Lead toxicity and the hypothalamic-pituitary-testicular axis

Moderate exposure to lead has been tied to dose-dependent changes in male endocrine functions, as indicated by the effects of lead on the hypothalamic-pituitary-testicular (HPT) axis (Ng et al., Reference Ng, Goh, Ng, Ong, Ong, Chia, Chia and Jeyaratnam1991). Studies suggest that the hypothalamus is the primary site of the neurotoxic action of lead (Sokol, Reference Sokol1987). A clinical study on boys undergoing sexual development showed that marginally increased BLL may delay puberty and ultimately limit final testicular volume (Staessen et al., Reference Staessen, Nawrot, Hond, Thijs, Fagard, Hoppenbrouwers, Koppen, Nelen, Schoeters, Vanderschueren, Van Hecke, Verschaeve, Vlietinck and Roels2001). The effect is potentially due to impaired gonadotropin-releasing hormone (GnRH) mRNA expression at low levels of long-term lead exposure (Sokol et al., Reference Sokol, Wang, Wan, Stanczyk, Gentzschein and Chapin2002). Lead may also disturb hypothalamic GnRH release by arresting prostaglandin E₂ synthesis or norepinephrine-stimulated release of prostaglandin E₂ (Sokol et al., Reference Sokol, Wang, Wan, Stanczyk, Gentzschein and Chapin2002). A study looking at moderate occupational exposure to lead found that serum LH decreased with increasing duration of lead exposure (Ng et al., Reference Ng, Goh, Ng, Ong, Ong, Chia, Chia and Jeyaratnam1991), which would ultimately impact testosterone production (Alexander et al., Reference Alexander, Checkoway, Faustman, van Netten, Muller and Ewers1998) in Leydig cells. Lead may act at the hypothalamic−pituitary unit by interfering with the calcium-dependent secondary messenger systems that regulate LH release from secretory granule storage (Ronis et al., Reference Ronis, Badger, Shema, Roberson and Shaikh1996); this disrupts the hormonal feedback loop and serve as an explanation for an increase in stored β-LH (causing vacuolization of gonadotropic cells). Increased LH mRNA and stored LH may also be caused by the interaction between lead and the metal-dependent testosterone receptor at the pituitary unit. At the testicular level, lead impairs testicular LH receptor concentrations and steroidogenesis at the pubertal stage (Ronis et al., Reference Ronis, Badger, Shema, Roberson and Shaikh1996). Another animal study with lead exposure during sexual development showed degenerative changes in gonadotropic cells of the pituitary gland, thus accounting for suppressed plasma LH and testosterone levels into adulthood (Ait Hamadouche et al., Reference Ait Hamadouche, Nesrine and Abdelkeder2013).

Decreased follicle-stimulating hormone (FSH) levels have also been observed in moderately exposed lead workers (Gustafson et al., Reference Gustafson, Hedner, Schutz and Skerfving1989), potentially affecting spermatogenesis. In contrast, increased (Ng et al., Reference Ng, Goh, Ng, Ong, Ong, Chia, Chia and Jeyaratnam1991) or unchanged (Assennato et al., Reference Assennato, Paci, Baser, Molinini, Candela, Altamura and Giorgino1987) FSH levels have also been reported. This disparity in reporting could be attributed to differing concentrations of lead, differing duration of exposure, and the physiological state of the reproductive axis and the testes. Metabolic function of Sertoli cells may also be impacted by lead exposure during pubertal development (Nathan et al., Reference Nathan, Huang, Pogach, Giglio, Bogden and Seebode1992), thus impairing spermatogenesis. Sertoli cell dysfunction from high lead exposure have been associated with inhibin B overproduction (Mahmoud et al., Reference Mahmoud, Kiss, Vanhoorne, De Bacquer and Comhaire2005), which may account for the decreased FSH levels previously discussed. In contrast, a lead exposure study in the primate model also reported a decreased inhibin/FSH ratio as a cause of Sertoli cell dysfunction. However most evidence, such as the lack of change in Sertoli cell morphology upon lead exposure (Nathan et al., Reference Nathan, Huang, Pogach, Giglio, Bogden and Seebode1992), points to reproductive axis dysfunction rather than Sertoli cell dysfunction in adult cases of lead poisoning.

Along with testosterone suppression, rat studies have shown increased epididymal androgen binding protein levels (Nathan et al., Reference Nathan, Huang, Pogach, Giglio, Bogden and Seebode1992). In vivo lead exposure studies on prepubertal rats have depicted decreased activity of 3β-hydroxysteroid dehydrogenase, an enzyme necessary for testosterone synthesis, as well as reduced gonadotropin-receptor binding and associated cyclic AMP production (Wiebe et al., Reference Wiebe, Salhanick and Myers1983). Impaired feedback response to plasma testosterone (Vigeh et al., Reference Vigeh, Smith and Hsu2011), pituitary LH dysregulation (Sokol et al., Reference Sokol, Madding and Swerdloff1985), and Leydig cell degeneration (Saxena et al., Reference Saxena, Hussain, Lal and Chandra1986) may also contribute to testosterone imbalance.

Many of the toxic effects we have presently discussed may be ascribed to the lead-induced generation of reactive oxygen species (ROS) (e.g. hydrogen peroxides and superoxides), which causes oxidative stress. ROS halts synthesis of sulfhydryl antioxidants and prevents enzymatic reactions (Vigeh et al., Reference Vigeh, Smith and Hsu2011). In fact, lead itself has high affinity to sulfhydryl residues in proteins, thus acting as a nonspecific enzyme inhibitor and arresting their antioxidant activity (Ait Hamadouche et al., Reference Ait Hamadouche, Nesrine and Abdelkeder2013). As oxidative stress disturbs chromatin condensation, it has been suggested that the under-protamination of sperm DNA could automatically lead to DNA fragmentation (Henkel et al., Reference Henkel, Bastiaan, Schuller, Hoppe, Starker and Menkveld2010). A study performed on Swiss mice found that lead-induced ROS was associated with a significantly reduced sperm count and increased sperm abnormalities. Intraperitoneal injections of lead acetate, used to induce lead poisoning in the mice, led to an increase in lipid peroxidation potential, an indicator of oxidative stress, in the testes of the study mice as compared with the control group. According to the study, the significant decrease in sperm count may be attributed to lead-induced ROS damage on the polyunsaturated fatty acids of cell membranes of germ cells, and subsequently the spermatozoa and mature sperms (Mishra & Acharya, Reference Mishra and Acharya2004). A computer-assisted semen analysis demonstrated that excessive ROS in semen damaged sperm concentration, motility, and other sperm motion parameters (Takeshima et al., Reference Takeshima, Yumura, Yasuda, Sanjo, Kuroda, Yamanaka and Iwasaki2016).

Brain catalase activity was found to be significantly decreased in a rat study looking at lead-induced HPT dysfunction; this will ultimately permit oxidative damage in cells (Ait Hamadouche et al., Reference Ait Hamadouche, Nesrine and Abdelkeder2013). Increased IL-7, IL-10, IL-12, and TNF-α cytokine levels have been found to accompany lead-related oxidative stress, particularly in semen (Kasperczyk et al., Reference Kasperczyk, Dobrakowski, Czuba, Horak and Kasperczyk2015). ROS may account for decreased serum testosterone through several means.

Lead toxicity and spermatogenesis

At various BLLs, men occupationally exposed to lead have exhibited decreased sperm concentration density per unit of seminal fluid, oligospermia, hypospermia, as well as teratozoospermia (e.g. widened, rounded, or shortened) and immature sperm (Vigeh et al., Reference Vigeh, Smith and Hsu2011). To circumvent the ethical dilemma of testing in humans, animal experimentation has demonstrated lead toxicity may account for decreased weight of testes, seminal vesicles, epididymis, and ventral prostate (Ronis et al., Reference Ronis, Badger, Shema, Roberson and Shaikh1996); this may be paralleled with chronic lead exposure affecting accessory gland development beginning in childhood. Animal studies have further established the effect of lead on aberrant testicular tissue morphology (Ait Hamadouche et al., Reference Ait Hamadouche, Nesrine and Abdelkeder2013; Saxena et al., Reference Saxena, Lal, Murthy and Chandra1984), testicular germ cell death (Adhikari et al., Reference Adhikari, Sinha, Narayan and Saxena2001), interstitial edema (Ait Hamadouche et al., Reference Ait Hamadouche, Nesrine and Abdelkeder2013), seminiferous tubule degeneration (Ait Hamadouche et al., Reference Ait Hamadouche, Nesrine and Abdelkeder2013; Graca, Ramalho-Santos & de Lourdes Pereira, Reference Graca, Ramalho-Santos and de Lourdes Pereira2004), and abnormal seminal cytology (Cullen, Singh, Dykeman, Rice & Foster, Reference Cullen, Singh, Dykeman, Rice and Foster1993). Developmental exposure has also revealed limitations in the number, size, shape of seminiferous tubules and spermatogonia (Garu et al., Reference Garu, Sharma and Barber2011).

In rats, dose-dependent reductions in the testicular and epididymal enzymatic activity of alkaline phosphatase and sodium-potassium ATPase were found with increased lead dosing (Batra et al., Reference Batra, Nehru and Bansal2001). The same study showed disrupted spermatogenesis with immature sperm deposited in the epididymal tubules, with complete arrest of spermatogenesis (i.e., empty epididymal tubules) at higher levels of lead (Batra et al., Reference Batra, Nehru and Bansal2001). The increased concentration of lead was found to not only vacuolize the cells, but also damage the basement membrane and epithelium of the caput and corpus epididymis (Batra et al., Reference Batra, Nehru and Bansal2001). Increased lipid peroxidation, another consequence of ROS that harms cell function, has been found in the testes and epididymis in rats chronically exposed to lead (Marchlewicz et al., Reference Marchlewicz, Wiszniewska, Gonet, Baranowska-Bosiacka, Safranow, Kolasa, Głabowski, Kurzawa, Jakubowska and Rac2007). A microanalysis study illustrated that though the blood−testes barrier generally protects the seminiferous epithelium from lead toxicity, the blood−epididymis barrier does not (Marchlewicz, Reference Marchlewicz1994). This study also identified lead accumulation in smooth myocytes, epithelial cells and in the lumen of epididymal duct, as indicated by diminished epididymal spermatozoa (Marchlewicz, Reference Marchlewicz1994).

Lead toxicity and sperm function

It is generally affirmed that a BLL of ≥40 µg/dl is associated with declined sperm quality and fecundity (Oliveira et al., Reference Oliveira, Spano, Santos and Pereira Mde2009). As mentioned before, the lack of sperm maturity may counteract sperm function. Asthenozoospermia and delayed semen liquefaction time have been found to compromise sperm quality in men with occupational lead exposure (Naha et al., Reference Naha, Bhar, Mukherjee and Chowdhury2005). Lead toxicity has also been associated with delayed semen melting, further negatively impacting quality (Xuezhi et al., Reference Xuezhi, Youxin and Yilan1992).

In addition to impaired sperm count and morphology, decreased sperm function is another potential consequence of lead toxicity (Hsu et al., Reference Hsu, Liu, Hsu, Chen and Guo1997; Taha et al., Reference Taha, Sayed, Ghandour, Mahran, Saleh, Amin and Shamloul2013). Generation of ROS may increase lipid peroxidation in sperm plasma membranes, causing declined fluidity essential for motility, integrity, and viability (Kasperczyk et al., Reference Kasperczyk, Kasperczyk, Horak, Ostalowska, Grucka-Mamczar, Romuk, Olejek and Birkner2008; Oliveira et al., Reference Oliveira, Spano, Santos and Pereira Mde2009). Lipid peroxides are capable of inducing DNA damage and reducing fertility during semen storage. The sperm plasma membranes are rich in polyunsaturated fatty acids containing double bonds vulnerable to free radical attack and initiation of the lipid peroxidation cascade, which is characterized by an autocatalytic, self-propagating reaction, giving rise to cell dysfunction accompanied by the loss of membrane functions and integrity. Decreased membrane functionality hinders binding with homologous and heterologous zona pellucida (i.e. sperm−oocyte fusion) and the ability to undergo acrosomal exocytose (Bansal & Bilaspuri, Reference Bansal and Bilaspuri2010). ROS may also reduce phosphorylation of sperm axonemes to further weaken motility (de Lamirande & Gagnon, Reference de Lamirande and Gagnon1992). ROS impact on motility may also be ascribed to ATP deficiency from the lack of axoneme protein phosphorylation, as glyceraldehyde-3-phosphate dehydrogenase in the fibrous sheath become limited to produce sufficient energy for motility through the glycolytic pathway (Armstrong et al., Reference Armstrong, Rajasekaran, Chamulitrat, Gatti, Hellstrom and Sikka1999; Gomes et al., Reference Gomes, Goncalves, Rocha, Sa, Alves, Silva, Barros, Pereira and Sousa2015). Even insignificant oxidative stress for broad periods could compromise the condensation state of sperm DNA, an effect that would not be observable before fertilization (de Lamirande et al., Reference de Lamirande, Jiang, Zini, Kodama and Gagnon1997; Gavriliouk & Aitken, Reference Gavriliouk and Aitken2015). In terms of direct effects on motility, studies show that lead impairs axonemal microtubule sliding and displaces the role of calcium in binding to calmodulin for tail protein tyrosine phosphorylation (Oliveira et al., Reference Oliveira, Spano, Santos and Pereira Mde2009). An in vitro study found an increase in flagellum abnormalities, with an increased presence of coiled tails (Gomes et al., Reference Gomes, Goncalves, Rocha, Sa, Alves, Silva, Barros, Pereira and Sousa2015). Lead may interact with the sulfhydryl groups on the proteins of the outer dense fibres and fibrous sheath, which are cytoskeletal components of the flagellum, causing its detachment from the plasma membrane (Gomes et al., Reference Gomes, Goncalves, Rocha, Sa, Alves, Silva, Barros, Pereira and Sousa2015). Finally, while still poorly understood, sperm apoptosis is the most fatal consequence of oxidative stress (Aitken et al., Reference Aitken, Baker and Nixon2015).

Previous studies indicate Cys₂His₂ zinc finger protein transcription factors as targets in lead-poisoning disease, in which low concentrations of lead are shown to replace zinc in the binding to closely spaced thiol groups within the zinc finger domains of proteins. This in turn irreversibly inhibits the binding of zinc to the zinc finger domain, causing a conformational change of the domain and a loss of specific DNA binding. Accumulation of lead ions has been shown to occur within cellular nuclei. Given the prevalence of Cys₂His₂ zinc finger factors near the amino terminus of proteins in eukaryotes, and their nucleic acid binding potential, it is suggested zinc finger protein transcription factors play a role in gene expression and regulation, in signal transduction, cell growth and differentiation, and chromosome structure (Hanas et al., 1999).

Inhibition of such regulatory proteins could be a potential cause for the disorders observed in spermatogenesis, under lead-poisoning conditions. A separate study exploring the role of Cys₂His₂ zinc finger protein transcription factors in spermatogenesis suggests they play an essential role during the meiosis prophase of the germ cells, possibly acting as regulatory factors during progression of meiosis in spermatogenesis (Ishizuka et al., Reference Ishizuka, Ohtsuka, Inoue, Odaka, Ohshima, Tamura, Yoshida, Sako, Baba, Kashiwabara, Okabe, Noguchi and Hagiwara2016). When testing the effects of varied lead doses on rat testis, it was found that with increasing dose, there was an accumulation of lead in the testis and epididymis, along with a respective decrease in the activities of enzymes alkaline phosphate and Na⁺/K⁺-ATPase, both known to play a role in spermiogenesis and spermatogenesis. At higher doses of lead, histological changes such as accumulation of immature cells in the lumen of seminiferous tubules, complete arrest of spermatogenesis, and damage to basement membrane of epididymis were observed (Batra et al., Reference Batra, Nehru and Bansal2001; Rafique et al., Reference Rafique, Khan, Perveen and Naqvi2009). However, with a concomitant administration of zinc, lower levels of lead accumulation occurred, accompanied with a significant improvement of enzyme activities, and prevention of histological damage to testis and epididymis. Therefore, it is thought that the significant competition of zinc and lead can help zinc serve a protective role during the event of lead poisoning (Batra et al., Reference Batra, Nehru and Bansal2001; Rafique et al., Reference Rafique, Khan, Perveen and Naqvi2009). Studies looking at oxidative stimulation and sperm injury in assisted reproductive technology have shown that zinc could inhibit hydrogen peroxide-induced impairment of sperm motility, vitality, membrane integrity, and DNA damage (Wu et al., Reference Wu, Wu, Xie, Wang, Wu, Cai, Luo, Huang and You2015). Zinc and lead measurements in the semen and serum of human males suffering from infertility for almost a year, suggest an optimal level of zinc serum (80 to <90 μg/dl) for best sperm parameters and which lowest values of lead in semen (Fatima et al., Reference Fatima, Hossain, Rahman, Rahman, Mugni, Sumon, Hossain and Hossain2015).

Lead appears to also play a role in displacing zinc from its binding sites on zinc-binding proteins and zinc-dependent proteins as indicated by an increase in free seminal zinc. As lead binds tightly to zinc outside of the sperm cells, zinc is prevented from entering the cell and playing its role in the sperm chromatin condensation−decondensation process (Hernandez-Ochoa et al., Reference Hernandez-Ochoa, Garcia-Vargas, Lopez-Carrillo, Rubio-Andrade, Moran-Martinez, Cebrian and Quintanilla-Vega2005). Another supporting hypothesis for unstable sperm chromatin structure is that lead binds to cysteine residues of protamines, thus preventing protamine−DNA binding complex (Hernandez-Ochoa et al., Reference Hernandez-Ochoa, Garcia-Vargas, Lopez-Carrillo, Rubio-Andrade, Moran-Martinez, Cebrian and Quintanilla-Vega2005). During the maturation stage at the epididymis, lead appears to increase chromatin condensation by interacting with adjacent sulfhydryl moieties during the formation of disulphide bonds (Johansson & Pellicciari, Reference Johansson and Pellicciari1988). Sperm DNA fragmentation, reportedly occurring at BLL of ≥45 µg/dl (Bonde et al., Reference Bonde, Joffe, Apostoli, Dale, Kiss, Spano, Caruso, Giwercman, Bisanti, Porru, Vanhoorne, Comhaire and Zschiesche2002), as well as elevated seminal ROS (Taha et al., Reference Taha, Sayed, Ghandour, Mahran, Saleh, Amin and Shamloul2013) attest to the reduced viability of sperm.

Aside from sperm motility, which is critical for sperm to manoeuver both male and female reproductive tracts pre- and post-ejaculation, increased seminal plasma lead has been shown to also impair the fertility potential of sperm in in vitro fertilization (Benoff et al., Reference Benoff, Centola, Millan, Napolitano, Marmar and Hurley2003; He et al., Reference He, Zou, Chen, Weng, Luo and Zeng2016). Lead causes premature acrosome breakdown of sperm. The pathogenesis of acrosome breakdown is attributed to lead infiltrating potassium channels of mature spermatozoa; susceptibility to potassium channel poisoning depends on ion channel polymorphisms which confer specific sensitivities to lead (Benoff et al., Reference Benoff, Jacob and Hurley2000). In vitro studies have shown that hydrogen peroxide, a ROS present from lead toxicity, may also initiate premature acrosome reaction (Hsu et al., Reference Hsu, Hsu and Guo1999). It should be noted that while excessive ROS is detrimental, normophysiologic ROS levels are important in the full maturation of spermatozoa, e.g., during capacitation, continuous production of ROS during sperm maturation is vital for the intracellular pathways of phosphorylation to remain functional (de Lamirande et al., Reference de Lamirande, Jiang, Zini, Kodama and Gagnon1997; Du Plessis et al., Reference Du Plessis, Agarwal, Halabi and Tvrda2015). Another in vitro study found that lead dose-dependently decreased the intracellular cAMP levels (explicated by potential lead/ROS-mediated breakdown of adenylate cyclase, which synthesizes cAMP (He et al., Reference He, Zou, Chen, Weng, Luo and Zeng2016), calcium levels, and progesterone-induced intracellular calcium increases, leading to decreased sperm protein tyrosine phosphorylation. This result explains reduced sperm motility, inhibited capacitation, and the inhibited progesterone-induced acrosome reaction (He et al., Reference He, Zou, Chen, Weng, Luo and Zeng2016). The latter appears to occur due to lead inhibition of CatSper channels (He et al., Reference He, Zou, Chen, Weng, Luo and Zeng2016). Premature acrosome breakdown reduces the capability of sperm to express sufficient mannose-binding lectins. This action impairs the mannose-stimulated acrosome reaction necessary for sperm penetration into the zona pellucida and corona radiata of the ovum (Benoff et al., Reference Benoff, Centola, Millan, Napolitano, Marmar and Hurley2003). Lead-induced ROS generation leading to impaired capacitation of sperm may also compromise penetrative ability (Hsu et al., Reference Hsu, Hsu, Liu, Chen and Guo1998). Subsequent capacitation, spermatozoa utilize chemotaxis to migrate through the lower female reproductive tract against a concentration gradient of progesterone, a sperm chemoattractant, secreted by oocytic cumulus cells. High levels of ROS produce a state of oxidative stress leading to a lower percentage of chemotactic spermatozoa (Sanchez et al., Reference Sanchez, Sepulveda, Risopatron, Villegas and Giojalas2010; Du Plessis et al., Reference Du Plessis, Agarwal, Halabi and Tvrda2015). The privation of ROS scavenging may also initiate spontaneous sperm hyperactivation, impairing sperm transport in the lower female reproductive tract or leading to premature capacitation (de Lamirande et al., Reference de Lamirande, Jiang, Zini, Kodama and Gagnon1997; Ichikawa, Oeda et al., Reference Ichikawa, Oeda, Ohmori and Schill1999). Furthermore, lead exposure studies in animals have shown a dose-dependent decrease of sperm adherence to the ova along with compromised DNA, RNA, and protein synthesis under the sperm−zona pellucida binding conditions (Chowdhuri et al., Reference Chowdhuri, Narayan and Saxena2001). Damaged nucleic acids and inhibited DNA repair has been shown to be caused by increased ROS in spermatozoa (Vigeh et al., Reference Vigeh, Smith and Hsu2011), which has been correlated with high lead levels in seminal plasma (Kiziler et al., Reference Kiziler, Aydemir, Onaran, Alici, Ozkara, Gulyasar and Akyolcu2007).

A study with male mice eating lead-exposed chow was associated with an increase in post-implantation embryo losses in utero, leading to below-average litter sizes (al-Hakkak et al., Reference al-Hakkak, Zahid, Ibrahim, al-Jumaily and Bazzaz1988). Similarly in humans, paternal lead exposure has been linked to increased risk of miscarriages in the men's wives (Lindbohm et al., Reference Lindbohm, Sallmen, Anttila, Taskinen and Hemminki1991). Lead has also been shown to affect male fecundity, quantitatively estimated by the amount of time to pregnancy, at higher exposure levels of ≥40 µg/dl (Apostoli et al., Reference Apostoli, Bellini, Porru and Bisanti2000). Despite the presence of epidemiologic studies, the etiologies for miscarriages, embryonic developmental complications, and similar scenarios have not been well studied (Gomes et al., Reference Gomes, Goncalves, Rocha, Sa, Alves, Silva, Barros, Pereira and Sousa2015).

Conflict of interest

The authors have no conflicts of interest to declare.