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Biology of Reproduction 62, 1052-1058 (2000)
© 2000 Society for the Study of Reproduction, Inc.

Articles

Olfactory Bulbectomy Blocks Mating-Induced Ovulation in Musk Shrews (Suncus murinus)1

Emilie F. Rissman2,a, and Xia Lia

a Biology Department, Gilmer Hall, University of Virginia, Charlottesville, Virginia 22903


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In many species, reproductive function can be modified by olfactory inputs. We employed bilateral olfactory bulbectomy (BULBX) to examine the effects of disruption of olfactory inputs on mating behavior and ovulation in female musk shrews. On several measures, sexual behavior was delayed in BULBX females compared to controls. When females were mated on five consecutive days, the majority of unoperated and sham-operated (SHAM) shrews ovulated; only one female subjected to BULBX ovulated. Administration of GnRH induced ovulation in the majority of females. We performed immunocytochemistry to assess the effects of bulbectomy on mating-induced responses of the neural GnRH system. In BULBX and SHAM females, the numbers of cells containing proGnRH immunoreactivity in the medial septum (MS)/diagonal band (DB) were significantly elevated 1 h after mating. Bulbectomy increased the numbers of GnRH-immunoreactive peptide-containing cells in the preoptic area, but it reduced neuron numbers in the MS/DB, as compared with those in SHAM controls. In addition, the GnRH-immunoreactive fiber area in the median eminence was greater in BULBX than in SHAM females. In sum, female musk shrews can display receptivity and engage in copulation without olfactory inputs. However, the olfactory system is essential for mating-induced ovulation.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In many mammals, olfactory inputs can regulate the timing of ovulation [1–4]. In rodents that have spontaneous ovulation during the estrous cycle, several findings link olfaction, GnRH, and mating. First, exposure to male pheromones can promote LH release and ovulation in mice [1, 2, 4]. Second, in ovariectomized female rats, exogenous GnRH can facilitate receptive behavior [5, 6]. Third, removal of the vomeronasal organ (VNO) can block receptive behavior in some rodents [7, 8]. Finally, mating can induce a Fos response in a subset of GnRH-immunoreactive (ir) cells [9, 10]. In rats, this response is blocked by VNO removal [11]. Taken together, these data suggest that olfactory cues stimulate GnRH; in turn, this neuropeptide can affect sexual behavior and ovulation.

In the rat and mouse, ovulation and mating are synchronized during the spontaneous ovarian cycle. However, many female mammals exhibit mating-induced ovulation [1]. The relationship between olfaction and ovulation in species with induced ovulation is less well understood. Several species of voles require mating to promote ovulation [1, 7, 8]. These females also require physical contact with males to come into behavioral estrus [1, 12]. Male-related pheromones can elevate estradiol levels in plasma, as evidenced by increased uterine weights [12]. Removal of the VNO blocks induction of behavioral estrus in the female prairie vole, and without sexual behavior, mating-induced ovulation is likewise prevented [7]. Likewise female gray short-tailed opossums come into estrus after exposure to male pheromones [13], and removal of the VNO blocks this response [14]. However, it is not clear if ovulation in this species is induced or spontaneous [15, 16]. In ferrets, mating, but not steroid hormone treatment, promotes ovulation [17]. Vaginal and cervical stimulation with a glass rod can induce ovulation. However, this procedure is effective only if females are simultaneously exposed to a male [18]. In rabbits, artificial vaginal/cervical stimulation in the absence of contact with a male can induce ovulation [19]. Bilateral olfactory bulbectomy does not block mating-induced ovulation in does [20]. These observations have lead to the formation of the working hypothesis that in spontaneously ovulating species, the trigger for ovulation is a hormonal event. In females that display induced ovulation, the trigger is a neural event [21]. In both cases, olfactory inputs may facilitate ovulation.

Female musk shrews are induced ovulators; unlike many other mammals they do not have an ovarian or behavioral estrous cycle [22, 23]. Typically, mating starts within an hour after introduction to a male [23]. At the onset of mating in virgin musk shrews, ovaries do not contain mature follicles, and plasma estradiol levels are low [24, 25]. Mating promotes both follicular development and ovulation [24]. At least two mating bouts, separated by at least 24 h, are required to trigger the first ovulation [26]. Because the first mating has direct and rapid effects on GnRH-ir neurons and on GnRH peptide content in brain, we have hypothesized that activation of the hypothalamic-pituitary-gonadal axis is induced by mating in this species [27]. For example, mating, or exposure to male-related pheromones, augments the numbers of GnRH-ir cells in the anterior forebrain [28]. Male-related pheromones also affect female sexual behavior in this species [29]. Pre-exposure to male-soiled bedding can facilitate rapid expression of sexual receptivity [29]. However, pre-exposure to pheromones does not augment the effects of mating on ovulation [26]. In the experiments described here, we asked whether olfactory inputs are required for display of mating behavior and/or ovulation in this species. We also asked if the effects of mating on GnRH-ir cells depend on olfactory inputs.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals

Virgin female musk shrews (Suncus murinus) 45–60 days of age were subjects in all experiments. Females were born in the breeding colony at the University of Virginia (Charlottesville, VA). At weaning (18–21 days of age), they were housed individually in clear plastic cages (28 x 17 x 12 cm) containing pine wood shavings and shredded paper toweling for nesting materials. Weaned females were housed in a room isolated from breeding females and males. Animals were fed a mixture of cat chow (Ralston-Purina Co., St. Louis, MO) and mink pellets (Milk Specialty Products, New Holstein, WI), and water was available ad libitum. Lights were on a 14L:10D cycle, with lights-on at 0700 h, and temperature was maintained at 23 ± 2°C.

Stereotaxic Surgery

Females were deeply anesthetized with sodium pentobarbital (4.5 mg/ml; 0.1 ml/10 g BW, i.p.). In addition, the cranial muscles along the top of the shrews' heads were anesthetized with lidocaine (1%, 0.03 ml). Females were fitted into a modified mouse stereotaxic apparatus (Kopf Instruments, Tujanga, CA). The skin on top of the head was cleaned and a single incision was made. A small hole was placed through the skull above the caudal portion of each olfactory bulb. A sterile probe (27-gauge) was lowered into the brain, and very gentle suction was applied to aspirate the olfactory bulbs. The disturbed area was packed with Gelfoam (Upjohn, Kalamazoo, MI), and the skin was sutured. Sham surgery consisted of anesthetizing the animal, placing her in the stereotaxic apparatus, and cutting the outer dermal layer, but not the skull. Sham surgery did not include opening the skull because, in preliminary studies, we found that in these small animals this procedure can easily damage the olfactory bulbs. Females were allowed 2–3 wk to recover from all surgeries. Survival rates for sham surgery was close to 100%; for BULBX the rate of survival was 65%.

Completeness of the surgery was confirmed after testing. Females were given an overdose injection of sodium pentobarbital (6 mg i.p./animal) before perfusion through the aorta. Females were perfused with 0.9% saline containing heparin (100 U/ml), followed by 4% paraformaldehyde. Brains from animals used in experiments 1–3 were embedded in egg/gelatin and cut in the horizontal plane at 50 µm. Slides were stained with cresyl violet [30]. Forebrains and olfactory bulbs were examined for any signs of damage in SHAM animals. Brains from bulbectomized animals were examined for completeness of surgery, and damage to the forebrain was noted. In cases of incomplete bulbectomy or frontal pole damage, the data from that female were excluded from the results.

Experiment 1: Effect of Bulbectomy on Sexual Behavior

Females were either subjected to bilateral olfactory bulbectomy (BULBX, n = 14) or given sham surgery (SHAM, n = 11). Females were tested twice, on two consecutive days. Testing took place between 0800–1200 h. Females were placed into a clean, new acrylic test box (measuring 39 x 19 x 10.5 cm) on a mirror stand for ventral viewing. The test box contained a rested stud male that had been placed there approximate 15 min previously. Behavior was observed for 60 min, or until the male achieved 5 placed intromissions (whichever occurred first). Sexually naive females are initially aggressive towards males, but after a brief period of interaction, females become sexually receptive. Receptivity is marked by a reduction in vocalizations and the onset of species-typical rump presenting and tail wagging behaviors, after which males begin mounting. Virgin females do not pause or stand still during mounting; males must achieve penile intromissions while females are moving. Males typically perform several placed intromissions before they ejaculate.

For the female, the latencies to rump presentation, tail wag, receipt of the first mount, first missed intromission, first placed intromission, and 5 placed intromissions were recorded. We scored frequency of male mounts, mounts with missed intromissions, and mounts with placed intromissions.

Experiment 2: Effect of Bulbectomy on Mating-Induced Ovulation

Three groups of females (n = 10–11 per group) were used. Females were BULBX, SHAM, or no surgery controls. Two to 3 wk after surgery, each female was paired on five consecutive days with a different male each day. Procedures were similar to those of experiment 1 except females were tested in the stud males' home cages. An observer watched the animals for up to 1 h, or until they noted an ejaculation. Ejaculation is very pronounced and stereotyped in this species. Males display a whole body freeze that lasts for up to 5 sec and includes the falling of the rigid male to one side of the female's body. After ejaculation, males stop mounting and typically follow the female, biting at her tail. Females were returned to their home cages after the mating encounter. Females that engaged in complete copulatory behavior (including receipt of an ejaculation) on at least 3 of the 5 daily pairings were checked for ovulation 24 h after the last mating encounter. At that time, females were anesthetized with sodium pentobarbital (4.5 mg/ml; 0.1 ml/10 g BW, i.p.), and both ovaries and oviducts were removed. Ovaries were examined for the presence of corpora lutea (CL), and oviducts were checked for ova. If both CL and ova were noted, an ovulation was scored [26]. In musk shrews, there is a preimplantational delay of up to 7 days postmating [22,25]; thus we can reliably find ova in the oviducts for up to 6 days postovulation.

Experiment 3: Effect of Bulbectomy on GnRH-Induced Ovulation

Females were BULBX (n = 9) or received SHAM (n = 9) surgery. Two to 3 wk after surgery, each female was given an s.c. injection of GnRH (Sigma, St. Louis, MO; 50 ng, as used in [31]). Twenty-four hours later, females were anesthetized (as described above), and both ovaries and oviducts were removed. Ovaries were examined for the presence of CL, and oviducts were checked for ova.

Experiment 4: Effect of Bulbectomy and Mating on GnRH Immunoreactivity

Females were BULBX (n = 14) or SHAM controls (n = 10). Two to 3 wk postsurgery, half of the females in each group were mated with a stud male, as described in experiment 1. The pairs remained together until females received an ejaculation (this took between 15 and 45 min). At that time, each female was returned to her home cage where she remained for 1 h until she was killed. Control females remained in their home cages until they were killed.

At the time the females were killed, they were deeply anesthetized with sodium pentobarbital (6 mg i.p./animal). Females were perfused through the aorta with 0.9% saline containing heparin (100 IU/ml), followed by Zamboni's fixative (4% paraformaldehyde in 0.1 M sodium phosphate buffer, pH 7.4, with 15% saturated picric acid). Brains were removed and cryoprotected overnight in 30% sucrose in 0.1 M phosphate buffer at 4°C, quickly frozen, then stored at -70°C until sectioned.

Serial coronal sections of 30-µm thickness were collected into a series of four wells. One well was processed for proGnRH using an antiserum made against the peptide sequence that spans from the end of the GnRH peptide into the gonadotropin-associated peptide (GAP) portion (amino acids 6–16, antiserum #1947, generously provided by Drs. R. Millar and J. King). Another quarter of the sections were processed using a GnRH monoclonal antiserum SMI-41 (produced by Sternberger Monoclonals Inc., Baltimore, MD). This antiserum was made against the 5 amino acids adjacent to the C terminal of the GnRH peptide, and the amidation site. Only mature GnRH peptide is likely to be recognized by this antiserum. We have previously validated the use of these two antisera to detect GnRH immunoreactivity in musk shrew brain [32].

All rinses and solutions were made in 0.02 M Tris-buffered saline (pH 7.8). Sections were pretreated in 1% sodium borohydride to remove residual aldehydes. Sections were incubated sequentially in avidin and biotin blocking solutions (Vector Blocking Kit; Vector, Inc., Burlingame, CA) to block endogenous biotin in musk shrew brains. A 48-h incubation in primary antiserum at 4°C followed. For sections incubated in anti-proGnRH (1:20 000), we used biotinylated goat anti-rabbit IgG (Vector; 1:500) as the secondary antiserum. When SMI-41 (1:25 000) was the primary antiserum, sections were incubated in biotinylated horse anti-mouse IgG (Vector; 1:500). In both cases, the secondary incubations lasted for 1 h. Next, sections were incubated for 60 min in avidin-biotin complex (Vector ABC Elite Kit, 1:1000). Immunoreactivity was visualized with nickel-intensified diaminobenzidine (DAB) solution (0.25% nickel ammonium sulfate and 0.05% DAB) activated by 0.001% hydrogen peroxide. All sections were developed in a single run to eliminate intra-run variability.

Tabulation of GnRH-ir cells was accomplished with the assistance of Jandel Mocha image analysis software (Jandel Scientific, Corte Madera, CA). The observer scored a neuron on the basis of the presence of a lighter region in the cytoplasm that we assumed to be the cell nucleus, and the existence of at least one immunoreactive fiber associated with the cell body. Immunoreactive cells were counted throughout the brain from the anterior forebrain (where most of the GnRH-ir cells are associated with the terminal nerve) to the few cells that reside in the hypothalamus. The immunoreactive fiber area in the median eminence was calculated on the basis of pixel intensity. All our methods have been described in previous publications [33].

Data Analysis

Analysis of behavioral data was conducted with a repeated-measures ANOVA. In the second and third experiments, chi-square analysis and Fisher's exact tests were used to compare the proportion of females that ovulated. Two-way ANOVA and pair-wise Student-Neuman-Keuls tests were employed to analyze the GnRH-ir data.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Experiment 1: Bulbectomy Delayed the Display of Some Aspects of Sexual Behavior

Four of the 14 BULBX females did not mate on both testing days, whereas all of the SHAM animals (n = 10) displayed sexual behavior. This difference was not significant (P = 0.079). When behavioral measures were compared between the BULBX and SHAM animals that display receptivity on both test days, a few differences were noted. BULBX females took longer to display tail wagging as compared with SHAM females (F[1,41] = 6.837, P < 0.017, Table 1). In addition, males paired with BULBX females took more time to obtain the first intromission and to achieve their fifth intromission, when compared with males paired with control females (F[1,41] = 8.368, 7.374, P < 0.02 at least for each comparison). Finally, the interval between the male's first mount and his final intromission was longer when mated with BULBX as compared with SHAM females (F[1,41] = 9.285, P < 0.007). No differences between the groups were noted on either test day in frequency of mounts or latencies for females to beginning rump presentation, receiving the first mount, or the first missed intromission. There were no differences in the interval between onset of tail wagging and the first mount, or the interval between the first and the fifth placed intromissions when BULBX and SHAM females were compared.


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  		TABLE 1. Mating behavior data for female musk shrews tested with males on two consecutive days (means in seconds; SEM in parentheses)

Experiment 2: Bulbectomy Blocked Mating-Induced Ovulation

As shown in Table 2, the majority of females that were unoperated or had a sham surgery ovulated after repeated mating. Only one of the females that received bilateral bulbectomy ovulated. There was a significant difference between the groups ({chi}2 = 8.61, P < 0.02).


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  		TABLE 2. Effects of bulbectomy on percentage of females ovulating after mating (experiment 2) or GnRH injection (experiment 3)

Experiment 3: GnRH Administration Induced Ovulation Despite Bulbectomy

Equal numbers of BULBX and SHAM females ovulated 24 h after a GnRH injection (see Table 2). In both groups, 7 of 9 individuals had CL on their ovaries and ova in their oviducts.

Experiment 4: Mating and Bulbectomy Affected proGnRH-ir Cells in the Medial Septum (MS)/Diagonal Band (DB)

As reported previously [33], we found greater proGnRH-ir cell numbers in the MS/DB region in brains of females mated 1 h before they were killed than in brains of unmated females (F[1,22] = 11.63, P < 0.003; Fig. 1). We also noted a main effect of bulbectomy in this region. Brains of BULBX females had fewer proGnRH-ir cells than did brains of SHAM females (F[1,22] = 6.32, P < 0.03). There was no interaction between these two factors (F[1,22] = 0.05). There were no effects of mating or surgery, nor was an interaction noted in proGnRH-ir cells in the preoptic area (POA; F[1,22] = 0.50, 0.43, 0.50, respectively).



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  		FIG. 1. Mean (+ SEM) numbers of proGnRH-ir cells in the medial septum/diagonal band (MS/DB). Females were either bulbectomized or underwent sham surgery. Half of the females in each condition (n = 5–7 per final group) were mated and killed 1 h later. The other females served as unmated controls. Significant effects of mating and of surgical condition were found. a, Significantly more cells than in unmated females. b, Significantly fewer cells than in SHAM females

Bulbectomy Affected GnRH-ir Peptide in Several Regions

As reported previously [33], there was no effect of mating on GnRH-ir peptide-containing cells in the MS/DB or the POA (F[1,23] = 0.27, 0.92, respectively; Fig. 2). However, there was an effect of bulbectomy in both regions. In the MS/DB, numbers of the peptide-containing GnRH-ir cells mirrored what we noted above for proGnRH-ir cells. Brains of BULBX females had fewer peptide-containing GnRH-ir cells than did brains of SHAM females (F[1,23] = 6.28, P < 0.03; Fig. 2). There was no interaction between these two factors (F[1,23] = 0.27). In the POA, the reverse effect was noted (Fig. 2). Brains of BULBX females contained significantly more GnRH-ir peptide neurons than did brains of SHAM females (F[1,23] = 7.87, P < 0.01). No interaction was noted in the POA (F[1,23] = 0.01).



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  		FIG. 2. Mean (+ SEM) numbers of GnRH-ir cells in the MS/DB and POA. Females were either bulbectomized or underwent sham surgery. Half of the females in each condition (n = 5–7 per final group) were mated and killed 1 h later. The other females served as unmated controls. A significant effect of surgical condition was found. In the MS/DB, BULBX females had fewer GnRH-ir cells than did SHAM females. In the POA, the reverse was true. *Significantly fewer GnRH-ir cells than in control females. **Significantly more GnRH-ir cells than in control females

The area of GnRH peptide-containing fibers in the median eminence (ME) displayed a pattern similar to that of GnRH-ir cells in the POA (Fig. 3). Fibers that were immunoreactive for GnRH peptide occupied a larger area in the ME of brains taken from BULBX females than that in SHAM females (F[1,23] = 6.34, P < 0.02). There were no effects of mating, nor was an interaction noted in ME the fiber area (F[1,23] = 0.16, 0.99, respectively).



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  		FIG. 3. Mean (+ SEM) GnRH-ir fiber area (mm2) in the ME. Females were either bulbectomized or underwent sham surgery. Half of the females in each condition (n = 5–7 per final group) were mated and killed 1 h later. The other females served as unmated controls. A significant effect of surgical condition was found. BULBX females had a larger immunoreactive area in the ME as compared with SHAM females. *Significantly smaller immunoreactive area than in BULBX brains

Bulbectomy Eliminated GnRH-ir Cells in the Terminal Nerve-Associated Forebrain

Animals that underwent bulbectomy had virtually no GnRH-ir cells in the region of the anterior forebrain associated with the infiltration of the terminal nerve (TN). This was true in tissues processed with either proGnRH or the GnRH peptide antisera (SMI). This difference was revealed by examining total brain GnRH-ir cell counts. Brains from BULBX females had fewer proGnRH- (F[1,22] = 10.88, P < 0.004) and peptide-containing cells (F[1,23] = 9.54, P < 0.006) as compared with those of SHAM controls. However, when GnRH-ir cells in the TN were not included in the total brain cell counts, there was no significant difference between BULBX and SHAM females (F[1,22] = 2.67 for proGnRH-ir; F[1,23] = 2.84 for GnRH peptide). Total cell counts (including the TN) did not differ as a function of mating (F[1,22] = 1.67 for proGnRH immunoreactivity and F[1,23] = 0.003 for GnRH peptide), nor was an interaction detected (F[1,22] = 0.004 for proGnRH immunoreactivity and F[1,23] = 0.007 for GnRH peptide).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Our findings demonstrate that female musk shrews do not require olfactory inputs to display receptive behavior and to mate. However, removal of the olfactory bulbs affects the timing of copulatory behaviors. Specifically females took longer to display some aspects of sexual receptivity if they lacked their olfactory bulbs. Moreover, olfactory bulbs were required for mating-induced ovulation. Because injections of exogenous GnRH promoted ovulation in BULBX females, and several aspects of the immunoreactive GnRH system were affected by this surgery, we hypothesize that normal olfactory inputs are required to maintain GnRH processing and/or release.

Our behavioral data are unlike those reported in other species in which removal of either the olfactory bulbs or the VNO blocks female receptivity [7,8]. Likewise, in sexually naive male hamsters, bulbectomy or VNO ablation blocks expression of sexual behavior [34]. In female rats, repeated exposures to males, during the evening of estrus, enhances receptivity [11]. Females mate after VNO removal, but display of the lordosis posture is not enhanced by repeated matings [11]. Bulbectomy in female musk shrews produced effects somewhat similar to those seen in female rats after VNO removal. Like rats, BULBX shrews still showed receptivity and mated with males; however, latencies to display of receptivity were longer in BULBX than in control females. These results suggest that olfactory (pheromonal) cues are part of a constellation of social signals that females employ to trigger receptivity.

The limited effect of olfactory input on female sexual behavior complements previously collected data. The transition from aggressive to mating interactions in musk shrews occurred more rapidly if virgin females were pre-exposed to male-related olfactory cues [29]. However, without pre-exposure to male pheromones, females did eventually mate; they just took longer to do so. Thus, olfactory cues facilitate female receptivity, yet these cues are not essential for the eventual display of copulatory behavior. During the heterosexual interactions that precede mating, physical contact and auditory exchanges, as well as chemo-investigation, occur. It is likely that other social cues could compensate for the lack of olfactory inputs when females were introduced to males. The fact that bulbectomized females still mated allowed us to examine the contribution of olfaction specifically to ovulation.

In the female musk shrew, ovulation is induced by mating; however, multiple mating bouts are required to stimulate the first ovulation [26]. During the course of these matings, the hypothalamic-pituitary system is activated. For example, within 15 min after females were first introduced to males, there was a significant increase in the numbers of GnRH-ir cells in the anterior forebrain [28]. When females received a single ejaculation, and 24 h later were divided into groups that did and did not ovulate, pronounced differences in GnRH were noted. Females that ovulated had significantly lower concentrations of GnRH protein in the ME eminence than did females that mated but did not ovulate [35]. We have hypothesized that the first mating acts to prime the GnRH system, increasing production of peptide and promoting its accumulation in the terminal field [33]. Bulbectomy increases the ir-GnRH accumulation in the ME. It is likely that GnRH release, in response to multiple matings, is diminished after bulbectomy. In female goats, multiple-unit neuronal activity, recorded from the medial basal hypothalamus, is correlated with pulsatile release of LH [36]. Females display rapid induction of multiple-unit activity and LH release when they are exposed to male goat hair [36]. The implication is that male pheromones trigger GnRH pulses in goats. We suggest that pheromones may act in a similar manner in female musk shrews. Validation of an LH assay for the musk shrew will allow us to test this hypothesis.

In the present study, we found an increase in proGnRH-ir cell numbers in the medial septum 1 h after mating. These data replicate and extend previous work done in our laboratory [33]. Mating per se, not olfactory stimulation from male pheromones, triggered this effect. As we have documented previously [33], GnRH-ir peptide was not modified within 1 h after mating, either in terms of cell numbers or fiber density in the ME. Because the mating-induced change in proGnRH was not accompanied by a change in GnRH peptide-containing cells, we hypothesize that mating induces either rapid translation and/or transcription of GnRH mRNA.

Bulbectomy, regardless of mating status, affected basal GnRH-ir cell numbers. Diametrically opposite changes in cell numbers occur in the MS/DB versus the POA. In the POA, GnRH peptide-containing cells increased in number after bulbectomy, whereas in the MS/DB, cell numbers declined. This may reflect a short-loop feedback connection between GnRH cells in these two adjacent regions [37].

Input from the TN, as well as from the olfactory system, is eliminated by bulbectomy. The TN is highly GnRH-immunoreactive in several species, including musk shrews [37–41]. TN lesion reduced the numbers of male hamsters that displayed sexual behavior [42]. But males with terminal nervectomy still responded to odors from females with a testosterone surge in plasma [43]. Thus, the hypothalamic (GnRH) pituitary (LH) pathway was not disrupted by lesions of this sensory input system. In the female musk shrew, it appears that TN and olfactory bulb inputs are essential for neuroendocrine regulation, particularly as related to ovulation. Perhaps the elimination of GnRH immunoreactivity in cells associated with the TN in the musk shrew disrupts GnRH release. Alternatively, in rats, the accessory olfactory bulb projects via the limbic system to a specialized population of cells in the anteroventral periventricular nucleus of the hypothalamus (AVPV), which in turn regulates LH release [44]. We have not yet examined the effects of bulbectomy on the AVPV or LH release, but it is possible that disruption of this circuit has pronounced consequences on ovulation in the musk shrew.

The GnRH neurons have a well-documented developmental relationship with the olfactory system. In all vertebrates examined to date, GnRH-containing neurons arise in the olfactory placode and migrate during fetal development into their adult positions in the forebrain [37, 45–47]. The data we and others have collected show that olfactory input is either essential, or plays an enhancing role, in ovulation [1–3]. Moreover, in some species, olfactory inputs also facilitate expression of sexual behavior [8, 11, 48]. In species with estrous cycles, the close temporal association of ovulation and receptivity may have allowed for the evolution of a second function for olfaction, to facilitate female sexual behavior [49]. This may be achieved via a direct influence of GnRH on neural circuits that regulate behavior. Shrews are considered to be evolutionarily primitive mammals. Given our data and the phylogenic status of shrew species, we speculate that the role olfaction plays in ovulation is a primitive one, which has changed little during the course of evolution. Most mammals are nocturnal and relatively asocial. Olfactory signals from males are extremely salient and may have direct facilitatory effects on ovulation, particularly in mammals with mating-induced ovulation.


   ACKNOWLEDGMENTS
 
We thank Aileen Wills for expert animal husbandry. We are also indebted to Dr. Sarah Winans for helping us develop and validate our bulbectomy protocol. We thank Drs. Robert Millar and Judy King for providing us with their proGnRH antiserum.


   FOOTNOTES
 
First decision: 1 November 1999.

1 This work was supported by NIH grants R01NS35429 and K02MH01349. Back

2 Correspondence: E.F. Rissman, Biology Department, Gilmer Hall, McCormick Road, University of Virginia, Charlottesville, VA 22903. FAX: 804 243 8433; rissman{at}virginia.edu Back

Accepted: November 29, 1999.

Received: October 11, 1999.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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J. Bakker, K. R. Kelliher, and M. J. Baum
Mating Induces Gonadotropin-Releasing Hormone Neuronal Activation in Anosmic Female Ferrets
Biol Reprod, April 1, 2001; 64(4): 1100 - 1105.
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