Non-canonical progesterone signaling in granulosa cell function

Abstract

It has been known for over 3 decades that progesterone (P4) suppresses follicle growth. It has been assumed that P4 acts directly on granulosa cells of developing follicles to slow their development, as P4 inhibits both mitosis and apoptosis of cultured granulosa cells. However, granulosa cells of developing follicles of mice, rats, monkeys, and humans do not express the A or B isoform of the classic nuclear receptor for P4 (PGR). By contrast, these granulosa cells express other P4 binding proteins, one of which is referred to as PGR membrane component 1 (PGRMC1). PGRMC1 specifically binds P4 with high affinity and mediates P4's anti-mitotic and anti-apoptotic action as evidenced by the lack of these P4-dependent effects in PGRMC1-depleted cells. In addition, mice in which PGRMC1 is conditionally depleted in granulosa cells show diminished follicle development. While the mechanism through which P4 activation of PGRMC1 affects granulosa cell function is not well defined, it appears that PGRMC1 controls granulosa cell function in part by regulating gene expression in T-cell-specific transcription factor/lymphoid enhancer factor-dependent manner. Clinically, altered PGRMC1 expression has been correlated with premature ovarian failure/insufficiency, polycystic ovarian syndrome, and infertility. These collective studies provide strong evidence that PGRMC1 functions as a receptor for P4 in granulosa cells and that altered expression results in compromised reproductive capacity. Ongoing studies seek to define the components of the signal transduction cascade through which P4 activation of PGRMC1 results in the regulation of granulosa cell function.

Figure 1

The expression of PGRMC1 in granulosa cells of antral (A) and preovulatory follicles (B) as assessed by immunohistochemistry. The antral follicle was obtained from an immature rat, while the preovulatory follicle was from an immature rat 48 h after PMSG injection. PGRMC1 was detected as a brown stain. The thecal layers at the base of each image show robust PGRMC1 expression. Data from Peluso et al (Peluso, et al. 2006).

Figure 2

The capacity of partially purified PGRMC1-GFP to bind P4 and R5020 (A) and the effect of PGRMC1 siRNA treatment on Pgrmc1 mRNA levels and specific ³H-P4 binding to SIGCs (B). Data in panel A taken from Peluso et al (Peluso, et al. 2009) and the data shown in panel B are unpublished observations (J Peluso, unpublished observations) that confirm our published data (Peluso, et al. 2008). The effect of progesterone (P4) on serum-induced SIGC mitosis is shown in panel C. Data from panel C from Peluso et al (Peluso 2013).

Figure 3

The inhibitory effect of progesterone (P4) on apoptosis of human granulosa/luteal cells, rat granulosa cells and rat luteal cells from PMSG-hCG primed immature rats 4 days after hCG treatment (A). Note that to detect the effects of P4 in luteal cells, endogenous P4 secretion was blocked with aminoglutethamide. The effect of P4 on the rate of spontaneously immortalized granulosa cells (SIGCs) apoptosis is shown in B. The anti-apoptotic action of P4 is ablated after PGRMC1 siRNA treatment. The * indicates a value that is significantly different from control (p < 0.05). In panel A all values under the bracket marked with an * are different from their respective control values. Panel A from data redrawn from the following publications: (Engmann, et al. 2006, Peluso, et al. 2009, Peluso, et al. 2005). Data for panel B is from Peluso et al (Peluso, et al. 2008).

Figure 4

The total number of antral follicles within the ovaries of controls (+/+), PGRMC1 heterozygous (+/−) and PGRMC1 conditional knockout (−/−) mice. Values are shown as means and SE with n of 6-8/treatment group. * indicates a value is different from control (p < 0.05) as demonstrated by an ANOVA followed by Dunnett’s Multiple Comparison Test. The percentage of atretic antral follicles is shown in the base of each column. The percentage of atretic antral follicles is greater in the ovaries of PGRMC1 +/− mice compared to the percentage in either the controls or PGRMC1 −/− mice as assessed by Fisher Exact/Chi-Square tests (p< 0.05). (Peluso 2013).

Figure 5

The effect of P4 and PGRMC1 on Tcf/Lef promoter in SIGCs as assessed by a luciferase reporter assay (A). In panel B, the ability of P4 to suppress Tcf/Lef activity in the presence or absence of PGRMC1-Flag protein was assessed by a luciferase reporter. In all graphs, * indicates a value that is significantly different than control (p < 0.05), ** indicates a value significantly less that cells transfected with an expression vector encoding PGRMC1-Flag (Peluso, et al. 2012b).

Figure 6

Expression of PGRMC family members in the immature mouse ovary and granulosa cells as assessed by real-time PCR. Note that the level of expression for the PGRMC family member is dramatically different with the lowest expression for PGRMC1, followed by PGRMC2 and then NENF, which has the highest level of expression (J Pru, unpublished observations).