Progesterone decreases tyrosine hydroxylase phosphorylation state and increases protein phosphatase 2A activity in the stalk-median eminence on proestrous afternoon

Abstract

The progesterone (P(4)) rise on proestrous afternoon is associated with dephosphorylation of tyrosine hydroxylase (TH) and reduced TH activity in the stalk-median eminence (SME), which contributes to the proestrous prolactin surge in rats. In the present study, we investigated the time course for P(4) effect on TH activity and phosphorylation state, as well as cAMP levels and protein phosphatase 2A (PP2A) activity and quantity, in the SME on proestrous morning and afternoon. P(4) (7.5 mg/kg, s.c.) treatment on proestrous afternoon decreased TH activity and TH phosphorylation state at Ser-31 and Ser-40 within 1 h, whereas morning administration of P(4) had no 1 h effect on TH. PP2A activity in the SME was enhanced after P(4) treatment for 1 h on proestrous afternoon without a change in PP2A catalytic subunit quantity, whereas P(4) treatment had no effect on PP2A activity or quantity on proestrous morning. cAMP levels in the SME were unchanged with 1 h P(4) treatment. At 5 h after P(4) treatment, TH activity and phosphorylation state declined coincident with an increase in plasma prolactin in both P(4)-treated morning and afternoon groups. PP2A activity in the SME was unchanged in 5 h P(4)-treated rat. Our data suggest that P(4) action on tuberoinfundibular dopaminergic (TIDA) neurons involves at least two components. A more rapid (1 h) P(4) effect engaged only on proestrous afternoon likely involves the activation of PP2A. The longer P(4) action on TIDA neurons is evident on both the morning and afternoon of proestrus and may involve a common, as yet unidentified, mechanism.

FIG. 1

Plasma prolactin levels in control, sham-operated, and ovariectomized rats treated with oil vehicle (1 ml/kg, sc) or P₄ (7.5 mg/kg, sc) on proestrus. Ovariectomy was performed at 1200h–1230h, and P₄ was injected at 1700h on proestrous afternoon. Sham-operation (n=7) did not influence prolactin secretion as compared with that of the control (n=6). P₄ administration (n=9) in the ovariectomized rats prolonged prolactin secretion at 3–5h later as compared to that of oil treatment (n=7). Each value represents a mean ± SEM. ^* p<0.05, ^*** p<0.001 vs. corresponding oil-treated ovariectomized rats.

FIG. 2

Plasma P₄ (A, C) and prolactin (B, D) levels at 0h, 1h, 3h and 5h after morning (A, B) or afternoon (C, D) administration of P₄ (7.5 mg/kg, sc) or sesame oil vehicle (1 ml/kg, sc). For the afternoon treatment groups (C, D), rats were ovariectomized at 1200h–1400h on proestrus. P₄ administered at 0930h (n=10) or 1700h (n=8) on proestrus increased plasma P₄ to 80 ng/ml within 1h and P₄ levels remained high for at least 5h. Oil treatment (n=6 for each group) did not affect the plasma P₄ level. After P₄ treatment at 0930h (B), circulating prolactin levels were unchanged at 1h and 3h after P₄ treatment, but increased 12.2-fold within 5h. After P₄ treatment at 1700h (D), circulating prolactin levels were increased by 2.5-fold at 3h and 6.6-fold at 5h after P₄ administration, compared to oil-treated control. Each value represents a mean ± SEM. ^*** p<0.001 vs. corresponding oil-treated control. ^# p<0.05 vs. oil treatment for 0h.

FIG. 3

TH activity in the SME of rats at 1h, 3h and 5h after P₄ or oil treatment at 0930h (A) or 1700h (B) on proestrus. TH activity in the SME was unaltered at 1h (n=8) after morning P₄ treatment, but was decreased by 16% at 3h (n=8) and 51% at 5h (n=9). TH activity was increased in the 3h (n=10) and 5h (n=9) oil-treated groups compared to the 1h control (n=8). Rats were ovariectomized at 1200h–1400h on proestrus for the afternoon P₄ treatment. P₄ administration at 1700h decreased TH activity by 17% at 1h (n=12) and by 32% 5h (n=10) later as compared to the corresponding 1h control (n=14 and n=10, respectively). TH activity was reduced in the oil-treated group at 2000h in the 3h group, but there was no significant difference between oil- and P₄-treated groups at 3h (n=11 each). Each value represents a mean ± SEM. ^* p<0.05, ^*** p<0.001 vs. corresponding oil-treated control. ^# p<0.05 vs. oil treatment for 1h.

FIG. 4

Phosphorylation state of TH at Ser-19, Ser-31 and Ser-40 within 1h (A–D) or 5h (E–H) after P₄ or oil treatment on proestrous morning. A representative immunoblot for each phosphorylation site of TH or TH protein is displayed on top of the bar graph. Similar to TH activity data, there were no differences in the phosphorylation state of TH at Ser-19, Ser-31 and Ser-40 within 1h after P₄ treatment at 0930h on proestrus. However, TH phosphorylation at Ser-19, Ser-31 and Ser-40 decreased by 14%, 26% and 31% at 5h later, respectively. Phospho-TH values were individually normalized to respective TH values. TH values were individually normalized to respective β-tubulin values. Each value represents a mean ± SEM of determinations of seven to eight rats. ^* p<0.05, ^*** p<0.001 vs. oil treatment.

FIG. 5

Phosphorylation state of TH at Ser-19, Ser-31 and Ser-40 at 1h (A–D) or 5h (E–H) after P₄- or oil- treatment on proestrous afternoon. A representative immunoblot for each phosphorylation site of TH, TH protein or β-tubulin is displayed on top of the bar graph. In contrast to morning administration, P₄ treatment at 1700h significantly decreased TH phosphorylation at Ser-31 by 19–21% and Ser-40 by 29–32% at 5h and 1h, respectively. There were no significant differences between the P₄- and oil-treated groups for Ser-19 phosphorylation, TH protein and β-tubulin levels. Phospho-TH values were individually normalized to respective TH values. TH values were individually normalized to respective β-tubulin values. Each value represents a mean ± SEM of determinations from seven (oil-treated) to eight (P₄-treated) rats. ^* p<0.05, ^** p<0.01 vs. oil treatment.

FIG. 6

PP2A activity in the SME of rats regulated by P₄ treatment for 1h (A, B) or 5h (C and D) on proestrous morning (A, C) and afternoon (B, D). P₄ administration at 0930h did not alter PP2A activity in the SME of rats at 1h (A, n=9) and 5h (C, n=6) after the treatment on proestrous morning. However, afternoon P₄ treatment significantly increased PP2A activity in the SME by 22% at 1h (B, n=9), but not 5h (D, n=6) later. Data are expressed as the mean percent change relative to oil-treated control ± SEM. ^* p<0.05 vs. oil-treated control.

FIG. 7

Catalytic subunit of PP2A (PP2Ac) level in the SME of rat at 1h after P₄ or oil treatment on proestrous morning and afternoon. A representative immunoblot for SME tissue reacted with PP2Ac and β-tubulin antibodies were displayed on top of the bar graph. PP2Ac values were individually normalized to respective β-tubulin values. Data are expressed as the mean percent change relative to oil- treated control ± SEM of determinations from six rats. No statistically significant difference was observed between the P₄- and oil- treated rats on proestrous morning and afternoon.