Aging-Related Increase of cGMP Disrupts Mitochondrial Homeostasis in Leydig Cells

Abstract

Since mitochondria play an essential role in the testosterone biosynthesis, serve as power centers and are a source of oxidative stress, a possible mitochondrial dysfunction could be connected with decreased activity of Leydig cells and lowered testosterone production during aging. Here we chronologically analyzed age-related alterations of mitochondrial function in Leydig cells correlated by the progressive rise of cGMP signaling and with respect to testosterone synthesis. To target cGMP signaling in Leydig cells, acute or long-term in vivo or ex vivo treatments with sildenafil (phosphodiesterase 5 [PDE5] inhibitor) were performed. Aging-related accumulation of cGMP in the Leydig cells is associated with mitochondrial dysfunction illustrated by reduced ATP and steroid production, lowered O2 consumption, increased mitochondrial abundance and mtDNA copies number, decreased expression of genes that regulate mitochondrial biogenesis (Ppargc1a/PGC1a-Tfam-Nrf1/NRF1), mitophagy (Pink1), fusion (Mfn1, Opa1), and increased Nrf2/NRF2. Acute in vivo PDE5 inhibition overaccumulated cGMP and stimulated testosterone but reduced ATP production in Leydig cells from adult, middle-aged, and old rats. The increased ATP/O ratio observed in cells from old compared to adult rats was diminished after stimulation of cGMP signaling. Opposite, long-term PDE5 inhibition decreased cGMP signaling and improved mitochondrial function/dynamics in Leydig cells from old rats. Mitochondrial abundance in Leydig cells decreased while ATP levels increased. Chronic treatment elevated Tfam, Nrf1, Nrf2, Opa1, Mfn1, Drp1, and normalized Pink1 expression. Altogether, long-term PDE5 inhibition prevented age-related NO and cGMP elevation, improved mitochondrial dynamics/function, and testosterone production. The results pointed on cGMP signaling in Leydig cells as a target for pharmacological manipulation of aging-associated changes in mitochondrial function and testosterone production.

Dependence of Leydig Cell’s Mitochondrial Physiology on Luteinizing Hormone Signaling

“…Mitochondrial abundance and mitochondrial membrane potential (ΔΨm) were determined by mitotrack green and tetramethylrhodamine (TMRE), respectively, staining for 20 min/34 °C/5% CO 2 were applied with subsequent fluorescence reading (Fluoroscan, Ascent, FL, USA). The excitation wavelength used for the each test was 485 and 550 nm while emission wavelengths were 510 and 590 nm respectively; after staining, cells were washed with 0.1% BSA-PBS [ 31 , 33 , 36 ]. Oxygen consumption by Leydig cells suspension was measured by Clark electrode at 34 °C and oxygen uptake and oxygraphic curves were obtained by digital multimeter VC820 (Conard Electronic, Hirsau, Bavaria, Germany) and software Digiscope for Windows (version 2.06) [ 31 ].…”

Multi‐ and transgenerational biochemical effects of low‐dose exposure to bisphenol A and 4‐nonylphenol on testicular interstitial (Leydig) cells

“…47,48 Testosterone synthesis occurs in the mitochondria of LCs; mitochondrial damage or dysfunction is an important cause of impaired testosterone secretion. 47,49 Exposure to BPA or NP can cause mitochondrial damage resulting in mitochondrial dysfunction and metabolic dysfunction in LCs. 4,42,50 The biosynthesis of 47 Consistent with previous studies, 4,22,50 we found that BPA, NP, and BPA&NP induced mitochondrial damage and endoplasmic reticulum stress in generations F0+ and F1+.…”

Growing Up Under Constant Light: A Challenge to the Endocrine Function of the Leydig Cells

“…The mtDNA decreases as well as Cytc (encoding subunit of respiratory protein), mitochondrial membranous potential, and ATP production. Since mitochondrial respiration produces around 80% of ATP in adult Leydig cells ( 22 ), decreased mitochondrial function significantly affects energy cell status. Decreased mitochondrial activity observed in Leydig cells from peripubertal and adult rats is supported by decreased Opa1 and Mfn2 with the potential to increase mitophagy due to increased Pink1 suggesting unbalanced mitochondrial dynamics connected with lower steroidogenesis.…”

“…Sample/standard and Luciferase reagent were mixed 1:1, and luminescence was measured by the Biosystems/luminometer (Fluoroscan, Ascent, FL). For DYm detection, as we described before (15,22), Leydig cells were placed in 96 well-plates (1 × 10 5 cells/well) and incubated with tetramethylrhodamine (TMRE) staining for 20 min/34°C/5% CO 2 . Fluorescence was measured on fluorimeter (Fluoroscan, Ascent, FL) on excitation wavelengths 485 and 550 nm, while emission wavelengths were 510 and 590 nm.…”