mRNA expression
When comparing subordinate values across the different reactions, there were no significant effects of sex or reaction on any gene examined. (Ar: reaction by sex interaction, F (3, 24) = 0.101, p = .959; main effect [ME] of reaction, F (3, 24) = 0.130, p = .942; and ME of sex, F (1, 8) = 0.007, p = .937. Cyp19a1: reaction by sex interaction, F (3, 24) = 0.089, p = .966; ME of reaction, F (3, 24) = 0.063, p = .979; and ME of sex, F (1, 8) = 0.117, p = .741. Esr1: reaction by sex interaction, F (3, 24) = 0.094, p = .962; ME of reaction, F (3, 24) = 0.042, p = .988; and ME of sex, F (1, 8) = 0.046, p = .836. Pgr: reaction by sex interaction, F (3, 24) = 0.053, p = .983; ME of reaction, F (3, 24) = 0.119, p = .948; and ME of sex, F (1, 8) = 0.055, p = .820). For further analyses, we therefore averaged subordinate mRNA values from all reactions and compared them to the other four groups (1-day, 1-week, 1-month, and breeder).
A significant interaction of group and sex was found for Ar, F (4, 43) = 6.398, p < .001, such that male breeders had significantly higher Ar mRNA levels than the subordinates, p = .002, 1-day males, p = .001, and 1-month males, p = .001, although they did not significantly differ from the 1-week males, p = .354. Conversely, the female groups did not differ in Ar mRNA expression (see Fig. 1). A significant main effect of sex, F (1, 43) = 16.76, p < .001, indicates that overall males have more Ar mRNA expression than females. Together, the results indicate changes in Ar mRNA in males, such that AR spikes at 1 week, followed by a decline at 1 month, and then again increases once breeder status is achieved.
A significant interaction of group and sex was also found for Cyp19a1, F (4, 43) = 8.712, p < .001; however, in this case, the male groups did not significantly differ from each other (all p > .05). The female breeders had significantly more Cyp19a1 mRNA compared to the 1-month females, p < .001, 1-day females, p < .001, and subordinates, p < .001, and was marginally increased compared to the 1-week females, p = .058. A main effect of sex indicates that females had more Cyp19a1 mRNA compared to males, F (1, 43) = 23.428, p < .001, and the main effect of group indicates that overall breeders had significantly more Cyp19a1 mRNA compared to all other groups (see Fig. 1).
Similar to Cyp19a1, a group by sex interaction, F (4, 43) = 14.131, p < .001, indicates that female breeders have significantly more Esr1 mRNA than all other female groups (1-month, 1-day, 1- week, and subordinate, all p < .001), in addition to a main effect of sex in which females have more Esr1 mRNA than males, F (2, 43) = 20.51, p < .001. The male groups did not significantly differ from each other (see Fig. 1). In our sample, Esr1 mRNA did not show any changes in females until after breeder status was achieved.
Pgr comparisons also revealed a significant group by sex interaction, F (4, 43) = 11.103, p < .001. The male breeders had significantly less Pgr mRNA compared to the subordinates, p = .015, and 1-day males, p = .026, but did not differ from the 1-month, p = .343, nor from the 1-week males, p = .829. Conversely, the female breeders had significantly more Pgr mRNA compared to all female groups (1-month, 1-day, 1-week, and subordinate, p < .01). The 1-month females did not differ from any group other than the female breeders, suggesting that Pgr does not increase in females until a litter is produced (see Fig. 1).
Hormone assays
T concentrations varied according to sex and group, F (4, 45) = 2.664, p = .044. The 1-month males had marginally more T compared to the subordinates, p = .091, whereas females did not show any differences across groups. Although other male groups are not significantly different from each other, there appears to be a trend for males to have an increase in T as early as 1 week after separation from the colony (see Fig. 2). A main effect of sex indicates that males have more T than females, F (1, 45) = 28.526, p < .001, and a main effect of group was trending, F (4, 45) = 2.215, p = .082, indicating a tendency for an increase in T over time away from the colony.
A sex by group interaction for E2 concentration, F (4, 43) = 6.896, p < .001, indicated that male breeders, but not female breeders, have increased E2 compared to the same-sex subordinate, p < .001; 1-day, p = .002, 1-week, p = .00, and 1-month groups, p < .001. Interestingly, the females showed an increase in E2 at 1 month, compared to the 1-week, p = .009, 1-day, p = .02, and subordinate groups, p = .024, but E2 declined once they produced a litter (see Fig. 2).
P concentrations also varied according to sex and group, F (4, 45) = 5.26, p = .001, such that female breeders, but not males, had increased P compared to the 1-week females, p = .01, 1-day, p = .027, and subordinate females, p = .03, but not the 1-month females, p = .835. There was a trend for the 1-month females to have more P than the 1-week females, suggesting that this hormone increases in females prior to producing a litter. A significant main effect of sex indicated that overall females have more P than males, F (1, 45) = 11.287, p = .002. A main effect of group, F (4, 45) = 4.909, p = .002, suggests that overall breeders have increased P compared to the 1-week, p = .02, 1-day, p = .024, and subordinate groups, p = .024, but not from the 1-month group, p = .967 (see Fig. 2).
Body weight and age
The animals ranged in age from 12 to 38 months of age (M = 23.10, SD = 7.17). No significant differences in age were found between the groups, F (4, 42) = 1.46, p = .231, nor between the sexes, F (1, 42) = 0.631, p = .431. Further, no significant interaction was found between group and sex, F (4, 42) = 0.161, p = .957.
Body weight significantly differed between the groups, F (4, 41) = 5.31, p < .001, but not between sexes, F (1, 41) = 1.41, p = .316 (see Fig. 3), indicating that the breeders weighed more than the subordinates, p < .001; 1-day, p < .001, and 1-week group, p = .001. The 1-month group also significantly differed in weight from the subordinates, p = .009, and 1-week group, p = .008, but did not significantly differ from the 1-day group, p = .104, nor from the breeders, p = .348.