Adverse Effects of Nicotine on Human Sperm Nuclear Proteins

Abstract

The effects of smoking on human health have long been documented. However, only a few studies have highlighted the direct effects of nicotine on sperm function. Nicotine, as a chemical compound found in tobacco, has been shown to modulate different aspects of spermatogenesis and sperm functions. Nicotine can lead to a reduction in the number of sperm, their motility and functionality. It can change the molecular expressions involved in sperm function, including genes encoding sperm nuclear proteins. The most important nuclear proteins that play a critical role in sperm function are known as H2B histone family, member W, testis-specific (H2BFWT), transition protein 1 (TNP1), transition protein 2 (TNP2), protamine-1 (PRM1), and protamine-2 (PRM2). These proteins are involved in sperm chromatin condensation, which in turn affects fertilization and embryonic development. Any alteration in the expression of these genes due to nicotine exposure/usage may lead to adverse implications in couples’ fertility and the health of future generations. Since research in this area is still relatively new, it underscores the importance of understanding the potential side effects of environmental factors such as nicotine on reproductive health.

INTRODUCTION

According to estimates, between 10% and 15% of couples globally experience infertility, a condition which is defined as the inability to conceive after a year or more of unprotected intercourse. A male factor is reported to account for 40% of cases of infertility [1,2,3,4]. Nonetheless, it is not always possible to pinpoint unique male causes of infertility, which is why lifestyle issues like smoking and alcohol consumption, as well as environmental factors like obesity and estrogenic pollution, have received a lot of attention [5]. Since smoking is known to have a significant negative impact on individual health, it has been suggested that smoking affects male fertility and, in turn, reproductive outcomes [6]. Smoking tobacco is one of the leading, preventable causes of death worldwide. Tobacco use is also linked to an increased risk of male sexual and reproductive diseases in modern times [7]. The precise mechanisms of damage to the male reproductive system appear to include direct effects on sperm function; however, they are still widely unclear [8]. Approximately four thousand distinct chemicals are produced when tobacco is burnt. Hydrocarbons, ammonia, nitric oxide, carbon monoxide, and other gases are among the substances in the gaseous phase. In the particle phase, other substances such as nicotine exist [9].

Nicotine is known as one of the most harmful and destructive compounds that may be detected in tobacco smoke. It is an alkaloid that is mostly found in plants, with tobacco (Nicotiana tabacum) having the highest concentration of it (10–14 mg). Nicotine is a very hazardous chemical that enters the body through the skin, oral mucosa, and respiratory tract fast [10] and has, in addition to chemical characteristics, also pharmacological activities that adversely affect human health [11]. It not only affects the lungs but also influences other physiological processes, including fertility [12]. According to data from the World Health Organization, around one-third of adults over 15 years old smoke tobacco [13,14]. Typically, people use hookahs, pipes, cigarettes and E-cigarettes to ingest tobacco. Research indicates that nicotine has more destructive effects on men’s and women's health than alcohol or caffeine use [13]. Regretfully, using nicotine compounds has grown rapidly around the world to the point that their use is blamed for the deaths of over 5 million people annually. In the next 20 to 30 years, this may rise to 10 million individuals [7].

Studies have suggested that the presence of nicotine in the body can have negative overall consequences on reproductive health, especially in males since it is more commonly used by them [7,15,16]. Miscarriages, low birth weight babies, and early births are linked to women who are exposed to nicotine [17]. Along with these effects, nicotine also lowers women's fertility and causes irregular menstruation [12] and may also lead to premature ovarian failure, which can cause infertility [18] as well as increased time to pregnancy [19]. In males, nicotine affects the testes where prolonged exposure changes the morphology of spermatogenic cells, reduces sperm production, decreased steroidogenesis and inhibits spermatogenesis resulting in decreased sperm counts, poor sperm motility and increased levels of DNA damage [20,21]. Early ejaculation and a diminished penile erection are additional negative consequences of nicotine intake; however, they vary depending on individual sensitivity or susceptibility [22]. All these negative effects directly contribute to male infertility and provide a stark reminder of the dangers of smoking and the importance of embracing a healthy lifestyle.

Since smoking is so prevalent in society and other nicotine-containing products such as vapes are increasingly consumed particularly by younger people, the aim of this narrative review is to compile the available information to better understand the processes by which nicotine alters sperm quality and sperm DNA/chromatin condensation.

EFFECTS OF NICOTINE ON SPERM QUALITY

A number of reports suggest that nicotine has detrimental effects on sperm quality including sperm DNA integrity, leads to decreased sperm motility, viability, concentration, and abnormalities in the offspring (Fig. 1) [23,24]. Nicotine can also disrupt hormonal pathways involved in male fertility thus affecting both sperm production and functioning of the male reproductive system as a whole [16,25]. Exposure to nicotine (in vivo) can cause disturbances in spermatogenesis leading to a reduction in sperm count with subsequent male infertility [26,27,28,29]. Mostafa et al (2018) [29] reported that heavy cigarette smokers have significantly lower sperm concentration compared to healthy non-smoking men. Similarly, Qutub et al (2022) [30] found that smoking reduces the number of sperm in infertile men in Saudi Arabia. Since nicotine may produce a number of reactive oxygen species, one theory contends that decrease in sperm count is caused by nicotine-mediated oxidative stress [31].

Fig. 1. Nicotine exposure’s adverse effect on sperm parameters. The side effects of nicotine on both sperm motility and sperm concentration is represented on the left and right side of the figure, respectively. nAChR: neuronal nicotinic acetylcholine receptor.

One other possible explanation might be gene expression change in sperm by nicotine exposure (in vitro and in vivo). For instance, "Neuronal nicotinic acetylcholine receptors" (nAChRs) are a class of ionotropic acetylcholine receptors that are bound by nicotine. Eight nAChR subunits (α1, α3, α4, α6, α7, β2, β4, and δ) present in the human body, although only α7 is detected in non-smoking human spermatozoa [32]. Consumption of nicotine products can alter the levels of the δ-subunit of nAChR as well as cell signaling of that receptors in placenta tissue [33]. The presence of receptors for nicotine on sperm [34] suggests it may disrupt processes within sperm, potentially leading to a decrease in sperm count, although this mechanism is not yet fully proven.

Sperm motility is also significantly reduced when exposed to nicotine [35]. Damage to the structure of the sperm flagellum after nicotine exposure is one of the factors contributing to reduced sperm motility [29,36,37,38]. Oyeyipo et al [12] demonstrated that exposure of rats (in vivo) to 0.5 mg/kg body weight nicotine for 30 days significantly altered sperm morphology in isolated sperm retrieved from the caudal epididymis. Deformed curved and rudimentary tails were the most noticeable abnormalities observed in the sperm [12]. Typically these abnormalities take place during sperm storage, maturation, and epididymal transit, when spermatozoa acquire motility [12,39].

The activation of different signaling pathways due to exposure to nicotine may be another factor in reducing sperm motility because several animal studies demonstrated that nicotine exposure (in vivo) increases the levels of proteins primarily implicated in the mitogen-activated protein kinase (MAPK) signaling pathway [40,41]. Hormonal pathway disruption is another potential mechanism that decreases sperm motility. Furthermore, nicotine is not only significantly decreasing the testicular activity of androgenic enzymes including testicular steroidogenic acute regulatory (StAR) protein resulting in decreased intratesticular and plasma testosterone and gonadotropin concentrations, but also reduced the testicular levels of glutathione, activities of antioxidative enzymes and mitochondrial membrane potential in testicular cells. This lead to significantly increased oxidative stress with elevated lipid peroxidation and increased plasma concentrations of corticosterone [42].

Since several in vitro studies have demonstrated how nicotine affects extracellular matrix disintegration, F-actin structure, and actin overexpression, it has long been considered that nicotine may disrupt the function of the cellular skeleton. This suggests that disruption of the sperm tail skeleton caused by the use of nicotine products might possibly be the cause of impaired sperm motility [43,44]. Contrary, another study has demonstrated that nicotine-containing products might boost sperm motility by demethylating the promoter of the pfn1 gene, which controls the actin skeleton [45]. However, this phenomenon might also be caused by other substances found in tobacco [46]. Nicotine exposure also lowers the levels of two important enzymes of the Krebs cycle, cytosolic malate dehydrogenase and aconitase 2 suggesting that the testicular energy metabolism is slowing down, which subsequently lowers the energy needed for sperm motility [47,48]. Taking together, this data underscores the significance of tackling nicotine addiction in men attempting to conceive. Nevertheless, there is a need for further investigations into the development of strategic approaches for male fertility.

SPERM NUCLEAR PROTEIN

Spermatogenesis is an epigenetically controlled process and any interruption at any phase might be the cause of male infertility. Proper packaging of chromatin, which is crucial for regulating genomes and gene expression, is one of the regulation mechanisms of spermatogenesis [49]. Sperm nuclear protein (SNP) genes regulate the production of proteins that are necessary for sperm cell development and function. Controlling chromatin structure, DNA replication, and repair in developing spermatozoa are functions of these genes, which are strongly expressed in the testes [50]. Together, histones and protamines make up SNPs, and each has a distinct function in the tight packing of the sperm cell [51].

Proper DNA segregation depends on a process known as "protamination" which occurs as the sperm genome progresses throughout spermatogenesis and results in the replacement of protamines with histones. In this process, the histones that are characteristic of somatic cells are replaced by protamines in a stepwise process where transition nuclear proteins (TNP) are intermediary proteins. As a result, of this exchange of histones by the alkaline protamines, the sperm DNA is much more densely packed than that in somatic cells [52]. However, in human sperm, around 15% of the DNA is still bound by histones in locations unique to certain sequences, suggesting a particular program of gene expression following fertilization [53,54]. Various mechanisms are involved in the regulation of protamination (Table 1) [55,56,57,58,59,60,61,62,63,64,65] and any disruption may lead to male infertility [66].

Table 1. Molecular factors involved in the transfer of histone to protamine in the process of sperm maturation.

Molecular factor	Role	Reference
BRD4 (Bromodomain-containing protein 4)	Protein with a bromodomain that interprets histone acetylation	[55]
BRDT (Bromodomain testis-specific protein)	Protein with a bromodomain that interprets histone acetylation	[56]
CDYL (Chromodomain Y Like)	Protein with a chromodomain that interprets histone methylation	[57]
DOT1L (DOT1-like, histone H3K79 methyltransferase)	Histone methyltransferase	[58]
EPC1 (Enhancer of polycomb homolog 1)	part of the histone acetyltransferase (HAT) complex NuA4	[59]
HDAC3 (Histone deacetylase 3)	Histone deacetylase	[60]
P300 ( Histone acetyltransferase p300)	Histone acetyl-transferase	[61]
PA200 (Proteasome activator PA200)	A proteasome component that selectively recognizes acetylated histones and promotes the breakdown of core histones	[62]
PHF7 (PHD Finger Protein 7)	Ubiquitin ligase	[63]
SirT1 ( NAD-dependent deacetylase sirtuin-1)	Histone deacetylase	[64]
SRPK1 (Serine/arginine-Rich Splicing Factor protein kinase-1)	Serine/threonine kinase	[65]

During the early stages of spermatogenesis, histones play a central role in DNA packaging. So far, two specific histones have been characterized in the human testis, including H2B histone family member W testis-specific (H2BFWT) and human testis/sperm-specific histone H2B (hTSH2B) which has been immunolocalized in 20%–30% of mature sperm [67]. Although the temporal accumulation of histone variants during spermatogenesis indicates their potential involvement in meiosis, spermiogenesis and fertilization, their function, however, is still mostly unknown [68]. The human Xq22.2 chromosome is home to the H2BFWT gene, which codes for the H2BFWT protein and has two introns [69]. Although its exact role is unknown, H2BFWT is known to be a histone variant whose main function is to help the transition histone–protamine pathway, which epigenetically regulates gene transcription. Its relationship with telomeres also implies that H2BFWT may have a role in fertilization during early chromatin remodeling [67].

Histones are replaced with TNPs, which have a strong affinity to DNA and are rich in arginine and lysine. While mammals are known to have at least four TNPs, TNP1 and TNP2 are the most abundant TNPs in human [70,71,72]. Protamines eventually take the place of TNPs [70,73]. TNP1 is a basic protein that is around 2.5-times larger than TNP2 [74]. TNP1 is probably able to destabilize DNA due to the presence of two tyrosine residues surrounded by basic amino acids. The possible roles of TNP1, revealed by in vitro investigations, include relaxation of the DNA in nucleosome core particles, decreasing the melting temperature of DNA, and stimulation of the DNA-relaxing activity of topoisomerase I [73,75,76,77,78]. TNP1 is expressed in the testes during spermatogenesis and undergoes post-translational modifications such as acetylation and phosphorylation [73]. TNP2 has a basic C-terminal domain and two zinc fingers for CpG island recognition [79]. It increases DNA compaction in nucleosome cores, resulting in higher melting temperatures, suggested to be a DNA-condensing protein [80].

In the final stages of spermatogenesis, protamines replace TNPs and allow mature spermatozoa to have a very stable and compact nucleus [70,81]. Protamines pack the paternal genome in a linear, not supercoiled array form instead of a supercoiled solenoid form like the histones [82,83,84], which function as a protective shell for chromatin against denaturation and are the most prevalent SNPs in many animals. Protamines contain numerous positively charged amino acids, especially arginine, which makes up 48% of human protamines [81,85]. Protamines occur in two forms: protamine-1 (PRM1), found in all mammalian species, and protamine-2 (PRM2), only identified in a few species including humans, horses, mice, and hamsters. During spermatogenesis, PRM1 and PRM2 attach to the sperm DNA and replace TNPs, thereby compacting the genome [86,87]. In a multigenic cluster on chromosome 16 (16p13.3) the PRM1 and PRM2 genes are situated next to TNP2, which suggests that they arose by gene duplication and might have retained common functions. Repetitive alanine elements found at methylation sites are found in the matrix attachment regions (MARs), which encircle this cluster. These MARs are critical, regardless of methylation status, for the proper control of protamine gene expression [81].

POSSIBLE MECHANISM OF NICOTINE ON SPERM NUCLEAR PROTEINS

The effects of nicotine on sperm include chromosomal abnormalities, aneuploidy, single- or double-strand breaks, and DNA cross-linking [88]. Inadequate repair of sperm DNA damage might lead to major mutations which can be inherited from one generation to the next and can take many different forms, ranging from structural alterations to single nucleotide variations and insertions/deletions (indels) [89]. Research has demonstrated that nicotine alters sperm DNA in a way that is detrimental and may cause male infertility. SNPs are one of the possible genes that nicotine can damage [88,90]. The mechanisms through which nicotine affects SNPs have been a subject of extensive research. The findings of several studies suggest that nicotine activates certain molecular pathways leading to oxidative stress, DNA damage, and abnormal hypermethylation of sperm DNA (Fig. 2) [7,16,25]. Another mechanism indicates that nicotine is causing DNA methylation [91]. Methylation is the mechanism by which gene expression is modulated and genes are silenced. Hypermethylation in turn can activate genes and facilitate chromatin connection to the nuclear matrix [92].

Fig. 2. Nicotine exposure’s impact on sperm nuclear proteins. Effects of nicotine exposure on protamines, transition proteins, and histones. H2BFWT: H2B histone family member W testis-specific, TNP1: transition protein 1, TNP2: transition protein 2, PRM1: protamine-1, PRM2: protamine-2, CRE: cAMP response element, MAR: matrix attachment region.

Transcription process disruption is another way by which nicotine modifies SNPs [93]. Because it makes it easier for transcription factors to bind to the promoter, the TATA-box found in SNPs genes is crucial for the commencement of transcription. Additionally, interaction between cAMP response elements (CREs) and many CRE proteins controls transcription [81]. Protamine transcripts are preserved after transcription as ribonucleoproteins, which are subsequently translated in elongated spermatids after being translationally inhibited [94]. Narcotic adhesion to nicotine cholinergic receptors has been observed to increase intracellular calcium and deactivate the CRE [95]. This transcription factor is the primary activator of several genes, including H2BFWT, TNP1, TNP2, PRM1, and PRM2 [95]. There is a need for further investigating the effects of nicotine on the functioning of SNPs in men attempting to conceive and more investigation into the influence of environmental factors on male fertility.

NICOTINE INFLUENCES DIFFERENT SPERM NUCLEAR PROTEINS

1. Histones

The impact of nicotine on H2BFWT and its functions has not yet been comprehensively investigated. However, some research that has been conducted indicates that exposure to nicotine alters the gene expression of H2BFWT (Table 2) [89,96]. Amor et al [96] showed that sperm from heavy smokers (in vivo) had a down-regulated copy of the H2BFWT gene as compared to the non-smokers who had not. The authors suggested that changes in the expression of H2BFWT may affect the structure and function of sperm cells, leading abnormalities in the sperm parameters and male infertility. Although Amor et al [89] demonstrated that the consumption of nicotine products changed sperm parameters and sperm DNA integrity, a connection between sperm parameters and the genetic alteration in the H2BFWT gene could not be demonstrated.

Table 2. Nicotine’s impact on sperm nuclear protein.

Protein	Nicotine's effect	Possible outcome	Reference
Histones (H2BFWT)	Reduced gene expression	May disrupt sperm structure and function, leading to abnormalities	[85]
	Lower transcript levels in hookah users	May be linked to decreased sperm motility	[86]
Transition Proteins (TNP1 & TNP2)	Significantly down-regulates expression	May impair sperm motility due to increased calcium levels	[85]
Protamines (PRM1 & PRM2)	Disrupts protamine packaging process	Alters the ratio of PRM1 to PRM2, potentially impacting fertility	[36,57,90,91,92]
	Decreases PRM2 levels	May contribute to structural changes in sperm DNA or oxidative stress damage	[92]
	Higher H2B to total nuclear protein ratio in infertile smokers	Suggests potential issues with sperm DNA packaging	[57]
	Increased PRM1/PRM2 ratio in heavy smokers	May be linked to abnormalities in histone-to-protamine transfer	[90]
	Reduced PRM1 & PRM2 mRNA levels in smokers	May contribute to sperm quality issues	[95]
	Hookah smoking also reduces PRM1 & PRM2 mRNA levels	May negatively impact sperm health through similar mechanisms	[86,94]

H2BFWT: H2B histone family member W testis-specific, PRM1: protamine-1, PRM2: protamine-2, TNP1: transition protein 1, TNP2: transition protein 2.

One of the most recent studies investigating the effects of nicotine on SNPs in human sperm showed significantly lower levels of H2BFWT transcripts in sperm samples of hookah users (in vivo) as compared to non-users [97]. This study also found a negative correlation between sperm mRNA levels of H2BFWT and non-progression/total motility while the transcript was positively correlate with non-motile sperm [97]. Further studies are required to completely understand the influence of nicotine on the H2BFWT alternation related to male fertility.

2. Transition proteins

TNPs and their involvement in sperm cell health have been subject in several studies [98,99]. To the best of our knowledge, the only study that investigated how nicotine affects TNPs demonstrates that exposure to nicotine (in vivo) dramatically down-regulates the expression of TNP1 and TNP2 (Table 2) [96]. This downregulation of TNPs with the resulting malfunction of CRE is due to increased intracellular calcium. Findings show that mutations in the recognition site of the circular CRE transcription factor in the promoter region of TNPs can alter these gene functions and expression [95,100].

3. Protamines

Unlike histones and transition proteins, the effects of nicotine on protamines have been extensively studied (Table 2). The findings of several in vivo studies indicate that the consumption of nicotine products is associated with abnormalities in the protamination process as well as changes in the mRNA expression of PRM1 and PRM2 in human spermatozoa [36,101,102]. Nicotine alters the ratio of PRM1/PRM2 which impacts male fertility [68,101,103]. Nicotine exposure had a detrimental effect on PRM2 levels as the PRM1/PRM2 ratio was significantly higher among people who use items containing nicotine and has been attributed to structural modifications in their capacity to attach DNA, or oxidative stress-induced sperm DNA damage [103]. Sperm of infertile smokers had a significantly greater ratio of H2B to the total nuclear protein (H2B+PRM1+PRM2) than in men not exposed to nicotine [68].

Another report shows a significantly higher PRM1/PRM2 ratio in heavy smokers that was closely linked to anomalies in histone-to-protamine transfer and changed protamine mRNA expression in human sperm [101]. These results indicate a negative relationship between male fertility and nicotine use and that the PRM1/PRM2 transcript ratio may be a helpful indicator of male infertility.

Following the discovery that nicotine alters the expression of the PRM1 and PRM2 genes, further studies were conducted to investigate whether various nicotine products, such as smoking and hookah, had a different impact on the expression of these protamines [86,96,97,104,105,106]. A study examining the relationship between protamine mRNA expression in smokers and semen quality revealed that the levels of PRM1 and PRM2 mRNA in smokers were considerably lower than those in non-smokers [104]. In two distinct investigations conducted in 2021 to examine the impact of heavy smoking on protamine transcripts in human sperm, Laqqan and Yassin [106], and Amor et al [96] found that sperm from heavy smokers had significantly lower levels of PRM1 and PRM2 gene expression than non-smokers. However, Laqqan and Yassin [106] found a distinct and positive correlation between the transcription levels of PRM1 and PRM2 and sperm chromatin condensation status and DNA fragmentation, and are consistent with the negative correlation between the ratio of PRM1/PRM2 and sperm parameters reported by others [101,107]. In contrast, Amor et al [89,96] was unable to detect any link between the changes in the transcription levels of PRM1 and PRM2 and sperm parameters.

The impact of hookah on the transcription of protamines in human sperm was examined by Laqqan and Yassin [97] Tofighi Niaki et al [105]. These authors found that hookah smoking significantly reduces the transcription levels of PRM1 and PRM2 genes; however, no correlation was found between the changes in protamine expression and sperm parameters [97,105] and can be explained by the fact that nicotine causes protamine dysfunction due to mutations and polymorphisms in MARs that may change the ratio and level of protamine [81].

FUTURE RESEARCH DIRECTIONS

Based on the current state of research, several future research directions can be proposed to further elucidate the relationship between nicotine and sperm nuclear protein genes.

1. Epigenetic effects

Investigating the long-term epigenetic effects of nicotine exposure on SNP genes, including DNA methylation patterns and histone modifications, to understand the transgenerational impact of nicotine on male fertility.

2. Molecular mechanisms

Elucidating the molecular mechanisms underlying the effects of nicotine on SNP genes, including the signaling pathways involved in nicotine-induced alterations in gene expression and chromatin structure. Some of the proposed molecular mechanisms that could be useful for future research include are as follows.

1) Alternative splicing and sperm nuclear protein genes

Barbazuk et al [108] discussed the importance of alternative splicing (AS) in eukaryotic genes, emphasizing its relevance in generating protein isoforms. Given the critical role of nuclear protein genes in sperm function, further exploration of AS in these genes could provide valuable insights into their regulation and function. Investigating the impact of nicotine compounds on AS patterns of SNPs genes may uncover novel mechanisms underlying male infertility.

2) Transcriptional co-repressors and male infertility

Blevins et al [109] highlighted the role of transcriptional co-repressors in oncogenesis and the potential for expanding the understanding of these proteins in the context of male infertility. Considering the intricate regulatory networks involved in SNP gene expression, exploring the involvement of co-repressors in modulating the effects of nicotine compounds on gene transcription could offer new avenues for research in male infertility.

3. Therapeutic interventions

Exploring potential therapeutic interventions to mitigate the adverse effects of nicotine on SNP genes, such as the use of natural compounds like thymoquinone or resveratrol, which have shown promise in animal models to counteract nicotine-induced reproductive damage [110,111]. Further research, including human trials, is warranted to explore the clinical efficacy and safety of these interventions.

4. Biomarkers of nicotine exposure

Identifying specific biomarkers of nicotine exposure in sperm, which could serve as indicators of male reproductive health and potential targets for intervention.

5. Social and developmental factors

Investigating the interplay between social and developmental factors, such as social functioning and context, in relation to nicotine use and its impact on SNP genes.

6. Evolutionary perspectives

Exploring the evolutionary origins and coevolution of essential sperm-egg binding partners, including the impact of nicotine exposure on the evolution of reproductive tract proteins in different species.

7. New smoking technologies

The effects of E-cigarettes on sperm health, is a developing area of research. Future studies could pinpoint how SNPs impacted by E-cigarettes. This would provide a clearer understanding of how E-cigarettes disrupt sperm function.

CONCLUSIONS

Nicotine consumption in any form (smoking, hooking, vaping etc.) has a detrimental impact on sperm motility, count, and its production. Any defect in the histone-to-protamine transition and any alternation in the protamine expression in human sperm are closely linked to the consumption of nicotine products. It should be advised that men who use various nicotine products be made aware of the harmful effects of those products on fertility, sperm quality, and its potential negative effect on the offspring via epigenetic modifications. The specific molecular mechanism of nicotine that mediates this action must be determined by further research.