Evidence for a dopamine intrinsic direct role in the regulation of the ovary reproductive function: in vitro study on rabbit corpora lutea

Abstract

Dopamine (DA) receptor (DR) type 1 (D1R) has been found to be expressed in luteal cells of various species, but the intrinsic role of the DA/DRs system on corpora lutea (CL) function is still unclear. Experiments were devised to characterize the expression of DR types and the presence of DA, as well as the in vitro effects of DA on hormone productions by CL in pseudopregnant rabbits. Immunoreactivity and gene expression for D1R decreased while that for D3R increased in luteal and blood vessel cells from early to late pseudopregnant stages. DA immunopositivity was evidenced only in luteal cells. The DA and D1R agonist increased in vitro release of progesterone and prostaglandin E2 (PGE2) by early CL, whereas the DA and D3R agonist decreased progesterone and increased PGF2α in vitro release by mid- and late CL. These results provide evidence that the DA/DR system exerts a dual modulatory function in the lifespan of CL: the DA/D1R is luteotropic while the DA/D3R is luteolytic. The present data shed new light on the physiological mechanisms regulating luteal activity that might improve our ability to optimize reproductive efficiency in mammal species, including humans.

Figure 1. In vitro effects of increasing concentrations of dopamine, D1R agonist (dihydrexidine hydrochloride), and D3R agonist (7-hydroxy-PIPAT maleate) on progesterone release by pseudopregnant rabbit CL collected at early, mid, and late luteal stages.

Values are the means ± SD of five replicates. Asterisks indicate a significantly different value (P<0.01) versus control (0).

Figure 2. Immunohistochemical demonstration of D1R, D3R, and DA in early, mid, and late CL of pseudopregnant rabbit.

A–C: D1R immunoreactivity in luteal and endothelial (arrows) cells. D–F: D3R immunoreactivity in luteal and endothelial (arrows) cells. G–I: DA immunosignals in luteal cells; endothelial cells (arrows) are immunonegative. Scale bars = 20 µm.

Figure 3. Immunodensitometry of D1R, D3R, and DA in early, mid, and late CL of pseudopregnant rabbit.

Insert: D1R/D3R ratio. Data are expressed in arbitrary units; values are the means ± SD of thirty replicates; np: not present; different letters above the bars indicate significant different values (P<0.01).

Figure 4. Immunohistochemical presence of D1R and D3R in rabbit brain striatum.

D1R (photo A) and D3R (photo B) immunosignals are localized in the cytoplasm of neuronal cells (NC) of the brain striatum. Note the immunonegativity of striosomes (S). Scale bars: photo A, C, E = 200 µm; B, D = 50 µm.

Figure 5. Immunoblot showing D1R and D3R in anterior pituitary (negative control), CL, and brain striatum (positive control) lysates.

Figure 6. Immunohistochemical presence of D2R, D4R, and D3R in rabbit kidney.

Photo A: D2R; photo B: D4R; photo C: D5R. Scale bars: 20 µm.

Figure 7. Expression of D1R and D3R mRNA by early, mid, and late CL of pseudopregnant rabbit.

Right panel: representative agarose gel electrophoresis stained with ethidium bromide to verify matching between expected and obtained PCR products. Left panel: the values, expressed in arbitrary fluorescence units, are means ± SD for 3 animals/group; different letters above the bars indicate significant different values (P<0.01).

Figure 8. Progesterone, PGF2α, and PGE2 in vitro releases by CL of pseudopregnant rabbit.

Early (upper panel), mid (middle panel B), and late (bottom panel) CL incubated with DA (100 nM), D1R agonist (D1Rag, dihydrexidine hydrochloride, 20 nM), D1R antagonist (D1Rant, SCH 23390 hydrochloride, 1 nM), D3R agonist (D3Rag, 7-hydroxy-PIPAT maleate, 2 nM), and D3R antagonist (D3Rant, GR 103691, 1 nM). Values are the means ± SD of ten replicates. Asterisks indicate significant different values (P<0.01) versus control.

Figure 9. Progesterone, PGF2α, and PGE2 in vitro releases by CL of pseudopregnant rabbit.

Early (upper panel), mid (middle panel B), and late (bottom panel) CL incubated with, D1R agonist (D1Rag, dihydrexidine hydrochloride, 20 nM), D3R agonist (D3Rag, 7-hydroxy-PIPAT maleate, 2 nM), EP2 antagonist (EP2ant, AH 6809, 1 µM); EP4 antagonist (EP4ant, AH 23848, 1 µM), and FP antagonist (FPant, AL 8810, 1 µM). Values are the means ± SD of ten replicates. Asterisks indicate significant different values (P<0.01) versus control.