Variations in the response of pituitary lactotrophs to oxytocin during the rat estrous cycle

Abstract

Although removal of dopamine inhibition is established as a major factor in prolactin (PRL) release, a large body of evidence suggests that hypothalamic oxytocin (OT) may serve as a PRL-releasing hormone in the rat. PRL release is modulated by estradiol (E2), which rises between diestrus and proestrus of the estrous cycle, causing a PRL surge in the afternoon of proestrus. Given that E2 strongly modulates OT actions in both central and peripheral tissues, OT action on lactotrophs might also be modulated by the stage of the estrous cycle. To test this hypothesis, we have monitored PRL release and intracellular calcium levels ([Ca(2+)](i)) induced by OT in pituitary lactotrophs obtained from female rats in either diestrus 1 or proestrus. We found that both secretory and [Ca(2+)](i) responses to OT are significantly increased in lactotrophs obtained on proestrus. Moreover, we show that these differences are due to an increase in both the number of OT-responding lactotrophs and the magnitude of their individual [Ca(2+)](i) responses. Both secretory and [Ca(2+)](i) responses were abolished by a specific OT antagonist. Finally, dose-dependent studies show that the increased PRL-releasing effect of OT on proestrus is significant over a wide range of concentrations, particularly those observed in hypophyseal portal plasma. These results suggest that the rising E2 titers that culminate on proestrus facilitate the stimulatory action of OT on lactotrophs and support the notion that OT is a PRL-releasing hormone with an important role in the production of the proestrous surge of PRL.

Figure 1

Comparison of the responses of lactotroph populations to OT at diestrus 1 and proestrus. A, OT application (100 nm for 10 min, horizontal bar) evokes a modest PRL increase in diestrus 1. Samples were collected every minute. B, OT application evokes a stronger PRL increase in proestrus. C, Box plots of the size of the PRL release in diestrus 1 (n = 7 experiments) and proestrus (n = 10). PRL release was normalized relative to the basal level and summed over 6 min (AUC). There is a significant difference between diestrus 1 and proestrus (*, P < 0.01). D, OT application (100 nm for 2 min, horizontal bar) evokes a modest [Ca²⁺]_i increase on diestrus 1. Ratios were collected every 2 sec. E, OT application evokes a stronger [Ca²⁺]_i increase on proestrus. F, Box plots of the size of the [Ca²⁺]_i increase in diestrus 1 (n = 9) and proestrus (n = 9). Ratios were averaged over all TRH-responsive cells in a given experiment. The mean increase relative to baseline, over the first 6 min of this averaged response, was then computed and expressed as a percentage. Box plots show the median (middle bar), interquartile range (box), and range (whiskers). The difference between diestrus 1 and proestrus is significant (*, P < 0.0005).

Figure 2

Specificity of the response to OT. A, In control conditions (upper panel), both OT (1 μm) and TRH (100 nm) evoke an increase in the rate of PRL release. In the presence of the OTA (lower panel), there is no increase in PRL release after OT application (significantly different from the control response to OT, P < 0.03; n = 4). Samples were collected every minute. B, In control conditions (upper panel), both OT (100 nm) and TRH (100 nm) evoke an increase in intracellular free calcium ([Ca²⁺]_i), measured as the fura-2 ratio. In the presence of OTA (lower panel), the calcium response to OT is abolished (average over 39 cells, response is significantly lower than in control, P < 0.0004). Cells were obtained from proestrus animals.

Figure 3

Comparison of the intracellular calcium responses of individual cells at diestrus 1 and proestrus. A, Fraction of lactotrophs (i.e. TRH-responding cells) responding to OT (100 nm) in lactotroph-enriched preparations. There is a significant difference between diestrus 1 and proestrus (*, P < 0.0005). B, Fractions of TRH-responding cells in lactotroph-enriched preparations. There is no significant difference between diestrus 1 and proestrus (P > 0.85). C, Response amplitude to OT (100 nm) relative to baseline, computed over the first 6 min for each individual OT-responding lactotroph and then averaged over the cells. The difference between diestrus 1 and proestrus is significant (*, P < 0.015). D, The response amplitude to TRH (100 nm) shows no significant difference between diestrus 1 and proestrus (P > 0.13). All panels were obtained from the same data set (diestrus 1, n = 9; proestrus, n = 9 experiments).

Figure 4

Dose-response curves of the PRL-releasing effect of OT in perifused anterior pituitary cells from female rats during diestrus 1 (○) and proestrus (•). Results are shown as normalized AUC means ± sem from at least six independent experiments, except for 10⁻² and 10⁻³ m OT (n = 4), which were pooled together to obtain the saturation level. *, Statistically significant differences (P < 0.05) between the two experimental groups for the same concentration of agonist. P values were as follows: OT 10⁻⁹ m, 0.012; 10⁻⁸ m, <0.0001; 10⁻⁷ m, 0.021; 10⁻⁶ m, 0.0031; 10⁻⁵ m, 0.027; 10⁻⁴ m, 0.022. Continuous lines represent the best fit curve for each experimental group. Regression curves were calculated and EC₅₀ values determined as described in Materials and Methods: for diestrus 1, EC₅₀ 4.24 μm (95% CI = 2.79–6.44 μm), for proestrus, EC₅₀ 0.57 μm (95% CI = 0.16–2.03 μm). Mean basal values were 38.8 ± 1.3 and 51.1 ± 1.8 ng/min for diestrus 1 and proestrus, respectively (P < 0.0001).