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Effect of a zinc-free diet from weaning through puberty on the structure of the testis in albino rats, with a special focus on the Leydig cells

Khalaf, Gehan; Fahmy, Hala F.; Saleh, Hanan A.

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The Egyptian Journal of Histology: June 2014 - Volume 37 - Issue 2 - p 304-315
doi: 10.1097/01.EHX.0000446584.81324.b0
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Abstract

Introduction

Zinc (Zn) deficiency is one of the most common deficiencies prevalent worldwide. The Food and Agricultural Organization suggested that about half of the world’s population was at risk for Zn deficiency. The WHO, however, estimated that Zn deficiency affected one-third of the world’s population (about two billion individuals), with the prevalence rates reaching 73% in certain regions (WHO, 2002) 1.

Zn deficiency could lead to a host of problems such as poor immune function 2, slow wound healing 3, DNA damage, cancer, and infertility 4.

Zn is found mainly in protein-rich food. Although the animal protein is expensive, the plant protein, however, is rich in phytate that would prevent Zn absorption 5. Food additives and chemicals used in food processing such as EDTA could also reduce Zn absorption. Furthermore, Zn is not well stored in the body; consequently, a reduction in dietary intake could lead to evident deficiency in a short time 6. Young children as well as populations in the developing countries who consume limited animal products are generally at an increased risk of Zn deficiency 1.

Many physiological processes would be impaired with low Zn intake. It has been considered an essential element in over 300 different biological processes. These included DNA transcription, protein translation, cell proliferation and differentiation, and apoptosis 7.

Zn is abundant in the male reproductive system. The highest Zn concentration in all organs of the body was found in the adult prostate, followed by the testes. The role of Zn in the testicular function has been to promote the maturation of testis germinal epithelium 8.

In vivo experiments in rodents have shown that Zn deficiency resulted in severe damage to the testes and inhibition of spermatid differentiation 9. In epidemiological studies in humans, the inhibition of spermatogenesis and sperm abnormalities have been observed in patients with Crohn’s disease and nutritional disorders, both of which induce Zn deficiency 10. Moreover, delayed sexual maturation and infertility have been related to dietary Zn deficiency 7.

In the literature, very few histological studies have focused on the effect of dietary Zn deficiency on puberty and sexual maturation. Hence, the aim of this work was to detect the structural changes in the testis following a Zn-free diet during the peripubertal period in male rats. In addition, special attention will be paid to the Leydig cells.

Materials and methods

Animals

Twenty weaned male albino rats (3-week-old, 40–50 g) were used in this study. They were obtained from the Animal House of the Scientific Research Centre at Ain Shams University. The animals were housed in wire mesh cages and were maintained under standard laboratory conditions (14 h light: 10 h dark, 24±2°C). They were fed on formulated diets and were allowed tap water ad libitum.

All experimental procedures were performed in accordance with the guidelines of the Institutional Animal Care and Use Committee of the Research Center, Faculty of Medicine, Ain Shams University, Cairo, Egypt.

Diet

Two diets were prepared in the Tests and Consulting Unit (food analysis and design for special groups) at the National Research Centre. One was a balanced diet and the other was a Zn-free diet (Table 1).

Table 1
Table 1:
Composition of the balanced and zinc-free diets

Experimental groups

Twenty weaned rats were divided randomly into three groups. Group I (the control group) included 10 rats that were fed a balanced diet. Five rats were sacrificed after 3 months (group IA) and five rats were sacrificed after 4 months (group IB). Group II (the Zn-free group) included five rats that were fed a Zn-free diet for 3 months. Group III (the recovery group) included five rats that were fed a Zn-free diet for 3 months, followed by a balanced diet for 1 month.

Before sacrifice, the rats were weighed and blood samples were collected to measure the serum testosterone hormone level. This was measured at the Laboratory Unit of the Biochemistry Department, Faculty of Medicine, Ain Shams University. The rats were sacrificed by decapitation. The left testes were dissected, removed, and weighed.

The right testes were dissected and processed for microscopic examination.

For light microscopic examination, specimens were fixed in 10% formol-saline, dehydrated, cleared, and embedded in paraffin. Thin sections (5 μm) were cut and stained with H&E.

Transmission electron microscope examination

Small testis specimens (1 mm3) were fixed in a 2.5% chilled gluteraldehyde solution. They were then postfixed in 1% osmium tetroxide, dehydrated, and embedded in Epon. Semithin sections were stained with 1% toluidine blue. Ultrathin sections were cut, stained with uranyl acetate and lead citrate, and then examined using transmission electron microscope (TEM) 1010-EXII (Joel, Tokyo, Japan) at the Regional Mycology and Biotechnology Unit, AL Azhar University, Cairo, Egypt.

Statistical study

Student’s t-test was used to compare between the mean values of the body weight, testis weight, and serum testosterone level of group II (Zn-free group) and group III (recovery group) with the corresponding control group (n=5).

Results

Body weight

There was a significant decrease in the mean body weight of the rats in group II (Zn-free group) compared with that of the control group. The recovery group (group III) showed a nonsignificant change in the body weight compared with that of the control group (Table 2).

Table 2
Table 2:
Mean body weight (g) and testis weight (g) of the different groups

Testis weight

The Zn-free group showed a significant decrease in the mean testis weight compared with that of the control group. A nonsignificant change was observed in the mean testis weight of the recovery group compared with the control group (Table 2).

Serum testosterone level

The mean serum testosterone level of the Zn-free group was significantly decreased compared with that of the control group, whereas that of the recovery group was nonsignificantly changed from that of the control rats (Table 3).

Table 3
Table 3:
Mean serum testosterone level (ng/ml) in the different groups

Light microscopic examination

Group I (the control group)

Examination of H&E and toluidine blue-stained sections of the control group showed that the testis was formed of seminiferous tubules (STs) separated by interstitial tissue. The STs were lined by several layers of spermatogenic epithelium and were surrounded by a basement membrane (Fig. 1).

Figure 1
Figure 1:
Seminiferous tubules (T) lined by several layers of spermatogenic cells. The tubules are separated by interstitial tissue (i). Control group, H&E, ×100.

The lining epithelium of the STs included spermatogenic cells and Sertoli cells. The spermatogenic cells included spermatogonia, primary and secondary spermatocytes, and early and late spermatids. Spermatogonia were detected basally on the basement membrane. They appeared rounded and showed rounded nuclei. Primary spermatocytes were the largest cells and showed various stages of chromatin coiling. Secondary spermatocytes were small and rarely detected. Early spermatids appeared rounded in the adlumenal compartment. They showed rounded nuclei with prominent acrosomal vesicles. Late spermatids were attached to the apices of Sertoli cells. Sertoli cells were situated on the basement membrane and showed large vesicular nuclei (Figs 2 and 3).

Figure 2
Figure 2:
Seminiferous tubules are lined by several layers of spermatogenic cells and the lumen contains spermatozoa. Note clusters of Leydig cells (L) with their vesicular nuclei in the interstitial tissue. Control group, H&E, ×640.
Figure 3
Figure 3:
Spermatogonia (g), primary spermatocytes (p), and early spermatids (d) with acrosomal vesicles (↑). Late spermatids ([Black up-pointing triangle]) are attached to Sertoli cells (s). Control group, toluidine blue, ×1000.

The interstitial cells of Leydig were detected in the interstitial tissue in between the tubules (Figs 1 and 2). Leydig cells showed rounded vesicular nuclei and vacuolated cytoplasm (Figs 3 and 4). Myoid cells were observed surrounding some of the STs.

Figure 4
Figure 4:
Interstitial tissue contains Leydig cells (L) with vesicular nuclei and vacuolated cytoplasm. Myoid cells (↑) can be seen surrounding the seminiferous tubule (T). Control group, toluidine blue, ×1000.

Group II (zinc-free group)

Examination of H&E and toluidine blue-stained sections showed marked alteration in the structure of the testis. Most of the STs appeared to be lined by few spermatogenic cells (Fig. 5). They were lined primarily by spermatogonia and Sertoli cells. Some STs showed more cells than others. The rest of the spermatogenic cells (primary and secondary spermatocytes, early and late spermatids) were absent in most of the tubules (Figs 6 and 7). Acidophilic cytoplasmic remnants were detected in the lumena of some STs (Fig. 6).

Figure 5
Figure 5:
Seminiferous tubules (T) lined by very few spermatogenic cells. C, capsule. Group II, H&E, ×100.
Figure 6
Figure 6:
Seminiferous tubules (T) are lined mainly by spermatogonia (g) and Sertoli cells (s). There is loss of spermatocytes. There are acidophilic cytoplasmic remnants (*) in the lumen. Group II, H&E, ×640.
Figure 7
Figure 7:
(a) A seminiferous tubule lined by very few cells. (b) Another seminiferous tubule lined by more layers. In both tubules, the cells are only spermatogonia (g) and Sertoli cells (s). Note a vacuolated Leydig cell (L) in the interstitium. Group II, H&E, ×1000.

Few STs showed marked disorganization of the spermatogenic cells. Few spermatogonia and Sertoli cells were identified on the basement membrane. The different types of spermatogenic cells were not distinguished. Most of the cells showed haphazard chromatin content and karyorrehxis and only a few cells showed mitosis as evidenced by anaphase (Fig. 8).

Figure 8
Figure 8:
Disorganized spermatogenic cells with haphazard chromatin content. Spermatogonia (g) were detected on the basement membrane. Other cells could not be identified. Note that a cell in anaphase (↑) and other cells show karyorrehxis ([Black up-pointing triangle]). Group II, H&E, ×1000.

Other STs showed arrested spermatogenesis at the primary spermatocyte stage. Some primary spermatocytes showed karyorrhexis (Fig. 9).

Figure 9
Figure 9:
Spermatogonia (g) and Sertoli cells (s). Few primary spermatocytes (p) can be detected. Note a large cell with karyorrhexis (↑). M, myoid cells. Group II, toluidine blue, ×1000.

The interstitial tissue showed areas of exudate and vacuolation. Some of the interstitial cells of Leydig showed pyknotic nuclei and vacuolated cytoplasm. Other cells showed markedly irregular nuclei (Figs 7 and 10).

Figure 10
Figure 10:
Areas of exudate (E) in the interstitial tissue. Most of the Leydig cells have irregular nuclei (↑) and some have pyknotic nuclei (thick arrows). Vacuolation (v) is also detected. Group II, toluidine blue, ×1000.

Group III (the recovery group)

Examination of the sections from group III showed a structure of the testis that was very similar to that of the control group. The STs were lined by several layers of spermatogenic epithelium and separated by interstitial tissue (Fig. 11). The spermatogenic epithelium included spermatogonia resting on the basement membrane. The primary spermatocytes appeared large, with rounded nuclei. Early spermatids were detected with the characteristic acrosomal vesicle. Late spermatids were also observed attached to the apices of Sertoli cells (Fig. 12a). Sertoli cells showed vesicular nuclei and rested on the basement membrane (Fig. 12b).

Figure 11
Figure 11:
Most of the seminiferous tubules (T) are lined by several layers of spermatogenic epithelium. In between the tubules is the interstitial tissue (i). Group III, H&E, ×100.
Figure 12
Figure 12:
(a) Spermatogonia (g) resting on the basement membrane. Primary spermatocytes (p) and early spermatids (d) with acrosomal vesicle (↑) can be detected. Late spermatids are attached to apices of Sertoli cells ([Black up-pointing triangle]). (b) Leydig cells (L) with their vesicular nuclei and vacuolated cytoplasm appear in the interstitial tissue. Note nearby Sertoli cells (s). Group III, toluidine blue ×1000.

The interstitial cells of Leydig appeared in the interstitium with vesicular nuclei and vacuolated cytoplasm (Fig. 12b).

Electron microscopic examination

Control group

By TEM examination, the ST appeared to be surrounded by a basement membrane and myoid cells. The spermatogonia rested on the basement membrane. More rounded cells, most probably type B, showed rounded nuclei with peripheral chromatin (Fig. 13). Type A cells were oval and more electron dense and showed oval nuclei (Fig. 14a).

Figure 13
Figure 13:
Basement membrane and myoid cells (m) surrounding the seminiferous tubules. Spermatogonia (g), primary spermatocyte (p), and early spermatids (d) can be detected. Note the electron-dense acrosomal cap (↑). Group I, TEM, ×2000.
Figure 14
Figure 14:
(a) Spermatogonia (g) and Sertoli cell (s) lying on the basement membrane. Primary spermatocytes (p) show heterochromatic nuclei. (b) An early spermatid shows the acrosomal cap ([Black up-pointing triangle]), prominent Golgi (G), and peripheral mitochondria (↑). (c) Middle piece of the sperm showing central microtubules, coarse fibers, and peripheral mitochondria (m). Control group, TEM, (a) ×4000, (b) ×10 000, (c) ×20 000.

Primary spermatocytes appeared with moderately electron-dense nuclei and dispersed heterochromatin. The cytoplasm showed dispersed mitochondria (Figs 13 and 14a). Early spermatids appeared with their characteristic acrosomal cap, prominent Golgi, and peripheral mitochondria (Figs 13 and 14b). Many sperm were detected. The middle piece of the sperm showed central microtubules surrounded by nine coarse fibers and peripheral mitochondria arranged circumferentially (Fig. 14c).

The interstitial tissue showed blood vessels and interstitial cells of Leydig (Fig. 15a). The Leydig cells showed euchromatic nuclei and prominent nucleoli. The cytoplasm showed mitochondria and smooth endoplasmic reticulum (SER) with narrow tubules (Figs 15b and 16).

Figure 15
Figure 15:
(a) An interstitial cell of Leydig (L) near a blood vessel (v) in the interstitial tissue. (b) The Ledyig cell shows a large euchromatic nucleus (N) with a prominent nucleolus. The cytoplasm shows mitochondria, smooth endoplasmic reticulum, and glycogen. Group I, TEM, (a) ×2500, (b) ×10 000.
Figure 16
Figure 16:
An interstitial cell of Leydig with a euchromatic nucleus (N) and a prominent nucleolus (n). The cytoplasm shows prominent mitochondria (m), small tubules of smooth endoplasmic reticulum (↑), lipid droplets (Lp), and few cisternae of rough endoplasmic reticulum. Group I, TEM, ×20 000.

Group II (the zinc-free group)

By TEM, most of the STs in this group appeared to be lined by spermatogonia and Sertoli cells. The spermatogonia appeared similar to those of the control group, with euchromatic nuclei (Figs 17 and 18). The Sertoli cells were more prominent. They showed highly indented nuclei. Most of their nuclei showed accumulation of the chromatin into small clumps (Fig. 18). The cytoplasm showed the characteristic crystals that appeared as filamentous structures. Some Sertoli cells were shed in the lumen (Fig. 19).

Figure 17
Figure 17:
A seminiferous tubule with a discontinuous basement membrane (↑). The lining cells are spermatogonia (g) and Sertoli cells (s). The Sertoli cells shows highly indented nuclei (N) and the cytoplasm shows vacuoles (v). Group II, TEM, ×3000.
Figure 18
Figure 18:
(a, b) Higher magnification of the previous section showing spermatogonia (g) and the highly indented nucleus of Sertoli cell (S). The cytoplasm of sertoli cell contains mitochondria (m) and the characteristic filamentous crystals (↑). Group II, TEM, (a) ×8000, (b) ×15 000.
Figure 19
Figure 19:
A seminiferous tubule lined by spermatogonia (g). A Sertoli cell (s) is shed in the lumen. The nucleus is highly indented and shows a very small clump of chromatin (↑). Inset: an early spermatid (d) is detected with a nucleus and little cytoplasm. The mitochondria are peripherally situated. Group II, TEM, ×4000, inset ×6000.

Early spermatids appeared in a few STs with a decrease in cytoplasm and electron-dense nuclei (Fig. 19, inset).

The interstitial cells of Leydig showed variable significant findings. Most of the Leydig cells showed irregular euchromatic nuclei. The cytoplasm was packed with organelles. These included extensive dilated tubules of SER, many mitochondria, vacuoles, and occasional peroxisomes. The mitochondria and the SER were in close proximity to each other at many sites (Fig. 20). There were also dilated elements of rough endoplasmic reticulum (RER) in many cells (Fig. 21). Other Leydig cells showed heterochromatic nuclei, dilated tubules of SER, and vacuolation in the cytoplasm (Fig. 22). In addition to the previous findings, a small number of Leydig cells showed few organelles and accumulation of glycogen granules in the cytoplasm and vacuolation (Fig. 23).

Figure 20
Figure 20:
A Leydig cell with an irregular nucleus (N). The cytoplasm shows dilated engorged tubules of smooth endoplasmic reticulum (SER) (sr). Many mitochondria (m) are in close contact with the SER (sr) at multiple sites (↑). Vacuoles (v) can also be detected. Note the peroxisome ([Black up-pointing triangle]). Group II, TEM, ×20 000.
Figure 21
Figure 21:
Two Leydig cells with euchromatic nucleus (N), dilated cisternae of rough endoplasmic reticulum (↑) and mitochondria (m). Group II, TEM, ×20 000.
Figure 22
Figure 22:
A Leydig cell with a hyperchromatic nucleus (N). The cytoplasm shows vacuolation (v), mitochondria (m), and dilated tubules of smooth endoplasmic reticulum (sr). Note elements of rough endoplasmic reticulum. Group II, TEM, ×20 000.
Figure 23
Figure 23:
A Leydig cell with a euchromatic nucleus (N). The cytoplasm shows abundant accumulation of glycogen (G) and vacuolation (v). Group II, TEM, ×20 000.

Group III (the recovery group)

The recovery group showed intact spermatogenic cells in different stages of maturation similar to the control group (Fig. 24). The Ledyig cells showed euchromatic nuclei with prominent nucleoli. The cytoplasm contained many mitochondria and SER (Fig. 25). Few Leydig cells showed variable elements of SER; some tubules were narrow, whereas others were dilated (Fig. 26).

Figure 24
Figure 24:
A seminiferous tubule with many layers of spermatogenic cells in different phases of maturation. Early spermatids (d) can be detected with acrosomal vesicles (↑). Late spermatids ([Black up-pointing triangle]) are attached to Sertoli cells. Group II, TEM, ×2000.
Figure 25
Figure 25:
A Leydig cell with a euchromatic nucleus (N) and prominent nucleoli. The cytoplasm shows many mitochondria (m), few lipid droplets P (Lp), and elements of smooth endoplasmic reticulum. Little exudate (E) can be detected in the interstitial tissue. Group III, TEM, ×6000.
Figure 26
Figure 26:
A Leydig cell with a euchromatic nucleus (N). The cytoplasm showed many mitochondria (m) and smooth endoplasmic reticulum that showed narrow tubules (↑) and dilated tubules ([Black up-pointing triangle]). Group III, TEM, 20 000.

Discussion

There is a general belief that Zn deficiency cannot occur in humans because Zn is assumed to be ubiquitous and plentiful in our diets. Today, Zn deficiency is recognized as a nutritional problem worldwide, pandemic in both developed and developing countries. Infants and children are particularly at a high risk 1.

It is known that Zn plays a key role in spermatogenesis from several perspectives. It is located primarily in the Leydig cells, the late type B spermatogonia, and the spermatids. Zn is essential for the production and secretion of testosterone from the Leydig cells, which, in conjunction with gonadotropins, is a key regulator of spermatogenesis 7. This study aimed to investigate the structural changes in the testis, particularly the Leydig cells, when exposed to a Zn-free diet in the peripubertal period in male rats.

Rats are considered to be pubertal at 50 days of age and sexually mature at 100 days of age 11. Accordingly, weaned rats were fed a Zn-free diet for 3 months in the present study. During this period, rats would have reached puberty and became sexually mature, which would be equivalent to 14–18 years of age in humans (http://www.rattyrat.com/guidebook/howold.html).

In the present work, the Zn-free group (group II) showed a significant reduction in body weight compared with the control group. The first recognizable clinical presentation of Zn deficiency was growth retardation 1. There was also a significant decrease in the testis weight compared with the control group. It appears evident that this decrease could be partly because of the generalized decrease in body weight and partly because of the direct effect of Zn deficiency on the testis.

In terms of the serum testosterone level, the Zn-free group (group II) showed a significant decrease in comparison with the control group. Similarly, dietary Zn restriction in normal young men was associated with a significant decrease in serum testosterone concentrations after 20 weeks of Zn restriction 12.

Group II also showed extensive structural alterations in both the STs and the interstitial cells of Leydig. The STs showed loss of spermatogenic cells, except for spermatogonia and Sertoli cells. The few primary spermatocytes and early spermatids detected in group II showed karyorrhexis and haphazard chromatin content. This is similar to previous studies in which Zn deficiency caused damage to the testes in the form of atrophy of the STs and inhibition of spermatid differentiation. The authors detected pyknosis, karyorrhexis, and karyolysis of the spermatogenic cells, with very few sperm in the lumen 9,13.

It is documented that Zn is highly concentrated in the developing spermatocytes as it is essential during DNA condensation and meiosis 14. Zn also facilitates the packaging of DNA in spermatids 15 and is considered to prolong spermatozoa life span once released after ejaculation 16,17.

The Zn concentration in the testis increases during spermatogenesis and accumulates mainly in germ cells. Zn deficiency causes inhibition of DNA synthesis in germ cells and induces an apoptotic response. It is suggested that Zn could be an essential trace element for the maintenance and regulation of both spermatogenesis and sperm motility 18.

In the present study, most of the interstitial cells of Leydig showed signs of overstimulation and hypertrophy in the Zn-free group. The cells showed irregular nuclei, dilatation of SER, and approximation between the SER and the mitochondria. These ultrastructural changes were described previously as characteristics of an active state of these cells 19. Another study reported that hypertyophied Leydig cells showed a marked increase in the quantities of SER, polymorphous mitochondria, and an enlarged Golgi complex 20.

The Leydig cells in group II also showed dilated cisternae of RER. Similarly, it was reported that the dilated and increased elements of SER are often accompanied by dilatation of the cisternae of RER 19.

It was reported that in young adult rats, the absolute volume of peroxisomes per Leydig cell correlated significantly with the absolute volume of SER per Leydig cell. These results, combined with the ultrastructural observations of close apposition of peroxisomes and SER, suggested that peroxisomes play a role in testosterone secretion by Leydig cells 21. This was not observed in the present study as the peroxisomes were only occasionally detected in the Zn-free group.

However, some Leydig cells in group II (the Zn-free group) showed widespread accumulation of glycogen with few organelles. In terms of this, Prince 22 detected cytoplasmic accumulation of glycogen in the immature human Leydig cells. He reported that glycogen was more prevalent in the cells with a reduced amount of SER 22.

Furthermore, glycogen granules are not normally observed in mature Leydig cells and may represent a storage form of carbohydrate in the absence of active secretion 23. Accordingly, it could be assumed that in the absence of Zn, some Leydig cells failed to enter the pubertal phase and retained their prepubertal glycogen.

In contrast to the present findings, previous studies reported severe Leydig cell damage in Zn-deficient rats 14,24. The authors detected decreased Leydig cell nuclear diameter, pyknosis, and apoptosis.

In this study, the low serum testosterone level detected in the Zn-free group would result in a feedback mechanism on the hypothalamopituitary axis. This would lead to gonadotropin release to stimulate the Leydig cells to secrete testosterone 25. The hyperactive Leydig cell could not compensate for the decreased level of testosterone in the absence of Zn. Therefore, it could be deduced that Leydig cell failure to increase the testosterone level was because of Zn deficiency.

In the recovery group, when the Zn-deficient rats were supplemented by a Zn-sufficient diet, the STs and the Leydig cells were very similar to those of the control group. In addition, the serum testosterone level was non significantly changed as compared with the control group. Leydig cells regained the ability to produce testosterone and subsequently spermatogenesis.

Zn-depletion studies in men reported oligospermia, which coincided with poor Leydig cell function and lower testosterone concentration, and was reversed by Zn supplementation. Therefore, Zn is an essential element for the synthesis of testosterone 26.

Furthermore, previous experiments have shown that treatment with intracellular Zn chelators caused germ cell death, which was prevented by the addition of Zn. It was also reported that Zn plays an important role in DNA synthesis involving mitotic cell proliferation and meiosis 17.

However, the exact and detailed mechanism of by which Zn affects the Leydig cells and spermatogenesis remains to be clarified in further studies.

Conclusion

Dietary Zn deficiency altered the structure of the testis including the STs and the interstitial cells of Leydig. Most of these changes were reversed when Zn was supplemented.

It is highly recommended to provide proper Zn supplementation in the diet during infancy and childhood to ensure proper gonadal maturation and sexual development.

Further studies are required to detect the exact mechanism by which Zn deficiency affects the testis.

Acknowledgements

Conflicts of interest

There are no conflicts of interest.

Table
Table:
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References

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Keywords:

Leydig cells; puberty; testis; ultrastructure; zinc

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