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.
Highlights
Many evidences attribute to cigarette smoking a negative effect on the reproductive health of both genders, but the mechanisms are not entirely clear
Many compounds derived from cigarette combustion may be responsible for the negative impact that cigarette smoke has on sperm parameters
We have shown that cigarette smoke extract reduces, in a dose- and time-dependent manner, sperm motility and the percentage of spermatozoa with normal mitochondrial function, whereas it increases the percentage of spermatozoa with abnormal chromatin and fragmented DNA (Calogero et al, 2009)
Summary
Many evidences attribute to cigarette smoking a negative effect on the reproductive health of both genders, but the mechanisms are not entirely clear. It has been shown that cigarette smoke is able to alter sperm density, motility, morphology and seminal fluid leukocyte concentration. Effects of the cigarette smoke on sperm DNA integrity, aneuploidy rate, production of free oxygen radicals (ROS) have been evaluated, but the results of these studies appear conflicting: some have shown a negative effect We have shown that cigarette smoke extract reduces, in a dose- and time-dependent manner, sperm motility and the percentage of spermatozoa with normal mitochondrial function, whereas it increases the percentage of spermatozoa with abnormal chromatin and fragmented DNA (Calogero et al, 2009). The effects of nicotine (NIC), an alkaloid present in the tobacco plant and the main constituent of cigarette smoke, on sperm parameters, have been studied, showing that it alters sperm parameters (Pacifici et al, 1995; Reddy et al, 1995; Gandini et al, 1997; Wong et al, 2000; Arabi and Shareghi, 2005)
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