Plasticity of the reproductive axis caused by social status change in an african cichlid fish: II. testicular gene expression and spermatogenesis

Abstract

Reproduction in all vertebrates is controlled by the brain-pituitary-gonad (BPG) axis, which is regulated socially in males of the African cichlid fish Astatotilapia burtoni. Although social information influences GnRH1 neurons at the apex of the BPG axis, little is known about how the social environment and dominance affects the cellular and molecular composition of the testes to regulate reproductive capacity. We created an opportunity for reproductively suppressed males to ascend in status and then measured changes in gene expression and tissue morphology to discover how quickly the perception of this opportunity can influence the testes. Our results show rapid up-regulation of mRNA levels of FSH receptor and several steroid receptor subtypes in the testes during social ascent. In contrast, LH receptor was not elevated until 72 h after ascent, but this increase was coincident with elevated circulating androgens and early stages of spermatogenesis, suggesting a role in steroidogenesis. The spermatogenic potential of the testes, as measured by cellular composition, was also elevated before the overall increase in testes size. The presence of cysts at all stages of spermatogenesis, coupled with lower levels of gonadotropin and steroid receptors in subordinate males, suggests that the BPG axis and spermatogenesis are maintained at a subthreshold level in anticipation of the chance to gain a territory and become reproductively active. Our results show that the testis is stimulated extremely quickly after perception of social opportunity, presumably to allow suppressed males to rapidly achieve high reproductive success in a dynamic social environment.

Figure 1

Relative mRNA levels of LHR and FSHR in the testes of male A. burtoni. A, LHR mRNA levels were elevated by 72 h after perception of social opportunity. B, FSHR mRNA levels were rapidly elevated at 30 min and were further increased by 120 h after ascent. Data are plotted as relative mRNA levels (mean ± se) referenced to the geometric mean of two housekeeping genes (18s and G3PDH) and corrected for testes size and cellular composition. Bars with different letters represent significant differences (P < 0.05), and sample sizes are indicated within each bar.

Figure 2

Relative mRNA levels of AR subtypes in the testes of male A. burtoni. A, ARα mRNA levels were elevated by 6 h after perception of social opportunity. B, ARβ mRNA levels were also elevated by 6 h after social ascent. mRNA levels of both ARs were also higher in stable dominant compared with stable subordinate males. Data are plotted as relative mRNA levels (mean ± se) referenced to the geometric mean of two housekeeping genes (18s and G3PDH) and corrected for testes size and cellular composition. Bars with different letters represent significant differences (P < 0.05), and sample sizes are indicated within each bar.

Figure 3

Relative mRNA levels of ER subtypes and aromatase in the testes of male A. burtoni. A, ERα mRNA levels were elevated by 6 h after ascent and were doubled at 120 h before being lowered to stable dominant male levels. B, ERβa mRNA levels did not differ between stable subordinate and stable dominant animals but were elevated during social ascent. C, ERβb mRNA levels were only higher in ascending males at 120 h after social ascent. D, Aromatase CYP19a mRNA levels were elevated only at 72–120 h after ascent and were then reduced in stable dominant males to levels that did not differ from stable subordinate animals. Data are plotted as relative mRNA levels (mean ± se) referenced to the geometric mean of two housekeeping genes (18s and G3PDH) and corrected for testes size and cellular composition. Bars with different letters represent significant differences (P < 0.05), and sample sizes are indicated within each bar.

Figure 4

Relative mRNA levels of corticosteroid receptor subtypes in the testes of male A. burtoni. A, GR2 mRNA levels were elevated by 30 min after social ascent, remained unchanged through 72 h, and then were further elevated at 120 h. B, GR1a mRNA levels were also elevated by 30 min, but were then lower at 24–72 h before showing a 5-fold increase at 120 h after social ascent. C, GR1b mRNA levels were elevated by 30 min and then showed a further increase at 120 h after social ascent. D, MR mRNA levels were elevated by 30 min after ascent, remained at this level through 72 h, and then showed a 3-fold increase at 120 h. All CRs were higher in stable dominant compared with stable subordinate males. Data are plotted as relative mRNA levels (mean ± se) referenced to the geometric mean of two housekeeping genes (18s and G3PDH) and corrected for testes size and cellular composition. Bars with different letters represent significant differences (P < 0.05), and sample sizes are indicated within each bar.

Figure 5

GSI and cell composition of the testes in male A. burtoni. Interstitial tissue was higher in stable subordinate males compared with stable dominant males but remained unchanged in ascending animals. Type B spermatogonia and spermatocytes were elevated by 72 h after ascent, whereas the percentage of spermatids was only higher in stable dominant males. There was no difference in the percentage of mature spermatozoa among stable phenotypes or during ascension. Data are plotted as the percentage of testis (mean ± se) composed of each cell type, and representative photomicrographs of each cell type are shown at right (cresyl violet stain; scale bars, 10 μm). GSI is overlaid on the top graph (red symbols and line), and increased to near stable dominant male levels by 120 h after ascent. Bars with different letters represent significant differences (P < 0.05), and sample sizes are indicated in parentheses within each bar. Pie charts show the percentage of each cell type represented in the testes of stable subordinate, stable dominant, and ascending males at each time point to illustrate the progressive increase in absolute spermatogenic potential during ascent.

Figure 6

Correlations between GSI and spermatogenic cell type in the testes of male A. burtoni. GSI was negatively correlated with interstitial tissue and positively correlated with type B spermatogonia, spermatocytes, and spermatids. There was no relationship between GSI and the percentage of mature spermatozoa in the testis. Correlation coefficients (r) and P values are shown.

Figure 7

Density of mature sperm within testicular lumens of male A. burtoni. Stable dominant males had a greater density of sperm within their lumens compared with stable subordinate males that were socially suppressed for 4–5 wk. Data are plotted as mean ± se, and sample sizes are indicated within each bar. Representative photomicrographs of mature sperm within the testicular lumen are shown.

Figure 8

Temporal summary of physiological changes along the reproductive axis during social ascent in male A. burtoni. Arrows indicate the time point at which the first significant increase (up arrows) or decrease (down arrows) from stable subordinate male values was observed. Note that any further significant differences after this initial change in each measure are not shown. These data were compiled from the present study, the companion manuscript, and Ref. . Schematic simplified diagram of the BPG axis is shown at left. 11-KT, 11-ketotestosterone; IT, interstitial tissue; SPC, spermatocytes; SPG B, type B spermatogonia; SPT, spermatids.