A novel role for progesterone and progesterone receptor membrane component 1 in regulating spindle microtubule stability during rat and human ovarian cell mitosis

Abstract

The present studies were designed to assess the roles of progesterone (P4) and Progesterone Receptor Membrane Component 1 (PGRMC1) in regulating mitosis of spontaneously immortalized granulosa cells (SIGCs) and ovarian cancer cells, SKOV-3 cells. Because PGRMC1 has been detected among the proteins of the human mitotic spindle, we theorized that P4 and PGRMC1 could affect mitosis through a microtubule-dependent process. The present study confirms that SIGC growth is slowed by either P4 treatment or transfection of a PGRMC1 antibody. In both cases, slower cell proliferation was accompanied by an increased percentage of mitotic cells, which is consistent with a P4-induced prolongation of the M phase of the cell cycle. In addition, P4 increased the stability of the spindle microtubules, as assessed by the rate of beta-tubulin disassembly in response to cooling. Also, P4 increased spindle microtubule stability of SKOV-3 cells. This effect was mimicked by the depletion of PGRMC1 in these cells. Importantly, P4 did not increase the stability of the microtubules over that observed in PGRMC1-depleted SKOV-3 cells. Immunofluorescent analysis revealed that PGRMC1 is distributed to the spindle apparatus as well as to the centrosomes at metaphase. Further in situ proximity ligation assay revealed that PGRMC1 interacted with beta-tubulin. Taken together, these results suggest that P4 inhibits mitosis of ovarian cells by increasing the stability of the mitotic spindle. Moreover, P4's actions appear to be dependent on PGRMC1's function within the mitotic spindle.

FIG. 1.

The effect of P4 treatment on the fold increase in cell number (A) and the percentage of mitotic figures (B) in SIGCs after 24 and 48 h of culture. The fold increase in cell number and the mitotic index were assessed as outlined in Materials and Methods. Values are means ± SEM. *Significantly different value from the appropriate control value (P < 0.05).

FIG. 2.

Immunodetection of PGRMC1 in SIGCs by Western blot analysis using the rabbit polyclonal anti-PGRMC1 antibody (A); values in kDa. The effect of the presence of the anti-PGRMC1 antibody or IgG on the fold increase in cell number (B) and the percentage of mitotic figures (C) in SIGCs after 21 h of culture. The fold increase in cell number and the percentage of mitotic figures were assessed as outlined in Materials and Methods. Values are means ± SEM. *Significantly different value from the appropriate control value (P < 0.05). Pro, prophase; Meta, metaphase; Ana, anaphase; Telo, telophase; Cyto, cytokinesis.

FIG. 3.

Validation of the assay to assess the process of spindle microtubule disassembly and reassembly in response to cold treatment and rewarming. A) Representative fluorescent images of β-tubulin-immunostained metaphase SIGCs at Time 0, and after 15–60 min of incubation on ice or 7 min of rewarming. B) The mean ± SEM total FI of the β-tubulin staining in the spindle is shown as an indicator of the quantity of polymerized tubulin in each spindle. The mean FI of the β-tubulin staining calculated in the Time 0 group was set as 100%, and the mean FI values for the other time points were expressed as a percentage of the Time 0 value. C) Effect of P4 treatment on the process of spindle microtubules disassembly after 3 and 7 min of cooling. Values are means ± SEMs. *Significantly different value from the Time 0 control value (P < 0.05). **Significantly less than the Time 0 control value but greater than all other groups (P < 0.05).

FIG. 4.

Immunodetection of PGRMC1 expression by Western blot analysis in dsRed SKOV-3 parental and PGRMC1-depleted dsRed SKOV-3 cells. A) GAPDH Western blot is shown to demonstrate equal protein loading. B) The effect of PGRMC1 depletion in dsRed SKOV-3 cells on the process of disassembly of the spindle microtubules after 7 min of incubation on ice. C) The effect of P4 treatment on spindle microtubule disassembly after 7 min of incubation on ice in the parental and PGRMC1-depleted dsRed SKOV-3 cells. Values are means ± SEMs. *Significantly different value from the appropriate control value (P < 0.05).

FIG. 5.

A) Representative images showing coimmunostaining of PGRMC1 (red) and β-tubulin (β-Tub) or γ-tubulin (γ-Tub; green) at metaphase in SKOV-3 cells. B) Representative images showing coimmunostaining of PGRMC1 and β-tubulin at anaphase/telophase and cytokinesis in SKOV-3 cells. DNA is counterstained with Hoechst 33342 (blue). The PGRMC1 and β-tubulin or PGRMC1 and γ-tubulin merged images are shown in the left panels. In B (lower row), the inset represents a 3× magnification of the midbody. Bar = 10 μm.

FIG. 6.

Representative images showing immunodetection of PGRMC1 expression by immunofluorescence analysis in parental and PGRMC1-depleted dsRed SKOV-3 cells. Bar = 10 μm.

FIG. 7.

Representative images showing the interaction between α-tubulin (α-Tub) and β-tubulin (β-Tub; A) and PGRMC1 and either β-tubulin or γ-tubulin (γ-Tub; B) in mitotic SKOV-3 cells, as revealed by in situ PLA. The presence of red fluorescent dots in the PLA assay indicates that two proteins are in close proximity (i.e., interacting). DNA is counterstained with 4′,6′-diamidino-2-phenylindole (DAPI; blue). In A, the lowest row is an example of a negative control obtained by omitting the anti-α-tubulin antibody. As a comparison of a standard immunofluorescent staining and the PLA assay, in A (top row) is shown the immunofluorescent staining of α-tubulin and β-tubulin (red and green, respectively). Bar = 10 μm.