immunohistochemical, histological and ultrastructural evaluation of the ...

Archives of Andrology, 51:395–405, 2005 Copyright # Taylor & Francis Inc. ISSN: 0148-5016 print/1521-0375 online DOI: 10.1080/014850190944375

IMMUNOHISTOCHEMICAL, HISTOLOGICAL AND ULTRASTRUCTURAL EVALUATION OF THE EFFECTS OF LEPTIN ON TESTES IN MICE

I. Kus and M. Sarsilmaz & Department of Anatomy, Faculty of Medicine, Firat University, Elazig, Turkey S. Canpolat, B. Yilmaz, and H. Kelestimur & Department of Physiology, Faculty of Medicine, Firat University, Elazig, Turkey N. Akpolat & Department of Pathology, Faculty of Medicine, Firat University, Elazig, Turkey C. Ozogul & Department of Histology & Embryology, Faculty of Medicine, Gazi University, Ankara, Turkey

& This study was undertaken to examine the effects of leptin on testes in mice. For this purpose, 12 male mice were divided into two groups. Animals in Group I were designated as control. Mice in Group II were injected daily with leptin for 5 days. All animals were decapitated at the end of the experiment. The testes were removed and weighed out. Testicular tissue specimens were processed for light and electron microscopic examination and semi-quantitative evaluation of immunohistochemical testosterone staining. Intensity of immunostaining was determined on a scale between 0 (no staining) and 5 (heavy staining). For morphometric comparison, diameters of seminiferous tubules from each group were measured. In the leptin injected group, testicular weights and diameters of seminiferous tubules were significantly increased in comparison to control values. In light microscopic examination, an increase in secretory granules in the cytoplasm of Leydig cells was observed after leptin treatment. In the same group, distinct changes indicative of increased cell activation were seen in the ultrastructure of Leydig cells. Amount of mitochondria, lysosomes and cytoplasmic secretory granules were increased. Furthermore, an increase in extensiveness of rough endoplasmic reticulum was noted in this group. Immunohistochemical testosterone staining of the cytoplasm of Leydig cells was heavy (5þ) in the leptin treated mice compared to mild score (2þ) in the control mice. Additionally, heavy immunostaining of testosterone was also observed in the interstitial space after injection of leptin. The present findings indicate that testicular functions and synthesis of testosterone increase after administration of leptin. Keywords immunohistochemistry, leptin, light and electron microscopy, testis

Address correspondence to Dr. Ilter Kus, Firat University, Faculty of Medicine, Department of Anatomy, 23119 Elazig, Turkey. E-mail: [email protected]

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Leptin is a 16-kD protein product of the obese gene (ob) produced primarily by adipose tissue. It is believed to play an important role in the regulation of metabolic efficiency, energy expenditure and food intake [17, 19]. Besides its well-known role in body weight homeostasis, increasing evidence suggests that leptin may also play a significant role in the regulation of neuroendocrine functions. Leptin has been implicated in the regulation of the somatotropic, thyroid, adrenal and reproductive axes. Among these, the proposed link between leptin and reproductive function seems to be of special physiological significance in the view of the established role of body fat, the source of leptin, in regulating the reproductive capability [8]. The success of puberty and reproduction is very much dependent on the plentiful availability of food and body energy stores, in particular distribution of white adipose tissue. As the leptin level is related to the status of body fat stores, it follows that leptin could influence fertility and reproductive functions [11, 14]. It has been reported that animals lacking the gene for leptin are sterile and unable to secrete sufficient amounts of gonadotropins [7, 9, 27]. Similarly, the Lepob=Lepob syndrome in mice, which results from a lack of biologically active leptin, is associated with infertility [32]. On the other hand, treatment of ob=ob mice, which exhibit a congenital deficiency in leptin, with leptin restores fertility in both sexes [3, 9, 20]. It has been shown that leptin treatment significantly increases testicular and seminal vesicle weights, and elevates sperm counts compared with controls [3, 20]. Furthermore, leptin has also been implicated in triggering the onset of puberty. In normal mice, leptin treatment at pharmacological doses accelerates the onset of puberty [1, 10]. Recent evidence that leptin is capable of stimulating lutenizing hormone-releasing hormone (LHRH) and luteinizing hormone (LH) release from adult male rat hypothalamic and pituitary tissues incubated in vitro [33] appears to support this hypothesis. However, the effects of leptin on testis morphology have not been well-documented yet. Therefore, in the present study, we aimed to examine the effects of leptin on testicular morphology in mice at light-electron microscopic and immunohistochemical level. MATERIAL AND METHODS Adult male mice (weighing 30–40 g) were used in this study. They were kept at a constant temp (21 1C) and controlled light conditions (light, 07.00–19.00). Food (standard pellet diet) and tap water were supplied ad libitum. All protocols in the present study were approved by the local ethics committee of the Medical School. The animals were divided into two groups. Mice in Group I (n ¼ 6) were designated as control. They were injected with the respective vehicle

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(0.5 ml 15 mM HCL and 0.3 ml 7.5 mM NaOH). Animals in Group II (n ¼ 6) were i.p. (intraperitoneally) injected with leptin (Sigma, mouse recombinant) in a dose of 25 mg=mouse in 0.1 ml 15 mM HCL for 5 days. The drugs were administered between 9:00–10:00 a.m. All animals were decapitated at the end of the experiment and the testes removed. The testes were dissected from the surrounding tissue and weighed out. Some of the testicular tissue specimens were used for light and electron microscopic examination. The remaining specimens were used for immunohistochemical examination. Light and Electron Microscopic Procedures Testicular tissue specimens were fixed in 2.5% glutaraldehyde in 0.2 M phosphate buffer (pH 7.4) at 4C. They were post-fixed in phosphate-buffered 1% osmium tetroxide. After dehydration in acetone, the specimens were embedded in Epon 812. Semi-thin sections were cut on ultramicrotome, stained with Toluidine blue and were used for light microscopy. Thin sections were cut on ultramicrotome, stained with uranyl acetate and lead citrate, and examined in Carlzeiss-900 electron microscope. Immunohistochemical Procedure Testicular tissue specimens were fixed in Bouin’s solution and embedded in paraffin. For immunohistochemical testosterone staining of the cytoplasm of Leydig cells, paraffin sections (thickness, 5 mm) were deparaffinized in xylene, hydrated and then placed in phosphate buffered saline (PBS; pH 7.6). Antigen retrieval was performed by boiling for 15 min in citrate buffer (0.01 M). Sections were treated with 3% hydrogen peroxide for 5 min to quench endogenous peroxidase activity, rinsed with deionized water and then washed with PBS. Sections were incubated first with 1% pre-immune rabbit serum to reduce non-specific staining and then with a monoclonal antibody against testosterone (Bio-Genex, San Ramon CA, USA) at 23C in a moist chamber for 1 h. Detection of the antibody was performed using a biotin-streptavidin detection system (Bio-Genex, San Ramon CA, USA) with 3-amino 9-ethyl carbazole (AEC) as chromogen (Dako, Carpinteria CA, USA). Sections were counterstained with Mayer’s hematoxylin, dehydrated and then cover-slipped with permount. Immunohistochemical testosterone staining of the cytoplasm of Leydig cells was semi-quantitatively evaluated. Intensity of immunostaining was scored as follows: no staining (0), minimal (1þ), mild (2þ), moderate (3þ), strong (4þ), heavy (5þ).

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I. Kus et al. TABLE 1 Mean SEM Weights of the Testes (gr=10 gr Body Weight) in Control Rats and Animals Treated with Leptin Groups Control (n ¼ 6) Leptin treatment (n ¼ 6)

Mean SEM 0.077 0.002 0.102 0.003

Significant difference as compared to control rats (p < 0.05) using Mann-Whitney U test.

Morphometric Measurement For morphometric comparison, in paraffin sections, diameters of 100 seminiferous tubules from each group were measured with ocular micrometer adapted to a microscope, and the mean diameter of seminiferous tubules of each group was determined.

Statistical Analysis Data (testicular weights and diameters of seminiferous tubules) were analyzed by using SPSS software for Windows. Repeated measure analysis of variance (Mann-Whitney U and Student-t) tests were utilized to test significance of differences between groups. Level of significance was set at p < 0.05.

RESULTS Light Microscopic Findings In leptin injected group, testicular weights and diameters of seminiferous tubules were significantly increased as compared to those in control mice (Tables 1 and 2). In light microscopic examination, when compared to control group (Figure 1), an increase in secretory granules in the cytoplasm of Leydig cells was observed after administration of leptin (Figure 2). TABLE 2 Mean SEM Diameters of Seminiferous Tubules (Micrometer) in Control Rats and Animals Treated with Leptin Groups Control (n ¼ 100) Leptin treatment (n ¼ 100)

Mean SEM 172.36 1.32 215.95 1.99

Significant difference as compared to control rats (p < 0.05) using Student-t test.


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FIGURE 1 Semi-thin section stained with toluidine blue of testis in control group. st: seminiferous tubule, arrow: leydig cell, c: capillary. Magnification, 40.

Immunohistochemical Findings In the process of immunohistochemical staining that was performed in order to have the testosterone hormone visible in testicular tissue and to investigate it, an evaluation was made according to the density of staining observed. The more testosterone antigen (testosterone hormone) present in the tissue or in the cell, the more binding will occur and as a result, the darker staining will be established. Immunohistochemical testosterone staining of the cytoplasm of Leydig cells was mild (2þ) and heavy (5þ) in the control (Figure 3) and leptintreated (Figure 4) mice, respectively. In addition, heavy immunostaining of testosterone in the interstitial space was observed after the administration of leptin (Figure 5).

FIGURE 2 An increase in secretory granules in the cytoplasm of Leydig cells (arrows) was observed after administration of leptin. Toluidine blue. Magnification, 40.

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FIGURE 3 Immunohistochemical staining of testosterone in control testis, showing mild levels of testosterone in the cytoplasm of Leydig cells (arrow). Magnification, 40.

FIGURE 4 Immunohistochemical staining of testosterone in mice that were treated with leptin, showing heavy testosterone staining in the cytoplasm of Leydig cells (arrow). Magnification, 40.

FIGURE 5 Immunohistochemical staining of testosterone in animals that were treated with leptin, showing heavy testosterone staining in interstitial space (arrows). Magnification, 40.


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FIGURE 6 Electron micrograph of the Leydig cell (Lc) in control mice. N: nucleus. Magnification, 3000.

Ultrastructural Findings Ultrastructure of Leydig cells in the control group had a normal appearance (Figure 6), distinct changes indicative of increased cell activation were seen in the ultrastructure of Leydig cells following administration of leptin. The amount of mitochondria, lysosomes and cytoplasmic secretory granules at various intensities were increased. Furthermore, an increase in extensiveness of rough endoplasmic reticulum in the cell cytoplasm was noted (Figures 7, 8).

FIGURE 7 Leptin treatment caused an increase of cytoplasmic secretory granules (large arrows) and lysosomes (small arrows) in the cytoplasm of Leydig cells. Additionally, an increase in extensiveness of rough endoplasmic reticulum (rER) in the cell cytoplasm was noticed. N: nucleus, m: mitochondrion. Magnification, 3000.

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FIGURE 8 An increase in the amount of mitochondria (m) was observed in the cytoplasm of Leydig cells after injection of leptin. Magnification, 3000.

In view of our findings, it is concluded that testicular functions and synthesis of testosterone increase after administration of leptin. DISCUSSION Leptin is produced by adipose tissue, and a rising level of leptin as triglyceride stores increase is proposed to serve as a negative feedback signal to the brain, resulting in decreased food intake, increased energy expenditure and resistance to obesity [5, 13, 23]. In addition to this primary role, circulating leptin appears to play an important role in the neuroendocrine axis [2], including regulation of reproductive functions [3, 4, 9, 22, 25]. However, in contrast to its well-documented effects in female fertility, the actual role of leptin in the regulatory network controlling male reproductive functions has been a matter of debate [28]. It has been reported that leptin has central and local effects on the male reproductive functions [28]. It is proposed that stimulatory effects of leptin on testicular functions are exerted at the hypothalamo-hypophysial-gonadal axis. In rats, mice and rhesus monkeys [12, 15, 24], leptin receptors (Ob-R) are localized in high concentrations within the hypothalamus, and reportedly leptin accelerates LHRH pulsatility in the arcuate hypothalamic neurons in a dose-dependent manner [18]. In addition to stimulatory actions of leptin at the hypothalamic level, its direct effects on the anterior pituitary have also been shown [16]. Leptin may directly stimulate LH release by the pituitary via NO synthase activation in gonadotropes. Results from pituitary tissue culture studies demonstrated that leptin induces a dose-related increase in LH release [33]. This appears to be consistent with studies demonstrating the ability of exogenous leptin to


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increase serum LH levels in fasted female mice and prevent reduced pulsatile LH secretion in fasted rats [2, 21, 30]. Furthermore, studies examining the effects of leptin on gonadotropin secretion in fasted male rhesus monkeys have shown that leptin prevents fasting-induced suppression of plasma LH and FSH (follicle-stimulating hormone) levels [12]. Similarly, Barash and colleagues [3] found that in ob=ob mice, leptin administration increases basal LH levels. In line with the results from experimental studies, in humans, the absence of endogenous leptin is associated to hypogonadism and absence of pubertal development [26, 31]. In the present study, testicular weights and diameters of seminiferous tubules were significantly increased after injection of leptin as compared to those in control mice. In light microscopic examination, an increase in secretory granules in the cytoplasm of Leydig cells was observed. Distinct changes in the ultrastructure of Leydig cells were also taken as an indication of increased cell activation following leptin administration. Additionally, our immunohistochemical findings showed that an increase in testosterone synthesis of Leydig cells in mice treated with leptin. Although LH and FSH were not determined in our study, all the testicular findings outlined above implicate an increased gonadotropic hormone activity as a result of leptin’s action at the hypothalamic and=or anterior pituitary level. Our findings thus appear to be in agreement with the reports suggesting a stimulatory role for leptin in the regulation of testicular functions. On the other hand, it has been reported that leptin has a direct local inhibitory effect on testicular function. Caprio and colleagues [6] have shown that a functional leptin receptor is expressed in rat Leydig cells and leptin can inhibit LH=hCG-stimulated testosterone and androstenedione production in the Leydig cells. In another in vitro study by TenaSempere and colleagues [29], inhibition of testosterone secretion by leptin in the rat testes was found. In contrast, the present immunohistochemical findings have shown an increase in synthesis of testosterone in the Leydig cells following administration of leptin. Therefore, we suggest that leptin plays a stimulatory role in testosterone secretion through actions exerted either locally and=or at the hypothalamo-pituitary level. In conclusion, the present immunohistochemical, histological and ultrastructural findings have provided further evidence that leptin has a stimulatory influence on testicular functions including the synthesis of testosterone in mice. REFERENCES 1. Ahima RS, Dushay J, et al. (1997): Leptin accelerates the onset of puberty in normal female mice. J Clin Invest 99:391–395. 2. Ahima RS, Prabakaran D, et al. (1996): Role of leptin in neuroendocrine response to fasting. Nature 382:250–252.

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