Mitochondrial metabolism determines the functional status of human sperm and correlates with semen parameters

Abstract

The diagnosis of male infertility is based essentially on the patient’s medical history and a standard semen analysis. However, the latter rarely provides information on the causes of a possible infertility, emphasizing the need to extend the analysis of the sperm function. Mitochondrial function has been associated with sperm function and dysfunction, the latter primarily through the production of excessive amounts of reactive oxygen species (ROS). We hypothesized that analysis of sperm mitochondrial metabolism together with sperm ROS production could be an additional tool to improve routine semen analysis, after appropriate validations. To test our hypothesis, we performed several experiments using a non-routine method (high-resolution respirometry, HRR) to access mitochondrial function. First, we investigated whether mitochondrial function is related to human sperm motility and morphology. When mitochondrial metabolism was challenged, sperm motility decreased significantly. Additionally, morphological abnormalities in the sperm mid-piece and mitochondria were associated with global sperm defects evaluated by routine methods. Subsequently, sperm mitochondrial function was assessed by HRR. Respiratory control ratio (RCR) was determined and evaluated in the context of classical sperm analysis. In parallel, sperm hydrogen peroxide (H2O2) production and seminal plasma (SP) antioxidant capacity were measured. The percentage of sperm with progressive motility correlated positively with RCR, SP antioxidant capacity, and negatively with the concentration of extracellular H2O2 production ([H2O2]). The percentage of normal sperm morphology correlated positively with RCR and negatively with [H2O2]. Sperm morphology did not correlate with seminal plasma antioxidant capacity. Furthermore, Receiver Operating Characteristic curves were used for the first time to test the diagnostic ability of RCR, [H2O2], and SP antioxidant capacity as binary classifiers. An RCR cut off value of 3.2 was established with a sensitivity of 73% and a specificity of 61%, using reference values considered normal or abnormal in routine semen analysis. The cut off value for [H2O2] was 0.2 μM/106 sperm (sensitivity = 65%, specificity = 60%). There were no reference values for SP antioxidant capacity that distinguished between abnormal and normal sperm samples. We conclude that sperm mitochondrial function indices in combination with [H2O2] may be useful tools to complement the routine semen analysis.

Bioenergetic changes in response to sperm capacitation and two-way metabolic compensation in a new murine model

“…Finally, these results may pave the way for future studies dealing with sperm metabolic variations and capacitation in human sperm, since these phenomena also appear to be related in this species [ 82 – 84 ]. However, it must be borne in mind that the high degree of phylogenetic relatedness between M. spicilegus and M. musculus suggests that significant differences in sperm metabolic features may arise quickly through evolutionary time.…”

Mitochondrial function and reactive oxygen species production during human sperm capacitation: Unraveling key players

“…52 In the present work, we demonstrated an increase in RCR and the spare mitochondrial capacity in human sperm, confirming similar behavior in mitochondrial mice and human sperm 6 . Notably, the respiratory control ratio reflects the most crucial points of mitochondrial function, e.g., the respiratory chain, mitochondrial membrane potential, and the amount of oxygen consumed dedicated to the production of ATP, 36,37,39 suggesting that an overall increase in sperm mitochondrial activity occurs during capacitation. In human sperm, a 34% increase in the spare mitochondria capacity was also observed (Table suggesting that during capacitation either recruitment of more active mitochondria occurs or more mitochondrial respiratory chain complexes are activated than in non-capacitated conditions.…”

“…In addition, the differential diffusibility of the reactive species makes them detectable inside the mitochondria, outside the organelle, or even outside the cells. To measure sperm ROS production we used a previously published assay 39,[60][61][62] based on the detection of extracellular H 2 O 2 . The measured H 2 O 2 represents the ROS generated by the sperm, but could not reflect the mitochondrial origin.…”

“…Fluorocitrate titration consisted of three steps adding 1.5 μL of 10 mM stock (final concentrations: 7.5, 15, and 22.5 μM). Aconitase-driven respiration was confirmed by recovered respiration after the addition of 5 mM isocitrate 38 2.8 | Evaluation of hydrogen peroxide production in sperm cells Extracellular H 2 O 2 production of sperm cells was measured by 10-acetyl-3,7-dihydroxyphenoxazine dye (Amplex™Red) in the presence of horseradish peroxidase (HRP) as previously described in Irigoyen et al 39 Previously, NC and CAP cells were centrifuged for 10 min at 500g and resuspended in HAM-F10 medium (126 mM NaCl, 3.8 mM KCl, 1.08 mM NaH 2 PO 4 , 0.6 mM MgSO 4 .7H 2 O, 0.3 mM CaCl 2 , 6 mM glucose, 20 mM HEPES and 6 mM NaHCO, pH 7.4). Briefly, between 2 and 6 × 10 6 spermatozoa were placed in 96-well Nunc F plates in a final volume of 100 μL and exposed to 8 μg/ mL HRP and 50 μM Amplex Red (Invitrogen, Waltham, Massachusetts, USA).…”

Mitochondrial uncouplers impair human sperm motility without altering ATP content

“…Human sperm are vigorously motile cells that require a wellregulated electrochemical potential across both the plasma membrane [1][2][3] and the mitochondrial membrane [4][5][6][7]. However, much is still unknown about the physiological importance of each of these potentials since it has been difficult to disentangle their roles.…”