. GnRH stimulation of PLA2 1. Stimulation of PLA2 pathway in the ovary 2. Stimulation of PLA2 pathway in the testis B. PGF2a stimulation of PLA2 C. LH stimulation of PLA2 VI. PLD Signaling Pathway A. GnRH stimulation of PLD VII. Other Potential Mediators of Hormone Action in the Gonads A. Tyrosine kinase 1. EGF 2. TGFa B. Hormone-mediated ion channel gating 1. LH 2. FSH VIII. Summary

I. Introduction

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HE OVARY and testis share homology of structure and function in the production of gametes and in the regulation of steroidogenesis. The dependence of gametogenesis on local synthesis of steroids necessitates a greater understanding of which endocrine factors are involved, the mechanisms by which they act, and how they affect biosynthetic pathways. In view of the abundance of hormones involved in the regulation of steroid synthesis, the gonads provide an ideal model for the study of endocrine, paracrine, autocrine, and intracrine mechanisms. The aim of this review is to provide an updated examination of our knowledge of the factors involved in gonadal function and the intracellular signaling mechanisms by which they act. Both the ovary and the testis are characterized by two functionally separate compartments that facilitate the production of gametes and the synthesis of steroids and other gonadal hormones. The similarities of structure and function between theca interna and Leydig cells, and granulosa and Sertoli cells are well recognized (1-3). As illustrated in Fig. 1, granulosa (in ovary) and Sertoli cells (in testis) are separated from the theca interna and Leydig cells, respectively, by a basal lamina. They share in the ability to synthesize and secrete proteins and steroid hormones in response to FSH and to form a diffusion barrier between germ cells and substances in blood and lymph. Theca interna and Leydig cells have the common ability to synthesize and secrete steroids in response to peripherally derived LH. The absolute interdependence of these neighboring cell types in both tissues is well recognized. In the testis, synthesis of testosterone by Leydig cells is crucial to the function of Sertoli cells in spermatogenesis. The concept of regulation of Leydig cell function by Sertoli cells is a relatively new one. As will be discussed later, several Address requests for reprints to: Peter C. K. Leung, Ph.D., Department of Obstetrics and Gynaecology, University of British Columbia, Grace Hospital, Vancouver, British Columbia, Canada. *This work was supported by the Medical Research Council of Canada.

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INTRACELLULAR SIGNALING IN THE GONADS

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peptides synthesized in Sertoli cells have been shown to modulate Leydig cell steroidogenesis. In the ovary, theca interna and granulosa cells are also dependent on one another for coordinated steroid synthesis. Androgens synthesized de novo in theca interna cells diffuse into granulosa cells where they are metabolized to estrogens (1, 4). Analogous to the testis, factors originating in granulosa cells are implicated in a feedback regulation of androgen production in neighboring theca interna cells. Thus, the combined steroidogenic capacity of theca interna and granulosa cells is crucial for gametogenesis. It allows for sustenance of a developing follicle and its transformation into a dominant follicle destined for ovulation (2). Normal function of both the ovary and the testis is long recognized to be dependent on the pituitary-synthesized gonadotropins (LH and FSH). These gonadotropic hormones are glycoprotein dimers, which share a common a-subunit (5). The regulation of steroidogenesis by LH and FSH in the ovary and testis is precisely coordinated with the steroidal milieu, which maintains an intricate balance of feedback interactions. Primarily, LH acts on theca interna and Leydig cells, while FSH regulates the function of Sertoli and granulosa cells (Fig. 1). In light of their responsiveness to common hormonal regulators, it is not surprising that the ovary and testis also share similarities in the distribution of receptors in their complementary cell types. Receptors for LH are present in both Leydig (6) and theca interna cells (7), while FSH receptors have been localized to Sertoli (8) and granulosa cells (9). At certain stages of the follicular cycle, granulosa cells also acquire LH receptors in response to stimulation by FSH and estrogen (10). Control

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of both ovarian and testicular hormonogenesis by LH and FSH has been well documented (1, 4,11). In addition to similarities between ovary and testis with regard to their endocrine regulation by peripherally synthesized hormones, there is considerable homology between these tissues in their intragonadal regulation of steroidogenesis. Many locally synthesized growth factors, peptides, and steroid hormones have been implicated in normal function of both the ovary and the testis. Some of these include GnRH, prostaglandin F 2a (PGF 2 J, prostaglandin E2 (PGE2), angiotensin II (Ang II), inhibin, activin, transforming growth factor (3 (TGF/3), epidermal growth factor (EGF), GRF, catecholamines, adenosine, insulin-like growth factor, and interleukin-1. It is obvious that such an abundance of regulators results in an intricate control of tissue function. Peripheral and local hormones involved in the regulation of gonadal steroidogenesis employ a number of recognized intracellular signaling mechanisms to initiate their effects. Some hormones have been reported to stimulate more than one signaling pathway. The recognized pathways and their second messengers include adenylate cyclase (cAMP), phospholipase C (PLC) [inositol phosphates, diacylglyceride (DAG), and arachidonic acid], phospholipase A2 (PLA2) (arachidonic acid and its metabolites), and phospholipase D (PLD) [phosphatidic acid (PA) and DAG]. Hormonal regulators of gonadal function will be discussed in the following sections with regard to their receptors, signaling mechanisms, and their effects on cellular function and steroidogenesis.

II. Gonadotropin Receptors in the Gonads A. LH receptors in the gonads

Endocrine Cells

Blood vessel Germ Cells

Ovarian Follicle Seminiferous Tubule FIG. 1. Similarities in the action of LH and FSH in the ovary and the testis. LH stimulates androgen production by the theca interna, cells lining the ovarian follicle which lie adjacent to the basal lamina. LH exerts a similar endocrine effect on the Leydig cells that line the seminiferous tubule. In both the ovary and the testis, FSH controls granulosa and Sertoli cell function.

The initial purification of LH receptors from rat ovary by different groups resulted in its characterization as either a 75 K (12), 90 K (13), or a 92 K polypeptide (14). Further characterization revealed the native receptor to be predominantly N-linked, glycosylated, and comprised of two identical hormone-binding subunits associated as a dimer by covalent interactions (15). Based on the predicted sequence of purified LH receptor, a DNA probe was generated in a polymerase chain reaction and a complementary DNA isolated from the rat luteal receptor (16). Sequence analysis revealed a 26-residue signal peptide, a 341-residue extracellular domain, and a 333residue region consisting of seven transmembrane segments. Interestingly, the extracellular domain contained an internal repeat structure characteristic of the leucinerich glycoprotein (LRG) family, while the transmembrane-spanning region bore sequence similarity to the G protein-coupled receptor genes (e.g. TSH receptor). Consequently, the LH receptor is postulated to have evolved


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from a recombination of the LRG and G protein-coupled receptor genes. Another distinctive feature of the LH receptor is that its gene has the apparent potential for alternative splicing, which results in diverse transcripts (17). In the rat testis, an LH receptor has been identified with a 266 base pair deletion resulting in the loss of the first transmembrane domain. Surprisingly, this receptor bound LH with high affinity and specificity when expressed in COS 1 cells. Accordingly, hormone binding may be associated with the amino-terminal extracellular region. Unlike the LH receptor, most other G protein-coupled receptors lack introns and contain their binding sites in the transmembrane region. Using probes specific for the LH receptor, multiple messenger RNA species of variable abundance have been identified in gonadal tissues of such closely related species as rat and mouse (18). In spite of this diversity, the size of the cell surface receptor expressed in these tissues has been shown to be identical. Comparison of rat ovarian, bovine luteal, and mouse Leydig LH receptors by ligand blotting indicates a high degree of receptor homology between tissues and among species (13). Purification of human LH receptor from corpus luteum has similarly revealed a polypeptide of comparable molecular weight to that of rat and bovine receptors (19). Simultaneous purification of ovarian and Leydig cell LH receptors revealed a 10 K size difference, which was accounted for by their property of existing as N-linked sialoglycoproteins (20). Evidently, the LH receptor gene is highly conserved in the mammalian world. After receptor binding by an agonist, a conformational change in the LH receptor has been shown to facilitate its phosphorylation during the initial stage of binding; prolonged exposure to the agonist reduced the rate of phosphorylation (21). The latter may act as a mechanism of receptor down-regulation. cAMP-dependent protein kinase A (PKA) catalyzes receptor phosphorylation at serine and threonine residues. Clustering and/or crosslinking of LH receptors has been postulated to favor the initial signaling mechanisms involved in the action of LH, which may be promoted by aggregation or dimerization of the receptor in the membrane (20). LH receptor content in the ovary and testis appears to be profoundly influenced by the hormonal milieu. Accordingly, there is variation in LH receptor content of these tissues at different stages of development and during various stages of the follicular and spermatogenic cycles. The significance of the variation is reflected in that LH receptor induction is a prerequisite for ovarian follicles to ovulate and form corpora lutea. In the rat ovary, small antral follicles have low expression of LH receptor, while the growth of preovulatory follicles is associated with an increase in all LH receptor mRNA transcripts (22, 23). The LH surge in vivo resulted in a

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rapid decline in LH receptor mRNAs in these follicles, an observation that could be mimicked in vitro. Corpora lutea exhibited low LH receptor mRNA content, which was increased markedly by exogenous treatment with PRL, or in the event of pregnancy. Similarly, receptor binding affinity of corpora lutea has been shown to increase significantly in response to human (h) CG treatment (24). Evidently, the regulation of LH receptors in the ovary during follicular growth, ovulation, and luteinization is due, at least in part, to modulation of receptor message (25, 26). In human corpus lutea, LH receptors exhibit a corresponding flux depending on the stage of the luteal phase (27). Using radiolabeled-hCG as a measure of receptor availability, both total and unoccupied LH receptor levels have been found to parallel progesterone secretion. While changes in the binding affinity of these receptors was suggested to be important for sustaining and/or rescuing the corpus luteum, receptor loss may be associated with luteolysis. Treatment of rat ovarian cells with various gonadotropins and growth factors in culture has proven to be an effective model for the study of LH receptor gene regulation. All gonadotropins tested (FSH, LH, and PRL) stimulated LH receptor formation in cultured granulosa cells, while EGF, fibroblast growth factor, and GnRH had inhibitory effects (28). The influence of these hormones on LH receptor content was again correlated with their regulation of receptor mRNA levels. Presumably, LH receptor content and availability are regulated by many hormonal factors, which may act by endocrine, paracrine, autocrine and/or intracrine mechanisms. In the testis, as in the ovary, the LH receptor appears to be similarly influenced by its hormonal milieu. LH receptors are induced by the agonist itself, an effect that is rapid, and are dependent upon preexposure to PRL (29,30). Further, a brief refractory period following PRL exposure has been identified during which time LH upregulation of the receptor does not occur. PRL mediation of LH receptor induction is specific and cannot be mimicked by FSH, GH, testosterone, or estrogen. However, both testosterone and estrogen demonstrated the ability to inhibit PRL-mediated LH induction of its homologous receptor (31). In addition to the stimulatory effect of LH on induction of its own receptor, LH also causes a refractoriness or desensitization of the steroidogenic response. This is at least partly attributed to the loss of LH receptors (32-35). Studies of the actual binding of LH to its membrane receptor in Leydig cells demonstrated a rapid internalization of the hormone-receptor complex (36). However, recycling of the receptor to the cell surface also occurred, which may be essential in maintaining the capacity to bind fresh hormone. Internalization of unoccupied receptors has also been demonstrated (37). This down-regulation of receptors occurs


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under the influence of physiological concentrations of the hormone and is likely necessary to maintain the normal response of Leydig cells to LH in vivo. In the fetal rat testis, regulation of LH receptors appears to differ from that of the adult (38). In both in vivo and in vitro studies, treatment with high doses of LH did not induce down-regulation of the LH receptor as seen in the adult animal. Similarly, neonatal rats treated with a single dose of hCG demonstrated a rapid proliferative response and LH receptor replenishment (39). B. FSH receptors in the gonads The FSH receptor purified from membranes of calf bovine testis was determined to be an oligomeric glycoprotein consisting of four disulfide-linked monomers, each 60 K (40). Recent progress in molecular biology has allowed for the cloning and sequencing of FSH receptors from both rat and human. The amino acid sequence of the FSH receptor has been deduced, allowing for the characterization of the FSH receptor as a 75 K polypeptide with a 348 residue extracellular domain (41). The latter is connected to a structure composed of seven putative transmembrane segments with homology to G protein-coupled receptors. Further, the rat FSH receptor displayed considerable sequence similarity to the rat LH receptor. The FSH receptor cloned and sequenced from a human cDNA library exhibited 89% homology with the cloned rat receptor, with the most highly conserved regions being the putative transmembrane segments (42). FSH receptors do not appear to be down-regulated by high levels of the ligand, and evidence points to the presence of a receptor pool in the adult rat testis (43). However, other factors may regulate ligand-hormone binding. Tight junctions formed between Sertoli cells during the peripubertal period may form a barrier to circulating FSH in the adult testis. Further, inhibitors of FSH receptor binding may exist. Low molecular weight inhibitors of FSH binding have been isolated from a variety of gonadal extracts, the release of which appears to be PLC-linked (44). Stimulation of FSH receptor binding and corresponding increases in adenylate cyclase activity appear to be achieved by some divalent cations (45). Enhanced FSH receptor binding in ovarian granulosa cells has been shown to be a result of an increase in apparent binding sites. Such intragonadal factors may allow for the local regulation of FSH activity. In the male rat, expression of FSH receptor mRNA has been localized to the Sertoli cell (46). Further, during the cycle of the seminiferous epithelium, the levels of receptor expression changed more than 3-fold. In the developing testis the number of FSH receptors increases markedly, an effect that may be partly attributed to the

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influence of testosterone and FSH itself (47). This increase in FSH receptors is partly reflected by a proliferation of Sertoli cells during the first 2 postnatal weeks, and a differentiation of these cells thereafter (48). Studies of FSH receptors in testes and ovaries of human and rhesus monkey fetuses have shown that primate fetal testis may be responsive to FSH stimulation during the first half and again at the end of gestation (49). However, this response did not involve the acute elevation of cAMP. In primate ovary, FSH receptors did not appear until late gestation, suggesting that this tissue is unresponsive to FSH during earlier stages of fetal development.

III. Intracellular Signaling: Adenylate CyclasecAMP Pathway The gonadotropins are critical regulatory hormones involved in gonadal steroidogenesis which are best recognized for their stimulation of the adenylate cyclasecAMP pathway via activation of a Gs protein. The G proteins are heterotrimers made up of a-, /?-, and ysubunits, but function as heterodimers because of a strong association of the 0- and 7-subunits (50-52). Activation of G8 occurs when agonist occupies the receptor, releasing GDP from the G protein and allowing GTP to bind in its place. Activation triggers the a-subunit to dissociate from the /37-subunits. When bound to GTP, the a-subunit of G8 is a potent activator of adenylate cyclase. The a-subunit also exhibits intrinsic GTPase activity which hydrolyzes the bound GTP to GDP. The hydrolysis of GTP may act as a timing mechanism that determines the duration of activation. The activation of adenylate cyclase results in the hydrolysis of ATP to cAMP, which subsequently activates PKA in the cytoplasm. The activation of this kinase represents the final step in the signalling pathway, resulting in the phosphorylation of proteins involved in steroidogenesis. A. LH-stimulated adenylate cyclase activity The participation of the adenylate cyclase-cAMP signalling pathway in the action of LH in the gonads is well accepted. LH activates steroidogenic enzymes which catalyze the conversion of cholesterol to C2i and C19 steroids (see Ref. 4 for review). The binding of LH receptors is associated with increased levels of cAMP (53-55), activation of PKA (56-58), and phosphorylation of at least six different proteins (59). Cells engineered to express LH receptor cDNA bind hCG and have been shown to increase cAMP when exposed to the hormone (16). The stimulatory G protein (Gs) is believed to mediate LH action on adenylate cyclase evidenced by increased cAMP production and steroidogenesis in response to cholera toxin in intact Leydig cells (60). Similarly, the


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presence of GTP enhances LH stimulation of these parameters. Direct evidence for the presence of both Gs and Gi in the testis has been obtained by the purification of membrane proteins from Leydig cells. Substrate for both cholera toxin and pertussis toxin were found, which corresponded with the known molecular weights of their respective G protein a-subunit substrates (61). Additional evidence for the involvement of cAMP in LH action lies in the stimulation of testicular cAMP generation and androgen biosynthesis by forskolin, a stimulator of the catalytic domain of adenylate cyclase (55). Further, the action of LH on steroidogenesis was amplified by forskolin. Contrary to the stimulatory effect of LH on adenylate cyclase activity, continued exposure of both ovarian and testicular receptors to this hormone results in a refractoriness or desensitization of the response. In addition to the down-regulation of receptors previously mentioned, there may be an uncoupling of LH from adenylate cyclase (62, 63), increased cAMP degradation by the action of phosphodiesterase (64) and/or a decrease in the activity of certain steroidogenic enzymes (65). Although somewhat paradoxical, the down-regulation of receptors in conjunction with desensitization of adenylate cyclase may be necessary to maintain the normal response to LH in view of the low number of LH receptors (37). Evidence suggests the desensitization of adenylate cyclase activity results from a lesion between the LH receptor and G8, while G8 remains coupled to the adenylate cyclase catalytic subunit (61). In luteinized rat ovary, several monovalent cations (sodium, lithium, and potassium) have been shown to inhibit adenylate cyclase activity, an effect that is enhanced by the presence of LH or GTP (66). This effect was attributed to a dissociation of G8 from the catalytic component of adenylate cyclase and an independent reduction of LH receptor affinity for its ligand. The divalent cation magnesium has also been shown to modulate transduction of the LH signal (67). This was particularly apparent in conjunction with structurally modified agonists. Second messengers have also been implicated in the control of steroidogenesis by LH. In theca cells of the hen, arachidonic acid has been shown to modulate both basal and LH-stimulated steroid production (68). LHinduced androstenedione synthesis was inhibited by exogenous arachidonic acid, due to effects both before and subsequent to cAMP formation. This inhibition could be mimicked by PLA2 and was not blocked by the addition of inhibitors of the cyclooxygenase or the lipoxygenase pathway (indomethacin and nordihydroguaiaretic acid, respectively). Others have observed a biphasic effect of arachidonic acid on LH-induced cAMP accumulation (69).

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Calcium is another modulator of LH action. This ion appears to play a significant permissive role in the stimulation of adenylate cyclase by LH in the ovary of several species (70-72). In human granulosa-luteal cells, a dependence on exogenous calcium may be somewhat variable depending on exposure of the cells to other hormones (71). In theca cells of the domestic hen, the mobilization of calcium is critical for agonist stimulation, as evidenced by the attenuation of LH steroidogenic activity by the addition of an extracellular calcium chelator (EGTA), an intracellular calcium inhibitor (TMB8), or the calcium channel blocker verapamil (72). Verapamil was also effective in inhibiting androstenedione production in response to forskolin and 8-bromo-cAMP. The permissive effect of calcium appears to be mediated by calmodulin, as demonstrated with the use of calmodulin inhibitors (73). The development of transmembrane signaling in response to LH has been investigated in the fetal Leydig cell (74). The ability of (Bu)2cAMP, cholera toxin, and hCG to stimulate testosterone production in fetal rat Leydig cells was evaluated at various stages of gestation in order to examine each moiety in the transmembrane signalling system. The LH receptor responded to LH with an increase in cAMP and testosterone production as early as 15.5 days. Despite the capacity of Leydig cells to respond to LH at this stage of development, the point at which the LH receptor becomes functional is not clear. B. FSH-stimulated adenylate cyclase activity The stimulation of aromatase activity and induction of progesterone synthesis by FSH has been comprehensively reviewed (4). The interaction of FSH with its receptor leads to increased cAMP levels in both granulosa (75, 76) and Sertoli cells (77-80). However, stimulation of the adenylate cyclase pathway by FSH may be modulated by a variety of other factors. In porcine granulosa cells, a partially purified fraction of follicular fluid, termed follicle regulatory protein, has been shown to inhibit FSH responsive adenylate cyclase activity (81). The characteristics of FSH stimulation itself can also affect the cAMP response. In immature porcine granulosa cells, the cAMP response has been shown to be dependent on the dose, duration, and frequency of exposure to FSH (82). Using a perifusion system to simulate the transient exposure of hormones to the ovary, short (15 min) pulses of the hormone (150 ng/ml) with a pulse interval of 2-3 h were found to be optimal. Under conditions of continuous exposure of the cells to high concentrations of FSH, a transient refractoriness to the hormone was observed. This was attributed to an alteration of the interaction between the FSH receptor and


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the guanine nucleotide regulatory component of adenylate cyclase. The cationic environment for granulosa cells has also proven to be critical to the function of the FSH receptor and adenylate cyclase function. Magnesium, calcium, manganese, and potassium, but not sodium, enhance the binding of FSH to its membrane receptor in porcine granulosa cells by an increase in the apparent number of binding sites (45, 83). Further, sodium, magnesium, and potassium were effective in enhancing FSH-induced cAMP production. In the rat granulosa cell, FSH regulation of steroidogenesis has been shown to be regulated itself by calcium and calmodulin-dependent mechanisms (84). Using each of the intracellular calcium inhibitors (lanthanum, verapamil, and EGTA) or the calmodulin inhibitor trifluoperazine, FSH-stimulated progesterone biosynthesis was inhibited. The dependence of FSH action on calcium and calmodulin was found to exist at sites both at the level of cAMP production as well as one(s) distal to its formation. The participation of a calcium-calmodulin system in FSH action appears to be independent of the stage of follicular maturation and cellular differentiation (85). In the testis, FSH receptors have been shown to be physically and functionally associated with a guanine nucleotide regulatory protein (G8) in rat, bovine, and human tissue (40,86). This G protein exhibits two classes of binding sites for GTP: high affinity and low capacity, as well as low affinity and high capacity binding sites. Although only the high affinity binding sites appear to be required for the activation of adenylate cyclase, both binding sites have the capacity to bind GTP (86). Therefore, the low affinity binding sites may regulate FSH binding despite their lack of involvement in the subsequent signaling pathway. The involvement of GTP and GDP in intracellular signaling via G protein modulation of adenylate cyclase has also been investigated (87). Thus, the activation of FSH-sensitive adenylate cyclase is dependent on several factors; the higher affinity of the G protein for GTP than GDP, enhanced release of GDP when FSH is present, and the hydrolysis of GTP coupled to a high rate of guanine nucleotide metabolism. In studies of cultured Sertoli cells, basement membrane substrates induced amplified cAMP levels in response to FSH, which was attributed to enhanced Gs complex of adenylate cyclase (88). Perhaps even the morphology of these cells is important for intracellular signaling mechanisms. The observation of a synergistic effect of pertussis toxin on FSH activity in hamster Sertoli cells suggests that the adenylate cyclase is under tonic inhibition (89). Endogenous adenosine has been implicated in this role due to the observed inhibitory effect of exogenous adenosine and the stimulatory effect of adenosine deaminase

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on FSH-induced cAMP accumulation. Further, cholera toxin and forskolin enhanced FSH-stimulated cAMP equally (90). In the same study, an inhibitory effect of the phorbol ester 12-O-tetradecanoyl phorbol-13-acetate (TPA) on FSH-stimulated cAMP was shown, suggesting a possible regulatory role for protein kinase C (PKC) in this pathway. Using various concentrations of a specific cAMP antagonist, dose-dependent decreases in FSH-stimulated progesterone accumulation have been demonstrated (91). However, the inability of this antagonist to completely arrest FSH action led to the hypothesis that other second messengers may also be involved. The possibility of additional FSH signaling mechanisms will be discussed in subsequent sections. C. Other factors stimulating adenylate cyclase in the gonads 1. Adenosine. There is evidence that adenosine may act both as substrate to intracellular metabolism, as well as an adenosine A2 receptor agonist in granulosa and luteal cells (92). Binding of the adenosine A2 receptor by adenosine stimulates adenylate cyclase (93, 94). In luteal and granulosa cells, adenosine increases cAMP only in the presence of gonadotropins, an effect that is blocked by a specific adenosine receptor antagonist. Further, adenosine increases granulosa cell ATP levels while gonadotropins decrease ATP in the presence or absence of adenosine. Accordingly, a positive feedback loop model has been suggested whereby gonadotropins stimulate extracellular adenosine accumulation which acts on membrane A2 receptors to replenish depleted intracellular ATP levels (92). Therefore, a dual role of adenosine in the adenylate cyclase-cAMP pathway may be critical for the action of both FSH and LH in the ovary. 2. Vasoactiue intestinal peptide (VIP). The finding that VIP-containing nerve fibers innervate the ovary (95, 96) has prompted investigations to postulate a role for this peptide in ovarian function. Indeed, there is evidence for the direct stimulation of progesterone, 20a-hydroxyprogesterone, and estradiol by VIP in isolated granulosa cells (97). It is interesting that VIP facilitates steroidogenesis early in granulosa cell development, based on the inability of most ovarian regulators (other than FSH, GnRH, or cAMP activators) to exert a similar stimulation at this time. The effects of VIP are specific; only the most homologous peptide from this family of glucagon-secretin peptides is able to partially reproduce VIP's actions (96, 97). The stimulation of cAMP accumulation in response to VIP is recognized in many other systems (98). This signaling mechanism appears to be functional in the ovary also, based on an observed enhancement of VIP


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activity in the presence of a phosphodiesterase inhibitor (97). Further, cAMP levels are increased in conjunction with VIP-induced steroid secretion (97, 99). The effects of VIP on steroidogenic enzymes (97,99,100) and steroid secretion (97, 99) may be reproduced by activation of adenylate cyclase, additional evidence for the involvement of this signaling pathway in the action of the hormone. 3. GRF. GRF is another hypthalamic peptide that appears to be synthesized locally in the gonads. hGRF gene expression was examined in transgenic mice using an anti-hGRF serum that does not recognize endogenous mouse GRF (101). Among other tissues, hGRF immunoreactivity was detected in both the ovary and the testis, localized specifically in the oocytes and Leydig cells, respectively. In ovaries from ovulating women, immunoperoxidase staining revealed GRF immunoreactivity in corpora lutea, while granulosa and theca cells did not stain (102). In the same study, GRF immunoreactivity in the testes of post-pubertal men was localized in the Leydig cells. In the rat testis, GRF may be localized in mature sperm forms, within the confines of the bloodtestis barrier (103). Immunocytochemical studies of GRF have been complemented with molecular studies, revealing potential differences between gonadal GRF and the hypothalamic peptide. Although GRF in the testis and hypothalamus share some structural and functional properties, there are differences in the electrophoretic mobility, HPLC retention time, and molecular size (103). The mRNA for testicular GRF is substantially larger than its counterpart in the hypothalamus, as revealed by Nothern blot analysis (104) and reverse phase HPLC (105). The identification of GRF in the gonads, both at the level of protein product and gene transcript, suggests a role for the peptide in paracrine or autocrine regulation of gonadal function. There is evidence for this in the ovary: GRF is believed to promote follicular maturation and ovulation by acting directly on granulosa cells. GRF is structurally related to VIP and shares a common receptor in the ovary (106). Receptor binding of GRF stimulates cAMP formation and potentiates FSH-induced cAMP and the subsequent actions of FSH in follicular development. Thus, GRF may add to the array of paracrine modulators involved in the regulation of ovarian steroidogenesis. Potential effects of GRF in testicular physiology have yet to be elucidated.

IV. PLC Pathway: Generation of Multiple Second Messengers Many hormone receptors are coupled to G proteins that promote the activation of PLC in the event of ligand binding. Molecular studies to date have revealed at least

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five distinct PLCs (107, 108). PLC hydrolyzes membrane-bound polyphosphoinositides, the products of which act as second messengers for the mobilization of calcium and activation of calmodulin-dependent enzymes and PKC. The topic of various second messengers derived from inositol lipids has been reviewed extensively (e.g. Ref. 109). Briefly, PLC activation results in a rapid metabolism of phosphatidlyinositol-4,5-bisphosphate [Ptdlns(4, 5)P2], resulting in the formation of inositol1,4,5-trisphosphate [Ins(l, 4, 5)P3] and DAG. Ins(l, 4, 5)P3 releases calcium from intracellular stores following binding to specific receptors (110), generally thought to be restricted to specialized regions of the endoplasmic reticulum (111). Controversy remains as to whether Ins(l, 4, 5)P3 may also bind to the plasma membrane (110), and to calsequestrin-containing organelles (termed "calciosomes") to stimulate calcium release (112). The observation of simultaneous generation of an Ins(l, 4, 5)P3 isomer, Ins(l, 3, 4)P3, raised questions as to the function of this apparently inactive metabolite (113). Moreover, a pathway involving the phosphorylation of Ins(l, 4, 5)P3 to Ins(l, 3, 4, 5)P4, and subsequent conversion to Ins(l, 3, 4)P3 was discovered (114). Ins(l, 3, 4, 5,)P4 may itself contribute to the generation of a calcium signal either independently (115) and/or in conjunction with Ins(l, 4, 5)P3 (116). However, the significance of this auxiliary pathway is not clear. Receptors for Ins(l, 4, 5)P3 bind with high specificity and affinity and have been estimated to stimulate the release of at least 20 calcium ions (117). The rapidity and extent of the initial calcium response may be, at least in part, attributable to a positive feedback action of calcium on the activity of PLC (118). Additional steps in the degradative and synthetic pathways of inositol metabolism include the generation of additional putative signal molecules, InsP 5 and InsP6, before recycling of inositol into membrane polyphosphoinositides (119). A second branch of the PLC pathway originates from the generation of DAG. DAG regulates the activity of the PKC family of calcium/phospholipid-dependent enzymes by controlling their affinity for calcium and for phosphatidylserine (120). At least seven subspecies of PKC have been reported (121). Simultaneous elevation of cytosolic calcium levels by Ins(l, 4, 5)P3 can enhance the activity of suboptimal concentrations of DAG (122). Phorbol esters, compounds that bear physical homology to DAG, are useful tools for the study of hormones thought to act through the stimulation of the PLC pathway. These compounds are tumor promoters, which exert their biological action by binding to the regulatory region of PKC, causing activation of the enzyme. In addition to the positive actions of PKC by the phosphorylation of cellular proteins, it may exert negative feedback through the inhibition of polyphosphoinositide hydrolysis and


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the promotion of Ins(l, 4, 5)P3 metabolism (122). Further, PKC may affect the response of the cell to hormonal stimuli over the long term through the phosphorylation of membrane receptors, altering their affinity for ligand binding. In addition to the activation of PKC, DAG may be hydrolyzed by DAG lipase resulting in the generation of arachidonic acid, another second messenger. Although arachidonic acid appears not to play a significant role in the PLC pathway, its regulatory capacity should not be overlooked. This second messenger will be discussed further with regard to PLA2. Different hormones may differentially stimulate inositol metabolism and DAG activity, resulting in variable signaling responses to a common receptor binding event. Cross-talk between these pathways, itself influenced by factors such as intracellular calcium levels, further negates a characteristic response of the PLC pathway to different hormonal stimuli. This complexity will be examined with relation to gonadal regulators thought to stimulate PLC activity. A. GnRH stimulation of PLC 1. Stimulation of the PLC pathway in the ovary. GnRH is primarily recognized for its regulation of LH and FSH release from the pituitary. However, it is also thought to be an important paracrine/autocrine regulator in the gonads. Although a GnRH-like peptide isolated from ovarian extracts (123) was identified as a histone protein (124), transcription of the GnRH gene has been confirmed in this tissue (125-127). The identification of ovarian GnRH receptors and evidence of direct effects on steroidogenesis (see Ref. 128 for review) lends credence to its putative role as an intraovarian hormone. GnRH has divergent effects on progesterone production in the ovary. Basal steroidogenesis is enhanced by GnRH while gonadotropin-induced cAMP and steroid production are attenuated by the hormone (128, 129). The response of granulosa cells to GnRH appears to be affected both by the duration of treatment and the maturation stage of the follicle (130). In porcine granulosa cells, GnRH receptors have been reported to be restricted to specific follicles, suggesting a role for the hormone only at certain stages of development (131). GnRHinduced blockade of FSH action has been attributed to both increased degradation and decreased production of cAMP (132, 133). Further, it may directly inhibit steroidogenic enzymes (134). These effects of GnRH appear to be mediated by the PLC pathway. The involvement of inositol metabolism in the action of GnRH in the ovary was first demonstrated using the technique of radiolabeling of phospholipids (135-137). Binding of the membrane receptor induced a rapid in-

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corporation of radioactivity in PA and phosphatidylinositol (PI), but not other phospholipids. A specific GnRH antagonist was an effective blocker, and other ovarian hormones (hCG, PRL, and FSH) did not appear to induce the same response. Concurrent exposure of granulosa cells to GnRH and 17/3-estradiol resulted in an augmented response, with respect to phosphoinositide turnover, suggesting a local autoregulatory effect of estrogens on their own production (138). Time course studies revealed the accumulation of labeled InsP3 and InsP2 as early as 30 sec after GnRH treatment, with significant labeling of PI only after 2 min (139). This is consistent with the chain of events in inositol lipid metabolism, proposed as the early intracellular signaling mechanism of GnRH (140-142) (Fig. 2). An involvement of the PLC pathway in the action of GnRH is further supported by the dependence of GnRH on calcium as an intracellular mediator (143). The introduction of fluorescent calcium probes enabled studies on the effect of GnRH on intracellular calcium levels [Ca2"1"];. Rapid increases in [Ca2"1"];, as measured by Quin II (144) and Fura II (145), were blocked by a specific GnRH antagonist. The stimulatory effect of GnRH on basal progesterone production, and its inhibitory effect on FSH- and cAMP-stimulated progesterone production, can be mimicked by the addition of calcium ionophore A23187 to granulosa cultures (146, 147). Further, a reversal of the GnRH-induced response was achieved using calcium channel blockers (verapamil and La3+), calcium chelators (EGTA and EDTA), and the calmodulin antagonist, trifluoperazine (143, 146, 148). The inhibitory effect of calcium channel blockers suggests an involvement of extracellular calcium in the GnRH action. The generation of DAG in granulosa cells in response to GnRH treatment has also been confirmed (142, 144). This allows for the stimulation of PKC and phosphorylation of proteins. The potential role for PKC in ovarian steroidogenesis is supported by the identification of PKC activity in ovarian tissue (149,150). Further, the phorbol ester TPA has been used to induce similar effects on steroidogenesis as observed with GnRH (151-153). An antagonist of PKC (H7) was partially effective in blocking the stimulatory effect of GnRH on steroidogenesis. In addition to the stimulation of basal progesterone production, TPA also acts synergistically with the calcium ionophore A23187 to enhance PGE2 production in rat granulosa cells (154,155). In addition to the generation of the calcium-mobilizing inositol phosphate (s) and the PKC-activator DAG, GnRH has also been reported to cause an accumulation of arachidonic acid in ovarian granulosa cells (156,157). Arachidonic acid may be generated via two different pathways: either by the consecutive action of PLC and DAG lipase, or through its liberation by PLA2 from the


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FIG. 2. Signaling mechanisms of GnRH action in the gonads. Three pathways appear to be involved: PLA2, PLC, and PLD. The stimulation of PLC activity results in the hydrolysis of polyphosphoinositides, generating IP3 and DAG. The former is responsible for the release of Ca2+ from intracellular sources, and the latter stimulates PKC activity. The stimulation of PLD activity results in the liberation of PA, which may contribute to PKC activation by its conversion to DAG. Further, PA has been implicated in Ca2+ regulation. The stimulation of PLA2 by GnRH results in the generation of arachidonic acid (AA). AA acts as substrate for the generation of lipoxygenase metabolites, which may also mediate the action of GnRH.

sn-2 position of several phospholipids (158). In many tissues, a single extracellular messenger may simultaneously stimulate the activity of PLC and PLA2. There is evidence that GnRH activates both pathways in the granulosa cell, resulting in the liberation of arachidonic acid (157,159). Stimulation of the PLA2 pathway will be discussed in a later section. 2. Stimulation of the PLC pathway in the testis. In the testis, GnRH receptors have been identified in Leydig but not Sertoli cells (160, 161), the latter being the proposed site of production for the receptor (162). As in the ovary, GnRH exerts a direct stimulatory effect on basal steroidogenesis in the testis, and an inhibitory effect on gonadotropin-stimulated androgen biosynthesis (163). The latter has been attributed to an effect on steps distal to cAMP and pregnenolone production and may be a result of decreased enzymic activity of 17-20 desmolase and 17a-hydroxylase (164). As in the ovary, binding of GnRH to the testicular GnRH receptor appears to be coupled to the stimulation of the PLC pathway. The first indication came from the observation of agonist-stimulated labeling of PA and PI in the Leydig cell (165). The specificity of the response was demonstrated using a GnRH antagonist. The changes in phospholipid metabolism were followed by increased testosterone production, suggesting that phospholipids may mediate the direct effects of GnRH in the testis (166). The effects of a GnRH agonist and PLC activators on protein phosphorylation and steroid production were similar in studies of testicular Leydig cells,

supporting a role for this pathway in the action of the hormone (167). There is indirect evidence for the involvement of calcium in the intracellular action of GnRH in the testis. Using Quin II, the stimulation of Leydig cell steroidogenesis by GnRH was shown to be accompanied by only a modest increase in [Ca2+]; (168). However, both the stimulation of steroidogenesis and the inhibition of LH-induced cAMP formation by GnRH were dependent on extracellular calcium levels (169171). Finally, the calcium ionophore A23187 effectively reproduced the actions of GnRH on steroidogenesis, independent of small increases in cAMP. Both phospholipid turnover and calcium dependency imply an involvement of the PLC pathway in GnRH action in the testis. Consequently, the generation of DAG may be active in the stimulation of PKC. Indeed, PKC activity is present in the seminiferous tubules and Leydig cells of the testis and has been shown to be involved in the regulation of these testicular compartments (172, 173). The phorbol ester TPA inhibited basal cAMP production by 50%, while basal testosterone synthesis was increased 2- to 3-fold. Notably, these actions of TPA are shared by GnRH. B. PGF2a stimulation of PLC

The important role of PGF 2a in corpus luteum regression is well documented (174, 175). In vitro studies of luteal tissue have demonstrated a rapid inhibition of both LH-stimulated cAMP formation (176) and steroidogenesis in response to the prostaglandin (177). It has


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also been shown to attenuate LH-induced LH receptor binding (178). PGF2a acts through specific membrane receptors (179) and inhibits cAMP accumulation in intact cells, but not in luteal membranes (180). This suggests the participation of an intracellular signaling mechanism. The signaling mechanism of PGF 2a in the ovary appears to be similar to that of GnRH, which is not unexpected considering their shared antigonadotropic effects. Radiolabeled [32P]orthophosphate is rapidly incorporated into both PA and PI (140, 181, 182), concomitant with enhanced inositol phosphates in response to PGF 2a treatment in rat luteal cells (183). Further, the addition of exogenous PLC caused a similar effect in identical cell cultures. Temporal studies of the stimulation of inositol metabolism in developing luteal tissue in response to PGF2a have shown a loss of responsiveness during the transformation of young corpora lutea (2 days) to those of maturity (7 days) (184). This may imply that additional signaling mechanisms may contribute to the acute luteolytic action of PGF2a in the rat. The induction of inositol phosphate metabolism in response to PGF2a has also been observed in bovine (185,186), ovine (187), and human (188) luteal cells. In bovine luteal cells, the identification of multiple polyphosphorylated inositol phosphates suggested a role for the inositol tris/tetrakisphosphate pathway (189). Analogous to the signaling mechanism of GnRH, calcium is thought to play an important role in the action of PGF2a. Transient increases in [Ca2+]i following PGF 2a treatment have been reported for ovine (190, 191), rat (192), and bovine (185) corpora lutea. In addition, using Fura-2 microspectrofluorimetry in single cells, PGF 2a has been shown to cause a transient increase in [Ca2+]i in rat (193) and human granulosa cells (194). Whether this mobilization of calcium originates from intracellular stores (195) or results from a flux across the membrane (191) remains controversial. This effect has been associated with the PGF2a-induced decrease in progesterone synthesis, providing evidence that calcium is involved in the physiological response (195). The observation of enhanced cAMP generation by LH in calcium-depleted media, calcium-dependent attenuation of LH-stimulated cAMP production by a calcium ionophore, and a direct inhibition by calcium of LH-sensitive adenylate cyclase activity in luteal membranes lends credence to the involvement of calcium in the inhibition of LH-stimulated adenylate cyclase activity (196). Further, progesterone production by dispersed primate luteal cells was suppressed by treatment with the calcium ionophore A23187 (197). The mechanism by which calcium affects LH may be through an interference of GTP activation of adenylate cyclase (198). However, there is also evidence that the PGF2a-induced [Ca2+]i increase is not involved in the

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inhibition of LH activity in rat luteal cells. Chelation of intracellular calcium with either dimethyl bis-(o-aminophenoxy)ethane-N, N,N' ,N' -tetraacetic acid or EGTA did not affect the inhibition of LH-stimulated cAMP accumulation by PGF2a, nor did a calcium ionophore mimic the effect of PGF2a on LH stimulation of cAMP (199). In another study, inhibition of [Ca2+]i with S-(N, JV-diethylamino)-octyl-3,4,5-trimethoxybenzoate (TMB-8), and inhibition of calmodulin with three different compounds were similarly ineffective in abolishing the inhibitory action of PGF2a on luteal cAMP production (200). Clearly, the involvement of calcium in the intracellular signaling of PGF2a remains to be clarified. Considering the stimulation of PLC by PGF2a, an accumulation of DAG would be expected in addition to the generation of inositol phosphates. DAG-activated PKC provides another potential signaling mechanism in the action of this prostaglandin, a topic that has been investigated with use of phorbol esters. Low concentrations of TPA mimicked the inhibitory, luteolytic effects of PGF2a on LH-induced cAMP and progesterone production, indicating a physiological role for PKC activity (201). The addition of PGF2a or TPA to isolated membranes was without effect, strengthening the notion that activation of PKC has an absolute requirement for calcium and membrane phospholipids. TPA, like PGF2co was also effective in the inhibition of 8-bromo-cAMPinduced progesterone production. Further, a PGF2a-induced calcium-dependent phosphorylation of endogenous proteins has also been identified in the luteal membranes of the rat ovary (202). This was suggested to be a result of the stimulation of PKC activity. In swine ovarian cells, a translocation of cytosolic PKC to the phospholipid-enriched membrane is observed in the response to PGF2a, indicating a role for PKC activation in the action of this prostaglandin (203). Contrary to these reports, the antigonadotropic actions of PGF2a and phorbol ester have also been reported to be mediated by separate processes (204). Comparison of the effects of TPA and PGF2« on various parameters revealed a differential inhibition and a cumulative effect of the two on LH-stimulated cAMP accumulation. Further, the PKC inhibitor staurosporine reversed the inhibition by TPA, but had no effect on PGF2a-induced inhibition. Clearly, our understanding of the intracellular signaling mechanisms involved in the action of PGF 2a is incomplete. C. LH stimulation of PLC 1. Stimulation of the PLC pathway in the ovary. There is evidence that the regulation of steroidogenesis by LH may be exerted through the stimulation of multiple pathways. The stimulation of the adenylate-cyclase pathway by LH is irrefutable. However, there are also implications


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for an involvement of the PLC pathway in progesterone production. Studies with a cloned murine LH receptor expressed in L cells have revealed a simultaneous activation of adenylate cyclase, phosphoinositide hydrolysis, and [Ca2+]j mobilization following hormone binding (205). The increase in [Ca2+]i, as determined by Fura II microspectrofluorimetry, was LH receptor-dependent and was not due to the accumulation of cAMP. Forskolin and PGEi, both of which stimulated cAMP, were ineffective in stimulating a similar increase in [Ca2+]i in these cells. In other studies, LH has been shown to increase both the synthesis (206, 207) and degradation (137) of phosphoinositides in rat granulosa cells isolated from mature follicles. In the same species, the accumulation of InsP3 was observed in response to LH treatment (137), despite the apparent lack of associated increase in [Ca2+]i (145, 155). LH has also been shown to induce a 5-fold accumulation of InsP and InsP2, and a 20-fold accumulation of InsP 3 in porcine granulosa cells (208, 209). Further, progesterone production was induced to the same extent by LH and InsP 3 in permeabilized cells. To the contrary, the activation of PLC by high doses of LH may provide a negative feedback on cAMP-induced steroidogenesis in bovine luteal cells (186, 210). In avian granulosa cells, LH receptor-mediated mobilization of [Ca2+]i was biphasic, unlike that of a nonreceptor-mediated (forskolin and 8-bromo-cAMP) mobilization (211). LH induced a rapid mobilization of [Ca2+]j which preceded, and appeared independent of, the delayed [Ca2+]i increase associated with the adenylate cyclasecAMP pathway. Using the fluorescent calcium indicator Indo I, the calcium response to LH has recently been shown to be dose-dependent, desensitizable, and independent of extracellular calcium (212). Further, simultaneous assessment of inositol phosphate metabolism and [Ca2+]i mobilization has provided additional evidence for an LH-sensitive PLC signaling pathway in chicken granulosa cells (213). Additional evidence for the involvement of the PLC pathway in the action of LH comes from reports of an apparent interregulation of PLC and adenylate cyclase cAMP pathways in some species. In swine luteal cells, a stimulatory coupling of PKC and cAMP-generating systems has been demonstrated, as determined by the effects of TPA on LH-stimulated cAMP formation (214). TPA and two synthetic diacylglycerols enhanced LH-, forskolin- and cholera toxin-activated cAMP formation. However, uncoupling of PLC and adenylate cyclase -cAMP pathways appears to occur in bovine cells, where a rapid and homologous desensitization of LH-sensitive adenylate cyclase is induced by LH itself, without inhibiting the LH-sensitive PLC pathway (215). This is presumed to involve a functional modification of the hormone receptor complex.

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2. Stimulation of the PLC pathway in the testis. The similarity of hormone action in the ovary and the testis is maintained in the action of LH, where there is evidence for the stimulation of the PLC pathway. The addition of LH or cAMP to rat Leydig cells provoked not only an increase in testosterone production, but a simultaneous accumulation of phospholipids (216). Mobilization of [Ca2+]i was also implicated in the action of LH. However, this effect was more likely associated with LH-stimulated cAMP levels, because cAMP analogs were equally effective in increasing [Ca2+]i (217). Both LH and cAMP have been shown to induce increases in [Ca2+]; in Leydig cells in synchrony with the stimulation of testosterone production (217). The involvement of calcium fluxes across the membrane has been postulated (171), which represents a similar mechanism in heart cells where these channels may be regulated by a cAMP-dependent phosphorylation (218). Low concentrations of LH may exclusively stimulate the PLC pathway, while the adenylate cyclase-cAMP pathway has been additionally implicated in maximal LH-stimulated steroidogenesis. This is based on the observation of a sole increase in [Ca2+]; in response to low concentrations of LH (73), and a recruitment of the c AMP -dependent pathway at higher concentrations (168). Further, the Ca2+-dependent effects of LH on testosterone production have been demonstrated to be modulated by calmodulin (73, 219). Although no direct stimulation of DAG and/or activation of PKC has been demonstrated in Leydig cells, the use of phorbol esters indicates an involvement of PKC in LH action. TPA mimicked the LH-induced desensitization of adenylate cyclase, an effect that was both dose- and time-dependent (220). An inhibition of the binding of [125I]hCG to rat Leydig cells after incubation with TPA also indicated the loss of cell-surface receptors. However, these observations have not been made by others. In murine Leydig cell tumor lines, TPA caused desensitization of hCG-stimulated adenylate cyclase activity without altering hCG receptor affinity (221, 222). Further, an inhibitory action of TPA on hCGinduced cAMP generation and steroidogenesis in rat Leydig cells did not affect [125I]hCG binding in the same cells (223). Therefore, although PKC appears to mediate desensitization of LH-induced activation of adenylate cyclase, an involvement of PKC in the desensitization of LH receptors remains speculative. Rather, it has been suggested that the actions of phorbol esters in Leydig cells are exerted at the level of the catalytic subunit of adenylate cyclase (223). The early stimulatory effects of LH on steroidogenesis may also be mimicked by phorbol esters, indicating an involvement of PKC activation. In porcine Leydig cells, basal testosterone and pregnenolone production were stimulated by TPA, while LH-stimulated steroidogenesis


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was inhibited by the phorbol ester (224). Combined treatment of phorbol esters with forskolin revealed a modulation of Leydig cell steroidogenesis by interactions of the PKC and PKA second messenger systems. However, conclusions regarding these observations require additional confirmation. D. Other potential activators of the PLC pathway 1. Ang II. The identification of Ang II-like immunoreactivity in human ovarian follicular fluid (225) led to the identification of an ovarian renin-angiotensin system (226). Although it is speculated that Ang II exerts a regulatory role on steroidogenesis (227-229) and ovulation (230), the physiological significance of this peptide remains in question. Ang II has been reported to increase estrogen secretion by rat ovarian tissue in vitro stimulated by PMSG (227, 231), and also increases gonadotropin-stimulated progesterone production in luteinized human granulosa cells (232). It is also speculated to play a role in angiogenesis in the ovary (233), either directly or .via fibroblast growth factor through induction of vasculafization of the thecal membrane (234). A functional role is indicated by the presence of specific type 2 Ang II receptors (235) on theca interna and granulosa cells in rat follicles, possibly those undergoing atresia (228, 229). In adrenal glomerulosa (236) and human trophoblast cells (237), there is evidence for a stimulation of inositol lipid metabolism by the hormone. An involvement of the PLC pathway in Ang II action in the ovary has not been shown. Ang II was found to stimulate a rapid, transient increase in [Ca2+]; in a discrete population of rat granulosa cells (238), but not in human granulosa cells (194). This response was completely blocked by an Ang II antagonist and was not mimicked by Ang I, indicating that the effect was mediated by specific receptors. The calcium response to Ang II in the ovary supports the notion of a [Ca2+];-mediated signaling pathway in the action of Ang II in this tissue. In cultured murine Leydig tumor cells, LH has been shown to stimulate the production of Ang II through the activation of renin (239). Ang II may be involved in the regulation of steroidogenesis in the testis. Functional, high affinity receptors for Ang II have been identified on rat Leydig cells (240). This hormone has been shown to exert an inhibitory effect on hCG-stimulated adenylate cyclase activity in Leydig cell membranes. Further, it reduces basal and hCG-stimulated cAMP pools and testosterone production in intact cells. The fact that this effect can be blocked by previous incubation of the cells with forskolin or pertussis toxin suggests that the Ang II inhibition is mediated by a pertussis toxin-sensitive guanine nucleotide inhibitory protein (241). However,

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there is little additional information regarding signaling mechanisms of Ang II in the testis. 2. Tumor necrosis factor-a (TNFa). TNFa has been localized in both granulosa and luteal cells of the human ovary (242, 243). It has been shown to stimulate progesterone production (244, 245) as well as hCG binding of granulosa cells (246). The potential regulatory role of this cytokine in steroidogenesis has sparked interest as to its mechanism of intracellular signaling. A stimulation of the PLC pathway was indicated by an apparent involvement of PKC and calcium in the action of TNFa (247). An activator of PKC (TPA) mimicked the biphasic increase in progesterone observed in response to TNFa, and a PKC inhibitor (H7) effectively blocked TNFa action. Further, the resulting stimulation of progesterone was not associated with an increase in either cAMP or cGMP. 3. CRF. An inhibitory effect of CRF on LH/hCG-induced cAMP generation and steroidogenesis has been demonstrated in rat Leydig cells (223). The action of CRF was unaffected by pertussis toxin and completely reversed by 8-bromo-cAMP. CRF also inhibited the stimulation of cAMP and testosterone production by cholera toxin and forskolin, indicating the interaction of CRF receptors with a pertussis toxin-insensitive G protein, possibly Gp (248). Studies of possible intracellular signaling mechanisms of CRF indicate the involvement of PKC. Like CRF, the phorbol ester TPA caused dose- and timedependent inhibition of hCG action. This effect was not through an attenuation of hCG binding and was reversed by the addition of cAMP. An inhibitory action of both CRF and TPA was lost in cells pretreated with PKC inhibitor and in cells with previous TPA-induced depletion of PKC. An involvement of PKC in the action of CRF is also supported by the observation of a rapid translocation of PKC in Leydig cells treated with CRF. This likely interferes with the catalytic subunit of adenylate cyclase, either through a direct or indirect action.

V. PLA2 Signaling Pathway Arachidonic acid is a second messenger implicated in the intracellular signaling of several gonadal hormones. Its generation through the consecutive actions of PLC and DAG lipase has been described, but it may also be liberated by its specific PLA2 from DAG-derived PA or directly from other phospholipids such as phosphatidylcholine (158). Subsequently, arachidonic acid acts as the precursor for the generation of eicosanoids via one of three pathways mediated by cyclooxygenase, lipoxygenase, and microsomal cytochrome P450-dependent epoxygenase. Cyclooxygenase generates endoperoxides through the oxygenation of arachidonic acid. Subse-


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quently, various metabolites of endoperoxides are generated including thromboxanes, and prostaglandins such as PGE2, PGD2, and PGF2a. The action of cyclooxygenase may be completely inhibited by acetylation with aspirin. Lipoxygenases are enzymes that oxygenate arachidonic acid to yield hydroperoxy derivatives. The hydroperoxy group could be in carbon 5, 11, 12, or 15 of arachidonic acid. Hydroxy analogs of hydroperoxy derivatives are subsequently generated through the action of a lipoxygenase-associated peroxidase activity. Of special interest is the 5-lipoxygenase, which generates leukotrienes that are implicated in signal transduction. Epoxidation of arachidonic acid by cytochrome P450-dependent epoxygenase yields several epoxides that can be converted to hydroxy acids by further oxidation. These add to the list of biologically active compounds generated by the PLA2 pathway. Eicosanoids may independently act as paracrine or autocrine agents and have been implicated in the action of several gonadal regulators. A. GnRH stimulation of PLA2 1. Stimulation of the PLA2 pathway in the ovary. Enhanced arachidonic acid in response to treatment of granulosa (156) and luteal cells (249) with GnRH has been observed. The functional significance of GnRHstimulated arachidonic acid accumulation is exemplified by an enhanced basal production of progesterone in response to both exogenous arachidonic acid and PLA2 activator (250, 251). Further, the effects of GnRH and arachidonic acid on steroidogenesis were additive. The contribution of arachidonic acid by PLC/DAG lipase vs. PLA2 is not clear, but there is evidence for its generation via both mechanisms. In view of the similarities of GnRH action in the gonads and the pituitary, and the demonstration of GnRH-induced PLA2 activity in the pituitary (252), it is not unreasonable to propose a similar mediatory role of PLA2 in the action of GnRH in the gonads. Indeed, the finding that luteal cells of different ages respond to GnRH with distinct inositol phosphate and arachidonic acid accumulation suggests that there may be independent coupling of the GnRH receptor to PLC and PLA2 (249). The involvement of lipoxygenase metabolites was demonstrated using the lipoxygenase inhibitor nordihydroguaiaretic acid (NDGA), which abolished arachidonic acid-stimulated progesterone production (251). In the same studies, NDGA treatment resulted in only a 50% inhibition of GnRH-stimulated progesterone levels, which is not surprising in view of its simultaneous activation of the PLC pathway. Further evidence in support of a role of lipoxygenase metabolites comes from the observation that ovarian progesterone and PGE2 production is stimulated by 5-hydroxyeicosatetraenoic acids (5-

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HETE), hydroperoxyeicosatetraenoic acids (5-HPETE), 12-HETE, 15-HETE, and 15-HPETE (253). Products of the cyclooxygenase pathway do not appear to be involved in the action of GnRH in the ovary, as demonstrated by a lack of effect of the cyclooxygenase inhibitor indomethacin on both arachidonic acid- and GnRH-stimulated progesterone production (251). Further, arachidonic acid does not appear to attenuate FSHstimulated steroidogenesis, as observed with GnRH (254). This may indicate an exclusive role for arachidonic acid in the stimulatory aspect of GnRH action. The mechanism by which arachidonic acid acts is not clear, but the additive effect on steroidogenesis of combined treatment with arachidonic acid and TPA may reflect an involvement of PKC stimulation (251). This interaction has been demonstrated in other systems (255). 2. Stimulation of the PLA2 pathway in the testis. The similarity of hormone action in the ovary and testis is maintained in the stimulation of PLA2 by GnRH. GnRH agonist-induced testosterone formation may be completely blocked by the PLA2 inhibitors chloroquin and quinacrine (256, 257). Exogenous arachidonic acid, like GnRH, directly stimulates basal steroidogenesis in the Leydig cell. It has been shown that lipoxygenase products of arachidonic acid metabolism may be required for LHstimulated steroidogenesis in rat Leydig cells (258), which appears also to apply to GnRH (73, 259). However, the involvement of the lipoxygenase pathway in GnRH action remains in contention; both arachidonic acid(257) and GnRH agonist-induced testosterone secretion (256) were potentiated by the addition of NDGA. The direct effects of treatment of cultured rat Leydig cells with GnRH have been reported to include a rapid increase in phospholipid metabolism, followed by increased PGE2 and testosterone production (166). However, cyclooxygenase inhibitors have no effect on either GnRH or arachidonic acid-stimulated steroidogenesis (256258). In fact, an inhibitor of all known pathways of arachidonic acid metabolism, 5,8,11,13-eicosatetraynoic acid, was also without effect suggesting that arachidonic acid itself may be the active second messenger. This idea is strengthened by the simultaneous activation of Ca2+dependent PKC and Leydig cell steroidogenesis by arachidonic acid (256). B. PGF2a stimulation of PLA2

The accumulation of arachidonic acid in response to PGF 2a has been described for cultured rat luteal cells (249). Whether arachidonic acid is generated by the action of PLC/DAG-lipase or PLA2 remains speculative. However, the observation that PGF2a consistently stimulated both inositol phosphate formation and arachidonic acid release in cells of different ages (249) suggests


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that if both pathways are operative, there is likely a cooperative coupling of the PGF2a receptor to both PLC and PLA2. This is in contrast to the GnRH receptor, which appears to be independently coupled to PLC or PLA2, possibly via different receptor subclasses. Treatment of rats with PGF2« in vivo activates PLA2 and decreases plasma membrane fluidity in the corpus luteum (260). Certainly, arachidonic acid mobilization by PGF 2a presents exciting prospects as an intracellular signaling mechanism, based on the potential positive feedback loop that would be created by the generation of additional eicosanoid substrate (Fig. 3). C. LH stimulation of PLA2 Although there is an apparent lack of evidence for the action of PLA2 regarding LH action in the ovary, there is some indication that such a signaling mechanism may be functional in the testis. This was first shown by the ability of various lipoxygenase inhibitors to block LHinduced steroidogenesis (73, 258). The cyclooxygenase inhibitor indomethacin was, however, without effect on LH-stimulated steroidogenesis (166). An involvement of the lipoxygenase pathway in LH action is supported by the stimulation of leukotriene B4 synthesis by LH and hCG in testicular Leydig cells (61). The kinetics of its production indicate that only short-lived precursors of the leukotriene are involved in the steroidogenic effects

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of LH. A dose- and time-dependent biphasic effect of arachidonic acid on LH-stimulated testosterone production in rat Leydig cells was recently reported (261). Further evidence of the involvement of PLA2 in the signaling of LH has been provided by the use of PLA2 inhibitors (262). The results reveal that PLA2 itself stimulates both basal and LH-induced testosterone production. The inhibition of LH-induced steroidogenesis by three different PLA2 inhibitors, however, was not accompanied by changes in cAMP accumulation. This effect was not exerted through an inhibition of steroidogenic enzymes, as evidenced by a lack of effect on 22a-hydroxycholesterol (262). These findings provide further evidence for the involvement of the PLA2 pathway in the action of LH, through a mechanism that is independent ofcAMP.

VI. PLD Signaling Pathway Recently, PLD has been implicated in intracellular hormone signaling in a number of mammalian systems (263). PLD, like PLC, is a phosphodiesterase that acts by attacking the head group of phospholipids. PLD primarily hydrolyzes phosphatidylcholine, but other phospholipids including PI have also been reported to act as substrate. The products of its action are PA and a head group alcohol. The importance of PLD in intracellular signaling may be at two levels: not only can PA partici-

FlG. 3. A hypothetical model of PGF2n and LH interaction in the corpus luteum. PGF2a stimulates PLC activity, resulting in the generation of IP3, which stimulates release of Ca2+ from intracellular stores, and DAG, which activates PKC. PGF2a-stimulated PLA2 activity results in the accumulation of AA. AA may serve as substrate either for the formation of additional prostaglandins, or for other metabolite pathways. The potential formation of PGF2a in this cascade may constitute a positive feedback mechanism for the amplification of PGF2a action in the ovary. LH stimulates the adenylate cyclase-cAMP pathway, resulting in the activation of PKA, and stimulation of the conversion of cholesterol (c) to steroid hormones. The potential interaction of these different intracellular signaling pathways is depicted.


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pate in the activation of PKC through its conversion to DAG, but it has also been implicated in Ca2+ regulation (264), cell proliferation (265), and the inhibition of a GTPase-activating protein (266). Activation of PLD may be receptor-coupled (263, 267), and there is evidence for cross-talk between the PLD and PLC pathways. A. GnRH stimulation of PLD GnRH receptors in preovulatory granulosa cells have been shown to activate PLD with a resultant increase in endogenous PA (268). Using a GnRH agonist, there was a 10-fold increase in phosphatidylethanol produced by PLD phosphatidyl transferase activity. This effect was dose-dependent and specific, as indicated by its inhibition by a GnRH antagonist. The PKC activator, TPA, enhanced the GnRH-stimulated phosphatidylethanol up to 30-fold. An additive effect of combined treatment of GnRH and TPA, however, suggested that they were acting through different mechanisms. This supports the notion of an independent regulation of PLD by PKC. The physiological relevance of PLD in GnRH action is substantiated by a similarity in progesterone production stimulated by PLD or GnRH agonist in cultured cells. Further, the stimulatory action of GnRH on steroidogenesis was mimicked by the addition of PA, a product of PLD activity. The mechanism by which PLD metabolites modulate cellular constituents involved in steroidogenesis remains to be examined.

VII. Other Potential Mediators of Hormone Action in the Gonads A. Tyrosine kinase 1. EGF. EGF is best recognized for its mitogenic activity and was first investigated for its effect on granulosa cell proliferation (269). In addition to many other tissues, it is produced in the ovary (270), and in vitro studies have revealed effects of EGF on a diversity of parameters of ovarian function. It attenuates FSH-, 8-bromo-cAMP-, and cholera toxin-mediated induction of LH/hCG receptors in rat granulosa cells, as well as the estrogen secretion induced by these components of receptor induction (271,272). A similar inhibition of steroidogenesis by EGF is observed in theca-interstitial cells where LH-induced androgen production is affected (273, 274). The effect of EGF on progestin remains controversial and is likely related to specific periods of cell differentiation or follicle development, or both (275). The mitogenic effect of EGF and its amplification by serum, however, is a consistent finding among bovine, porcine, human, and rabbit granulosa cells. Clonal agar cultures of bovine granulosa cells respond to the growth factor with increasing colony formation and growth (276). Interestingly, this action of EGF is absent during the LH surge and ovulation.

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The intracellular events associated with receptor binding of EGF have been studied in other cellular systems, revealing a distinctive signaling mechanism. Specifically, the EGF receptor exhibits cytoplasmic tyrosine kinase activity (277). Ligand binding is functionally heterogeneous, in that both low- and high-affinity binding occurs. Monomeric receptors allow for low-affinity binding, and a corresponding low activity tyrosine kinase. However, binding of the receptor additionally induced receptor oligomerization, generating high-affinity binding and a corresponding enhanced tyrosine kinase activity (278, 279). Autophosphorylation of the receptor tyrosine is one of the biochemical changes that is essential for the receptor's biological activity. Moreover, the kinase may mediate receptor internalization and down-regulation, although subsequent steps in intracellular signaling are not understood. 2. TGFa. TGFa is a polypeptide sharing a high degree of homology with EGF, which also shares in the binding of EGF receptors (280). It similarly stimulates an intrinsic tyrosine kinase resulting in autophosphorylation of the receptor. Based on these observations, it is not surprising that the bioactivities of the two growth factors in the granulosa cell are very similar. Both act as mitogens and attenuate FSH-stimulated estrogen secretion in a timeand dose-dependent manner. Further, its actions appear to be exerted at a point distal to cAMP production. However, the potency of TGFa and EGF action may vary, and interactions between the two may be disparate depending on whether the cell is in a proliferative or differentiating stage of development (275). In the porcine theca cell, TGFa has been shown to exert an inhibitory effect on both androstenedione and progesterone production in response to hCG (281). This observation, in conjunction with a simultaneous stimulation of cell growth, suggests a role for TGFa in the inhibition of theca cell differentiation. B. Hormone-mediated ion channel gating 1. LH. The possibility of physiological effects of agonist/ receptor-regulated ion channels in the gonads is a relatively novel finding. In both the ovary and testis, there is evidence for the existence of ion currents and their modulation by the action of LH. Specific outward potassium currents have been recorded in rat Leydig cells using the patch-clamp technique in the whole cell configuration (282). Membrane depolarization in response to LH has been reported in Leydig and Sertoli cells of the testis (283). In adrenocortical cells, depolarization in response to various endocrine stimuli has been shown to be dependent on altered potassium conductance (284). It has been postulated that a hormone-induced inhibition of outward potassium current, resulting in membrane


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depolarization, may result in the opening of voltage-gated calcium channels. A subsequent increase in [Ca2+]i could be the signal affecting cell function. Indeed, inward calcium currents have been identified in Leydig cells of the mouse and shown to be activated by physiological changes in membrane potential (285). In the same cell type, a calcium-dependent potassium channel appears to be dependent on increases in [Ca2+]; for its activation. Conceivably, intercellular coupling of these cells allows their endocrine activities to be synchronized or amplified. Further, there may be a dual origin of calcium, as has been postulated for ovarian luteal cells (286). The initial rapid increase may be attributed to intracellular calcium, while the second increase is sustained and dependent on an influx of extracellular calcium. Accordingly, LH may initially stimulate release of calcium by the action of InsP3, triggering a further increase through membrane channels. Presumably, both sources are important for the regulation of endocrine processes. 2. FSH. Although fast membrane potassium movements have been implicated in membrane voltage regulation and modulation of steroidogenesis in ovarian cells (287), our knowledge of this area is limited. Only recently have transmembrane ion currents been directly identified in the ovary; using whole cell patch clamp technique, both Ca2+ and K+ currents were confirmed in avian granulosa cells (288). The stimulation of estrogen secretion by FSH from avian theca cells has been shown to be highly dependent on calcium, possibly of an extracellular source (289). Both calcium-free media and the addition of EGTA, a calcium chelator, abolished the FSH-stimulated estradiol and estrone secretion. Further, the calcium ionophore A23187 mimicked the effect of FSH on steroidogenesis, provided calcium was present in the media. The involvement of calcium flux in the action of FSH is also supported by studies of rat granulosa cell progesterone production (84). The biosynthesis of progesterone in response to stimulation with (Bu)2cAMP is significantly attenuated by the addition of the calcium channel blocker, verapamil. Further, a calmodulin inhibitor reduced FSH-induced cAMP formation and progesterone production, providing additional evidence for a role of calcium in FSH action. Using the fluorescent calcium indicator dye, Fura II, FSH treatment has been shown to elicit specific and sustained increases in [Ca2+]i in swine granulosa cells (290) but not in rat granulosa cells (142). This effect could not be attributed to InsP3-stimulated release due to the distinctive temporal pattern. Further, it was blocked by the absence of extracellular calcium. Although there is strong evidence for an involvement of calcium flux in the action of FSH on steroidogenesis, the mechanism by which this gonadotropin might affect membrane channels remains unclear.

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There is also evidence for an FSH-stimulated calcium flux in the testicular Sertoli cell. Both voltage-independent and voltage-dependent calcium channel blocking agents were effective in reducing the FSH-induced 45Ca2+ flux in rat Sertoli cells, which was associated with an inhibition of aromatase activity (291). The purported influx of calcium through FSH receptor-regulated calcium channels was independent of both cholera toxinand pertussis toxin-sensitive G proteins and did not require adenylate cyclase activity (292). Further, FSH receptor binding may be associated with changes in Na + / Ca2+ exchange, also implicated in the regulation of [Ca2+]; (293). As in the ovary, these FSH-induced changes in [Ca2+]; are probably not the result of a stimulation of the phosphoinositide pathway (294, 295). VIII. Summary The regulation of steroidogenesis in both the ovary and testis involves a complex interaction of a diversity of hormones and intracellular signaling pathways. The recent cloning of LH and FSH receptors has paved the way for an increased understanding of the mechanisms of receptor conformation, ligand-receptor interaction, and facilitation of post-receptor activity. The dominant role played by LH in the regulation of steroid production appears to be mediated by more than one intracellular signaling pathway. In addition to the stimulation of the adenylate cyclase-cAMP pathway, also known to be stimulated by FSH, the actions of LH may be additionally mediated by other intracellular messengers, such as those derived from the PLC pathway. Steroidogenesis in the gonads appears to be modulated by a variety of factors in addition to the gonadotropins. In this review, those factors of intracellular signaling mechanisms of which we have some understanding have been discussed. These include GnRH, PGF2a, Ang II, VIP, GHRH, TNFO, CRF, EGF, and TGFa. Many of these factors have been shown to be locally synthesized, and specific receptors have been identified in the gonads. Many gonadal factors have the capacity to exert effects on steroidogenesis independent of the gonadotropins. Alternately, they have been demonstrated to alter the gonadal response to the gonadotropins via autocrine, paracrine, and intracrine mechanisms. As yet, our understanding of the intracellular signaling mechanisms used by novel gonadal regulators is limited. The involvement of the PLC, PLA2, and PLD pathways in this regard has been reviewed. It is becoming apparent that multiple signaling pathways may be stimulated by a single hormone, as in the case of GnRH, PGF2a, and LH. The complexity of intracellular signal transduction in the gonads is enhanced by the potential cross-talk at numerous steps in the signaling cascades.


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Modeling the intracellular organization of calcium signaling.

INTRACELLULAR SIGNALING BY BILE ACIDS.

Intracellular Signaling Pathways in Rheumatoid Arthritis.

Intracellular signaling of cardiac fibroblasts.

Nonsteroidal signals originating in the gonads.

Intracellular Zn(2+) signaling in cognition.

Receptors and intracellular signaling in human neutrophils.

Intracellular calcium signaling in the fertilized eggs of Annelida.

The intracellular carboxyl tail of the PAR-2 receptor controls intracellular signaling and cell death.

Extracellular and Intracellular Signaling for Neuronal Polarity.

Intracellular Ca2+ signaling and preimplantation development.

Intracellular leptin signaling following effective weight loss.

Optogenetic control of intracellular signaling pathways.

Translating intracellular calcium signaling into models.

MicroRNA: master controllers of intracellular signaling pathways.

Analysis of intracellular PTEN signaling and secretion.

Evolution of the Calcium-Based Intracellular Signaling System.

Multiple intracellular signaling pathways of the neuropeptide substance P receptor.

Fatty acid signaling: the new function of intracellular lipases.

The intracellular location, mechanisms and outcomes of NOD1 signaling.

The phosphatidylethanolamine derivative diDCP-LA-PE mimics intracellular insulin signaling.

The growth hormone secretagogue receptor: its intracellular signaling and regulation.

Role of the gonads in hypertension-prone rats.

Modeling of progesterone-induced intracellular calcium signaling in human spermatozoa.