Social regulation of gonadotropin-releasing hormone
Program in Neuroscience, Stanford University, Stanford, CA
94305-2130, USA
* Present address: Department of Physiological Science, UCLA, Los Angeles, CA
90095-1606, USA
(e-mail: swhite{at}physci.ucla.edu)
Accepted 27 May 2002
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Summary |
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Key words: behaviour, gonadotropin-releasing hormone, cichlid, Haplochromis burtoni, gonad
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Introduction |
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In many fish, social factors regulate growth and reproduction throughout
life (Berglund, 1991;
Borowsky, 1973
;
Fraley and Fernald, 1982
;
Hofmann et al., 1999
;
Schultz et al., 1991
). In the
African cichlid Haplochromis (Astatotilapia)
burtoni (Günther), the species studied here, gonadal maturation
is suppressed when juvenile males are reared in the presence of adult males
(Davis and Fernald, 1990
).
Suppressed juveniles have small, unspermiated testes and GnRH-containing
neurons in hypothalamo-preoptic area that are, on average, eight times smaller
in volume than those of unsuppressed age-mates
(Davis and Fernald, 1990
;
Fraley and Fernald, 1982
). In
contrast, juvenile females attain sexual maturity irrespective of the presence
of adults. This shows that social stimuli produced by adult males suppress
sexual maturation in juvenile males via preoptic GnRH neurons, but
that other cues must regulate sexual maturation in females.
In their natural environment, the shore-pools of Lake Tanganyika,
reproductive opportunity for H. burtoni males depends upon defense of
a territory containing food, which ensures access to females that enter the
territory to feed and spawn (Fernald and Hirata,
1977a,b
).
Territorial males are brightly colored and socially dominant. Since food
resources are limited, only a fraction of H. burtoni males hold
territories and reproduce at any given time. The remaining males are
non-territorial, and their coloration, like that of females, matches the lake
bottom. These socially subordinate males postpone sexual maturation until a
habitat becomes available, at which time they undergo a transformation to the
territorial state.
Mature males can change territorial status in either direction. Switches
from territorial (T) and reproductively active to non-territorial (NT) and
reproductively inactive (TNT), or vice versa (NT
T), occur
in the wild and in aquaria (Hofmann et
al., 1999
). Switches can be achieved experimentally by moving
individuals to new communities where the social constellation influences the
direction of the change (Francis et al.,
1993
). A male introduced to a novel community quickly adopts
behaviors and body colors that reflect his new social status. Remarkably,
plasticity is also found in the brain, where GnRH-containing neurons within
the hypothalamo-preoptic area change size, reversibly, depending on the
direction of social change (Francis et
al., 1993
), with T males having larger preoptic immunoreactive
GnRH (irGnRH) neurons than NT males. Preoptic irGnRH neurons in female H.
burtoni also change size with reproductive state, but these changes are
independent of social interactions (White
and Fernald, 1993
).
The pronounced social control of reproduction in H. burtoni males and the apparent lack of it in females provide an opportunity to investigate the mechanisms through which social interactions alter reproductive status via the brain. Such mechanisms must ultimately control GnRH delivery to the pituitary. To begin to explore these mechanisms, we tested the hypotheses that increased transcription of GnRH mRNA contributes to reproductive capacity in both males and females, while the signals that drive this upregulation are sexually dimorphic.
In many species, multiple cDNAs code for multiple GnRH peptides
(Gestrin et al., 1999;
Kasten et al., 1996
;
Latimer et al., 2000
;
White et al., 1994
), and in
H. burtoni three genes for GnRH are expressed in distinct neuronal
populations (Bond et al., 1991
;
White et al., 1994
,
1995
; for nomenclature, see
White and Fernald, 1998
). One
of these genes, GnRH1, is expressed in preoptic neurons that project to the
pituitary (Bushnik and Fernald,
1995
) and have been shown to change size in response to social
change (White et al., 1995
).
The other two, one localized in the midbrain (GnRH2) and one expressed in
cells located along the forebrain terminal nerve (GnRH3), have unknown
functions. We used molecular probes specific for each form to test whether
social cues alter reproductive capacity via gene expression of any
GnRH form. We found that social opportunity initiates a cascade of responses
in males, including heightened aggressiveness, increased expression of only
GnRH1 and enlargement of preoptic GnRH neurons and of gonads. Further, the
cortisol levels of ascending males dropped, consistent with idea that social
suppression of reproductive capacity in NT males results from stressful
behavioral interactions with aggressive T males.
The biological changes in GnRH that occur during social ascent happen faster than those that accompany social descent. Interestingly, behavioral changes show the reverse pattern: aggressive behaviors emerge more slowly in socially ascending animals than they disappear in socially descending animals. This time course of physiological and molecular change fits well with the life history pattern of male H. burtoni in their natural habitat. For comparison, we also measured GnRH and stress indices in female H. burtoni, which do not undergo changes in social status but do experience cyclical changes in reproductive state. Although female gonads and preoptic irGnRH neurons exhibit changes that are comparable in magnitude with those in males, in females these changes are regulated via nutritional not social cues.
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Materials and methods |
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Subjects
The Haplochromis (Astatotilapia) burtoni
(Günther) used in this study were bred from wild-caught stock and
maintained at Stanford University under laboratory conditions that simulate
those of their natural environment in Lake Tanganyika, Africa
(Fernald, 1977): pH 7.8-8.2,
temperature 29 °C, 12h:12h light:dark cycle with full-spectrum
illumination (Duralight 30 W; Bob Corey Associates, Merrick, NY, USA). Gravel
and terracotta pot-shards provided visual isolation, allowing dominant males
to establish and maintain territories, an integral component of their
reproductive and social behavior (Fernald,
1977
). Fish were fed once daily at 09:00-09:30h with cichlid
formula pellets and flakes (Aquadine, Healdsburg, CA, USA). All work was
performed in compliance with the animal care and use guidelines at Stanford
University.
Behavioral observations and analysis
To allow behavioral observations of specific individuals, males were tagged
near the dorsal fin with a unique combination of colored beads. Focal
observations were made as follows. Each male was observed for 3 min between
14:00 and 16:00 h, three times per week (or, where noted, daily), and its
social and reproductive state were recorded. Males were classified as T or NT
on the basis of their behavior and coloration as follows. NT males are
cryptically colored and resemble females. Their behavior consists primarily of
schooling and fleeing from attacking T males. T males are either bright blue
or yellow and have a dark lachrymal stripe (eye-bar) and orange humeral
patches. Their behavior consists of territorial defense, solicitation and
courtship of females and feeding. Behaviors were scored using established
criteria (Fernald, 1977;
Fernald and Hirata, 1977b
) and
were categorized as aggression towards subordinates (chasing or biting females
or NT males), aggression towards T males (chasing or biting T males, threat
and border displays and fighting), reproductive acts (digging, courting and
spawning) or submissive acts (fleeing). The number of instances of each
behavior was recorded, and the presence of an eye-bar was noted.
Males were ranked for aggression using an index of dominance (DI). DI was calculated as the sum of the number of aggressive acts minus the number of submissive acts that occurred during a given observation period. DIs were averaged over the number of days an animal spent in one social setting, as were the number of reproductive displays. These daily mean values were used for comparison.
Experimental design
Three different experimental protocols (I, II and III) were used to examine
responses of males to social change: undisturbed animals (study I), males
ascending in social status (study II) and males ascending and descending in
social status (study III). These protocols are depicted in
Fig. 1 and described below.
Study I: GnRH gene expression levels in H. burtoni males living in
undisturbed communities
To measure the effect of ongoing social interactions on GnRH gene
expression in mature males, social communities were assembled that had 3-4 T
males, 9-12 NT males and 12-16 females
(Fig. 1A, study I). Communities
were established in three tanks (91 cmx61 cmx46 cm; width x
length x height) that were left undisturbed for 4 months except for
feeding, behavioral observations and occasional removal of a female for a
separate experiment (see below). Behavioral observations were used to select
males that had maintained a consistent social status (e.g. T or NT) for a
minimum of two (2) and up to 10 consecutive weeks prior to being killed.
Blood samples were obtained immediately prior to killing the fish for
measurement of serum cortisol levels. Body and gonad masses were obtained for
calculation of the gonosomatic index (GSI, see below). Brains were removed and
frozen in liquid nitrogen for subsequent measurement of mRNA levels for the
three forms of GnRH using ribonuclease protection assays (RPA; see below).
Study II: changes in GnRH gene expression during social ascent
(NTT)
To determine whether GnRH gene expression changes during an ascent in
social status, 13 pairs of young NT males were matched for size and social
history and then either induced to change into T males or kept as NT males. To
do this, the paired NT males were removed from their home tanks and introduced
into one half of a new tank (13 halves, each half 46 cmx46 cmx30
cm; width x length x height) that also contained two larger T
males and several females (Fig.
1B, study II). The other half of the tank contained only females.
After 2 weeks of daily observations to obtain baseline behavioral values, one
member of each pair was chosen at random and moved to the opposite side of the
tank where, in the absence of tactile interactions with males, it had the
maximum chance of becoming territorial. At the time of transfer, both NT males
were caught and blood was taken for subsequent cortisol measurement (see
below). One male (NTT) was then placed in the all-female half of the
tank. The other NT (control NT) in each pair was returned to the first side of
the tank containing the larger T males. Behavioral observations of both
subjects continued daily for 3 or 7 days. Blood samples were then taken, and
the experimental males were killed by rapid cervical transection. Body and
gonad mass were measured, and the brain tissues were processed for analysis of
GnRH mRNA expression levels.
Study III: comparison of the time course for social ascent
(NTT) versus social descent (T
NT)
Although study II provided information about how quickly social change can
lead to physiological change during social ascent, we wanted to know the time
course of changes in GnRH expression during social ascent or descent under
conditions that more closely paralleled the social situation of animals in
their natural habitat (i.e. in the presence of other males, rather than alone
with a group of females). To do this, we induced changes in social status
within community settings. Groups of males were observed as they established
social communities, after which those designated as controls (15 T and 11 NT
males) were killed (Fig. 1C,
study III). The social status of the remaining T males was then changed from T
to NT (TNT) by moving them to new tanks inhabited by a community of
older, larger fish. Conversely, the remaining NT males were induced to become
territorial (NT
T) by moving them to tanks containing younger, smaller
fish communities. Animals remained in their new tank communities, and
behavioral observations continued for a period based on pilot studies (see 3-
and 7-day data above; Nguyen,
1996
).
Briefly, a pilot study (Nguyen,
1996) was conducted to assess the time course of structural
changes in preoptic irGnRH neurons. This study used identical procedures to
those used here except that neuronal soma sizes were measured only at 2 weeks
following the induced change in social status. These revealed that, in NT
males that had ascended in social status, structural changes had already
occurred by 2 weeks. At this time, preoptic irGnRH neurons were similar in
size to those of control T males. In contrast, in T males that were descending
in social state, no change in irGnRH neuron size was observed at 2 weeks.
Thus, for the present study, new time points were added to establish when the
structural differences accompanying social change happened. Accordingly,
NT
T males were killed 1 (N=8) or 2 weeks (N=9) after
entry into the new tank community, while T
NT males were killed after 2
(N=8) or 3 (N=6) weeks following the social transition. For
the 2-week time points, preliminary analysis (see Statistical
analysis) revealed that data obtained from the pilot study and study II
were identical when values were reported as the percentage of control T values
for each study. These data were therefore combined and plotted (see
Fig. 5). To measure GnRH mRNA
expression levels, males were killed 1 (N=7) or 3 weeks
(N=5) after being switched to new communities. Brains were removed
and processed either for immunocytochemistry for measurement of preoptic
irGnRH neuronal soma size or for the ribonuclease protection assay to quantify
GnRH transcript levels.
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Study IV: regulation of GnRH gene expression in females
In female H. burtoni, preoptic irGnRH neurons change size
depending on whether the individual is spawning or brooding fry
(White and Fernald, 1993). To
determine whether GnRH transcript levels also fluctuate during the
reproductive cycle, females were observed until the act of spawning
(Fig. 2). Some were caught,
blood was drawn and the fish were killed (Sp; N=6). For comparison,
other spawning females (N=7) were allowed to brood their fry for the
normal brooding period (2 weeks; Br), at which time blood was drawn and the
animals were killed. Body and gonad masses were measured, and brains were
processed for comparison of GnRH transcript levels.
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To identify endogenous cues implicated in GnRH production in female H. burtoni, the effect of brooding fry on GnRH levels was measured. H. burtoni are mouth-brooders, so females do not eat during the 14 days when they maintain their fry in their mouths. To distinguish whether the presence of fry or the lack of food regulates GnRH production, females (N=38) in stable communities were observed until the act of spawning. Approximately half were allowed to brood fry for 2 weeks prior to being killed (Br; N=18). The remainder were induced to release their eggs and moved to tanks without males. Over the next 2 weeks, some of these `broodless' females were fed (Br-F+; N=12), while the others were not (Br-F-; N=8). The fish were killed, and body and gonad masses were measured. Brains were processed to measure preoptic irGnRH neuronal soma size and GnRH mRNA transcript levels. Preliminary analysis revealed that GnRH1 expression in the two groups of brooding females, probed on two different gels, were not significantly different from one another (mean normalized, optical density, gel A 0.62±0.11, N=7; gel B, 0.66±0.05, N=6, MannWhitney U-test, P=0.72; see Measurement of mRNA levels). These data were subsequently pooled for further statistical comparisons.
Cortisol measurement
Serum cortisol levels were used as an index of stress on the basis of our
previous analyses that established control values for serum cortisol levels in
animals of different social states (Fox et
al., 1997). To compare the magnitude and time course of changes in
cortisol levels during the present studies, cortisol levels were measured in
serum samples following our established procedures
(Fox et al., 1997
). Only
samples that were obtained within 3 min of approaching a given fish's tank
were used to avoid confounding increases in cortisol level due to
capture-associated stress.
Body and gonad mass
All animal subjects were killed by rapid cervical transection and then
weighed. The brains were removed and frozen in liquid nitrogen. The gonads
were removed and weighed, and the gonosomatic index (GSI) was calculated
[GSI=(gonad mass/body mass)x100, where mass is in g].
Quantification of preoptic irGnRH neuronal soma size
To compare irGnRH cell sizes, brain tissue was processed and immunostained
for detection of preoptic irGnRH neurons using a commercial antibody
(anti-LHRH; ImmunoStar; Hudson, WI, USA), as previously described
(White and Fernald, 1993).
Previous measurements showed that the mean soma sizes of GnRH-containing
neurons in the terminal nerve and the diencephalon do not differ in animals of
different social status (Davis and
Fernald, 1990
). Therefore, only the preoptic irGnRH cell sizes
were analyzed. Cross-sectional areas were measured on computer-captured video
images (NIH Image 1.40 by Wayne Rasband) from a microscope (Zeiss,
600x), as described by White and Fernald
(1993
).
Measurement of mRNA levels
To measure the effects of change in social status on GnRH gene expression,
a ribonuclease protection assay (RPA) was developed. Total RNA was isolated
from brain tissue using Ultraspec II/RNA tack resin (BioTecx, Houston, TX,
USA) following standard protocols. Approximately 30 µg of total RNA was
obtained from 30 mg of tissue.
For each of the three different GnRH mRNAs in H. burtoni, labeled
antisense riboprobes (White et al.,
1995) were transcribed from vectors (T7 RNA polymerase; Promega,
Madison, WI, USA) incorporating [
-32P]UTP (NEN, Boston, MA,
USA) to a specific activity of approximately 3x108 cts
min-1 µg-1. Probe reactions were size-separated on a
4.8% acrylamide gel, and full-length transcripts were identified by
autoradiography. Bands were cut from the region of the gel containing the
longest transcripts, and these were eluted either at 37 °C for 3 h or at 4
°C overnight prior to the RPA. To control for loading error, an antisense
18S riboprobe with low specific activity was generated from a T7 vector
(Ambion, Austin, TX, USA) using an Ampliscribe transcription kit (Epicentre,
Madison, WI, USA). Reactions were run at 42 °C, incorporating radiolabeled
UTP together with unlabeled ATP, GTP, CTP and UTP nucleotides and typically
generated approximately 50 µg of probe.
RPAs were performed on samples of total brain RNA (Hybespeed, Ambion) according to the protocol provided. Pilot studies revealed non-specific interactions when RNA samples were simultaneously hybridized with all three GnRH riboprobes and the 18S control. Accordingly, for each experiment, three separate hybridizations were performed from identical pools of total RNA. For detection of GnRH1, 2.5 µg of total RNA from each subject was used. Because the signals for GnRH2 and GnRH3 were weaker, the blots used to detect these transcript levels contained 10 µg of total RNA per subject. All blots were probed with 2x104 cts min-1 of the respective labeled probe. A loading control of 500 ng of the 18S riboprobe was used to saturate binding to 10 µg of total RNA. Following size separation on a non-denaturing 4.8% acrylamide gel, the amount of labeled RNA was measured using phosphodetection of the signal (Molecular Dynamics, Sunnyvale, CA, USA) and subsequent quantification of the protected signal optical density (IPLabGel, Signal Analytics, Vienna, VA, USA). Each GnRH optical density (OD) was normalized to that of its corresponding 18S loading control. These individual normalized signals were averaged for each group, and the means ± S.E.M. are reported.
Pilot studies confirmed that the amounts of GnRH and 18S ribosomal probes
used for hybridization reliably saturated endogenous transcripts, thereby
allowing quantification of GnRH mRNAs. Samples of total brain RNA (i.e. either
2.5 µg for GnRH1 or 10 µg for the other two GnRH transcripts) as well as
twice these amounts were hybridized to 2x104 cts
min-1 of the respective probes. Following ribonuclease digestion,
gel electrophoresis and OD analysis, the intensity of the protected signals
reliably reflected the twofold increase (data not shown), indicating that
sufficient amounts of each probe had been used. To confirm the consistency of
quantitative results across RPAs, sets of samples were processed twice for
each of the three GnRH probes. These demonstrated high reliability since
correlations of the quantified protected signals between duplicate samples run
on separate gels averaged 0.80 (Spearman , P<0.005).
Statistical analysis
Non-parametric tests were used because the data could not be assumed to be
normally distributed. The minimum significance level was determined by a
one-way 2 approximation and set at P<0.05.
Two-tailed tests were used throughout. Wilcoxon/KruskallWallace rank
sum tests were used for non-parametric comparisons across more than two
groups. When warranted, MannWhitney U-tests were used to
compare behavioral frequencies, GSIs, GnRH mRNA levels, preoptic irGnRH
neuronal soma sizes and serum cortisol concentrations between two experimental
groups. Repeated measures of serum cortisol levels and behaviors on the same
individual were performed using Wilcoxon signed-rank difference tests.
Spearman rank tests were used to assess correlations between measures. Values
are reported as the mean ± the standard error of the mean (S.E.M.). All
statistical analyses were performed using JMP IN software (SAS Institute,
Inc., Cary, NC, USA).
To obtain the values reported for preoptic irGnRH neuronal soma sizes shown
in Fig. 5, mean soma sizes for
individual fish were normalized with respect to the mean for the control T
fish. This was performed for a pilot study (described in study III, above), in
which males underwent a 2-week transition (NTT or T
NT) in social
status and were compared with control T and NT males, and for the follow-up
study (study III) in which controls and males with 2-week transition times
were repeated and 1-week and 3-week time points were added. In both the pilot
study and study III, identical behavioral manipulations were used except that
1-week and 3-week time points were also sampled in study III. In each study,
individual soma size means were normalized to their respective control T mean.
Because the normalized values for the 2-week transitions did not differ
between studies, these were pooled, converted to percentages of the control T
value and plotted in Fig. 5.
Statistical comparisons for Fig.
5 are reported in Table
1.
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Results |
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Social status was reflected in brain levels of mRNA encoding GnRH1. As
predicted, T males had higher levels of this transcript than did NT males (OD,
T males=1.77±0.46, N=5, versus NT
males=0.72±0.13, N=6; MannWhitney U-test,
P<0.03; Figs 3B,C,
4A). Comparison of gonadal size
showed that the mean GSI of T males was greater than that of NT males (GSI, T
males=0.74±0.05 versus NT males=0.43±0.06,
MannWhitney U-test, P<0.02;
Fig. 4B). GSIs and levels of
GnRH1 mRNA were positively correlated (Spearman =0.86,
P<0.0005; Fig. 4C).
Mean serum cortisol levels in NT males were three times those in T males
(NT=3.19±2.20 ng ml-1, N=4, versus
T=0.89±0.31 ng ml-1, N=4, not significant). The
high variance in measured cortisol levels, particularly for NT males, and the
small number of blood samples successfully obtained rendered this difference
not statistically significant.
Stability of social status, as measured by the number of weeks a fish
remained dominant, was strongly correlated with GnRH1 mRNA level (Spearman
=0.77, P<0.005) and with GSI (Spearman
=0.86,
P<0.0005; Fig. 4C).
As shown in Fig. 3, the case of
the one NT male (NT6) that was territorial at the beginning of behavioral
observations highlights this trend. This individual had the most GnRH1 mRNA
and largest gonads of all NT males. There were no significant correlations
between levels of the other GnRH transcripts (GnRH2 and GnRH3) and any other
measure.
The onset of aggressive behavior and GnRH1 gene expression during
social ascent
To determine how quickly behavioral and physiological changes reflect a
change in social status, one member in a pair of NT males was induced to
become T (NTT) by moving it to a social setting with only females. In
their new social setting, NT males responded by exhibiting color and
behavioral patterns characteristic of T males. Behavioral responses, cortisol
levels and mRNA levels of all three GnRH forms were compared between NT
T
males and their NT counterparts 3 days after the move. Prior to experimental
manipulation, paired NT males exhibited uniformly low levels of aggressive
behavior seen in their mean dominance index scores (pre-switch DI for
NT
T males=-2.4±0.5, N=8 versus control NT
males=-1.4±0.5, N=6, not significant). Moving one NT male to
the opposite half of the tank containing only females produced a switch in his
social status. Three days after being moved, these NT
T males had higher
DIs than those of control NT males (post-switch DI for
NT
T=4.3±1.4 versus control NT=-1.2±0.4,
MannWhitney U-test, P<0.002). NT
T males also
exhibited increased aggressive behavior relative to their own baseline levels
as NT males (Wilcoxon signed-rank test, P<0.01).
The reproductive measures for study II are shown in
Fig. 4B. Three days following
the switch, GnRH1 gene expression increased by approximately 20% in NTT
males relative to control NT males, but this did not reach significance (OD,
NT
T=1.06±0.09 versus NT control=0.80±0.07,
MannWhitney U-test, P=0.07;
Fig. 4A). Gonad size did not
differ between the two groups (GSI, NT
T=0.53±0.10 versus
NT control=0.49±0.08, not significant,
Fig. 4B) nor were GSIs
correlated with GnRH1 expression at this time, when all males were included
(Spearman
=0.07, not significant, Fig.
4C). Among NT
T males, however, GSI and GnRH1 transcript
levels were positively correlated (Spearman
=0.74, P<0.05).
There were no differences in expression levels of the other two GnRH
forms.
Prior to moving males to the all-female compartment, NT males had similar
cortisol levels (NTT prior to switch=11.3±3.1 ng ml-1,
N=7, versus control NT=9.7±4.9 ng ml-1,
N=4, not significant). Three days after being moved to the opposite
side of the barrier, cortisol levels had decreased in the NT
T males
relative to their own baseline levels. This shows that the new territorial
status was associated with lower stress levels within an animal (NT
T
post-switch=2.4±1.3 ng ml-1, N=7; Wilcoxon
signed-rank, P<0.01). In addition, there was a non-significant
decrease in cortisol levels between NT
T and control NT males at 3 days
(MannWhitney U-test, P=0.09). It is worth noting that
repeated cortisol measurements within an individual animal provide a more
useful analysis of change than measurements made between individuals because
of the high individual variability of this measure
(Fox et al., 1997
).
To extend the time course for observing changes in GnRH1 transcript levels
and cortisol levels, the above experiment was repeated, but fish were killed 7
days following transfer of one male in each pair of NTs to the all-female side
of the tank. Baseline behaviors of paired NT males were equivalent until one
in each pair was moved. Amidst only females, NT males that were moved quickly
adopted T behaviors, as exhibited by elevated DIs relative to their own
baseline levels and to matched control NT males (post-switch DI for NTT
males=9.6±1.44, N=8, versus pre-switch
DI=-2.6±1.4, Wilcoxon signed-rank test, P<0.001;
versus control NT males=-2.4±0.59, N=6;
MannWhitney U-test P<0.005). Seven days after
being moved into an all-female setting, NT
T males had elevated GnRH1
mRNA levels relative to control NT values (OD, 1.19±0.15
versus 0.85±0.06, MannWhitney U-test,
P<0.03; Fig. 4A). No differences were detected in expression levels of the other two GnRH
forms.
The gonads of NTT males at 7 days were larger than those of control
NT males (GSI, NT
T=0.61±0.08, N=6, versus
control NT=0.29±0.07, N=6; MannWhitney U-test,
P<0.03; Fig. 4B),
in contrast to the non-significant difference seen at 3 days (see above). At 7
days, GnRH1 mRNA levels were positively correlated with gonad size in the
entire population (Spearman
=0.61; P<0.04;
Fig. 4C). Surprisingly,
cortisol levels of NT
T males did not differ from their own preswitch
values (pre-switch=4.3±2.5 ng ml-1, N=5,
versus post-switch=5.7±1.1 ng ml-1, N=5;
Wilcoxon signed-rank test, P=0.16), in contrast to the difference
observed after 3 days.
Effects of the direction of social change on the speed of behavioral,
cellular and molecular change
To determine the time course of changes in GnRH expression in community
settings and during both social ascent and descent, the social status of
experimental animals was changed from NT to T, or vice versa, by
manipulating their social situation (study III, see Materials and methods).
Experimentally manipulated animals were remarkably different in both their
behavioral and physiological responses to changes in social status. These
manipulations resulted in overall significant differences in behavior
(Wilcoxon/KruskallWallace rank sum, P<0.0001), preoptic
irGnRH soma size (P<0.0001) and GnRH1 mRNA levels
(P<0.02) across experimental groups
(Fig. 5). Not only were T and
NT males significantly different in every comparison but, surprisingly, the
time course of the behavioral and physiological changes depended on the
direction of status change. All comparisons made between groups are outlined
below, and the significance level for each comparison is reported in
Table 1.
As expected, control T males were more aggressive than control NT males
(DI, T males=9.0±1.8, N=15 versus NT
males=-1.4±0.2, N=11; MannWhitney U-test,
P<0.0001; Fig. 5A).
The behaviorally inferred social status of males corresponded to indices of
preoptic GnRH expression. As anticipated, T males had larger preoptic irGnRH
neurons than those of NT males [T=147±6 µm2,
N=14, versus NT=102±8 µm2,
N=11; MannWhitney U-test, P<0.002;
normalized values (see Materials and methods) are shown in
Fig. 5B] replicating earlier
findings (Francis et al.,
1993). T males also had larger amounts of GnRH1 transcript (OD, T
males 1.16±0.14, N=7, versus NT males
0.82±0.04, N=7; MannWhitney U-test,
P<0.03; Fig. 5C)
than NT males.
Among NTT males, the fundamental behavioral patterns characteristic
of T males emerged within 1 day (Fig.
5A) but were quantitatively similar to those in control T males
only after 2 weeks. That is, within 1 day after being moved to tanks with
smaller males, previously NT males began aggressive acts (DI, NT
T at 1
day after move=2.4±3.8, N=9). However, the level of aggressive
behaviors by these newly T males did not match typical T behavior until 2
weeks after the move (DI, NT
T at 2 weeks=6.9±1.0 versus
NT=-1.4±0.02; MannWhitney U-test, P<0.02;
versus T=9.0±1.8, not significant). In contrast to this
relatively cautious change in the quantitative behavioral activity, neuronal
structure changed rapidly during social ascent. The size of preoptic irGnRH
neurons in NT
T males was equivalent to that of control T males within
just 1 week after the change in social status (NT
T at 1
week=140±9 µm2, N=8, versus
T=147±6 µm2, not significant;
Fig. 5B). During social
decline, behavioral changes were relatively fast compared with biological
ones. T males descending to NT status assumed subordinate behaviors equivalent
to those of control NT males within 1 day (DI, T
NT at 1
day=-0.57±0.57 versus NT, not significant), but their preoptic
irGnRH neuronal soma sizes did not match those of control NT males until 3
weeks later (T
NT at 3 weeks=113±5 µm2,
N=6, versus NT, not significant;
Fig. 5B).
Changes in GnRH1 mRNA expression associated with social transitions
generally paralleled the changes in neuronal soma size
(Fig. 5C). During social
ascent, the mean GnRH1 mRNA level in NTT males increased to T levels and
beyond that of control NTs within 1 week (OD, NT
T at 1
week=1.49±0.16, N=4, versus control
NT=0.82±0.04, N=7; MannWhitney U-test,
P<0.01). While the mean GnRH1 transcript level of NT
T males
at 3 weeks was also equivalent to T levels, it was no longer significantly
different from NT values (OD, NT
T at 3 weeks=1.05±0.18,
N=3; versus T or NT, not significant). One week into social
descent, no change was evident in GnRH1 expression levels (OD, T
NT at 1
week=1.32±0.15, N=3, versus control
T=1.16±0.14, N=7, not significant). Although levels of GnRH1
mRNA in T
NT males 3 weeks after being moved to new tanks were less than
70 % of those of control T males (OD, T
NT at 3 weeks=0.77±0.12),
the difference was not significant, probably because of the small number of
animals (two) that achieved and maintained T status during this part of the
study.
As with all other social manipulations tested, the expression levels for GnRH2 and GnRH3 remained the same among animals whatever their social or reproductive state. In summary, during social ascent, GnRH1 indices upregulate more quickly than do aggressive behaviors. In contrast, during social decline, GnRH1 expression persists well after aggressive behaviors have vanished.
Effects of endogenous cues on GnRH I gene expression in females
Female H. burtoni have a uniformity in both behavior and color
(but see White and Fernald,
1993), yet their preoptic irGnRH neurons undergo cyclical changes
in soma size comparable in magnitude with those in males. Spawning females
have larger preoptic irGnRH neurons than those of brooding females, similar to
the relationship between T and NT males. Internal reproductive signals rather
than external social cues are hypothesized to be critical for irGnRH neuronal
soma size regulation in females (White and
Fernald, 1993
). To determine whether these structural changes
reflect differences in GnRH mRNA expression, GnRH transcript levels were
compared between spawning (Sp) and brooding (Br) females. Reproductive
measures for study IV are shown in Fig.
6. Spawning females had twice as much GnRH1 transcript as brooding
females (OD, Sp=1.4±0.18, N=6, versus
Br=0.66±0.06, N=13; MannWhitney U-test,
P<0.005; Fig. 6A),
while levels of GnRH2 and GnRH3 mRNA did not change between these two
reproductive states.
|
During mouth-brooding of fry, female H. burtoni do not eat. Therefore, the source of the signals that produce small preoptic irGnRH neurons and low GnRH1 mRNA levels in brooding females could either be endogenous, as previously hypothesized, and due to nutritional needs, or exogenous due to `social', e.g. tactile, chemical or other cues provided by fry. To distinguish between these possibilities, indices of GnRH expression were measured in females that had brooded their fry for 2 weeks (Br) and in `broodless' females, i.e. females that had spawned but whose eggs had been removed from their mouths and were either fed (Br-F+) or fasted (Br-F-; see Fig. 2).
Levels of GnRH1 mRNA varied among female groups when all groups were compared (Wilcoxon/KruskallWallace rank sum, P<0.002). To determine which variables contributed to these overall differences, we examined two groups at a time. The presence of broods was not a major contributor to changes in GnRH1 mRNA levels because brooding females and broodless females that were not fed had similarly low GnRH1 levels (OD, Br=0.66±0.06, N=13, versus Br-F-=0.86±0.12, N=6, not significant; Fig. 6A). Thus, irrespective of the presence of broods, GnRH1 mRNA levels in these two groups were low, indicating that fasting contributes to GnRH1 regulation. Indeed, GnRH1 levels of broodless females that were fed were high, similar to those of spawners (OD, Br-F+=1.24±0.23, N=8, versus Sp=1.4±0.18, N=6; not significant; Fig. 6A). Thus, despite distinct reproductive states, these two groups of feeding females had similarly high levels of GnRH1 transcript. However, nutritional state did not completely account for differences in GnRH1 expression in females: the OD value of GnRH1 mRNA tended to be higher in broodless females that were feeding than in their fasting counterparts, but this difference was not significant (OD, Br-F+=1.24±0.23, N=8, versus Br-F-=0.86±0.12, N=6, not significant). No differences were observed in GnRH2 or GnRH3 expression levels between females in the differing reproductive states.
As expected, ovarian mass changed with reproductive state and largely
paralleled changes in GnRH1 mRNA levels. Spawning females had large ovaries of
variable mass, probably because of the range in number of eggs released during
spawning, prior to capture (White and
Fernald, 1993). Despite this variability, the ovaries of spawning
females were larger than those of brooding females (GSI, Sp=2.1±0.3
versus Br=0.94±0.06, MannWhitney, P<0.005;
Fig. 6B), replicating earlier
findings (White and Fernald,
1993
). Removal of fry and restoration of food to females caused
their ovaries to increase in size: broodless females that were fed had larger
gonads than those of either brooders or broodless females that were
food-deprived (GSI, Br-F+=5.3±0.5 versus Br,
P<0.001; versus Br-F-=2.4±0.6, MannWhitney
U-test, P<0.005; Fig.
6B). This suggests that nutritional status plays a role in
regulating gonad size in H. burtoni females, perhaps via
regulating GnRH1 expression. However, there was some influence of social cues
on GSI. Broodless females that were fasted had significantly larger ovaries
than brooding females (GSI, Br-F-=2.4±0.6 versus
Br=0.94±0.06, MannWhitney U-test, P<0.01).
Thus, ovary size began to increase in females that had had their broods
removed compared with females whose broods were intact, even though both these
groups were fasting.
Preoptic irGnRH neuronal soma size also differed across brooding and
broodless groups (Wilcoxon/KruskallWallace rank sum,
P<0.05). As previously reported, brooding females in this study
had small preoptic irGnRH neurons with a mean cross-sectional area of
94±8 µm2 (N=5), approximately one-third of the
size previously reported for spawning females
(White and Fernald, 1993).
Unfortunately, we were unable to obtain sufficient soma size measurements from
the two broodless groups to draw conclusions about the role of nutritional
versus social cues in regulating preoptic irGnRH soma size. However,
a trend towards increasing soma size following the removal of broods and
restoration of food is evident (Br-F+=115±7 µm2,
N=4, versus Br=94±8 µm2, N=5;
MannWhitney U-test, P=0.09, not significant).
In contrast to most reproductive measures, cortisol levels did not differ between spawning and brooding females (Sp=10.3±3.1 ng ml-1, N=5, versus Br=7.7±2.1 ng ml-1, N=9; not significant), with the caveat that the comparison was made between groups rather than via repeated measures from the same individual in different reproductive states.
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Discussion |
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The profile of change in preoptic GnRH indices was remarkably consistent across the many different social settings examined here. For example, during social ascent in males, changes in both GnRH1 mRNA levels and irGnRH soma sizes occurred within 1 week for both the 3- and 7-day transitions in study II and for the transitions induced within a community in study III. Socially induced decreases in preoptic GnRH levels took longer, as demonstrated by the slow, 3-week decline in mRNA levels in study III and the decrease in neuron size in this same study and a pilot study (see Materials and methods). There is even a hint of this slow decline in study I, in which males of stable social status were compared. There, the one NT male that only became NT 2 weeks before being killed (having been T for 8 weeks previously) had the highest levels of GnRH1 mRNA among NT males (Fig. 3), consistent with a time course of 3 weeks or more for preoptic GnRH downregulation. This consistency in GnRH changes may reflect the fact that the animals have been placed in a new, unambiguously different social milieu, causing an irreversible change from their prior social state. Given this clear social change, it makes sense to change the physiology needed either to start or to stop successful reproduction.
The asymmetries in the responses of behavioral and physiological systems to
social change observed in study III were unexpected. During an ascent in
social status, the swift increases in GnRH1 expression are in contrast to
behavioral changes, which require more than 2 weeks to attain levels
equivalent to those of control T males. In contrast, when a previously T male
loses his territory, his aggressive behavior stops immediately (<1 day),
while indices of GnRH expression decline more slowly. Thus, somewhat
surprisingly, animals in social ascent are relatively slow to display their
rise in status despite physiological changes that would allow them to exploit
it, while animals in social decline immediately exhibit subordinate behaviors
despite the persistence of large GnRH neurons within their preoptic areas for
several weeks. Why is the rate of cellular processes oppositely biased to the
rate of behavioral change in response to altered social status? Ultimately,
both behavioral and neuronal outcomes make sense for H. burtoni on
the basis of observations of their life in Africa
(Fernald and Hirata, 1977b).
In their natural habitat, physical conditions change unpredictably as a result
of winds and intrusions of large animals, including Hippopotamus
amphibius, into the shore-pools. This uncertainty probably increases the
chances that a male will reproduce since animals exploit these chance
occurrences to change social status
(Hofmann et al., 1999
).
Considered in this way, the trajectories of biological change measured here
fit well with both field and laboratory data on the rate at which territories
become available. For example, the median time for which animals occupy a
territory in a fluctuating environment is 3 weeks
(Hofmann et al., 1999
). Those
animals gaining social status and, hence, reproductive access, have an
accelerated physiological switch to afford reproduction quickly. Conversely,
new opportunities for establishing a territory, either for the first time or
after having been `deposed', occur at an average of 4 weeks
(Hofmann et al., 1999
). Thus,
a male losing his territory and hence reproductive opportunity may maintain a
vestige of his prior prowess since a return to that opportunity might soon
occur. Covertly, the fish's biology is biased towards reproductive capacity,
whereas his overt aggressive behavior is more tentative within new social
circumstances. Although neuronal and behavioral strategies differ temporally,
together they reflect a delicate balance between pragmatism and optimism
needed for survival.
Our data demonstrate that social interactions between male H.
burtoni change neuronal gene expression and not vice versa. To
show this, animals in study II were induced to change social status through a
manipulation of the social situation. This transition to a higher social
status was performed over 3 or 7 days. Pairs of NT males were matched for body
size, social history, social setting and level of submissive behavioral acts.
One male of each pair was then given the opportunity to ascend in social
status (NTT) by being moved to a tank with only females. Subsequent
behavioral observations confirmed that these individuals became dominant
rather quickly. GnRH1 mRNA levels began to increase in these NT males within 3
days of the move and were higher at 1 week relative to expression in socially
suppressed counterparts. Since the default state for H. burtoni males
is territorial (Fraley and Fernald,
1982
), NT males are socially suppressed by T males, and relief
from this suppression triggers the increase in GRH1 indices.
Our data suggest that social suppression of reproductive capacity in NT
males results from stressful behavioral interactions with aggressive T males.
Consistent with this interpretation, NT males in study II had higher levels of
cortisol than did T males. Moreover, cortisol levels declined within 3 days of
the NT being moved to an all-female setting. Stress has been shown in other
systems to inhibit reproduction at many levels. For example, in the amphibian
Taricha granulosa, a rapid and direct suppressive effect of stress on
male reproductive behavior is mediated by glucocorticoid receptors on neuronal
membranes (Moore and Miller,
1984; Orchinik et al.,
1991
). Cortisol might mediate similarly rapid behavioral changes
in H. burtoni, in which dominant behaviors can disappear within
minutes and such changes are independent of androgens
(Francis et al., 1992
). It
seems likely that high cortisol levels in NT males act via
conventional intracellular glucocorticoid receptors to suppress GnRH1 gene
expression and keep GnRH1 neurons small and gonads immature. This potential
for direct genomic regulation by both cortisol and androgens is supported by
the discovery of six putative consensus sequences for glucocorticoid
regulation and one for androgens within the GnRH1 gene promoter of H.
burtoni (White and Fernald,
1998
). We have previously shown that differences in cortisol
levels between males of high and low social rank arise only after new social
groups stabilize (Fox et al.,
1997
). This supports the hypothesis that social interactions
result in a shift in basal cortisol levels, which then alters GnRH1
transcription. Since cortisol levels in NT males were an average of 10 times
higher than those of T males, this seems a likely scenario.
In study II, we measured differences in cortisol level between T and NT
males that were less than the 10-fold values previously reported. What might
account for this apparent difference? One important component of social stress
is the nature of the social community
(Sapolsky, 1993). In our
previous study (Fox et al.,
1997
), the aquarium was arranged to mimic the natural situation
closely. The available space was relatively large for the number of animals,
and food was delivered only to territorial areas, similar to the African
habitat (Fernald and Hirata,
1977a
). In this situation, NT males had to enter defended
territories to feed, where they were typically chased by resident T males. In
contrast, for the data reported here, animals were kept at a higher density
and food was scattered across the surface of the aquarium, possibly reducing
the stress of feeding in NT males. Supporting this notion, Fox et al.
(1997
) also measured cortisol
levels in fish kept in conventional tanks and reported that the difference
between T and NT levels was only twofold, similar to the present study. From
both studies, it is clear that interactions between males produce differences
in cortisol levels and that, once a community is stable, T status is
associated with low basal cortisol levels. Cortisol could provide the link
between social interactions and preoptic GnRH indices, but this study does not
reveal whether cortisol also acts via social interactions to induce
or facilitate changes in social state.
Somewhat surprisingly, in study IV, we found no significant decrease in cortisol level when NT males were moved to all-female settings for 7 days, whereas at 3 days, NT males had high cortisol levels (approximately 10 ng ml-1), which dropped when they were moved. This is not likely to be due to differences in the social community since this was controlled experimentally. One possibility is that the endocrine profile accompanying social change has distinct phases, reflected in immediate, intermediate and long-term changes. Unfortunately, the frequent blood sampling required to track such profiles creates chronic stress, preventing multiple samples from a single fish and perhaps the resolution to the discrepancy between the 3- and 7-day results.
In contrast to the social regulation of GnRH1 in male H. burtoni,
results from study IV showed that endogenous cues, including nutritional
state, regulate GnRH1 mRNA levels, preoptic irGnRH neuronal soma size and
gonad size in females. We have previously reported that spawning females have
larger preoptic irGnRH cells than brooding females
(White and Fernald, 1993).
Here, we show that these differences reflect the amounts of GnRH1 mRNA. Two
possible regulatory signals were examined, the presence of a brood and
nutritional state. The first is a social cue from fry in the brooders' mouths.
The second is a physiological cue reflecting changes in nutritional state
associated with the obligate fasting of brooding females. Our data suggest
that GnRH1 mRNA level and preoptic neuronal soma size both depend on
nutritional cues. Brooding females and BrFfemales, two groups
that were fasting, had low GnRH indices, while females that were fed (Sp,
BrF+) had higher GnRH1 mRNA levels and larger preoptic irGnRH neurons
(present study; White and Fernald,
1993
).
In other systems, nutritional status has been shown to regulate fertility
in both sexes (Cunningham et al.,
1999; Foster and Nagatani,
1999
). Here, fasting females (Br, BrF) had smaller
ovaries than fed females. However, a complete absence of social cues in
regulating GnRH in females cannot be ruled out because BrF
females had slightly larger ovaries than brooding females. Further, this
change may have been triggered by the non-significant increase in GnRH1 mRNA
levels following removal of the brood. Although small, this increase resulted
in expression levels that were equivalent between BrF and
BrF+ females. Thus, the absence of a brood, a social cue, triggers a
change in reproductive state in females, but on a smaller scale than in males
and of lesser impact than endogenous nutritional cues.
The role of stress as a mediator of GnRH expression is less evident in
female H. burtoni, since no differences were observed between
cortisol levels of brooding versus spawning females. This lack of
correlation may be real or could be due to our inability to gather repeated
cortisol measures from individual females when they were either brooding or
spawning. Sampling the blood of spawning or brooding females often ends those
activities. In H. burtoni, in which cortisol levels in males reflect
the stress of social interactions, a lack of correlation between cortisol
levels and reproductive state in females underscores the role of endogenous
regulators of their GnRH expression. However, the interplay between
reproductive and nutritional state in vertebrates is complex and probably
includes the stress axis. For example, leptin, a 16kDa protein produced by the
white adipocytes of vertebrates, has been postulated to link nutritional and
reproductive state and is also thought to reduce the stress response
(Foster and Nagatani,
1999).
An intriguing result of these experiments is that no changes in mRNA levels
were detected for either GnRH2 or GnRH3 despite the diverse set of
manipulations performed on the animals. The GnRH peptides encoded by these
transcripts have been postulated to play a role in reproduction because of
their similarity to GnRH1 and because of the remarkable conservation of all
three peptide sequences over evolutionary time
(Fernald and White, 1999).
However, there are actually few data to support any role for GnRH2 or GnRH3 in
reproductive function. For example, although the distribution of GnRH2 is
known in several species (Gestrin et al.,
1999
; Kasten et al.,
1996
; Latimer et al.,
2000
; Lescheid et al.,
1997
; White et al.,
1994
), the only functional data about its role suggests that it
may act as a neurotransmitter and neuromodulator at specific synapses in
amphibian sympathetic ganglia (Jan et al.,
1979
; Jones, 1987
;
Kuffler and Sejnowski, 1983
).
For GnRH3, there are no functional studies, but its wide distribution in the
terminal nerve area of the forebrain and its projection to the retina of
non-mammalian vertebrates (Münz et
al., 1982
) are consistent with a putative role as a neuromodulator
(Oka, 1992
).
While release of GnRH from the terminal nerve in the aquatic salamander
Necturus maculosus appears to modulate odorant sensitivity according
to seasonal reproductive cycles, the GnRH form contained in amphibian terminal
nerve neurons is GnRH1 (Eisthen et al.,
2000). Analysis of mRNA expression levels of a GnRH2 homolog in
the musk shrew Suncus murinus using both males and females in
different developmental and reproductive states failed to find evidence for
differences in gene expression (White,
1997
). Obviously, failure to detect mRNA regulation in response to
changes in reproductive state does not preclude a reproductive role for these
peptides. Our sampling protocol may not have been appropriate to detect a
change in transcript levels of GnRH2 and GnRH3, or their reproductive
regulation might occur at the translational level or via their
receptors. However, reproductive regulation of GnRH peptides in another
teleost fish, the grass rockfish Sebastes rastrelliger, is limited to
the form found in the pituitary (Collins
et al., 2001
), suggesting that the lack of transcriptional change
in GnRH2 and GnRH3 observed in the present studies is borne out at the peptide
level. Perhaps the functions of GnRH peptides have diverged such that GnRH2
and GnRH3 now have non-reproductive roles in the animal. Divergent functions
for peptides with similar sequences are well-documented, with oxytocin and
vasopressin being prime examples (Mohr et
al., 1995
; Venkatesh and
Brenner, 1995
). Unveiling the roles of the other GnRH-like
peptides must await identification and mapping of their receptors as well as
specific functional tests.
Many features of a functioning organism, such as setting the circadian
clock by the influence of light, are subject to environmental regulation. The
pathways by which these influences change gene expression and concomitant
protein abundance are becoming understood, although the complexity is
daunting. Similarly, there are many reports documenting the effects of social
interactions on the behavior of individuals. In contrast, however, much less
is known about how these social influences cause changes in the brain through
changes in gene expression. This work documents that change in social status
directly influences the mRNA levels of GnRH1, which is crucial for
reproductive behavior (Mason et al.,
1986). Just how social exchanges are transduced into molecular
events remains to be determined.
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Acknowledgments |
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