Index

1. INTRODUCTION

Human, animal and aquatic lives are exposed to certain metals, through different sources. Some of these metals and their oxide are lethal to health inclosing reproductive health and few are important in trace quantity for numerous physiological functions of the body. In 1746, Zinc (Zn) was discovered by German chemist and it is reported as the second most abundant transition metal in the animal body and it is the only element which seems in all enzyme classes [1]. It cannot be synthesis in the body [2] and needs daily dietary intake to meet the physiological requirements. Zn importance has been recognized many years ago. In biological system, It plays structural, regulatory or catalytic roles in more than 200 enzymes [3]. Zn is an essential element of unique biologic and public health significance [4]. Zn as an important mineral is essential for animals [5] plants [6] and microorganisms [7].

It is reported that Zn is necessary for immune function, DNA replication, RNA polymerases and some other metabolic processes [4]. Another structural role of Zn is in protein synthesis, cell replication and growth processes.  Zn is necessary for testicular maturation, wound healing, immunocompetence and neurological function [8]. It is also required for reproduction [9] gene expression [10] ossification [10] photochemical procedures of vision [11]  and antibody production [12].  In addition, Zn is counted in many nutritional supplements [13]. Studies reported that Zn is also required for normal condition of epidermis, epithelium, skin and hooves [14]. Another study reported that in mammalian genomes about 3 to 10 % of all proteins are considered to bind zinc for holding, activity and conformational changes [15].

In 1934, first time it was reported that Zn was essential for normal growth in rats [16]. After 25 year, some authors reported that Zn deficiency were detected in chicks hatched from hens, fed Zn-deficient (ZD) diets and were died 4 days after birth [17]. The chicks were described by various skeletal defects and structural abnormalities of brain [18]. Several authors [19] stated that rats fed a Zn-deficient (ZD) feed had a fewer live fetuses/litter, and were low body weight, and several skeletal and soft tissues anomalies [19]. There were common abnormalities of development, deformities of the brain, lungs, heart, and urogenital system. In addition, it comprised the misshaped heads and fused or misplaced digits of the feet.

Zn deficiency has been recognized in small laboratory animals, domestic animals, minks, and monkeys. Zn deficiency in mammals reported reduced food intake, growth retardation, testicular atrophy, swelling of feet, skin lesions, hair loss and hindered reproduction [18, 20]. Another study in human reported that chronic Zn deficiency is connected with immature testes, delayed sexual maturity, lack of appetite, immunosuppression, dwarfism, night blindness, spleen and liver abnormalities, reduced secretion of luteinizing hormone (LH) and folate deficiency [14, 21, 22].

On the other hand, Zn is thought to be an important ingredient in male reproductive system of the human and animals. Therefore, the aim of this review was to analyze the effect of Zn deficiency on reproductive profile of male rat and their promising consequences.

1.1. Zn Deficiency and its Adverse Effects

The surprising increase in the demand of Zn is mostly ascribed due to its superior antibacterial properties [22]. It has been reported that due to antimicrobial properties Zn is being utilized in the food industry as packaging and additives agent [23]. On the other hand, Zn deficiency has been documented in humans and a wide range of animals (Table 1). It shows severe effects on all phases of growth and male reproductive system in an adult. In marine invertebrates, Zn plays a significant role in pH regulation of sperm. A report of semen examination described that Zn deficiency less than 6.5 µg/L badly affected sperm motility and pH in horseshoe crab (Limulus polyphemus), sea urchins (Strongylocentrotus purpuratus), and starfish [7]. Zn deficiency causes injurious effects on esophagus, kidney, bone and testes of rats, and it is reported that in bone and plasma, alkaline phosphatase (ALP) activity is reduced in ZD pigs, cows and rats [22, 24].

Table-1. Effects of Zn deficiency on different species

Source:  Kumari, et al. [27]

Zn deficiency is associated with abnormal testicular function in humans and animals and appears to be necessary for spermatogenesis and sperm production [27]. An adult male fed ZD diet, reported testicular lesions and decreased weight of male accessory sex gland [18]. Hypogonadism and testicular atrophy is reported in ZD rats and it causes the spermatic arrest, atrophy of seminiferous tubules and interstitial cells, decreased testosterone (T) levels and serum testicular Zn concentrations [28]. In Leydig cells, Zn is necessary for normal testosterone metabolism. Calcitonin hinders flow of Zn in the isolated rat Leydig cell, but these sound effects take more than 2 days and are reported only in Zn deficiency [6]. During maturity of rats, Zn deficiency reduces the action of dipeptidyl carboxypeptidase (Dcp) enzyme in the testes and epididymis; this Dcp is essential for maturation of sperm cells and decreased activity of Dcp may cause delay in sexual maturity [29].

The literature findings demonstrated that both Zn higher and lower concentration may act as testicular toxicant in mice [30]. But, it also has been observed that excess dietary Zn may cause subnormal growth, anemia and reproductive failure in rats.

1.2. Effect on Male Reproductive System

Nutritional status plays an energetic role in human and animal reproduction (Table 2). There are some trace elements especially Zn is considered an essential ingredients for the optimal performance and normal productivity in male reproductive system. Hypogonadism is noticeable characteristic of severe Zn deficiency detected in the young and growing male rats [5]. In 1961 and 1963, Prasad reported the first case of Zn deficiency in humans [22, 31]. Hypogonadic dwarfism was recorded in Iranian and Egyptian children. Prasad proved that this was due to Zn deficiency. Another study reported that mild Zn deficiency causes the oligospermic sterility and inability in rats and men. These effects have been observed in subjects with drepanocytosis [32] and dialyzed uremia [5]. Several authors examined that oligospermia was observed in 4/5 men on a limited 30-week diet containing only 4 mg of Zn [24]. In all these anomalies, it is detected that ZD diet below the average supplementation has deleterious effects on reproductive profile of human and male rats.

Table-2. Role of Zn in Spermatogenesis

Source: Cuevas and Koyanagi [5]

1.3. Effect on Testicular Growth

The testicles have main role in sperm production (Table 3). The seminiferous tubules are considered as the structural and functional unit of testicles and weight of testes depends on the number of seminiferous tubules. Fertility is highly associated with testicles size and poor fertility often being linked with small testicles, because the process of spermatogenesis take place in the seminiferous tubules and less testicles weight is considered due to lower number or length of seminiferous tubules [33].

Table-3. Function of male reproductive system

Source:  Ibtisham, et al. [21]

It is reported that in rats Zn deficiency decreases the diameter of seminiferous tubules and also both the basal and luminal areas. Severe atrophic variations in spermatogenic cells has been described in ZD animal and deterioration of the cellular layer of the seminiferous tubules [13]. According to previous literature, ZD rats may cause the decrease in tubular diameter and decrease in the number of germinal cells and Sertoli cells. In rats [26] and men [5] Zn deficiency has been found to be associated with low testosterone (T) levels. In rats, testicular atrophy was recorded due to Zn deficiency [5]. In rats and boars, Zn deficiency may involve in decreasing the growth and development of accessory sex glands. Scrotal size has been shown to be correlated to testicular weight and testicular weight has also been associated to sperm production [23]. Taken together, In ZD rats, it has been found that there is an obvious decrease in testosterone (T) levels, seminiferous tubular diameter and in the weight of male sex organs.

1.4. Effect on Spermatogenesis

Previous studies in hypophysectomized rats clearly explained that follicle stimulating hormone (FSH) and T are main factors for the regulation of spermatogenesis [34]. FSH requires for the development of the undeveloped testes by proliferation of Sertoli cells and later development of A and B spermatogonial stem cells [6]. T alone can play an important role in spermatogenesis, but the synergistic role of FSH is essential to stabilize the characteristics of spermatogenesis [34].

Zn deficiency impairs the process of spermatogenesis by different pathways. It has been reported that in testes of rats, Zn deficiency has been linked with testicular atrophy, destruction of testicular cells and testicular function of steroidogenesis [4].  Zn deficiency also has been associated with low T level, which provides a strong proof that Zn plays a key role in the production of T, because T is produced and secreted from leydig cells, so Zn deficiency may be involve in apoptosis of the Leydig cells. T is considered an important hormone for spermatogenesis. As a result, in primary stages of spermatogenesis, Zn deficiency may be linked with apoptosis of testicular cells, as well as spermotocyte maturation phase, causing impairment in the late phase of spermatogenesis [33].

Another study reported that increased oxidative stress, high serum malondialdehyde (MDA) and high tumor necrosis factor-alpha (TNF-α) and low levels of total antioxidant activity, serum superoxide dismutase (SOD) and alpha-tocopherol was associated in ZD animals. Some factors that incidentally increase the oxidative stress by reducing the capacity of cells to resist oxidation will also causes the apoptosis [35]. In light microscopy, Zn deficiency has described the spermatogenic arrest at the phase of round and elongated spermatids. This displays that high lipid peroxidation hinder the consequence of spermatogenesis, as confirmed by microscopic examination of apoptosis in the Sertoli cell linked with oxidative stress and Zn deficiency.

It has been reported in rat study, that Zn deficiency is associated with stress and elevated cortisol levels. Stress can affect the spermatogenesis due to variable T secretion. A new invented gonadotropin-inhibitory hormone has an inhibitory effect on the hypothalamic–pituitary gonadal axis (Fig. 1). Decrease in T levels has been reported due to inhibition of the hypothalamic–pituitary gonadal axis, which may causes the variations in Sertoli cells and tightest barrier (blood-testis barrier), as a result which may affects the spermatogenesis.

Therefore, due to Zn deficiency, oxidative stress, apoptosis and reduced T production is connected with impaired process of spermatogenesis. These outcomes propose that Zn deficiency influence the reproductive system of male rats.

1.5. Effect on Sperm Morphology

The main factor in the semen examination is the sperm morphology. Sperm motility plays a fundamental role in the fertilization, because abnormal sperm impaired the fertility and leading conception more difficult. Zn is considered an important ingredient in the quality; quantity and growth of sperm, but their deficiency produce several sperm morphological abnormalities.

Zn deficiency causes the reduction of T [5] and may also involve in the degenerative variations. A recent study reported that abnormal morphology is due to proliferation of the axonemedense fibre-mitochondria complexes. In rats, the same variation was found after Fe treatment [13]. In rats testes, high Fe content and lipid peroxidation was recorded due to Zn deficiency [36]. Hence, this structural variation can be described by high oxygen free radicals formation, which is induced by Fe [7].

Another study detected that structural abnormalities are due to irregular arrangement of outer dense fibres (ODF) like uncoiling and flattening shape (fig 2). These abnormalities were not present in Fe-treated rats testes. 90% of the sperm Zn is attach to SH-groups of ODF [37]. Thus, due to Zn deficiency, Zn may be detached from SH-groups of ODF followed by their structural alteration leading to uncoiling and flattening. In addition, ODF consist of two [37] or more than two proteins [36] with diverse molecular mass and amino acid composition. The proteins are connected by SS-bond and display a keratin-like structure [37]. ODF may provide elasticity in sperm circulation [37] and may provide safety in mammalian sperm against impairment [38]. The uncoiled and flattened dense fibres in Zn deficiency may also considered as functionally damaged.

From clinical point of view, these results have more importance. Infertile men show a high quantity of sperm with abnormally developed ODF [36] and orally Zn intake has enhanced sperm motility in infertile men with astenospermia and oligospermia [39]. But, in another research this outcome of Zn treatment was not observed [20]. In male, sperm morphology exhibited a minor increase in round sperm head due to Zn deficiency [23]. Sperm with increased axonemedense fibre-mitochondria complexes were not explained. But, it is detected that it may be due to mild Zn deficiency [23] or abnormal sperm which were reduced during maturation and so could not be seen.

A similar study showed that essential fatty acid composition is influenced by Zn deficiency in rats testes. This altered fatty acid may involve in reduction of testicular function, spermatogenesis and androgenesis. These results suggest that Zn deficiency linked with abnormal testicular function is followed by alterations in membrane fatty acid composition, and thus affects the sperm integrity. However, the specific function of this process is remained unclear. It is thought that it can be due to arrested spermatogenesis or testicular necrosis. Additionally, abnormal sperm motility was found  in the Japanese eel and goat due to decreased Zn concentration in feed [11, 40]. Sertoli and Leydig cells showed insignificant morphological changes. In control study, rats testes did not show any morphological changes.

1.6. Effect on Histopathology

of androgen, like testosterone (T). FSH involves in the development of the immature testis by proliferation of Sertoli cell and later spermatogonial stem cells [7]. T alone can play an important role in spermatogenesis, but the synergistic role of FSH is essential to normalize the process of spermatogenesis [35, 42].

Deficiency of Zn has negative effect on the reproductive profile of humans and animals and it is an enormous problem to nutritionist. Normal histology of testes depends on normal testicular functions (spermatogenesis and steroidogenesis). In histopathological examination, it has been reported that ZD rat presented a number of abnormalities, compared to control group [41] which may affect sperm physiology.

A study on ZD rat showed cytological degenerative changes in germ cells of seminiferous tubules, leading to less number of germ cell, sertoli cells, interstitial cells, primary and secondary spermatogonia, and spermatocytes (sperm cells). Histological study carried by Ashrafi, et al. [3] reported that Zn deficiency in rats, explained the atrophy of seminiferous tubules in some ZD rats. An 8-week feeding trials have been conducted, containing a total of 22 ZD (Zn-deficient) and 19 ZC (Zn-control) male rats.

In histological examination, the testes of rats after 8-weeks on ZD diet again verified the previous results of several authors [3]. Complete or Partial degeneration of the germ cells epithelium had examined in 17/22 rats. It was clear that the matured testes composed of healthy tubule and consists of a less number of sperms in the epididymis. Apparently healthy but immature testicular tissue was present in 3 ZD rats and the remaining 2 exposed all degenerative phases of spermatogenesis. However, one of these, which was recently matured were examined no sperms in the epididymis. The other numerous sperms were present in the epididymis were less than the controls group. Normal mature testes including the large numbers of sperms in the epididymis was recorded in control rats. In all ZD rats, the interstitial cells were detected in the testes, except for reduced size, but looked normal. In ZD rats, decreased size of epithelial layer, small acini, less secretion were observed in all accessory sex organs but no additional pathological variations were detected in these tissues. In ZD rats, the dorsolateral prostate glands

associated to Zn- specific stain were negative or slightly positive.

In the control groups, the lateral edges of the glands revealed a clear positive reaction. In ZD rats, gonadotrophs and thyrotrophs were characterized in pituitary glands stained with a glycoprotein stain compared with the control groups. In ZD rats, testes demonstrated atrophy of the tubular epithelium. Taken together, ZD group influence the male reproductive system of the rats and cause the reduction in all sex organs, testicular atrophy, clear atrophy of the tubular epithelium, and reduced Zn concentrations in testes, epididymis, and dorsolateral prostate glands.

2. CONCLUSION

The overall consequences of this overview provide a confirmation that certain essential elements especially Zn deficiency may affect the male rat reproductive system inclosing the function of spermatogenesis, sperm motility, secretion of accessory glands, fertility, T-level and antioxidant defense system. Experimental studies on rats explained that Zn deficiency depends on dose and time duration and their effects are also variable. The available data reported that 5 mg/day per rat Zn supplementation is considered as optimum level without causing any harmful effects on libido and sexual ability of male rats. Therefore, it is recommended that optimum amount of Zinc supplementation should be used to avoid the deleterious effects on male reproductive physiology.

Abbreviation

Zn; Zinc, T; Testosterone, FSH; Follicle stimulating hormone, LH; Luteinizing hormone, ZD; Zinc deficient, ZC; Zinc control , ALT; Alanine aminotransferase, ALP; Alkaline phosphatase , LDH; Lactate dehydrogenase, ZnO nP; Zinc oxide nanoparticle, ZnO mP; Zinc oxide microparticle, MDA; Malondialdehyde , TNF-α; Tumor necrosis factor-alpha,  SOD; Serum superoxide dismutase , GnIH; Gonadotropin-inhibitory hormone , HPG; hypothalamic–pituitary gonadal, ODF; Outer dense fibres, ZnCl2; Zinc chloride, ZnSO4; Zinc sulfate, Fe; Iron, SH; Sulfhydral, S-S bond; Disulfide bond, DCP; Dipeptidyl carboxypeptidase

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Fig-1. FSH and LH plays a key role in sperm production and in the process of spermatogenesis. But, various studies reported that gonadotropin-inhibitory hormone (GnIH) negatively affects the hypothalamic–pituitary gonadal axis (HPG axis) and disturb the testosterone level and ultimately hinder the spermatogenesis.

Source: Shahraki, et al. [1]

Fig-2. Sperm morphology plays an important role in ova fertilization. Healthy sperm consists of normal head, middle and tail piece. By electron microscopic examination of rat sperm, it is reported that Zn deficiency affects the middle piece (ODF) of sperm and produce the sperm structural abnormalities. As consequences it may lead to infertility.

Source: Yatoo, et al. [2]