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Department of Neuroscience, Albert Einstein College of Medicine, Bronx, New York 10461
Address all correspondence and requests for reprints to: Dr. Anne M. Etgen, Department of Neuroscience, F113, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, New York 10461. E-mail: etgen{at}aecom.yu.edu.
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Abstract |
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Introduction |
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1B-adenergic receptor, the oxytocin receptor, and a variety of other signaling molecules involved in neurotransmission (4, 5). However, of these regulated gene products, only a minority are known to be regulated by the classical mechanism of ER action, in which ligand-activated ER dimerizes, binds to estrogen response elements in the promoter of the target gene, and increases gene transcription (6, 7). Hence, elucidating the behaviorally relevant molecular events that occur in neurons subsequent to E2 binding to ERs is still an active area of investigation.
In both the brain and in peripheral reproductive tissues, E2 and growth factors often work together to promote tissue remodeling, synaptic plasticity, and cell survival (8, 9, 10, 11, 12, 13, 14). In particular, cross-talk between ERs and IGF-I receptors (IGF-IRs) mediates changes in synaptic structure in the arcuate nucleus (15), protects hippocampal hilar neurons from kainate-induced degeneration (16), and influences ER-dependent gene transcription in rat uterine cells (17). We recently demonstrated that administration of behaviorally relevant doses of E2 to gonadectomized female rats increases the density of IGF-I binding sites and promotes IGF-IR enhancement of 1-adenergic receptor signal transduction in the hypothalamus (18). We then used intracerebroventricular (icv) infusions of the highly selective IGF-IR antagonist JB-1 (19) to determine whether brain IGF-IR signaling participates in E2 regulation of female reproductive physiology. Pharmacological blockade of IGF-IRs during 2 d of estrogen priming abolished steroid hormone-induced LH release and the induction of 1B-adenergic receptor binding in the hypothalamus and preoptic. In addition, the expression of hormone-dependent lordosis behavior was significantly reduced (20). These findings provide strong evidence that brain IGF-IRs interact with ERs to regulate the neural mechanisms that govern female reproductive physiology and behavior.
The purpose of the present experiments was to identify downstream signal transduction pathways that underlie the E2/IGF-I facilitation of lordosis behavior. Both E2 and IGF-I can act independently or synergistically to stimulate serine-threonine kinases associated with growth factor action (21, 22, 23, 24, 25, 26). The two most intensely studied pathways are those involving MAPK, especially the p42/44 MAPKs (also known as ERK1/2), and phosphatidyl-inositol-3-kinase (PI3K). Therefore, we used icv infusion of MAPK and PI3K inhibitors during a 2-d estrogen priming period to test the hypothesis that one or both of these signal transduction pathways mediates E2 facilitation of lordosis. The data implicate both p42/44 MAPK and PI3K signaling pathways in the brain as mediators of hormone-dependent lordosis behavior.
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Materials and Methods |
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Animals and surgery
Female Sprague Dawley rats weighing 175–200 g were purchased from Taconic Farms (Germantown, NY), housed individually, and maintained on a 14-h light, 10-h dark reversed light/dark cycle (lights off at 1100 h) with food and water ad libitum. Animals were anesthetized with ketamine/xylazine (40 and 7 mg/kg, im, respectively), placed into a Kopf stereotaxic apparatus, and secured with ear bars and a nosepiece set at + 5.0 mm. A 26-gauge guide cannula (Plastics One, Roanoke, VA) was implanted into the third ventricle (A/P, +0.2 mm; medial/lateral, 0.0 mm; D/V, -9.8 mm with respect to Bregma; from Ref.27). The cannula assembly had a 28-gauge dummy insert that extended 1 mm below the outer cannula to prevent obstruction of the guide. The 28-gauge infusion cannula was inserted only during drug administration. Immediately after stereotaxic surgery animals were bilaterally ovariohysterectomized (OVX) to remove the principal source of E2 and P. Behavior testing began 1 wk after surgery. All procedures used in these experiments followed the National Institutes of Health Guide for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee at Albert Einstein College of Medicine.
Drug administration
Experiment 1: effect of icv infusions of the IGF-IR antagonist JB-1 on lordosis behavior.
One week after surgery, rats were given two sc injections of 2 µg of E2B 48 and 24 h before behavioral testing. As this estrogen-priming regimen produces very low levels of lordosis responding in the absence of P, animals received 500 µg of P 4 h before behavioral testing. We first varied the timing of icv JB-1 infusions to determine the most effective duration of IGF-IR blockade needed to maximally inhibit lordosis. JB-1 is a synthetic peptide analog of IGF-I whose amino acid sequence is closely related to the C-terminal, D domain of endogenous IGF-I, the domain that mediates IGF-I binding to IGF-IRs. This peptide is a potent, highly selective, competitive antagonist of IGF-I-dependent autophosphorylation of IGF-IRs and cellular proliferation with no activity at IGF-II, insulin, or epidermal growth factor receptors (19). The information obtained with JB-1 was then used to establish the time at which MAPK and PI3K blockers would be infused in subsequent experiments. Initially, icv infusions of JB-1 were based on our prior study (Quesada and Etgen, Ref.20) and consisted of an initial infusion 1 h before the first E2B injection and three additional infusions at 12-h intervals, with the last infusion approximately 13 h before lordosis testing (Fig. 1
). Each infusion consisted of 10 µg of JB-1 in 2 µl of sterile saline or 2 µl of sterile saline vehicle injected over a 1-min period with a Hamilton syringe. Animals were randomly assigned to receive drug or vehicle on the first test and were used twice. After the first test, hormone treatment and behavioral testing were repeated 1 wk later with the infusion treatment reversed. That is, those females previously receiving JB-1 received vehicle and vice versa.
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Experiment 2: effects of icv infusions of MAPK and PI3K inhibitors on lordosis behavior.
One week after cannula implantation and OVX, female rats were injected with E2B and P as described above. To inhibit MAPK kinase (MEK1), the selective antagonists PD98059 (0.2 µg or 0.4 µg) and U0126 (38 or 76 ng) were used. To inhibit PI3K, LY294002 (2 µg) and wortmannin (2 µg) were used. These drugs exhibit good selectivity for the target kinases (28, 29), and the drug doses were chosen based on their reported ability to attenuate various forms of synaptic plasticity in the rat brain (see Discussion). In addition, a separate group of animals was infused with the selective inhibitor of p38 MAPK, SB203580 (0.4 µg). As illustrated in Fig. 1
, these drugs were infused icv 1 h before the first E2B injection and three additional times at 12-h intervals. The infusion volume was always 2 µl, and the vehicle was 1.0% DMSO.
To investigate the effect of simultaneous inhibition of both MAPK and PI3K on lordosis behavior, inhibitors for these enzymes were coadministered into the third ventricle of OVX rats primed with E2B and P. Drug combinations included: U0126 (76 ng) plus wortmannin (2 µg), PD98059 (0.4 µg) plus wortmannin (2 µg), and PD98059 (0.4 µg) plus LY294002 (2 µg). For each drug dose or combination, animals were used twice; after the first test, behavioral testing was repeated 1 wk later with drug and vehicle treatment reversed. The only exception was the experiment using PD98059 alone, in which case animals were used three times (vehicle, 0.2 µg and 0.4 µg of PD98059 per infusion).
Experiment 3: effect of acute icv administration of MAPK and PI3K inhibitors on lordosis behavior.
We previously showed that acute administration of JB-1 either 12 h before behavior testing or concurrently with P does not inhibit lordosis (20). This suggests that the behavioral effects of JB-1 are not attributable to blockade of IGF-IRs by the drug at the time of behavioral testing. To verify that the effects of the kinase inhibitors on lordosis behavior were not due to residual carry over from the fourth drug infusion, which occurred approximately 13 h before behavioral testing, single drug infusions were performed 13 h before behavioral testing (+35 h relative to the first E2B injection in Fig. 1). Drug concentrations were the same ones used in experiment 2.
Behavioral testing
For lordosis testing, experienced stimulus males were allowed to adapt to the testing chamber (20-gallon glass-walled observation tank) for at least 10 min before the introduction of an experimental female. Males were permitted to mount the female rats 10 times, and the number of lordosis responses as well as the quality of each lordosis were recorded. A lordosis quotient (LQ; number of lordosis/number of mounts x 100) was derived and served as a measure of estrous responsiveness. The intensity of lordosis (a subjective estimate of the lordosis posture) was scored according to the scale of Hardy and DeBold (30) as follows: 0, no lordosis; 1, a shallow arching of the back; 2, a definite dorsiflexion of the spine; and 3, an exaggerated lordosis posture.
Verification of cannula placements
After the final behavioral test, animals were anesthetized with sodium pentobarbital, and the dye methyl blue was infused through the cannula. After 1 min, animals were decapitated and brains were removed. Verification of correct placements was made by observing the dispersion of the dye throughout the brain.
Statistical analysis
LQ data were analyzed using a Wilcoxon signed-rank test. In experiments in which animals were used three times a Friedman test was used. Lordosis intensity scores were analyzed using paired t test or ANOVA for experiments in which animals were used three times. Differences were considered significant if P < 0.05.
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reduced LQ scores in OVX rats primed with E2B and P by about 30% (20). To investigate if a higher dose of JB-1 would produce a more dramatic suppression of receptivity, we used 10 µg of JB-1 per infusion in the present study. Figure 2A shows that infusion of the higher dose of JB-1 significantly suppressed lordosis behavior (P = 0.03), as measured by LQ scores, by approximately 50%. However, JB-1 treatment did not completely abolish lordosis responding. Next, we investigated whether infusion of the same total dose of JB-1 during the first 12–18 h of E2B priming would be sufficient to suppress lordosis behavior. Figure 2B shows that icv infusion of JB-1 (20 µg per infusion) 1 h before and either 4 or 11 h after the first E2B injection had no effect on hormone-dependent receptive behavior. In addition, our previous work showed that acute administration of JB-1 either 12 h before behavior testing or concurrently with P (i.e. 4 h before behavior testing) does not inhibit lordosis (20). Therefore, continuous blockade of IGF-IRs throughout the 2 d of estrogen priming seems necessary to significantly reduce lordosis behavior in female rats. Based on this finding, subsequent experiments with MAPK and PI3K inhibitors used the multiple infusion procedure shown in Fig. 1.
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; P = 0.007). Doses of 0.2 and 0.4 µg per infusion were equally effective in suppressing receptivity by about 40%. U0126, another MAPK inhibitor with selectivity for p42/44 MAPK, also inhibited lordosis behavior after chronic icv infusions (Fig. 3B). The effect of U0126 was dose dependent; 76 but not 38 ng per infusion significantly reduced receptivity (P = 0.18 and P = 0.02 for the lower and higher dose, respectively). That the magnitude of lordosis suppression induced by multiple infusions of 76 ng of U0126 was almost identical to that caused by 3- to 5-fold higher doses of PD98059 most likely reflects the greater potency of U0126 as a p42/44 MAPK inhibitor (28, 29). In contrast with the effects of PD98059 and U0126, chronic infusions of the p38 MAPK inhibitor SB203580 did not affect lordosis behavior (n = 6; data not shown).
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) and LY2940002 (Fig. 4B) were chronically infused into the third ventricle of OVX, hormone-primed rats. Multiple icv infusions of either compound significantly suppressed lordosis behavior when compared with vehicle (P = 0.02 and P = 0.01 for wortmannin and LY2940002, respectively). Wortmannin was a more effective inhibitor of lordosis than LY294002 (42% and 30% suppression by wortmannin and LY294002, respectively). None of the MAPK or PI3K inhibitors had any measurable effects on locomotor activity at the time of behavioral testing (data not shown). Moreover, the inhibitory effects of these drugs on lordosis were reversible. Animals that received drug on their first test did not show lower levels of lordosis during their vehicle test than did animals that received vehicle first.
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). The suppression produced by combined blockade of both PI3K and MAPK was significantly greater than that seen after infusion of either drug by itself, and the inhibition appeared to be additive (79% inhibition by the drug combination vs. 38–42% when the same drugs were individually infused). However, when other combinations of MAPK and PI3K inhibitors were used, the inhibition of lordosis was not as great as that produced by wortmannin and PD98059 (Fig. 5).
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). However, a modest but statistically significant (P < 0.05) inhibition of lordosis was observed following coinfusions of both MAPK and PI3K inhibitors (Table 1). The inhibition of receptivity produced by the single coinfusion of these drugs was similar to that caused by chronic coinfusions, with the exception of wortmannin and PD98059. Chronic coadministration of these two drugs virtually abolished lordosis (Fig. 5), whereas a single coinfusion 35 h after the onset of E2B priming had much more modest effects (Table 1).
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). Similarly, multiple infusions of specific MAPK and PI3K inhibitors significantly reduced the quality of lordosis when the drugs were administered alone or in combination (Table 2). Single icv infusions of individual inhibitors 35 h after the first estrogen injection did not affect the intensity of lordosis (data not shown). However, combinations of MAPK and PI3K inhibitors modestly decreased the intensity of lordosis behavior when infused 35 h after the first E2B treatment (data not shown).
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Discussion |
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To demonstrate causal relationships between specific signal transduction pathways and hormonal facilitation of lordosis behavior, two criteria must be fulfilled. First, the hormones that regulate the behavior must activate the signaling cascade in relevant brain regions. Second, activation of the signal transduction machinery must be necessary (although not necessarily sufficient) for hormonal facilitation of the behavior. A considerable body of data documents the ability of E2 and IGF-I to activate both the MAPK and PI3K pathways in brain (21, 22, 23, 24, 25, 37) and other tissues [e.g. vascular endothelial cells (38)]. Indeed icv administration of E2 and IGF-I, both independently and synergistically, promotes sustained increases in Akt activity, a downstream target of PI3K, in female rat hypothalamus (23). We showed that IGF-I activates MAPK signaling, as evidenced by increased phosphorylation of extracellular signal-regulated kinases, in hypothalamic slices in vitro (20). Ovariectomy reduces and E2 treatment increases MAPK signaling in many regions of the adult female rat brain [including hypothalamus (37)], and the ovarian steroid rapidly activates MAPK in cortical explants (24, 39) and in primary cultures of cortical neurons (25). Thus, the first criterion has, for the most part, been met. Here we provide the first evidence that both MAPK and PI3K signaling in the brain are also necessary for the full expression of hormone-dependent lordosis behavior, thereby fulfilling the second criterion. Furthermore, our data suggest that complete inhibition of estrogen-facilitated receptivity may require concurrent inhibition of both MAPK and PI3K signaling. Thus, activation of both pathways may be necessary, but neither pathway alone may be sufficient, for the full expression of lordosis.
In many respects, the actions of the MAPK and PI3K blockers parallel those of JB-1, the highly specific IGF-IR antagonist (19). When administered icv during estrogen priming, JB-1 attenuates estrogen-dependent lordosis behavior in a specific and reversible manner (Fig. 2 and Ref.20). The IGF-I antagonist also fails to inhibit lordosis when given either 35 h after the onset of estrogen treatment or concurrently with P [4 h before lordosis testing (20)], demonstrating that the presence of JB-1 at the time of testing does not account for the suppression of lordosis. Taken together, these observations suggest that E2 and IGF-I work together in the brain, possibly via prolonged activation of MAPK and PI3K, to regulate the expression of female reproductive behavior. They also extend previous demonstrations of a role for PI3K and MAPK in E2-dependent processes in cultured cells (e.g. Refs.38, 40) to a physiologically significant, in vivo situation.
It is intriguing to consider the cellular mechanisms by which E2 and IGF-I activation of MAPK and PI3K signaling in the brain might regulate female reproductive function. In addition to stimulating tissue growth, differentiation and cell survival, growth factors such as IGF-I also participate in tissue remodeling (see Ref.21). There is also compelling evidence that E2 (and P) can initiate anatomical and biochemical synaptic remodeling in the adult brain. E2 increases synaptic spine density in the ventromedial hypothalamus (41, 42), the major neural site at which E2 acts to facilitate lordosis (1, 2, 46) and in the CA1 subfield of the hippocampus (43, 44, 45). These changes in synaptic morphology might require the activation of growth factor signaling pathways. E2 and IGF-I act together to promote synaptic remodeling in the arcuate nucleus (15), an action that may explain the ability of icv infusions of the IGF-IR antagonist JB-1 to block hormone-dependent LH release (20).
Both MAPK and PI3K have also been implicated in the regulation of enzymes that synthesize and take up norepinephrine in cultured neurons (47). As hypothalamic norepinephrine release is important for hormone-dependent reproductive behavior and the preovulatory gonadotropin surge (48, 49, 50), these enzymes may be relevant downstream targets of MAPK and/or PI3K. Behaviorally relevant E2 and P treatments also extensively reconfigure the molecular and biochemical pathways mediating norepinephrine synaptic transmission in the hypothalamus and preoptic area (5, 51). At least one aspect of E2 regulation of noradrenergic signaling, the induction of 1B-adrenergic receptor expression in the hypothalamus and preoptic area, is abrogated if IGF-IRs are blocked by icv administration of JB-1 during estrogen priming (20). This adrenergic receptor subtype is thought to mediate norepinephrine facilitation of both lordosis behavior and LH release (52, 53). Perhaps concurrent activation of PI3K, MAPK, and their downstream targets by E2 and IGF-I modifies synaptic structure and function in brain regions that govern reproductive function, thereby increasing the probability that females are behaviorally sexually receptive in the periovulatory period.
At present we cannot attribute the inhibitory effects of MAPK and PI3K blockade on lordosis solely to interference with estrogen priming. P facilitation of lordosis behavior, which requires estrogen priming, might also involve biochemical cascades that intersect with growth factor signaling pathways. However, the inability of JB-1 to influence lordosis behavior when given concurrently with P (20) indicates that brain IGF-IR activation is unlikely to be required for progestin facilitation of lordosis. Likewise, we cannot rule out the possibility that some level of MAPK and/or PI3K activity in the brain is necessary at the time female reproductive behavior is expressed. For example, infusions of kinase inhibitors only once, 13 h before behavior testing (Table 1), sometimes produced modest inhibition of lordosis.
The pharmacological strategy used in the present study does not permit us to define the temporal duration or neuroanatomical specificity of PI3K and MAPK activity that underlie hormonal facilitation of estrous behavior. E2 facilitation of lordosis is not evident until 18–24 h after hormone administration, regardless of hormone dose or route of administration (see Refs.1, 2). Because multiple but not single icv infusions of JB-1 or kinase blockers are needed to interfere with estrogen-dependent lordosis, the latency to onset of behavioral response to E2 may reflect a complex and dynamic pattern of kinase activation. Hoffmann et al. (54) recently demonstrated such complexity in the regulation of gene expression by the transcription factor NF-
B. Ligand-dependent transcription of some target genes required persistent NF-B activation, whereas other genes were reliably expressed regardless of the duration of ligand stimulation. This complexity makes it difficult to determine either the optimal time course for in vivo drug delivery or the time after drug administration that one should examine kinase activity to verify drug efficacy. Similarly, as we chose icv infusions as the route of drug delivery, we cannot conclude at this time that the ventromedial hypothalamus is the only relevant site of action of the IGF-IR antagonist or the kinase inhibitors.
An interesting question is whether our present observations are related to a previous report that icv infusions of selected doses of epidermal growth factor and IGF-I produce short latency (e.g. 1–4 h) facilitation of lordosis behavior in ovariectomized rats that have not been primed with E2 (55). The temporal features of lordosis inhibition by JB-1 and blockers of MAPK and PI3K in hormone-treated females suggest that this is not the case. When the IGF-IR antagonist is infused either concurrently with P (3–4 h before behavioral testing) or approximately 12 h before testing, lordosis behavior is unaffected (see Ref.20). This suggests that brain IGF-IR signaling need not be active at the time of lordosis expression. JB-1 is also unable to attenuate lordosis behavior when it is administered only during the first 12 h of estrogen priming (Fig. 2). Thus, in contrast with the short latency, estrogen-independent facilitation of lordosis produced by epidermal growth factor and IGF-I (55), the temporal features of JB-1 inhibition imply that sustained activation of brain IGF-IR signaling is necessary for estrogen-dependent lordosis. In support of this interpretation, the MAPK and PI3K blockers used in the current study had little or no effect on lordosis when infused only once, in close proximity to lordosis testing (Table 1). Nonetheless, because the drugs used to interfere with MAPK and PI3K are not selective for IGF-IR and/or ER activation of these kinases, it is entirely possible that other growth factors normally contribute to hormone-dependent lordosis. Indeed, this may be why prolonged antagonism of both PI3K and MAPK produces a more complete inhibition of lordosis than does JB-1 (compare Figs. 2 and 5).
Although we did not carry out an extensive battery of neurological tests to detect possible nonspecific effects of the drug infusions, we consider it unlikely that general drug toxicity accounts for the observed inhibition of lordosis. First, drug-infused rats did not exhibit either lethargy or hyperresponsiveness to handling. Second, when locomotion and general activity were observed before and during lordosis testing, the experimental animals were indistinguishable from vehicle-infused controls. Third, vehicle-infused animals were always highly receptive even if they had received drug infusions the previous week. Fourth, it is also unlikely that vascular effects of the drugs are responsible for the attenuation of lordosis behavior. If the inhibition of lordosis resulted from reduced arterial pressure, one might predict that the single drug infusions given closest in time to behavioral testing would have been as effective as the multiple drug infusions (56). Finally, a number of the pharmacological agents used in the present study have been used in vivo to examine the cellular mechanisms involved in synaptic plasticity and neuronal survival, with no evidence of cellular toxicity (32, 33, 34, 36).
The degree of behavioral inhibition produced by the different agents may be related to a variety of factors. Of the two PI3K inhibitors used, wortmannin tended to be more effective than LY294002 when given alone and was considerably more effective than LY294002 when combined with the MAPK inhibitor PD98059. Perhaps this reflects the greater efficacy of wortmannin in blocking IGF-I-stimulated phosphorylation of the downstream kinase Akt (protein kinase B), which was recently shown in skeletal muscle cells (57). In that report, LY294002 was much more effective in blocking IGF-I activation of p70S6K, another downstream target of PI3K, than in reducing Akt activity. In view of the reported ability of in vivo administration of E2 and IGF-I to promote sustained increases in Akt phosphorylation in rat hypothalamus (23), it is tempting to speculate that Akt is a key downstream kinase mediating hormonal facilitation of lordosis behavior. It is also interesting that the combination of wortmannin and U0126 was not nearly as effective as the combination of PD98059 and wortmannin. These differences may reflect the relatively (at least 10-fold) greater ability of PD98059 to block raf-dependent activation of MAPK kinase 1 than of MAPK kinase 2 (also referred to as MEK1 and MEK2, respectively) (29). In contrast to the clear inhibition of lordosis produced by both PD98059 and U0126, icv infusions of SB203580 failed to modulate receptivity. As both PD98059 and U0126 target the p42/44 MAPKs, whereas SB203580 inhibits p38 MAPKs (29), our data implicate the p42/44 rather than the p38 MAPKs in hormonal priming of lordosis. This conclusion also agrees with observations from another group that E2 increases p42/44 MAPK but not p38 MAPK activity (37).
In summary, the present findings demonstrate for the first time that stimulation of both MAPK and PI3K signal transduction in the brain is necessary for estrogen facilitation of female reproductive behavior. The data also confirm earlier observations that blockade of brain IGF-IRs interferes with estrogen priming of lordosis. Because both E2 and IGF-I can independently, and sometimes synergistically, initiate sustained signaling through these downstream kinases, it is possible that the combined activation of MAPK and PI3K by estrogen and IGF-I are key molecular events underlying the neuroendocrine regulation of female reproductive function.
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Acknowledgments |
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Footnotes |
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Abbreviations: DMSO, Dimethylsulfoxide; E2, estradiol; E2B, estradiol benzoate; ER, estrogen receptor; icv, intracerebroventricular; IGF-IR, IGF-I receptor; LQ, lordosis quotient; MEK, MAPK kinase; P, progesterone; PI3K, phosphatidyl-inositol-3-kinase; OVX, ovariohysterectomized.
Received February 4, 2003.
Accepted for publication May 13, 2003.
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  P. Dewing, A. Christensen, G. Bondar, and P. Micevych Protein Kinase C Signaling in the Hypothalamic Arcuate Nucleus Regulates Sexual Receptivity in Female Rats Endocrinology, December 1, 2008; 149(12): 5934 - 5942. [Abstract] [Full Text] [PDF]
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 L. M. Garcia-Segura, B. Lorenz, and L. L DonCarlos The role of glia in the hypothalamus: implications for gonadal steroid feedback and reproductive neuroendocrine output Reproduction, April 1, 2008; 135(4): 419 - 429. [Abstract] [Full Text] [PDF]
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T. Jover-Mengual, R. S. Zukin, and A. M. Etgen MAPK Signaling Is Critical to Estradiol Protection of CA1 Neurons in Global Ischemia Endocrinology, March 1, 2007; 148(3): 1131 - 1143. [Abstract] [Full Text] [PDF]
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 P. Mendez, I. Azcoitia, and L. M. Garcia-Segura Interdependence of oestrogen and insulin-like growth factor-I in the brain: potential for analysing neuroprotective mechanisms J. Endocrinol., April 1, 2005; 185(1): 11 - 17. [Abstract] [Full Text] [PDF]
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O. Gonzalez-Flores, J. Shu, I. Camacho-Arroyo, and A. M. Etgen Regulation of Lordosis by Cyclic 3',5'-Guanosine Monophosphate, Progesterone, and Its 5{alpha}-Reduced Metabolites Involves Mitogen-Activated Protein Kinase Endocrinology, December 1, 2004; 145(12): 5560 - 5567. [Abstract] [Full Text] [PDF]
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