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Social regulation of gonadotropin-releasing hormone

Stephanie A. White^*, Tuan Nguyen and Russell D. Fernald

Program in Neuroscience, Stanford University, Stanford, CA 94305-2130, USA
^* Present address: Department of Physiological Science, UCLA, Los Angeles, CA 90095-1606, USA

View larger version (27K): [in a new window]   Fig. 1. Timeline of experimental manipulations used for studies I, II and III of Haplochromis burtoni males. Three experiments (I, II and III) examined how social opportunity changes behaviors and gonadotropin-releasing hormone (GnRH) expression. These are shown schematically by the three types of aquaria used and the corresponding time courses of behavioral observations and physiological and molecular measures. T, territorial male; NT, non-territorial male; F, female. (A) Study I: undisturbed aquaria were used to identify males that maintained a stable social status of either T or NT for more than 2 weeks. Six NT males and five T males fulfilled this behavioral criterion. After 10 weeks of observation, fish were captured, and blood was sampled for measurement of serum cortisol levels. Gonad and body masses were obtained for calculating the gonosomatic index (GSI; see Materials and methods), used to confirm the behaviorally defined social state. Brains were processed for the detection of GnRH gene expression. DI, index of dominance (see Materials and methods). (B) Study II: to determine how fast GnRH gene expression changes during social ascent (NTT), half-tanks were prepared with distinct communities on either side of a Plexiglas barrier. A pair of NT males, matched for age and size, were housed in one half of an aquarium containing two T males and a few females. After 2 weeks of baseline behavioral observations, blood samples were obtained, and one member of each pair (NTT) was then moved to the opposite half of the tank, where there were only females. The other (control NT) was returned to the first side. Behavioral observations continued for 3 or 7 days, when blood was again sampled and NTT and control NT males were killed for analysis of GSI and GnRH mRNAs. (C) Study III: to examine changes in GnRH expression during both social ascent (NTT) and descent (TNT), males were observed for 3 weeks to ascertain their social state. Control males were killed, and experimental males were moved to new tanks containing communities designed to induce a change in their social status. Only males for whom behavioral observations confirmed that the intended social change had taken place were selected for measurement of GSI, GnRH mRNAs and preoptic immunoreactive GnRH neuronal soma sizes at the time points indicated. Asterisks indicate when serum cortisol levels were measured.

View larger version (18K): [in a new window]   Fig. 5. Study III: asymmetry of behaviors and gonadotropin-releasing hormone (GnRH) expression during social change. Changes in behavior (A, index of dominance, DI), immunoreactive GnRH neuronal soma cross-sectional area (B) and GnRH1 mRNA levels (C) during changes in social status. The x-axis indicates whether males were control territorial (T) or non-territorial (NT) or were undergoing a transition in social status for the indicated number of weeks. In all graphs, data from control NT males, 1- and 2-week NTT transitions, control T males, and 2- and 3-week TNT transitions are shown by open columns. Data for control NT males are plotted twice for clarity. Aggressive behaviors (A) were also measured at 1 day (filled triangles) following a transition in social status. (A) Mean aggressive behaviors are plotted as DI scores + S.E.M. (N=11 for control NT; 9 for 1 day observations, and 8, 14, 14, 12 and 6 for remaining groups, respectively), which increase slowly during social ascent (NTT) and disappear within 1 day (filled triangle) during social descent (TNT). (B) Preoptic immunoreactive GnRH neuronal soma cross-sectional areas are shown as a percentage of control T area, indicated by the dotted line. When NT males become territorial, these neurons grow to T size within 1 week. It takes 3 weeks for neurons to return to NT sizes during social descent. Values are means + S.E.M. (N=11 for control NT; N=8, 14, 14, 12 and 6 for remaining groups, respectively). (C) GnRH1 transcript levels, normalized for loading, are plotted as optical densities. The time course of changes in GnRH1 levels is also asymmetrical and generally parallels changes in neuron size. Values are means + S.E.M. (N=7 for control NT; 4, 3, 7, 3, 2 and 2 for remaining groups, respectively). See Results for statistical comparisons and P-values.

View larger version (20K): [in a new window]   Fig. 2. Experimental timeline used for Haplochromis burtoni females. Different reproductive and nutritional states were examined for levels of gonadotropin-releasing hormone (GnRH) expression in females (study IV). Females were observed until the act of spawning. A subset of these (Spawners; Sp) was removed for blood sampling and then killed for determination of body and gonad masses and GnRH mRNA levels. The remainder were divided into three groups. Brooders (Br) were females that brooded fry in their mouths for 2 weeks after spawning. Broodless females (Br-) were females whose eggs were removed immediately following spawning. Of these, half were fed (Br-F+) and the other half were food-deprived (Br-F-). At the end of 2 weeks, brooders were removed for blood sampling, and all three groups were killed for determination of relative gonadal masses and of GnRH mRNA levels. GSI, gonosomatic index (see Materials and methods).

View larger version (65K): [in a new window]   Fig. 3. Study I: gonadotropin-releasing hormone 1 (GnRH1) gene expression differs with social state in undisturbed territorial (T) and non-territorial (NT) males. (A) The behavior of males was examined for 10 weeks to monitor social state. The social status of six males used for the NT group plotted as a function of time is shown on the left; that of T males (N=5) is shown on the right. Note that NT6 initially displayed territorial behaviors. NT/T on the vertical axis indicates that the fish exhibited a mixture of aggressive and subordinate behaviors. (B) Preoptic GnRH1 mRNA levels were measured for each individual and are plotted as optical density (OD), described in the Materials and methods and below. Note that NT6 has the highest levels of all NTs. When averaged, T males had higher levels of preoptic GnRH1 mRNA than NT males (see Fig. 4A, and Results). (C) Only GnRH1 exhibits differential expression according to social status. Phosphoimages show protected bands from three separate ribonuclease protection assays of whole-brain RNA for these 11 males. Each assay was simultaneously probed with an 18S probe (data not shown) as well as a specific probe for GnRH1, GnRH2 or GnRH3. The optical densities of the protected GnRH bands were normalized against the signal for an 18S loading control to obtain the group means reported in the text and plotted in B.

View larger version (21K): [in a new window]   Fig. 4. Study II: gonadotropin-releasing hormone 1 (GnRH1) gene expression increases within 3 days following removal of suppressive tactile cues from dominant conspecifics. Mean GnRH1 mRNA levels (A), plotted as normalized optical density (see Materials and methods), and gonosomatic indices (GSIs) (B) are plotted for males undergoing a 3- or 7-day transition from non-territorial (NT) to territorial (T) and are compared with those of the NT and T males of stable social status (2 weeks) from study I. Values are means + S.E.M. (N=6 NT, 8T for 3- and for 7-day transmissions; N=6 NT, 5 T for >2 weeks). Open columns show control NT values. Filled columns show NTT and undisturbed T male values. (C) Gonad size, plotted as the gonosomatic index (GSI; see Materials and methods) and GnRH1 gene expression (plotted as normalized optical density) are positively correlated. The strength of the correlation (Spearman ) increased with the time spent in the T state. Asterisks indicate P-values: *P<0.05; **P<0.0005.

View larger version (13K): [in a new window]   Fig. 6. Study IV: preoptic gonadotropin-releasing hormone 1 (GnRH1) expression is regulated by endogenous cues in females. Spawning (Sp), brooding (Br) and broodless (Br-) females were used to test the relative influence of social versus endogenous regulation of GnRH levels. Experimental groups are indicated on the lower x-axis and include two groups of broodless females, either fed (Br-F+) or fasted (Br-F-). (A) Normalized GnRH1 mRNA signals are plotted as their optical density (OD). Values are high in females that eat (Sp and B-/F+) and low in fasted females (Br and Br-F-). Soma sizes from spawning females are from White and Fernald (1993). (B) Brooding females have small ovaries indicated by their small gonosomatic index (GSI). Feeding increases ovary size, as does the absence of broods. The intermediate Sp value reflects egg release during capture while spawning. Values are means ± S.E.M. (N=6 Sp, 13 Br, 8 Br-F+, 6 Br-F-). Asterisks indicate P-values: *P<0.05; **P<0.005; ***P<0.0005.