Progesterone directly and rapidly inhibits GnRH neuronal activity via progesterone receptor membrane component 1

Abstract

GnRH neurons are essential for reproduction, being an integral component of the hypothalamic-pituitary-gonadal axis. Progesterone (P4), a steroid hormone, modulates reproductive behavior and is associated with rapid changes in GnRH secretion. However, a direct action of P4 on GnRH neurons has not been previously described. Receptors in the progestin/adipoQ receptor family (PAQR), as well as progesterone receptor membrane component 1 (PgRMC1) and its partner serpin peptidase inhibitor, clade E (nexin, plasminogen activator inhibitor type 1) mRNA binding protein 1 (SERBP1), have been shown to mediate rapid progestin actions in various tissues, including the brain. This study shows that PgRMC1 and SERBP1, but not PAQR, are expressed in prenatal GnRH neurons. Expression of PgRMC1 and SERBP1 was verified in adult mouse GnRH neurons. To investigate the effect of P4 on GnRH neuronal activity, calcium imaging was used on primary GnRH neurons maintained in explants. Application of P4 significantly decreased the activity of GnRH neurons, independent of secretion of gamma-aminobutyric acidergic and glutamatergic input, suggesting a direct action of P4 on GnRH neurons. Inhibition was not blocked by RU486, an antagonist of the classic nuclear P4 receptor. Inhibition was also maintained after uncoupling of the inhibitory regulative G protein (G(i/o)), the signal transduction pathway used by PAQR. However, AG-205, a PgRMC1 ligand and inhibitor, blocked the rapid P4-mediated inhibition, and inhibition of protein kinase G, thought to be activated downstream of PgRMC1, also blocked the inhibitory activity of P4. These data show for the first time that P4 can act directly on GnRH neurons through PgRMC1 to inhibit neuronal activity.

Fig. 1.

PgRMC1 and SERBP1 are expressed in GnRH neurons. PCR on cDNA from three single-cell GnRH neurons maintained in nasal explants shows bands of appropriate size for PgRMC1 but not PGR (A). Controls, Adult mouse brain cDNA shows bands for both PgRMC1 and PGR (B). A ×40 bright-field images of mouse uteri sections (C–F) used as a positive control for PgRMC1, SERBP1, and PGR ICC/IF staining. In panels C₁–F₁, the box represents the ×100 field shown in C₂–F₂; 1, Endometrial stroma, 2, endometrial glands, and 3, epithelium of the endometrium. GnRH neurons in explants express PgRMC1 (G₁–G₃) and SERBP1 (H₁–H₃). Scale bars, 25 μm in panels C₁–F₁; 10 μm in panels C₂–F₂; 5 μm in panels G and H. DAPI, 4′,6′-Diamino-2-phenylindole.

Fig. 2.

P4 rapidly inhibits GnRH neurons. Application of 10 nm progesterone to GnRH neurons in explants caused rapid inhibition of calcium oscillations (A). Analysis of the data revealed a significant decrease in PPM after P4 application (B). Population analysis indicated that only 56% of the GnRH cells responded to P4 and in this subpopulation PPM decreased by approximately 50% (C). In panels B and C, N = number of explants and n = number of GnRH neurons. *, Significantly different from previous period.

Fig. 3.

P4-mediated inhibition is direct and does not involve PAQR or PGR. Rapid P4-mediated inhibition of GnRH neuronal activity occurred in the presence of the GABA_A antagonist Bic (A) and AMPA/NMDA antagonists CNQX/AP5 (B). Neither PTX, which uncouples G_i/o from membrane-bound receptors (C), nor RU486, a competitive inhibitor of the nuclear progesterone receptor (D), prevented the rapid P4-mediated inhibition in GnRH neurons. Left panels, Representative trace from a single GnRH neuron in the treatment group. Right panels, Grouped PPM data from the responder population. N and n, Number of explants and number of GnRH neurons, respectively. *, Significantly different from previous period.

Fig. 4.

P4-mediated inhibition is through PgRMC1. The PgRMC1 ligand and inhibitor AG-205 caused a sharp rise in intracellular calcium in the micromolar (A, shaded area). In the nanomolar range (B, boxed area), the effect was attenuated and cells were able to return to normal PPM values. P4 rapidly inhibits GnRH neuronal activity after the application of DMSO vehicle control (C and E), but inhibition is blocked by AG-205 treatment (D and F). C and D, Representative traces from single GnRH neurons. E and F, Grouped PPM data from the responder population (E) and total population (F). N and n, Number of explants and number of GnRH neurons, respectively.

Fig. 5.

P4-mediated inhibition is dependent on PKG activation. The PKG inhibitor RP8 blocked P4-mediated inhibition. Left panel, Calcium oscillation trace in a single representative cell. Right panel, Grouped PPM data from total population. N and n, Number of explants and number of GnRH neurons, respectively.

Fig. 6.

PgRMC1 and SERBP1 are expressed by GnRH cells in adult mouse hypothalamus. GnRH neurons in adult GnRH-GFP mice express PgRMC1 (A) and SERBP1 (B). Scale bars, 5 μm in panels A and B.