Testicular apoptosis after dietary zinc deficiency: Ultrastructural and TUNEL studies

Abstract

The present study was conducted in Wistar rats to determine whether prepubertal dietary zinc deficiency causes apoptotic changes in testes. Prepubertal male Wistar rats (40–50 gm) were divided into 3 groups: zinc control (ZC), pairfed (PF), and zinc deficient (ZD). Control and pairfed groups were given a 100 ppm zinc diet while the deficient groups received 1 ppm zinc diet for 2 and 4 weeks (w), respectively. Ultrastructural studies revealed several apoptotic features such as wavy basement membrane, displaced nuclei, chromatin condensation, plasma membrane blebbing, nuclear membrane dissolution, loss of inter-Sertoli cell junctional complexes, and intercellular bridges and deformed mitochondria. A variable spectrum of sperm defects had also been visualized e.g., acrosomal deformities such as decapitation and a ring of condensed chromatin around the nuclear periphery, deformed sperm heads with a condensed nucleus, tail-elements with superfluous cytoplasm, and damage to the mitochondrial sheath and aggregation of spermatozoa within the membrane. This was further supported by TUNEL studies. Apoptotic index, epididymal sperm concentration, motility, and fertility index also revealed a significant (P < 0.05) decrease in zinc deficient groups (2 and 4 w) when compared with their respective control and pairfed groups. All the above findings are indicative that changes observed in the testes after dietary zinc deficiency are due to the onset of apoptosis. Increased apoptotic degeneration in testes may cause irreversible changes in the germ cells associated with decreased epididymal sperm concentration, motility, and fertility index which contributes to the low efficiency of spermatogenesis thereby indicating a possible role of zinc in fertility.

Keywords:

• apoptosis
• male fertility
• spermatogenesis
• testes
• zinc deficiency

Introduction

Zinc, an essential trace element, is ubiquitously present throughout all biological systems and has abundant and varied functions within these systems [Kaji 2001; Auld 2009; Maret 2009, Tuerk and Fazel 2009]. Zinc deficiency, a global problem, contributes negatively to human health and development [Maret and Sandstead 2008; Walingo 2009]. Moderate and marginal zinc deficiency, due to inadequate intake, is by far more common throughout the world than severe deficiency [Al-Timimi 2009], especially in those stages of life when requirements are increased such as infancy, childhood, and pregnancy [Fesharakinia et al. 2009, Hess and King 2009; Uriu-Adams and Keen 2010]. Zinc affects different aspects of mammalian reproduction [Dissanayake et al. 2009; Björndahl and Kvist 2010]. Low zinc intake by young males of several species including humans, interferes with normal sexual development [Bedwal et al. 1991; Saxena et al. 1993; Bedwal and Bahuguna 1994; Kaji 2001; Dissanayake et al. 2009] and leads to gonadal dysfunction. Zinc also plays an essential role in the synthesis and secretion of luteinizing hormone (LH), follicle-stimulating hormone (FSH) and prolactin, gonadal differentiation, testicular steroidogenesis, and genetic expression of steroid hormone receptors [Bedwal et al. 1991; Bedwal and Bahuguna 1994; Ali et al. 2007]. Insufficient zinc initiates programmed cell death in hepatocytes, glioma, monocytes, fibroblasts, kidney, brain, prostate, and testicular cells [Fraker 2005; Yan et al. 2008; Adamo and Oteiza 2010]. Multiple studies have demonstrated the essential role of Zn²⁺ for cell survival. Zinc deficiency accelerates apoptosis both in vitro and in vivo [Zalewski et al. 1991; Duffy et al. 2001]. The ability of Zn²⁺ to prevent DNA fragmentation has been attributed to its inhibitory effect on Ca²⁺ and Mg²⁺-dependent endonucleases [Giannakis et al. 1991; Zhivotovsky et al. 1994], although there are numerous targets of zinc in the apoptotic cascade such as caspase-3, caspase-6, bcl-2, p53, etc. [Takahashi et al. 1996; Stennicke and Salvesen 1997; Wolf et al. 1997; Fukamachi et al. 1998; Chai et al. 1999; Jankowski-Hennig et al. 2000; Fanzo et al. 2001; Ho and Ames 2002; Ho et al. 2003], imbalance of either would lead to the initiation of apoptosis. Abnormally accelerated apoptosis of germ cells may lead to an imbalance of cell proliferation and death resulting in spermatogenic impairment [Hassan et al. 2009]. Studies have shown that germ cell degeneration in testes involves an apoptotic mechanism [Billig et al. 1995; Henriksen et al. 1995]. The present study was therefore conducted on Wistar rats to determine whether prepubertal dietary zinc deficiency causes apoptotic changes in testes.

Results

Ultrastructural Studies

Normal ultrastructural details of basement membrane, spermatogonia, spermatocytes, spermatids, spermatozoa, and Sertoli and Leydig cells are evident in the control groups after 2 and 4 weeks (Figs1A and 2A). The PF group after 2 and 4 w also showed somewhat normal features except for the appearance of lipid droplets, phagocytic vacuoles, and abundant multivesicular bodies in the cytoplasm (Figs 1B and 2B). The 2 w ZD group testes displayed a wavy basement membrane with basal lamina, endothelial cells, and disrupted collagen fibres. Cytoplasm showed extensive vacuolization, disruption of intercellular bridges, and abundant multivesicular bodies (Fig. 1C). The ultrastructure of primary spermatocyte nuclei of the 2 w ZD group illustrated early apoptotic changes with chromatin condensation on the periphery. Cytoplasm revealed extensive damage with a large number of multivesicular bodies (Fig. 1D). The nuclei of the 2 w ZD group Leydig cell exhibited peripheral heterochromatin but the juxtanuclear region of cytoplasm showed extensive vacuolization, dilated smooth endoplasmic reticulum (ER), and abundant multivesicular bodies (Fig. 1E). The variable degree of degeneration seems to be more pronounced after 4 w of zinc deficiency. Testes from the 4 w ZD group revealed several apoptotic features such as wavy and thickened basement membrane, dissolution of collagen fibres, dense, irregular, and displaced nuclei, chromatin condensation, plasma membrane blebbing, nuclear membrane dissolution, vacuolization, loss of inter-Sertoli cell junctional complexes and intercellular bridges, and deformed mitochondria (Fig. 3A, B, C, and H). A variable spectrum of sperm defects like acrosomal deformities such as decapitation and a ring of condensed chromatin around the nuclear periphery, deformed sperm heads with a condensed nucleus, tail elements with superfluous cytoplasm, damaged mitochondrial sheath, and aggregation of spermatozoa enclosed by cytoplasm and a common membrane (Fig. 3D, E, F, and G) were also visualized.

Figure 1.  Electronmicrograph of control, pairfed, and zinc deficient groups after 2 weeks. The scale bar = 2 µm. (A) Electronmicrograph of 2ZC group testes illustrating normal features of basement membrane (BM) showing basal lamina (bl), collagen fibres (cf), and endothelial cells (ed). Leydig cell nucleus (Nu) revealed characteristic peripheral heterochromatin (Hc). Presence of Type-A Spermatogonia (Sg) with its distinctive spherical nucleus and dense nucleolar body in association with the inner aspect of the nuclear envelope (Ne) is evident along with a large number of mitochondria (mt) with tubular cristae, rough endoplasmic reticulum (rER), ribosomes (r) and lysosomes (Ly) in the cytoplasm. (B) Electronmicrograph of 2PF group testes showing disrupted basement membrane (BM) with basal lamina (bl), collagen fibres (cf), and myoid cell process (My). Sertoli cell nucleus (Sc) exhibiting indentation and cytoplasm showing a large number of multivesicular bodies (mvb), lipid droplets (Ld), dilated smooth endoplasmic reticulum (sER), rough endoplasmic reticulum (rER), inter-Sertoli cell junctional complexes (Is), and parts of spermatocyte nuclei (Sp) with synaptonemal threads (St). (C) Ultrastructure of testes of 2ZD group testes displaying wavy basement membrane (BM) with basal lamina (bl), endothelial cells (ed), and disrupted collagen fibres (cf). Cytoplasm showing extensive vacuolization (V) and disruption of intercellular bridges (Icb), polyribosomes (pr), ribosomes (r), smooth endoplasmic reticulum (sER), rough endoplasmic reticulum (rER), abundant multivesicular bodies (mvb), and displaced Type A spermatogonium nucleus (Nu) showing chromatin conglomerates (Ch) with distinct nuclear envelope (Ne). (D) Ultrastructure of primary spermatocyte nuclei (Sp) of 2ZD group testes illustrating early apoptotic change viz. chromatin condensation (Cc) on the periphery. Extensive cytoplasmic damages with a large number of multivesicular bodies (mvb) are also distinct. (E) Electronmicrograph of 2ZD group testes illustrating juxtanuclear region of cytoplasm showing extensive vacuolization (V), dilated smooth endoplasmic reticulum (sER), and aggregation of multivesicular bodies (mvb). Peripheral heterochromatin (Hc) of Leydig cell nucleus (Nu) are evident.

Figure 2.  Electronmicrograph of control and pairfed groups after 4 weeks. The scale bar = 2 µm. (A) Electronmicrograph of testes of 4ZC group illustrating normal features of basement membrane (BM) with basal lamina (bl), collagen fibres (cf), and endothelial cells (ed). Cytoplasm shows characteristic peripheral arrangement of mitochondria (pmt) with dilated intra-cristal space, rough endoplasmic reticulum (rER), and inter-Sertoli cell junctional complexes (Is). Sertoli cell (Sc) and Leydig cell (Lc) are distinct. The nucleus of Leydig cells (Lc) reveals peripheral heterochromatin (Hc). (B) Electronmicrograph of testes of 4PF group showing basement membrane (BM) with basal lamina (bl), collagen fibres (cf), and endothelial cells (ed). Sertoli cell (Sc) with lobulated nucleus (Nu), mitochondria (mt), smooth endoplasmic reticulum (sER), lysosomes (Ly), phagocytic vacuoles (pgv), and mutivesicular bodies (mvb) seen in large number in the cytoplasm.

Figure 3.  Electronmicrograph of zinc deficient groups after 4 weeks showing several apoptotic features. The scale bar = 2 µm (A, B, C, D, E, and G); The scale bar = 1 µm (F and H). (A) Electronmicrograph of testes of 4ZD group showing basement membrane (BM) with layer of basal lamina (bl), collagen fibers (cf) and cytoplasmic extensions of endothelial cells (ed). Degenerative changes are evident in the Sertoli cell (Sc) cytoplasm. Multivesicular bodies (mvb), vacuolization (v), and few mitochondria (mt) are distinct. (B) Electronmicrograph of 4ZD group testes showing disrupted spermatogenic epithelium (BM), dissolution of collagen fibres (cf) and displaced Type B spermatogonium nucleus (Nu) with condensed chromatin (Cc) associated with the nuclear envelope (Ne). Cytoplasm reveals vacuolization (V) with complete loss of junctional complexes. Lysosomes (Ly), deformed mitochondria (Amt), polyribosomes (pr), smooth endoplasmic reticulum (sER), and lipid droplets (Ld) are evident. (C) Ultrastructure of 4ZD group testes displaying primary spermatocyte nucleus (Nu) showing dissolution of nuclear envelope (Ne) and chromatin conglomerates (Ch). Cytoplasm reveals ovoid mitochondria (mt) and interrupted intercellular bridges (Icb). Cytoplasmic dissolution (CD) is also evident in some areas. (D) Electronmicrograph of testes of 4ZD group displaying detachment of acrosomal cap (ac) from head region of spermatid nucleus (Nu) just above the sub-acrosomal space (sas). One of the apoptotic spermatid (Asd) showing ring of condensed chromatin (Cc) around the nuclear periphery. Presence of mitochondria (mt) and lysosomes (Ly) are distinct. Degeneration of cytoplasm (CD) between the two spermatids is evident. (E) Electronmicrograph of testes of 4ZD group revealing deformed sperm heads (DS) with condensed nucleus (Nu), and disintegrated nuclear envelope (Ne). Cytoplasm shows few deformed mitochondria (Amt), multivesicular bodies (mvb), and dissolution of cytoplasm (CD) in some areas. (F) Cross sectional view of mid-piece (mp), principal piece (pp), and end piece (ep) of 4ZD spermatozoa tail revealing outer dense fibres (df) and central fibres (cf), mitochondrial sheath degeneration (mts) and retention of superfluous cytoplasm (sfc). (G) Aggregation of spermatozoa enclosed by a cytoplasm and common membrane (M). (H) Electronmicrograph of testes of 4ZD group displaying late apoptotic stage of Leydig cell nucleus (Nu) in which both chromatin (Cc) and cytoplasm have condensed. Peripheral heterochromatin (Hc) is distinct. Blebs (B) in the nuclear membrane of Leydig cell is also evident.

TUNEL Assay

The 2 and 4 w ZC control groups exhibited negative TUNEL staining in the majority of tubules although few tubules revealed TUNEL positive germ cells (Figs 4A and 5A). The PF 2 and 4 w groups only showed positive staining in a few germ cells (Figs 4B and 5B). An increase in the frequency of TUNEL positive cells was evident in the case of the 2 w ZD group as compared to the control group (13.85 ± 0.21 vs. 1.90 ± 0.08) (Fig. 4C; Table 1). As illustrated in Figure 5C and Table 1, a higher apoptotic rate of germ cells were also observed in the case of the 4 w ZD group when compared with its respective control group (31.47 ± 0.20 vs. 2.62 ± 0.12).

Figure 4.  Microphotograph of TUNEL staining after 2 weeks for control, pairfed, and zinc deficient groups. Note: 400X. (A) Microphotograph of testes of 2ZC group revealing negative TUNEL staining in all the germ cells. (B) Microphotograph of testes of 2PF group showing few TUNEL positive germ cells (arrows). (C) Microphotograph of testes of 2ZD group exhibiting few TUNEL positive apoptotic spermatogonia and primary spermatocytes (arrows).

Figure 5.  Microphotograph of TUNEL staining after 4 weeks for control, pairfed, and zinc deficient groups. Note: 400X. (A) Microphotograph of testes of 4ZC group exhibiting negative TUNEL staining in all the germ cells. (B) Microphotograph of testes of 4PF group showing few TUNEL positive cells (arrows). (C) Microphotograph of testes of 4ZD group revealing increase in the number of apoptotic cells. Positive staining seen in spermatogonia, primary spermatocytes, and giant cells (Gi) (cap-phase spermatids) (arrows).

Table 1. Apoptotic Index (%) in testes of Wistar rats.

Groups	2 Weeks	4 Weeks
ZC	1.90 ± 0.083	2.62 ± 0.12
PF	5.70 ± 0.17^a*	10.34 ± 0.16^a*
ZD	13.85 ± 0.21^b*,c*	31.47 ± 0.20 ^b*,c*

^aZC vs. PF; ^bZC vs. ZD; ^cPF vs. ZD; *Significant (P < 0.05). Mean ± SEM; Note: multiple comparisons of means were performed separately for 2 week and 4 week sub-groups.

Epididymal Sperm Concentration, Sperm Motility, and Fertility Index

A significant (P < 0.05) decrease in epididymal sperm concentration, sperm motility, and fertility index were evident in zinc deficient groups after 2 and 4 w when compared with their respective control and PF groups. Parallel observations are summarized in Tables 2 and 3 when comparisons were made between ZC and PF and PF and ZD groups in both sets of experiments.

Table 2. Epididymal sperm concentration (x 10⁶/ ml) and motility (%) of Wistar rats.

Groups	Sperm Concentration (× 10^6/ ml)		Sperm Motility (%)
	2 Weeks	4 Weeks	2 Weeks	4 Weeks
ZC	4.66 ± 0.10	14.83 ± 0.21	33.00 ± 0.25	64.00 ± 0.36
PF	1.10 ± 0.043^a*	5.92 ± 0.13^a*	20.00 ± 0.39^a*	40.00 ± 0.47^a*
ZD	0.82 ± 0.038^b*,c*	2.84 ± 0.20^b*,c*	10.00 ± 0.25^b*,c*	7.00 ±0.42^b*,c*

^aZC vs. PF; ^bZC vs. ZD; ^cPF vs. ZD; *Significant (P < 0.05). Mean ± SEM; Note: multiple comparisons of means were performed separately for 2 week and 4 week sub-groups.

Table 3.  Fertility Index of male Wistar rats.

Groups	Number of females cohabited with males	Number of males siring offspring	Fertility Index (%)
2ZC	20	9	99.80 ± 0.13
2PF	20	7	68.00 ± 1.33^a*
2ZD	20	5	44.00 ± 2.66^b*,c*
4ZC	20	10	99.00 ± 1.00
4PF	20	6	57.00 ± 1.52^a*
4ZD	20	2	16.00 ± 1.63^b*,c*

n = 10 for each group; ^aZC vs. PF; ^bZC vs. ZD; ^cPF vs. ZD; *Significant (P <0.05). Mean ± SEM; Note: multiple comparisons of means were performed separately for 2 week and 4 week sub-groups.

Discussion

Morphological alterations of the nucleus, cell-membrane, cytosol, and mitochondria are apparent during apoptotic cell death. Ultrastructurally, apoptosis is characterized by cell volume shrinkage, chromatin condensation, blebbing of the nuclear envelope, fragmentation of the nucleus, cytoplasmic vacuolization, condensation of the cytoplasm, and disintegration of the cell into membrane bound remnants termed apoptotic bodies [Kerr et al. 1972; Widlak et al. 2002; Hassan et al. 2009]. Most organelles in the cytoplasm retain their normal appearance only during the early stages of apoptosis, but at later stages cytoplasmic organelles are no longer identifiable [Erkkilä et al. 1998]. The present study demonstrated severe atrophic changes which increased with the duration of zinc deficiency. Electron microscopy revealed several apoptotic features. The tunica propria of seminiferous tubules from control and PF groups were comprised of elongated endothelial cells surrounded by extracellular matrix (ECM) consisting of basal lamina and thin collagen fibres. The basement membrane of the 2 w ZD group was wavy with disrupted collagen fibres and peritubular myoid cells, a significant component of blood-testes barrier that ensures the structural cohesion and contraction of seminiferous tubules [Dym and Fawcett 1970]. After 4 w, the degeneration was more prominent. Disruption of lamina propria at several areas and dissolution of collagen fibres were also observed. Bahuguna and Bedwal [1998] also reported alterations in the microenvironment of seminiferous tubules owing to perturbations in the boundary layer. Dirami et al. [1995] reported that basement membrane plays a crucial role in Sertoli cell survival in vitro. Apoptosis is induced by disruption of the interactions between normal epithelial cells and ECM [Frisch and Francis 1994]. It is possible that the cooperation between Sertoli cells and peritubular myoid cells might have been disrupted in zinc deficient groups and as a result an abnormality in the synthesis of ECM could have occurred, leading to the disruption in the boundary tissues of the seminiferous tubules.

In the present study, spermatogonia of ZC and PF group showed normal structure. In comparison the deficient groups, presented dislocated spermatogonia with irregular cell boundary and dense chromatin material associated with the nuclear envelope. Cytoplasm showed vacuolization and loss of junctional complexes. Electron microscopic (EM) studies by others [Erkkilä et al. 1998; Pentikäinen et al. 1999] have revealed dense nuclear chromatin with numerous vacuoles in the cytoplasm during the early stages of apoptosis in spermatogonia.

The primary spermatocytes of control groups exhibited normal architecture. The 2 and 4 w PF groups also exhibited normal features, although degeneration was evident around the nucleus that could be due to low intake of essential nutrients as a result of a restricted food supply. In comparison, primary spermatocytes of the 2 w deficient group showed early apoptotic change i.e., chromatin condensation in crescent form on the periphery of nucleus with a few scattered clumps, a feature also reported by authors [Kerr et al. 1972; Erkkilä et al. 1997]. After 4 w of zinc deficiency, primary spermatocytes showed nuclear envelope disruption and chromatin conglomerates. The intercellular bridges were disrupted and the number of healthy mitochondria reduced. Intercellular bridges are necessary for sharing molecular signals between rat spermatids. They may function in a similar way in spermatogonia and primary spermatocytes [Ventelä et al. 2003]. The disruption of intercellular bridges in zinc deficiency is consistent with the alteration in pro-apoptotic signals in spermatogonia and primary spermatocytes. Erkkilä et al. [1998] also reported that during the later stages of primary spermatocyte apoptosis, the structure of nuclear envelope started to disperse and finally disappeared. The presence of chelatable zinc in pre-leptotene spermatocytes has been suggested to play an essential role at the onset of meiosis [Sorensen et al. 1998]. Zinc deficiency might affect spindle formation hence cell division through microtubules which are required for the formation of spindle fibres where zinc is one of the essential ions for polymerization [Hesketh 1984].

Ultrastructurally, spermatids of the 4 w ZC and PF groups presented an acrosomal complex with an intact outer- and inner-acrosomal membrane and homogenous acrosomal content. However, the 4 w ZD spermatids showed acrosomal deformities such as decapitation (i.e., detachment of the acrosomal cap from the head region) and a ring of condensed chromatin around the nuclear periphery. Their cytoplasm revealed loss of intercellular bridges, lysosomes, and deformed mitochondria. These features have been observed by others [Merker and Günther 1997; Erkkilä et al. 1998]. The final stages of spermatid differentiation, which include cytoplasmic modeling and the formation of the residual body [Russell 1979], were adversely affected by zinc-deprivation. Barney et al. [1968] also reported that the transformation of spermatids into spermatozoa is inhibited in zinc deficient rats. Dramatic biochemical and ultrastructural changes in nuclei occur as mammalian spermatids develop into spermatozoa. CREM (cyclic AMP response element modulator) knock-out mice are sterile due to arrest of spermatogenesis at the round spermatid stage [Nantel et al. 1996]. Infertility is due to lack of expression of key post-meiotic genes i.e., protamines 1 and 2, transition proteins TNP1 and TNP2, proacrosin, and calspermin [Sassone-Corsi 1998]. CREM activity in post meiotic-germ cells is enhanced by its association with LIM only protein, ACT (Activator of CREM) in the testis [Fimia et al. 1999; Palermo et al. 2001]. ACT is composed of four and a half LIM domains each containing two zinc finger motifs that facilitate protein-protein interactions [Fimia et al. 1999], thus suggesting a role of zinc in spermatogenesis. The present study displayed abnormalities in the condensation of nuclei of mature spermatids and spermatozoa which suggests that zinc deficiency might alter the transition process by causing changes in nucleoproteins of spermatozoa as it has been reported that zinc is also involved in histone displacement in the process of nuclear condensation during spermiogenesis [Hunt and Johnson 1990].

The cross sectional structural view of the mid piece of the spermatozoa tails of the 4 w ZC and PF groups appears normal although the degeneration of the mitochondrial sheath with blebs in the membrane of the principal piece and intermediate filaments were observed in 4 w PF groups. The mitochondrial sheath of mid piece and fibrous sheath of tail regions of spermatozoa of the 4 w ZD groups showed degeneration along with superfluous cytoplasm. This group also showed deformed sperm heads with condensed nuclei, a disintegrated nuclear envelope, cytoplasmic dissolution, and aggregation of 5–10 spermatozoa enclosed by cytoplasm and a common membrane. These structures presented a process involved in the separation of spermatids into individual spermatozoa that might have also been affected by zinc deficiency and hence might increase the frequency of abnormal, double, triple, or even more spermatozoa with defective structures in the ejaculate. Parallel were the findings of Dinsdale and Williams [1980], Wallace et al. [1984], Bedwal and Bahuguna [1994], Merker and Günther [1997], and Merrells et al. [2009]. Zinc is known to influence the motility, viability, and morphology of spermatozoa all of which are vital for normal sperm physiology [Ebisch et al. 2007; Merrells et al. 2009]. Omu et al. [2008] reported that zinc therapy reduces asthenozoospermia through several mechanisms preventing oxidative stress, apoptosis, and sperm DNA fragmentation. In the sperm nucleus, zinc contributes to stability of the quarternary structure of chromatin and preserves genomic integrity [Wallace et al. 1984; Kvist et al. 1988; Bedwal and Bahuguna 1994; Ho et al. 2003; Song et al. 2009; Björndahl and Kvist 2010]; a model of which has been proposed [Björndahl and Kvist 2011]. Production of reactive oxygen species (ROS) by spermatozoa is a normal physiological process [Ebisch et al. 2007; Shamsi et al. 2008] but high levels of ROS are potentially toxic to sperm quality and function [Saleh and Agarwal 2002]. Mammalian spermatozoa are rich in polyunsaturated fatty acids (PUFA) which are vulnerable to attack by ROS and membrane lipid peroxide ion mediated by lipid peroxidation [Dandekar et al. 2002; Zalata et al. 2004]. Previous reports from our laboratory showed an increased lipid peroxidation (TBARS) concentration in testes, and caput and cauda epididymis of zinc deficient Wistar rats [Nair et al. 2005]. Lipid peroxidation of PUFA molecules of plasma membrane of spermatozoa is largely responsible for defective spermatozoa and male infertility [Aitken et al. 1993]. Lipid peroxidation causes leakiness of sperm plasma membrane which in turn losses its fluidity and integrity [Aitken et al. 1993], damaging the mid-piece and axonemal complex leading to decreased concentration and loss of motility as observed in the present study, since disruption of the testicular homeostatic mechanism influences considerably the epididymal reserves and decreases capacitation [Tramer et al. 1998; Dandekar et al. 2002]. Zinc deficiency adversely affects sperm integrity and modulates testes fatty acid composition by interrupting essential fatty acid metabolism thereby affecting overall testicular function [Merrells et al. 2009]. Ultrastructural observations of the present study exhibited several sperm defects such as tail-elements with superfluous cytoplasm and degeneration in mitochondrial sheath which further supported that zinc deficiency affects sperm motility. Henkel et al. [1999] reported the importance of zinc during epididymal sperm maturation for functional competence of the outer dense fibers and, therefore, motility. A low content of nuclear zinc would impair the structural stability of chromatin and thereby increase the vulnerability of the male genome [Kvist et al. 1988]. This may be one of the reasons for affecting the fertilizing capacity of spermatozoa. This is further supported by a decrease in fertility index in the 2 and 4 w ZD groups as observed in the present study by cohabiting the zinc deficient males with proven females.

Leydig cell nuclei of the 2 and 4 w ZC and PF groups were more or less elongated with heterochromatin masses present towards the periphery. The 2 w ZD groups displayed degeneration of cytoplasm along with the remnants of cell organelles, dilated smooth endoplasmic reticulum (sER), lipid droplets, vacuolization, and abundant multivesicular bodies (MVBs). Further, the degeneration of Leydig cell was more prominent after 4 w of zinc deficiency as evident by condensation of both cytoplasm and chromatin with larger heterochromatin areas. Hesketh [1982] reported that Leydig cells from zinc-deficient animals were smaller with abnormal endoplasmic reticulum.

The highly coordinated nature of spermatogenesis requires intimate functional and junctional communication between Sertoli and germ cells [Sluka et al. 2006]. In the present study, the ovoid nucleus of Sertoli cells in the 2 and 4 w ZC and PF groups was characterized by a deep indentation with a well-defined dense nucleolus. The Sertoli cells of the 2 w ZD group revealed normal structural features, whereas the 4 w ZD group nucleus exhibited small indentations with loss of specialized intercellular junctions. The degenerative changes of Sertoli cells accounts for the increased apoptotic cell number which may be due to loss of its phagocytic activity. Detachment of epithelial cells from the matrix [Dirami et al. 1995] and the absence of basement membrane for Sertoli cells [Frisch and Francis 1994] results in apoptosis. The present study emphasized that altered Sertoli cell structures i.e., appearance of vacuoles due to cytoplasmic degeneration and morphological disruption of Sertoli cell junctional specializations might be the major cause of extensive testes deterioration. This supports the notion that Sertoli cells are sensitive to zinc deficiency that mediates the disruption of spermatogenic epithelium. These alterations may be the main cause of germ cell loss by apoptosis and tubular atrophy in zinc deficient rats.

The major hallmark of apoptosis in the nucleus is the internucleosomal fragmentation of double stranded DNA into fragments of 180 to 200 base pair length [Saraste and Pulkki 2000]. During apoptosis, caspase-3 cleaves the inhibitor allowing the nuclease to cut the chromatin [Rao et al. 1996]. Using terminal deoxynucleotidyl transferase (TdT) dUTP nick end labelling (TUNEL) method, degenerating cells could probably be identified as primary spermatocytes and round spermatids located in the adluminal compartment. Ultrastructural studies also confirmed degenerative changes in primary spermatocytes and round spermatids. TUNEL positive stained cells were equivalent to the observations of ultrastructural studies enabling the determination of cells undergoing apoptosis. Rare apoptotic spermatogonia displaced from the basal lamina were evident in ultrastructural studies. Further, both in the 2 and 4 w ZD groups primary spermatocytes and cap phase spermatids were prominently affected. Li et al. [1998] observed a significant (P < 0.01) increase in apoptotic number of spermatogenic cells after zinc deficiency. Nodera et al. [2001] also reported the appearance of TUNEL positive cells after 3 w of zinc deficiency in testes and thymus of 7 w old Sprague Dawley rats. Abnormally accelerated apoptosis of germ cells may lead to an imbalance of cell proliferation and death resulting in spermatogenic impairment [Takagi et al. 2001; Kimura et al. 2003]. The correct balance between the apoptotic pathways is important for the maintenance of genomic integrity while preventing unnecessary cell death [Clarke and Allan 2009]. In the present study, apoptotic frequency was more pronounced after 4 w of the experiment, hence it can be concluded that prolonged zinc deficiency enhances apoptosis of testicular cells.

Increased apoptotic degeneration in testes may cause irreversible changes in the germ cells associated with decreased epididymal sperm concentration, motility, and fertility index. Together, the above findings are indicative that the onset of apoptosis observed in the testes can be induced by dietary zinc deficiency, which contributes to the low efficiency of spermatogenesis thereby indicating a possible role of zinc in fertility.

Materials and Methods

The composition of basal experimental diet (per Kg) was prepared as follows: egg albumin - 180 g, corn oil – 100 g, corn starch – 443g, sucrose – 200 g, cellulose – 30 g, choline chloride – 2g, AIN-76 salt mixture – 35g; AIN-76C vitamin-antibiotic mixture – 10 g; DL-methionine – 7g; D-Biotin - 20 mg. Zinc contents of basal diets from each lot was estimated at 213.9 nm in air-acetylene flame on GBC 902 Atomic Absorption Spectrophotometer (AAS) and zinc concentrations were adjusted to 1.0 ppm and 100 ppm by addition of appropriate amounts of zinc sulphate. Sixty colony bred male pre-pubertal Wistar rats (30 – 40 d; 40–50 gm) were divided into three groups (each comprised of 10 animals in a 2 and 4 w sub group: control, pairfed, and zinc deficient): (i) ZC (100ppm zinc diet), (ii) PF (100ppm zinc diet but the amount of feed was equal to the (average) amount consumed by the zinc deficient group the previous day, this group was run so as to study starvation effects caused by reduced intake of diet and stress effects of synthetic diet), and (iii) ZD (1.00ppm zinc diet).

The animals were housed individually in polypropylene cages with stainless steel grills. The polypropylene cages, grills, and bottles were washed with detergent solution, demineralized water and finally were rinsed in 10% EDTA so as to avoid any contamination and subsequent removal of zinc. The experiments were set for 2 and 4 w and were approved by the Departmental Ethics Committee, Department of Zoology, University of Rajasthan, Jaipur, India. Animals were anaesthetized under light ether anesthesia and testes were excised, cleaned off of extraneous tissues, and processed.

Ultrastructural Studies

Testes were removed and immersed quickly in Karnovsky's fixative, cut into small pieces under a drop of fixative and again kept in fresh fixative for 4 h. After 4 h, tissues were washed frequently in cacodylate buffer (pH – 7.2) and kept overnight at 4°C. The pieces were osmicated with Caulfield's fixative for 2 h at 4°C and then washed in cold TDW (giving 4–5 changes) and brought to room temperature. They were then dehydrated in ethanol series, cleared in dry acetone, and embedded in araldite CY 212. Semithin sections were cut, stained with toludine blue, and observed under a light microscope. Ultrathin sections (30 - 40nm) were stained with lead citrate and uranyl acetate. Twenty loci of testicular tissue of different groups were sampled and 10 were taken into account. Observations and micrographs were taken on a Transmission Electron Microscope (Morgagni 268D, FEI Company, The Netherlands; All India Institute of Medical Sciences, New Delhi).

TUNEL

In situ detection (TA 4625, R and D Systems, Inc, Minneapolis, USA) was used to identify apoptotic cells by detecting DNA fragmentation through a combination of enzymology and immunohistochemistry techniques. Bouin's fixed (5 µm) sections of testes were used. In brief, after deparaffinization and rehydration, tissue sections were incubated with proteinase K, washed in distilled water, and then incubated in phosphate buffered saline solution (PBS) containing 3% hydrogen peroxide for 10 min to quench endogenous peroxidase activity. Next, biotinylated nucleotides were detected by using streptavidin-horseradish peroxidase conjugate followed by the substrate diaminobenzidine (DAB). The enzyme reaction generates an insoluble coloured precipitate where DNA fragmentation has occurred.

Quantification of apoptotic cells

Nine testes from each experimental group were used for the study. The apoptotic index was calculated by dividing the number of TUNEL positive germ cells per seminiferous tubule by the total number of germ cells per seminiferous tubule and the result was multiplied by 100. Mean AI of each case was calculated. At least 20 apoptotic tubule cross sections (TCS) within 200 TCS in each testis were taken into account.

Epididymal sperm concentration and sperm motility

Epididymal sperm concentration and motility were assessed by the method of Linder et al. [1986]. The epididymal sperm were collected by cutting the epididymis into small pieces and flushing the sperm in normal saline. The sperm collected was centrifuged at 225 × g for 10 min. The pellet was resuspended in 2.0 ml of normal saline. An aliquot of this solution was placed in a Neubauer hemocytometer and sperm counts, motile and non-motile sperms were counted. Sperm motility was expressed as a percent of motile sperm of the total sperm counted.

Fertility index

Ten males from each experimental group were reared until adulthood on standard rat feed (Aashirwad Ltd., Chandigarh, India) and tap water ad libitum and assessed for mating and fertility. Each male was placed with an adult female rat in the ratio of 1:2. Females were checked for plugs and if found were caged separately till offspring were born and counted. If a plug was not found after 7 d, the male was scored as not having mated and was removed from the cage, the female was monitored to confirm the pregnancy.

Statistical analysis

Data were expressed as mean ± SEM. Further analysis of 2 and 4 w treatment groups was carried out separately using One way Analysis of Variance (ANOVA) and if the difference was found to be significant then post-hoc test (Duncan's Multiple Comparison Test) was applied. P < 0.05 was considered to be significant.

Abbreviations

ZC:	=	zinc control
PF:	=	pairfed
ZD:	=	zinc deficient
LH:	=	luteinizing hormone
ER:	=	endoplasmic reticulum
FSH	=	follicle-stimulating hormone
ECM:	=	extracellular matrix
CREM:	=	cyclic AMP response element modulator
ACT:	=	activator of CREM
ROS:	=	reactive oxygen species
PUFA:	=	polyunsaturated fatty acids
TUNEL:	=	terminal deoxynucleotidyl transferase [TdT] dUTP nick endlabelling
MVBs:	=	multivesicular bodies.

Acknowledgments

Author Ms. Deepa Kumari, thanks the Department of Science and Technology (DST), Government of Rajasthan, Jaipur [F [5/2] vipro/ DST/ R and D/07-1937] for providing financial assistance, the Department of Zoology, University of Rajasthan, Jaipur [F-41/08/DRS-81/91] for providing Departmental Research Fellowship and the University Grants Commission (UGC), New Delhi [F.4-3/2006 [BSR] 5- 96/2007[BSR] for providing Meritorious Research Fellowship.

Declaration of Interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.