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NEUROENDOCRINOLOGY |
Unité Mixte de Recherches Physiologie de la Reproduction et des Comportements, 6073 (Institut National de la Recherche Agronomique/Centre National de la Recherche Scientifique/Université), 37380 Nouzilly, France
Address all correspondence and requests for reprints to: Dr. A. Caraty, Unité Mixte de Recherches Physiologie de la Reproduction et des Comportements, 6073 (Institut National de la Recherche Agronomique/Centre National de la Recherche Scientifique/Université), 37380 Nouzilly, France. E-mail: caraty{at}tours.inra.fr
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Abstract |
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Our results demonstrate for the first time that GnRH is involved in the control of receptivity in a ruminant species and suggest that in the cycling ewe the sustained preovulatory GnRH secretion plays a physiological role in extending the duration of estrous behavior. They also indicate that it is possible to dissociate a direct effect of E2 on estrous behavior from its effect via stimulation of GnRH secretion.
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Introduction |
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In sheep, as in many other species, behavioral changes and preovulatory LH secretion are triggered by an increasing concentration of E2 secreted by the developing follicle at the end of the follicular phase. Sheep are very sensitive to E2, and a 15-µg injection is sufficient for a 50-kg ewe to display estrus. Another factor implicated in the control of estrous behavior is the progesterone (P) present during the luteal phase, which increases the number of E2 receptors in the mediobasal hypothalamus and therefore increases sensitivity to E2 (17). P priming is therefore necessary for normal estrus to be displayed (18, 19). Progesterone levels must fall to baseline, and the latency of endocrine and behavioral events depends upon the balance between the triggering effect of E2 and the inhibitory role of P (20, 21).
Estrous duration in ovariectomized (OVX) ewes depends both on the dose and duration of E2 (22, 23). However, in intact ewes E2 concentrations decrease abruptly within 4 h after the onset of the GnRH and LH surge and are basal within 12 h, which is also well before the end of the GnRH surge (24). This suggests that E2 might partially control estrous duration via stimulation of GnRH. Taken together, these observations suggest that in a physiological context, GnRH functions to maintain receptive behavior by extending the initial triggering effect of E2. To test this hypothesis, a series of experiments was conducted using OVX ewes in which various intensities of estrus were induced and the GnRH system was manipulated by icv administration of either a GnRH antagonist or the endogenous peptide.
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Materials and Methods |
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Behavioral observations
Female sexual behavior in sheep is first expressed by a discrete
proceptive behavior with search of a partner and a few motor patterns
(movement of the head, the tail, immobilization; 25).
Occurrence of these events was recorded, but in the present experiments
proceptivity could not be quantified in a standardized way. When the
ram starts to be sexually active and courts the ewe (a typical
manifestation is a lateral approach called "nudge"; see Ref.
25 for a complete description), the ewe expresses its
receptivity by a tonic immobilization in front of the male (the
"standing posture" characteristic of estrus) that permits the mount
and intromission. The "nudge" may be repeated several times.
Ejaculation generally occurs at the first intromission and is followed
by a marked decrease in activity. Mounting is not frequent enough to be
used to quantify receptivity but, as shown in a previous study
(26), the response to nudges is closely linked to the
response to mounting. Receptivity can therefore be quantified by a
receptivity index (RI) similar to the rodent lordosis quotient and
obtained by measuring in a standardized procedure the proportion of
immobilization in response to male nudges over a total number of
nudges. To achieve this, each ewe is introduced in a pen (4
m2) adjacent to her home pen for a minimum of ten
interactions with a ram free to court and mount and kept active by
wearing an apron preventing intromission for a minimum test duration of
2 min. This allows a quantitative and reliable measurement of the
intensity of female sexual behavior independently of possible
variations in male stimuli as shown by studying intact ewe
(27).
Experimental design
Exp 1: can a GnRH antagonist decrease E2-induced estrous
behavior? The experiment was conducted using an endocrine model
that mimicked the normal changes in ovarian steroids during the estrous
cycle (see Fig. 1
). Ewes (n =
10) were OVX at random stages of the estrous cycle and treated
immediately with an intravaginal progesterone-releasing
controlled internal drug release dispenser (CIDR; InterAg, Hamilton,
New Zealand) and a 10-mm sc SILASTIC brand silicon tubing (Dow Corning Corp., Midland, MI) implant containing E2 (inserted
under the skin in the internal face of one of the back leg) to mimic
the steroid milieu of the mid-luteal phase of the estrous cycle. After
12 d, the progesterone implants were removed to simulate luteal
regression, and 24 h later two additional 30-mm E2 implants were
inserted for 24 h (sc). This treatment raises circulating E2
concentrations to a peak follicular phase level and for a duration
similar to that observed in gonad intact, cycling ewes. It has been
shown to reliably induce a preovulatory-like surge of GnRH and LH
(3), as well as estrous behavior (5).
Twenty-four hours later, the 2 x 30-mm E2 implants were removed
and a polyethylene catheter (Biotrol, Paris, France; od: 0.7 mm; id:
0.3 mm) was inserted through the guide cannula so that the distal tip
ended at the tip of the guide cannula. Animals (n = 10), were then
treated icv via the catheter connected to a portable syringe pump
(Graseby Medical, Watford, Hertsfordshire, UK) for 20 h either
with a GnRH antagonist (Teverelix, Europeptides, Argenteuil, France)
diluted at a concentration of 0.5 mg/ml in sterile, pyrogen free
distilled water (flow rate 3 µl/min) or with the vehicle. Blood
samples were taken by venipuncture every 2 h for 48 h
starting at the time of E2 insertion. Receptivity of animals was
quantified 0, 12, 24, 32, 36, 40, 44, 48, 52, 56, and 72 h after
E2 administration. Using a cross-over design, the same ewes were then
tested during a second artificial cycle with the opposite
treatments.
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Animals (n = 14) were tested during an artificial estrous cycle as for Exp 1, but the E2 implants (2 x 30-mm) were left for 48 h. GnRH antagonist was infused into 7 ewes and vehicle into the other 7 for 20 h starting 24 h after E2 insertion. Blood samples as well as behavioral tests were performed as for Exp 1.
Exps 3 and 4. The previous experiments demonstrate that GnRH antagonist administration decreases estrous behavior if E2 is not present (Exps 1 and 2), suggesting a sequential role of E2 and GnRH for the full duration of estrous behavior. The goal of the following experiments was to determine if administration of exogenous GnRH could prolong (Exp 3) or even induce (Exp 4) estrous behavior in ewes.
A preliminary study was conducted to determine the smallest E2 signal (duration and quantity) necessary for induction of the LH surge and estrous behavior in this breed and with this type of treatment (sc implants). Twenty-four mature ewes were OVX and immediately treated with a CIDR and a 10-mm E2 implant as previously described. Twelve days later, the CIDR was removed and 24 h later animals were assigned in a random order to one of the following groups (n = 6): 2 x 30-mm E2 implants were inserted for 3, 6, 9 or 12 h. One week later, 21 of these 24 ewes were again run through a second artificial cycle and were treated in a random order with a 1 x 30-mm E2 implant for 3, 6 or 9 h (7 animals per group). For the two cycles, blood samples and receptivity tests were performed respectively every 2 and 4 h, from 0–48 h after E2 insertion.
Results of this preliminary experiment are shown in Table 1. The 2 x 30-mm E2 implants
stimulated the onset of estrous behavior in all females even when left
for only 3 h. However, 6 h of E2 duration were needed to
induce an LH surge in all females. This is in line with previous
results showing that the LH surge had a higher E2 threshold than
stimulation of estrous behavior (21). Estrous behavior and
an LH surge were also observed in most females after treatment with a
1 x 30-mm E2 left for 9 h, but the proportion of females
responding fell markedly when shorter durations were tested. Only one
female treated with a 1 x 30-mm E2 for 3 h displayed estrous
behavior with a receptivity index above 50%, but no LH surge.
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Exp 3: can GnRH prolong E2 induced estrous behavior? Twelve of the 24 OVX ewes of the preliminary experiment were prepared for icv administration and tested during an artificial cycle as for Exp1 except that E2 implants (2 x 30-mm) were only inserted for 6 h, thereby inducing a short estrus. Twenty hours after insertion of the E2 implants, receptivity tests were performed every 4 h until a significant decrease of the receptivity index was observed. Six animals were then infused for 18 h (from 34 to 52 h post E2) with GnRH (Ferring Research, San Diego, CA) diluted at a concentration of 0.5 mg/ml (flow rate 3 µl/min), whereas the 6 remaining ewes were treated with the vehicle (sterile, pyrogen free distilled water). Behavioral tests were then conducted at 44, 48, 52, and 56 h post E2.
Exp 4: can GnRH induce sexual behavior? The objective of this experiment was to determine if administration of GnRH can induce estrous behavior in animals treated by a dose of E2 below the threshold for inducing an LH surge and estrous behavior. Ten of the 12 remaining OVX ewes of the preliminary experiment were prepared for icv administration and tested during two successive artificial cycles in a cross over design. Twenty-four hours following progesterone removal, animals were treated with a 1 x 30-mm E2 implant for 3 h. Six hours post E2 insertion, animals were treated icv with GnRH (0.5 mg/ml, flow rate 3 µl/min) or with the vehicle (sterile, pyrogen-free distilled water) for 18 h (the treatment being reversed for each animal during the second cycle). For this experiment, behavioral tests were performed before E2 insertion and 6, 8, 10, 14, 18, 24, 28, and 32 h afterward.
Hormone assays
Blood samples were assayed for LH in duplicate 100-µl aliquots
of plasma using a previously described RIA method (28, 29). All samples from an experiment were run in a single assay.
Intraassay coefficient of variation averaged 9% and assay sensitivity
was 0.16 ± 0.05 ng/ml (4 assays) of standard 1051-CY-LH
(i.e. 0.31 ng/ml of NIH-LH-S1).
Data analysis
An LH surge (Exp1, Exp 2, and pre-experiment) was defined as a
sustained increase in LH level (above 10 ng/ml in two consecutive
samples), and it was considered to begin (surge onset) when the LH
level exceed the presurge baseline by 3 SD (calculated from
the samples collected during the first 8 h of the experiment).
During the luteal phase or seasonal anestrous, intact females or induced OVX females actively avoid approaches by males [RI between 0 and 10% (26)]. Females with a receptivity index above 25% were therefore considered as showing partial estrous behavior.
Data were analyzed by ANOVA adapted to each experimental design using contrast analysis as post hoc test (30) after arc sinus square root transformation of the variable (using SAS, SAS Institute, Inc., Cary, NC). Furthermore, results were confirmed by ANOVA on row data and nonparametric Friedman or Kruskal-Wallis tests.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). ANOVA did reveal a
significant effect of treatment (P < 0,0001). GnRH
antagonist infusion was followed by a significant reduction of
receptivity 40–48 h post E2 compared with vehicle infusion
(P < 0.001 at 40 and 44 h post E2, and
P < 0.01 at 48 h post E2). There is no difference
in response to treatment whether the ewes were used in cycle 1 or cycle
2. Proceptive patterns were displayed during both cycles.
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) as well as proceptive behavior.
ANOVA analysis did not reveal any effect of the treatment. However, a
transitory decrease of receptivity was observed just after removal of
E2 in the antagonist-treated group (P < 0.05 between
48 and 52 h post E2).
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, RI significantly lower than during the
three preceding tests, P < 0.01). Following exogenous
GnRH administration, receptivity remained higher in these animals
compared with those treated with the vehicle (P < 0.05
at 44 h post E2, P < 0.001 at 48 h post E2
and P < 0.01 at 52 h post E2).
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; RI above
25% at least twice in 8 of 12 vs. 3 of 12 ewes,
respectively), and the mean level of receptivity was significantly
higher 18 and 24 h after E2 insertion (P < 0.05
for both). However, receptivity was much weaker than in Exps 1–3: RI
was above 75% in only one female, and three females never showed any
sign of receptivity during the two cycles.). Proceptive patterns were
presented by 7 of 12 ewes after GnRH vs. 3 of 12 during the
control cycle.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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GnRH antagonist administration greatly decreased sexual activity in estrogen-primed OVX ewes when E2 was no longer present. Moreover, administration of the natural GnRH peptide was able to prolong estrous behavior in animals treated with a short E2 signal. Collectively, these results demonstrate that GnRH is able to maintain receptive behavior in the ewe by extending the initial triggering effect of E2. Moreover, as the duration of elevated plasma E2 levels achieved in OVX ewes by 24 h insertion of E2 implant duplicates the duration typical of gonadally intact cycling ewes (31, 32), our results suggest that the role of the extended preovulatory GnRH secretion during the late follicular phase is to prolong receptivity after E2 has disappeared from the peripheral circulation.
A facilitatory action of GnRH on sexual behavior has been shown in various mammalian species including rats, hamsters [reviewed by Moss and Dudley (8), Pfaff (9)], and monkeys (13) as well as in birds (14), reptiles (15), and fish (16). However, to our knowledge, this is the first time that this effect has been shown in relation to the extended preovulatory GnRH secretion. This demonstration of a sequential role of E2 and GnRH has been facilitated by the length of estrus in sheep (36–48 h; 21), allowing well-timed sequential treatments. However, because an extended duration of preovulatory GnRH secretion has also been reported in rats (33), monkey, (34), and cows (35), similar sequential mechanisms may exist in other species.
While GnRH seems able to stimulate estrous behavior after E2 removal, the inhibitory effect of the GnRH antagonist cannot be detected if E2 is still present. It can therefore be concluded that hypothalamic GnRH receptor activation is not compulsory for the expression of estrous behavior in ewes. Similarly, hypogonadal mice that do not have detectable GnRH in the forebrain, display normal lordosis behavior after E2 and progesterone treatment (36). Thus, in both species, and possibly in others, GnRH has a facilitatory role, but this action is not essential for the neuroendocrine control of female sexual behavior. Our results also indicate that E2 can overcome the absence of the stimulatory effect of GnRH. In sheep as in goats, estrus duration can be extended far beyond the normal span if high blood levels of E2 are maintained (21, 37). In contrast, increasing the duration of the E2 signal from 14 to 21 h does not increase the duration of the GnRH surge (38). Therefore, changes in estrous behavior by manipulation of the GnRH system can only be seen in a window of time relative to the E2 priming. This may also explain why previous reports indicate no effect of the peptide on estrous behavior of large farm animals like cattle (10, 11) and pigs (12). As species specificity seems unlikely to explain the lack of effect of the peptide, a more probable explanation is that either the dose, the route of administration or the physiological state were not appropriate in these studies. Finally, our data suggest that the overriding effect of E2 must involve a mechanism different from GnRH stimulation. In rats, a link has been established between the facilitatory role of GnRH and the stimulatory action of progesterone, which is reduced by an LHRH antagonist (39), the effect of GnRH itself being reduced by RU 486, an antagonist to progesterone receptor (40). However, the proceptive behavior expressed after E2 and P treatment, is never observed after E2 plus GnRH administration (41). This means that E2 plus GnRH does not mimic all the E2 + P effects. In sheep, E2 and GnRH stimulation can also be dissociated in time, as shown by the delay between the onset of estrus and the preovulatory increase in GnRH (42, 43, 44). Clearly steroids have some effects that are independent of GnRH.
Although E2 seems able to stimulate estrous behavior independently of GnRH activity, in all mammalian species studied to date, E2 priming is necessary for the facilitating role of GnRH on sexual behavior (14, 45). In many cases, the responses to GnRH are variable among animals and most easily detectable when the amount of estrogen was just below that necessary for increased receptivity alone (14, 45). In our experiments, we increased estrus duration by giving GnRH at the end of a short E2 treatment (Exp 3) but only induced a low level of receptivity in females primed with a subthreshold E2 treatment (Exp 4). This low response observed in Exp 4 is unlikely to be due to insufficient GnRH stimulation because the same treatment was clearly effective in expanding receptivity duration in Exp 3. More likely, this low response is due to low sensitivity of these females to E2, which made our E2 priming insufficient. Such individual variation in sensitivity to E2 has been demonstrated in other studies in sheep (5, 46). Moreover, because several GnRH molecules coexist in many mammals (human, monkey, bovine; 47), the mammalian GnRH that we used in sheep may not be the one that has the greatest influence on behavior. This needs to be tested in further studies.
Our findings that the GnRH surge is an important component in the control of estrous behavior in ewes are interesting in regard to the facilitator role of progesterone in this species. In sheep, progesterone priming is necessary for the display of normal estrous behavior (21, 48). In intact females, the first ovulation of the breeding season is not accompanied by estrous behavior and OVX ewes only respond to physiological doses of E2 if pretreated with progesterone (21). This is partly explained by an increase in E2 receptors in the mediobasal hypothalamus after progesterone treatment (17). However, we have demonstrated recently that progesterone priming also greatly increases the magnitude of the E2induced GnRH surge into the cerebrospinal fluid (49). Moreover, in cycling rats or in OVX rats treated with E2 and progesterone, dynamic changes in the amount of GnRH receptor mRNA have been reported in the ventromedial nuclei during the morning of proestrus (50). Thus, another component of the facilitatory effect of progesterone on estrous behavior in sheep, could be the increase in GnRH secretion and/or hypothalamic GnRH receptors.
Using the third ventricle route of administration, we demonstrated a sequential role of GnRH and E2 according to the timing and duration of the GnRH secretion in portal blood. However, it is very likely that the major effects of antagonists or agonists are exerted on GnRH receptors that are the targets of nonhypophysiotropic GnRH cells. In sheep, as in other species, a number of GnRH cells do not project to the median eminence (51, 52, 53). Moreover, as there is nearly no GnRH in the peripheral circulation (54), it is obvious that the portal GnRH cannot act on the brain. The more probable explanation for the sequential effect of GnRH on estrous behavior is that both nonhypophysiotropic and hypophysiotropic cells work in parallel. In support to these hypotheses, it has been demonstrated in rat (55) as in sheep (56) that fos expression in relation to the GnRH surge is not restricted to particular sites in the hypothalamus. Thus, a system of dual release of GnRH into the hypothalamo-hypophyseal portal blood and into the brain could provide an efficient mechanism by which endocrine events can be coupled to intracerebral actions, such as facilitation of lordosis. However, very high GnRH concentrations in the CSF (6, 49) concomitant with the increase of sexual receptivity in ewe, as well as active transport of GnRH from the third ventricle toward the fourth ventricle (57) also suggest that hypophysiotropic GnRH cells may also be involved in sexual behavior. GnRH molecules diffusing from the median eminence into the CSF may reach nuclei close to the ventricular system and involved in estrous behavior, such as the ventromedial nucleus (58) or the mesencephalic central gray (59, 60). However as concentrations of GnRH antagonist or agonist achieved by icv infusion in the present studies, similar to that administered in rodent (8, 9), were certainly above a physiological range, this hypothesis remain to be tested.
In summary, our results demonstrate that during the estrous cycle of
the ewe at least two mechanisms are necessary for the expression of
sexual behavior around the time of ovulation: one involving E2 and one
involving E2-induced GnRH secretion. A schematic of the events leading
to the preovulatory LH secretion and estrous behavior is given in Fig. 6. The behavioral effect of GnRH, long
after its effect on LH, that allows sexual interaction to occur
as long as partners are available, has obvious adaptive value,
i.e. increasing coordination between endocrine and
behavioral changes and therefore reproductive success. Our results also
suggest that one factor involved in the facilitatory role of
progesterone is the magnitude of GnRH secretion. The role of the CSF
GnRH as a possible candidate in this regulation remains to be
determined.
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Acknowledgments |
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Footnotes |
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Received April 17, 2001.
Accepted for publication September 28, 2001.
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