Evidence of a role for cAMP in mitochondrial regulation in ovarian granulosa cells

Abstract

In the ovary, proliferation and differentiation of granulosa cells (GCs) drive follicular growth. Our immunohistochemical study in a non-human primate, the Rhesus monkey, showed that the mitochondrial activity marker protein cytochrome c oxidase subunit 4 (COX4) increases in GCs in parallel to follicle size, and furthermore, its intracellular localization changes. This suggested that there is mitochondrial biogenesis and trafficking, and implicates the actions of gonadotropins, which regulate follicular growth and ovulation. Human KGN cells, i.e. granulosa tumour cells, were therefore used to study these possibilities. To robustly elevate cAMP, and thereby mimic the actions of gonadotropins, we used forskolin (FSK). FSK increased the cell size and the amount of mitochondrial DNA of KGN cells within 24 h. As revealed by MitoTracker™ experiments and ultrastructural 3D reconstruction, FSK treatment induced the formation of elaborate mitochondrial networks. H89, a protein kinase A (PKA) inhibitor, reduced the network formation. A proteomic analysis indicated that FSK elevated the levels of regulators of the cytoskeleton, among others (data available via ProteomeXchange with identifier PXD032160). The steroidogenic enzyme CYP11A1 (Cytochrome P450 Family 11 Subfamily A Member 1), located in mitochondria, was more than 3-fold increased by FSK, implying that the cAMP/PKA-associated structural changes occur in parallel with the acquisition of steroidogenic competence of mitochondria in KGN cells. In summary, the observations show increases in mitochondria and suggest intracellular trafficking of mitochondria in GCs during follicular growth, and indicate that they may partially be under the control of gonadotropins and cAMP. In line with this, increased cAMP in KGN cells profoundly affected mitochondrial dynamics in a PKA-dependent manner and implicated cytoskeletal changes.

Keywords: KGN; cAMP; granulosa cells; mitochondrial dynamics; ovary.

Figure 1.

Immunohistochemical staining of COX4 in Rhesus monkey ovaries. (A) COX4 is readily detected in oocytes (asterisks), granulosa (GC) and thecal cells (TC) of growing follicles, as well as in interstitial cells (IC). Inset: Corresponding control (IgG instead of primary antibody). (B) COX4 protein is barely seen in flat pre-granulosa cells of primordial follicles (1) but increased staining is seen in granulosa cells of primary (2) and secondary follicles (3). Asterisks mark oocytes. (C) COX4 levels in mural granulosa cells (GC; arrows) of an antral follicle were increased. Asterisks mark oocytes. (D) COX4 in luteinized granulosa cells (GLC) and luteinized thecal cells (TLC). Inset: Corresponding control (primary antibody omitted). Sections were slightly counterstained with haematoxylin.

Figure 2.

Effects of FSK treatment on morphology, number, diameter and mtDNA amount of granulosa tumour (KGN) cells. (A) Representative microscopic images of KGN cells treated with the solvent (ethanol) control (EtOH ctrl., left panel) or 50 µM FSK (right panel) for 24 h. FSK treatment resulted in altered cellular morphology compared to the solvent control. Scale bar 50 µm. (B) FSK treatment did not affect the cell number (n = 3), but significantly increased both the (C) cellular diameter and the (D) mtDNA amount (n = 6 each). Individual values and means ± SEM are given; **P < 0.01; n.s., not significant; FSK, forskolin.

Figure 3.

Mitochondrial morphology in granulosa tumour (KGN) cells and effect of FSK treatment. (A) Representative MitoTracker™ Orange images of KGN mitochondria with fragmented (left panel), intermediate (middle panel) or network-like morphology (right panel). (B) Representative MitoTracker™ Orange images of 24 h solvent control or 50 µM FSK-treated KGN cells (B, left). Percentage distribution of the mitochondrial morphology after the treatments (n = 4 individual experiments, 526 evaluated cells). Means ± SEM are given; *P < 0.05; **P < 0.01; n.s., not significant (B, right). For a detailed statistical evaluation, see Supplementary Table SI. (C) FIB/SEM tomography of KGN cells treated with solvent control or 50 µM FSK for 24 h. Selected cells were re-localized with SEM and longitudinally sectioned with FIB. In the control cell, the mitochondria were rather egg-shaped with large diameter (C, top left). The mitochondria of the FSK-treated cell had a small diameter and were roundish (C, top right). 3D reconstruction of the mitochondria confirmed egg-shaped mitochondria in the control cells (C, lower left). Mitochondria of the FSK-treated KGN cell shown were rather thin and elongated (C, lower right). FSK, forskolin.

Figure 4.

Effect of FSK and H89 individually and in combination on mitochondrial morphology in granulosa tumour (KGN) cells. (A) Representative MitoTracker™ Orange images of untreated (untr.), ethanol solvent (EtOH ctrl.), 50 µM FSK, 30 µM H89 or FSK and H89 (in combination) treated KGN cells. (B) Changes in percentage distribution of mitochondrial morphology after 24 h treatment with 50 µM FSK, 30 µM H89, the combination of both, or in untreated cells (n = 3 experiments, 138 evaluated cells). Means ± SEM are given; *P < 0.05; **P < 0.01; n.s., not significant. For detailed statistical evaluation, see Supplementary Table SI. FSK, forskolin; H89, a protein kinase A (PKA) inhibitor.

Figure 5.

Volcano plot of proteomic analysis of granulosa tumour (KGN) cells treated with FSK or solvent control. Proteomic analysis of KGN cells treated with 50 µM FSK or solvent control for 24 h (n = 3). Volcano plot of significantly decreased (left, 1–5) and significantly increased (right, 6–17) proteins due to FSK treatment. Proteins altered in abundance of FSK-treated KGN cells are listed in Table II. FSK, forskolin.

Figure 6.

Effect of FSK and H89 individually and in combination on mRNA levels in granulosa tumour (KGN) cells. (A, B) 24 h treatment of KGN cells with 50 µM FSK (n = 7) showed no changes in mRNA expression levels of (A) the fusion genes MFN1, MFN2, OPA1 and of the fission gene DNM1L, as well as COX4, but the mRNA levels of (B) the steroidogenic enzymes StAR, CYP19A1 and CYP11A1 were significantly increased. (C) 3 h treatment with 50 µM FSK resulted in significantly increased expression levels of StAR, CYP19A1 and CYP11A1, which could be blocked by 1 h preincubation with 30 µM of the PKA-inhibitor H89 and subsequent treatment with both agents in combination (n = 4). mRNA levels were normalized to solvent treated control cells; individual levels and means ± SEM are given; **P < 0.01: ***P < 0.001; n.s., not significant; FSK, forskolin.