Molecular perspective concerning fluoride and arsenic mediated disorders on epididymal maturation of spermatozoa: A concise review

Abstract

Epididymis is a complex tubular structure of male reproductive system where spermatozoa undergo maturation and gain the fertilizing ability. Epididymal pseudostratified columnar epithelium with different cell types play imperative role by their secretory properties and enrich the luminal microenvironment necessary for achieving spermatozoal motility. During epididymal transit several secretory proteins like P26h, SPAG11, HSPD1 and many others are deposited on spermatozoal surface. At the same time spermatozoal proteins are also modified in this intraluminal milieu, which include cyritestin, fertilin, CE9 and others. Natural and anthropogenic activities disclose various environmental pollutants which affect different physiological systems of animals and human being. Likewise, reproductive system is also being affected. Fluoride causes structural alterations of caput and cauda segments of epididymis. Redox homeostasis and functional integrity are also altered due to diminished activities of SOD1, GR, Crisp2, Lrp2 and other important proteins. On the contrary arsenic affects mostly on cauda segment. Redox imbalance and functional amendment in epididymis have been observed with arsenic revelation as evidenced by altered genomic appearance of SOD, GST, catalase, Ddx3Y, VEGF and VEGFR2. This review is dealt with structure-function interplay in normal epididymal spermatozoal maturation along with subsequent complications developed under fluoride and arsenic toxicities.

Keywords

Epididymis spermatozoal maturation reprotoxicity fluoride arsenic

Introduction

Spermatogenesis is exclusively dependent on close liaison in-between Sertoli and Leydig cells of testis.¹ However, the spermatozoa formed by the testis are immature and infertile.^2,3 The spermatozoa need to be transported via epididymis to mature entirely and thus attain the competence to fertilize.^4,5 The epididymis is sustained by an array of homeostatic control systems operated by hormonal and non-hormonal features, which coalesce to ensure spermatozoal maturation and fortification. It is a unique and fascinating accessory sex organ of the male reproductive system made up of super coiled and convoluted tubule restraining a nutritious luminal fluid required by the spermatozoa for the attainment of fertility.^6,7 Structural orientation has sub-divided epididymis into three different regions like caput, corpus and cauda.⁶ They are composed of various types of cells which in turn by their secretory activities help to create and maintain the luminal fluid composition for spermatozoal nourishment.⁷ The cauda portion of the epididymis stores the matured spermatozoa and also plays an active role in spermatozoal nutrition and transportation. Thus, epididymal maturation of spermatozoa is considered crucial for the establishment of male fertility.

Quite a lot of natural and anthropogenic bustles bring about environmental pollution through exposure of several organic, inorganic, metallic and non-metallic compounds present in nature which lead to blemish of both terrestrial and aquatic ecosystems. Among the inorganic contaminants, as per the present day scenario, the increased presence of fluoride and arsenic in the environment is one of the key concerns of developing and developed countries. Both the geogenic and anthropogenic sources are responsible for simultaneous increase of fluoride and arsenic. Several industries such as pharmaceutical, microelectronics, mining, beverages, pesticide, glass and food are the major anthropogenic sources liable for environmental contagion with fluoride and arsenic.⁸ It has been reported that above 50 percent population through the entire globe utilize ground water for household and drinking purposes⁹ and most of people from various parts of the world are suffering from the toxic effects of fluoride and inorganic arsenic through the consumption of groundwater.^10,11 Worldwide coexistence of fluoride and arsenic has been reported in Asia (Japan, Pakistan, Korea, China and India), Africa (Tanzania, Ethiopia, Nigeria and Ghana) and also in Latin America (Mexico, Argentina, Peru, Bolivia, Colombia and Chile).¹² Extensive survey revealed that fluoride and arsenic are the two major groundwater contaminants causing a set of health symptoms known commonly as fluorosis and arsenicosis respectively.⁸ Reproductive toxicities are also associated with the health hazards also caused by these two pollutants. Waste water discharge in an indecent manner enhances the possibility of combined existence of fluoride and arsenic in the ground water.⁸ The present literature summarizes the toxic effects of these two contaminants on the epididymal spermatozoal maturation which is considered as a very important feature in connection with fertility issue. Fluoride is the 13th most abundant element on earth crust and considered as potent environmental toxicant but unlike other toxicants it acts as di-sword weapon because at low concentration it has mitogenic effects but increased exposure might result in serious toxic effects on animals and human beings.¹³ Arsenic being 20th most abundant element on earth crust is also considered as human carcinogen, exposure of which causes unpleasant human health consequences, which include hepatic, renal, cardiovascular disorders and also cancer in lung, liver, kidney, skin and urinary bladder.¹⁴

Various reports have suggested that individually these elements are reprotoxic and cause reproductive disorders in both male and female systems. Fluoride has been found to impede with the reproductive system of animals. In male animals the consequences include diminished activity of the steroidogenic enzymes, reduced serum testosterone levels, decreased sperm count and motility, testicular damages, structural defects in spermatozoa along with oxidative stress.^15–21 In female Sprague-Dawley rats, administration of sodium fluoride in drinking water notably reduces in the number of viable fetuses and increases in the resorption rate were observed.²² Fluoride induced free radical toxicity was also reported in mice ovary.^23,24 Some experimental studies on animals have testified that arsenic exposure is linked with spermatozoal toxicities,^25,26 diminished testicular steroidogenic activity, reduction of testicular weight and also weight of accessory reproductive organs.²⁷ Arsenic intoxication is also associated with the inhibition of ovarian steroidogenesis and as well as secretion of gonadotrophins,²⁸ elevated adrenocortical steroidogenesis and level of plasma corticosterone.²⁹ Epidemiological study demonstrated that chronic arsenic exposure through drinking water causes adverse pregnancy conditions like premature delivery, neonatal death and spontaneous abortion.³⁰ Literature reports even suggested that exposure to high fluoride concentrations in drinking water is also linked with decreased human birth rates.³¹ But the knowledge of the joint action of these two elements is lacking under male reproduction involving epididymis and the results derived from previous studies were inconclusive on epididymis regarding spermatozoal maturation under such reprotoxic environment. This review emphasizes normal structural and functional integrity of epididymis and altered spermatozoal maturation under toxic effects of fluoride and arsenic.

Biology of epididymis

Spermatozoa after development within testis go through one long, narrow and convoluted tubule which is known as epididymis. Spermatozoa undergo maturation after interacting with secretory proteins in the epididymal lumen and obtain progressive motility to fertilize ovum. According to histomorphological and ultrastructural features epididymis has been differentiated into three regions, which include head (caput), body (corpus) and tail (cauda). In some species such as mouse, the most proximal segment of epididymis is known as initial segment which is in addition to the earlier nomenclature.³²

Cellular distribution

Epididymis is lined by pseudostratified epithelium having different cell types, namely apical cell, basal cell, clear cell, halo cell, narrow cell and principal cell. The principal cells constitute about 80% of epithelium and responsible for maximum secretion within lumen.³² The thickness of epididymal epithelium gradually decreases along the tubule³³ and due to water reabsorption; sperm concentration subsequently increases down the epididymal segment. All these factors are responsible for gradually increasing luminal diameter along the epididymis.³⁴ The other notable features along with functions of different cells of epididymal epithelium have been well characterized and are presented here (Figure 1).

Figure 1.

Functions of Epididymal cells and their secretory proteins.

Principal cell

The major constituents of epididymal epithelium are principal cells having secretory function in nature. Nuclei of principal cells of first portion of caput are spindle shaped but become gradually infolded in distal region of caput, corpus and especially in cauda. The intense nuclear lobation is an important characteristic feature of cauda region. Often a nuclear lobe is connected to another lobe by attenuated fold in nuclear envelope and in some instances, nuclear envelope displays continuation with endoplasmic reticulum, and thereby nucleus is invested with endoplasmic reticulum profile.³⁵ Numerous stereocilia, extended several micrometer, cover apical surface of the principal cells. Well developed rough endoplasmic reticulum and different sized mitochondria with distinctive lamellar cristae are other characteristic features of these cells.³⁵ Claudin and occludin are transmembrane proteins, responsible for formation of tight junctions, are also present between principal cells and thus form blood-epididymis barrier, which offers immunoprotective region in epididymal lumen necessary for maturation of spermatozoa.³⁶ Connexin, an integral protein forms gap junctions at apical and lateral surfaces of neighboring principal cells and permit the transportation of molecules having <1 kDa molecular weight.³²

Apical cell

Apical cells, found consistently in epididymal epithelium but not in huge number, extend to the lumen from basal lamina. It is narrow throughout its length but become wider near the lumen and luminal surface contains few short microvilli. Apically located nucleus appears as round or oval shaped having shallow infolding on nuclear envelope. One salient feature of apical cells is the presence of huge number of mitochondria, located in apical region.³⁵

Basal cell

The base of epididymal epithelium contains small cells, which reside on basal lamina throughout epididymal tubule and cytoplasm of these cells do not extend into the lumen. Round or ovoid nuclei are having with or without invaginations, few nucleolar and small heterochromatin elements. One key feature of these cells is the interdigitations of its plasma membrane with bordering principal cells.³⁵ The cells also contain coated pits on its apical plasma membrane and which are responsible for receptor mediated endocytosis of molecules derived from principal cells and blood. Accumulation of secretory materials in the Golgi saccules is the other characteristic feature of basal cells.³⁵

Halo cell

The cells contain round or intended nuclei, which are highly infolded occasionally having abundant heterochromatin. Cytoplasm possesses little amount of endoplasmic reticulum and few mitochondria. The key feature of this cell type is presence of cytoplasmic pseudopodia protruding between principal cells.³⁵ In adult animals, these cells are having helper T lymphocytes, cytotoxic T lymphocytes and as well as monocytes but not the B lymphocytes. Through the advancement with age, region specific enhancement of the number of each immune cell is observed along with occasional appearance of B lymphocytes and eosinophils.³⁷

Clear cell

The cells are prevalently present in cauda segment and are considered as primary immune cells in epididymis.³² These cells are described by appearance of endosomes, lysosomes, coated pits, vesicles and multivesicular bodies in apical region whereas basal region contains nucleus and lipid droplets. Endocytic activity of these cells is much greater than adjacent principal cells, particularly in cauda region as executed by endosomes and lysosomes. Cytoplasmic droplets as released from spermatozoa during transmission through lumen are taken up by these cells.³³

Narrow cell

These cells are narrower than that of neighboring principal cells and are attenuated. The cells send fine processes of cytoplasm to reach the basement membrane. The key characteristic feature of these cells are the presence of numerous apically positioned cup-shaped vesicles which are involved in endocytosis and secretion of H⁺ ions into lumen.³⁸

Spermatozoal maturation in epididymal lumen

During epididymal transition from proximal to distal part, spermatozoa undergo several biochemical, morphological and physiological alterations, finally attain the progressive motion and also fertilizing ability.³² Spermatozoal remodeling includes changes in dimension and the internal appearance of acrosome, alterations in the dimension of nucleus, migration of cytoplasmic droplets along sperm tail and structural changes in the intracellular organelles.³⁹ Phospholipid and cholesterol contents of spermatozoal plasma membrane are also changed.⁴⁰ Spermatozoa obtain the capacity for regulating cell volume by uptake of several luminal components, which function as osmolyte reserves, so that after exposure in female reproductive tract, spermatozoa do not undergo osmotic shock.⁴¹

Secretory proteins and their roles

Numerous luminal secretory proteins and a number of spermatozoal proteins, which are modified during luminal transport and also the signaling event in caudal segment are documented here (Figure 1). Apocrine secretions through exosome and epididymosome are the main mode of transfer of luminal proteins to sperm plasma membrane.⁴² Apical region of principal cells release blebs, known as epididymosome and it has been reported to occur in rat, bovine, mice, horse and human.⁴³ Caput being the most metabolic active region of epididymis, secrets about 70–80% of total protein released in epididymal lumen and during transit through this region about 99% of fluid is resorbed.⁴⁴ Most secretory proteins undergo trafficking through Golgi apparatus then undergo packaging and ultimately released from secretory granules to be delivered to sperm surface.³²

Proteins, secreted in luminal microenvironment are responsible for conformational change of the sperm proteins from non-native, misfolded and denatured form to native form, which include tumor rejection antigen 1 (TRA1), a member of HSP 90 family and heat-shock protein 1 (HSPD1 and HSP60).³²

A number of proteins, associated with epididymosome are transported to spermatozoa during their luminal transit and these are P26h (zona pellucida binding protein), HE5, ubiquitin, macrophage migration inhibitory factor and glutathione peroxidase.⁴⁵ Beside these proteins, SPAM1 protein, secreted from epididymal epithelial cells is incorporated in spermatozoal head through epididymosome and this protein helps to break the cumulus layer of egg.⁴⁶ Apart from SPAM1, Other two proteins serine protease PRSS21 and acrosin (ACR) play pivotal role in fertilization process by promoting spermatozoal motility and acrosome reaction.⁴⁶ Other transmembrane proteins, CD9 and CD81, which also represent epididymosome and promote signal transduction. MFGE8 is also present in epididymosome and is responsible for maturation of spermatozoa.⁴⁶

Quite a few secretory proteins interact with spermatozoa and provoke sperm maturations, which include SPAG11, P34h, P26h, CRISP family proteins, Eppin, SED1 and Clusterin.^47–53 Other important proteins, secreted through the epididymis are Low-density lipoprotein receptor-related protein 2 (Lrp2), involved in endocytosis and sorbitol dehydrogenase, which controls epididymal secretory function.⁵⁴ Some proteins play important role in sperm maturation, which include Ddx3y, vascular endothelial growth factor (VEGF) and VEGF receptor 2 (VEGFR2).^55,56

Several other secretory proteins, identified in human epididymal lumen are albumin, α1-antitrypsin, prostaglandin D2 synthase, clusterin, lactoferrin, extracellular matrix protein, transferrin NCP2/CTP/HE1 and actin binding protein.⁵⁷

Many novel genes had been first identified in human epididymis and then studied in many other mammalian species, which include HE1, encodes cholesterol binding protein; HE2, a β-defensin; HE3, having unidentified function; HE4, a proteinase inhibitor family; HE5 and HE6.⁵⁸

Conformational changes of spermatozoa proteins in epididymal lumen

Some spermatozoal proteins, which are synthesized during spermatogenesis, undergo modification during epididymal transit in terms of proteolytic processing or deglycosylation. These proteins include ADAM family members cyritestin and fertilin, CE9, α mannosidase, endoplasmin, β-galactosidase, Grp78/Hsp70, phosphatidylethanolamine binding protein, basigin, α-enolase, lactate dehydrogenase, cytokeratin, testis lipid-binding protein, and β-subunit of F1-ATPase (Figure 2).^59–64

Figure 2.

Conformational changes of epididymal spermatozoal proteins.

During proteolytic processing in epididymal lumen, some spermatozoal proteins undergo new localization pattern, which include β-galactosyltransferase and SPAM1(Figure 2).⁶⁵

Final maturation in cauda and molecular cascade

Study on spermatozoal motility clearly indicated that asymmetrical beating of flagella as observed in caput was seen to be changed to symmetrical forward motion in cauda (Figure 3).⁶⁶

Scientific data regarding the achievement of spermatozoal motility is still unclear but the available information pointed out that intracellular cyclic adenosine monophosphate (cAMP) generation by activating the enzyme adenylate cyclase and subsequent phosphorylation of protein kinase A and many other proteins are the key factors.⁶⁷ It was also observed that the concentration of cAMP in spermatozoa increases gradually from corpus to cauda⁵⁷ that leads to the increase of metabolic competence.⁶⁸ Presence of calcium and bicarbonate in the luminal fluids directly control spermatozoal cAMP concentration and subsequent activation of phosphorylation of other proteins and also spermatozoal motility.⁶⁹ Another In-vitro study explains that the combined presence of calcium and bicarbonate help in attainment of flagellar motion of mature spermatozoa.⁷⁰

Mass spectrometry analysis has revealed alterations of protein profile between immature spermatozoa from caput and mature spermatozoa from cauda. Presence of phosphorylated proteins like heat-shock protein 70, β-tubulin, actin, glucose-regulating protein, lactic acid dehydrogenase, mitochondrial protein aconitase and also β subunit of F1 ATPase as observed only in caudal spermatozoa which may be considered as the marker of matured sperm.⁷¹ Hence the presence of regulatory molecules in cauda and phosphorylation of sperm proteins symbolize a key maturational step of spermatozoa. Another vital protein, predominantly secreted from cauda region is beta-defensin 126, absorbed in spermatozoa.⁷²

Figure 3.

Molecular cascades involved in maturation caudal spermatozoa.

Effects of fluoride toxicity

Structural amendments

Fluoride is responsible for a lot of changes in testicular structure and also has a large impact on histomorphometry of entire epididymis. The associated changes in both caput and cauda are documented (Figure 4).

Figure 4.

Effects of fluoride and arsenic toxicity on structure—function of epididymis.

Caput: The first portion of epididymis from untreated animals structurally characterized by many tubules covered with pseudostratified columnar epithelial cells having numerous stereocilia in the luminal surface and intertubular interstitium present between tubules. Secretory granules are abundantly present in epithelial cells and tubular lumen is filled with bundles of sperm. An experimental study by intoxication with sodium fluoride to mice at 10 and 20 mg/kg body weight/day for 30 days revealed increased tubular diameter, nuclear pyknosis of secretory epithelial cells and absent of tubular spermatozoa.⁷³ When rabbits were exposed with NaF at 10 mg/kg body weight/day for the duration of 23 months it results drastic changes such as presence of fewer stereocilia or complete absent of stereocilia in many areas on epithelial cell line, presence of less abundant secretory granules in tubule and even in epithelial cells, increase of luminal diameter and decrease of height of epithelium⁷⁴ but the same experimental study for the duration of 20 months results no significant structural alterations.

Cauda: This region of untreated animals are morphologically characterized by presence of circular and regular tubules having larger diameter than caput, presence of pseudostratified columnar epithelium in tubular lining, presence of intertubular interstitium, presence of blood vessels in extratubular space, dense layer of stereocilia protruding from apical surface of principal cells toward luminal side and tubular lumen occupied by healthy spermatozoa having well defined head and tail. According to Chinoy et al., mice from treated group (10 and 20 mg/kg /day for 30 days) showed a massive changes of caudal structure in terms of decreased height of epithelial cells, larger tubules having denudation of cells within lumen and tubules are devoid of spermatozoa.⁷³ Extensive morphological alterations also found in sodium fluoride treated (10 mg/kg body weight/day for 23 months) rabbits and these alterations has been exemplified as bald appearance of pseudostratified epithelium due to less dense or absence of stereocilia, fewer spermatozoa in the tubular lumen, reduced number of secretory granules, decreased epithelial height and increased tubular diameter but animals having same treatment dose for 20 months devoid of such alterations⁷⁴. Fluoride is also responsible for alterations the caudal structure in a dose dependent manner (100, 200, 300 ppm of NaF/day for 40 days) in rats. Structural evaluation by histology and Scanning Electron Microscopy (SEM) revealed that treatment with 100 ppm of NaF results disruption of basement membrane, vacuole formation in germinal epithelial cells, reduced sperm number and less stereocilia in lumen. Structural analysis of cauda in second treated group (200 ppm) showed epididymal hyperplasia, less spermatozoa and presence of cellular debris, disintegrated connective tissues and presence of very less stereocilia. Ultrastructural analysis by SEM divulges same outcome. Severe alterations have been noted in highest treated group (300 ppm) such as reduced lumen diameter with very few spermatozoa and huge exfoliated germ cells, inflammatory infiltration having lymphoid aggregates, distortion of basement membrane, vacuolation in germinal epithelium, focal infolding in epithelium with pseudo-glandular appearance and hyperplasia was also noted in caudal region. SEM analysis revealed bald appearance due to absence of stereocilia and presence of fragments in epithelial surface.⁷⁵

Functional alterations

A recent experimental study has explored altered epididymal protein expression, which in turn impedes epididymal functions (Figure 4). Lrp2 is expressed through the entire length of epididymis, which participates endocytosis and as well as lysosomal degradation of several ligands and this protein is known to be expressed anomalously upon fluoride exposure.⁵⁴ Sorbitol dehydrogenase, encoded by a key gene Sord is involved to maintain epididymal secretory functions and fluoride has been known to alter the expression of Sord gene and disrupts its function.⁵⁴ An important protein, secrets preferentially from cauda epididymis is beta-defensin 126, which is absorbed through post acrosomal region and tail of spermatozoa⁷² and regulates spermatozoa activity by increasing the swimming ability in cervical mucus and also ensure protection from immune system within female reproductive tract.⁷⁶ Fluoride also amends the expression of beta-defensin 126.⁵⁴ Crisp1 and Crisp2 are family members of cysteine rich secretory protein (Crisp). Crisp1 glycoprotein secreted from epididymal epithelium inhibits sperm capacitation and also suppresses sperm-egg fusion⁷⁷; Crisp2 is also involved in male fertility.⁷⁸ Altered expression of these epididymal proteins has been reported upon sodium fluoride intoxication and that leads to male infertility.⁵⁴ Recent report also revealed that fluoride downregulates the expression of SPAM1, CD9 and CD81 proteins in entire cauda epididymis, which cause obstruction of the sperm maturation and as well as inhibition of sperm egg binding.⁴⁶ This study has revealed that spermatozoa deficient with ACR protein show decreased progressive motility toward the cumulus cells than wild type spermatozoa and PRSS21 disrupted spermatozoa exhibit fragile neck in addition to slower motility rate. The study further states that fluoride exposure down-regulates both mRNA and protein expression of ACR and PRSS21 protein in cauda epididymis ⁴⁶ and this might be one possible mechanism by which fluoride affects spermatozoal progressive motility and fertilizing ability. CRISP2, a CRISP family protein which promotes spermatozoal motility and sperm egg fusion has known to be declined after fluoride exposure.⁷⁹

Redox balance

It is significantly approved that redox balance is crucial for standard physiological function of a living system and this balance is sustained following combat between internal antioxidant mechanisms (both enzymatic and nonenzymatic) with reactive oxygen species (ROS) generated in the living system. However, in living system, interactions with any kind of xenobiotic compound triggers potentially damaging events, which can directly or indirectly confront redox homeostasis. Fluorine, being a highly ionized element can interrupt oxygen metabolism and promotes increased generation of O₂^- free radicals, which in turn promotes the formation of other free radicals like hydroxyl, hydrogen peroxide and peroxynitrite.⁸⁰ In case of mammalian tissues super oxide dismutase (SOD) is a central antioxidant enzymatic component having three different isozymes, CuZn-SOD (SOD1), Mn-SOD (SOD2), and EC-SOD (SOD3).⁸⁰ Among these SOD1 is crucial scavenging enzyme catalyzes O₂^- into H₂O₂ and another vital enzyme to remove free radicals is catalase (CAT).⁸¹ An experimental study by intoxication with sodium fluoride through drinking water to mice at different doses (25, 50 and 100 mg NaF/L) for 10 weeks results significant decreased mRNA expression of SOD1 and CAT in epididymis at all three treated groups.⁵⁴ From this it can be concluded that fluoride alters normal homeostasis between oxidative stress and antioxidant enzymes in epididymal tissues (Figure 4).

Glutathione subsists in thiol-reduced (GSH) and disulfide-oxidized (GSSG) forms and both are found in about all mammalian tissues and defend against oxidative stress.⁸² When glutathione is converted from reduced (GSH) to oxidized (GSSG) form, GSH peroxidase (GPx) reduces lipid peroxide and hydrogen peroxide and during conversion from GSSG to GSH, GSSG reductase (GR) is involved at the expense of NADPH.⁸² Organic peroxides (ROOH) are reduced by either GPx or by another enzyme glutathione S-transferase (GST).⁸² An experimental study by exposure of NaF at 25 and 100 mg/L for 60 days to mice results decreased mRNA and protein expression of GPx, GST and GR in epididymis in a dose dependent manner and this study has explored that fluoride affects epididymal antioxidant status by changing glutathione related enzymes.⁵⁴

Effects of arsenic toxicity

Structural amendments

Several studies have stated that arsenic toxicity is responsible for the decrease in the weight of testes along with epididymis at the organ level.^83–85 Structural detoriation of epididymis has also been reported upon arsenic threat (Figure 4). Histological observations in arsenic affected epididymis of goat (obtained from arsenic prone zone) revealed altered histoarchitecture in terms of irregular and dense connective tissue, elastic fibers and also collagen fibers as compared to control animal group. Lesser number of spermatozoa in the lumen has also been noted in treated animals.⁸⁶ Photomicrographs of cauda epididymis from sodium arsenate (25 ppm for 70days) exposed rat highlighted architectural disarray of pseudo-stratified columnar epithelium with fewer or devoid of spermatozoa in the lumen.⁸⁴ A recent study on arsenic exposed epididymis of pre-pubertal rats (52 and 82 days age) reported cribiform appearance in the epithelium, lack of spermatozoa in the lumen, presence of vacuoles and cell debris indicating disruption of luminal microenvironment leading to impairment of spermatozoal maturation.⁸⁷

Functional alterations

It is well established that arsenic after entering the normal homeostatic system, increases ROS generation and oxidative stress.⁸⁸ Literature survey revealed that apoptosis is triggered by high levels of ROS in the testis and accessory reproductive organs⁸⁹; eventually cause low sperm count, motility and finally triggering altered spermatozoal quality.^27,83 It has been reported that arsenic accumulates in male reproductive tissues, including prostate, seminal vesicles, testis and also in epididymis.^83,90 Epididymal arsenic accumulation leads to low spermatozoal viability⁹⁰ as a consequence of spermatozoal DNA damage coupled with spermicidal activity.^91,92 According to the report of Li et al.,⁵⁵ the sub-chronic exposure of arsenic significantly down-regulates the expression of Ddx3y gene and protein in the epididymis of mice, suggesting that this may affect maturation of sperms in the epididymis. Although their results showed that arsenic exposure had detrimental effects on spermatogenesis and sperm development through down-regulating the expression of Ddx3y gene but its effect on fertility of the exposed animals are yet to be confirmed. Further investigation is required to justify adverse effect of sub-chronic exposure of arsenic on fertility of the male animals through down-regulating the expression of this gene and also to know the precise molecular mechanisms on suppression of this gene.

VEGF being involved in reproductive system endorses angiogenesis and also plays a vital role to resistance apoptosis. VEGF and VEGFR2 are involved in sperm maturation and expressed in testis, epididymis, prostate and seminal vesicle. It has been reported that the expression of mRNA and protein level has been declined in epididymis of rat upon arsenic threat, which alter epididymal microenvironment and sperm maturation.⁵⁶ This study suggested that it might be one of the mechanisms of male infertility caused by arsenic poisoning (Figure 4).

Redox balance

Expression of genes, which instruct antioxidant enzymes are used to assess the extent of toxicity of any environmental pollutant. It has been reported that sodium arsenite causes declined mRNA expression of epididymal SOD1, SOD2, CAT and GST, which undoubtedly impede epididymal redox status and leads to reproductive disorders (Figure 4).⁹³ A recent finding has further elucidated that arsenic enhances the NO2/NO3 levels in the epididymis and thereby promotes nitrosative stress.⁹⁴ Another study on arsenic exposed epididymis of post-natal rats (52 days age) has reported a significant up-regulation of SOD1, CAT and GSTK1 genes in response to combat with the arsenic-induced oxidative stress.⁸⁷ Increase of MDA level and decrease of enzymatic CAT activity have also been observed also suggesting altered redox state.⁸⁷ Recently Souza ACF ⁹³ has reported that arsenic exposure on post-natal rats of 14 and 28 days caused increased activity of CAT to neutralize arsenic induced reactive species but exhaustion occurred finally reducing the activity below normal level. Souza ACF⁹³ has also stated that arsenic induced reduction of epididymal GST activity in post-natal rats of 28 days validating the malfunction of antioxidant enzyme that act against arsenic mediated oxidative stress.

It is well known that micronutrients are essential for production of antioxidant enzymes. Manganese is required for the activity of SOD in mitochondria, whereas zinc and copper are essential for the same enzyme activity in cytosol. Similarly iron is essential for catalase activity and selenium is required for GPx activity. Imbalance of these trace elements has also been reported in rat epididymis upon arsenic exposure, which might be probable reason underlying altered antioxidant defense.⁹⁴

Conclusion

Descendent of testicular spermatozoa through the epididymis is prerequisite to gain progressive motility and fertilizing ability. The functional maturation of spermatozoa is accomplished by incessant interaction with epididymal intraluminal milieu. Secretory activities of epithelial cells endow with several proteins which are deposited on spermatozoa and a number of spermatozoal proteins are also modified during luminal transportation. Over exposure of fluoride and arsenic affects structural integrity as well as intraluminal microenvironment of epididymis resulting distorted epithelial lining along with decreased luminal spermatozoa. Altered redox homeostasis is another disadvantageous outcome as evidence by anomalous genomic and proteomic expression of antioxidant molecules. Expression of crucial biomarkers, responsible for spermatozoal maturation gets altered under such reprotoxic state. Cumulative outcome of these experimental findings clearly pointed out that excessive exposure of fluoride and arsenic decline the structure-function entity of epididymis which gradually impair male fertility.

Footnotes

Abbreviations

Author contributions

PP and SB equally contributed in literature search and writing of the manuscript. PKM is responsible for revision of the article. All authors read and approved the final manuscript.

Acknowledgment

The author’s would like to acknowledge the FRPDF grant of Presidency University.

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) received no financial support for the research, authorship, and/or publication of this article.

ORCID iD

Prabir Kumar Mukhopadhyay

References

Chojnacka

Zarzycka

Mruk

. Biology of the Sertoli cell in the fetal, pubertal, and adult mammalian testis. Results Probl Cell Differ 2016; 58: 225–251.

Dong

Chen

Wang

, et al. Rictor regulates spermatogenesis by controlling Sertoli cell cytoskeletal organization and cell polarity in the mouse testis. Endocrinology 2015; 156: 4244–4256.

Jiang

Yie

Zhen

, et al. Effect of You Gui Wan on mouse sperm fertilising ability in vivo and in vitro. Andrologia 2014; 46: 283–289.

Peña

Summers

Gummow

, et al. Oviduct binding ability of porcine spermatozoa develops in the epididymis and can be advanced by incubation with caudal fluid. Theriogenology 2015; 83: 1502–1513.

Arroteia

Barbieri

Souza

GHMF

, et al. Albumin is synthesized in epididymis and aggregates in a high molecular mass glycoprotein complex involved in sperm-egg fertilization. PLoS One 2014; 9(8): 103566.

Robaire

Hinton

. The epididymis. In: Plant

Zeleznik

(eds) Knobil and Neill’s physiology of reproduction. San Diego: Academic Press, 2015, pp. 691–771.

Zhou

De Iuliis

Dun

, et al. Characteristics of the epididymal luminal environment responsible for sperm maturation and storage. Front Endocrinol; 9. Epub ahead of print 28 February 2018. DOI: 10.3389/fendo.2018.00059.

Chouhan

Flora

SJS

. Arsenic and fluoride: two major ground water pollutants. Indian J Exp Biol 2010; 48(7): 666–678.

Jadhav

Bringas

Yadav

, et al. Arsenic and fluoride contaminated groundwaters: a review of current technologies for contaminants removal. J Environ Manag 2015; 162: 306–325.

10.

Ruiz-Payana

Ortizb

Duarte-Gardea

. Determination of fluoride in drinking water and in urine of adolescents living in three counties in Northern Chihuahua, Mexico using a fluoride ion selective electrode. Microchem J 2005; 81: 19–22.

11.

Gebel

. Arsenic and drinking water contamination. Science (80-) 1999; 283: 1455e–1455.

12.

Mondal

Chattopadhyay

. Environmental exposure of arsenic and fluoride and their combined toxicity: a recent update. J Appl Toxicol 2020; 40: 552–566.

13.

Spittle

. Halting the inertia of indifference: fluoride and fertility revisited. Fluoride 2009; 42: 159–161.

14.

Kitchin

. Recent advances in arsenic carcinogenesis: modes of action, animal model systems, and methylated arsenic metabolites. Toxicol Appl Pharmacol 2001; 172: 249–261.

15.

Narayana

Chinoy

. Effect of fluoride on rat testicular steroidogenesis. Fluoride 1994; 27: 7–12.

16.

Huang

Nui

Wang

. Toxic effects of sodium fluoride on reproductive function in male mice. Fluoride 2007; 40: 162–168.

17.

Zhang

Liang

, et al. Effects of sodium fluoride and sulfur dioxide on sperm motility and serum testosterone in male rats. Fluoride 2006; 39: 126–131.

18.

Chinoy

Sharma

. Amelioration of fluoride toxicity by vitamins E and D in reproductive functions of male mice. Fluoride 1998; 31: 203–216.

19.

Krasowska

Włostowski

Bonda

. Zinc protection from fluoride-induced testicular injury in the bank vole (Clethrionomys glareolus). Toxicol Lett 2004; 147: 229–235.

20.

Kumar

Susheela

. Ultrastructural studies of spermiogenesis in rabbit exposed to chronic fluoride toxicity. Int J Fertil 1994; 39: 164–171.

21.

Ghosh

Das

Maiti

, et al. Testicular toxicity in sodium fluoride treated rats: association with oxidative stress. Reprod Toxicol 2002; 16: 385–390.

22.

Al-Hiyasat

Elbetieha

Darmani

. Reproductive toxic effects of ingestion of sodium fluoride in the female rats. Fluoride 2000; 33: 79–84.

23.

Chinoy

Patel

. Influence of fluoride on biological free radicals in ovary of mice and its reversal. Env Sci 1998; 6: 171–184.

24.

Jhala

Chinoy

Rao

. Mitigating effects of some antidotes on fluoride and arsenic induced free radical toxicity in mice ovary. Food Chem Toxicol 2008; 46: 1138–1142.

25.

Waalkes

Ward

Liu

, et al. Transplacental carcinogenicity of inorganic arsenic in the drinking water: induction of hepatic, ovarian, pulmonary, and adrenal tumors in mice. Toxicol Appl Pharmacol 2003; 186: 7–17.

26.

Pant

Murthy

Srivastava

. Male reproductive toxicity of sodium arsenite in mice. Hum Exp Toxicol 2004; 23: 399–403.

27.

Sarkar

Chaudhuri

Chattopadhyay

, et al. Effect of sodium arsenite on spermatogenesis, plasma gonadotrophins and testosterone in rats. Asian J Androl 2003; 5: 27–31.

28.

Chattopadhyay

Ghosh

Chaki

, et al. Effect of sodium arsenite on plasma levels of gonadotrophins and ovarian steroidogenesis in mature albino rats: duration-dependent response. J Toxicol Sci 1999; 24: 425–431.

29.

Ghosh

Chattopadhyay

Debnath

. Effect of sodium arsenite on adrenocortical activity in immature female rats: evidence of dose-dependent response. J Env Sci 1999; 11: 419–422.

30.

Milton

Smith

Rahman

, et al. Chronic arsenic exposure and adverse pregnancy outcomes in Bangladesh. Epidemiology 2005; 16: 82–86.

31.

Freni

. Exposure to high fluoride concentrations in drinking water is associated with decreased birth rates. J Toxicol Environ Health 1994; 42: 109–121.

32.

Cornwall

. New insights into epididymal biology and function. Hum Reprod Update 2009; 15: 213–227.

33.

Hermo

Robaire

Epididymal cell types and their functions. In: Robaire

Hinton

(ed.) The epididymis, from molecules to clinical practice. New York, NY: Kluwer Academic/Plenum Publishers, 2002, pp. 81–101.

34.

Moore

HDM

. The influence of the epididymis on human and animal sperm maturation and storage. Hum Reprod 1996; 11: 103–110.

35.

Ramos

Dym

. Fine structure of the monkey epididymis. Am J Anat 1977; 149: 501–531.

36.

Cyr

Gregory

Dubé

, et al. Orchestration of occludins, claudins, catenins and cadherins as players involved in maintenance of the blood-epididymal barrier in animals and humans. Asian J Androl 2007; 9: 463–475.

37.

Robaire

Hinton

Orgebin-Crist

. The Epididymis. In: Jimmy

(ed.) Knobil and Neill’s physiology of reproduction. Amsterdam: Elsevier, 2006, pp. 1071–1148.

38.

Kujala

Hihnala

Tienari

, et al. Expression of ion transport-associated proteins in human efferent and epididymal ducts. Reproduction 2007; 133: 775–784.

39.

Olson

NagDas

Winfrey

. Structural differentiation of spermatozoa during post-testicular maturation. In: Robaire

Hinton

(eds) The epididymis: from molecules to clinical practice. New York, NY: Kluwer Academic/Plenum Publishers, 2002, pp. 371–387.

40.

Jones

James

Howes

, et al. Supramolecular organization of the sperm plasma membrane during maturation and capacitation. Asian J Androl 2007; 9: 438–444.

41.

Cooper

. Sperm maturation in the epididymis: a new look at an old problem. Asian J Androl 2007; 9: 533–539.

42.

Sullivan

Saez

Girouard

, et al. Role of exosomes in sperm maturation during the transit along the male reproductive tract. Blood Cells, Mol Dis 2005; 35: 1–10.

43.

Binato De Souza

Schorr-Lenz

Lucca

, et al. The epididymis and its role on sperm quality and male fertility. Anim Reprod, v 2017; 14: 1234–1244.

44.

Clulow

Jones

Hansen

, et al. Fluid and electrolyte reabsorption in the ductuli efferentes testis. J Reprod Fert Suppl 1998; 53: 1–14.

45.

Saez

Frenette

Sullivan

. Epididymosomes and prostasomes: their roles in posttesticular maturation of the sperm cells. J Androl 2003; 24: 149–154.

46.

Liu

Liang

Gao

, et al. Fluoride interferes with the sperm fertilizing ability via downregulated SPAM1, ACR, and PRSS21 expression in rat epididymis. J Agric Food Chem 2019; 67: 5240–5249.

47.

Cohen

Rochwerger

Ellerman

, et al. Relationship between the association of rat epididymal protein “DE” with spermatozoa and the behavior and function of the protein. Mol Reprod Dev 2000; 56: 180–188.

48.

Légaré

Bérubé

Boué

, et al. Hamster sperm antigen P26h is a phosphatidylinositol-anchored protein. Mol Reprod Dev 1999; 52: 225–233.

49.

Sylvester

Morales

Oko

, et al. Localization of sulfated glycoprotein-2 (clusterin) on spermatozoa and in the reproductive tract of the male rat. Biol Reprod 1991; 45: 195–207.

50.

Yenugu

Hamil

Grossman

, et al. Identification, cloning and functional characterization of novel sperm associated antigen II (SPAG II) isoforms in the rat. Reprod Biol Endocrinol 2006; 4: 23.

51.

Richardson

Sivashanmugam

Hall

, et al. Cloning and sequencing of human Eppin: a novel family of protease inhibitors expressed in the epididymis and testis. Gene 2001; 270: 93–102.

52.

Hamil

Liu

Sivashanmugam

, et al. Cystatin 11: a new member of the cystatin type 2 family. Endocrinology 2002; 143: 2787–2796.

53.

Ensslin

Shur

. Identification of mouse sperm SED1, a bimotif EGF repeat and discoidin-domain protein involved in sperm-egg binding. Cell 2003; 114: 405–417.

54.

Sun

, et al. Alterations in epididymal proteomics and antioxidant activity of mice exposed to fluoride. Arch Toxicol 2018; 92: 169–180.

55.

Wang

Piao

, et al. Subchronic exposure to arsenic inhibits spermatogenesis and downregulates the expression of Ddx3y in testis and epididymis of mice. Toxicol Sci 2012; 128: 482–489.

56.

Yan-Ping

Xiao-Qin

Xiao Ping

, et al. Effects of chronic exposure to sodium arsenite on expressions of VEGF and VEGFR2 proteins in the epididymis of rats. Biomed Res Int 2017; 9: 2597256.

57.

Dacheux

Paquignon

. Relations between the fertilizing ability, motility and metabolism of epididymal spermatozoa. Reprod Nutrit Develop 1980; 20: 1085–1099.

58.

Kirchhoff

Specific gene expression in the human and non-human primate epididymis. In: Robaire

Hinton

(eds) The epididymis: from molecules to clinical practice. New York, NY: Kluwer Academic/Plenum Publishers, 2002, pp. 201–218.

59.

Zhu

Myles

Primakoff

. Testase 1 (ADAM 24) a plasma membrane-anchored sperm protease implicated in sperm function during epididymal maturation or fertilization. J Cell Sci 2001; 114: 1787–1794.

60.

Tulsiani

DRP

NagDas

Skudlarek

, et al. Rat sperm plasma membrane mannosidase: localization and evidence for proteolytic processing during epididymal maturation. Dev Biol 1995; 167: 584–595.

61.

Nehme

Cesario

Myles

, et al. Breaching the diffusion barrier that compartmentalizes the transmembrane glycoprotein CE9 to the posterior-tail plasma membrane domain of the rat spermatozoon. J Cell Biol 1993; 120: 687–694.

62.

Scully

Shaper

Shur

. Spatial and temporal expression of cell surface galactosyltransferase during mouse spermatogenesis and epididymal maturation. Dev Biol 1987; 124: 111–124.

63.

Saxena

Oh-oka

Kadomatsu

, et al. Behaviour of a sperm surface transmembrane glycoprotein basigin during epididymal maturation and its role in fertilization in mice. Reproduction 2002; 123: 435–444.

64.

Baker

Witherdin

Hetherington

, et al. Identification of post-translational modifications that occur during sperm maturation using difference in two-dimensional gel electrophoresis. Proteomics 2005; 5: 1003–1012.

65.

Phelps

Koppel

Primakoff

, et al. Evidence that proteolysis of the surface is an initial step in the mechanism of formation of sperm cell surface domains. J Cell Biol 1990; 111: 1839–1847.

66.

Dacheux

. New insights into epididymal function in relation to sperm maturation. Reproduction 2014; 147: 27–42.

67.

Turner

. Moving to the beat: a review of mammalian sperm motility regulation. Reproduct Fert Develop 2006; 18: 25–38.

68.

Inskeep

Hammerstedt

. Changes in metabolism of ram sperm associated with epididymal transit or induced by exogenous carnitine. Biol Reprod 1982; 27: 735–743.

69.

Xia

Reigada

Mitchell

, et al. CATSPER channel-mediated Ca²⁺ entry into mouse sperm triggers a tail-to-head propagation. Biol Reprod 2007; 77: 551–559.

70.

Carlson

Hille

Babcock

. External Ca²⁺ acts upstream of adenylyl cyclase SACY in the bicarbonate signaled activation of sperm motility. Dev Biol 2007; 312: 183–192.

71.

Aitken

Nixon

Lin

, et al. Proteomic changes in mammalian spermatozoa during epididymal maturation. Asian J Androl 2007; 9: 554–564.

72.

Fernandez-Fuertes

Narciandi

O’Farrelly

, et al. Cauda epididymis-specific beta-defensin 126 promotes sperm motility but not fertilizing ability in cattle. Biol Reprod 2016; 95: 1–12.

73.

Chinoy

Sequeira

. Effects of fluoride on the histoarchitecture of reproductive organs of the male mouse. Reprod Toxicol 1989; 3: 261–267.

74.

Kumar

Susheela

. Effects of chronic fluoride toxicity on the morphology of ductus epididymis and the maturation of spermatozoa of rabbit. Int J Exp Pathol 1995; 76: 1–11.

75.

Shashi

Khan

. Mitigating effect of Punarnava (Boerhaavia diffusa L.) on light and scanning electron microscopic alterations in cauda epididymis of fluorotic rats. ejbps 2017; 11: 503–509.

76.

Narciandi

Fernandez-Fuertes

Khairulzaman

, et al. Sperm-coating beta-defensin 126 is a dissociation-resistant dimer produced by epididymal epithelium in the bovine reproductive tract. Biol Reprod 2016; 95: 1–9.

77.

Roberts

Johnston

Nolan

, et al. Structure and function of epididymal protein cysteine-rich secretory protein-1. Asian J Androl 2007; 9: 508–514.

78.

Gibbs

Bianco

Jamsai

, et al. Cysteine-rich secretory protein 2 binds to mitogen-activated protein kinase kinase kinase 11 in mouse sperm. Biol Reprod 2007; 77: 108–114.

79.

Xuhua

Zilong

Baijuan

, et al. Fluoride reduced CRISP2 expression in testis and epididymal sperm of rats. Fluoride 2020; 53: 239–248.

80.

Zelko

Mariani

Folz

. Superoxide dismutase multigene family: a comparison of the CuZn-SOD (SOD1), Mn-SOD (SOD2), and EC-SOD (SOD3) gene structures, evolution, and expression. Free Rad Biol Med 2002; 33: 337–349.

81.

Nagae

Nakata

Takahashi

. Identification of negative cis-acting elements in response to copper in the chloroplastic iron superoxide dismutase gene of the moss Barbula unguiculata. Plant Physiol 2008; 146: 1687–1696.

82.

. Glutathione synthesis. Biochimica et Biophysica Acta—General Subjects 2013; 1830: 3143–3153.

83.

Pant

Kumar

Murthy

, et al. Male reproductive effect of arsenic in mice. Biometals 2001; 14: 113–117.

84.

Prathima

Pavani

Sukeerthi

, et al. α-Lipoic acid inhibits testicular and epididymal oxidative damage and improves fertility efficacy in arsenic-intoxicated rats. J Biochem Mol Toxicol 2018; 32: 2.

85.

Biswas

Kumar Mukhopadhyay

. Casein- and pea-enriched high-protein diet can take care of the reprotoxic effects of arsenic in male rats. Andrologia 2020; 52: 5–13560.

86.

Wares

Awal

Das

, et al. Chronic natural arsenic exposure affecting histoarchitecture of gonads in Black Bengal goats (Capra aegagrushircus). J Adv Vet Anim Res 2015; 2: 128–133.

87.

Couto-Santos

Souza

ACF

Bastos

DSS

, et al. Prepubertal exposure to arsenic alters male reproductive parameters in pubertal and adult rats. Toxicol Appl Pharmacol; 409. Epub ahead of print 15 December 2020. DOI: 10.1016/j.taap.2020.115304.

88.

Chang

Jin

Youn

, et al. Arsenic-induced toxicity and the protective role of ascorbic acid in mouse testis. Toxicol Appl Pharmacol 2007; 218: 196–203.

89.

Das

Ghosh

Manna

, et al. Taurine protects rat testes against NaAsO(2)-induced oxidative stress and apoptosis via mitochondrial dependent and independent pathways. Toxicol Lett 2009; 187: 201–210.

90.

Danielsson

Dencker

Lindgren

, et al. Accumulation of toxic metals in male reproduction organs. Arch Toxicol Suppl 1984; 7: 177–180.

91.

Recio

Robbins

Borja-Aburto

, et al. Organophosphorous pesticide exposure increases the frequency of sperm sex null aneuploidy. Env Heal Perspect 2001; 109: 1237–1240.

92.

Sakkas

Urner

Bizzaro

, et al. Sperm nuclear DNA damage and altered chromatin structure: Effect on fertilization and embryo development. Human Reprod 1998; 13: 11–19.

93.

Souza

ACF

Machado-Neves

Bastos

DSS

, et al. Impact of prenatal arsenic exposure on the testes and epididymides of prepubertal rats. Chem Biol Interact 2021; 333: 109314.

94.

Souza

ACF

Bastos

DSS

Sertorio

, et al. Combined effects of arsenic exposure and diabetes on male reproductive functions. Andrology 2019; 7: 730–740.