Gonadotropin-Releasing Hormone II Stimulates Female Sexual Behavior in Marmoset Monkeys
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《内分泌学杂志》
National Primate Research Center (D.K.B., T.M.B., D.H.A.), University of Wisconsin, Madison, Wisconsin 53715
Department of Obstetrics and Gynecology (D.H.A.), University of Wisconsin, Madison, Wisconsin 53792
Endocrinology-Reproductive Physiology Training Program (D.H.A.), University of Wisconsin, Madison, Wisconsin 53706
Medical Research Council Human Reproduction Sciences Unit (R.P.M.), EH3 9ET Edinburgh, Scotland, United Kingdom
Abstract
GnRH II (pGlu-His-Trp-Ser-Try-Gly-Leu-Arg-Pro-GlyNH2), an evolutionarily conserved member of the GnRH family, stimulates reproductive behavior in a number of vertebrates. To explore a role for GnRH II in regulating primate sexual behavior, eight adult female common marmosets, each fitted with an indwelling intracerebroventricular (icv) cannula, were ovariectomized, implanted subcutaneously with empty (n = 4) or estradiol-filled (n = 4) SILASTIC brand capsules, and pair housed with an adult male mate. After icv infusion of vehicle or peptides, females were placed in an observation cage for 90 min, out of visual contact with other marmosets, before the 30-min behavioral test with their male partner. Compared with vehicle, GnRH II (1 and 10 μg) increased the total number of proceptive (sexual solicitation) behaviors (tongue flicking, proceptive stares, and frozen postures) exhibited by females toward their pair mates and specifically increased the frequency of freeze postures. Effects were maximal at 1 μg and not dependent upon estradiol supplementation. GnRH II agonists/GnRH I antagonists 135-18 (1 μg) and 132-25 (1 μg), which stimulate inositol phosphate production via the marmoset type II receptor, increased the frequency of total proceptive behavior but did not specifically stimulate freeze-posture behavior. In contrast, GnRH I, at 1 μg, did not alter the frequency of proceptive behaviors. Female receptivity (female compliance with male sexual behavior) was not altered by any of the peptides tested. These findings implicate a role for GnRH II and the cognate GnRH type II receptor in stimulating female marmoset sexual behavior.
Introduction
GnRHs STIMULATE female sexual behavior in many different species (see Table 1). An understanding of the role of GnRHs in primate sexual behavior is, however, lacking. Compared with other mammalian and nonmammalian groups, female sexual behavior in primates is less synchronized with the timing of ovulation and less strictly regulated by ovarian hormones (1, 2). In a variety of primate species, copulations frequently increase around the time of ovulation, but they also occur throughout the menstrual cycle (1), as well as after ovariectomy (3), suggesting a degree of emancipation of primate sexual behavior from ovarian control that is not exhibited by other spontaneously ovulating, nonprimate species (2, 4, 5). Increased likelihood of male ejaculation during copulations with periovulatory females, however, suggests that ovarian influences on male-female sexual interactions are still present in primates and may include nonbehavioral changes in female attractiveness as well as hormonally mediated behavioral changes in female sexual behavior and motivation (2, 6, 7). Female primates can thus exhibit sexual behavior independently of gonadal hormones in addition to ovarian-cycle influences on sexual behavior expression.
GnRH peptides are obvious candidates for extraovarian stimulators of primate sexual behavior. Because they also act as potent neuroendocrine regulators of ovarian function (see Ref.8 for review), GnRHs can potentially drive ovarian influences on female sexual behavior. To date, 24 GnRH decapeptides have been identified (9, 10), and most vertebrates express two or more GnRH variants [e.g. in primates, GnRH I and GnRH II have been identified (9, 11)]. The common mammalian form, GnRH I (pGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2), is thought to be primarily released by the hypothalamus to stimulate FSH and LH release from pituitary gonadotropes that regulate the ovarian or menstrual cycle (12, 13, 14). In contrast, GnRH II, evolutionarily conserved from bony fish to humans (pGlu-His-Trp-Ser-His-Gly-Trp-Tyr-Pro-Gly-NH2) (8, 15, 16), appears to function more as a neurotransmitter than a hypothalamic neurosecretory product (10, 11, 17). Both GnRH peptides can stimulate female sexual behavior in a variety of vertebrate species (see Table 1); however, GnRH II has received more recent interest (18).
Although GnRH II regulation of primate sexual behavior has until now been only a matter of speculation (19), such a role is supported by neural localization of GnRH II and its highly specific receptors in brain areas involved in the expression of sexual behavior in both common marmosets (20) and rhesus macaques (21, 22), including the preoptic area and ventro- and dorsomedial nuclei (11). Currently, two distinct GnRH receptors (type I and type II; see Ref.9 for discussion of their characteristics) are candidates to mediate GnRH II action (see Table 1 for species distribution). Type I receptors, which are found in all mammals (15, 23) and mediate GnRH I neurosecretory stimulation of pituitary gonadotropes, are also capable of binding GnRH II with high affinity (9) and may be able to differentially respond to both forms of GnRH (9, 24). In contrast, type II receptors, have high ligand selectivity for GnRH II (20, 23) and are unlikely mediators of GnRH I effects (9).
In studies of GnRH effects on sexual behavior, it is generally unknown which receptor(s) mediates GnRH action. The existence of multiple GnRH receptors in some species (Table 1) (9, 11), as well as the ability of GnRH receptors to bind to multiple GnRH ligands with differing affinities (9), may explain early observations that GnRH analogs incapable of stimulating FSH or LH release from pituitary gonadotropes increase lordosis in female rats similarly to GnRH I (25). Such realization, however, complicates interpretation of GnRH action. Characterization of GnRH analogs with specific receptor-type specificities, such as the mammalian GnRH type I receptor antagonists 135-18 and 135-25 that can act as agonists at other receptors (20, 26, 27), make these analogs useful tools with which to investigate GnRH II receptor type involvement.
Marmosets are an ideal model for testing the effects of GnRH II in a primate because of the identification of GnRH II ligand and receptor in the preoptic area and hypothalamus (20), areas of the brain functionally implicated in the regulation of female marmoset sexual behavior (28, 29, 30). The availability of a well-characterized behavioral ethogram (Table 2) (31, 32, 33, 34, 35, 36) of marmoset sexual proceptivity (solicitation behaviors that invite mounting by males) and sexual receptivity [reflecting female compliance with male mounting/intromission attempts (6)] permits validated quantification of marmoset sexual behavior.
In female marmosets, sexual proceptivity is strongly influenced by the ovarian cycle and generally enhanced by estradiol (31). Female invitational behaviors, which can play a crucial role in determining whether copulation will occur in primates (see Refs.34 and 35), indicate the sexual motivation of a female (4). In contrast, sexual receptivity changes little across the ovarian cycle, and the effects of estrogen stimulation are inconsistent (1, 31). Female marmosets are attractive to the male throughout the ovarian cycle, as well as after ovariectomy and after adrenalectomy (1, 3, 33, 34, 36). The ability of a male to achieve intromission is a marker of female receptivity, because the male marmoset usually engages in a single penile intromission before ejaculation (31). Typical of many primate and nonprimate species, female compliance is necessary for successful intromission by the male.
In this study, GnRHs I and II, as well as analogs that acted as antagonists on the marmoset type I GnRH receptor, but agonists at the type II receptor, were tested in ovariectomized female common marmosets to determine whether GnRH II stimulates female sexual behavior via the type II receptor.
Materials and Methods
Study animals
This research was conducted in accordance with the recommendations of the Guide for the Care and Use of Laboratory Animals and the Animal Welfare Act and its subsequent amendments. All animal procedures were reviewed and approved by the Graduate School Animal Care and Use Committee of the University of Wisconsin-Madison. The National Primate Research Center at the University of Wisconsin-Madison (WPRC) is accredited by the Association for Assessment and Accreditation of Laboratory Animal Care as part of the University of Wisconsin-Madison Graduate School.
Eight adult (age 2–4 yr) nulliparous captive-born common marmoset (Callithrix jacchus) females were pair housed with similarly aged male partners at the WPRC for at least 6 months before the onset of this study. Females were housed with the same male partner for the entire study. Marmosets were housed indoors at the WPRC in aluminum and welded wire cages, measuring 61 x 91 x 183 cm, and had visual, olfactory, and auditory access to conspecifics in other cages. Lights were on from 0600–1800 h, and animals were fed at 1230–1330 h daily. Additional information on animal housing and husbandry has been published previously (38).
Surgical procedures
To implant intracerebroventricular (icv) cannulae, females were anesthetized with ketamine (15 mg/kg, im) and placed into a stereotaxic apparatus. Anesthesia was maintained with isoflurane (0.5–2%; 0.6 liter/min). Each animal was given 5 mg/kg dexamethasone im and 5 ml of 5% dextrose sc 14–18 h before surgery and 5 ml of 5% dextrose sc, 0.02–0.04 mg/kg atropine im, and 0.01 mg/kg buprenorphine im after anesthesia induction. Fluid replacement was maintained throughout the surgery by sc administration of 5 ml of 5% dextrose/h. Vital signs were monitored via pulse oximetry. Body temperature was maintained by a wrap-around body heating apparatus thermostatically controlled at body temperature. At the start of the procedure, 2% lidocaine was injected id to the scalp. Presurgery x-rays of the head of each marmoset were compared with x-ray ventriculograms from previous animals with comparable head size and shape to enable accurate estimation of cannula length and coordinates for icv placement. Guide cannulae (22 gauge, 11–13 mm; Plastics One, Roanoke, VA) were implanted into the third ventricle, and placement was verified by x-ray after infusion of radiopaque dye (20 μl infused over 1 min; Omnipaque, Nycomed Inc., Princeton, NJ). After the position of the guide cannula was confirmed, the guide cannula was anchored in place using dental acrylic (Justi Products, Oxnard, CA). The infusion cannula was removed from the implanted guide cannula and replaced with a stylet.
For bilateral ovariectomy, females were anesthetized with ketamine (15 mg/kg, im) and maintained on isoflurane (2%; 0.6 liter/min oxygen). At the time of ketamine administration, 0.02–0.04 mg/kg atropine im and 0.01 mg/kg buprenorphine im were administered. Each ovary was isolated through a ventral midline incision and exteriorized for visualization of the fallopian tube and ovarian pedicle. Subsequent histological examination confirmed complete ovarian removal.
Estradiol replacement
Two SILASTIC brand capsules (inner diameter, 0.058 in.; outer diameter, 0.077 in.; length, 11-mm; Dow Corning, Midland, MI) were implanted into each female sc via a small dorsal midline incision between the scapulae under ketamine anesthesia (10 mg/kg, iv) after 2% lidocaine (2 mg/kg) sc injection. Four females were implanted with 17-estradiol-filled capsules (Sigma, St. Louis, MO) previously shown to maintain approximately mid-follicular-phase levels of estradiol (Abbott, D. H., unpublished observations), whereas the remaining females were implanted with empty SILASTIC brand capsules.
Experimental design
The study tested GnRH II and related peptides against vehicle in three experiments: experiment 1 tested GnRH II (0, 1, and 10 μg; four replicate behavioral tests per dose per eight females), experiment 2 tested the GnRH type II receptor-specific activator 135-18 (0 and 1 μg; three replicate behavioral tests per treatment per eight females), and experiment 3 tested the GnRH type II receptor-specific activator 135-25 and GnRH I (0 and both peptides at 1 μg; three to four behavioral tests per treatment per seven females). In each experiment, females received treatments in a counterbalanced order with respect to GnRH-related peptides and vehicle, with more than 2 d between successive infusions. Treatment and behavioral testing for experiment 1 started 7 wk after ovariectomy and 9–10 wk after cannula implantation. Experiments 2 and 3 started 15 and 28 wk after ovariectomy, respectively (i.e. 7 wk after removal of capsules from the prior experiment). One week before each experiment, females were implanted sc with estradiol-filled (n = 4 for experiments 1 and 2; n = 3 for experiment 3) or empty (n = 4 for experiments 1–3) SILASTIC brand capsules; each female received similarly filled capsules for all experiments. SILASTIC brand capsules were removed at the end of each experiment, and no testing was performed between experiments. Estradiol levels in the blood were monitored every 2 wk whenever SILASTIC brand capsules were implanted.
For each behavioral test, subjects were removed from their home cage between 0700 and 1100 h, were blood sampled to assess baseline circulating cortisol levels (37), received peptide treatment or vehicle icv, were separated from their male partner for 90 min, and were observed in a 30-min sexual behavioral test. After the behavioral test, subjects were blood sampled again to assess changes in circulating cortisol levels resulting from treatment and testing.
Blood sampling and hormone assays
Marmosets were manually captured from the home cage or test cage and briefly restrained in a marmoset restraint tube (38) for collection of blood into heparinized syringes by femoral puncture (0.1 ml for cortisol; 0.4 ml for estradiol). To minimize blood cortisol responses to the sampling procedures (39), and pretest blood samples were collected within 3 min of home-cage entry (except for 11% of samples that were collected between 3 and 4 min and 3% collected between 4 and 6 min; without effect on cortisol values, data not shown) or within 4 min after the end of a behavioral test. Blood samples were refrigerated at 4 C for 1–5 h until they were centrifuged for 10 min at 2000 rpm. Plasma was separated and stored at –20 C until assay.
Plasma cortisol concentrations were measured in duplicate aliquots using an antibody-coated tube RIA kit, coat (Incstar Corp., Stillwater, MN) (39). The assay sensitivity was 0.1 ng/tube, and the intra- and interassay coefficients of variation for a plasma pool assayed in duplicate were 5.26% and 7.19%, respectively.
Estradiol was measured by RIA as previously described (37). The intra- and interassay coefficients of variation were 5.11% and 13.89%, respectively.
Intracerebroventricular infusions
After blood sampling, the stylet plugging the indwelling cannula was removed and a sterile infusion cannula, attached to a primed 25-μl Hamilton syringe, was inserted after effusion of cerebrospinal fluid was observed. Using aseptic technique, 8 μl of artificial cerebrospinal fluid (vehicle) or 8 μl of peptide treatment (in artificial cerebrospinal fluid) was infused into the third ventricle over 1 min. The infusion cannula was left in place for 30 sec to prevent backflow and replaced with a sterile stylet. The marmosets readily adapted to the icv infusions and, by the end of experiment 1 and throughout experiments 2 and 3, seven of the eight animals would voluntarily bow their heads for the procedure.
Behavioral testing
Behavioral responses to GnRH II and related peptides were tested in ovariectomized female pair-bonded common marmosets under laboratory conditions designed to minimize baseline levels of sexual behavior. To this end, motivators of sexual behavior, such as overnight separation before testing (40) or separate housing of partners between tests (1, 3), were avoided. After icv infusion of treatment, females were placed in a test cage (61 x 46 x 61 cm) out of visual contact from their male partner for 90 min. The behavioral test then occurred from 90–120 min after the GnRH II or vehicle infusion because, in female rats, sexual behavior is stimulated 90–180 min after icv infusion of GnRH I or certain fragments or analogs of GnRH I (41) and, in female marmosets, iv infusion of GnRH I stimulates female sexual behavior at 120 min (42).
At the start of the behavioral test, the male partner was introduced to the female by remote door operation, and behaviors were recorded by observers from behind a one-way mirror. Frequencies of sexual behaviors, classified as either proceptive (sexual solicitation behaviors: tongue flicking, proceptive stares, and freeze postures) or receptive (compliance with male sexual behavior: accepting male mount, intromission, or ejaculation; see Table 2 for descriptions), and latencies to those behaviors were recorded for 30 min using a laptop computer with an elapsed-time tag and videotaped for archival on DVDs for re-review. After testing, a blood sample was taken from the female within 4 min to permit assessment of posttest female cortisol levels, and the pair was reunited in their home cage. The 90-min separation of pair mates before each 30-min behavioral test produced frequencies of sexual behavior similar to those observed between pair-bonded animals in their home cages (35), in contrast to the higher frequencies of sexual behavior displayed during behavioral tests between newly paired or singly housed animals.
Behavioral tests were reanalyzed in a random fashion by two observers from the DVD recordings for all three experiments. Each observer was blind with respect to experiment and treatment details. The reanalyzed data were compared with data taken on the day of the test. Interobserver reliability scores for behavioral data collection averaged 97.3%, and within-observer reliability scores averaged 88.9%.
Data analysis
Frequency of proceptive stares, freeze postures, and tongue flicking provided assessment of proceptivity. Receptivity was quantified by frequencies of female mount acceptance and male intromission and ejaculation, as well by latencies to exhibit those behaviors from the onset of the test and the first male mount or mount attempt, respectively. Analyses were performed on transformed behavioral frequencies [square root (1 + x)] and the ratio of posttest to pretest plasma cortisol levels [log] to achieve homogeneity of variance and to increase linearity of data (43, 44). Both transformations normalized data distributions. Values for each female for each dose were compared by two-way ANOVA with repeated-measures design using estradiol and treatment as factors to determine the independent effects of these variables and their interaction. Post hoc univariate analysis determined variable differences when statistical interactions were significantly different (P < 0.05). Results are presented as back-transformed least-square means + 95% confidence intervals, as validated by Bland and Altman (45). Analyses of plasma estradiol and pre- and posttest cortisol values were performed on untransformed data.
Results
Steroid hormone levels during behavioral testing
Estradiol.
Ovariectomized females implanted with empty SILASTIC brand capsules (n = 3–4) exhibited lower (P < 0.001) circulating estradiol levels of 76 ± 34 pg/ml during periods of behavioral testing compared with mid-follicular-phase estradiol levels (278 ± 628 pg/ml) achieved in females implanted with estradiol-filled capsules (n = 4). Mean values for circulating estradiol in individual females are shown for each experiment in Fig. 1 and are similar within each female throughout all experiments. There were no correlations between any parameter of female sexual behavior and circulating plasma estradiol levels (data not shown).
Cortisol.
Pretest basal plasma cortisol levels (Table 3) were within expected values (37) for female marmosets receiving no estradiol replacement after ovariectomy (n = 3–4) and for ovary-intact females receiving estradiol replacement that reflected follicular-phase circulating levels (n = 4). There was no effect of estradiol replacement on either pre- or posttest plasma cortisol levels (Table 3). This lack of effect of estradiol was not surprising because, compared with ovariectomized females, plasma cortisol levels in ovary-intact female marmosets are elevated only during the periovulatory period (37). During stressful procedures, such as new social group formation, plasma cortisol levels can be expected to increase approximately 2-fold over values from undisturbed animals (46). The ratio of posttest circulating cortisol to pretest baseline values was similar for all peptides infused when compared with their vehicle controls (all females combined; n = 8): experiment 1 (posttest cortisol was 115 ± 15% of pretest levels after vehicle vs. 120 ± 16% and 128 ± 17% after 1 and 10 μg of GnRH II, respectively); experiment 2 (120 ± 21% after vehicle vs. 120 ± 21% after 135-18); and experiment 3 (134 ± 21% after vehicle vs. 133 ± 21% after 135-25 and 130 ± 21% after GnRH I). In experiments 1 and 2, there was no effect of estradiol on the ratio of pretest to posttest cortisol (P = 0.174 and P = 0.243). In experiment 3, however, the ratio of posttest cortisol to baseline was unexpectedly higher (P = 0.011) in females without estradiol supplementation compared with females with estradiol implants, regardless of treatment. There were no correlations between any of the measures of female sexual behavior and either pretest or posttest plasma cortisol levels (data not shown).
Baseline levels of sexual behavior
After icv infusion of vehicle, female partners failed to exhibit proceptive, sexual soliciting behavior in 47% of tests, and their male partners failed to achieve a mount with intromission and ejaculation in over half of these vehicle tests. Baseline levels (vehicle treatments) of proceptive and receptive behaviors were consistent between the three experiments (see Fig. 1 and Table 4).
Stimulation of female marmoset sexual proceptivity (sexual solicitation of males by females)
Experiment 1.
There were no significant test-replicate effects indicating that over the 6 wk of behavioral testing, subjects did not alter their responses to either GnRH II or vehicle treatment. GnRH II stimulated female proceptivity, as reflected by a doubling of the mean total number of proceptive behaviors (1.89 ± 1.13 behaviors/GnRH II test) compared with vehicle control (0.81 ± 0.92 behaviors/vehicle test) when all females were combined (n = 8). Effects of GnRH II were maximal at 1 μg (treatment, P = 0.001) but were not estradiol dependent. There were no effects of estradiol (P = 0.408) and no estradiol x GnRH II interaction (P = 0.057) involving the total number of proceptive behaviors. GnRH II specifically increased the frequency of freeze postures (from 0.4 ± 0.36/vehicle test to 0.67 ± 0.4/1-μg GnRH II test and 0.74 ± 0.4/10-μg GnRH II test; P = 0.042; for all females combined; n = 8) without affecting the frequency of tongue-flicking bouts (0.37 ± 0.51/vehicle test to 1.08 ± 0.62/1-μg GnRH II test and 0.81 ± 0.58/10-μg GnRH II test; P = 0.065) or proceptive stares (0.12 ± 0.23/vehicle test, 0.15 ± 0.23/1-μg GnRH II, and 0.20 ± 0.24/10-μg GnRH II test; P = 0.684) for all females combined (n = 8). Frequencies of sexual behavior induced by GnRH II were comparable to levels observed in periovulatory females studied in their home cage with their partner (34).
Experiment 2.
There were no significant test-replicate effects indicating that over the 6 wk of behavioral testing, subjects did not alter their responses to either 135-18 or vehicle treatment. The GnRH II type II receptor agonist 135-18 approximately doubled the mean total number of proceptive behaviors (1.85 ± 1.29/135-18 test vs. 0.97 ± 1.10/vehicle test) similarly to native GnRH II for all females combined (n = 8). In contrast to GnRH II, there was a treatment-by-estradiol interaction (P = 0.023), with stimulation of proceptivity only in females that did not receive estradiol replacement (see Fig. 1). This GnRH type II receptor selective agonist did not specifically stimulate freeze-posture proceptive frequency (P = 0.312) in either female group.
Experiment 3.
There were no significant test-replicate effects indicating that over the 6 wk of behavioral testing, subjects did not alter their responses to 135-25, GnRH I, or vehicle treatment.
The GnRH type II receptor agonist 135-25 approximately tripled the total number of proceptive behaviors (3.85 ± 2.07/135-25 test vs. 1.28 ± 1.48/vehicle test; for all females combined; n = 7), exaggerating the behavioral effect induced by native GnRH II (Fig. 1). In contrast to GnRH II, but similar to 135-18, there was a treatment-by-estradiol interaction (P = 0.008), with stimulation of proceptivity specifically in females that did not receive estradiol replacement (Fig. 1). The analog 135-25 failed to specifically stimulate freeze-posture proceptive frequency (P = 0.332) in either female group but more than tripled the frequency of tongue-flicking bouts in females without estradiol (n = 3; P = 0.001) compared with vehicle control tests. The effect of 135-25 on tongue flicking was present in the females without estradiol supplementation (estradiol x treatment interaction P = 0.017), with 135-25 increasing the number of bouts of tongue flicking in these females to 6.73 ± 2.89 from 1.91 ± 1.86 in vehicle control (P = 0.001).
GnRH I did not increase the frequency of any individual proceptive behavior or the combined total frequency (2.56 ± 1.80 behaviors/GnRH I test; all females combined; n = 7) of proceptive behaviors (P = 0.107) over vehicle control values. GnRH I did not have an estradiol-dependent effect in either females receiving (n = 3) or not receiving (n = 4) estradiol replacement (Fig. 1; estradiol, P = 0.162; estradiol x treatment, P = 0.202).
Regulation of female marmoset sexual receptivity (females accepting male sexual behavior)
There were no GnRH-mediated effects on female sexual receptivity (experiments 1–3 combined), nor were there effects of estradiol supplementation on female sexual receptivity (data not shown). There was no increase in the frequency of females permitting male intromission and ejaculation after peptide compared with vehicle infusion (Table 4). Male marmosets, like human males, usually exhibit only a single penile intromission before ejaculation (31). When male marmosets achieve intromission, ejaculation occurs quickly. Thus intromission reflects female marmoset receptivity or compliance. The frequencies with which males attempted to mount their partners, and achieved a mount position, were not affected by infusion of any peptide, suggesting that male sexual motivation and attraction to their female partner was not altered by peptide treatment of the female. The latencies to male sexual behaviors were not reduced by any of the treatments (data not shown). There were no significant test-replicate effects indicating that behavioral responses to treatment did not change over the course of testing.
Discussion
The highly conserved sequence of the GnRH II decapeptide from bony fish to humans (47, 48, 49, 50, 51, 52, 53, 54, 55) and its ability to stimulate sexual behavior in fish, birds, and nonprimate mammals (Table 1) suggest that GnRH II may be an early-evolved regulator of reproductive behavior in vertebrates (16, 17, 56). In this report, we extend these findings to primates and functionally implicate GnRH II in the regulation of female primate sexual behavior in a manner similar to that demonstrated in nonprimate taxa (Table 1). Specifically, GnRH II is able to stimulate female marmoset solicitation or sexual initiation behaviors (proceptivity), behaviors that are elevated by estradiol in primates and that represent relevant measures of female sexual motivation (4). Because the behavioral testing occurred 90–120 min after GnRH II infusion and because this peptide has been shown to elicit behavioral effects from 15–90 min after administration (56, 57, 58) (Fig. 1), additional studies are needed to determine whether the action of GnRH II directly or indirectly stimulates proceptive behaviors.
Multiple GnRH receptors have arisen with differing affinities for GnRH II, including the goldfish type Ib receptor (59), the mammalian type I receptor (16) and the type II GnRH receptor (see Table 1 for species distribution). Both GnRH type I and type II receptors are present in the common marmoset and, together, the two receptor forms may serve as potential candidates to mediate the observed effects of GnRH II on female sexual behavior (16, 20). Although GnRH II has only 10% affinity for the type I receptor compared with that for the type II receptor, at the relatively high doses (1 and 10 μg) used in this study, it is plausible that either receptor could mediate the effects of GnRH II on female sexual behavior. The failure of GnRH I infusion, however, to stimulate proceptivity in female marmosets at the same dose as GnRH II (1 μg) echoes findings from the goldfish (56), sparrow (57), and food-restricted musk shrew (58), in which only GnRH II stimulated female sexual behavior. In this context, our marmoset findings suggest that GnRH II action on female sexual behavior may be mediated through type II receptors alone. Previous findings of GnRH I stimulation of female marmoset sexual proceptivity (43) may reflect the extremely high dose (25 μg) used and potential activation of GnRH type II receptors.
Stimulation of female sexual proceptivity using the GnRH type II receptor selective activators 135-18 (20) and 135-25 (Mamputha, S., R. N. Roeske, Z. L. Lu, R. P. Millar, A. A. Katz, and C. A. Flanagan, unpublished data) demonstrates that activation of GnRH type II receptors increases marmoset sexual solicitation and implicates specific involvement of the type II receptor, because these analogs are both antagonists at the type I receptor, and is similar to a recent report showing 135-18 mimics GnRH II action in musk shrews (60). Type II receptors are located within marmoset brain areas that are functionally associated with the activation of female sexual responses including the preoptic area and ventro- and dorsomedial nuclei (20). In female marmosets, damage to the anterior (30) and dorso- (28) and ventromedial (28) hypothalamus specifically reduces female proceptive sexual behavior.
Whereas both 135-18 and 135-25 stimulation of the total number of female marmoset proceptive behaviors was similar to that of GnRH II, differences emerged when these analogs were compared with GnRH II for specific proceptive behavior activation. GnRH II alone specifically increased freeze-posture behavior. In contrast, 135-25 alone enhanced tongue-flicking behavior, and 135-18 did not stimulate any individual proceptive behavior above frequencies observed after vehicle control treatment. Although it is intriguing to speculate that structural differences in the analogs, and the presumed altered ligand-receptor interactions, may be responsible for differential analog activation of female marmoset sexual solicitation compared with native ligand, a more parsimonious explanation might be the relatively lower activity of both 135-18 and 135-25 at the type II receptor.
Marmoset female sexual proceptivity increases during the periovulatory phase of the ovarian cycle, as well as after estradiol treatment in ovariectomized females (1, 33). In this study, however, estradiol supplementation of ovariectomized marmosets was not required for stimulatory GnRH II or analog action on female sexual solicitation, suggesting that such peptide-induced behavioral activation may not require ovarian steroids for efficacy. Rather, the ability of estradiol to increase GnRH II immunoreactivity in food-restricted musk shrews (61), as well as increase expression of GnRH II mRNA in the hypothalamus of ovariectomized rhesus macaques (62), and human neuronal cells (TE-6781 cells) (63) suggests that, under normal physiological conditions (e.g. ovary intact females), estradiol may activate endogenous GnRH II without being required to mediate its action.
In contrast to previous studies, estradiol-replacement treatment alone failed to enhance female marmoset sexual behavior. The circulating levels of estradiol induced by estradiol implants in this study approximated levels found during the mid-follicular-phase levels in ovary-intact females (37, 64) and not those found during the preovulatory peak, as used in previous studies (33). Plasma estradiol levels in this study were also chronically maintained over the 6–8 wk of behavioral testing and thus lacked the normal cyclical changes observed during the course of the 28-d marmoset ovarian cycle (37, 64). In previous studies, female marmoset sexual proceptivity was shown to be 1) under ovarian cycle regulation in ovary-intact females (1) and 2) enhanced by estradiol supplementation to preovulatory peak levels (940 pg/ml) in ovariectomized animals (33). The latter preovulatory estradiol supplementation stimulated proceptive tongue flicking within 4.38 ± 1.52 d but was inconsistent in its effect over the remaining 5-wk testing period (33). The acute rise in levels of circulating estradiol before ovulation may well be responsible for inducing periovulatory female marmoset proceptive behavior (62), as found in other female primates and nonprimates (5). Chronically maintained estradiol levels may diminish biological effectiveness in stimulating female sexual behavior (4, 65) by as early as 2 wk after onset of treatment (Wallen, K., personal communication).
It is not surprising that changes in receptivity were not detected after GnRH treatment in this study, because female marmosets are highly receptive to the sexual advances of males both throughout their ovarian cycle (1) and after ovariectomy (3, 33). Whereas medial hypothalamic lesions in the female common marmoset virtually abolishes proceptivity, no corresponding change in receptivity to the sexual approach of a male occurs (28, 29, 30), suggesting a neuroanatomical distinction between brain structures influencing proceptivity vs. receptivity. Proceptivity in female marmosets, as determined by species-specific behaviors such as tongue flicking, freeze posturing, and staring, reflects female marmoset sexual motivation that may be analogous to sexual initiation and sexual desire in women (4).
The highly conserved sequence of GnRH II, its conserved role in regulating sexual behavior from fish to anthropoid primates, and its ability to stimulate female sexual behavior without estradiol supplementation suggest that this peptide is an early-evolved regulator of sexual behavior. The presence of GnRH II in the human brain (54) suggests that it may regulate female sexual function in a similar fashion to that demonstrated in the marmoset and thus may have application in improving sexual motivation in women. Sexual dysfunction has been reported to be a considerable health problem for as many as 43% of women in the United States (66), although definitions of female sexual dysfunction are under debate (67). Nevertheless, increasing concern that hormonal treatment with estrogen and progestogens may impair the health and wellbeing of women (68) has produced a preference for treatments to involve nonsteroidal pharmacotherapy. The action of GnRH II in stimulating female sexual solicitation behavior in the common marmoset, without the need for estradiol supplementation, encourages exploration of its role in restoring normal sexual motivation in women with reduced sexual desire. Although GnRH II function is conserved in humans, it is not clear how the action of this peptide is mediated in the brain because the type II receptor may be nonfunctional (23, 69, 70), similar to its homolog in chimpanzees (69), cattle (9), and sheep (71). There may be an additional functional GnRH receptor specific for GnRH II, however, possibly via production of 5 or 7 transmembrane GnRH II-responsive complex (70), because the antiproliferative effects of GnRH II in human endometrial and ovarian cancer cells are not mediated by the type I GnRH receptor (72, 73). Alternatively, it has recently emerged that GnRH II is a more potent activator of Gi than GnRH I at the type I GnRH receptor, although the converse is true for Gq activation through the phenomenon of ligand-induced selective signaling (74). Because the homolog of Gi in the brain is Go, this suggests that GnRH II could have similar effects on reproductive behavior in humans through the type I receptor. Until the existence of a functional type II receptor is determined, understanding of GnRH II action in humans will be limited. Nevertheless, GnRH II analogs that can cross the blood-brain barrier may have application in understanding the neural mechanisms underlying sexual motivation in women.
Acknowledgments
We thank the veterinary staff and animal care staff at the WPRC including Dr. Iris Bolten, Dr. Kevin Brunner, Denny Mohr, Deb Werner-Kelln, Vicky Carter, and Dr. Nancy Schultz-Darken for assistance with animal care and Ann Carlson and Anna Wagner for technical assistance with some of the icv infusions. We are grateful to Dan Wittwer and Fritz Wegner for technical assistance with cortisol and estradiol assays.
Footnotes
This work was supported by Ardana Bioscience Limited (to D.H.A.) and National Institutes of Health Grant P51 RR000167 to the WPRC. This research was conducted at a facility constructed with support from Research Facilities Improvement Program Grants RR15459-01 and RR020141-01.
First Published Online September 22, 2005
Abbreviations: icv, Intracerebroventricular; WPRC, National Primate Research Center at the University of Wisconsin-Madison.
Accepted for publication September 15, 2005.
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Department of Obstetrics and Gynecology (D.H.A.), University of Wisconsin, Madison, Wisconsin 53792
Endocrinology-Reproductive Physiology Training Program (D.H.A.), University of Wisconsin, Madison, Wisconsin 53706
Medical Research Council Human Reproduction Sciences Unit (R.P.M.), EH3 9ET Edinburgh, Scotland, United Kingdom
Abstract
GnRH II (pGlu-His-Trp-Ser-Try-Gly-Leu-Arg-Pro-GlyNH2), an evolutionarily conserved member of the GnRH family, stimulates reproductive behavior in a number of vertebrates. To explore a role for GnRH II in regulating primate sexual behavior, eight adult female common marmosets, each fitted with an indwelling intracerebroventricular (icv) cannula, were ovariectomized, implanted subcutaneously with empty (n = 4) or estradiol-filled (n = 4) SILASTIC brand capsules, and pair housed with an adult male mate. After icv infusion of vehicle or peptides, females were placed in an observation cage for 90 min, out of visual contact with other marmosets, before the 30-min behavioral test with their male partner. Compared with vehicle, GnRH II (1 and 10 μg) increased the total number of proceptive (sexual solicitation) behaviors (tongue flicking, proceptive stares, and frozen postures) exhibited by females toward their pair mates and specifically increased the frequency of freeze postures. Effects were maximal at 1 μg and not dependent upon estradiol supplementation. GnRH II agonists/GnRH I antagonists 135-18 (1 μg) and 132-25 (1 μg), which stimulate inositol phosphate production via the marmoset type II receptor, increased the frequency of total proceptive behavior but did not specifically stimulate freeze-posture behavior. In contrast, GnRH I, at 1 μg, did not alter the frequency of proceptive behaviors. Female receptivity (female compliance with male sexual behavior) was not altered by any of the peptides tested. These findings implicate a role for GnRH II and the cognate GnRH type II receptor in stimulating female marmoset sexual behavior.
Introduction
GnRHs STIMULATE female sexual behavior in many different species (see Table 1). An understanding of the role of GnRHs in primate sexual behavior is, however, lacking. Compared with other mammalian and nonmammalian groups, female sexual behavior in primates is less synchronized with the timing of ovulation and less strictly regulated by ovarian hormones (1, 2). In a variety of primate species, copulations frequently increase around the time of ovulation, but they also occur throughout the menstrual cycle (1), as well as after ovariectomy (3), suggesting a degree of emancipation of primate sexual behavior from ovarian control that is not exhibited by other spontaneously ovulating, nonprimate species (2, 4, 5). Increased likelihood of male ejaculation during copulations with periovulatory females, however, suggests that ovarian influences on male-female sexual interactions are still present in primates and may include nonbehavioral changes in female attractiveness as well as hormonally mediated behavioral changes in female sexual behavior and motivation (2, 6, 7). Female primates can thus exhibit sexual behavior independently of gonadal hormones in addition to ovarian-cycle influences on sexual behavior expression.
GnRH peptides are obvious candidates for extraovarian stimulators of primate sexual behavior. Because they also act as potent neuroendocrine regulators of ovarian function (see Ref.8 for review), GnRHs can potentially drive ovarian influences on female sexual behavior. To date, 24 GnRH decapeptides have been identified (9, 10), and most vertebrates express two or more GnRH variants [e.g. in primates, GnRH I and GnRH II have been identified (9, 11)]. The common mammalian form, GnRH I (pGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2), is thought to be primarily released by the hypothalamus to stimulate FSH and LH release from pituitary gonadotropes that regulate the ovarian or menstrual cycle (12, 13, 14). In contrast, GnRH II, evolutionarily conserved from bony fish to humans (pGlu-His-Trp-Ser-His-Gly-Trp-Tyr-Pro-Gly-NH2) (8, 15, 16), appears to function more as a neurotransmitter than a hypothalamic neurosecretory product (10, 11, 17). Both GnRH peptides can stimulate female sexual behavior in a variety of vertebrate species (see Table 1); however, GnRH II has received more recent interest (18).
Although GnRH II regulation of primate sexual behavior has until now been only a matter of speculation (19), such a role is supported by neural localization of GnRH II and its highly specific receptors in brain areas involved in the expression of sexual behavior in both common marmosets (20) and rhesus macaques (21, 22), including the preoptic area and ventro- and dorsomedial nuclei (11). Currently, two distinct GnRH receptors (type I and type II; see Ref.9 for discussion of their characteristics) are candidates to mediate GnRH II action (see Table 1 for species distribution). Type I receptors, which are found in all mammals (15, 23) and mediate GnRH I neurosecretory stimulation of pituitary gonadotropes, are also capable of binding GnRH II with high affinity (9) and may be able to differentially respond to both forms of GnRH (9, 24). In contrast, type II receptors, have high ligand selectivity for GnRH II (20, 23) and are unlikely mediators of GnRH I effects (9).
In studies of GnRH effects on sexual behavior, it is generally unknown which receptor(s) mediates GnRH action. The existence of multiple GnRH receptors in some species (Table 1) (9, 11), as well as the ability of GnRH receptors to bind to multiple GnRH ligands with differing affinities (9), may explain early observations that GnRH analogs incapable of stimulating FSH or LH release from pituitary gonadotropes increase lordosis in female rats similarly to GnRH I (25). Such realization, however, complicates interpretation of GnRH action. Characterization of GnRH analogs with specific receptor-type specificities, such as the mammalian GnRH type I receptor antagonists 135-18 and 135-25 that can act as agonists at other receptors (20, 26, 27), make these analogs useful tools with which to investigate GnRH II receptor type involvement.
Marmosets are an ideal model for testing the effects of GnRH II in a primate because of the identification of GnRH II ligand and receptor in the preoptic area and hypothalamus (20), areas of the brain functionally implicated in the regulation of female marmoset sexual behavior (28, 29, 30). The availability of a well-characterized behavioral ethogram (Table 2) (31, 32, 33, 34, 35, 36) of marmoset sexual proceptivity (solicitation behaviors that invite mounting by males) and sexual receptivity [reflecting female compliance with male mounting/intromission attempts (6)] permits validated quantification of marmoset sexual behavior.
In female marmosets, sexual proceptivity is strongly influenced by the ovarian cycle and generally enhanced by estradiol (31). Female invitational behaviors, which can play a crucial role in determining whether copulation will occur in primates (see Refs.34 and 35), indicate the sexual motivation of a female (4). In contrast, sexual receptivity changes little across the ovarian cycle, and the effects of estrogen stimulation are inconsistent (1, 31). Female marmosets are attractive to the male throughout the ovarian cycle, as well as after ovariectomy and after adrenalectomy (1, 3, 33, 34, 36). The ability of a male to achieve intromission is a marker of female receptivity, because the male marmoset usually engages in a single penile intromission before ejaculation (31). Typical of many primate and nonprimate species, female compliance is necessary for successful intromission by the male.
In this study, GnRHs I and II, as well as analogs that acted as antagonists on the marmoset type I GnRH receptor, but agonists at the type II receptor, were tested in ovariectomized female common marmosets to determine whether GnRH II stimulates female sexual behavior via the type II receptor.
Materials and Methods
Study animals
This research was conducted in accordance with the recommendations of the Guide for the Care and Use of Laboratory Animals and the Animal Welfare Act and its subsequent amendments. All animal procedures were reviewed and approved by the Graduate School Animal Care and Use Committee of the University of Wisconsin-Madison. The National Primate Research Center at the University of Wisconsin-Madison (WPRC) is accredited by the Association for Assessment and Accreditation of Laboratory Animal Care as part of the University of Wisconsin-Madison Graduate School.
Eight adult (age 2–4 yr) nulliparous captive-born common marmoset (Callithrix jacchus) females were pair housed with similarly aged male partners at the WPRC for at least 6 months before the onset of this study. Females were housed with the same male partner for the entire study. Marmosets were housed indoors at the WPRC in aluminum and welded wire cages, measuring 61 x 91 x 183 cm, and had visual, olfactory, and auditory access to conspecifics in other cages. Lights were on from 0600–1800 h, and animals were fed at 1230–1330 h daily. Additional information on animal housing and husbandry has been published previously (38).
Surgical procedures
To implant intracerebroventricular (icv) cannulae, females were anesthetized with ketamine (15 mg/kg, im) and placed into a stereotaxic apparatus. Anesthesia was maintained with isoflurane (0.5–2%; 0.6 liter/min). Each animal was given 5 mg/kg dexamethasone im and 5 ml of 5% dextrose sc 14–18 h before surgery and 5 ml of 5% dextrose sc, 0.02–0.04 mg/kg atropine im, and 0.01 mg/kg buprenorphine im after anesthesia induction. Fluid replacement was maintained throughout the surgery by sc administration of 5 ml of 5% dextrose/h. Vital signs were monitored via pulse oximetry. Body temperature was maintained by a wrap-around body heating apparatus thermostatically controlled at body temperature. At the start of the procedure, 2% lidocaine was injected id to the scalp. Presurgery x-rays of the head of each marmoset were compared with x-ray ventriculograms from previous animals with comparable head size and shape to enable accurate estimation of cannula length and coordinates for icv placement. Guide cannulae (22 gauge, 11–13 mm; Plastics One, Roanoke, VA) were implanted into the third ventricle, and placement was verified by x-ray after infusion of radiopaque dye (20 μl infused over 1 min; Omnipaque, Nycomed Inc., Princeton, NJ). After the position of the guide cannula was confirmed, the guide cannula was anchored in place using dental acrylic (Justi Products, Oxnard, CA). The infusion cannula was removed from the implanted guide cannula and replaced with a stylet.
For bilateral ovariectomy, females were anesthetized with ketamine (15 mg/kg, im) and maintained on isoflurane (2%; 0.6 liter/min oxygen). At the time of ketamine administration, 0.02–0.04 mg/kg atropine im and 0.01 mg/kg buprenorphine im were administered. Each ovary was isolated through a ventral midline incision and exteriorized for visualization of the fallopian tube and ovarian pedicle. Subsequent histological examination confirmed complete ovarian removal.
Estradiol replacement
Two SILASTIC brand capsules (inner diameter, 0.058 in.; outer diameter, 0.077 in.; length, 11-mm; Dow Corning, Midland, MI) were implanted into each female sc via a small dorsal midline incision between the scapulae under ketamine anesthesia (10 mg/kg, iv) after 2% lidocaine (2 mg/kg) sc injection. Four females were implanted with 17-estradiol-filled capsules (Sigma, St. Louis, MO) previously shown to maintain approximately mid-follicular-phase levels of estradiol (Abbott, D. H., unpublished observations), whereas the remaining females were implanted with empty SILASTIC brand capsules.
Experimental design
The study tested GnRH II and related peptides against vehicle in three experiments: experiment 1 tested GnRH II (0, 1, and 10 μg; four replicate behavioral tests per dose per eight females), experiment 2 tested the GnRH type II receptor-specific activator 135-18 (0 and 1 μg; three replicate behavioral tests per treatment per eight females), and experiment 3 tested the GnRH type II receptor-specific activator 135-25 and GnRH I (0 and both peptides at 1 μg; three to four behavioral tests per treatment per seven females). In each experiment, females received treatments in a counterbalanced order with respect to GnRH-related peptides and vehicle, with more than 2 d between successive infusions. Treatment and behavioral testing for experiment 1 started 7 wk after ovariectomy and 9–10 wk after cannula implantation. Experiments 2 and 3 started 15 and 28 wk after ovariectomy, respectively (i.e. 7 wk after removal of capsules from the prior experiment). One week before each experiment, females were implanted sc with estradiol-filled (n = 4 for experiments 1 and 2; n = 3 for experiment 3) or empty (n = 4 for experiments 1–3) SILASTIC brand capsules; each female received similarly filled capsules for all experiments. SILASTIC brand capsules were removed at the end of each experiment, and no testing was performed between experiments. Estradiol levels in the blood were monitored every 2 wk whenever SILASTIC brand capsules were implanted.
For each behavioral test, subjects were removed from their home cage between 0700 and 1100 h, were blood sampled to assess baseline circulating cortisol levels (37), received peptide treatment or vehicle icv, were separated from their male partner for 90 min, and were observed in a 30-min sexual behavioral test. After the behavioral test, subjects were blood sampled again to assess changes in circulating cortisol levels resulting from treatment and testing.
Blood sampling and hormone assays
Marmosets were manually captured from the home cage or test cage and briefly restrained in a marmoset restraint tube (38) for collection of blood into heparinized syringes by femoral puncture (0.1 ml for cortisol; 0.4 ml for estradiol). To minimize blood cortisol responses to the sampling procedures (39), and pretest blood samples were collected within 3 min of home-cage entry (except for 11% of samples that were collected between 3 and 4 min and 3% collected between 4 and 6 min; without effect on cortisol values, data not shown) or within 4 min after the end of a behavioral test. Blood samples were refrigerated at 4 C for 1–5 h until they were centrifuged for 10 min at 2000 rpm. Plasma was separated and stored at –20 C until assay.
Plasma cortisol concentrations were measured in duplicate aliquots using an antibody-coated tube RIA kit, coat (Incstar Corp., Stillwater, MN) (39). The assay sensitivity was 0.1 ng/tube, and the intra- and interassay coefficients of variation for a plasma pool assayed in duplicate were 5.26% and 7.19%, respectively.
Estradiol was measured by RIA as previously described (37). The intra- and interassay coefficients of variation were 5.11% and 13.89%, respectively.
Intracerebroventricular infusions
After blood sampling, the stylet plugging the indwelling cannula was removed and a sterile infusion cannula, attached to a primed 25-μl Hamilton syringe, was inserted after effusion of cerebrospinal fluid was observed. Using aseptic technique, 8 μl of artificial cerebrospinal fluid (vehicle) or 8 μl of peptide treatment (in artificial cerebrospinal fluid) was infused into the third ventricle over 1 min. The infusion cannula was left in place for 30 sec to prevent backflow and replaced with a sterile stylet. The marmosets readily adapted to the icv infusions and, by the end of experiment 1 and throughout experiments 2 and 3, seven of the eight animals would voluntarily bow their heads for the procedure.
Behavioral testing
Behavioral responses to GnRH II and related peptides were tested in ovariectomized female pair-bonded common marmosets under laboratory conditions designed to minimize baseline levels of sexual behavior. To this end, motivators of sexual behavior, such as overnight separation before testing (40) or separate housing of partners between tests (1, 3), were avoided. After icv infusion of treatment, females were placed in a test cage (61 x 46 x 61 cm) out of visual contact from their male partner for 90 min. The behavioral test then occurred from 90–120 min after the GnRH II or vehicle infusion because, in female rats, sexual behavior is stimulated 90–180 min after icv infusion of GnRH I or certain fragments or analogs of GnRH I (41) and, in female marmosets, iv infusion of GnRH I stimulates female sexual behavior at 120 min (42).
At the start of the behavioral test, the male partner was introduced to the female by remote door operation, and behaviors were recorded by observers from behind a one-way mirror. Frequencies of sexual behaviors, classified as either proceptive (sexual solicitation behaviors: tongue flicking, proceptive stares, and freeze postures) or receptive (compliance with male sexual behavior: accepting male mount, intromission, or ejaculation; see Table 2 for descriptions), and latencies to those behaviors were recorded for 30 min using a laptop computer with an elapsed-time tag and videotaped for archival on DVDs for re-review. After testing, a blood sample was taken from the female within 4 min to permit assessment of posttest female cortisol levels, and the pair was reunited in their home cage. The 90-min separation of pair mates before each 30-min behavioral test produced frequencies of sexual behavior similar to those observed between pair-bonded animals in their home cages (35), in contrast to the higher frequencies of sexual behavior displayed during behavioral tests between newly paired or singly housed animals.
Behavioral tests were reanalyzed in a random fashion by two observers from the DVD recordings for all three experiments. Each observer was blind with respect to experiment and treatment details. The reanalyzed data were compared with data taken on the day of the test. Interobserver reliability scores for behavioral data collection averaged 97.3%, and within-observer reliability scores averaged 88.9%.
Data analysis
Frequency of proceptive stares, freeze postures, and tongue flicking provided assessment of proceptivity. Receptivity was quantified by frequencies of female mount acceptance and male intromission and ejaculation, as well by latencies to exhibit those behaviors from the onset of the test and the first male mount or mount attempt, respectively. Analyses were performed on transformed behavioral frequencies [square root (1 + x)] and the ratio of posttest to pretest plasma cortisol levels [log] to achieve homogeneity of variance and to increase linearity of data (43, 44). Both transformations normalized data distributions. Values for each female for each dose were compared by two-way ANOVA with repeated-measures design using estradiol and treatment as factors to determine the independent effects of these variables and their interaction. Post hoc univariate analysis determined variable differences when statistical interactions were significantly different (P < 0.05). Results are presented as back-transformed least-square means + 95% confidence intervals, as validated by Bland and Altman (45). Analyses of plasma estradiol and pre- and posttest cortisol values were performed on untransformed data.
Results
Steroid hormone levels during behavioral testing
Estradiol.
Ovariectomized females implanted with empty SILASTIC brand capsules (n = 3–4) exhibited lower (P < 0.001) circulating estradiol levels of 76 ± 34 pg/ml during periods of behavioral testing compared with mid-follicular-phase estradiol levels (278 ± 628 pg/ml) achieved in females implanted with estradiol-filled capsules (n = 4). Mean values for circulating estradiol in individual females are shown for each experiment in Fig. 1 and are similar within each female throughout all experiments. There were no correlations between any parameter of female sexual behavior and circulating plasma estradiol levels (data not shown).
Cortisol.
Pretest basal plasma cortisol levels (Table 3) were within expected values (37) for female marmosets receiving no estradiol replacement after ovariectomy (n = 3–4) and for ovary-intact females receiving estradiol replacement that reflected follicular-phase circulating levels (n = 4). There was no effect of estradiol replacement on either pre- or posttest plasma cortisol levels (Table 3). This lack of effect of estradiol was not surprising because, compared with ovariectomized females, plasma cortisol levels in ovary-intact female marmosets are elevated only during the periovulatory period (37). During stressful procedures, such as new social group formation, plasma cortisol levels can be expected to increase approximately 2-fold over values from undisturbed animals (46). The ratio of posttest circulating cortisol to pretest baseline values was similar for all peptides infused when compared with their vehicle controls (all females combined; n = 8): experiment 1 (posttest cortisol was 115 ± 15% of pretest levels after vehicle vs. 120 ± 16% and 128 ± 17% after 1 and 10 μg of GnRH II, respectively); experiment 2 (120 ± 21% after vehicle vs. 120 ± 21% after 135-18); and experiment 3 (134 ± 21% after vehicle vs. 133 ± 21% after 135-25 and 130 ± 21% after GnRH I). In experiments 1 and 2, there was no effect of estradiol on the ratio of pretest to posttest cortisol (P = 0.174 and P = 0.243). In experiment 3, however, the ratio of posttest cortisol to baseline was unexpectedly higher (P = 0.011) in females without estradiol supplementation compared with females with estradiol implants, regardless of treatment. There were no correlations between any of the measures of female sexual behavior and either pretest or posttest plasma cortisol levels (data not shown).
Baseline levels of sexual behavior
After icv infusion of vehicle, female partners failed to exhibit proceptive, sexual soliciting behavior in 47% of tests, and their male partners failed to achieve a mount with intromission and ejaculation in over half of these vehicle tests. Baseline levels (vehicle treatments) of proceptive and receptive behaviors were consistent between the three experiments (see Fig. 1 and Table 4).
Stimulation of female marmoset sexual proceptivity (sexual solicitation of males by females)
Experiment 1.
There were no significant test-replicate effects indicating that over the 6 wk of behavioral testing, subjects did not alter their responses to either GnRH II or vehicle treatment. GnRH II stimulated female proceptivity, as reflected by a doubling of the mean total number of proceptive behaviors (1.89 ± 1.13 behaviors/GnRH II test) compared with vehicle control (0.81 ± 0.92 behaviors/vehicle test) when all females were combined (n = 8). Effects of GnRH II were maximal at 1 μg (treatment, P = 0.001) but were not estradiol dependent. There were no effects of estradiol (P = 0.408) and no estradiol x GnRH II interaction (P = 0.057) involving the total number of proceptive behaviors. GnRH II specifically increased the frequency of freeze postures (from 0.4 ± 0.36/vehicle test to 0.67 ± 0.4/1-μg GnRH II test and 0.74 ± 0.4/10-μg GnRH II test; P = 0.042; for all females combined; n = 8) without affecting the frequency of tongue-flicking bouts (0.37 ± 0.51/vehicle test to 1.08 ± 0.62/1-μg GnRH II test and 0.81 ± 0.58/10-μg GnRH II test; P = 0.065) or proceptive stares (0.12 ± 0.23/vehicle test, 0.15 ± 0.23/1-μg GnRH II, and 0.20 ± 0.24/10-μg GnRH II test; P = 0.684) for all females combined (n = 8). Frequencies of sexual behavior induced by GnRH II were comparable to levels observed in periovulatory females studied in their home cage with their partner (34).
Experiment 2.
There were no significant test-replicate effects indicating that over the 6 wk of behavioral testing, subjects did not alter their responses to either 135-18 or vehicle treatment. The GnRH II type II receptor agonist 135-18 approximately doubled the mean total number of proceptive behaviors (1.85 ± 1.29/135-18 test vs. 0.97 ± 1.10/vehicle test) similarly to native GnRH II for all females combined (n = 8). In contrast to GnRH II, there was a treatment-by-estradiol interaction (P = 0.023), with stimulation of proceptivity only in females that did not receive estradiol replacement (see Fig. 1). This GnRH type II receptor selective agonist did not specifically stimulate freeze-posture proceptive frequency (P = 0.312) in either female group.
Experiment 3.
There were no significant test-replicate effects indicating that over the 6 wk of behavioral testing, subjects did not alter their responses to 135-25, GnRH I, or vehicle treatment.
The GnRH type II receptor agonist 135-25 approximately tripled the total number of proceptive behaviors (3.85 ± 2.07/135-25 test vs. 1.28 ± 1.48/vehicle test; for all females combined; n = 7), exaggerating the behavioral effect induced by native GnRH II (Fig. 1). In contrast to GnRH II, but similar to 135-18, there was a treatment-by-estradiol interaction (P = 0.008), with stimulation of proceptivity specifically in females that did not receive estradiol replacement (Fig. 1). The analog 135-25 failed to specifically stimulate freeze-posture proceptive frequency (P = 0.332) in either female group but more than tripled the frequency of tongue-flicking bouts in females without estradiol (n = 3; P = 0.001) compared with vehicle control tests. The effect of 135-25 on tongue flicking was present in the females without estradiol supplementation (estradiol x treatment interaction P = 0.017), with 135-25 increasing the number of bouts of tongue flicking in these females to 6.73 ± 2.89 from 1.91 ± 1.86 in vehicle control (P = 0.001).
GnRH I did not increase the frequency of any individual proceptive behavior or the combined total frequency (2.56 ± 1.80 behaviors/GnRH I test; all females combined; n = 7) of proceptive behaviors (P = 0.107) over vehicle control values. GnRH I did not have an estradiol-dependent effect in either females receiving (n = 3) or not receiving (n = 4) estradiol replacement (Fig. 1; estradiol, P = 0.162; estradiol x treatment, P = 0.202).
Regulation of female marmoset sexual receptivity (females accepting male sexual behavior)
There were no GnRH-mediated effects on female sexual receptivity (experiments 1–3 combined), nor were there effects of estradiol supplementation on female sexual receptivity (data not shown). There was no increase in the frequency of females permitting male intromission and ejaculation after peptide compared with vehicle infusion (Table 4). Male marmosets, like human males, usually exhibit only a single penile intromission before ejaculation (31). When male marmosets achieve intromission, ejaculation occurs quickly. Thus intromission reflects female marmoset receptivity or compliance. The frequencies with which males attempted to mount their partners, and achieved a mount position, were not affected by infusion of any peptide, suggesting that male sexual motivation and attraction to their female partner was not altered by peptide treatment of the female. The latencies to male sexual behaviors were not reduced by any of the treatments (data not shown). There were no significant test-replicate effects indicating that behavioral responses to treatment did not change over the course of testing.
Discussion
The highly conserved sequence of the GnRH II decapeptide from bony fish to humans (47, 48, 49, 50, 51, 52, 53, 54, 55) and its ability to stimulate sexual behavior in fish, birds, and nonprimate mammals (Table 1) suggest that GnRH II may be an early-evolved regulator of reproductive behavior in vertebrates (16, 17, 56). In this report, we extend these findings to primates and functionally implicate GnRH II in the regulation of female primate sexual behavior in a manner similar to that demonstrated in nonprimate taxa (Table 1). Specifically, GnRH II is able to stimulate female marmoset solicitation or sexual initiation behaviors (proceptivity), behaviors that are elevated by estradiol in primates and that represent relevant measures of female sexual motivation (4). Because the behavioral testing occurred 90–120 min after GnRH II infusion and because this peptide has been shown to elicit behavioral effects from 15–90 min after administration (56, 57, 58) (Fig. 1), additional studies are needed to determine whether the action of GnRH II directly or indirectly stimulates proceptive behaviors.
Multiple GnRH receptors have arisen with differing affinities for GnRH II, including the goldfish type Ib receptor (59), the mammalian type I receptor (16) and the type II GnRH receptor (see Table 1 for species distribution). Both GnRH type I and type II receptors are present in the common marmoset and, together, the two receptor forms may serve as potential candidates to mediate the observed effects of GnRH II on female sexual behavior (16, 20). Although GnRH II has only 10% affinity for the type I receptor compared with that for the type II receptor, at the relatively high doses (1 and 10 μg) used in this study, it is plausible that either receptor could mediate the effects of GnRH II on female sexual behavior. The failure of GnRH I infusion, however, to stimulate proceptivity in female marmosets at the same dose as GnRH II (1 μg) echoes findings from the goldfish (56), sparrow (57), and food-restricted musk shrew (58), in which only GnRH II stimulated female sexual behavior. In this context, our marmoset findings suggest that GnRH II action on female sexual behavior may be mediated through type II receptors alone. Previous findings of GnRH I stimulation of female marmoset sexual proceptivity (43) may reflect the extremely high dose (25 μg) used and potential activation of GnRH type II receptors.
Stimulation of female sexual proceptivity using the GnRH type II receptor selective activators 135-18 (20) and 135-25 (Mamputha, S., R. N. Roeske, Z. L. Lu, R. P. Millar, A. A. Katz, and C. A. Flanagan, unpublished data) demonstrates that activation of GnRH type II receptors increases marmoset sexual solicitation and implicates specific involvement of the type II receptor, because these analogs are both antagonists at the type I receptor, and is similar to a recent report showing 135-18 mimics GnRH II action in musk shrews (60). Type II receptors are located within marmoset brain areas that are functionally associated with the activation of female sexual responses including the preoptic area and ventro- and dorsomedial nuclei (20). In female marmosets, damage to the anterior (30) and dorso- (28) and ventromedial (28) hypothalamus specifically reduces female proceptive sexual behavior.
Whereas both 135-18 and 135-25 stimulation of the total number of female marmoset proceptive behaviors was similar to that of GnRH II, differences emerged when these analogs were compared with GnRH II for specific proceptive behavior activation. GnRH II alone specifically increased freeze-posture behavior. In contrast, 135-25 alone enhanced tongue-flicking behavior, and 135-18 did not stimulate any individual proceptive behavior above frequencies observed after vehicle control treatment. Although it is intriguing to speculate that structural differences in the analogs, and the presumed altered ligand-receptor interactions, may be responsible for differential analog activation of female marmoset sexual solicitation compared with native ligand, a more parsimonious explanation might be the relatively lower activity of both 135-18 and 135-25 at the type II receptor.
Marmoset female sexual proceptivity increases during the periovulatory phase of the ovarian cycle, as well as after estradiol treatment in ovariectomized females (1, 33). In this study, however, estradiol supplementation of ovariectomized marmosets was not required for stimulatory GnRH II or analog action on female sexual solicitation, suggesting that such peptide-induced behavioral activation may not require ovarian steroids for efficacy. Rather, the ability of estradiol to increase GnRH II immunoreactivity in food-restricted musk shrews (61), as well as increase expression of GnRH II mRNA in the hypothalamus of ovariectomized rhesus macaques (62), and human neuronal cells (TE-6781 cells) (63) suggests that, under normal physiological conditions (e.g. ovary intact females), estradiol may activate endogenous GnRH II without being required to mediate its action.
In contrast to previous studies, estradiol-replacement treatment alone failed to enhance female marmoset sexual behavior. The circulating levels of estradiol induced by estradiol implants in this study approximated levels found during the mid-follicular-phase levels in ovary-intact females (37, 64) and not those found during the preovulatory peak, as used in previous studies (33). Plasma estradiol levels in this study were also chronically maintained over the 6–8 wk of behavioral testing and thus lacked the normal cyclical changes observed during the course of the 28-d marmoset ovarian cycle (37, 64). In previous studies, female marmoset sexual proceptivity was shown to be 1) under ovarian cycle regulation in ovary-intact females (1) and 2) enhanced by estradiol supplementation to preovulatory peak levels (940 pg/ml) in ovariectomized animals (33). The latter preovulatory estradiol supplementation stimulated proceptive tongue flicking within 4.38 ± 1.52 d but was inconsistent in its effect over the remaining 5-wk testing period (33). The acute rise in levels of circulating estradiol before ovulation may well be responsible for inducing periovulatory female marmoset proceptive behavior (62), as found in other female primates and nonprimates (5). Chronically maintained estradiol levels may diminish biological effectiveness in stimulating female sexual behavior (4, 65) by as early as 2 wk after onset of treatment (Wallen, K., personal communication).
It is not surprising that changes in receptivity were not detected after GnRH treatment in this study, because female marmosets are highly receptive to the sexual advances of males both throughout their ovarian cycle (1) and after ovariectomy (3, 33). Whereas medial hypothalamic lesions in the female common marmoset virtually abolishes proceptivity, no corresponding change in receptivity to the sexual approach of a male occurs (28, 29, 30), suggesting a neuroanatomical distinction between brain structures influencing proceptivity vs. receptivity. Proceptivity in female marmosets, as determined by species-specific behaviors such as tongue flicking, freeze posturing, and staring, reflects female marmoset sexual motivation that may be analogous to sexual initiation and sexual desire in women (4).
The highly conserved sequence of GnRH II, its conserved role in regulating sexual behavior from fish to anthropoid primates, and its ability to stimulate female sexual behavior without estradiol supplementation suggest that this peptide is an early-evolved regulator of sexual behavior. The presence of GnRH II in the human brain (54) suggests that it may regulate female sexual function in a similar fashion to that demonstrated in the marmoset and thus may have application in improving sexual motivation in women. Sexual dysfunction has been reported to be a considerable health problem for as many as 43% of women in the United States (66), although definitions of female sexual dysfunction are under debate (67). Nevertheless, increasing concern that hormonal treatment with estrogen and progestogens may impair the health and wellbeing of women (68) has produced a preference for treatments to involve nonsteroidal pharmacotherapy. The action of GnRH II in stimulating female sexual solicitation behavior in the common marmoset, without the need for estradiol supplementation, encourages exploration of its role in restoring normal sexual motivation in women with reduced sexual desire. Although GnRH II function is conserved in humans, it is not clear how the action of this peptide is mediated in the brain because the type II receptor may be nonfunctional (23, 69, 70), similar to its homolog in chimpanzees (69), cattle (9), and sheep (71). There may be an additional functional GnRH receptor specific for GnRH II, however, possibly via production of 5 or 7 transmembrane GnRH II-responsive complex (70), because the antiproliferative effects of GnRH II in human endometrial and ovarian cancer cells are not mediated by the type I GnRH receptor (72, 73). Alternatively, it has recently emerged that GnRH II is a more potent activator of Gi than GnRH I at the type I GnRH receptor, although the converse is true for Gq activation through the phenomenon of ligand-induced selective signaling (74). Because the homolog of Gi in the brain is Go, this suggests that GnRH II could have similar effects on reproductive behavior in humans through the type I receptor. Until the existence of a functional type II receptor is determined, understanding of GnRH II action in humans will be limited. Nevertheless, GnRH II analogs that can cross the blood-brain barrier may have application in understanding the neural mechanisms underlying sexual motivation in women.
Acknowledgments
We thank the veterinary staff and animal care staff at the WPRC including Dr. Iris Bolten, Dr. Kevin Brunner, Denny Mohr, Deb Werner-Kelln, Vicky Carter, and Dr. Nancy Schultz-Darken for assistance with animal care and Ann Carlson and Anna Wagner for technical assistance with some of the icv infusions. We are grateful to Dan Wittwer and Fritz Wegner for technical assistance with cortisol and estradiol assays.
Footnotes
This work was supported by Ardana Bioscience Limited (to D.H.A.) and National Institutes of Health Grant P51 RR000167 to the WPRC. This research was conducted at a facility constructed with support from Research Facilities Improvement Program Grants RR15459-01 and RR020141-01.
First Published Online September 22, 2005
Abbreviations: icv, Intracerebroventricular; WPRC, National Primate Research Center at the University of Wisconsin-Madison.
Accepted for publication September 15, 2005.
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