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Endocrinology Vol. 147, No. 5 2544-2549
Copyright © 2006 by The Endocrine Society

Facilitation of Lordosis in Rats by a Metabolite of Luteinizing Hormone Releasing Hormone

T. J. Wu, Marc J. Glucksman, James L. Roberts and Shaila K. Mani

Program in Neuroscience and Department of Obstetrics and Gynecology (T.J.W., S.K.M.), Uniformed Services University of the Health Sciences, Bethesda, Maryland 20814; Midwest Proteome Center and Department of Biochemistry and Molecular Biology (M.J.G.), Rosalind Franklin School of Medicine and Science, Chicago, Illinois 60064; Department of Pharmacology (J.L.R.), The University of Texas Health Science Center, San Antonio, Texas 78229; and Department of Molecular and Cellular Biology, Psychiatry and Behavioral Sciences (S.K.M.), Baylor College of Medicine, Houston, Texas 77030

Address all correspondence and requests for reprints to: T. John Wu, Ph.D., Department of Obstetrics and Gynecology, Room B2020, Uniformed Services University of the Health Sciences, 4301 Jones Bridge Road, Bethesda, Maryland 20814. E-mail: twu{at}usuhs.mil; or Shaila Mani, Ph.D., Departments of Molecular and Cellular Biology, Psychiatry and Behavioral Sciences, Baylor College of Medicine, Houston, Texas 77005. E-mail: smani{at}bcm.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
In the female rat, ovulation is preceded by a marked increase in the release of the decapeptide, LHRH, culminating in a preovulatory LH surge, which coincides with a period of sexual receptivity. The decapeptide, LHRH, is processed by a zinc metalloendopeptidase EC 3.4.24.15 (EP24.15) that cleaves the hormone at the Tyr5-Gly6 bond. We have previously reported that the autoregulation of LHRH gene expression can also be mediated by its metabolite, LHRH-(1–5). Given the central function of LHRH in reproduction and reproductive behavior, we examined the role of the metabolite, LHRH-(1–5), in mediation of LHRH-facilitated reproductive behavior. Intracerebroventricular administration of LHRH-(1–5) facilitated sexual behavior responses, similar to those facilitated by the decapeptide LHRH, in ovariectomized estradiol-primed female rats. Furthermore, immunoneutralization of EP24.15 resulted in the inhibition of the LHRH-facilitated lordosis but had no inhibitory effects on LHRH-(1–5)-facilitated lordosis. The LHRH antagonist, Antide, was capable of inhibiting LHRH-facilitated lordosis, without affecting LHRH-(1–5)-facilitated lordosis. Collectively, these results suggest a role for LHRH metabolites in the facilitation of female receptive behavior in rats.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE OVARIAN STEROID hormones estrogen (E) and progesterone (P) act within the brain to coordinate the release of pituitary gonadotropins that trigger ovulation. In the female rodent, the preovulatory discharge of gonadotropins is triggered by the release of LHRH on proestrous day and is followed by the onset of a sexually receptive phase when the female displays reproductive behavior (1). The LHRH neurons that comprise the final common pathway for the control of gonadotropin secretion are widely scattered in the basal forebrain region where their axons are directed to the median eminence. Regulation of the anterior pituitary is mediated by the release of LHRH into the hypophyseal portal vessels and its subsequent delivery to the target. Not only does LHRH release LH from the pituitary to trigger ovulation, it also facilitates sexual behavior in the rat. This synchronization of behavioral and peripheral preparations for reproduction maximizes the probability that a female will contact, and be inseminated, by a con-specific male at the optimal time to achieve pregnancy (1, 2).

Lordosis is a reflex that is a characteristic component of sexual behavior produced in response to copulatory stimulation by the male (3, 4). Intracerebroventricular (icv) administration of LHRH in estrone-primed ovariectomized and estradiol-primed ovariectomized and adrenalectomized female rats has been shown to increase proceptive behavior and the lordosis response (5, 6, 7, 8, 9). This effect of LHRH on facilitating lordosis that was initially shown in the rat has also been shown in other species and may serve as a common mechanism underlying this behavior (10, 11, 12, 13, 14, 15). The pathway involving the facilitation of lordosis behavior appears to involve the activation of P receptor, nitric oxide synthase, N-methyl-D-aspartic acid (NMDA) receptor, LHRH receptor (5, 6, 7, 8, 9, 16, 17), as well as a host of neurotransmitters, including serotonin, opioids, norepinephrine, and acetylcholine (16, 18, 19, 20).

The studies above underscore the complexity of the coordination of the reproductive physiology and behavior. We have reported previously that LHRH and its metabolite, LHRH-(1–5), are important in the autoregulation of its own gene expression (21). This has led us to hypothesize that this metabolite of LHRH, LHRH-(1–5), may also mediate the LHRH-facilitated lordosis in the estradiol-primed ovariectomized rat. To that end, the present experiments were designed to examine the effect(s) of LHRH and LHRH-(1–5) on the sexual behavior of female rats and to demonstrate the role of LHRH-(1–5) as a mediator of LHRH function in sexual behavior.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Ovariectomized Sprague Dawley rats (160–180 g body weight) were obtained from Harlan (Indianapolis, IN). The animals were housed in a controlled environment of 24 C on a 12-h light, 12-h dark reversed light cycle with lights off at 1200 h. Food and water were available ad libitum. All animal use and care was conducted in compliance with Federal guidelines and approved by the Institutional protocol and care committee. Proven male breeders were also purchased from Harlan. All rats were handled daily from the time of arrival to facilitate animal handling and to reduce stress.

Hormonal treatments and behavior testing
All behavior tests were conducted during the dark phase of the reversed light-dark cycle. Hormonal treatments and behavioral testing were performed according to established protocols in the laboratory (9, 10). All ovariectomized female rats were prescreened for their behavioral response in the presence of males by sequential treatments with the ovarian steroids, estradiol benzoate (EB) and P. Briefly, 2 µg EB in a sesame oil vehicle was injected sc followed by 100 µg P (sc) 48 h later. Four hours after the P injection, the animals were tested for sex behavior in the presence of sexually active males housed in a 50 x 45 x 24-cm polystyrene arena. Proceptive behavior was analyzed by determining the incidence of hopping, darting, and ear-wiggling across the entire test period. The proportion of animals displaying any of these behaviors was used as a measure of proceptivity. Receptive behavior of each female rat in the presence of the male was evaluated as an expression of the lordosis response of the female rat upon mounting by the male rat (22, 23). Lordosis response of each female was observed for 10 mounts by the male for a total of 30 min, or whichever came first, scored, and recorded. A lordosis quotient (LQ), calculated as a percentage of the total number of lordosis responses divided by the total number of mounts, was used as a measure of sexual receptivity (22). The observer was blind to the treatment conditions. All experiments described here were performed a minimum of six times each, and the observations were recorded. Subsequent to the initial prescreen, the rats displaying the most robust response (>80% LQs) were used in this study.

Stereotaxic surgery
The rats that displayed the most robust response to the sexual behavior test were implanted with chronic indwelling cannula in their third cerebral ventricle (3V). The rats were anesthetized using a combination anesthetic (0.5 ml/kg body weight) containing ketamine (42.8 mg/ml), xylazine (8.6 mg/ml), and acepromazine (1.4 mg/ml) by im injection. A stainless steel cannula (23 gauge) was implanted into the third ventricle adjacent to the ventromedial hypothalamic. The coordinates used were antero-posterior, bregma –3.3 mm; lateral, on the midline (above the superior longitudinal sinus); and dorsoventral, –8.5 mm, and were defined by Paxinos and Watson (24). The procedure for cannulation was similar to that described by Antunes-Rodrigues and McCann (25) and published previously (9, 26). Animals were allowed to recover from surgery for 1 wk before experimentation.

Central administration of test substances
The rats with chronic indwelling steel cannula in their 3V were randomly divided into treatment groups according to each experiment. All rats were EB-primed as before and administered (icv) the test substances indicated below. LHRH and LHRH-(1–5) were initially purchased from Calbiochem (La Jolla, CA). Subsequently, LHRH-(1–5) was synthesized on-site (Mr. M. Flora, Bioinstrumentation Center, Uniformed Services University of the Health Sciences, Bethesda, MD). The potent LHRH receptor antagonist, Antide (Ac-D-2-Nal-4-chloro-D-Phe-b-(3-pyridyl)-D-Ala-Ser-Lys(nicotinoyl)-D-Lys(nicotioyl)-Pro-D-Ala-NH2) (27, 28), was purchased from Bachem Bioscience (catalog no. H-9215; King of Prussia, PA). Antide was used for this study because it is known to have relatively low histamine-releasing activity relative to other antagonists (29). The polyclonal antiserum (no. 48) used in the study has been previously characterized and demonstrated to be specific against the zinc metalloendopeptide EC 3.4.24.15 (EP24.15) (30), an important enzyme in the metabolism of LHRH (30, 31).

Forty-eight hours after EB priming, the test substances, LHRH, LHRH-(1–5), or P, were microinjected into the 3V in 2 µl artificial cerebral spinal fluid (aCSF; catalog no. AH59-7316; Harvard Apparatus, Holliston, MA) over 60 sec using a programmable infusion pump (Stoelting Co., Wood Dale, IL). The initial experiment tested the effect of 35 and 70 pmol LHRH and LHRH-(1–5); P was injected at a concentration of 2 µg/2 µl. The antagonist, Antide, and/or antibody to EP24.15 were administered 60 min before the administration of LHRH or LHRH-(1–5) in EB-primed rats. The antagonist, Antide, was used initially tested at 50 and 100 pmol to determine the optimal dose that would effectively block the LHRH-facilitated lordosis behavior. The rationale for these initial doses was that affinity constant of Antide for the LHRH receptor I is an order of magnitude greater than LHRH (32, 33). Proceptive and receptive behaviors were observed in the presence of a sexually active male 30 min postinjection. Proceptivity was quantified by the number of hops and darts and ear wiggles displayed by the female for the duration of testing. Control animals received aCSF instead of a test substance. In the studies with antiserum, 2 µl of the antiserum (1:10 dilution in aCSF) was administered into the third ventricle with LHRH or LHRH-(1–5). For the antibody experiments, normal rabbit serum was used in place of the antibody in control animals.

Verification of cannula placement and data analyses
Upon completion of the behavioral tests, all the females were anesthetized and transcardially perfused with 4% paraformaldehyde in PBS (pH 7.4); brains were isolated and postfixed in the same fixative overnight and stored in 30% sucrose in PBS at 4 C until they were sectioned. Forty-micrometer sections were made through the region of cannula placement using a vibrating microtome and the sections were Nissl-stained to verify the cannula placement. Data from the animals with correctly placed cannulae were considered for further analysis.

Statistics
A one-way ANOVA followed by Fisher’s least significant difference post hoc test (significance at P < 0.05) were conducted to determine differences between the mean LQ of each treatment group.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
LHRH-(1–5) facilitates sexual behavior in female rats
icv administration of the pentapeptide LHRH-(1–5) facilitated (P < 0.05) lordosis response in the ovariectomized EB-primed rats (Fig. 1AGo). This effect of LHRH-(1–5) was dose-dependent and maximal at 70 pmol with the animals showing high levels of lordosis, similar to the P-treated controls. EB-primed ovariectomized rats that received icv injections of LHRH also showed high levels of lordosis response (P < 0.05) consistent with previously reported studies (10, 26) (Fig. 1BGo). Interestingly, it is also noted that LHRH and LHRH-(1–5) showed significantly enhanced hop/darting proceptive behavior compared with vehicle-treated controls (P < 0.05). However, although ear wiggling proceptive response was only slightly increased (P < 0.05) by both LHRH and LHRH-(1–5) compared with vehicle-treated controls, their effects were not as dramatic as those seen in P treatment (Table 1Go).


Figure 1
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  		FIG. 1. Effect of icv administration of LHRH-(1–5) (35 and 70 pmol/2 µl) (A) and LHRH (35- and 70-pmol/2 µl) (B) on the lordosis response of ovariectomized E-primed (sc) female rats. Microinjection of LHRH-(1–5) and LHRH resulted in a dose-dependent increase in the LQ. P treatment, icv, served as the positive control. There were eight rats per group. Values in this and subsequent figures are the mean ± SEM of the LQ scores (expressed as a percentage of the total number of lordosis responses divided by the total number of mounts). Bars with different letters (a, b, and c) indicate differences (P < 0.05).

 

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  		TABLE 1. Proceptive behaviors following icv injections of LHRH, LHRH-(1–5), and P in EB-primed ovariectomized rats

 
EP24.15 antiserum inhibits LHRH- but not LHRH-(1–5)-facilitated lordosis
To examine the importance of LHRH metabolism on its ability to facilitate lordosis response, we treated rats with the antiserum to the endopeptidase EP24.15 to immunoneutralize the enzyme activity, and examined LHRH-facilitated lordosis. Administration of the EP24.15 antiserum icv 60 min before the injection of LHRH-(1–5) did not inhibit or prevent the LHRH-(1–5)-facilitated lordosis (Fig. 2AGo), suggesting that the antiserum by itself did not have any effect on the EB-primed rat receptive behavior. In contrast, the EP24.15 antiserum LHRH prevented lordosis response in EB-primed rats (P > 0.10 compared with vehicle treatment; Fig. 2BGo). Furthermore, EP24.15 antiserum by itself had no effect on lordosis compared with vehicle treatment (P > 0.10) (14.3 ± 5.5 vs. 11.7 ± 2.1, respectively). An added control showed that normal rabbit serum (10.2 ± 1.6) did not significantly (P > 0.10) affect lordosis behavior in rats when compared with vehicle-treated rats (11.7 ± 2.1).


Figure 2
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  		FIG. 2. Effect of EP24.15 antiserum on LHRH-(1–5)- (A) and LHRH-facilitated (B) sexual behavior of ovariectomized E-primed female rats. Normal rabbit serum was used in lieu of the antiserum as a negative control (Vehicle). The peptides, 70 pmol LHRH-(1–5) and 70 pmol LHRH, were administered 1 h after the administration of the antiserum. There were eight rats per group. *, P < 0.05 vs. vehicle. LHRH-(1–5) is abbreviated as (1 2 3 4 5 ) on the horizontal axis.

 
The LHRH antagonist, Antide, inhibits LHRH-facilitated, but not LHRH-(1–5)-facilitated lordosis
To examine a role of the LHRH receptor in the facilitation of lordosis behavior, the potent LHRH antagonist, Antide, was used. Administration of LHRH receptor antagonist, Antide, icv, 60 min before the icv administration of LHRH, inhibited lordosis response in EB-primed ovariectomized rats (Fig. 3BGo). Antide at 50 and 100 pmol significantly inhibited the LHRH-facilitated lordosis (P < 0.05). In contrast, the lower dose of Antide (50 pmol), capable of inhibiting lordosis in LHRH-treated rats was unable to inhibit LHRH-(1–5)-facilitated lordosis (Fig. 3AGo). Antide by itself did not affect lordosis behavior (data not shown).


Figure 3
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  		FIG. 3. Effect of the LHRH receptor antagonist, Antide, on LHRH-(1–5) (A) and LHRH-facilitated (B) sexual behavior of ovariectomized E-primed female rats. Both peptides, 70 pmol LHRH-(1–5) and 70 pmol LHRH were administered 1 h after the administration of the Antide. In the experiment with LHRH (b), 50 and 100 pmol Antide (in 2 µl) was administered and was effective in blocking the ability of LHRH to facilitate lordosis behavior. In the experiment with LHRH-(1–5), only the 50 pmol Antide (in 2 µl) was used, which did not block the ability of LHRH-(1–5) to facilitate lordosis. *, P < 0.05 vs. vehicle. LHRH-(1–5) is abbreviated as (1 2 3 4 5 ) on the horizontal axis.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The current observations support our hypothesis that the metabolite of LHRH, LHRH-(1–5), is biologically active and is an integral part of the LHRH-facilitated lordosis in the female ovariectomized EB-primed rat. To our knowledge, this is the first time that the cleavage product of LHRH, LHRH-(1–5), has been shown to facilitate a behavioral effect in an animal. Previous studies have suggested that fragments of LHRH may facilitate lordosis behavior and may be biologically active (13, 34, 35, 36, 37, 38). However, the studies focused on a fragment, Ac-LHRH-(5–10), that to date, has not been identified endogenously (37, 38). In contrast, for LHRH, the primary cleavage site appears to be the peptide bond linking the fifth and sixth amino acids (Tyr-Gly) (39, 40, 41) and is cleaved by the zinc metalloendopeptidase, EP24.15, a peptidase that has been identified and cloned (30, 42).

It is well established that the hypothalamic hypophysiotropic peptide hormone, LHRH, not only releases LH from the pituitary, but also induces sexual behavior (1, 43, 44). LHRH facilitates lordosis after mounting by the male rat in estrone-primed ovariectomized (7) and EB-primed adrenalectomized female rats (2). Interestingly, injection of both LHRH-(1–5) and LHRH into the third ventricle facilitated lordosis behavior in the EB-primed ovariectomized rats. This effect of LHRH-(1–5) is dose-dependent and equivalent to the effect of the LHRH dose previously shown to be potent in facilitating lordosis behavior (7, 9). Furthermore, the onset of proceptive behavior and lordosis appears to occur faster for those rats treated with LHRH-(1–5) than LHRH (T. J. Wu and S. K. Mani, unpublished data). More detailed studies are underway to address the timing of the onset of these behaviors. Nevertheless, the result from this study suggests that LHRH-(1–5) is an important component of the LHRH-facilitated lordosis behavior.

To further test this possibility that LHRH-(1–5) mediates the LHRH-facilitated lordosis behavior, we blocked the metabolism of LHRH by immunoneutralizing the enzyme, EP24.15, previously shown to be important in the breakdown of LHRH (31, 45, 46). The results show that blocking the metabolism of LHRH by immunoneutralizing EP24.15 during the injection of LHRH eliminated its ability to facilitate lordosis behavior. In contrast, immunoneutralization of EP24.15 during the injection of LHRH-(1–5) did not affect the ability of this pentapeptide to facilitate lordosis. This observation further supports our hypothesis that the LHRH facilitated lordosis behavior in the female is mediated by its metabolism to produce LHRH-(1–5). EP24.15 has been shown to cleave LHRH at the bond linking Tyr and Gly, the fifth and sixth amino acids, to produce LHRH-(1–5), and is known to modulate LHRH signal to the pituitary (31, 45, 46). A systemically administered EP24.15 inhibitor to the rat during the steroid-induced LH surge enhanced the magnitude of this surge. Finally, EP24.15 immunoreactivity not only coincides with regions of the brain where LHRH neurons and fibers are localized but also fluctuates on the proestrous day of the rat estrous cycle within the median eminence in a phase-dependent manner (31). Our laboratory has recently shown that the mechanism underlying the autoregulation of LHRH also involves this metabolite, LHRH-(1–5). Although LHRH negatively regulates its own gene expression, LHRH-(1–5) positively regulates LHRH gene expression in the GT1 cells (21).

Previous studies have shown that Antide is a highly specific LHRH receptor antagonist (27, 28). In this study, we sought to block the ability of LHRH and LHRH-(1–5) to facilitate lordosis behavior using this LHRH receptor antagonist. In this experiment, Antide blocked LHRH-facilitated lordosis behavior but not LHRH-(1–5) effect. This implies that, like Ac-LHRH-(5–10) (35), the LHRH-(1–5) effect may not act through the LHRH receptor. Yet, the LHRH receptor would appear to be involved in the lordosis pathway because Antide blocked the LHRH-facilitated lordosis behavior. Interestingly, this observation is similar to a previous study showing that Ac-LHRH-(5–10) is not blocked by the LHRH antagonist ([Ac-dehydro,Pro1,pCl,D-Phe2,D-Trp3,6]-LHRH; Ref.35). This paradox may be resolved in part by the complexity of the cellular events leading to lordosis behavior. Previously, P has been demonstrated to facilitate sexual behavior sequentially by nitric oxide and LHRH (5, 9). Other groups have also suggested that antagonism of the LHRH receptor reversed the effects of the LHRH-facilitated lordosis (5, 6, 35, 47). It may be possible that the LHRH receptor may be required in the steps preceding the metabolism of LHRH and that its inhibition (through an antagonist) may impede this process. Another plausible explanation is that Antide may directly inhibit EP24.15. GnRH analogs were shown to directly inhibit EP24.15 activity in vitro (48). It is interesting to note from structure-activity relationships that a tendency exists in that the greater the inhibition of EP24.15 activity with GnRH analogs results with greater the volume and the more hydrophobic the residue in the sixth position of the GnRH analog (48); Antide as well as all other clinical LHRH analogs have a D-amino acid at the sixth position where EP24.15 would cleave LHRH. The use of Antide may have a similar effect of inhibiting EP24.15 activity as in the immunoneutralization experiments shown in the present study attenuating production of LHRH-(1–5) from LHRH. Thus, inhibiting LHRH-(1–5) production can prevent the effect of LHRH on lordosis behavior.

The results from this study suggest that LHRH-(1–5) is not likely to act through the LHRH receptor because its blockade did not affect LHRH-(1–5) facilitation of lordosis behavior. A similar finding suggested that not only is an LHRH antagonist unable to inhibit mating behavior facilitated by the LHRH fragment, Ac-LHRH-(5–10) (35), but that the same LHRH fragment is also unable to interfere with the binding of an LHRH agonist to the receptor (49). Since the discovery of multiple forms of LHRH (over 20 in mammals) and some of their cognate receptors, it is possible that LHRH-(1–5) may act through one of the alternative LHRH receptors. However, it remains controversial as to whether there exists a homolog to the marmoset LHRH receptor-II in the rat. There is evidence that this gene has been inactivated from the genome (50, 51). Nevertheless, the presence of a second form of LHRH, LHRH-II (pGlu-His-Trp-Ser-His-Gly-Trp-Tyr-Pro-Gly-NH2), across vertebrates, including the rodent, suggests that this second form of LHRH may be functional (51, 52, 53, 54, 55, 56, 57). It is interesting to note that, whereas LHRH-II may promote LH secretion in mammals, its effect is blocked with an antagonist to LHRH receptor-1 (58, 59). In contrast to the ability of the LHRH receptor-1 antagonist to block the LHRH-II-induced LH secretion, the antagonist, Antide, does not have the same ability to block LHRH-II facilitation of sexual behavior in the mouse (60). It is certainly possible that LHRH-(1–5) action may be mediated by LHRH receptor-II. A preliminary report from our laboratory suggests that LHRH-(1–5) may function through a pathway similar to LHRH-II but not LHRH-I, in the human Ishikawa cell-line (61). This is supported by the similarity in the first 4 amino acids of LHRH-II and LHRH-(1–5). Yet, in the studies with Ac-LHRH-(5–10), Moss and co-workers (37, 38) found that, whereas Ac-LHRH-(5–10) may facilitate sexual behavior in the rat and stimulate CA1 pyramidal neuronal activity in in vitro rat hippocampal slice preparations, LHRH-(1–6) had no activity in the same assays. These studies underscore the importance of the tyrosine residue in LHRH-(1–5) for rendering biological activity. To resolve these issues, biochemical studies involving the down-regulation of the LHRH receptor-II gene expression is currently in progress.

Bourguignon and co-workers (62) suggested that LHRH-(1–5) may serve as an antagonist to the NMDA receptor but not the kainate receptor in a rat hypothalamic perifusion model measuring the secretion of LHRH. Although this may be a likely scenario for LHRH secretion, it is not likely in the current behavioral model. A number of studies have now shown that inhibition of the NMDA receptor with their respective antagonists would block the P- and LHRH-facilitated lordosis behavior (17, 63, 64, 65, 66). In the lordosis model, if LHRH-(1–5) acts as an antagonist to the NMDA receptor, then it should not facilitate lordosis unless it blocks an upstream site from the LHRH-(1–5) action. Further studies are warranted to examine this possibility.

Previous studies showed not only that LHRH could induce lordosis in response to sexual stimulation in E-primed rat (7) but also that LHRH antiserum and antagonists could block facilitation by P (5, 8, 9). We may deduce that the P-facilitated lordosis is, at least in part, mediated by LHRH and its subsequent metabolism to produce LHRH-(1–5). It is interesting to note that P may induce a 6-fold increase in expression of the metalloendopeptidase EP24.15 in the rat hypothalamus and mid-brain (Salvit, C., T. J. Wu, M. J. Glucksman, and J. L. Roberts, unpublished observation). In addition to facilitating lordosis, P facilitates proceptive behavior. Earlier studies have shown that, in the primate (67) but not the rodent (6, 7, 37), LHRH may enhance proceptivity. This is in contrast to the results from the current study. The reason will require further studies that specifically address the potential difference in observation.

It is evident that the induction of sexual behavior and the regulation LHRH neuronal function is highly dependent on a large number of inputs including nitric oxide synthase, NMDA, and {gamma}-aminobutyric acid (for review, see Refs.15 and 67). Precisely how the metabolism of LHRH and the role of the LHRH receptor fits into this complex sequence of events remains to be determined by mapping the spatial and temporal activation of cellular and intracellular structures. Data from this study show that blocking the production of the metabolite LHRH-(1–5) blocks the LHRH facilitation of lordosis provides evidence that LHRH-(1–5) may be exerting a biologic effect to facilitate lordosis.


   Acknowledgments
 
The opinions or assertions contained herein are the private ones of the authors and are not to be construed as official or reflecting the views of the Department of Defense or the Uniformed Services University of the Health Sciences. We thank Andrea Reyna for expert technical assistance in animal surgery and behavioral testing. We also thank Drs. Richard Mills and Heather Fugger and Ms. Emily Baldwin for their editorial assistance, and Drs. Greg Mueller and Bill Driscoll for helpful discussions regarding peptide and enzyme chemistry.


   Footnotes
 
This work was supported by the National Science Foundation, IBN-0315923 (to T.J.W.), United States Public Health Service Grants MH63954 and MH57442 from the National Institutes of Health (to S.K.M.)., and HRSA C76 HF03610-01-00 (to M.J.G).

Disclosure Statement: The authors have nothing to disclose.

First Published Online February 23, 2006

Abbreviations: aCSF, Artificial cerebral spinal fluid; E, estrogen; EB, estradiol benzoate; icv, intracerebroventricular; LQ, lordosis quotient; NMDA, N-methyl-D-aspartic acid; NRS, normal rabbit serum; P, progesterone; 3V, third cerebral ventricle.

Received December 23, 2005.

Accepted for publication February 13, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Moss RL, Cooper KJ 1973 Temporal relationship of spontaneous and coitus-induced release of luteinizing hormone in the normal cyclic rat. Endocrinology 92:1748–1753[Medline]
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