In vitro and in vivo studies on the effect of a mitochondrial fusion promoter on Leydig cell integrity and function

Abstract

Background: The interstitial testicular Leydig cells are responsible for the production of testosterone, which functionally deteriorate with normal aging. Decreased expression of mitochondrial steroidogenic interactome proteins and diminished mitochondrial function in aging Leydig cells suggest that mitochondrial dynamics play a role in maintaining adequate levels of testosterone. Optic atrophy 1 (OPA1) protein regulates mitochondrial dynamics and cristae formation in many cell types. Previous studies showed that increasing OPA1 expression in dysfunctional Leydig cells restored mitochondrial function and recovered androgen production to levels found in healthy Leydig cells. These findings suggested that mitochondrial dynamics may be a promising target to ameliorate diminished testosterone levels in aging males. Methods: We used twelve-month-old rats to explore the relationship between mitochondrial dynamics and Leydig cell function. Isolated Leydig cells from aged rats were treated ex vivo with the cell-permeable mitochondrial fusion promoter 4-Chloro-2-(1-(2-(2,4,6-trichlorophenyl)hydrazono)ethyl) phenol (mitochondrial fusion promoter M1), which enhances mitochondrial tubular network formation. In parallel, rats were treated with 2 mg/kg/day M1 for 6 weeks before Leydig cells were isolated. Results: Ex vivo M1-treated cells showed enhanced mitochondrial tubular network formation by transmission electron microscopy, enhanced Leydig cell mitochondrial integrity, improved mitochondrial function, and higher testosterone biosynthesis compared to controls. However, in vivo treatment of aged rats with M1 not only failed to re-establish testosterone levels to that of young rats, it also led to further reduction of testosterone levels and increased apoptosis, suggesting M1 toxicity in the testis. The in vivo M1 toxicity seemed to be tissue-specific, however. Conclusion: Promoting mitochondrial fusion may be one approach to enhancing cell health and wellbeing with aging, but more investigations are warranted. Our findings suggest that fusion promoters could potentially enhance the productivity of aged Leydig cells when carefully regulated.

Keywords: aging; bioenergetics; hypogonadism; mitochondrial fusion; steroidogenesis; testosterone; toxicity.

FIGURE 1

Characterization of MACS-isolated Leydig cells. (A) Time course of testosterone formation levels of MACS-isolated cells under basal and hormone-stimulated (hCG) conditions. (B) Gating strategy and flow cytometry analysis for PRLR + cells. (C) Staining for 3β-hydroxysteroid dehydrogenase enzymatic activity. Data are presented as mean ± SEM. *p < .05, **p. < .01, and ***p < .001 by ANOVA.

FIGURE 2

Promoting mitochondrial fusion in Leydig cells isolated from aged rats enhances bioenergetics. (A) Oxygen consumption, ATP production, and steroid hormone production of MACS-isolated Leydig cells from M1-treated and control (basal) aged rats. (B) TEM imaging of MACS-isolated Leydig cells from treated and untreated aged rats highlighting mitochondrial morphology. (C) Characteristics of mitochondrial health, mitochondrial biogenesis and mitochondrial size for mitochondria from MACS-isolated Leydig cells from M1-treated and control (wild-type) aged rats. Data are presented as mean ± SEM (n = 4). *p < .05, **p. < .01, and ***p < .001 by Student’s t-test. Scale bar, 500 nm. TEM, transmission electron microscopy.

FIGURE 3

Administration of M1 leads to decreases in weight and testosterone levels in aged rats. (A) Rat weight and serum testosterone levels with M1 treatment over the experimental time course. (B) Representative immunoblots and comparative protein expression levels in Leydig cells from M1-treated and control aged rats. Data are presented as mean ± SEM. *p < .05, **p. < .01, and ***p < .001 by Student’s t-test.

FIGURE 4

Leydig cell mitochondrial integrity is compromised in M1-treated aged rats. (A) TEM imaging of mitochondria from untreated basal and isolated primary Leydig cells from M1-treated aged rats. (B) Mitochondrial biogenesis and size of Leydig cell mitochondria. Data are presented as mean ± SEM. *p < .05, **p. < .01, and ***p < .001 by Student’s t-test. Scale bar, 1 μm. TEM, transmission electron microscopy.

FIGURE 5

Effect of M1 treatment varies with tissue type. Representative immunoblot and protein expression values in adrenal (A), liver (B), and heart (C) samples from M1-treated aged rats. Data are presented as mean ± SEM. *p < .05, **p. < .01, and ***p < .001 by Student’s t-test.

FIGURE 6

GastroPlus and ADMET Predictor modeling of the physicochemical, metabolism, and pharmacokinetic properties of M1. (A) Predicted enzymatic properties of M1 with glucuronosyltransferases and cytochrome P450 enzymes. (B) Molecular structure of M1 and its glucuronidated metabolite and its predicted exposure to tissues of interest over the course of the study. (C) Predicted plasma concentrations of M1 after injection over 6 weeks.