Nicotine Effects and Receptor Expression on Human Spermatozoa: Possible Neuroendocrine Mechanism

Abstract

The aim of this experimental study was to investigate the mechanism by which nicotine (NIC) alters spermatozoa and to evaluate the expression of nicotinic receptors (nAChR) subunits in human spermatozoa. We analyzed 30 healthy normozoospermic men. Spermatozoa were incubated with NIC 100 ng/ml and the nAChR antagonist, hexamethonium (HEX) (0, 100, 1,000, 10,000 ng/ml) for 3 and 24 h. The following sperm parameters evaluated: (a) progressive motility; (b) mitochondrial membrane potential (MMP); (c) chromatin compactness; (d) externalization of phosphatidylserine (PS); (e) late apoptosis; (f) viability; (g) DNA fragmentation; (h) degree of lipid peroxidation (LP) by flow cytometry; (i) nAChR subunits expression by quantitative Real Time PCR and (j) protein expression evaluation by Western blot analysis. HEX fully antagonized the effects of NIC both after 3 and 24 h of incubation with significant improvement (p < 0.05) of sperm progressive motility, MMP, abnormal chromatin compactness, PS externalization, late apoptosis and DNA fragmentation, already at the concentration of HEX 100 ng/ml. The degree of LP increased after incubation with NIC in raw semen but this effect was fully antagonized (p < 0.05) by HEX after 3 and 24 h of incubation. Finally, 8 nAChR subunits mRNA (α1, α3, α4, α6, α7, β2, β4, and δ) were found expressed in all samples examined, but only α7 subunit is translated, making an homomer receptor, in non-smokers subjects. The effects of NIC on sperm function are mediated by interaction with a specific nicotinic receptor. The presence of nAChR subunits suggests the presence of a neuroendocrine mechanism on human spermatozoa.

Substance Abuse and Male Hypogonadism

“…Indeed, four meta-analyses have confirmed that smoking is a risk factor for oligozoospermia [44,[49][50][51]. Testicular damage is probably due to smoke-induced oxidative stress, but also a neuroendocrine mechanism of nicotine has been hypothesized [31]. Conversely, tobacco does not decrease testosterone levels in men; rather, it would seem to increase androgen concentrations with mechanisms not entirely clear [43].…”

“…Harmful cigarette smoke constituents include carbon monoxide, nitrogen oxide, ammonia, heavy metals, various polycyclic aromatic hydrocarbons and aldehydes, such as hydroquinone, catechol, acrolein, crotonaldehyde, and formaldehyde [30]. Recently, even nicotine, the major psychoactive substance in cigarette smoke, has been called into question in the pathogenesis of smokers sperm alterations with a possible neuroendocrine mechanism [31]. Indeed, it has been demonstrated that nicotine and its metabolites are capable to cross the blood-testis barrier [32].…”

“…Nicotine binds to a class of ionotropic acetylcholine receptors, the nicotinic receptors (nAChR), made up of five subunits, whose expression has been demonstrated also on human spermatozoa. Since hexamethonium, the main antagonist for the neuronal nAChR, is able to reverse the detrimental effects of nicotine on non-conventional sperm parameters, including apoptotic signs, a possible neuroendocrine mechanism has been postulated for nicotine-related sperm damage [31]. In semen of non-smokers, only a homomeric nicotinic receptor consisting of five α7 subunits is present; however, it has been demonstrated that cigarette smoking may stimulate the expression of some other subunits, such as α9 subunit, that in pregnant smoker women is involved in the vasoconstriction, disepithelization, and apoptosis of the placenta.…”

Epigenetics of Male Fertility: Effects on Assisted Reproductive Techniques

“…Accordingly, impaired sperm protamination has been related to increased DNA fragmentation [4850]. Furthermore, several factors negatively affecting male fertility and sperm quality have been found to impair sperm chromatin compactness, such as overweight and obesity [51], nicotine [5253], male accessory gland infections [54] and varicocele [55]. Lower chromatin compactness and poor sperm quality have been described in patients with low testicular volume [56].…”

Mitochondrial Membrane Potential Predicts 4-Hour Sperm Motility

“…[ 13 ]; some life styles (cigarette smoking, alcohol abuse, drug addiction, etc.) [ 14 , 15 , 16 ]; environmental pollution [ 17 , 18 ], other endocrine diseases (i.e., diabetes mellitus type 1 and 2; hyper- or hypo-thyroidism) [ 19 , 20 , 21 ] and all the causes that increase oxidative stress [ 22 ]. MMP may therefore be used as an index of sperm quality/function [ 8 , 23 ].…”