**Abstract**

Endocrine disruptors (EDs) are chemical substances that affect physiological processes in the body via hormonal regulation. They are often detected in food, plastic water bottles, cosmetics, and many other daily need items. Thereafter, EDs are detected in many bodily fluids, pointing out the real exposure to even very low doses. Permanent and long-term utilization of EDs has harmful effects on male reproductive health mainly due to interference with sex hormone synthesis and mechanism of action. However, with decreasing dosage of EDs, the possibilities of unpredictable modes of action arise. In addition to various molecular actions of individual EDs, the interference of individual ones represents another dimension of the ED issue. This review provides an overview of the EDs and their possible impact on reproductive health in males, with focus on sperm quality with the mighty potential of epigenetic transmission to further generations. The "posttranslational" effect of EDs in really low doses in real exposure routes is stigmatized in this review, being strongly considered as creeping molecular action of individual EDs as well as amplifications of their copresence in the environment.

**Keywords:** endocrine disruptor, bisphenol, regretable substitution, posttranslational modification

## **1. Introduction**

Maturation of sperm cells (spermatogenesis) is a continuous process starting in puberty. The process is stimulated by follicle-stimulating hormone (FSH) and luteinizing hormone (LH). Until the onset of puberty, spermatogonia are quiescent and their quantity does not change significantly. After sexual maturity is reached, an expressive activation of mitochondrial activity and the process of spermatogenesis begin, leading to the formation of spermatids. They are then transformed into spermatozoa by the spermiohistogenesis process, when a round spermatid changes into a sperm cell with a tail, middle section, and head. However, whole spermatogenesis, including gonadal ridge colonization and differentiation of primordial germ cells (PGCs), followed by further development, begins during early embryogenesis. In light of this fact, there are several exposure windows when environmental noxi can hit spermatogenesis along the entire process.

Considering the transmission of extraneous agents, the hemotesticular barrier (HTB) represents the morphological division of the seminiferous tubulus into two compartments: basal and adluminal. The barrier is crucial for full functionality of germinal epithelium, as well as for the elimination of negative impacts of environmental pollutants. Physiologically, this strict division ensures free paracellular movement of substances among the compartments, such as water, nutrients, electrolytes, hormones, and paracrine factors. HTB provides protection of the emerging sperm cells from autoimmune damage by antibodies to sperm cells produced if the barrier was impaired, and the immune system would be in contact with spermatozoa during puberty when the body has already reached immunocompetence. Therefore, the cross talk of the immune system and HTB is potentially another sensitive target to a pollutant impact.

There is a basal compartment in close proximity to the basal membrane. This segment contains vessels and nerves, and spermatogenesis is initiated here. Spermatogonia and spermatocytes up to the proleptotene stage are present here. This segment is necessary for nutrition. The spermatogonia are subsequently transported through tight intercellular junctions to the adluminal compartment which is a place of spermatogenesis completion and subsequent metamorphosis of spermatids to spermatozoa. Both compartments are demarcated by the blood-testis barrier (BTB). Vessels and nerves are no longer present in this segment, and the nutrition of the germinal epithelium cells is covered by the Sertoli cells. The impact of various doses and concentrations of EDs on the male reproductive system can affect the functions of this barrier. The differentiation and development of the male reproductive system depends on elementary estrogen/ androgen ratio, and the antagonistic and agonistic effects of EDs often disrupt their balance. The development of testicular tissue is crucial for further development of the entire reproductive system, as the endocrine activity of testicles determines overall masculinization of the body. Any disruption in the development of the testicles can therefore impair the overall masculinization process and sperm production.

Sperm concentration in men decreases worldwide, and spermiogram parameters deteriorate mainly in the Western world population [1]. Among others, huge amounts of endocrine disruptors (EDs) in our environment can cause this state. This final manifestation of the noxious effect of EDs has an unknown background, such as dose, kinds of EDs, interactions, and crosstalk of individual EDs and/or the timing of the exposure. Therefore, biomonitoring data represents significant input for experimental designing, leading to the description of molecular action in simulated conditions. Based on the newest findings, the record of the biological impact of individual EDs is an ongoing research issue leading to indicating the found compounds as endocrine disruptors.

Many cases of impaired sexual development due to the effects of EDs are also known from the animal kingdom. For example, reduction of penile length was observed in crocodiles living in waters contaminated with EDs [2]. EDs can significantly influence not only the process of spermatogenesis but also the development of testicular tissue. It has been documented that increased exposition of pregnant mice to BPA caused alterations of organelles, that is, mitochondria and lysosomes, in Sertoli and Leydig cells, respectively. These alterations led to maturation disorders in spermatocytes and androgen synthesis inhibition [3].

### **2. Spermatogenesis, epigenetics, biochemical status of spermatozoa, and implications for male reproduction**

The creation of the spermatozoon leads to the terminally differentiated cell with an extremely high level of chromatin methylation and silencing. The final shape of the spermatozoon, often species-specific, requires many morphological and biochemical changes, in particular, dynamic remodelation of the chromatin [4].

**39**

*Endocrine Disruptors: Very Low Doses with Genuinely High Impacts on Male Reproduction*

Protamination, histone-protamine exchange in elongating spermatids, represents a drastic, expressible change of sperm chromatin [5]. A tight protamine-derived DNA package protects sperm chromatin against damage and, interestingly, even the ratio of protamines PRM1 (sperm protamine P1) and PRM2 (sperm protamine P2) is decisive about sperm quality [6]. In accordance with the tight chromatin package, DNA is strongly methylated, and, therefore, general chromatin silencing is required for sperm stability [7, 8]. Protamination represents a tool for the protection of paternal gene imprinting [9]. Temporal protamine-packaged sperm DNA undergoes a second exchange of chromatin proteins after fertilization, and then maternal histones are incorporated into the paternal pronucleus. Both protaminehistone transition events, first and second in testicular seminiferous tubuli and fertilized oocyte, respectively, are obviously sensitive to environmental influences

Although most core histone is substituted by protamines, a residual speciesspecific amount of histones resists in the sperm head. In addition to DNA methylome, epigenetic hallmarks of mature spermatozoa include the epigenetic code of residual histones, based on many posttranslational modifications (PTMs) of individual amino acids [12, 13]. These chromatin-repressive histone marks positively correlate with DNA methylome and accompany imprinted genes. Moreover, the sperm histone code shows an exact physiological role in fertilization and early embryonic development [14]. The histone code establishment is highly orchestrated [15] and, therefore,

Following comprehensive demethylation of parental chromatin after fertilization, the total erasure of the methylation pattern, including gene imprinting on paternal and maternal alleles, is needed for the re-establishment of gene imprinting adequate to the paternal pattern in the sperm cell. This erasure comes early after gonadal ridge colonization, and primordial germ cells (PGCs) occur, at human embryonic days E32 and E10.5 in mice [16]. The recurrent "writing" of the epigenetic pattern into imprinted loci occurs in the late prenatal period when the spermatogonia are formed. This period between erased PGCs and remethylated spermatozoa represents a highly sensitive and quite extensive exposure window, when the epigenetic status can be changed by environmental factors during embryonic development in utero. There is another dynamic chromatin demethylation, many years later, when sperm chromatin remodeling occurs when paternal and maternal pronuclei are developed in the early zygote. This methylation erasure is not complete and excludes parent-of-origin methylation, that is, erasure-resistant loci, such as IAPs, LINEs, and transposon-related loci. Taken together, the transgenerational and intergenerational inheritances of epigenetic shifts (i.e., non-genomic or non-Mendelian inheritance) are based on these two exposure windows, when epigenetic erasure, including gene imprinting in PGCs and imprinted gene-excluding erasure, occur, respectively [17]. The renewal of gene imprinting between PGCs and mature gamete is another power of transgenerational epigenetic inheritance [18]. The dynamics of the epigenetic code is subjected to a well-tuned orchestra of "erasures" (TET oxygenases, histone deacetylases, and demethylases) and "writers" (DNA methyltransferases, histone methyl transferases, and acetyl transferases) (reviewed in [19]). It is assumed that, via EDs, they change the epigenetic code through these upstream factors (the possible methods of exposure are summarized in **Figure 1**). Doubtless, a properly established epigenetic code plays an extremely important role, in particular in imprinted genes in epimutation-prone gametes. The epigenetic code of the spermatozoon is highly protected by the protamination, determining the stability of the genome and gene imprinting. Otherwise, epigenetic disorders arise: Prader-Willi syndrome, Angelman syndrome, or Silver-Russell syndrome. Moreover, residual histones bring the epigenetic information via histone PTMs.

enforces spermatid sensitivity to exposure to environmental pollutants.

*DOI: http://dx.doi.org/10.5772/intechopen.88142*

and represent susceptible exposure windows [10, 11].

### *Endocrine Disruptors: Very Low Doses with Genuinely High Impacts on Male Reproduction DOI: http://dx.doi.org/10.5772/intechopen.88142*

Protamination, histone-protamine exchange in elongating spermatids, represents a drastic, expressible change of sperm chromatin [5]. A tight protamine-derived DNA package protects sperm chromatin against damage and, interestingly, even the ratio of protamines PRM1 (sperm protamine P1) and PRM2 (sperm protamine P2) is decisive about sperm quality [6]. In accordance with the tight chromatin package, DNA is strongly methylated, and, therefore, general chromatin silencing is required for sperm stability [7, 8]. Protamination represents a tool for the protection of paternal gene imprinting [9]. Temporal protamine-packaged sperm DNA undergoes a second exchange of chromatin proteins after fertilization, and then maternal histones are incorporated into the paternal pronucleus. Both protaminehistone transition events, first and second in testicular seminiferous tubuli and fertilized oocyte, respectively, are obviously sensitive to environmental influences and represent susceptible exposure windows [10, 11].

Although most core histone is substituted by protamines, a residual speciesspecific amount of histones resists in the sperm head. In addition to DNA methylome, epigenetic hallmarks of mature spermatozoa include the epigenetic code of residual histones, based on many posttranslational modifications (PTMs) of individual amino acids [12, 13]. These chromatin-repressive histone marks positively correlate with DNA methylome and accompany imprinted genes. Moreover, the sperm histone code shows an exact physiological role in fertilization and early embryonic development [14]. The histone code establishment is highly orchestrated [15] and, therefore, enforces spermatid sensitivity to exposure to environmental pollutants.

Following comprehensive demethylation of parental chromatin after fertilization, the total erasure of the methylation pattern, including gene imprinting on paternal and maternal alleles, is needed for the re-establishment of gene imprinting adequate to the paternal pattern in the sperm cell. This erasure comes early after gonadal ridge colonization, and primordial germ cells (PGCs) occur, at human embryonic days E32 and E10.5 in mice [16]. The recurrent "writing" of the epigenetic pattern into imprinted loci occurs in the late prenatal period when the spermatogonia are formed. This period between erased PGCs and remethylated spermatozoa represents a highly sensitive and quite extensive exposure window, when the epigenetic status can be changed by environmental factors during embryonic development in utero. There is another dynamic chromatin demethylation, many years later, when sperm chromatin remodeling occurs when paternal and maternal pronuclei are developed in the early zygote. This methylation erasure is not complete and excludes parent-of-origin methylation, that is, erasure-resistant loci, such as IAPs, LINEs, and transposon-related loci. Taken together, the transgenerational and intergenerational inheritances of epigenetic shifts (i.e., non-genomic or non-Mendelian inheritance) are based on these two exposure windows, when epigenetic erasure, including gene imprinting in PGCs and imprinted gene-excluding erasure, occur, respectively [17]. The renewal of gene imprinting between PGCs and mature gamete is another power of transgenerational epigenetic inheritance [18]. The dynamics of the epigenetic code is subjected to a well-tuned orchestra of "erasures" (TET oxygenases, histone deacetylases, and demethylases) and "writers" (DNA methyltransferases, histone methyl transferases, and acetyl transferases) (reviewed in [19]). It is assumed that, via EDs, they change the epigenetic code through these upstream factors (the possible methods of exposure are summarized in **Figure 1**).

Doubtless, a properly established epigenetic code plays an extremely important role, in particular in imprinted genes in epimutation-prone gametes. The epigenetic code of the spermatozoon is highly protected by the protamination, determining the stability of the genome and gene imprinting. Otherwise, epigenetic disorders arise: Prader-Willi syndrome, Angelman syndrome, or Silver-Russell syndrome. Moreover, residual histones bring the epigenetic information via histone PTMs.

*Male Reproductive Health*

sensitive target to a pollutant impact.

found compounds as endocrine disruptors.

ders in spermatocytes and androgen synthesis inhibition [3].

**and implications for male reproduction**

compartments: basal and adluminal. The barrier is crucial for full functionality of germinal epithelium, as well as for the elimination of negative impacts of environmental pollutants. Physiologically, this strict division ensures free paracellular movement of substances among the compartments, such as water, nutrients, electrolytes, hormones, and paracrine factors. HTB provides protection of the emerging sperm cells from autoimmune damage by antibodies to sperm cells produced if the barrier was impaired, and the immune system would be in contact with spermatozoa during puberty when the body has already reached immunocompetence. Therefore, the cross talk of the immune system and HTB is potentially another

There is a basal compartment in close proximity to the basal membrane. This segment contains vessels and nerves, and spermatogenesis is initiated here. Spermatogonia and spermatocytes up to the proleptotene stage are present here. This segment is necessary for nutrition. The spermatogonia are subsequently transported through tight intercellular junctions to the adluminal compartment which is a place of spermatogenesis completion and subsequent metamorphosis of spermatids to spermatozoa. Both compartments are demarcated by the blood-testis barrier (BTB). Vessels and nerves are no longer present in this segment, and the nutrition of the germinal epithelium cells is covered by the Sertoli cells. The impact of various doses and concentrations of EDs on the male reproductive system can affect the functions of this barrier. The differentiation and development of the male reproductive system depends on elementary estrogen/ androgen ratio, and the antagonistic and agonistic effects of EDs often disrupt their balance. The development of testicular tissue is crucial for further development of the entire reproductive system, as the endocrine activity of testicles determines overall masculinization of the body. Any disruption in the development of the testicles can

therefore impair the overall masculinization process and sperm production.

Sperm concentration in men decreases worldwide, and spermiogram parameters deteriorate mainly in the Western world population [1]. Among others, huge amounts of endocrine disruptors (EDs) in our environment can cause this state. This final manifestation of the noxious effect of EDs has an unknown background, such as dose, kinds of EDs, interactions, and crosstalk of individual EDs and/or the timing of the exposure. Therefore, biomonitoring data represents significant input for experimental designing, leading to the description of molecular action in simulated conditions. Based on the newest findings, the record of the biological impact of individual EDs is an ongoing research issue leading to indicating the

Many cases of impaired sexual development due to the effects of EDs are also known from the animal kingdom. For example, reduction of penile length was observed in crocodiles living in waters contaminated with EDs [2]. EDs can significantly influence not only the process of spermatogenesis but also the development of testicular tissue. It has been documented that increased exposition of pregnant mice to BPA caused alterations of organelles, that is, mitochondria and lysosomes, in Sertoli and Leydig cells, respectively. These alterations led to maturation disor-

**2. Spermatogenesis, epigenetics, biochemical status of spermatozoa,** 

The creation of the spermatozoon leads to the terminally differentiated cell with an extremely high level of chromatin methylation and silencing. The final shape of the spermatozoon, often species-specific, requires many morphological and biochemical changes, in particular, dynamic remodelation of the chromatin [4].

**38**

### **Figure 1.**

*Endocrine disruptors induce non-genomic inheritance through posttranslational modifications (PTMs) of various epigenetic factors. (A) Environmentally impacted posttranslational modifications of proteins incoming into intergenerational and transgenerational effects. (B) Endocrine disruptors are able to affect developing gonads through transplacental transmission in utero. Gonad activity is changed and hormonal levels, puberty onset, and sperm quality are affected. Sperm quality contributes to embryonic development and can influence the health of an offspring, and, therefore, the intergenerational transmission of the ED effect to F2 generation is obvious. Gene imprinting and epigenetic erasure are assumed to be the tools of this effect. The epigenetic code of erasure-resistant loci is possibly affected by ED, and the transgenerational effect appears. Nonspecific symptoms accompany these epigenetic faults, and many disorders are classified as idiopathic. (C) From the molecular point of view, inadequate changes in DNA and chromatin proteins, including PTMs of core histones and/or RNA polymerases, are responsible for the epigenetic record and gene manifestation, and ED becomes potentially dangerous for these protein modifications through "posttranslational" effect. Obviously, male reproduction is endangered through several exposure windows during gamete formation, including epigenetic code erasure and re-establishment. Therefore, in addition to direct modification of chromatin, responsible "erasures" and "writers" (responsible for de-differentiation and gene imprinting, respectively) undergo regulation via PTMs when the EDs' effect is considered.*

Obviously, in addition to the genetic information, the sperm head carries a package of epigenetic notice, very sensitive to the disruption through its establishment throughout the spermatogenesis.

In addition to the establishment of epigenetic code of sperm histones, achievement of other PTMs of regulating proteins is required. Frequently, the loss of a PTM leads to protein activity lacking, sometimes leading to fatal clinical manifestations, for example, the inability of PARKIN1 S-sulfhydration of cysteine followed by sporadic Parkinson's disease [20]. During post-ejaculation the sperm changes, such as capacitation and acrosomal reaction; there are many PTMs of key proteins necessary for the achievement of fertilization ability. Therefore, protein kinase A (PKA)-driven phosphorylation of Arg-X-X-(Ser/Thr) motifs is required, as the result of upstream regulation by soluble adenylyl cyclase and cAMP production [21]. However, acetylation of ε-amino group of lysine residues arises as regulatory tool for

**41**

**Table 1.**

*Endocrine Disruptors: Very Low Doses with Genuinely High Impacts on Male Reproduction*

likely manner in which disruptors (EDs) work in their real doses.

**3. Endocrine disruptors: mode of action and nonlinear effect**

There are many shared features of EDs, such as spatiotemporal omnipresence, exposure to very low doses, and, therefore, often a nontoxic effect [28]. Nevertheless, the affection of hormonal balance represents a major sign of them, giving the name to endocrine disruptors [29]. Indeed, there is an increasing number of observations of exposure to EDs, across all age, race, profession, lifestyle, and health status categories [30]. These findings are in accordance with the ubiquity of

**Compound Phenotype of filial generation Species Reference**

Reduction of lipid accumulation *Caenorhabditis* 

*Drosophila melanogaster*

*elegans*

Dawley)

*rerio*)

Rat (Sprague Dawley)

Rat (Sprague Dawley)

Dawley)

Reproduction failure Mouse (CD-1) [114]

Medaka (*Oryzias latipes*)

Mouse (C57BL/6 J) [111]

[108]

[109]

[110]

[112]

[113]

[115]

[116]

[117]

responsible for basic metabolism, cell cycle, stress response, and

responsible for steroidogenesis, and

PKA, and, accordingly, the hyperacetylation of sperm proteins is needed for sperm capacitation [22], essential for sperm hyperactivation in female reproductive tract. Versatile role of protein acetylation is obvious, including aforementioned residual histones as well as protein kinases. Taken together, the impact of endocrine disruptors on histone PTMs [23, 24] as well as sperm phosphorylation [25–27] has been described, and, therefore, the modifications of proteins (protein PTMs) become the

*DOI: http://dx.doi.org/10.5772/intechopen.88142*

EDs through the presence in daily need items.

Benzylisoquinoline alkaloids

Di(2-ethylhexyl)phthalate

*due to PTM-driven epimutations [106, 107].*

(DEHP)

Antibiotics (Geneticin) Up-/downregulation of genes

development

DNA methylation

social interactions

DDT Pathology of gonads, obesity Rat (Sprague

Dioxin Testicular tissue abnormalities Zebrafish (*Danio* 

kidney failure; birth abnormalities

disease susceptibility of gonads, ancestry glands, *mamma*, and kidney

*Representative studies are included, testing different compounds (in toxic and sub-toxic doses) on various biomodels, mostly exposed during the establishment of germ cells and gonad maturation. These exposures lead to changed phenotype of filial generation through the epimutation of germ cells. In addition to pregnant exposure, PTMs of epigenetic factors and/or histone code represent a molecular tool of endocrine disruptor-inherited impact along generations, even though the exposure is during adulthood. Although direct human evidence is lacking, there are several indications of the effect of transmission of endocrine disruptors in very low doses, on further generations* 

Methoxychlor Obesity, ovary, and kidney diseases Rat (Sprague

*Overview of recent knowledge of environmental inheritance of endocrine disruptor effects.*

Atrazine Reproduction, altered transcriptome

BPA Affected neurogenesis and damaged

Glyphosate Obesity, prostate, and ovary diseases;

Vinclozolin Alterations transcriptome with

*Endocrine Disruptors: Very Low Doses with Genuinely High Impacts on Male Reproduction DOI: http://dx.doi.org/10.5772/intechopen.88142*

PKA, and, accordingly, the hyperacetylation of sperm proteins is needed for sperm capacitation [22], essential for sperm hyperactivation in female reproductive tract. Versatile role of protein acetylation is obvious, including aforementioned residual histones as well as protein kinases. Taken together, the impact of endocrine disruptors on histone PTMs [23, 24] as well as sperm phosphorylation [25–27] has been described, and, therefore, the modifications of proteins (protein PTMs) become the likely manner in which disruptors (EDs) work in their real doses.
